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|Publication number
|Publication type
|Application number
|Publication date
|Jul 8, 1997
|Filing date
|Aug 15, 1994
|Priority date
|Feb 4, 1988
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|Publication number
|Christian Klein, Christa Hubner-Parajsz, Hans-Georg Batz, Wolfgang Rollinger, Ulrich Essig, Lorenz Kerscher
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|Boehringer Mannheim Gmbh
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|Patent Citations (9), Non-Patent Citations (6), Referenced by (8), Classifications (31), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 08/061,830, filed May 14, 1993 (now abandoned) which is a continuation of application Ser. No. 07/873,663 filed Apr. 23, 1992 (now abandoned), which is a continuation of application Ser. No. 07/304,808, filed Jan. 31, 1989 (now abandoned).
The present invention is concerned with an immunogen for obtaining HbA1c -specific antibodies, as well as a process for obtaining these antibodies.
Hemoglobin, which brings about the transport of inhaled oxygen and of carbon dioxide and is localized in the red blood cells, consists of four chains, of which, in each case, two have the same structure. It consists preponderantly of two α-chains and two β-chains. Hemoglobin is present in blood in an amount of more than 90% in this form, which is referred to as HbA0.
Glycosylated hemoglobin is formed in vivo by the non-enzymatic reaction of hemoglobin with glucose. The glycosylation proceeds via the formation of a Schiffs base between the aldehyde group of the glucose and the amino group of the hemoglobin. The aldimine formed rearranges by an Amadori rearrangement to give the N-(1-desoxy-D-fructos-1-yl) radical. The glycosylated hemoglobin is stable in this rearranged form.
The glycosylated hemoglobins see designated with HbA1 and the most important one of this group with HbA1c. HbA1c is formed by the glycosylation of the free amino group of the valine residue which is present on the amino end of the β-chain of the hemoglobin. There is thereby formed an N-(1-desoxy-D-fructos-1-yl)-L-valine radical which, in the following, is referred to as "fructose valine".
The concentration of HbA1c in the blood is dependent upon the sugar concentration of the blood. In the case of adults, the proportion of HbA1c in the total hemoglobin is normally in the range of from 3 to 6%. In the case of an increased blood sugar level, the proportion of glycosylated hemoglobins in the total hemoglobin increases and to as much as 15% of total hemoglobin. Therefore, the determination of the proportion of HbA1c is s dependable parameter for monitoring sugar metabolism. Since an erythrocyte and, with it, the stable HbA1, has an average life span of 120 days, the determination of the proportion of glycosylated hemoglobins in the blood offers a good parameter for monitoring the carbohydrate metabolism, which is especially important in the case of diabetic patients. This parameter is independent of a brief increase of the blood sugar level after a carbohydrate-rich meal and serves, therefore, as a long-term parameter.
For the diagnosis of diabetes and for monitoring diabetic patients, it is, therefore, important specifically to determine the proportion of HbA1c in the blood.
There are a number of processes for the analysis of glycosylated hemoglobins. The most common methods depend upon the loss of positive charges in the hemoglobin molecule when the free amino groups of the β-chains are reacted with glucose. In particular, column chromatographic and electrophoretic processes are used. Other methods attempt to determine colorimetrically the incorporated glucose molecules or the fructose molecules after the Amadori rearrangement (thiobarbituric acid method).
The previously known methods are, in part, very time consuming and laborious and, in part, are not sufficiently specific for glycosylated hemoglobin.
Therefore, there is a need for s simple process of determination which very specifically determines this glycosylated proportion of the total hemoglobin.
Such simple processes are the various variants of immunoassays which are known to the expert. In the case of homogeneous competitive immunosssays, for example, a labelled HbA1c derivative competes with HbA1c from the sample to be measured for the antibodies. As labelled HbA1c derivative there can be used a short synthetic peptide, which represents an epitope of the HbA1c, which is bound to a label. In order to be able to carry out the determination reproducibly with precise results, an antibody must be used which binds the labelled peptide and the HbA1c from the sample with substantially the same high affinity in order to make possible a competitive reaction. Therefore, for carrying out the immunosssays, antibodies must be available which recognise HbA1c very specifically, i.e. which specifically bind the glycosylated N-terminal end of the HbA1c β-chain but do not bind the corresponding non-glycosylated N-terminal end of the HbA0 β-chain.
A further variant of the competitive immunoassay is carried out in hererogenous phase. In this case, for example, a solid phase-bound synthesized peptide, which contains an HbA1c epitope, competes with the HbA1c from the sample for the specific antibody. The solid phase-bound peptide can be, for example, a conjugate of bovine serum albumin and a synthetic glycosylated peptide which has the sequence of an epitope of HbA1c. For the carrying out of the variant, too, the antibody should bind the peptide and the HbA1c from the sample with substantially equally high affinity.
In order to be able to carry out a sensitive and specific process for the detection of HbA1c, it was important to obtain an antibody which specifically binds HbA1c but not HbA0.
Processes for the detection of HbA1c are already known, as well as antibodies suitable for the process, for example from published European Patent Application No. 0,185,870. However, all known antibodies suffer from the disadvantage that they show a very low affinity for the HbA1c molecule. Therefore, as a rule, before carrying out s determination, denaturing must be carried out in order to liberate the antigenic determinants of the HbA1 to such an extent that the antibodies can be bound to a sufficient extent. Such s process is laborious.
Furthermore, the immunogens used in the case of the process according to published European Patent Application No. 0,185,870 have the disadvantage that they bring about an immune response of only a low specificity. Thus, according to the process described therein, only polyclonal sheep and mouse sera could be obtained which have no measurable specificity with regard to the differentiation of HbA1c and HbA0.
From published Federal Republic of Germany Patent Specification No. 34 39 610, a process is known for obtaining antibodies against HbA1c in which, as immunogen, there is used a conjugate of a sugar, a peptide residue of the β-chain of the hemoglobin and an immunogenic carrier. However, the antibodies obtained with this process do not show sufficient selectivity and their relative affinity is low.
Therefore, it is an object of the present invention, starting from this prior art, to provide a process which yields highly specific polyclonal and monoclonal antibodies against HbA1c with high affinity.
A further object of the present invention is to provide antibodies which display molar relative affinities towards the native HbA1c molecule and a peptide which has the N-terminal sequence of the β-chain of the hemoglobin which differ by a factor which is as small as possible.
Thus, according to the present invention, there is provided an immunogen for obtaining HbA1c -specific antibodies which immunogen has the formula: ##STR3## wherein m is 1 to 40, T is a carrier protein and A is a spacer with a chain length of 10 to 20 atoms which has the general formula: ##STR4## wherein X is a sulphur atom or an --NH-- group, n is 1 to 4 and B is an organic radical which contains a succinimide group.
Surprisingly, we have found that in the case of the use of the immunogens according to the present invention, high specific antibodies can be obtained, the selectivity and affinity of which far exceeds those of the previously known HbA1c antibodies.
The immunogen according to the present invention consists of three parts: a hapten part which corresponds to the N-terminal end of the HbA1c protein, a spacer and an immunogenic protein. The hapten part of the immunogen according to the present invention contains the first 4 amino acids of the N-terminal end of the β-chain of hemoglobin and, additionally, a fructose molecule. As in the native HbA1 protein, in the immunogen according to the present invention, the amino acids valine, histidine, leucine and threonine are bound to the C1 atom of the fructose molecule.
The preparation of the hapten part takes place in known manner. Solid phase synthesis (survey in G. Barany and R. W. Merrifield in Gross, Meienhofer, The Peptides, Vol. 2, pp. 3-285, New York, 1978) has proved to be especially suitable. For this purpose, the α-amino groups are advantageously protected with Nα-fluorenylmethoxycarbonyl radicals. The side chain functional groups are protected as tert.-butyl ether, tert.-butyl ester or as tert.-butoxycarbonyl radicals. In the case of the acidolytic splitting off of the finally synthesized peptide from the carrier, these protective groups are removed at the same time. The N-(1-desoxy-D-fructos-1-yl) radical is introduced in known manner by reaction of the peptide with glucose.
By subsequent Amadori rearrangement (cf. K. Heyns and H. Paulsen, J. Liebigs Ann. Chem., 627, 160-174/1959 and H. Roper et al., Carbohydrates, 116, 183-195/1983), there is obtained the desired N-(1-desoxy-D-fructos-1-yl)-peptide.
A further component of the immunogenic conjugate, which is important for the present invention, is the spacer which binds the hapten with the immunogenic protein. According to the invention, the spacer has a chain length of 10 to 20 atoms, whereby the chain can be formed from carbon, oxygen, nitrogen and/or sulphur atoms. For the chain length, only the chain-forming atoms are counted but not the hydrogen atoms or the side group atoms.
The spacer is bound to the carboxyl group of the threonine.
The spacer has the following general formula: ##STR5## wherein X is a sulphur atom or an --NH-- group and n is a whole number of from 1 to 4. Preferably, X is a sulphur atom and n is 1 or X is an --NH-- group and n is 4.
The group B of the above general formula represents a known spacer molecule which contains a succinimidyl group. The construction of the group B is in itself not critical but it must have so many chain molecules that, together with the rest of the spacer part, it has a chain length of 10 to 20 atoms. A succinimidyl-hexanoyl radical is preferably used as group B.
The spacer is introduced in known manner. It contains an amino acid which can be bound precisely on the C-terminal end of the peptide and which carries an NH2 -- or SH-- group. The succinimidyl radical bringing about the binding with the carrier protein can be introduced via this amino or mercapto group. As amino acid,it is preferred to use cysteine, homocysteine, lysine or ornithine. If, as amino acid, the spacer contains cysteine or homocysteine, then there can first be used cysteine or homocysteine with a protected mercapto group, in which case a tert.-butylsulphenyl radical is preferably used as protective group, as starting amino acid for the solid phase synthesis of the peptide. Subsequently, the carrier protein is reacted with a bifunctional linker which provides the succinimidyl radical, for example maleimidohexanoyl-N-hydroxy-succinimide, and then the spacer-carrier protein conjugate is coupled to the liberated mercapto group of the cysteine or homocysteine. If, as amino acid, the spacer contains lysine or ornithine, then the amino acid with protected α-amino group is preferably first used as starting amino acid for the solid phase synthesis of the peptide, in which case, as protective group, there is preferably used a carbobenzoxy radical, and subsequently the liberated α-amino group of the peptide is reacted with the bifunctional linker. The peptide-amino acid-spacer conjugate is then coupled to the carrier protein, the binding taking place via the mercapto groups of the carrier protein.
As carrier protein, there can be used known proteins. Examples of appropriate proteins include albumins, such as bovine serum albumin and ovalbumin, haemocyanines, such as keyhole limpet haemocyanine, polyamino acids, such as poly-L-Lys and poly-L-(Lys:Glu) or enzymes, such as galactosidase. Edestin or galactosidase is preferably used as carrier protein.
Several hapten-spacer groups are preferably bound to the carrier protein. The number of bound groups depends upon the size of the carrier protein. As a rule, a maximum of 25% of the weight of the carrier protein can be bound to hapten-spacer groups. If edestin is used as protein, 10 to 30 hapten spacer groups are preferably bound.
With the immunogens provided by the present invention, there are obtained highly active, specific antibodies against HbA1c. These antibodies display very little cross-reactivity with HbA0.
The present invention also provides a process obtaining antibodies directed against HbA1c. For this purpose, an immunogen according to the present invention is injected several times into an appropriate organism and then the antibodies are obtained in known manner. For obtaining antibodies, as a rule mammals are immunized. Examples of appropriate mammals include mice, sheep, rabbits, rats and guinea pigs. The immunogen preferably dissolved in buffer and with the addition of a conventional adjuvant, for example Freund's adjuvant, is injected into the host animal. In order to achieve high antibody titers, the injection is repeated at regular intervals of time, for example every two to four weeks. The antisera which contain the polyclonal antibodies are obtained in the usual way from the blood of the host animals.
With the immunogens according to the present invention, there can also be obtained highly specific monoclonal antibodies with the use of the known hybridization process of G. Kohler and C. Milstein, which is described, for example, in Nature, 266, 495/1977 and in Science, 208, 692 et seq./1980. For this purpose, after immunization of the host organism, B-lymphocytes are isolated from the spleen of the immunized animal, fused with myeloma cells and the hybridoma cells formed are cloned. From the clones formed there are then isolated the cell lines which produce the antibodies which react specifically with HbA1c and show practically no cross-reaction with other molecules. The isolation of these cell lines is simple since a very high proportion of the clones produce specific antibodies. These cell lines are further cultured and the desired monoclonal antibodies cam then be obtained therefrom. The antibody activities are determined in known manner in the serum or in the hybridoma supernatants, usually with the use of an enzyme immunoassay.
The antibodies obtained according to the present invention are characterised by a high specificity and high affinity to HbA1c. The cross-reaction with non-glycosylated hemoglobin and with other proteins present in body fluids is negligibly small. Therefore, they are outstanding for use in processes for the determination of HbA1c in body fluids. Especially appropriate monoclonal antibodies are MABs3.609.325, 3.51.56 and 3.230.140. The corresponding hybridoma cell lines have been deposited with the European Collection of Animal Cell Cultures PHLS CAMR, Porton Down Salisbury, Wilts, SP4 OIG, Great Britain, under the numbers ECACC 87120801, 88122302 and 88122301 on Dec. 8, 1987 (first designated cell line) and Dec. 23, 1988 (last 2 designated cell lines) respectfully.
The following Examples are given for the purpose of illustrating the present invention, reference being made to the accompanying drawings, in which
FIG. 1 shows a standard curve of the activity against HbA1c of monoclonal anti-HbA1c antibodies (MAB 3.609.325)
FIG. 2 shows the titer determination (double determination) for an antiserum obtained according to Example 14; and
FIG. 3 shows a diagram with the relative affinities of the antibodies towards peptides and hemoglobin.
The peptide fructose Val-His-Leu-Thr-Cys-OH was synthesized. The solid phase synthesis was carried out in a semi-automatic peptide synthesiser of the firm Labortec, Bubendorf, Switzerland.
The Fmoc group (fluorenylmethoxycarbonyl radical) was used as Nα-amino protective group. A description of this method of peptide synthesis is given by J. Meienhofer et al. in Int. J. Peptide Protein Res., 13, 35-42/1979.
The C-terminal Fmoc amino acid was coupled, as described by Meyerhofer, on to p-alkyloxybenzyl alcohol resin (firm Bachem, Bubendorf, Switzerland).
Synthesis protocol for s synthesis cycle:
______________________________________step time reagent/solvent______________________________________1 2 � 1 min. dimethylformamide (DMF)2 1 � 3 min. piperidine/DMF 1:4 v/v3 1 � 7 min. piperidine/DMF 1:4 v/v4 4 � 0.5 min. DMF5 2 � 0.5 min. isopropanol6 stop ninhydrin test7 2 � 1 min. DMF8 stop addition of the next Fmoc amino acid and HOBt (1-hydroxybenzo- triazole) in DMF9 2 min. shaking10 stop addition of dicyclohexylcarbo- diimide (DCC) in dichloro- methane (DCM)11 90 min. coupling12 3 � 1 min. DMF13 2 � 1 min. isopropanol14 stop ninhydrin test______________________________________
For the coupling according to steps 8 to 11, the Fmoc amino acids and DCC are, in each case, used in threefold molar amount, referred to the loading of the starting resin. HOBt is used in a 4.5 fold molar amount.
After the coupling of the last N-terminal Fmoc amino acid, steps 1 to 5 of the synthesis cycle are followed for splitting off the Fmoc protective group. Thereafter, the resin is shaken for 2 hours at ambient temperature in a 15 fold volume of dichloromethane (DCM)/trifluoroacetic acid (TFA) (1:1 v/v). After filtering, the resin is again washed twice with DCM/TFA (4:1 v/v), all the filtrates are combined and evaporated in a vacuum at 25� C. with the addition of toluene. The residue is mixed with diethyl ether. The solid material is filtered off and dried.
According to the above-described scheme, fructose-Val-His-Leu-Thr-Cys(StBu)OH is synthesized. As starting resin, 10 g. Fmoc Cys(StBu)-p-alkoxybenzyl alcohol resin loaded with 0.6 mMole/g Fmoc amino acids. In the synthesis cycle, the following Fmoc amino acids are used successively:
1. 6.2 g. Fmoc Thr (tBu)
2. 6.4 g. Fmoc Leu
3. 11 g. Fmoc His (Trt)
4. 6.1 g. Fmoc Val.
The following abbreviations are used:
tBu; tertiary butyl ether
StBu: tertiary butyl thioether
OtBu: tertiary butyl ester
Trt: trityl
Z: benzyloxycarbonyl
-Cys(StBu): tertiary butyl sulphenylcysteine.
The crude yield after splitting off of resin were 3.29 g. HVal-His-Leu-Thr-Cys (StBu)OH. TLC: (silica gel HPTLC Merck, elution agent: ethanol/glacial acetic acid/water 6:2:2 v/v/v) Rf =0.62. After spraying with 1% ninhydrin spray (Merck) and developing at 120� C. for 5 minutes, there was obtained a red-violet coloration. In the case of spraying with a mixture of 200 ml. 0.4% methanolic resorcinol solution and 40 ml. 5N sulphuric acid (resorcinol sulphuric acid) and developing at 120� C. for 5 to 10 minutes, no coloration was obtained.
1 g. of the crude peptide was mixed with 540 mg. glucose and 50 ml. pyridine/glacial acetic acid and stirred for 5 days at ambient temperature. The reaction mixture was then evaporated in a vacuum at ambient temperature and subsequently the residue was taken up three times with 50 ml. of water and again evaporated to dryness. The residue was taken up in 50 ml. of water, applied to a column of Dowex 50 WX8 (H+ form, 50�3.5 cm.) and washed with water until all of the glucose was eluted. Thereafter, the product was eluted with 1N ammonia and lyophilized. There were obtained 810 mg. of lyophilisate which were taken up in 0.1M triethylammonium acetate buffer (pH 8.5) and washed on a column of Affigel 601 (Biorad, 5�26 cm.) with 2 liters of 0.1M triethylammonium acetate buffer (pH 8.5) and thereafter with 2 liters of water. The product was then eluted with 0.1% formic acid and lyophilized. The lyophilisate this obtained was chromatographed on Polygosil C18, 5μ (Macherey and Nagel) (gradient 0.1% TFA in water to 65% isopropanol in water, 0.1% TFA). There were obtained 283 mg. fructose Val-His-Leu-Thr-Cys (StBu)OH. TLC: (silica gel: elution agent as above): Rf =0.58. Coloration with ninhydrin: brownish. Coloration with resorcinol sulphuric acid: red-brown. Fab-MS (fast atom bombardment MS)(positive): MH+ =822.
For splitting off the cysteine protective group, the above-obtained peptide was dissolved in 130 ml. 0.1M potassium phosphate buffer (pH 8.5), degassed several times and again aerated with nitrogen. The solution was then mixed with 778 mg. dithiothreitol and left to stand for 24 hours under nitrogen. Subsequently, it was acidified with hydrochloric acid to pH 5 and purified by chromatography on Polygosil C18 (gradient as above). There were obtained 176 mg. fructose Val-His-Leu-Thr-CysOH. FabMS; positive: MH+ =734.
The peptide fructose ValHisLeuThrLysOH was synthesised according to the synthesis scheme given in Example 1. As start resin, there were used 10 mg. Fmoc Lys(Z) p-alkoxybenzyl alcohol resin loaded with 0.48 mMole/g. The following Fmoc amino acids were used successively in the synthesis cycle:
1. 5 g. Fmoc Thr (tBu)
2. 5.1 g. Fmoc Leu
3. 8.9 g. Fmoc His (Trt)
4. 4.9 g. Fmoc Val.
Crude yield after splitting from the resin: 3.2 g. TLC: (silica gel HPTLC, elution agent see Example 1.3): Rf =0.58.
The crude peptide was stirred with 1.6 g. glucose at ambient temperature in 150 ml. pyridine/glacial acetic acid (1:1 v/v). The reaction solution was evaporated, the residue purified as described Example 1.3 on Dowex 50EX8 H+ and Affigel 601. There were obtained 1.7 g. fructose ValHisLeuThrLys(Z)OH.
TLC: (silica gel HPTLC, elution agent see Example 1): Rf =0.58; coloration with sulphuric acid spray reagent: red-brown.
1 H-NMR (300 Mhz, D2 O): δ=0.85 (d, J=5.1 Hz, 3H,); 0.89 (d, J=4.9 Hz, 3H); 0.96 (d, J=7.1 Hz, 3H); 1.04 (d, J=6.8 Hz, 3H); 1.20 (d, J=1.20, 3H); 1.3-1.9 (m, 9H); 2.3 (m, 2H); 3.0-3.3 (m, 6H); 3.63-4.1 (m, 5H); 4.1-4.29 (m, 4H); 4.32 (d, J=5.4 Hz, 1H); 4.45 (m, 1H); 4.48 (m, 2H); 5.09 (s, 2H); 7.32 (s, 1H); 7.4 ("s", 5H); 8.63 ppm (s, 1H).
400 mg. of the above-obtained product were hydrogenated in 50 ml. methanol/water (5:1 v/v) with the use of palladium-active charcoal catalyst. The catalyst was filtered off, the filtrate evaporated and the residue chromatographed on Polygosil C18. Yield: 300 mg. TLC (silica gel HPTLC, elution agent see Example 1): Rf =0.09; coloration with sulphuric acid spray reagent: red-brown).
10 mg. Fructose ValHisLeuThrLysOH from Example 2 were taken up in 1 ml. 0.1M potassium phosphate buffer (pH 7). 6.2 mg Maleimidohexanoic acid N-hydroxysuccinimide ester in 2 ml. ethanol were added thereto. The reaction solution was stirred at ambient temperature for 14 hours and purified on Polygosil C18. After lyophilization of the pure fractions, there were obtained 4.3 mg. fructose ValHisLeuThrLys(maleimidohexsnoyl)OH.
FAB-MS, positive: MH+ : 952.
1 H-NMR (300 MHz, DMSO (D6)/CD3 OD): 0.83-1.06 (m, 12H; Leu-CH3, Val-CH3); 1.04 (d, J=6.5 Hz, 3H; Thr-CH3); 2.01 (t, J=6.5 Hz, 2H; MH--CO--CH2) and 6.92 ppm (s, 2H; MH--CH═CH).
5 g. Edestin from hemp seed (Roth) were stirred in 500 ml. 0.1M potassium phosphate buffer (pH 7.0) and mixed with s solution of 500 mg. maleimidohexanoic acid N-hydroxysuccinimide ester in 100 ml. ethanol. The solution was stirred for 90 minutes at ambient temperature, the solid material was filtered off with suction, washed twice with, in each case, 100 ml. of water, four times with, in each case, 100 ml. ethanol and again twice with, in each case 100 ml. of water. The solid material was slurried in 150 ml. of water and lyophilized Yield: 4.34 g.
Maleinimido groups/mole edestin: 17.
The number of maleinimido groups is determined as follows:
Solution A: 1 mM cysteine, 0.5 mM EDTA in water
Solution B: 10 mM 5,5'-dithio-bis-(2-nitrobenzoate) (Ellmann's reagent) in 0.1M potassium phosphate buffer (pH 8.0)
Solution C: 1M Tris.HCl (pH 8.2)
8 mg. Maleimidohexanoyl-edestin are taken up in 39 ml 0.05M potassium phosphate buffer end treated in an ultrasonic bath for 15 minutes. Thereafter, 1 ml. of Solution A is added thereto, followed by incubation for 10 minutes at 25� C. After stirring with 3.2 ml. of Solution C for 2 minutes at 25� C., 200 μl. of Solution B are added thereto, followed by incubation for 10 minutes at 37� C. The sample is then centrifuged for 15 minutes at 25� C. The extinction is measured in the supernatant at 405 nm. The blank value is obtained by proceeding analogously but with the use of the maleimidohexanoyl-edestin sample. The number of maleimido groups per nmole is given by: ##EQU1## wherein ΔE is the extinction difference between the blank value and the sample. The molar amount of edestin is obtained with the precisely weighed amount and the relative molecular weight of the edestin of 310,000: ##EQU2## 250 mg. of the so obtained maleimidohexanoyl-edestin were mixed under an atmosphere of argon with 20 ml. 0.1M potassium phosphate buffer (pH 6.5). 34 mg. fructose ValHisLeuThrCysOH, which has been obtained according to Example 1, were also added thereto with the exclusion of oxygen and the mixture stirred for 29 hours at ambient temperature. The solution was centrifuged, the precipitate washed three times with water and in each case again centrifuged. The solid residue was slurried in 10 ml. water and lyophilized. There were obtained 175 mg. of the conjugate of fructose ValHisLeuThrCysOH and maleimidohexanoyl-edestin, which is called immunogen 1.
The still free maleimido groups were determined as described above. From the difference as compared with the original loading, it follows that the immunogen contains 14.6 mole peptide per mole of edestin.
48 mg. β-galactosidase (EIA quality, Boehringer Mannheim GmbH) were dissolved under an atmosphere of argon in 2 ml. 0.1M potassium phosphate buffer (pH 7.0) gassed with argon. 5 mg. Fructose ValHisLeuThrLys(MH)OH, which had been obtained according to Example 3, were added thereto with the exclusion of oxygen and the mixture stirred at ambient temperature for 1 hour.
An AcA 202 column (2�24 cm.) was equilibrated with argon-saturated 0.9% aqueous sodium chloride solution. The total reaction solution was applied to this column. Elution was carried out with argon-saturated 0.9% sodium chloride solution and the protein fraction collected. 15 ml. immunogen solution were obtained (c=3.1 mg./ml.). Loading with immunogen can be determined by reaction of a sample with Ellmann's reagent: per mole of SH group, 1 mole carboxynitrothiopyridone is liberated (λmax =412 nm., ε=13,600 at pH 8.0).
β-Galactosidase of EIA quality contains 14 SH groups per molecule. After the reaction with the peptide, there are found 2 free SH groups, i.e. the loading is with 12 mole of peptide per mole of β-galactosidase.
The conjugate obtained from fructose ValHisLeuThrLys(MH)OH and β galactosidase is called immunogen 2.
For comparison, an immunogen was prepared with a comparatively long peptide chain. For this purpose, there was first prepared the peptide ValHisLeuThrProGluGluCysOH according to the synthesis scheme described in Example 1.
According to the synthesis scheme of Example 1 was prepared the peptide HValHisLeuThrProGluGluCys(StBu)OH. As start resin, there was used 10 g. of FmocCys(StBu)-p-alkoxybenzyl alcohol resin with a loading of 0.5 mMole/g. The following amino acids were used in the synthesis cycle:
1. 6.4 g. Fmoc Glu (OtBu)
2. 6.4 g. Fmoc Glu (OtBu)
3. 5.1 g. Fmoc Pro
4. 6 g. Fmoc Thr (tBu)
5. 5.3 g. Fmoc Leu
6. 9.3 g. Fmoc His (Trt)
7. 5.1 g. Fmoc Val; the coupling was repeated once.
Crude yield after splitting off from the resin: 4.2 g. The crude product was chromatographed on Polygosil C18 as described in Example 1. The yield was 880 mg. HValHisLeuThrProGluGluCys(StBu)OH; TLC (silica gel, elution agent as in Example 1): Rf =0.53.
800 mg. of this peptide were then reacted with glucose as described in Example 1 and purified on Dowex 50WX8 H+ form and thereafter on Affigel 601. After chromatography on Polygosil C18, there were obtained 150 mg. of product. FabMS, positive: MH+ =1177. Analogously to Example 1, the cysteine protective groups were then split off by the addition of dithiothreitol. There were obtained 78 mg. of fructose ValHisLeuThrProGluGluCysOH (FabMS positive: MH+ =1087. The peptide thus obtained was reacted with 322 mg. maleidohexanoyl-edestin as described in Example 4. There were thus obtained 200 mg. of immunogen which was used as comparative immunogen VI.
As further comparative immunogen, there was prepared a conjugate of fructose ValHisLeuThrCys and pyridyldithiopropionyledestin. The peptide was prepared as described in Example 2.
For the preparation of the pyridyldithiopropionyledestin, 1 g. of edestin from hemp seed was taken up in 150 ml. 0.1M potassium phosphate buffer (pH 7.5). To this solution were added, with stirring, 10 mg. succinimidyl-3-(2-pyridyldithio)-propionate, dissolved in 12.5 ml. ethanol. The reaction mixture was stirred for 2 hours, the solid material then filtered off with suction, washed three times with, in each case, 100 ml. water, 100 ml. ethanol and again 100 ml. water. The solid material was taken up in 100 ml. water and lyophilized.
352 mg. of the pyridyldithiopropionyledestin thus obtained were mixed under an atmosphere of argon with 100 ml. 0.05M potassium phosphate buffer (pH 6.0). 49.5 mg. of the peptide fructose ValHisLeuThrCys were added thereto, also with the exclusion of oxygen, and the reaction mixture then stirred for 23 hours at ambient temperature. It was subsequently ceutrifuged, the precipitate was washed three times with, in each case, 50 ml. of water, twice with, in each case, 50 ml. ethanol and again once with water and, in each case, again centrifuged off. The solid residue was taken up in 50 ml. of water and lyophilized. For the determination of the loading, the liberated thiopyridone in the supernatant of the first centrifuging was measured. There were obtained 300 mg. of the conjugate of fructose ValHisLeuThrCys and pyridyldithiopropionyledestin as comparative immunogen called V2. The loading was 39 mole of peptide per mole of edestin.
Polyhaptens were prepared for carrying out the screening test on anti-HbA1c antibodies. Polyhapten 1 was prepared from HValHisLeuThrProGluGluCysOH and pyridyldithiopropionyl-bovine serum albumin.
1 g. of bovine serum albumin was dissolved in 30 ml. 0.1M potassium phosphate buffer (pH 7.5). To this solution were added 226 mg. succinimidylpyridyldithiopropionate, dissolved in 15 ml. ethanol. The reaction mixture was stirred for 40 minutes at ambient temperature and the whole reaction solution chromatographed over ACA 202 (31�3 cm.); eluate 0.1M potassium phosphate buffer (pH 6.0). There were obtained 152 ml. of protein solution (c=6.6 mg./ml.). The solution was mixed with 280.5 mg. dithiothreitol and made up with 0.1M potassium phosphate buffer (pH 7.5) to 10 ml. This solution was then diluted to 1/6 of its initial concentration. The concentration of the liberated thiopyridone corresponded to the concentration of the dithiopyridyl groups used. From the extinction coefficient for thiopyridone at 340 nm of 8080 and the relative molecular weight of edestin of 310,000, there can be determined the loading: it is 36 mole/mole of protein.
10.5 ml. of the solution obtained of pyridyldithiopropionyl-bovine serum albumin and 47 mg. HValHisLeuThrProGluGluCysOH, which was obtained according to Example 6, was stirred for 1 day under argon (exclusion of oxygen). The loading was calculated by determination of the liberated thiopyridone at 33 mole peptide/mole protein. Remaining reactive groups were saturated by the addition of 1.7 mg. cysteine. The total reaction solution was chromatographed over an ACA 202 column (31�3 cm.), the protein fraction dialyzed overnight against water and lyophilized. The product obtained is called polyhapten 1. Yield: 85 mg.
For the preparation of polyhapten 2 (cf. Example 8), there were used 7 ml. pyridyldithiopropionyl-bovine serum albumin solution which was obtained in the manner described in Example 6. As peptide component, there were used 7.8 mg. of the peptide prepared according to Example 6 which carried a fructose on the amino acid Val. For this purpose, 800 mg. of the peptide HValHisLeuThrProGluGluCysOH obtained according to Example 6 were reacted as described in Example 1 with glucose and purified on Dowex 50 WX H-form and thereafter on Affigel 600. After chromatography on Polygosil C18, there were obtained 150 mg. of the glycosylated peptide; FabMS positive, MH+ =1177. After splitting off the cysteine protective group with dithiothreitol, from 100 mg. of the glycosylated peptide there were obtained 78 mg. of the desired product; FabMS positive MH+ =1089. The product so obtained is called polyhapten 2.
In order to recognise the presence and specificity of antibodies against HbA1c in the sera of immunized mice or in the culture supernatant of the hybrid cells or in ascites, an Elisa process was used as test principle:
Microtiter plates were coated with 10 μg. of polyhapten 2 or 10 μg. polyhapten 1/ml. of coating buffer (0.2M sodium carbonate/bicarbonate; pH 9.3 to 9.5) at ambient temperature for 1 hour with shaking. After-treatment was then carried out for 10 minutes with 0.9% sodium chloride solution and 1% albumin solution. Subsequently, washing was carried out with 0.9% sodium chloride solution. Thereafter, incubation with 100 μl. of sample was carried out at ambient temperature for about 1 hour and again washed with 0.9% sodium chloride solution. There followed a further incubation for 1 hour with 200 U/ml. of a sheep fab-anti-mouse Fcγ-peroxidase conjugate. After a further washing step with 0.9% sodium chloride solution, the peroxidase activity was determined in the usual way by reacting with ABTS for 15 minutes at ambient temperature. Thereafter, the extinction difference i.e., the mE difference, was determined at 405 nm.
The antisera which were obtained after immunization with the immunogen 1 according to the present invention obtained according to Example 4 gave, in the case of all 100 immunized mice, binding with polyhapten 2 and no or only weak binding with polyhapten 1. Thus, there were obtained antibodies binding selectively with polyhapten 2.
Of the antisera which were obtained after immunization with the immunogen 2 according to the present invention prepared according to Example 6, 6 of 18 sera obtained from immunized mice reacted with polyhapten 2 and not with polyhapten 1. The binding to polyhapten 2 was just as good as in the case of immunogen 1. Thus, here, too, selective antibodies were obtained.
In the case of the immunization carried out for comparison with the comparative immunogen V1, from 100 mice one antiserum was obtained which reacted only preferentially with polyhapten 2, all the other 99 antisera reacting equally well with both polyhaptens. Thus, in this case, it was not possible to obtained antibodies binding selectively with polyhapten 2.
In the case of immunization with the comparative immunogen V2, no differentiating mouse antisera could be obtained. All antisera obtained from 100 mice reacted equally well with both polyhaptens. The binding to the polyhaptens was weaker by a factor of 10 than in the case of the sera obtained with the immunogens 1 and 2 according to the invention.
Balb/c and B10.D2 mice, 8 to 12 weeks old, were first immunized intraperitoneally (i.p.) with 100 μg. HbA1c (β 1-4Cys, MHS)-edestin (immunogen 1) in complete Freund's adjuvant (CFA). After 6 and 10 weeks, two further immunizations were carried out 100 μg. of immunogen in incomplete Freund's adjuvant (IFA) were thereby administered. 10 days after the last immunization, the mice were bled in order to determine the antibody titer from the serum. 4 and 3 days before the fusion, the mice were again immunized intravenously with, in each case, 100 μg. immunogen in buffer.
For the fusion, with reference to Galfre, Methods in Enzymology, 73, 3/1981, 108 spleen cells of an immunised mouse were mixed with 2�107 myeloma cells (P3x63Ag8-653, ATCC-CRL 8375) and subsequently centrifuged for 10 minutes (300 g, 4� C.). The cells were again washed once with BSS (balanced salt solution) and centrifuged at 400 g in a 50 ml pointed test tube The supernatant was removed. The cell sediment was loosened and mixed with 1 ml. 60% PEG solution (MW 4000, Merck). After 1 minute in a waterbath, 5 ml. of RPMI 1640 medium (RPM=Roswell Park Memorial Institute) without fetal calf serum (FCS) were added dropwise at ambient temperature over a period of time of 4 to 5 minutes, mixed up, made up with medium to 50 ml. and subsequently centrifuged for 10 minutes at 400 g. and 4� C. The sedimented cells were taken up in RPMI 16400 medium with 10% FCS. In each case, 105 spleen cells were introduced into 24 well cell culture plates (firm Nunc). To each culture were added 5�104 peritoneal exudate cells as feed cells. On the following day, hypoxanthine-azaserine selection medium (100 mM hypoxanthine, 1 μg./ml. azaserine) was added thereto.
After about 7 to 10 days, many clones were already visible. The supernatant of the primary cultures was tested according to the ELISA process described in Example 10. Primary cultures which showed no or only small cross-reaction with HbA0 were further cloned with the help of a fluorescent-activating cell sorter (FACS) on 96 well cell culture plates (form Nunc). 1�104 peritoneal exudate cells serves as feed cells.
In this way, there could be isolated, for example, the hybridoma cell lines which have been deposited with the ECACC (European Collection of Animal Cell Cultures) under the deposit number 87120801, 88122301 and 88122302. The monoclonal antibodies MAB 3609325, MAB 3.230.140 and MAB 3.51.56 can be obtained respectively from these cell line.
For the production of ascites, mice were injected intraperitoneally with 5�106 hybrid cells, which had previously been pre-treated once or twice with 0.5 ml. pristane. 1 to 3 weeks thereafter, ascites fluid could be obtained from the mice with an IgG concentration of 5 to 20 mg./ml. The antibodies could be isolated therefrom in the usual way. These monoclonal antibodies are specifically directed against HbA1c and show no or only a slight cross-reactivity with HbA0.
microtiter plates: A: NUNC 4-42404 II B: NUNC 2-69620
12 canal pipette: Dynstech, catalogue No. 77-887-00
plate shaker: Flow Laboratories, Titertek, catalogue No. 77-471-00
covering films: Dynatech Plate Sealers, catalogue No. M 30
ELISA reader: Dynatech MR700
coating buffer: 50 mM sodium carbonate (pH 9.6)
sample buffer: 10 mM sodium phosphate (pH 7.4), 0.9% sodium chloride, 0.1% Tween 20, 1% crotein C
wash buffer: 0.9% sodium chloride, 0.1% Tween 20
antibody-enzyme conjugate: conjugate of peroxidase and the Fab fragment of a polyclonal antibody from sheep which is directed against the Fcγ part of mouse IgG, 25 mU/ml. in sample buffer
substrate: 100 mMole/l. phosphate-citrate buffer (pH 4.4) 3.2 mMole/l. sodium perborate 1.9 mMole/l. ABTS (2,2'-azino-di-[3-ethylbenzthiazolin-6-sulphonic acid] diammonium salt)
antibody: MAB 3.609.325 (ECACC 87120801)
polyhaptens: polyhapten 1 according to Example 8 polyhapten 2 according to Example 9
antigens: HbA1c native HbA0 native
In a preliminary experiment, there were determined the amounts of antibody to be used in the actual specificity experiments. For this purpose, microtiter plates were coated with polyhapten 1 or polyhapten 2. Per 100 μl. well, 1 μg. of polyhapten per ml. of coating buffer were incubated for 1 hour at ambient temperature. Thereafter, the solution was sucked out and washed three times with wash buffer.
Subsequently, to the solid phase-bound polyhapten were added dilution series of the antibody 3.609.325 (ascites), the dilution being accomplished with sample buffer from 1:100 in the fourth stage. In each case, incubation was carried out with 100 μl./well for 1 hour at ambient temperature and subsequently washed.
After addition of a conjugate of peroxidase and the Fab fragment of a polyclonal antibody from sheep which is directed against the Fc γ part of mouse IgG, the antibody bound to the hapten is determined via the reaction of the peroxidase with the added substrate. 100 μl. of conjugate/well are added and incubated for 1 hour at ambient temperature. The detection reaction is started by the addition of 100 μl. of substrate/well into all of the wells. The measurement takes place in the ELISA reader at 405 nm (reference wavelength 490 nm).
"Titer", as defined hereafter, refers to the antibody dilution at which half of maximum bonding occurs. This amount of antibody is used in the following experiment.
The specificity of the monoclonal antibodies was investigated. For this purpose, the reactivities of individual antibodies with different components present in solution were compared.
Into microtiter plates pre-coated with 1% crotein C were, in each case, pipetted 50 μl. of a solution of the monoclonal antibody in double titre concentration and 50 μl. of antigen solution (dilution series, see below), mixed and incubated for 30 minutes at ambient temperature. Thereafter, 100 μl. aliquots of the mixture were transferred into the microtiter plates coated with polyhapten.
______________________________________Dilution series:fructose ValHisLeuThrCys(StBu)OH from 5 μg./ml.(according to Example 1 sample buffer withfructose ValHisLeuThrProGluGluCys sample buffer in(StBu)OH(according to Example 6) the fourth stagefructose valineHValHisLeuThrProGluGluCys(StBu)OH(according to Example 6) from 100 μg./ml.HbA1c sample buffer inHbAO fourth stage______________________________________
The concentration in mole/liter of s substance to be determined associated with the half-maximum binding is defined as molar relative affinity.
For the comparison of the reactivity of a monoclonal antibody with various components, the relative affinity of the monoclonal antibody for the component fructose ValHisLeuThrProGluGluCys(StBu)OH is taken as being equal to 100%. The reactivities, which are also referred to as cross-reactions, with other components are then given from the quotients of the relative affinities as follows: ##EQU3##
The values obtained for the individual components are to be seen from the following Table 1.
Results were also evaluated which were obtained with different antibodies obtained in an analogous manner.
Table 1 shows that with the immunogens according to the present invention, a plurality of monoclonal antibodies can be produced with bind HbA1c very specifically but not HbA0. Compared with the antibodies which have been obtained with the use of the comparative immunogen V1 or with the antibodies known from published European Patent Specification No. 0,185,870, the antibodies obtained according to the present invention recognize HbA1c without special denaturing with substantially higher affinity. Therefore, they are very well suited for competitive immunoassays.
TABLE 1__________________________________________________________________________Specificity investigation of <HbA1c > clones MAB 1 MAB 2 KMC19.2 KMC19.3 MAB MAB MABreactivity with .sup.⊕ HB8639 (ATCC) HB8869 (ATCC) 3.51.56 3.230.140 MAB 3 MAB 4 MBA 5 3.609.325 MAB MAB__________________________________________________________________________ 7polyhapten 2 + + + + + + + + + +peptide A 100 100 100 100 100 100 100 100 100 100HbA1c native 4.4 9.4 15.3 20.0 20.7 40.4 25.0 40.0 4.1 7.1 22.0fructose-valine <0.29 <0.03 <0.01 n.d. <0.56 <0.11 n.d n.d. <0.04 <0.1polyhapten 1 -- -- -- n.d. -- -- n.d. n.d. n.d. n.d.peptide B <0.06 <0.01 <0.01 <0.03 <0.1 <0.02 <0.04 <0.24 3 5.1HbAO native <3.5 <0.4 <0.13 <0.95 <6.0 <1.3 <1.1 <6.7 <0.5 <0.13__________________________________________________________________________ n.d. = not determined + clone with the designation KMC: pub. Europ. Pat. Appln. No. 0,185,870 (equivalent issued U.S. Pats. Nos. 4,647,654, 4,727,036 and 4,658,022) MAB 1 to MAB 5 and MAB 3.609,325: prepared with immunogen from Example 4 MAB 6 and MAB 7: prepared with comparative immunogen V1 from Example 6 peptide A: fructose ValHisLeuThrProGluGluCys(StBu)OH peptide B: HValHisLeuThrProGluGluCys(StBu)OH
As described in Example 12, into microtiter plates pre-coated with 1% crotein C are, in each case, pipetted in 50 μl. of a solution of MAB 3.609.325 in double titer concentration and 50 μl. HbA1c solution, mixed and incubated at ambient temperature for 30 minutes. Various concentrations of HbA1c are used.
Thereafter, 100 μl. aliquots of the mixtures are transferred into microtiter plates coated with polyhapten 2. The antibodies bound to the polyhapten 2 are, as described in Example 12, detected with the help of a conjugate of peroxidase and the Fab fragment of a polyclonal antibody from sheep which is directed against the Fcγ part of mouse IgG.
The more HbA1c is present in the sample solution, the less antibodies bind to the polyhapten, i.e. the smaller is the measured extinction. In FIG. 1 of the accompanying drawings is illustrated the curve obtained in the case of the determination of HbA1c.
In each case, 10 sheep were immunized with immunogen 1 or 2 according to the present invention in complete Freund's adjuvant. In each case, the dosage amounted to 200 μg. of immunogen per animal for the first and each subsequent immunization. The immunizations took place at monthly intervals.
The sera obtained were investigated for the presence and specificity of antibodies against HbA1c as described in Example 12. For this purpose, microtiter plates were coated with 0.04 μg./ml. of polyhapten 2. As antibody-enzyme conjugate there was used a conjugate of peroxidase and a rabbit anti-sheep immunoglobulin. The concentration used of this conjugate was 150 mU/ml. As antigens there were used HbA1c and HbA0 native and as peptides HValHisLeuThrProGluGluCysOH and fructose ValHisLeuThrProGluGluCysOH.
The process was carried out in the manner described in Example 12.
The following samples were investigated:
Pool 1: mixture of aliquots of serum samples of all 10 animals which had been treated with the immunogen from Example 4 according to the present invention (blood sampling 45 days after first immunization).
Pool 2: mixture of aliquots of serum samples of all 10 animals which had been treated with the immunogen from Example 5 according to the present invention (blood sampling 45 days after first immunization).
Sample a: serum sample from sheep 3227 which had been treated with the immunogen from Example 4 according to the present invention (blood sampling 165 days after first immunization).
Sample b: serum sample from sheep 3233 which had been treated with the immunogen from Example 4 according to the present invention (blood sampling 165 days after first immunization).
Sample c: serum sample from sheep 3272 which had been treated with the immunogen from Example 5 according to the present invention (blood sampling 75 days after first immunization).
The titre determination gave the following results:
Pool 1: 1:5400
Pool 2: 1:8400
Sample a: 1:3700
Sample b: 1:3600
Sample c: 1:2600
FIG. 2 of the accompanying drawings shows an Example for the course of the extinction depending upon the antiserum concentration.
In the manner described in Example 14, the affinities and specificities of antibodies to glycosylsted and non-glycosylated antigens were determined. The results are given in the following Tables 2 and 3, as well as in FIG. 3 of the accompanying drawings. In FIG. 3:
______________________________________ maximum concentrationcurve antigen used (nmole/1.)______________________________________1 peptide A 53402 peptide B 160003 HbA1c 26704 HbAO 2670______________________________________
The Tables show that, with the immunogens according to the present invention, there can be produced highly specific polyclonal antibodies which bind very specifically HbA1c but not HbA0. It is also shown that the polyclonal antibodies recognised HbA1c, without denaturing, with high affinity. Furthermore, from the results of the various pools, which represent a mixture of the polyclonal antibodies obtained from, in each case, 10 sheep after treatment with the immunogens according to the present invention, it can be seen that, in the case of the use of the immunogens according to the present invention, a very high percentage of the immunised animals produce suitable antibodies.
TABLE 2______________________________________Affinity of the antibodies to glycosylated andnon-glycosylated antigens relative affinity (nmole/1.) peptide peptide cross-reactionsample A+ B++ HbA1c HbAO HbAO /HbA1c______________________________________Pool 1 21 >>16000 30 >>2670 <<1.1%Sample a 33 >>16000 115 >>2670 <<4.3%Pool 2 15 >>16000 18 >>2670 <<0.7%Sample c 21 >>16000 134 >>2670 <<5.0%______________________________________ + peptide A: fructose ValHisLeuThrProGluGluCys(StBu)OH ++ peptide B: HValHisLeuThrProGluGluCys(StBu)OH
TABLE 3______________________________________Specificity investigations of polyclonalantibodies against HbA1cResults:reactivity with + pool 1 sample a pool a sample c______________________________________polyhapten 2 + + + +peptide A 100 100 100 100HbA1c native 70 28.6 83.3 15.7polyhapten 1 -- -- -- --peptide B <<0.13 <<0.2 <<0.09 <<0.13HbAO native <<0.8 <<1.2 <<0.5 <<0.8______________________________________ + Pool 1, sample a: prepared from immunogen 1 of Example 4 Pool 2, sample c: prepared from immunogen 2 of Example 5
|Cited Patent
|Filing date
|Publication date
|Jan 25, 1982
|Oct 23, 1984
|Sherwood Medical Company
|Method of obtaining antibodies
|Aug 8, 1985
|Mar 3, 1987
|Molecular Diagnostics, Inc.
|Peptides useful in preparing hemoglobin A1c immunogens
|Sep 27, 1985
|Feb 23, 1988
|Molecular Diagnostics, Inc.
|Antibodies for use in determining hemoglobin A1c
|Feb 5, 1987
|Feb 21, 1989
|Becton, Dickinson And Company
|Measurement of glycosylated hemoglobin by immunoassay
|Oct 30, 1984
|Apr 30, 1986
|Boehringer Mannheim Gmbh
|Immunogens, process for their preparation and antibodies against glycosylated haemoglobin obtained therewith
|Nov 23, 1983
|Jun 20, 1984
|Miles Laboratories, Inc.
|Immunoassay for nonenzymatically glucosylated proteins and protein fragments - an index of glycemia
|Sep 6, 1986
|Mar 25, 1987
|Boehringer Mannheim Gmbh
|Method and composition for determining glycosylated hemoglobin as well as compounds suited therefor and methods for their preparation
|Title not available
|Title not available
|Chou et al., "Development of a Laser Nephelometric Method For The Quantitation of Human Glycohemoglobins", Anal. Lett. 14 (B13): 1071-1087 (1981).
|Chou et al., Development of a Laser Nephelometric Method For The Quantitation of Human Glycohemoglobins , Anal. Lett. 14 (B13): 1071 1087 (1981).
|Javid et al., "Immunologic Characterization and Quantification of Hemoglobin A1c", Br. J. Haematol. 38: 329-337 (1978).
|Javid et al., Immunologic Characterization and Quantification of Hemoglobin A1c , Br. J. Haematol. 38: 329 337 (1978).
|Tanaka, et al. (1985) Proc. Natl. Acad. Sci. USA 82: 3400 3404.
|Tanaka, et al. (1985) Proc. Natl. Acad. Sci. USA 82: 3400-3404.
|Citing Patent
|Filing date
|Publication date
|Feb 23, 1998
|Dec 12, 2000
|Roche Diagnostics Gmbh
|Analytical test element with a blister filled with liquid
|Sep 18, 1998
|Sep 24, 2002
|Serex, Inc.
|Methods to improve immunogenicity of antigens and specificity of antibodies
|Aug 21, 2002
|Dec 12, 2006
|Serex, Inc.
|Methods to improve immunogenicity of antigens and specificity of antibodies
|Aug 21, 2002
|Dec 4, 2007
|Serex, Inc.
|Methods to improve immunogenicity of antigens and specificity of antibodies
|Aug 21, 2002
|Feb 26, 2008
|Serex, Inc.
|Methods to improve immunogenicity of antigens and specificity of antibodies
|Aug 21, 2002
|Apr 24, 2003
|Serex, Inc.
|Methods to improve immunogenicity of antigens and specificity of antibodies
|Aug 21, 2002
|May 22, 2003
|Serex, Inc.
|Methods to improve immunogenicity of antigens and specificity of antibodies
|Aug 21, 2002
|Jun 19, 2003
|Serex, Inc.
|Methods to improve immunogenicity of antigens and specificity of antibodies
|U.S. Classification
|530/389.6, 424/139.1, 530/387.5, 530/322, 530/387.9, 435/7.25, 424/173.1, 530/330
|International Classification
|Cooperative Classification
|European Classification
|Dec 26, 2000
|Fee payment
Year of fee payment: 4
|Dec 3, 2004
|Fee payment
Year of fee payment: 8
|Sep 30, 2008
|Fee payment
Year of fee payment: 12


text:
Unless you've been living in a cave this past week—or at least not reading this blog—you've heard about Microsoft's offer to buy up Yahoo! for $31 a share, for a total of $44.6 billion. John Markoff, of the New York Times, called the moved the "firing the final shot of yesterday's war"—the war in question being one we all know well: the battle for search advertising.
Nothing could be further from the truth. Buying Yahoo is firing the first futuristic shot in tomorrow's war; Yahoo! is just the beginning for Microsoft. And the war is not for search advertising space alone; even a combined 34.6% for Microsoft/Yahoo! (assuming no one drops off during the merger) doesn't compete with Google's 61% (including its major partners). This deal is about much more.
For one, it is about mobile, one of the last frontiers in which Google's dominance is still tenuous. Google has spent time and made waves developing the Android mobile operating system, but its mobile offerings are relics compared with the new Yahoo! Go 3.0 and Microsoft's recently-acquired TellMe service. It's about emerging ad space, like Facebook—of which Microsoft owns a piece and has a 10-year exclusive advertising deal, beating out a Google bid—and advertising exchanges like Right Media, which was acquired by Yahoo!
Google may have video covered online, as it owns most video streams via its acquisition of YouTube and most video searches via its new integrated search bar and rumors of true frame-by-frame video search being developed. Photos, however, are still fair game, with Yahoo! holding on to more than 2 billion photos via its acquisition of Flickr. Blogging is worth fighting for as well. Blogger.com has been given over to spammers, but Windows Live Spaces and Yahoo! 360 both do well, with 45 million users between the two of them.
Microsoft wants Yahoo! for search, but it recognizes the need to lead in every other emerging online market where advertising dollars can be spent. And that means more acquisitions. And lots more shots fired.
Related reading
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Four key digital transformation trends you must pay attention to right now
Last week ClickZ Intelligence held its webinar on The What, Why and How of Digital Transformation in association with Marketo.
Google launches imported call conversions
If the internet killed the phone call, the smartphone has revived it.
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Nintendo's Pokémon Go, a free augmented reality (AR) mobile game, is on fire.

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Aroona Television Services also Installs Digital Radio and HDMI distribution & Remote Control systems.
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Awesome choice for Best Stylist
I do have a question Elizabeth. Do you ever watch any other comedians in the Low Country or just the one's downtown? Roland Jay Moore, Sean Jones, Dave Evans and myself started doing comedy (and we all live here BTW) here over 20 years ago. We all have performed here locally, everywhere from Mt. P., James Island, Downtown, Summerville, Goose Creek, West Ashley, North Charleston as well as all over The USA ( I performed for the troops in Japan last June) Jay is Princess Cruise Lines top comic, Sean has been seen in movies & TV, Dave not only performs but has had one of the biggest comedy competitions (Put your money where your mouth is) this area has ever seen. He had comics come from Fla., NC, Mrytle Beach, Ga. I just did a show at Ms. Rose's in November and they had to turn people away, I've got it on video!! I'm sorry, I don't want to sound bitter and I like Dusty, he has been a middle act for me several times (that means he opened up the show and I followed him) and I've judged several competitions he's been in and won, I voted for him. I just think it's lazy to just put his name out there again when guys such as the folks I mentioned have been making people laugh in the area for years get overlooked just because they're not in the right clique. I'll talk about this on my radio show and in my WEB series that shows what life is like being a road comic called "BTS On the Road" (24 episodes so far, shooting 25 later this month). How about we show support for ALL the great comedians in our area and not just the peninsula crowd or maybe they should distribute the City Paper only in the downtown area where they should be limited to getting they're sponsors from? As for me, I don't care, I live local, but I'm worldwide.
What a joke.
Best Tour Company Ever!
'All Books' was an absolute mind-blower, I couldn't believe the owners let it happen. I caught the very tail end of it, but the little dose I got opened up so much. Many friendships came from it and a most important moment hanging out with Andrew Barranca in the parking lot after a 'Puke Attack' performance and talking about making a film together.
Thanks to all involved!
Way to go Bulldog Tours!!
Hell yeah!
Brad and Ann are amazing people. Great Friends to have. They did so much.
PJ is awesome he always opened his doors to everyone, keep it up.
Buying a boat can be one of the best things ever. I remember going out on trips to lakes in Utah and Idaho with my friend's family. It would be so much fun to be able to do that on my own. If I lived near the ocean it would make even more sense to buy something like this.
Brother Bill
Please come and be apart of the New STONO Breeze off maybank rd. Nice bar and restraunt off of maybank rd and is an awesome bar located on the STONO River. I think you would be an awesome guest that would love the johns island people. I also think you would be an awesome Addition to the most awesome people around.
Just saying
Please come and see us
Don and Tonya
Going Coastal Limo Service. Professional Drivers providing the utmost in service and reliability.
the link says this will be about jlinsnider, but the description is dumas
Best of all of South Carolina!!
Not sure who T. Rav is, but it is really easy to renounce your U.S. Citizenship. For example, check out this book: http://glr.com/80ae
Wagener Terrace is the biggest and best neighborhood downtown!
Ken Lam Appointed New CSO Music Director
The collection of lion pictures is because the restaurant opened in 1972 as the Hungry Lion Restaurant, owned by Paul Kemerle and Jack Sewell. Paul sold his interest to Jack in 1992 and Jack renamed it but kept the pictures and theme.
Circular is second only to the Unitarian Church!
As first time visitors to South Carolina, we had experience with fresh water fishing, but none with inshore fishing, so we were looking forward to our charter with Captain Jamie. Unfortunately, our excitement was short lived. Our "lovely" afternoon started with Captain Jamie being 15 minutes late to meet us at the dock, and digressed from there. He spent the majority of our 4 hour tour making and receiving personal phone calls, he was surly, and short tempered, and he had the audacity to yell, yes literally yell, at us when he thought we were scaring his fish away. He equated our photo taking to Japanese tourists, which as tourists from out of state, it seemed only natural for us to take pictures of what we were seeing and doing. To make matters worse, he got our boat anchor hooked to another 2 boats, and as he tried to speed off to get our anchor unhooked, he nearly sunk the other 2 boats; and gosh, those other tourists and their guides looked mighty happy with Captain Jamie over that. The charter was awful, and when we finally got around to fishing and catching fish, no one wanted to participate after 3 hours of verbal abuse and disrespect. We were his customers and he treated us terribly. Captain Jamie not only lacks 'Southern Charm,' but basic respect, and he should stick to fishing my himself in tournaments where he doesn't have to inflict his poor behavior on tourists who are willing to pay for a happy experience on the South Carolina coast.


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Lacrosse Magazine, owned and published by US Lacrosse, the national governing body of men's and women's lacrosse, is circulated to more than 430,000 members of the organization as one of the benefits of membership. Join Today!
By Paul Ohanian
John Strohsacker
US Lacrosse, the national governing body of men's and women's lacrosse, announced today that the organization has awarded 29 new grants to encourage and empower lacrosse participation in underrepresented communities. These awards, made through the Diversity & Inclusion Grant Program, will be utilized by local organizations to initiate and continue programs that promote lacrosse diversity participation and education.
US Lacrosse believes that broad representation and participation add significant value to the lacrosse experience, and seeks to provide sustained leadership and resources for the growth of the game in all communities. Now in its second year, the D&I Grant Program is providing $78,000 in grants to this year's community partners.
"We had a large number of qualified applicant programs that are really making a significant impact around the country, and therefore we increased the number of grants this year in order to provide support for as many deserving organizations as possible," said Eboni Preston-Laurent, senior manager for diversity and inclusion at US Lacrosse. "We've selected some phenomenal groups that are committed to supporting the underrepresented lacrosse community in a truly positive way."
The majority of the D&I grants range from $1,000 to $3,000, and are awarded in six separate categories: community impact, disability access, girls' initiatives, urban outreach, event support and BRIDGE programs. The 29 organizations receiving grants this year represent 14 different states across all regions of the country.
The National Diversity and Inclusion Grant Program offers grants in the following categories: Girls' Initiative, Urban Outreach, Community Impact, Event Support, Disability Access and BRIDGE Programs. Learn more about the grants and about US Lacrosse's commitment to diversity and inclusion by clicking on the link below.
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US Lacrosse has awarded 24 new grants to support lacrosse participation opportunities for underrepresented youth.


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Narrow your search
Jane's Addiction fans, wireless industry executives, and the media converge for an event promoting LG's upcoming Thrill 4G.
Natali Morris takes a first look at the LG 3D Mobile DTV from CES 2011.
Watch the 84-inch LG 3D television in action at CES 2012, as well as gesture control on the company's smart TVs.
Sky TV has bought 15,000 LG 3D TVs to go in bars around the country. Football might just have gotten more interesting.


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- a low structure projecting from the doorway of a house and forming a covered entrance
- US Canadian an exterior roofed gallery, often partly enclosed; veranda
WordReference Random House Learner's Dictionary of American English © 2016
porch /pɔrtʃ/USA pronunciation n. [countable]WordReference Random House Unabridged Dictionary of American English © 2016
- Architecturean outside addition to a building, forming a covered approach or entrance to a doorway.
- Architecturea raised platform on the outside of a building, usually level with a door;
a veranda.
porch (pôrch, pōrch),
porch′like′, adj.
- Architecturean exterior appendage to a building, forming a covered approach or vestibule to a doorway.
- Architecturea veranda.
- Ancient History, Philosophythe Porch, the portico or stoa in the agora of ancient Athens, where the Stoic philosopher Zeno of Citium and his followers met.
- Architecture[Obs.]a portico.
Etymology:porch′less, adj.
- Latin porticus porch, portico
- Old French
- Middle English porche 1250–1300
porch′like′, adj.
Collins Concise English Dictionary © HarperCollins Publishers::
porch /pɔːtʃ/ n
'porch' also found in these entries:


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Book your room at Americas Best Value Inn & Suites
Get an overview of this hotelHotel in Bismarck with free breakfast and 24-hour front desk
Situated in Bismarck, this hotel is within 1 mi (2 km) of North Dakota Heritage Center & State Museum and North Dakota State Capitol. Belle Mehus Auditorium and Bismarck Civic Center are also within 3 mi (5 km).
Hotel Features
This hotel features coffee/tea in a common area, laundry facilities, and a 24-hour front desk. Free continental breakfast, free WiFi in public areas, and free self parking are also provided.
Room Amenities
All 90 rooms offer free WiFi, cable TV, and free local calls.Would you like to give us feedback? Share it! Opens in a new window
Americas Best Value Inn & Suites1505 Interchange Avenue Bismarck ND 58501
Hotel Amenities
Hotel Amenities
A complimentary breakfast is offered. High-speed wireless Internet access is complimentary. This Bismarck hotel also offers coffee/tea in a common area and laundry facilities. Onsite self parking is complimentary.
- 24-hour front desk
- Coffee/tea in common areas
- Free breakfast
- Free self parking
- Free WiFi
- Laundry facilities
- Number of buildings/towers - 1
- Number of floors - 2
- Total number of rooms - 90
Family Friendly Amenities
- Cribs/infant beds (complimentary)
- Free Wi-Fi
- Laundry facilities
- Rollaway/extra beds available
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking
Room Amenities
- Air conditioning
- Cable TV service
- Free cribs/infant beds
- Free local calls
- Free WiFi
- Hair dryer
- Rollaway/extra beds available
Where to Eat
Guests are offered a complimentary continental breakfast.
To make requests for specific accessibility needs, complete the "Special or Accessibility Requests" section of "Trip Preferences" when you book your hotel.
- Accessible bathroom
- Roll-in shower
- In-room accessibility
Hotel Policies
Check-in time starts at 3 PM
Check-out time is 11 AM
Payment types
- Pets allowed for an extra charge of USD 10 per pet, per night
- Specific rooms only
- Pet-friendly rooms can be requested by contacting the property at the number on the booking confirmation
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No elevators
Optional extras
- Pet fee: USD 10 per pet, per night
- Rollaway beds are available for an additional fee
Hotel Name
- Americas Best Value Inn Bismarck
- Americas Best Value Inn Hotel Bismarck
We should mention
This property allows pets in specific rooms only (surcharges apply and can be found in the Fees section). Guests can request one of these rooms by contacting the property directly, using the contact information on the booking confirmation.


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From Our Editors
The stylists at Shear Madness in Stockton will take your needs seriously and provide you with the best hair remedies possible. For beauty services that are sure to impress, such as make up application, head to Shear Madness. Shear Madness provides a wide variety of hair removal options, such as laser treatments and waxing. Take care of your hair with a personalized color and cut treatment.
This season, try something new! Find your new style at Shear Madness.


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Product Type
Accessories(15)Balloons(8)Banners(3)Bracelets(2)Decorations(38)Drinkware(9)Eyewear(2)Games & activities(3)Invitation Cards(10)Jewelry(13)Leis & boas(8)Necklaces & beads(11)Paint & Markers(1)Paper goods(13)Party favors(29)Party kits(1)Tableware(15)Toys(3)
Luau Party Supplies
Guests won't know where they are with our Luau Party backgrounds. Plan you entire Luau Party party from start to finish with our Luau Party invitations to announce the party, to the Luau Party party favors. Cool Glow is a one-stop shop for all your Luau Party party decorations, Luau Party tableware, Luau Party party favors, Luau Party banners, Luau Party invitations and more! Stock up now with these fabulous Luau Party party supplies.
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A collection of things you think only you do. Go ahead and confess. You probably aren't the only one.
"The word of today is "leg's",no whom are i kidding, bird is the word!."
via: Anti-Pickup Line
"Jeremy on their challenge when James was gonna be piloting a plane: "So it'll be Captain Captain Slow and his Hammond hand luggage!"."
via: Clarksonisms
via: Pointless Inventions
"The power to withstand camel ****.."
©2008-2014 Things You Think Only You Do. All rights reserved.
A Horse Head Huffer Production.
Rails Hosting provided by BlueBox


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Featuring a different MGPL staff member every week!
I love spending time with my children, grandchildren, and husband. My second love is my sewing room.
To learn more about Sue, visit our Staff Spotlight page!
This presentation will discuss the city's greatest single-event loss of life, the SS Eastland rolling over in the Chicago River. Ted Wachholz from the Eastland Disaster Historical Society will talk about the tragic event, which will feature newly discovered video footage, photographs, and more.
Saturday, August 6 at 2:00 PM
Saturday, July 30 at 1:00-3:00PM
You know you were jealous last summer when the little ones were tie-dying their T's. Here's your chance, you lucky teen! Bring your own item to tie-dye. Keep it practical and smallish. A white shirt, pillowcase, or scarf is perfect! Siblings, pets, and king-sized comforters cannot be tie-dyed!
To register for this groovy event.
"How easy it was to disappear: A thousand trains a day entered or left Chicago"
- The Devil in the White City: Murder, Magic, and Madness at the Fair that Changed America by Erik Larson
To read this fascinating book about Chicago's history, find it here!
Get out and about this summer and don't forget to snap and share pix that fit the theme of this year's #MGPLTEEN Instagram Contest - "The Great Outdoors." To enter the contest, just tag your Instagram photos with #MGPLTeen or email them to firstname.lastname@example.com.
The contest ends on August 28th, so get started now taking Great Outdoors photos and tagging #MGPLTeen.
Stop in at the library each week on Fridays at noon for a different family-friendly sports movie. Bring your lunch with you, and we will provide the dessert!
The Summer Lunch Movies Series continues with Little Giants (1994, PG, 1 hr 47 min) on July 22nd because who doesn't love an underdog sports movie? Make sure to come back on July 29th for Angels in the Outfield (1994, PG, 1 hr 42 min)
Children under 8 must be accompanied by a parent/caregiver.
The Library has added Lynda.com to its list of online resources! This is a great resource because it includes thousands of step-by-step video tutorials on software, business management, accounting, social media, coding, game design and so much more. Interested? Watch this video tutorial.
Find Lynda.com through the library's website. Please note, the mobile app is not set up for library accounts.
In partnership with Driving-Tests.org, the Morton Grove Public Library offers free online practice tests for anyone preparing for their Illinois driver's license test. Included are: 11 car practice tests, 8 motorcycle practice tests, 4 CDL (Commercial Driver's License) practice test, and 3 online driver's manuals (car, motorcycle, and CDL).
6140 Lincoln Ave.
Morton Grove, IL 60053-2989


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No Field Goals Made for this player
No Field Goals Made for this player
College: Syracuse
Weighted Career AV (100-95-...): 93 (197th overall since 1960)
High School: White Plains (NY)
Draft: Washington Redskins in the 1st round (18th overall) of the 1980 NFL Draft.
Learn about our new design. Give us your feedback.
The design is optimized for your phone. Give it a try.
No Field Goals Made for this player
All logos are the trademark & property of their owners and not Sports Reference LLC. We present them here for purely educational purposes. Our reasoning for presenting offensive logos.
Logos were compiled by the amazing SportsLogos.net.
Copyright © 2000-2016 Sports Reference LLC. All rights reserved.
Most data provided by 24-7 Baseball, creators of the ESPN Pro Football Encyclopedia.


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Nexus Watch? All the Google Smartwatch Rumors So Far
If CES and MWC are anything to go by, wearable tech is the next big thing, and smartwatches are front of the line in terms of hot new trends. Samsung has one, Pebble has one, Apple's apparently working on one, and even Google is said to be developing a watch of its own. This Nexus watch from Google has been in the news since last year, and there's been plenty of rumors since then (even some renders, as evidenced by the picture above). We've rounded them all up in one tidy spot and will update as time goes on.
Unlike Samsung, Apple, or Pebble, one of the major questions with Google's rumored smartwatch is who's going to make the device. Google rarely makes its own hardware. In the past, it's contracted Samsung, Asus, HTC and LG to build its Nexus electronics (aside from the Nexus Q, but let's not go near that). The first question you have to ask when you consider a Nexus-branded smartwatch is who's going to be slapping their logo right next to Google's own? Recent reports suggest Google was going to go with a device from Motorola, and that this watch was codenamed Gem. However, February brought talk that the Gem had been scrapped in favor of a device from LG, which has made the last two Nexus Phones. It's important to remember that Google owned Motorola last year, so it could have had the team working on a watch. Now that Motorola is gone to Lenovo; it's possible Google preferred to go with its current preferred partner.
Form Factor
This is arguably one of the most important aspects of wearable tech. While a lot of it comes down to personal taste, no one is going to want to wear it if it looks like garbage. Period. Samsung has one bulkier offering, the Galaxy, Gear and one more sleek and polished smartwatch that comes in the form of the Gear Fit. Apple's also thought to be working on a curved wristwatch. If leaked images of the Gem are anything to go by, this Nexus smartwatch will look more like the Galaxy Gear and the Pebble smartwatch than the Galaxy Fit or any slap bracelet-style device Apple comes up with. But if we're to believe Google has made the switch from Motorola to LG, then what can we expect? That's hard to say, but if we look to the past, LG has already played the smartwatch game. What you see below is the GD910. It cost roughly a grand and was actually a wristwatch phone as opposed to just a companion device.
While this device isn't too dissimilar to the Galaxy Gear in terms of form factor, it's important to note that the GD910 was announced way back in 2007 and released in 2009, so LG may have a different idea of what passes for fashionable these days. Patents filed by Google feature illustrations of a watch with a round face, as opposed to a rectangular or square face.
According to our favorite leaker, @evleaks, the Google smartwatch will pack a yet to be determined processor as well as a 1.65-inch IPS LCD with a 280 × 280 resolution, 512 MB RAM, and 4 GB internal storage. No mention of whether or not it will have a camera (Apple's offering apparently will), and given the specs, it looks like this will definitely be a companion device as opposed to a standalone piece of consumer electronics. A patent filed by Google last year mentions a smartwatch with touch sensitive areas built into the band. These areas would be used to control the device as opposed to the small touch screen, where swiping and scrolling might feel cramped.
Of course, this being a Google product, you can expect it to be an Android device, though it likely won't run a full version of Android. Google's probably going to push Google Now pretty hard when it comes to the Nexus watch, which makes a lot of sense. Google Now is an integral part of the Google Glass experience, conveniently providing users with location- and time-specific information without forcing them to take out their phone and unlock the device. No doubt the idea behind the Google watch is the same. A quick glance at your wrist will tell you if your brother has answered your email or if you need to bring a coat when you leave for work. CNet cites a source that says Google will be developing an OS specifically for its smartwatch.
Release Date
Last year, there was talk that the Google smartwatch would debut alongside KitKat. Obviously, that didn't happen. Now, rumors say we can expect to see the device at Google I/O in June. This comes via CNet's source as well as our old faithful, @evleaks. Google is apparently not going to show off a new version of Android at I/O this year. Could that be because it wants to focus on its watch? The developer conference kicks off on June 25 and runs for two days.
Follow Jane McEntegart @JaneMcEntegart. Follow us @tomshardware, on Facebook and on Google+.
Christopher ShafferThe Galaxy watch that Samsung has integrated with their phones so that you can actually answer and talk with it intrigues me as a Dick Tracy fan, but that novelty isn't enough for me to buy one. I think Samsung's take on this is the most relevant I've seen.
THAT said, I don't see this smart watch thing actually taking off. I think Apple with put out their obligatory offer, but ultimately I think this is where we'll see the smart accessory market taper off, and as such, it's tie-in with the smart phone market. Wearable tech only makes sense when it's actually practical (that sounds like a an oxymoron, I know) and serves a purpose.
So far, every suggestion I've seen seems to be that this is basically an extension of my phone. Like the LG pic above, I don't need to see pics on my watch. If I have time to pull up pics on my watch, I'll pull them up on my phone instead.
What I'd like to see is the integration of a *small* smart watch with something like the Jawbone band. I don't need pics and video and crap, just give me a Dick Tracy watch that can monitor my stats and optionally send them via bluetooth to an app on my phone and also stores them locally to download later.
I think instead these smart phone manufacturers are trying to do the phone tie-in to force propriety, which goes completely against the Android philosophy and what makes it competitive with Apple.
BranFlake5Not saying that I'm particularly in the market for a smart watch, but this design is so far the best I have seen. I'd be interested in this.Imagine the ultimate Nexus experience when you can access things from your wrist and view them on your glasses. The whole thing powered by a fast, cheap Nexus phone.I feel that if battery life is addressed these could become popular.


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About Us
Peaceforce Christian Academy was founded in 2000 by Senior Pastor, Zoila J. Quintero, Th.D., as a ministry of Iglesia Cristiana Obreros De Paz Church. ICODP Church has served in ministry in Orlando, Florida since 1975; located in the West Colonial area since 1980.
Our Mission is to glorify God by equipping children for real success in life and present them perfect in Christ.
Our Vision is to develop a secured generation with moral values, not fearing the future, well socio-academically equipped to face all life challenges. To train youth of every ability in the highest principles of moral character, personal integrity, self-discipline, individual responsibility, Christian character and good citizenship.
Registration Special! The Registration Fee for grades K5-8 will be waived until Thursday, August 11, 2016. Contact the school to schedule an appointment.
We are currently enrolling Kindergarten [K5] through Grade 8. For more information, contact our office at
Parent Orientation is on Thursday, August 11, 2016 at 12:00 pm.
June 27 - July 27 OPEN [Mon-Wed]
July 28 - August 5 CLOSED
August 8 - 10 OPEN [Mon-Wed]
OPEN Office Hours: 10am - 2pm
"Whom we preach, warning every man, and teaching every man in all wisdom; that we may present every man perfect in Christ."
-Colossians 1:28
Peaceforce Christian Academy
501 Wilmer Avenue
Orlando, Florida 32808


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Chance to Lose Fair and Square is All the Bills Want
A team that loses honestly is still losing. Conversely, beating the stupid Patriots counts no matter who stands behind center. Tom Brady is officially going to miss one-quarter of the season… if you thought there could be no awesome football Continue reading


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Four years ago, MedMates was conceived at a brainstorming session about making economic growth happen in Rhode Island. David Goldsmith, co-founder of the group focused on providing a voice and opportunities for businesses in the state's … more
By Lori Stabile | firstname.lastname@example.com | 5/12/16
Leonard Lardaro said Rhode Island's economy showed more strength in June, and appears to be transitioning to a more rapid rate of economic growth. more
By Lori Stabile
If you have a customer-loyalty program, you'd be wise to re-evaluate it. Pending accounting changes may soon make your program too risky. Incentives to returning customers already require a careful balancing act between risk and reward. … more
A psychology degree is not the typical credential associated with running a business, but then, Stephanie Additon is not a traditional entrepreneur. Additon launched the primarily wholesale business Java Skincare in 2014 from the back of … more
What makes the U.S. economy grow? A look at consumer spending data offers a simple if perhaps troubling answer: Increasingly, it's people buying stuff they don't need. Any effort to separate wants from needs involves subjectivity. One … more
Mark Whitehouse guest column | 11/6/15
Ocean State Job Lot will receive $7.8 million in state assistance to expand its distribution and logistics center at Quonset Business Park, if it moves ahead with the planned $49.1 million facility and creates more than 100 new jobs. more
By Mary MacDonald
Magnus Jonsson, chief marketing officer for A.T. Cross Co. in Lincoln, discusses the venerable company's recent announcement that it will distribute Sheaffer pens in the global corporate gift sector. A.T. Cross, known for fine writing instruments, also recently launched a Star Wars pen line. Jonsson talks about Cross' plans going forward. more
By Lori Stabile
One gets the impression that Swipely is ahead of the curve simply by checking out its website: the company's interactive and engaging videos and animation leave the impression of a company on the cutting edge. Which it is. Swipely processes … more
BY JAIME LOWE | Contributing Writer | 9/17/15
The owner of Providence Place mall plans to replace two levels of the vacant J.C. Penney Co. retail store with additional parking, part of an estimated $20 million renovation package that also will modernize the parking payment system for the mall. more
By Mary MacDonald
(Updated, March 8, 12:54 p.m.) Two projects that would involve new construction of downtown buildings will come before a city design review panel this month for initial discussion. more
By Mary MacDonald


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Pair arrested in Rutland street disruptionBy Brent Curtis
STAFF WRITER | January 21,2013Two men were arrested Sunday night in Rutland after police from three agencies were called to disperse a crowd that gathered over a dispute on School Street.
More than 40 people were reportedly involved in a series of heated verbal and physical confrontations in the city's southwest neighborhood, Rutland Police Sgt. John Sly said.
"A lot of people came out to watch or get involved," Sly said.
In what he described as a "convoluted sequence of events" police were initially called to 36 Meadow Street at 4:23 p.m. for a reported fight.
At the scene, Sly said police found about 10 people arguing about the theft of a cell phone and a missing hat.
Sly said police left the scene believing that the parties would stay separated. But more than three hours later they were called back when some of the same people involved in the earlier dispute moved to 79 School St. where a crowd had
The size of the gathering was so great that city police called for backup from Vermont State Police and the Rutland County Sherriff's Department as well as another shift of officers coming on duty in the city.
Police also arrested two participants — 19-year-old Tiler Cormia and Nicholas Prior, 20, both of Rutland — on charges of disorderly conduct.
For the complete story, see Tuesday's Rutland Herald.MORE IN This Just InPARIS You cant put a guard in every church and patrol every beach. Full StoryA New Hampshire man has been arrested for possession of heroin following a traffic stop on I-91... Full StoryGROTON State police have arrested a Groton man for a drug-related death that occurred at his... Full Story
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Power Tools
Expansion of the multi-tool system: Bosch AMW 10 now with leaf blower attachment Efficient removal of leaves using a focused air flow
- 1000 watt power unit for high performance in every application
- Powerful, concentrated air flow for controlled operation
- Easy, convenient handling and storage of the tool
Save text
- August 26, 2014
- Power Tools
- Press releases
The Power Tools Division of the Bosch Group is one of the worlds leading providers for power tools, power tool accessories and measuring tools. In 2015, its roughly 20,000 associates generated sales of 4.5 billion euros, about 85 percent of which outside of Germany. With brands such as Bosch, Skil and Dremel, the division stands for customer focus and great engineering progress. The core success factors are innovative strength and pace of innovation. The division generated about one third of its sales in 2015 with products that have been on the market for less than two years. In 2016, Bosch Power Tools will again launch more than 100 new products onto the German market in its four business segments power tools, accessories, measuring tools and garden tools.
PI8657 - August 26, 2014


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Logan Couture is frustrated.
From Comcast SportsNetNEW YORK (AP) -- So much for a two-week break.
From Comcast SportsNetTORONTO (AP) -- Negotiations had already hit a wall in the ongoing hockey labor fight, and now the NHL has suggested the sides take an official two-week break before getting b
With the world thinking this morning of Barack Obama and the local, national and global ramifications of the election, its nice to know that some things remain unchanged.Like Gary Bettman and Don F
According to multiple reports, the NHLPA on Thursday held a conference call with its executive committee as the lockout reached its 46th day.


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Ocean Reef Club, Key Largo, FL
To book your room at our specially discounted group rate, please call the on-site reservations department at 305-367-5923 and reference the Search Insider Summit, or book your room online, using this link. Please note, the room block expires on April 27th.
Ocean Reef Club is located at the northern tip of Key Largo, approximately 50 miles by road southeast of Miami Airport. Its two thousand lush tropical acres are bordered on three sides by water. The Club can be reached by land, sea and air. An exclusive getaway for business or leisure, Ocean Reef is world-renowned for its gigantic meeting spaces and numerous services. Lodging options abound, with the Inn, as well as rentable condos, studios and villas for groups of any size. With tons of dining and recreation options, work won't be all you're thinking about. You can sail away on a yacht, lounge by the sea with the only living coral reef in North America or golf at one of their immaculate courses. There's also a spa, fishing opportunities, a fitness center, tennis courts and more. There's something for everyone. Come to work, stay to play.


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my grandson is supposed to have adhd and the prescriptions are tenax and strattera, tenax lowers the blood pressure,is it safe for him take these together? I am not comfortable with having to give my grandson any kind of the adhd drugs, do you have any suggestions?
Tenex - can you take tenax with strattera?
- Posted:
- 26 Oct 2009 by dups
- Topics:
- strattera, tenex, attention-deficit hyperactivity disorder (adhd), blood disorders
Answers (1)
8 Nov 2009
Yes, it should be fine for him to take Tenex and Strattera together. There are no known interactions between these medicines. However, it does not mean that no interactions exist so if he does experience any odd symptoms please get him to see a doctor.
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