Abstract
The multipin peptide synthesis technique has been used to map antigenic sites of proteins (1,2). Antibodies raised to the whole protein are screened on pin-synthesized overlapping octapeptides homologous with the protein of interest, and the peptides that bind antibodies clearly identify the epitopes.
What is described in this study is a method using pin-synthesized peptides to generate specific antibodies to many peptides. Cleavable linkers have been developed (3) that, used together with the multipin peptide synthesis technique, allow the synthesis and cleavage of many thousands of peptides into aqueous solutions at physiological pH. This technique is useful for assays requiring peptides in solution, e.g., mapping of T-cell determinants.
A technique has been developed for the cleavage of many peptides from pins and simultaneous coupling to immunogenic carriers (4). The conjugates produced are suitable for the generation of antipeptide antibodies.
This procedure is illustrated using several 15 amino acid long peptides (15-mers), homologous with the sequence of a model antigen, myohemerythrin (MHr). The resulting antipeptide sera generated were tested by ELISA for titer and specificity on pinsynthesized peptides and β-amide peptides and the protein antigen coated to microtiter plates.
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Abbreviations
- AcOH:
-
acetic acid
- b-Ala:
-
b-alanine
- BSA:
-
bovine serum albumin
- Boc:
-
t-butoxycarbonyl
- DCC:
-
dicyclohexylcarbodiimide
- DKP:
-
diketopiperazine
- DMAP:
-
4-(dimethylamino) pyridine
- DMF:
-
dimethylformamide
- DT:
-
diphtheria toxoid
- EDT:
-
ethanedithiol
- ELISA:
-
enzyme-linked immunosorbent assay
- Fmoc:
-
9-fluorenylmethoxycarbonyl
- HEMA:
-
hydroxyethylmethacrylate
- HMD:
-
1,6-diaminohexane
- HOBT:
-
1-hydroxybenzotriazole
- HPLC:
-
high-pressure liquid chromatography
- ip:
-
intraperitoneal
- MCS:
-
6-maleimido-caproic acidN-succinimide ester
- MeOH:
-
methanol
- MHr:
-
myohemerythrin
- PBS:
-
phosphate-buffered saline
- PITC:
-
phenylisothiocyanate
- TEA:
-
triethylamine
- TFA:
-
trifluoroacetic acid
- THF:
-
tetrahydrofuran
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Triantafyllou, B., Tribbick, G., Maeji, N.J. et al. Use of the multipin peptide synthesis technique for the generation of antipeptide sera. Cell Biophysics 21, 33–52 (1992). https://doi.org/10.1007/BF02789476
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DOI: https://doi.org/10.1007/BF02789476