TY - JOUR
T1 - An avidin-biotin-peroxidase assay to detect synthetic peptides bound to polystyrene plates
AU - Griesmann, Guy E.
AU - McCormick, Daniel J.
AU - Lennon, Vanda A.
N1 - Funding Information:
Correspondence to: G. Griesmann, Neuroimmunology Laboratory, 828 Guggenheim Building, Mayo Clinic, Rochester, MN 55905, U.S.A. * This work was supported by grants from the National Institutes of Health (NS 15057 (V.A.L.) and NS 24694 (D.J.M.)) and the William K. Warren Foundation. Abbreviations: ABC, avidin-hiotinylated horseradish peroxidase complex; ABTS, 2,2'-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ELISA, enzyme-linked immunosorbent assay; Hepes, N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid; MHC, major histocompatibility complex; NHS-LC-Biotin, sulfosuccininidyl-6-(biotinamido) hexanoate.
PY - 1991/4/8
Y1 - 1991/4/8
N2 - The simple chemical method described for detecting synthetic peptides bound to polystyrene should facilitate interpretation of studies involving interactions of antibodies or solubilized major histocompatibility complex (MHC) molecules with immobilized peptide antigens. After its application to an ELISA plate, the peptide is biotinylated in situ and avidin-biotinylated horseradish peroxidase complex and substrate are added sequentially. For 36 of 43 peptides tested (11-27 residues long), a colored reaction product confirmed that the peptide was bound. In three of the seven instances of a negative result, peptides were positively detected by binding of antibody. Four instances remained in which it could not be determined whether the peptide did not bind to the plate or whether it was not biotinylated. On the other hand, the biotin-ABC assay positively detected peptide in 18 of 22 instances without evidence of antibody binding, implying seronegativity or a loss of antigenic conformation in the bound peptide. This general method should be applicable to assays of microbial antigens, autoantigens and allergens. Modification of the technique by use of biotin hydrazide should enable monitoring of the binding to polystyrene of carbohydrates, glycolipids or DNA.
AB - The simple chemical method described for detecting synthetic peptides bound to polystyrene should facilitate interpretation of studies involving interactions of antibodies or solubilized major histocompatibility complex (MHC) molecules with immobilized peptide antigens. After its application to an ELISA plate, the peptide is biotinylated in situ and avidin-biotinylated horseradish peroxidase complex and substrate are added sequentially. For 36 of 43 peptides tested (11-27 residues long), a colored reaction product confirmed that the peptide was bound. In three of the seven instances of a negative result, peptides were positively detected by binding of antibody. Four instances remained in which it could not be determined whether the peptide did not bind to the plate or whether it was not biotinylated. On the other hand, the biotin-ABC assay positively detected peptide in 18 of 22 instances without evidence of antibody binding, implying seronegativity or a loss of antigenic conformation in the bound peptide. This general method should be applicable to assays of microbial antigens, autoantigens and allergens. Modification of the technique by use of biotin hydrazide should enable monitoring of the binding to polystyrene of carbohydrates, glycolipids or DNA.
KW - Avidin-biotin complex
KW - Biotinylation
KW - ELISA
KW - Major histocompatibility complex
KW - Synthetic peptide
KW - solubilized
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U2 - 10.1016/0022-1759(91)90060-S
DO - 10.1016/0022-1759(91)90060-S
M3 - Article
C2 - 2019744
AN - SCOPUS:0025829514
SN - 0022-1759
VL - 138
SP - 25
EP - 29
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1
ER -