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Synthetic peptide conjugates with horseradish peroxidase and β-galactosidase for use in epitope-specific immunocytochemistry and ELISA

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Author: Laman, J.D. · Eertwegh, A.J.M. van den · Deen, C. · Vermeulen, N. · Boersma, W.J.A. · Claassen, E.
Type:article
Date:1991
Institution: Medisch Biologisch Laboratorium TNO
Source:Journal of Immunological Methods, 1-2, 145, 1-10
Identifier: 231703
doi: doi:10.1016/0022-1759(91)90304-X
Keywords: β-galactosidase · Alkaline phosphatase · Conjugation · EL ISA · Epitope · Glutaraldehyde · Immunocytochemistry · Periodate · Peroxidase · Synthetic peptide · Alkaline phosphatase · Beta galactosidase · Epitope · Horseradish peroxidase · Peroxidase · Synthetic peptide · Animal experiment · Animal tissue · Conjugation · Enzyme linked immunosorbent assay · Female · Immunocytochemistry · Mouse · Nonhuman · Priority journal · Animal · Antibody-Producing Cells · beta-Galactosidase · Enzyme-Linked Immunosorbent Assay · Epitopes · Glutaral · Horseradish Peroxidase · Immunohistochemistry · Mice · Mice, Inbred BALB C · Peptides · Periodic Acid · Spleen

Abstract

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-β-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to β-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.