Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA.
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-beta-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 beta-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.
Laman
J D
JD
Department of Immunology and Medical Microbiology, TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
van den Eertwegh
A J
AJ
Deen
C
C
Vermeulen
N
N
Boersma
W J
WJ
Claassen
E
E
D000818:Animals; D000921:Antibody-Producing Cells / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes; D005976:Glutaral / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry*; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D010455:Peptides / Q000737:chemistry; D010504:Periodic Acid / Q000737:chemistry; D013154:Spleen / Q000166:cytology; D001616:beta-Galactosidase / Q000737:chemistry*
D000939:Epitopes; D010455:Peptides; D010504:Periodic Acid; D006735:Horseradish Peroxidase; D001616:beta-Galactosidase; D005976:Glutaral