Optimization and Validation of an ELISA to Measure Specific Guinea Pig IgG1 Antibody as an Alternative to the in Vivo Passive Cutaneous Anaphylaxis Assay

Abstract

Optimization and Validation of an ELISA to Measure Specific Guinea Pig IgG1 Antibody as an Alternative to the in Vivo Passive Cutaneous Anaphylaxis Assay. Kawabata, T. T., Babcock, L. S., Gauggel, D. L., Asquith, T. N., Fletcher, E. R., Horn, P. A., Ratajczak, H. V., and Graziano, F. M. (1995). Fundam. Appl. Toxicol. 24, 238-246.Assessment of the allergenic potency of enzymes involves the use of a guinea pig model in which specific IgG1 antibody titers are used as the endpoint. The in vivo passive cutaneous anaphylaxis (PCA) assay is used to measure specific IgG1 antibody. This report describes the development and validation of an enzyme-linked immunosorbent assay (ELISA) to measure guinea pig specific IgG1 antibody as an in vitro alternative to the PCA assay. Cross reactivity of various rabbit and mouse (monoclonal) anti-guinea pig IgG1 preparations were evaluated using purified IgG1 and IgG2 from serum of guinea pigs immunized with ovalbumin. The two subclasses of guinea pig IgG were purified by first using Protein A affinity chromatography, followed by anion exchange chromatography and fluid phase isoelectric focusing. Affinity-purified rabbit anti-guinea pig IgG1 was shown to have minimal cross reactivity toward IgG2, while providing a strong signal with IgG1. The ELISA was designed as an antigen capture system in which the following are added in sequence: (1) enzyme antigen (passively adsorbed to the plate), (2) diluted serum samples from guinea pigs immunized with enzyme, (3) affinity-purified rabbit anti-guinea pig IgG1, (4) alkaline phosphatase-conjugated donkey anti-rabbit IgG, and (5) p -nitrophenyl phosphate substrate. Three replicate ELISA and PCA analyses were conducted on sera samples of varying titers from guinea pigs immunized with either Alcalase (protease), BPN' (protease), and Termamyl (amylase) enzyme. The correlation coefficients (r2) between the ELISA and PCA assay for Alcalase, BPN', and Termamyl were 0.826, 0.945, and 0.755, respectively. These findings demonstrate that the specific antibody ELISA developed in this study is a valid in vitro alternative to the PCA assay to measure anti-enzyme guinea pig IgG1.