ATLA::Alternatives to Laboratory Animals

Volume 24, Number 1

The development and interlaboratory validation of a quantitative antibody capture ELISA for the measurement of specific guinea-pig IgG1: an alternative to the passive cutaneous anaphylaxis assay.

ATLA 24, 73-79, January/February 1996

Laura S. Babcock,¹ Thomas T. Kawabata,¹ Victor S. Moore,² Catherine M. Condo² and Annette Prentø³

¹Corporate Professional and Regulatory Services Division, Human Safety Department, The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45253-8707; ²Battelle, Columbus Laboratories, Columbus, OH 43201; ³Department of Enzyme Toxicology, NOVO Nordisk, Novo Allé, 2880 Bagsvaerd, Denmark

SUMMARY

Specific guinea-pig IgG1 has traditionally been measured by using an in vitro guinea-pig passive cutaneous anaphylaxis (PCA) assay. This paper describes the development and validation of a quantitative enzyme-linked immunosorbent assay (ELISA) for specific IgG1 against two protein antigens (Alcalase and Enzyme B) as an alternative to the PCA assay. The ELISA format involved a rabbit antibody bound to microtitre plates to capture the antigen. The test sera is added to this, followed sequentially by goat anti-guinea-pig IgG1 and rabbit antigoat-lgG-alkaline phosphatase conjugate. Aliquots of each serum sample from immunised guinea-pigs were analysed with the ELISA for specific IgG1 titres in three laboratories and were compared to titres determined by using the PCA assay. The findings demonstrate that there is a good correlation between the ELISA and the PCA assay and that the ELISA shows good interlaboratory reproducibility. Thus, the antibody capture ELISA described in this report is a valid and robust replacement for the guinea-pig PCA assay.

Keywords: guinea-pig, immunoassay, ELISA, hypersensitivity, passive cutaneous anaphylaxis assay, alternative, validation