Typing of serum‐soluble HLA‐B27 antigen by ELISA

Abstract

An ELISA using serum as soluble HLA antigen source was developed for HLA-B27 typing. Two sandwich assays were run in parallel. The first assay utilized a monoclonal antibody (mAb) reacting with a determinant expressed by both HLA-B7 and B27 antigens; the other assay utilized a mAb reactive with HLA-B7 antigens but not with HLA-B27 antigens. After incubation with serum samples, bound HLA antigen was detected using an anti-beta 2m antibody conjugated to peroxidase and a chromogenic substrate. Absorbance of each well was measured at 490 nm. Based on analysis of absorbances obtained with panels of specimens of known HLA phenotypes, a mathematical algorithm was developed to derive the specimen HLA-B27 phenotype from its ELISA absorbance values. Despite the lack of monospecific mAb, an accurate HLA-B27 typing was possible. 362 specimens (including 151 HLA-B27-positive) were tested. Agreement between microlymphocytotoxicity and ELISA was 99.2%. No correlation between the level of HLA-B27 antigen reactivity and the amount of total HLA class I antigen in serum was observed. This report demonstrates the possibility of using serum-soluble HLA antigen and ELISA technology for histocompatibility testing. The assay offers several significant advantages over microlymphocytotoxicity: no need for cell preparation, batch testing capabilities and objective, reproducible interpretation of results.