Development of an indirect competitive ELISA for the detection of ABO blood group antigens

We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of ABO blood group antigens in human samples; in particular for blood stains. ABO blood group antigens conjugated to polyacrylamide were used for immobilized antigen. ABO blood group antigens were extracted from blood stains using a novel method involving pre-incubation with proteinase K (PK), followed by heat treatment. The extracts (analytes) were combined with either anti-A or -B monoclonal antibodies (mAbs), and added directly to the antigen-coated wells. The anti-A and -B mAbs were captured by either ABO blood group antigens present in the analyte or by the immobilized blood group antigens. Peroxidase-conjugated anti-mouse IgM antibody was used to detect anti-A and -B mAbs complexed with immobilized blood group antigens, and a colorimetric reaction using o-phenylenediamine/H2O2 used for its measurement. The ELISA developed in this study was able to detect blood group antigens in blood, saliva and blood stains. The detection limit for unknown blood, saliva and blood stain were determined as 1:200, 1:32 and 1:16. Overall the ABO blood grouping ELISA can be used with relative ease for the high throughput screening of biological samples for the detection of ABO blood group antigens.