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.