Detection of blood group antigen in stroma-free hemoglobin by enzyme immunoassay

An inhibition enzyme immunoassay (EIA) was developed to quantify blood group antigen in stroma-free hemoglobins (SFH). Glycophorin A bearing type A antigenicity was purified and used for coating EIA plates and as the standard. The lower detection limit was 0.3 ng/well, which value was 10-fold more sensitive than that by the hemagglutination inhibition test (HAI). Both assays were applied to compare six representative SFHs. The Rabiner's conventional SFH and heat-treated SFH were shown to contain a significant amount of antigen at 8.40-8.70 ng/mg of hemoglobin by EIA, and also positive by the HAI. In contrast, other SFHs prepared by acid-treatment, chloroform extraction, or ultrafiltration were negative by HAI, but EIA successfully revealed that the SFHs still contained the antigen in the range of 0.46-0.84 ng/mg. These results indicate that these SFHs are 10 times purer than the conventional SFH. EIA is sensitive, fast and easy to perform, and is therefore a useful tool for the quality control of SFH.