Reverse passive hemagglutination assay for human chorionic gonadotropin in urine using monoclonal antibody

Aiming to find a urinary hCG immuno-assay which is specific, sensitive and easy to perform, a reverse passive hemagglutination reaction was studied by using sheep red blood cells (SRBC) coupled with monoclonal antibodies (Mab) to hCG. Three Mabs (5D4, 6E4, 2F8) with different specialty were used for the study. Mab 5D4 reacted to hCG, hCG-beta, and LH but not to hCG-alpha. Mab 6E4 reacted to hCG, hCG-alpha and LH, but not to hCG-beta. Mab 2F8 reacted to hCG but not to hCG-alpha, hCG-beta, or LH. All three Mabs were IgG1. SRBC were treated with glutaraldehyde and then with tannic acid. These treated SRBC were coupled with IgG(2mg/ml) of each anti hCG-Mab. For assays, 30 microliters of 1:1 mixtures of two different Mab-coupled SRBC and 30 microliters of standard hCG or urine samples were mixed in wells of microtiter plates and reacted for 60 min at room temperature. Among three different combinations, the couple 5D4-SRBC and 2F8-SRBC were most sensitive and specific for hCG assays and the minimum amount of hCG and LH detected in this combination assays was 12.5 mIU/ml and 800 mIU/ml, respectively. Some clinical data obtained by applying this assay were presented.