DEVELOPMENT OF AN ANTIGEN DETECTION METHOD FOR BRUGIA MALAYI BmR1 RECOMBINANT ANTIGEN USING POLYCLONAL/MONOCLONAL ANTIBODIES

Abstract

Brugian filariasis infects 13 million people in Asia. The reference test, microscopic detection of microfilariae in peripheral night blood, is unpopular, inconvenient and relatively insensitive. To ensure the elimination of these infections in the context of the Global Programme for Elimination of Lymphatic Filariasis (GPELF), assays which are more sensitive than night blood examination must be employed. New Zealand white rabbit (polyclonal antibody production) and BALB/c mice (monoclonal antibody production) were immunized with BmR1 recombinant antigen. Splenocytes from immunized mice were fused with myeloma cells NS1. Positive hybridomas were selected by HAT medium, screened using ELISA method and sub-cloned twice using limiting dilution method. The monoclonal antibodies were upscaled by growing them into various volumes of culture flasks. Ascites fluid was obtained using BALB/c mice. The monoclonal antibodies so obtained were then purified and characterized. A Brugia malayi antigen detection test using sandwich ELISA system (optimized by checkerboard titration method) was developed using the polyclonal/monoclonal antibodies that were obtained. The system was then tested using „spiked‟ normal human sera. The sensitivity and specificity of this system was determined as well. Out of 814 wells with hybridomas, nine hybridomas (F1P1A4, F1P2G1, F3P1E9, F3P2F3, F3P2F5, F3P2G9, F3P4F8, F3P7C2 and F3P7D4) were tested positive for BmR1 recombinant antigen. FIP1A4 (11%) produced IgG1 antibody, while the rest (89%) produced IgM antibody. All these monoclonal antibodies expressed kappa light chain. F1P2G1 was used as capture antibody at the optimum dilutions 1:400; polyclonal antibody as detection antibody at 1:1000 and conjugate at 1:1000. The sensitivity of this assay was good as it could detect 1ng/mL of BmR1 recombinant antigen under optimal assay conditions. The linear range of this assay was from 1ng/mL to 625ng/mL of BmR1 recombinant antigen concentrations. It did not cross-react with BmSXP (recombinant antigen of Wuchereria bancrofti), Toxoplasma gondii, Dirofilaria immitis, Toxocara canis and Trichuris sp. but reacted slightly with Syphacia muris antigens. Further tests with clinical specimens will be carried out in the future. In conclusion, an antigen detection enzyme immunoassay based on a polyclonal/monoclonal antibody system against the Brugia malayi BmR1 recombinant antigen was successfully developed.