Production of monoclonal antibodies against the Protozoa, Perkinsus marinus: estimation of parasite multiplication in vitro

Abstract

Among the different stages in the protozoan Perkinsus marinus life cycle, the trophozoite stage is known to be the most infective stage in marine molluscs. To develop a direct method for in vitro studies of P. marinus proliferation under various environmental conditions, monoclonal antibodies (MAbs) specific for this pathogen were produced. Inbred strains of mice BALB/c were immunised with a trophozoite prepared from cloned isolate Perkinsus 1 cultured on JLODRP1 medium. The mouse polyclonal antiserum showing the highest antibody titre for pathogen trophozoites was chosen for lymphocyte hybridisation. The screening of positive hybridoma by indirect enzyme-linked immunosorbent assay (ELISA) revealed two probes (17B₂D₅ and 19G₃H₆) detecting P. marinus trophozoites and their protein lysates but also trophozoites from P. atlanticus. These MAbs belonged to the immunoglobulin IgG1 subclass. Their binding specificity was investigated by ELISA and fluorescein (FITC) methods. Both immunoreacted with trophozoite stage as well as hypnospore and zoospore stages of P. marinus, but neither with hemolymph and tissues of oysters, Crassostrea gigas and C. virginica, nor with parasites, Bonamia ostreae and Marteilia refringens. A competitive ELISA method was developed, using 17B₂D₅ MAb to evaluate parasite multiplication in culture media and to estimate the parasite burdens from infected oysters. This method is sensitive enough to detect 10³ trophozoites in 50 μL assay sample.