Production and characterization of monoclonal antibodies against asexual stages of Plasmodium yoelii nigeriensis.

Swiss mice vaccinated with Plasmodium yoelii nigeriensis-soluble antigen and saponin, following a homologous 100% lethal challenge, showed 60% protection (6 out of 10 mice survived). Monoclonal antibodies (MAbs), generated by hybridizing the Sp2/0 myeloma cells with the splenocytes of each of these ten mice, separately, were screened using enzyme-linked immunosorbent assay (ELISA), and were characterized by using merozoite (Mz) invasion inhibition assay in vitro, immunofluorescence assay (IFA), passive transfer of protection and ELISA-based isotyping. Curiously, purified MAbs from each of the six protected mice showed a distinct dichotomy: only two or three of them inhibited >86% Mz invasion, whereas the remaining six to nine showed <58% Mz invasion inhibition. However, none of the purified MAbs from the nonprotected mice could inhibit >58% Mz invasion. Furthermore, the ability of the MAbs to inhibit Mz invasion appeared to correlate with their IFA-reactivity with the free-Mz, suggesting that these MAbs were directed against the Mz surface antigens involved in invasion. In passive transfer of protection experiments, pooled purified MAbs from protected mice, that inhibited >86% Mz invasion, transferred 60% protection from challenge; the remaining pooled purified MAbs from protected mice, and those from nonprotected mice, when transferred separately, imparted only 30 and 10% protection, respectively. Isotypically, the MAbs belonged to IgG(1), IgG(2a), IgG(2b), and IgG(3) subclasses. Our results indicate that purified MAbs against P. yoelii nigeriensis, produced from the hybrids generated using the splenocytes of vaccinated and protected mice, belonged to two distinct groups: a small group that inhibited >86% Mz invasion, strongly cross-reacted with free-Mz, transferred up to 60% passive protection, and belonged to IgG(2a) and IgG(3) subclasses, whereas the other relatively larger group inhibited <58% Mz invasion, weakly cross-reacted with free-Mz, and transferred only 30% passive protection.