Production and Characterization of Monoclonal Antibodies against Asexual Stages of Plasmodium yoelii nigeriensis

Abstract

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 IgG₁, IgG_2a, IgG_2b, and IgG₃ 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 IgG₃ subclasses, whereas the other relatively larger group inhibited <58% Mz invasion, weakly cross-reacted with free-Mz, and transferred only 30% passive protection.