Abstract
We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one V(H) family-derived germ-line gene (V(H)1) and two V(L) family segments (V(L)2 and V(K)I).
Highlights
Large panel of anti-Pfs48/45 monoclonal antibodies (mAbs) was needed for a number of reasons: 1) to elucidate the relationship between Pfs48/45 and Pfs230 and 2) to measure and characterize anti-Pfs48/45 antibodies in experimental and field sera
Clones SB12 to SG10 showed competition, suggesting specificity for epitope III of Pfs48/45, whereas the clones for Pfs48/45unrelated antigen did not compete. These results show that phage antibodies from the eight competition ELISA-positive clones (Table II) were capable of inhibiting the interaction of single-chain Fv fragments (scFv) antibody fragments to Pfs48/45 by more than 65%
In the present study we employed a novel strategy for generating recombinant human monoclonal antibody fragments from peripheral B cells from malaria immune patients
Summary
Large panel of anti-Pfs48/45 mAbs was needed for a number of reasons: 1) to elucidate the relationship between Pfs48/45 and Pfs230 and 2) to measure and characterize anti-Pfs48/45 antibodies in experimental and field sera. Phages were selected from these libraries by panning on Pfs48/45 antigen and phages bound to the antigen eluted by competition with mAbs [8]. This method resulted in human scFv antibodies directed against epitope III of Pfs48/45. A panel of murine and rat monoclonal antibodies (mAbs) has been produced against Pfs48/45 and has recognized at least five different epitopes. Some of these mAbs showed transmissionblocking activity [3,4,5,6]. A ʈ A research fellow of the Royal Netherlands Academy of Arts and Sciences (KNAW)
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