Differential antibody screening of cloned Plasmodium falciparum sequences expressed in Escherichia coli: procedure for isolation of defined antigens and analysis of human antisera.
We describe a procedure that uses polyspecific human sera for screening Escherichia coli colonies expressing cloned Plasmodium falciparum cDNA sequences in order to detect colonies that react differentially with different sera. This procedure can be used for two distinct purposes. First, it enables the isolation of clones encoding specified antigenic sequences present in the complex mixture, without purification of either antigens or antibodies by conventional procedures. This requires that the antigen can be expressed in E. coli and that antisera are available that differ substantially in their reactivities to the component of interest. To develop the procedure, we used two polyspecific sera that shared many anti-P. falciparum specificities but differed in that only one was reactive to the isolate-specific S antigen of P. falciparum strain FCQ27/PNG (called FC27). Differential screening with the two sera identified 30 cDNA clones, and colony hybridization confirmed that 25 of these express S-antigen sequences. Second, the procedure identifies defined antibody specificities within polyspecific human sera by virtue of their ability to react with any given cDNA clone. The procedure has been used here to identify antibody specificities that increase dramatically in titer between the acute and convalescent phases of malaria in certain individuals and, hence, to isolate clones encoding the corresponding antigens.
D000818:Animals; D000941:Antigens / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D007106:Immune Sera; D007118:Immunoassay; D010963:Plasmodium falciparum / Q000235:genetics* / Q000276:immunology
D000941:Antigens; D007106:Immune Sera; D004247:DNA