The SICA[ − ] or non-agglutinable phenotype of Plasmodium knowlesi schizont-infected erythrocytes has been defined serologically but not biochemically. Similarly, non-cloned SICA[ + ] or agglutinable parasites have been shown serologically to express SICA or variant antigen(s) but the number and nature of such antigens have not been defined. Here we describe the immunochemical analysis of surface antigen expression on [125I]lactoperoxidase-labelled erythrocytes infected either with a SICA[ − ] elone or with non-cloned SICA[ + ] parasites using the methods developed for identification of variant antigens with cloned SICA[ + ] parasites. No 125I-labelled antigens in the size range Mr 190000–225000 were specifically immunoprecipitated from erythrocytes infected with the SICA[ − ] elone, even using homologous antisera produced by multiple infections or immunizations. Further, no125I-labelled proteins of this size were seen in detergent extracts of the SICA[ −] parasites that were not also seen with uninfected cells. We conclude that the SICA[ − ] phenotype reflects the absence of a variant antigen at the erythrocyte surface, as predicted by the serological assays. In contrast, with the non-cloned SICA[ + ] parasites, a complex group of proteins, Mr 195000–225000, was identified by [125I]lactoperoxidase labelling of intact infected erythrocytes. These proteins are SICA antigens since they not only share the characteristic detergent solubility properties and size range of SICA antigens identified previously with SICA[ + ] clones, but they were only immunoprecipitated by antisera which reacted specifically with the surface of infected erythrocytes. Agglutinating sera immunoprecipitated several of these 125I-labelled antigens. Sera specific for clones derived from this non-cloned SICA[ + ] population failed to agglutinate, but did react by indirect immunofluorescence with 10–16% of infected cells. These sera specifically immunoprecipitated single, quantitatively minor 125I-labelled antigens in this size range. The results suggest that a population of non-cloned SICA[+] parasites contains at least 10 different variant-antigen phenotypes. Indirect immunofluorescence was also performed against a non-cloned SICA[ + ] population derived by antigenic variation of a SICA[ + ] clone in vivo. The variant population contained at least 3 antigenically distinct SICA phenotypes, indicating that antigenic variation of clones may produce populations as antigenically heterogenous as antigenic variation of uncloned lines. It is therefore likely that natural malaria isolates contain a large number of different variant antigens.
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