Isolation and partial characterisation of a 26 kilodalton antigen from Plasmodium falciparum recognised by an inhibitory monoclonal antibody.

A 26 kDa protein, present in trophozoites and schizonts of Plasmodium falciparum, has been identified as the target of a monoclonal antibody that weakly inhibits parasite growth in vitro. The antigen has been purified to homogeneity by immuno-affinity chromatography and electrophoresis. The sequence of 19 amino acids at the N-terminus of the protein has been determined.

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides.

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofluorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeled parasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent.

Inhibitory monoclonal antibody against a (myristylated) small-molecular-weight antigen from Plasmodium falciparum associated with the parasitophorous vacuole membrane.

A small-molecular-weight antigen that occurs in asexual blood stages in synchronized cultures of Plasmodium falciparum was detected by a monoclonal antibody which inhibits parasite growth in vitro. This antigen, QF116, showed a molecular weight of 15,000 in parasite strain FCR-3K+ from The Gambia and 19,000 in strain FCQ-27 from Papua New Guinea. The protein did not show significant glycosylation by galactose or glucosamine labeling but was found to be acylated by myristic acid. By using immunogold labeling and electron microscopy, the location of the antigen could be attributed to the parasitophorous vacuole membrane and to inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell.

The parasitophorous vacuole membrane of Plasmodium falciparum: demonstration of vesicle formation using an immunoprobe.

We have applied several immunolabeling techniques using a monoclonal antibody to a Plasmodium falciparum antigen to differentiate morphologically dissimilar membranous structures present in infected erythrocytes. Evidence is presented that cytoplasmic clefts, multimembranous structures and vesicles within the infected cell originate from the parasitophorous vacuole membrane by a process described as budding off. The parasitophorous vacuole membrane and related structures in infected, parasitized erythrocytes reacted with the cyanine dye Merocyanine 540, demonstrating that they are accessible to molecules from the extracellular environment. Immunogold labeling of freeze-fractured preparations and of thin sections of parasitized cells using pre- and post-embedding techniques revealed that each of the membranous structures carried a common parasite antigen, QF 116, which was identified by monoclonal antibody 8E7/55.

An antigenic complex in the rhoptries of Plasmodium falciparum.

A previously identified putative rhoptry antigen of Plasmodium falciparum is composed of two major components, one of 80 kDa and a doublet at 42/40 kDa. An inhibitory monoclonal antibody immunoprecipitated both the 80 kDa protein and the 42/40 kDa doublet, but immunoblotted only the 80 kDa component. A second monoclonal antibody, raised against the affinity purified complex, immunoblotted only the 42 kDa band under non-reducing conditions. Electron microscopic examination of thin sections of parasites immunolabeled with these monoclonal antibodies and colloidal gold anti-mouse conjugate has confirmed that this antigen is localised in the rhoptry organelles of mature schizonts and free merozoites. The antigen is associated with apparent membranous structures released from free merozoites. Immunoblotting and immunoprecipitation with two different monoclonal antibodies, and protease digestion experiments, have clearly demonstrated that this antigen is a complex composed of two separate and distinct proteins, and does not represent a monomer/dimer pair. The 80 kDa protein is synthesised as an 84 kDa precursor.

An epitope recognised by inhibitory monoclonal antibodies that react with a 51 kilodalton merozoite surface antigen in Plasmodium falciparum.

Monoclonal antibodies designated 8G10/48 and 9E3/48 raised against mature asexual blood stages of Plasmodium falciparum inhibit parasite growth in vitro. Both antibodies bind to an epitope which includes the linear sequence Ser Thr Asn Ser and which is present in a cDNA clone from a P. falciparum expression library. These antibodies recognise a glycosylated antigen of approximately 51 kDa which is located on the merozoite surface membrane.

Localisation of internal antigens of Plasmodium falciparum using monoclonal antibodies and colloidal gold.

By the examination of several defined malarial antigens, we have demonstrated the necessity for etching pretreatments to be used in conjunction with post-embedding immunolabelling of LR White-embedded parasite material. In general, etching procedures markedly enhanced immunolabelling of the various antigens, while in some cases etching was essential for obtaining positive immunolabelling. Of the etching pretreatments evaluated, a combination of an alcoholic solution of sodium hydroxide followed by sodium metaperiodate gave optimal labelling with minimal background. A number of fixation regimes were also compared for their applicability to immunolabelling of malaria-infected erythrocytes. Generally, fixation with low concentrations of glutaraldehyde was found to be appropriate. We have also successfully used paraformaldehyde fixation coupled with etching to localise a rhoptry-associated antigen, which is presumably sensitive to glutaraldehyde fixation. Due to the high specificity of monoclonal antibodies, however, different fixation regimes may need to be considered for various combinations of antigen and antibody.