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Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates
D Camus1, J A Lyon, T Reaud-Jareed
1Department of Immunology, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100.
Abstract:
Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.
Insights
Researchers purified malaria parasite Plasmodium falciparum antigens using monoclonal antibodies. They found that epitopes could be regenerated after acid elution, which has implications for malaria antigen purification and function studies.
Area of Science:
- Parasitology
- Immunology
- Biochemistry
Background:
- Plasmodium falciparum malaria parasites synthesize a 195 kDa surface glycoprotein (gp195).
- gp195 undergoes sequential processing into smaller products, including 83 kDa, 73 kDa, and 67 kDa fragments.
- These processed fragments are crucial for understanding parasite biology and immune responses.
Purpose of the Study:
- To investigate the purification and characterization of Plasmodium falciparum gp195 processing products.
- To explore the use of monoclonal antibodies (McAbs) for affinity purification of specific antigen fragments.
- To assess the stability and regeneration of epitopes on purified malaria antigens.
Main Methods:
- Utilized three monoclonal antibodies (McAbs) to precipitate 83 kDa and 73 kDa gp195 products from culture supernates.
- Employed affinity purification using McAb 7B2 coupled to Affigel 10.
- Eluted purified antigens using either 0.2 N acetic acid (pH 2.8) or 3 M potassium isothiocyanate (KSCN).
- Investigated epitope regeneration by treating acid-eluted antigens with 8 M urea followed by dialysis.
Main Results:
- The 83 kDa and 73 kDa gp195 processing products were successfully precipitated by McAbs.
- Affinity purification yielded the 83 kDa and 73 kDa fragments.
- Acid elution denatured the epitope recognized by McAb 7B2, but it could be regenerated.
- Regeneration of the epitope was achieved through treatment with 8 M urea and subsequent dialysis.
Conclusions:
- Monoclonal antibodies are effective tools for purifying specific Plasmodium falciparum antigen fragments.
- Epitope regeneration is feasible after harsh elution conditions, such as acid treatment.
- These findings have significant implications for the purification, functional studies, and immunogenicity assessment of malaria antigens.