A dual expression system for the generation, analysis and purification of antibodies to a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA.
A novel dual expression system for the generation and analysis of immune responses to recombinant protein is described. The two expression systems are based on the IgG-binding domains (ZZ) of staphylococcal protein A (SpA) and the human serum albumin (HSA) binding domains (BB) of streptococcal protein G, respectively. Products of fusions with the ZZ region are used to generate an immune response against the recombinant peptide and the corresponding peptide fused to the BB region is used for analysis and purification of the specific antibodies. The protein A and protein G expression systems were used to produce fusion proteins with the repeated C terminal octapeptide subunit EENVEHDA of the Plasmodium falciparum merozoite derived protein Pf155/RESA. Rabbits were immunized with the protein A-derived fusion protein (designated ZZ-M1) and the antibody response was analyzed using the protein G-derived fusion protein (designated BB-M1). The rabbit antisera reacted with BB-M1 in both ELISA and immunoblotting. In addition, BB-M1 proved to be an efficient ligand for affinity purification of antibodies specific for the malaria peptide. Furthermore, the rabbit antisera reacted with Pf155/RESA both in merozoite extracts and when deposited in the membrane of parasite infected erythrocytes.
Ståhl
S
S
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Sjölander
A
A
Nygren
P A
PA
Berzins
K
K
Perlmann
P
P
Uhlén
M
M
D000818:Animals; D000913:Antibodies, Protozoan / Q000096:biosynthesis* / Q000302:isolation & purification; D000917:Antibody Formation; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000276:immunology; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D007114:Immunization; D015151:Immunoblotting; D008288:Malaria / Q000276:immunology; D010963:Plasmodium falciparum / Q000235:genetics / Q000276:immunology*; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology; D012091:Repetitive Sequences, Nucleic Acid; D013205:Staphylococcal Protein A / Q000235:genetics
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C050256:ring-infected erythrocyte surface antigen (RESA), Plasmodium falciparum