Fast immunopurification of small amounts of specific antibodies on peptides bound to ELISA plates.
ELISA is widely used as a means to detect antibodies, but the potential of ELISA plates as an immunosorbent for the purification of specific antibodies does not seem to have been evaluated. In this study, ELISA plates coated with peptides representing short sequences of various antigens from Plasmodium falciparum, the etiologic agent of human malaria, have been successfully used as a means to purify small amounts of the corresponding antibodies. ELISA plates, identical to those used for antibody detection, also permitted the evaluation of various elution conditions for each pairing of peptide and serum; we tested four eluting buffers (0.2 M glycine, pH 2.5; 0.2 M lysine, pH 11.5; 3.0 M MgCl2, 0.075 M Hepes, 25% ethylene glycol, pH 7.1-7.2 and 4 M NH4SCN in 0.1 M NaH2PO4, pH 6.0) with four pairs of peptides and sera. The ELISA plates could also be used to estimate the affinity of the eluted antibodies by the technique of Pullen et al. (1986). The eluted antibodies were compared to those obtained by immunopurification on recombinant proteins adsorbed on nitrocellulose filters. In contrast to the latter, they were not contaminated by antibodies directed against the carrier moiety of the recombinant protein. When used in immunofluorescence assays with various stages of the parasite the antibodies immunopurified on peptides bound to ELISA plates were able to react with the native antigens in the parasite.
Parasitologie Bio-Médicale, Institut Pasteur, Paris, France.
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Brahimi
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JL
Pérignon
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Bossus
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Gras
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Tartar
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Druilhe
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000913:Antibodies, Protozoan / Q000302:isolation & purification; D000915:Antibody Affinity; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007163:Immunosorbent Techniques; D010455:Peptides; D010963:Plasmodium falciparum / Q000276:immunology
D000906:Antibodies; D000913:Antibodies, Protozoan; D002021:Buffers; D010455:Peptides