Expression, purification, and characterization of an anti-RBCFab-p24 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood.
A fusion protein A41Fabp246 consisting of Fab of an anti-RBC MAb A41 and amino acids 8-153 of HIV-1 p24 (p246) fused at the C-terminus of A41Fd was purified following assembly of A41Fdp246 with A41 LC in vitro, using a denaturation- renaturation protocol and a 4-step column chromatography procedure. The highly purified, monomeric A41Fabp246 was then evaluated for hemagglutination-based detection of anti-p24 antibodies using sera from HIV-infected individuals. This derivative of p24 is devoid of maximum homology region and the C-terminal domain of p24, which is responsible for oligomerization of p24, but retains full complement of immunodominant epitopes. This new fusion protein in combination with fusion proteins consisting of monovalent fragment of another anti-human RBC antibody fused to immunodominant regions of envelope glycoproteins of HIV-1 and HIV-2 should be useful in preparing a cocktail of reagents for highly sensitive detection of anti-HIV antibodies in whole blood.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002460:Cell Line; D015870:Gene Expression; D015483:HIV Antibodies / Q000097:blood* / Q000276:immunology; D016655:HIV Core Protein p24 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology; D006386:Hemagglutination Tests / Q000379:methods*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D002479:Inclusion Bodies; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D016655:HIV Core Protein p24; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins