Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood.
Recombinant fusion proteins, consisting of a monovalent anti-human RBC monoclonal antibody B6, and conserved immunodominant peptide of HIV-1 envelope glycoprotein gp41 or HIV-2 envelope glycoprotein gp36, have been designed and purified after over-expression in E. coli. These fusion proteins are Fab-based and were obtained by assembling the light chain with Fd (variable domain and the first constant domain of the heavy chain) or Fd fusions containing HIV-derived peptide, and following a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation to assemble functional Fab. Using a multistep column chromatographic procedure, monomeric Fab and Fab fusion proteins containing HIV-derived peptide were purified to high degree, free of aggregates. The yield of various proteins on the laboratory scale (1-2 l of shake flask culture) was in the range of tens of milligram. Purified anti-human RBC Fab fusion proteins containing sequences derived from HIV-1 gp41 and HIV-2 gp36 were highly specific for detection of antibodies to HIV-1 and HIV-2, respectively. The described design, expression and purification protocols will make it possible to produce specific recombinant reagents in large quantities for agglutination-based rapid detection of antibodies to HIV in whole blood.
Gupta
A
A
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi-110 021, India.
Gupta
S
S
Chaudhary
V K
VK
D000372:Agglutination Tests / Q000379:methods*; D017123:Bacteriophage T7 / Q000235:genetics; D004912:Erythrocytes / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D015686:Gene Products, env / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D005822:Genetic Vectors; D015483:HIV Antibodies / Q000097:blood*; D015488:HIV Antigens / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D015658:HIV Infections / Q000175:diagnosis*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D014170:Transformation, Genetic; D054299:env Gene Products, Human Immunodeficiency Virus
D015686:Gene Products, env; D015483:HIV Antibodies; D015488:HIV Antigens; D015700:HIV Envelope Protein gp41; D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; D054299:env Gene Products, Human Immunodeficiency Virus; C059763:env protein gp36, Human immunodeficiency virus