Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood

被引:12
作者
Gupta, A [1 ]
Gupta, S [1 ]
Chaudhary, VK [1 ]
机构
[1] Univ Delhi, Dept Biochem, New Delhi 110021, India
关键词
agglutination; diagnosis; HIV;
D O I
10.1016/S0022-1759(01)00435-5
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 1 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. (C) 2001 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:121 / 140
页数:20
相关论文
共 30 条
[1]   IMMUNOTOXINS AGAINST CANCER [J].
BRINKMANN, U ;
PASTAN, I .
BIOCHIMICA ET BIOPHYSICA ACTA-REVIEWS ON CANCER, 1994, 1198 (01) :27-45
[2]  
BROLIDEN PA, 1991, J ACQ IMMUN DEF SYND, V4, P952
[3]   RENATURATION OF A SINGLE-CHAIN IMMUNOTOXIN FACILITATED BY CHAPERONES AND PROTEIN DISULFIDE ISOMERASE [J].
BUCHNER, J ;
BRINKMANN, U ;
PASTAN, I .
BIO-TECHNOLOGY, 1992, 10 (06) :682-685
[4]   A METHOD FOR INCREASING THE YIELD OF PROPERLY FOLDED RECOMBINANT FUSION PROTEINS - SINGLE-CHAIN IMMUNOTOXINS FROM RENATURATION OF BACTERIAL INCLUSION-BODIES [J].
BUCHNER, J ;
PASTAN, I ;
BRINKMANN, U .
ANALYTICAL BIOCHEMISTRY, 1992, 205 (02) :263-270
[5]  
BUCHNER J, 1991, BIOTECHNOLOGY, V9, P158
[6]   A RAPID METHOD OF CLONING FUNCTIONAL VARIABLE-REGION ANTIBODY GENES IN ESCHERICHIA-COLI AS SINGLE-CHAIN IMMUNOTOXINS [J].
CHAUDHARY, VK ;
BATRA, JK ;
GALLO, MG ;
WILLINGHAM, MC ;
FITZGERALD, DJ ;
PASTAN, I .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (03) :1066-1070
[7]   A RECOMBINANT IMMUNOTOXIN CONSISTING OF 2 ANTIBODY VARIABLE DOMAINS FUSED TO PSEUDOMONAS EXOTOXIN [J].
CHAUDHARY, VK ;
QUEEN, C ;
JUNGHANS, RP ;
WALDMANN, TA ;
FITZGERALD, DJ ;
PASTAN, I .
NATURE, 1989, 339 (6223) :394-397
[8]  
CHAUDHARY VK, 1990, J BIOL CHEM, V265, P16306
[9]   Single-chain Fv/folate conjugates mediate efficient lysis of folate-receptor-positive tumor cells [J].
Cho, BK ;
Roy, EJ ;
Patrick, TA ;
Kranz, DM .
BIOCONJUGATE CHEMISTRY, 1997, 8 (03) :338-346
[10]  
CHOE M, 1994, CANCER RES, V54, P3460