Overexpression and purification of a recombinant chimeric HIV type 2/HIV type 1 envelope peptide and application in an accelerated immunobased HIV type 1/2 antibody detection system (AIBS): a new rapid serological screening assay.
A chimeric HIV-2/HIV-1 envelope sequence containing an immunodominant region of HIV-2 gp36 and the corresponding region of HIV-1 gp41 was constructed and overexpressed in Escherichia coli. The recombinant product (rp21/18) was purified and applied in a novel antibody-screening assay. Characteristics in the design of this new principle are as follows: (1) the overall assay time is about 30 min; (2) the assay procedure includes three manipulation steps; and (3) the test shows a reliable performance with respect to sensitivity and specificity. The diluted serum sample and the protein G-horseradish peroxidase conjugate are added simultaneously into a coated (hybrid antigen HIV-1/2) and blocked microtiter plate well. The in-batch incubation of serum sample with protein G-horseradish peroxidase saves two manipulation steps that are normally necessary in the five-step procedure of a classical ELISA. AIBS was evaluated with commercially available seroconversion panels and with random negative serum samples from a blood bank. Seroconversion results demonstrated that AIBS has equivalent sensitivity to ELISAs and the third generation assays. The specificity was determined on a total blood donor population of 5012 (Red Cross Vienna, Austria). The repeat reactive rates for donor population were 0.02%. AIBS represents a general immunometric system (not only HIV antibodies). The entire assay procedure of AIBS evaluated for HIV-1/2 screening, including result reporting, can be performed automatically by several commercially available systems. Depending on these systems AIBS is potentially useful in laboratories or blood banks that have both high- and low-volume testing.
Ecker
B
B
University of Vienna, Institute of Biochemistry and Molecular Cell Biology, Austria.
Vollenhofer
S
S
Bares
T
T
Schalkhammer
T
T
Schinkinger
M
M
Pittner
F
F
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology*; D015700:HIV Envelope Protein gp41 / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification*; D015498:HIV-2 / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007118:Immunoassay; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012698:Serologic Tests / Q000379:methods*
D000914:Antibodies, Viral; D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins