Diagnosis of HIV-1 infection by detection of antibody IgG to HIV-1 in urine with ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens

✍ Scribed by Seiichi Hashida; Kazuya Hashinaka; Atsushi Saitoh; Akihisa Takamizawa; Hideo Shinagawa; Shinichi Oka; Kaoru Shimada; Kouichi Hirota; Takeyuki Kohno; Setsuko Ishikawa; Dr. Eiji Ishikawa

• Publisher: John Wiley and Sons
• Year: 1994
• Tongue: English
• Weight: 785 KB
• Volume: 8
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.1860080410

✦ Synopsis

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p i 7 and p24 as antigens, and p-o-galactosidase from Escherichia coli as label. Anti-HIV-I IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant prOtein-P-D-galaCtOSidaSe conjugate. The immune complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinitypurified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of &N-2,4-dinitrophenyl-~-lysine and transferred to clean polystyrene balls coated with aff inity-purified (anti-human IgG ychain) IgG.

Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 1 OO%, and the lowest signal for 60 asymptomatic carriers was 8.2-fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p l 7 and p24 as antigens. The sensitivity could be improved by a longer assay of bound p-o-galactosidase activity, by using concentrated urine samples and by the combined use of recombinant RT, p l 7, and p24.Thus, reliable diagnosis of HIV-1 infection was possible for asymptomatic carriers. Q 1994 Wiley-Liss, inc.