PubMed1997
Ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) were developed for antibody IgGs to HIV-1 using recombinant reverse transcriptase (rRT), p17 (rp17), and p24 (rp24) as antigens. Antibody IgGs were reacted with 2,4-dinitrophenyl-recombinant antigens and recombinant antigen-beta-D-galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complexes were eluted from the polystyrene beads with excess of epsilon N-2,4-dinitrophenyl-L-lysine and were transferred to clean polystyrene beads coated with (antihuman IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the last polystyrene beads was assayed by fluorometry. By transfer of the immune complexes from one solid phase to another, the nonspecific binding of the beta-D-galactosidase conjugates was minimized and the sensitivity was markedly improved. The immune complex transfer enzyme immunoassays using rRT, rp17, and rp24 as antigens were 300-1,000-fold, 1,000-3,000-fold, and 30-100-fold, respectively, more sensitive than Western blotting for the corresponding antigens and 10-300-fold more sensitive than a conventional ELISA and a gelatin particle agglutination test. For urine (100 microliters), whole saliva (1 microliter), and serum (1 microliter) samples, the sensitivity and specificity of the immune complex transfer enzyme immunoassay using rRT as antigen were both 100%. However, for urine samples in which the specific activities of antibody IgG to RT, p17, and p24 were much lower than those in serum samples probably due to degradation by the kidney, a longer assay of bound beta-D-galactosidase activity or/and a concentration process for urine was required. The use of more than 1 microliter of whole saliva was recommended for reliable diagnosis of the infections, whereas 1 microliter of serum was sufficient for the purpose. The positivity with rRT as antigen could be confirmed by demonstration of antibody IgGs to p17 and p24 in most of the urine, whole saliva, and serum samples. In HIV-1 seroconversion serum panels, antibody IgG to p17 was detected as early as or even earlier than antibodies to HIV-1 by a conventional ELISA or/and a gelation particle agglutination test, whereas antibody IgGs to RT and p24 were detected as early as or later than antibody IgG to p17. Thus the uses of rRT and rp17 as antigens were advantageous over that of the other antigens for randomly collected serum samples probably long after the infection and serum samples at early stages of the infection, respectively. On the basis of these results and other reports, the immune complex transfer enzyme immunoassay was developed for simultaneous detection of p24 antigen and antibody IgGs to RT and p17 in a single assay tube, and the window period (8 weeks, although widely variable), during which diagnosis of HIV-1 infection is not possible due to the absence of detectable antibodies to HIV-1, was shortened by 2 weeks. As a result, the simultaneous detection made possible not only as early diagnosis as that by detection of p24 antigen, but also as reliable diagnosis as that by detection of antibodies to HIV-1. Finally, the immune complex transfer enzyme immunoassay has been recently improved so as to be performed within shorter periods of time (2-3 hr) with higher sensitivity, and testing many samples has become easy.
Acquired Immunodeficiency Syndrome↗