Immune complex transfer enzyme immunoassay that is more sensitive and specific than western blotting for detection of antibody immunoglobulin G to human immunodeficiency virus type 1 in serum with recombinant pol and gag proteins as antigens

Hashida, Seiichi; Hashinaka, Kazuya; Nishikata, Ichiro; Oka, Shinichi; Shimada, Kaoru; Saito, Atsushi; Takamizawa, Akihisa; Shinagawa, Hideo; Yano, Saburo; Kojima, Hiroshi

doi:10.1128/cdli.2.5.535-541.1995

Cited by 28 publications

(23 citation statements)

References 26 publications

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“…When increasing volumes of two serum samples from HIV-1 seropositive subjects were tested by the immune complex transfer enzyme immunoassays using rRT, rp17 and rp24 as antigens and β-D-galactosidase from E. coli as label, the signal was enhanced almost linearly with up to 5 µl, and only slight interference was observed with 10 µl of serum (19).…”

Section: Effect Of Serum Volume On Signal (Serum Interference)mentioning

confidence: 99%

“…Serum samples from 79 HIV-1 seropositive subjects (50 asymptomatic carriers, 9 patients with AIDS-related complex (ARC) and 20 patients with AIDS) and 100 HIV-1 seronegative subjects were tested by the immune complex transfer enzyme immunoassays (Fig. 11) (19). The volume of serum samples used was 10 µl.…”

Section: Sensitivity Of Immune Complex Transfer Enzyme Immunoassay Comentioning

confidence: 99%

“…Two serum samples from HIV-1 seropositive subjects were serially diluted with serum from an HIV-1 seronegative subject and were tested by the immune complex transfer enzyme immunoassays, Western blotting (Ortho kit), the conventional ELISA using five recombinant proteins of HIV-1 as antigens (Abbott HTLV-III EIA kit), and the gelatin particle agglutination test using a lysate of HIV-1 as antigen (Serodia-HIV kit, Fujirebio, Tokyo, Japan) ( Fig. 12) (19). The maximal dilutions of the serum samples to show the positivity were compared.…”

Section: Sensitivity Of Immune Complex Transfer Enzyme Immunoassay Comentioning

confidence: 99%

“…Fifty serum samples were collected from healthy subjects with low risk for HIV infection and tested by the conventional ELISA, the gelatin particle agglutination test, and Western blotting using two commercial kits (Ortho Diagnostic Systems and Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) (Table 4) (19). All the serum samples were negative by both the conventional ELISA and the gelatin particle agglutination test.…”

Section: Specificity Of Immune Complex Transfer Enzyme Immunoassay Comentioning

confidence: 99%

“…This article reviews the application of ultrasensitive and highly specific enzyme immunoassays (immune complex transfer enzyme immunoassays) for antibody IgGs to pol (reverse transcriptase (RT)) and gag (p17 and p24) proteins of HIV-1 and for p24 antigen of HIV-1, which significantly overcome the above drawbacks of conventional methods in diagnosis of HIV-1 infection (12,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24).…”

Section: Introduction: Review Of Enzyme Immunoassay Applicationsmentioning

confidence: 99%

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More reliable diagnosis of infection with human immunodeficiency virus type 1 (HIV‐1) by detection of antibody IgGs to pol and gag proteins of HIV‐1 and p24 antigen of HIV‐1 in urine, saliva, and/or serum with highly sensitive and specific enzyme immunoassay (immune complex transfer enzyme immunoassay): A review

Hashida¹,

Hashinaka²,

Ishikawa³

et al. 1997

J. Clin. Lab. Anal.

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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 wee...

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Section: Effect Of Serum Volume On Signal (Serum Interference)mentioning

confidence: 99%

Section: Sensitivity Of Immune Complex Transfer Enzyme Immunoassay Comentioning

confidence: 99%

Section: Sensitivity Of Immune Complex Transfer Enzyme Immunoassay Comentioning

confidence: 99%

Section: Specificity Of Immune Complex Transfer Enzyme Immunoassay Comentioning

confidence: 99%