Simple and more sensitive immune complex transfer enzyme immunoassay for antibody IgG to reverse transcriptase of HIV-1 using microplates, modified polystyrene solid phase, and fluororeader

Hashida, Seiichi; Ishikawa, Setsuko; Nakamoto, Hidetaka; Tanaka, Satoshi; Kojima, Masaaki; Ishikawa, Eiji

doi:10.1002/(sici)1098-2825(1996)10:5<294::aid-jcla10>3.0.co;2-s

Cited by 6 publications

(2 citation statements)

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“…By the immune complex transfer, the nonspecific signal, that is, the signal in the absence of antigens and antibodies to be measured is reduced to greater extents than the specific signal due to the presence of the analytes, and the detection limit of the analytes is lowered to various extents from case to case. Three examples shown in Tables 1-3 are HIV-1 p24 antigen (9,10) and antibody IgGs to HIV-1 p17 and reverse transcriptase (RT) antigens (8,(11)(12)(13)(14)(15)(16)(17)(18).…”

Section: Potential Of the Immune Complex Transfer Enzyme Immunoassay mentioning

confidence: 99%

Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

Ishikawa

Hashida

et al. 1998

J. Clin. Lab. Anal.

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In order to reduce the nonspecific signal of noncompetitive solid phase immunoassays and to improve their sensitivities, the immune complex transfer enzyme immunoassay has been developed. Antigens to be measured were reacted with 2,4-dinitrophenyl-biotinyl-antibody Fab' and antibody Fab'-beta-D-galactosidase conjugate, and antibody IgGs to be measured were reacted with 2,4-dinitrophenyl-antigen and antigen-beta-D-galactosidase conjugate. The immune complexes formed comprising the three components were trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complexes were eluted from the colored polystyrene beads with epsilonN-2,4-dinitrophenyl-L-lysine, and the eluates were incubated with white polystyrene beads coated with streptavidin for antigens and coated with affinity-purified (anti-human IgG gamma-chain) IgG for antibody IgGs to transfer the immune complexes. By this method, ultrasensitive enzyme immunoassays have been developed for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and reverse transcriptase (RT). The nonspecific signals in the absence of the antigen and the antibody IgGs were reduced 300 to 15,000-fold by the immune complex transfer process, but the amounts of the immune complexes decreased only 1.8 to 3.1-fold by the immune complex transfer. As a result, the sensitivities for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and RT were improved 100 to 5,600-fold by the immune complex transfer. The detection limit of HIV-1 p24 antigen by 20 hr assay of beta-D-galactosidase activity (10 zmol) was 4,000 to 17,000-fold lower than those obtained with currently available commercial kits. The improved sensitivities for antibody IgGs to p17 and RT by 20 hr assay of beta-D-galactosidase activity were 1 x 10(5) to 3 x 10(5)-fold higher than those of Western blotting for p17 and p66 bands. However, the nonspecific signals in the absence of antigens and antibody IgGs were enhanced to various degrees by two factors. In order to transfer the immune complexes more efficiently within shorter periods of time, the colored polystyrene beads were incubated with the white polystyrene beads in the presence of epsilonN-2,4-dinitrophenyl-L-lysine. Such direct contact between solid phases for trapping and transferring of the immune complexes significantly enhanced the nonspecific signals. In addition, the presence of human serum samples containing neither antigens to be measured nor antibody IgGs to be measured also enhanced the nonspecific signals to various extents. Namely, these two factors limited the effect of the immune complex transfer to improve the sensitivity by 20 hr assay of beta-D-galactosidase activity. By 1 hr assay of beta-D-galactosidase activity, the detection limit of HIV-1 p24 antigen using 10 microl of serum samples (0.24 pg/ml) was 40 to 80-fold lower than those obtained with currently available commercial kits using 100 to 200 microl of serum samples (10 to 20 pg/ml) and the detection limits of antibody IgGs to HIV-1 ...

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Section: Potential Of the Immune Complex Transfer Enzyme Immunoassay mentioning

confidence: 99%

Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

Ishikawa

Hashida

et al. 1998

J. Clin. Lab. Anal.

Self Cite

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“…16) for test tubes, a spectrofluorophotometer, and polystyrene beads, respectively (Fig. 17) (59)(60)(61). Polystyrene sticks (Fig.…”

Section: Improvement Of Immune Complex Transfer Enzyme Immunoassaymentioning

confidence: 99%

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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More sensitive immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV-1 with shorter incubation time for immunoreactions and larger volumes of serum samples

Ishikawa

Hashida

Hashinaka

et al. 1997

J. Clin. Lab. Anal.

Self Cite

View full text Add to dashboard Cite

Simple and more sensitive immune complex transfer enzyme immunoassay for antibody IgG to reverse transcriptase of HIV-1 using microplates, modified polystyrene solid phase, and fluororeader

Cited by 6 publications

References 15 publications

Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

More sensitive immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV-1 with shorter incubation time for immunoreactions and larger volumes of serum samples

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