Earlier diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgGs to p17 and reverse transcriptase in serum with enzyme immunoassay

Hashida, Seiichi; Hashinaka, Kazuya; Nishikata, Ichiro; Saito, Atsushi; Takamizawa, Akihisa; Shinagawa, Hideo; Ishikawa, Eiji

doi:10.1002/(sici)1098-2825(1996)10:4<213::aid-jcla7>3.0.co;2-a

Cited by 11 publications

(5 citation statements)

References 26 publications

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“…As our first test case we have synthesized an excimer-based PB directed against an antibody diagnostic of HIV infection . The human immunodeficiency virus type 1 (HIV-1) matrix protein p17, an important component in the viral life cycle, is among the more antigenic proteins in the retrovirus.…”

mentioning

confidence: 99%

“…As our first test case we have synthesized an excimer-based PB directed against an antibody diagnostic of HIV infection. 12 The human immunodeficiency virus type 1 (HIV-1) matrix protein p17, an important component in the viral life cycle, 13 is among the more antigenic proteins in the retrovirus. We have employed a highly antigenic, 14 six-residue epitope present in the protein as the basis of our first PB.…”

mentioning

confidence: 99%

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Excimer-Based Peptide Beacons: A Convenient Experimental Approach for Monitoring Polypeptide−Protein and Polypeptide−Oligonucleotide Interactions

Cash

Plaxco

2006

J. Am. Chem. Soc.

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While protein-polypeptide and nucleic acid-polypeptide interactions are of significant experimental interest, quantitative methods for the characterization of such interactions are often cumbersome. Here we described a relatively simple means of optically monitoring such interactions using excimer-based peptide beacons (PBs). The design of PBs is based on the observation that, whereas short peptides are almost invariably unfolded and highly dynamic, they become rigid when complexed with macromolecular targets. Using this binding-induced folding to segregate two pyrene moieties and therefore inhibit excimer formation, we have produced PBs directed against both anti-HIV antibodies and the retroviral transactive response (TAR) RNA hairpin. For both polypeptides, target recognition is accompanied by a roughly 2-fold decrease in excimer emission, thus allowing the detection of their respective targets at concentrations of a few nanomolar. Because excimer emission requires the formation of a tight, precisely oriented pyrene dimer, even relatively trivial binding-induced segregation reduces fluorescence significantly. This suggests that the PB approach will be suitable for monitoring a wide range of peptide-macromolecule recognition events. Moreover, the synthesis of excimer-based PBs utilizes commercially available modified pyrenes in a simple and well-established protocol, making the approach well suited for routine laboratory applications.

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confidence: 99%

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confidence: 99%

Excimer-Based Peptide Beacons: A Convenient Experimental Approach for Monitoring Polypeptide−Protein and Polypeptide−Oligonucleotide Interactions

Cash

Plaxco

2006

J. Am. Chem. Soc.

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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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“…14), and 11 HIV-1 seroconversion serum panels were tested ( Table 5 and Fig. 15) (20,21). For detection of p24 antigen, monoclonal mouse anti-p24 Fab´-β-D-galactosidase conjugate was used.…”

Section: Simultaneous Detection Of Both P24 Antigen and Antibody Iggsmentioning

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%

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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Earlier diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgGs to p17 and reverse transcriptase in serum with enzyme immunoassay

Cited by 11 publications

References 26 publications

Excimer-Based Peptide Beacons: A Convenient Experimental Approach for Monitoring Polypeptide−Protein and Polypeptide−Oligonucleotide Interactions

Excimer-Based Peptide Beacons: A Convenient Experimental Approach for Monitoring Polypeptide−Protein and Polypeptide−Oligonucleotide Interactions

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

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