Measurement of human immunodeficiency virus type 1 p24 in serum by an ultrasensitive enzyme immunoassay, the two-site immune complex transfer enzyme immunoassay

Hashida, Seiichi; Hashinaka, Kazuya; Nishikata, Ichiro; Oka, Shinichi; Shimada, Kaoru; Saitoh, Atsushi; Takamizawa, Akihisa; Shinagawa, Hideo; Ishikawa, Eiji

doi:10.1128/jcm.33.2.298-303.1995

Cited by 31 publications

(18 citation statements)

References 26 publications

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“…In addition, the total assay time was shortened 10-fold compared to the two-site immune complex transfer enzyme immunoassay described previously. 31,32 Furthermore, specimens were treated with pretreatment reagents to detect HBsAg even in the presence of anti-HBs antibodies in this assay. The ingredients in the pretreatment reagents were optimized for sensitive HBsAg detection by releasing HBsAg from immune complexes consisting of HBsAg and anti-HBs.…”

Section: Discussionmentioning

confidence: 99%

Novel monitoring of hepatitis B reactivation based on ultra‐high sensitive hepatitis B surface antigen assay

Shinkai

Kusumoto

Murakami

et al. 2017

Liver International

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Section: Discussionmentioning

confidence: 99%

Novel monitoring of hepatitis B reactivation based on ultra‐high sensitive hepatitis B surface antigen assay

Shinkai

Kusumoto

Murakami

et al. 2017

Liver International

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“…Since detection of p24 by a highly sensitive ELISA-based method can play a critical role in diagnosis of new HIV-1 infections, many sensitive p24 detection assays have been developed [ 33 – 36 ]. However, the complex procedures, high cost, and the inherently inaccurate quantification of target molecules become major hurdles for their wide use.…”

Section: Discussionmentioning

confidence: 99%

“…However, this window period was not significantly shortened while the complexity of testing was increased [ 32 ]. The ultrasensitive immunoassay (immune complex transfer enzyme immunoassay) was more sensitive for detection of p24 (> 630-fold), but this procedure was tedious and the serious serum interference limited its application [ 33 ]. Recently, nanoparticle based assays were reported to be able to significantly lower the detection limit of p24 than the conventional ELISA [ 34 – 36 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

Fan

et al. 2015

PLoS ONE

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The relatively high detection limit of the Enzyme-linked immunosorbent assay (ELISA) prevents its application for detection of low concentrations of antigens. To increase the sensitivity for detection of HIV-1 p24 antigen, we developed a highly sensitive nuclease-linked fluorescence oligonucleotide assay (NLFOA). Two major improvements were incorporated in NLFOA to amplify antibody-antigen interaction signals and reduce the signal/noise ratio; a large number of nuclease molecules coupled to the gold nanoparticle/streptavidin complex and fluorescent signals generated from fluorescent-labeled oligonucleotides by the nuclease. The detection limit of p24 by NLFOA was 1 pg/mL, which was 10-fold more sensitive than the conventional ELISA (10 pg/mL). The specificity was 100% and the coefficient of variation (CV) was 7.8% at low p24 concentration (1.5 pg/mL) with various concentrations of spiked p24 in HIV-1 negative sera. Thus, NLFOA is highly sensitive, specific, reproducible and user-friendly. The more sensitive detection of low p24 concentrations in HIV-1-infected individuals by NLFOA could allow detection of HIV-1 infections that are missed by the conventional ELISA at the window period during acute infection to further reduce the risk for HIV-1 infection due to the undetected HIV-1 in the blood products. Moreover, NLFOA can be easily applied to more sensitive detection of other antigens.

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“…The recombinant proviral clone used was pNL4-3 (28), which contained DNA from HIV-1 isolates NY5 (GenBank accession number HIVNL43) and LAV (29), and the sequences for rRT, rp17, and rp24 derived from NY5. By applying the same principle as used for the detection of antibodies, p24 antigen of HIV-1 has been measured with high sensitivity (22).…”

Section: Immune Complex Transfer Enzyme Immunoassay For Antibody Igg mentioning

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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Measurement of human immunodeficiency virus type 1 p24 in serum by an ultrasensitive enzyme immunoassay, the two-site immune complex transfer enzyme immunoassay

Cited by 31 publications

References 26 publications

Novel monitoring of hepatitis B reactivation based on ultra‐high sensitive hepatitis B surface antigen assay

Novel monitoring of hepatitis B reactivation based on ultra‐high sensitive hepatitis B surface antigen assay

Enhanced Sensitivity for Detection of HIV-1 p24 Antigen by a Novel Nuclease-Linked Fluorescence Oligonucleotide Assay

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