Optimal conditions of immune complex transfer enzyme immunoassay for p24 antigen of HIV-1

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

doi:10.1002/(sici)1098-2825(1998)12:2<115::aid-jcla7>3.0.co;2-9

Cited by 9 publications

(4 citation statements)

References 15 publications

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“…Similarly, immune complex transfer EIA of HIV-1 p24 antigen has been developed (11,14) and improved in sensitivity (16). Simultaneous detection of HIV-1 p24 antigen and antibody IgGs to RT and p17 antigens by immune complex transfer EIAs has shortened the window period after HIV-1 infection, during which diagnosis of the infection is not possible due to the absence of detectable antibodies to HIV-1 in the circulation (13).…”

mentioning

confidence: 83%

“…The previous immunoassay of HIV-1 p24 antigen in serum was modified as follows (16). An aliquot (50 l) of serum samples was incubated for 16 h with 100 l of buffer A containing 0.5 M NaCl, 100 fmol of 2,4-dinitrophenyl-biotinylbovine serum albumin-affinity-purified rabbit anti-p24 FabЈ conjugate, 5 fmol of monoclonal mouse anti-p24 FabЈ-␤-D-galactosidase conjugate, 50 g of inactive ␤-D-galactosidase, and 10 l of nonspecific rabbit serum and subsequently for 2 h with two colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG.…”

Section: Methodsmentioning

confidence: 99%

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Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

Hashida

Ishikawa

Hashinaka

et al. 2000

Clin Diagn Lab Immunol

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For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25-and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.The positive rates of immunoglobulin G (IgG) antibodies to human immunodeficiency virus type 1 (HIV-1) antigens in serum samples from HIV-1-infected subjects have been reported by the conventional enzyme-linked immunosorbent assay (ELISA) and Western blotting, and it has been generally accepted that the positive rates are high with gp160, gp41 and reverse transcriptase (RT), or p66 (a subunit of RT) as antigens (98 to 100% in asymptomatic carriers, 86 to 100% in patients with AIDS-related complex [ARC], and 77 to 100% in patients with AIDS) but low with gag proteins as antigens (63 to 92% in asymptomatic carriers, 50 to 97% in patients with ARC, and 21 to 77% in patients with AIDS using p24 as antigen and 41% in asymptomatic carriers, 30% in patients with ARC, and 14% in patients with AIDS using p17 as antigen) (2,4,6,8). In seroconversion serum panels, the earliest positive band seen by Western blotting has been observed with p24 in some cases (26,29,34) and with gp160 in other cases (30, 32, 35) but not with p17 (13).Recently, ultrasensitive enzyme immunoassays (EIAs) (immune complex transfer EIAs) of antibody IgGs to HIV-1 antigens have been developed using recombinant RT, p24, and p17 antigens (10,12,18). Antibody IgGs were allowed to react simultaneously with 2,4-dinitrophenyl antigens and antigenenzyme conjugates. The immune complexes of the three components formed were trapped on (anti-2,4-dinitrophenyl group) IgG-coated solid-phase (first solid phase) and, after wash...

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mentioning

confidence: 83%

Section: Methodsmentioning

confidence: 99%

Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

Hashida

Ishikawa

Hashinaka

et al. 2000

Clin Diagn Lab Immunol

Self Cite

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“…That is, our method also has the potential to reach limits of detection of 10 −20 mol/assay. Ishikawa and Hashida 30 , 31 have achieved the limit of detection of 10 −20 mol/assay for HIV-1 p24 by their own method, the ‘immune complex transfer enzyme immunoassay’. This method, however, requires time and effort for the detection compared with our method.…”

Section: Methodsmentioning

confidence: 99%

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Watabe¹,

Kodama

Kaneda

et al. 2014

BIOPHYSICS

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An ultrasensitive method for the determination of proteins is described that combines an enzyme-linked immunosorbent assay (ELISA) and a thionicotinamide-adenine dinucleotide (thio-NAD) cycling method. A sandwich method using a primary and a secondary antibody for antigens is employed in an ELISA. An androsterone derivative, 3α-hydroxysteroid, is produced by the hydrolysis of 3α-hydroxysteroid 3-phosphate with alkaline phosphatase linked to the secondary antibody. This 3α-hydroxysteroid is oxidized to a 3-ketosteroid by 3α- hydroxysteroid dehydrogenase (3α-HSD) with a cofactor thio-NAD. By the opposite reaction, the 3-ketosteroid is reduced to a 3α-hydroxysteroid by 3α-HSD with a cofactor NADH. During this cycling reaction, thio-NADH accumulates in a quadratic function-like fashion. Accumulated thio-NADH can be measured directly at an absorbance of 400 nm without any interference from other cofactors. These features enable us to detect a target protein with ultrasensitivity (10−19 mol/assay) by measuring the cumulative quantity of thio-NADH. Our ultrasensitive determination of proteins thus allows for the detection of small amounts of proteins only by the application of thio-NAD cycling reagents to the usual ELISA system.

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“…For example, an immune complex transfer enzyme immunoassay showed a limit of detection of 0.24 pg/mL (i.e., ca . 1 x 10 −17 moles/mL) [ 6 , 7 ]. A cytometric bead assay was developed to detect p24 at 0.4 pg/mL (i.e., ca .…”

Section: Introductionmentioning

confidence: 99%

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

et al. 2015

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To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10-18 moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10-18 moles of the p24/assay corresponds to ca. 103 copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (102 copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.

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Optimal conditions of immune complex transfer enzyme immunoassay for p24 antigen of HIV-1

Cited by 9 publications

References 15 publications

Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

Ultrasensitive enzyme-linked immunosorbent assay (ELISA) of proteins by combination with the thio-NAD cycling method

Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling

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