Ultrasensitive enzyme immunoassay

Ishikawa, Eiji; Hashida, Seiichi; Kohno, Takeyuki; Hirota, Kouichi

doi:10.1016/0009-8981(90)90303-a

Cited by 62 publications

(42 citation statements)

References 43 publications

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“…White and colored polystyrene beads (3.2 mm in diameter; Immuno Chemical, Inc., Okayama, Japan) were coated with proteins by physical adsorption (20). Colored polystyrene beads were coated with affinity-purified (anti-2,4-dinitrophenyl-bovine serum albumin) IgG (0.05 g/liter), which had been eluted from 2,4-dinitrophenyl-bovine serum albumin-Sepharose 4B (9) with 3.2 mM HCl (pH 2.5) (24).…”

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

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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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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

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“…7 The amount of the conjugate was calculated from peroxidase activity. 8 Immune complex transfer enzyme immunoassay for detection of anti-ovalbumin IgA in serum (See Fig. 1.)…”

Section: Ovalbumin--d-galactosidase Conjugatementioning

confidence: 99%

Immune complex transfer enzyme immunoassay for anti-ovalbumin IgA in serum

Hirota

Hashida

et al. 2002

Ann Clin Biochem

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“…White polystyrene beads (3.2 mm in diameter, Immuno Chemical, Okayama, Japan) were coated with rabbit anti-p24 IgG (0.1 g/L) by physical adsorption (13). The antigen, p24 or rp24, in 10 pl of serum mixed with 140 pl of buffer A containing 0.4 mol/L NaCl was incubated with a polystyrene bead coated with anti-rp24 IgG at room temperature overnight.…”

Section: Conventional Two-site Enzyme Lmmunoassay For P24 Antigenmentioning

confidence: 99%