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, Setsuko; Hashida, Seiichi; Hashinaka, Kazuya; Kojima, Masaaki; Saito, Atsushi; Takamizawa, Akihisa; Shinagawa, Hideo; Oka, Shinichi; Shimada, Kaoru; Ishikawa, Eiji

doi:10.1002/(sici)1098-2825(1997)11:5<244::aid-jcla2>3.0.co;2-7

Cited by 18 publications

(15 citation statements)

References 17 publications

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“…The colored polystyrene beads after washing were incubated for 1 h with two white polystyrene beads coated with affinity-purified (anti-human IgG ␥-chain) IgG in the presence of εN-2,4-dinitrophenyl-L-lysine. The incubations were performed at room temperature with 180 shakings per min throughout (21). Bound ␤-D-galactosidase activity was assayed at 30°C for 2 h as described above.…”

Section: Methodsmentioning

confidence: 99%

“…The specific signal was calculated by subtraction of the negative signal (N) obtained with serum of an HIV-1-seronegative subject from the positive signal (P) obtained with serum of an HIV-1-seropositive subject and was divided by the negative signal as follows: (P Ϫ N)/N. The improved immunoassay of antibody IgG to HIV-1 p17 antigen with shaking using 100-l serum samples (21) in the present study was approximately 90-fold more sensitive than the previous one without shaking using 10-l serum samples (13).…”

Section: Methodsmentioning

confidence: 99%

“…More recently, immune complex transfer EIA of antibody IgG to p17 antigen has been improved in sensitivity by performing immunoreactions with shaking and increasing the serum sample volume (21), and the conditions for this immunoassay have been optimized (15). Similarly, immune complex transfer EIA of HIV-1 p24 antigen has been developed (11,14) and improved in sensitivity (16).…”

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

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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“…Immune complex transfer enzyme immunoassay of antibody IgG to HIV-1. Immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 was performed essentially in the same ways as described previously (11,21). An aliquot (10 l) of serum samples was incubated with 140 l of buffer J containing 0.4 M NaCl, 50 g of inactive ␤-D-galactosidase (␤-galactosidase-mutein; Boehringer Mannheim), 100 fmol each of 2,4-dinitrophenyl-antigen and antigen-␤-Dgalactosidase conjugate at room temperature for 2 h. Inactive ␤-D-galactosidase was used to eliminate interference by anti-␤-D-galactosidase antibodies (24).…”

Section: Labeling Of Recombinant Proteins With ␤-D-galactosidase (I)mentioning

confidence: 99%

Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1

Hashinaka

Hashida

Nishikata

et al. 2000

Clin Diagn Lab Immunol

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An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in E. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to ␤-D-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y ‫؍‬ 0.99 log X ؉ 0.23; r ‫؍‬ 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.

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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

Cited by 18 publications

References 17 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

Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1

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