More sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum

A more sensitive and simpler immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum and its use for the measurement of antithyroglobulin IgG in healthy subjects and patients with thyroid diseases are described. Antithyroglobulin IgG in test serum was reacted simultaneously with dinitrophenylthyroglobulin and thyroglobulin-beta-D-galactosidase conjugate. The complex formed of antithyroglobulin IgG, dinitrophenyl thyroglobulin, and thyroglobulin-beta-D-galactosidase conjugate was trapped onto two polystyrene balls coated with affinity-purified rabbit (antidinitrophenyl bovine serum albumin) IgG. The polystyrene balls were washed to eliminate nonspecific IgG in the test serum, and the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to two polystyrene balls coated with affinity-purified rabbit (anti-human IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the polystyrene balls was assayed by fluorimetry using 4-methylumbelliferyl-beta-D-galactoside as substrate. As compared with the previous immune complex transfer enzyme immunoassay, the procedure was simplified by reducing one incubation step and one washing step, and the detection limit of antithyroglobulin IgG in serum (0.1 microgram/liter) was lowered 100-fold. More careful and extensive examination than in a previous study revealed the presence of antithyroglobulin IgG in a large proportion of healthy subjects and in all patients with Graves' disease and chronic thyroiditis.     

Use of inactive beta-D-galactosidase for elimination of interference by anti-beta-D-galactosidase antibodies in immune complex transfer enzyme immunoassay for anti-thyroglobulin IgG in serum using beta-D-galactosidase from Escherichia coli as label

A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG using beta-D-galactosidase from Escherichia coli as label was reported previously. This immunoassay was highly sensitive in demonstrating anti-thyroglobulin IgG not only in all patients with Graves' disease and chronic thyroiditis but also in a large proportion of healthy subjects. However, the detection of anti-thyroglobulin IgG at low levels in some serum samples was difficult, probably due to the presence of anti-beta-D-galactosidase antibodies. In the present study, the use of inactive beta-D-galactosidase was tested for elimination of interference by anti-beta-D-galactosidase antibodies. Preincubation of serum samples with excess of inactive beta-D-galactosidase resulted in sufficiently low backgrounds to detect low levels of anti-thyroglobulin IgG with little effect on the dose-response of anti-thyroglobulin IgG. As a result, it was revealed that anti-thyroglobulin IgG was present in almost all healthy subjects as well as all patients with Graves' disease and chronic thyroiditis.     

Development and applications of sensitive enzyme immunoassay for antibodies: a review

The sensitivity of the conventional enzyme immunoassay methods is seriously limited by the presence of nonspecific immunoglobulins at high concentrations in test serum. In order to overcome this difficulty, various methods have been developed, and the detection limit of serum antibodies has been lowered to 50-100 ng/l, which is 300,000-fold lower than that by the conventional enzyme immunoassay method using antigen-coated solid phases and anti-immunoglobulin antibody-enzyme conjugates. As a result, anti-insulin antibodies have been demonstrated in most of patients treated with insulin for 0.5-10 months, and anti-thyroglobulin IgG has been demonstrated not only in all patients with Graves' disease and chronic thyroiditis, but also in a large proportion of healthy subjects.     

Novel enzyme immunoassay (Immune Complex Transfer Enzyme Immunoassay) for anti-thyroglobulin IgG in human serum

A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglobulin IgG in human serum is described. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. After eliminating nonspecific IgG in test serum by washing, the complex was eluted from the polystyrene ball with dinitrophenyl-L-lysine and transferred to a clean polystyrene ball coated with rabbit anti-thyroglobulin IgG. The amount of human anti-thyroglobulin IgG in the complex on the rabbit anti-thyroglobulin IgG-coated polystyrene ball was estimated using rabbit (anti-human IgG 7-chain) Fab'-horse-radish peroxidase conjugate. The present enzyme immunoassay was 1,000–3,000-fold more sensitive than the conventional enzyme immunoassay, in which a thyroglobulin-coated polystyrene ball was incubated with serum containing anti-thyroglobulin IgG and subsequently with rabbit (anti-human IgG 7–chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-thyroglobulin IgG was demonstrated in all patients with Graves' disease and chronic thyroiditis.     

Measurement of anti-thyroglobulin IgG in urine of patients with autoimmune thyroid diseases by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay)

Anti-thyroglobulin IgG in urine of patients with Graves' disease and chronic thyroiditis and healthy subjects was measured by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). Anti-thyroglobulin IgG in dialyzed urine was reacted simultaneously with 2,4-dinitrophenylated thyroglobulin and thyroglobulin-βT-D -galactosidase conjugate. The immune complex formed consisting of the three components was trapped onto polystyrene balls coated with (anti-2,4, dinitrophenyl group) IgG, eluted with ∈N-2,4-dinitrophenyl-L-lysine, and transferred onto polystyrene balls coated with (antihuman IgG γ-chain) IgG. β-D -Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. Anti-thyroglobulin IgG was detected in most of the patients, but not in most of the healthy subjects; levels of anti-thyroglobulin IgG in urine of the patients were well correlated to those in serum of the same patients. The measurement of anti-thyroglobulin IgG in urine by the immune complex transfer enzyme immunoassay was suggested to be useful as a diagnostic aid for autoimmune thyroid diseases. The conventional standard ELISA was not sufficiently sensitive for measuring anti-thyroglobulin IgG in urine. © 1993 Wiley-Liss, Inc.     

Improved measurement of anti-thyroglobulin IgG in urine of patients with autoimmune thyroid diseases by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay)

Previously, antithyroglobulin IgG was assayed in dialyzed urine from patients with autoimmune thyroid diseases by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay), and most of the assay results were useful as a diagnostic aid for autoimmune thyroid diseases. However, dialysis of urine was laborious and time-consuming, and some results were less reliable due to low levels of anti-thyroglobulin IgG in urine. This paper describes some improvements of the assay. Useful assay results could be obtained for most of urine samples without dialysis, although some interfering substance(s) was suggested to be present in some urine samples before dialysis. Accurate assay results with no interference could be obtained after gel filtration by only two min centrifugation in place of dialysis. More reliable assay results for urine samples containing low levels of antithyroglobulin IgG were obtained after concentration using a molecular sieve. © 1993 Wiley-Liss, Inc.     

Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using recombinant gag p24(14-214) as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using recombinant gag p24(14-214) of HTLV-I is described. The recombinant gag p24(14-214) is soluble in the absence of detergents and allows the use of enzymes other than horseradish peroxidase as a label in the assays. The usefulness of recombinant gag p24(14-214) was examined with 305 sera characterized by other methods including gelatin particle agglutination, enzyme-linked immunosorbent assay (ELISA) using HTLV-I, and Western blotting. This assay was more sensitive than other methods using HTLV-I as antigen. The specificity could be tested by preincubation of test serum with excess of the recombinant protein. Most of negative and positive sera were discriminated. However, some results appeared to be false-positive or false-negative, and recombinant gag p24(14-214) was suggested to be useful, when used with other recombinant proteins and/or peptides, for improving the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant gag p24(14-214) conjugate and recombinant gag p24(14-214)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-env gp46(188-224), as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, Cys-env gp46(188-224) of HTLV-I, is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Cys-env gp46 (188-224) conjugate and Cys-env gp46 (188-224)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was more sensitive and useful than the immune complex transfer enzyme immunoassay using Cys-Arg-env gp46(188-209) and other methods using HTLV-I as antigen.     

Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-Gag p19(100-130), as antigen.

An immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, cys-gag p19(100-130), is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl bovine serum albumin-cys-gag p19(100-130) conjugate and cys-gag p19(100-130)-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was 100-fold more sensitive than the conventional enzyme immunoassay, in which a cys-gag p19(100-130)-bovine serum albumin-coated polystyrene ball was incubated with test serum and, after washing, with (anti-human IgG gamma-chain) Fab'-peroxidase conjugate. The degree of inhibition by preincubation of test sera with excess of cys-gag p19(100-130) in combination with an appropriate cut-off value for the fluorescence intensity of bound beta-D-galactosidase activity discriminated almost all seropositive samples from seronegative ones.     

Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (antihuman T-cell leukemia virus type I) immunoglobulin G in serum using a synthetic peptide, Ala-Cys-Env gp46(237-262), as antigen.

A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (antihuman T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a synthetic peptide, Ala-Cys-env gp46(237-262), of HTLV-I is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Ala-Cys-env gp46(237-262) conjugate and Ala-Cys-env gp46(237-262)-beta-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. beta-D-galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was more sensitive than other methods using HTLV-I as antigen, and most negative and positive sera were discriminated. However, some results appeared to be false positive or false negative, and the peptide, Ala-Cys-env gp46(237-262), was suggested to be useful, in combination with other peptides, for improving the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible.     

Principle and applications of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibodies in body fluids

The sensitivity and specificity of enzyme immunoassay for antibodies in body fluids have been improved considerably by transferring the complex of labelled antigen and antibody to be detected from one solid phase to another to eliminate interfering substance(s) in the samples (immune complex transfer enzyme immunoassay). Usefulness of the new method has been tested for antibodies in serum as well as in urine. Anti-thyroglobulin IgG could be measured not only in serum of all patients with autoimmune thyroid diseases and almost all healthy subjects but also in the urine of most of the patients. Anti-HTLV-I IgG was unequivocally demonstrated in some of sera, which were indeterminate or negative by Western blotting, and diagnosis of HIV infection by detecting anti-HIV IgG in urine and saliva would be possible with higher reliability than by conventional methods. © 1993 Wiley-Liss, Inc.     

Time-resolved fluorometric sandwich immunoassay for human growth hormone in serum and urine

A specific and sensitive time-resolved fluorometric sandwich immunoassay for human growth hormone (hGH) in serum and urine is described. A monoclonal anti-hGH IgG1 (5802)-coated polystyrene ball was incubated with serum or dialyzed urine and subsequently with europium ion-labeled monoclonal anti-hGH IgG1 (5801). Europium ion bound to the polystyrene ball was measured by time-resolved fluorometry. The detection limit of hGH was 0.3 pg/tube, which was 15-fold higher than that by sandwich enzyme immunoassay using horseradish peroxidase as label. The assay range of serum hGH was 15-15,000 ng/liter using 20 microliters of serum. The assay range of urinary hGH was 2-2,000 ng/liter using 150 microliters of dialyzed urine. The detection limits of hGH in serum and urine by this immunoassay were satisfactory for measuring hGH levels in serum and urine of healthy children and in serum of healthy adults and higher levels but not in urine of healthy adults and in serum and urine of patients with hGH deficiency.     

Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Env gp46(188-209), as antigen

An immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG (anti-HTLV-I IgG) in serum using a chemically and safely synthesized peptide, env gp46(188-209), is described. Anti-HTLV-I IgG in test serum, which had been incubated with excess of inactive beta-D-galactosidase to eliminate interference by anti-beta-D-galactosidase antibodies, was reacted simultaneously with dinitrophenyl bovine serum albumin-env gp46(188-209) conjugate and env gp46(188-209)-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified (anti-dinitrophenyl group) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the beta-D-galactosidase conjugate, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the (anti-human IgG gamma-chain) IgG-coated polystyrene balls was assayed by fluorometry. This assay was sensitive and detected anti-HTLV-I IgG in serum samples which were negative by the conventional enzyme immunoassay and Western blotting. And the specificity of this assay was confirmed by preincubation of test serum with excess of env gp46(188-209). However, some disadvantages were also noted.     

Novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide.

A novel and ultrasensitive noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay) for alpha-human atrial natriuretic peptide (alpha-hANP) in plasma is described. alpha-hANP was biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-alpha-hANP [6-28] IgG-coated polystyrene ball. After washing, biotinylated alpha-hANP was eluted from the polystyrene ball with HCI and was reacted with 2,4-dinitrophenyl-fluorescein-bovine serum albumin-disulfide-rabbit anti-alpha-hANP [6-28] IgG conjugate. The complex formed was trapped onto (anti-2,4-dinitrophenyl group) IgG-coated polystyrene balls and, after washing, reacted with avidin-beta-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with epsilon N-2, 4-dinitrophenyl-L-lysine and transferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to (anti-rabbit IgG) IgG-coated polystyrene balls. beta-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of alpha-hANP [1-28] was 3 fg (1 amol)/tube. Interference by plasma proteins was eliminated by separation of peptides from proteins using a molecular sieve. The assay range of plasma alpha-hANP [1-28] was 0.04-120 ng/L, and plasma levels of hANP in healthy subjects (11-56 ng/L) were measured without concentration.     

Sensitive enzyme immunoassay of human growth hormone for clinical application: A review

Radioimmunoassay, a useful tool to measure human growth hormone (hGH) in serum, cannot measure very low hGH levels in serum and urine under some conditions. This work reviews recent studies on the development and applicability of sensitive enzyme immunoassay technique for hGH, which can measure very low levels of hGH in serum and urine that have not been measured by radioimmunoassay.     

Immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 gp41 antigen using synthetic peptides as antigens

The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 gp41 antigen was developed using two synthetic peptides. An aliquot (10 μl) of serum samples from HIV-1 seropositive subjects was incubated simultaneously with 2,4-dinitrophenyl-bovine serum albumin-synthetic HIV-1 gp41 peptide conjugates and synthetic HIV-1 gp41 peptide-β-D -galactosidase conjugates and subsequently with colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG to trap the immune complexes formed comprising the three components. After washing, the colored polystyrene beads were incubated with white polystyrene beads coated with affinity-purified (anti-human IgG γ-chain) IgG in the presence of ϵN-2,4-dinitrophenyl-L -lysine to transfer the immune complexes to the white polystyrene beads. β-D -Galactosidase activity bound to the white polystyrene beads was assayed by fluorometry. The formation, trapping and transferring of the immune complexes were completed within 0.5, 0.5 and 1.5 hr, respectively. Since each peptide appeared to react with its own specific antibody IgG, serum samples were tested by the equimolar combination of the two peptides. The lowest signals (fluorescence intensities for bound β-D -galactosidase activity) for serum samples from HIV-1 asymptomatic carriers, patients with AIDS-related complex and patients with AIDS were 1490-, 2210- and 1460-fold, respectively, higher than the highest signal for serum samples from HIV-1 seronegative subjects. In five seroconversion serum panels, antibody IgG to HIV-1 gp41 antigen was detected as early as antibodies to HIV-1 detected by three currently commercially available methods. J. Clin. Lab. Anal. 12:197–204, 1998. © 1998 Wiley-Liss, Inc.     

Use of microplates and fluororeader for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HTLV-I IgG

Previously, an ultrasensitive solid phase enzyme immunoassay (immune complex transfer enzyme immunoassay) was described to detect low levels of anti-HTLV-I IgG in serum below those detectable by conventional methods. In this method, polystyrene balls as solid phase were transferred from test tube to test tube with tweezers. This was not only tedious but also causative of false-positivity by carryover, unless tips of the tweezers were washed carefully after each transfer of polystyrene balls. Bound enzyme activities for many samples were measured one by one by fluorometry using a spectrofluorophotometer. As a result, the assay of many samples was difficult. In the present study, microplates and a fluororeaderwere used in place of test tubes and a spectrofluorophotometer. Polystyrene balls were transferred quickly and easily from well to well by placing a microplate upside down on that containing polystyrene balls, and turning the two plates together upside down. Tweezers were not used for transfer of polystyrene balls, minimizing the possibility of false-positivity. Fluorescence intensities of bound enzyme activities for 96 samples were measured within a minute by using a fluororeader. Thus, it became easy to test many samples, although the sensitivity was lowered to some extent. © 1994 Wiley-Liss, Inc.     

Anti-HTLV-I IgG in urine detected by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide,Cys-env gp46(188-224), as antigen

Antibody IgG to human T-cell leukemia virus type I (HTLV-I) in urine was detected by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide, Cys-env gp46(188–224), as antigen, the sensitivity and specificity of which were 100 and 98.5%, respectively, using serum samples. Anti-HTLV-I IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin–Cys-env gp46(188–224) conjugate and Cys-env gp46(188–224)-β-D -galactosidase (Escherichia coli) conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with ?N-2,4-dinitrophenyl-L -lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG γ-chain) IgG. Finally, bound β-D -galactosidase activity was assayed by fluorometry. Thirty-one urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tested. The sensitivity and specificity were 87 and 100%, respectively, with unconcentrated urine samples and 94 and 100%, respectively, with approximately 10-fold concentrated urine samples. These results were superior to those by the conventional ELISA and gelatin particle agglutination test. © 1994 Wiley-Liss, Inc.     

Detection of antibody IgG to HIV-1 in urine by ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 as antigen for diagnosis of HIV-1 infection

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant p24 gag protein (p24) of HIV-1 as antigen and β-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2, 4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate and recombinant p24-β-D-galactosidase conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2, 4-dinitrophenyl group) IgG, eluted with ?N-2, 4-dinitrophenyl-L-lysine, and transferred to polystyrene balls coated with affinity-purified (anti-human IgG γ-chain) IgG. Bound γ-D-galactosidase activity was assayed by fluorometry. This assay was at least 3, 000-fold more sensitive than conventional methods. The lowest signal among 49 asymptomatic carriers was 3.1-fold higher than the highest nonspecific signal among 100 seronegative subjects. The sensitivity and specificity were both 100%. The positivity could be confirmed by preincubation of urine samples with excess of the antigen. Thus, this assay would be a powerful tool for detecting IgG antibody to HIV-1 in urine. © 1994 Wiley-Liss, Inc.