Ultrasensitive and rapid enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for antibody IgG to HIV-1 p17 antigen

The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 p17 antigen was performed in two different ways (the present immunoassays I and II) within shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, antibody IgG to HIV-1 p17 antigen in 10 μL of serum samples was incubated simultaneously with 2,4-dinitrophenyl-maltose binding protein-recombinant p17(rp17) fusion protein and rp17-β-D -galactosidase conjugate in a total volume of 22 μL for 10 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min in a styrol test tube (13.3 × 54 mm and 2.1 g) to trap the immune complex. After washing, the polystyrene bead was incubated with 30 μL of ϵN-2,4-dinitrophenyl-L -lysine solution in a polystyrene tube (12 × 75 mm) coated with affinity-purified (antihuman IgG γ-chain) IgG for 10 min to transfer the immune complex. In the present (sequential) immunoassay II, a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-maltose binding protein-rp17 fusion protein was incubated in a styrol test tube (13.3 × 54 mm and 2.1 g) sequentially with antibody IgG to HIV-1 p17 antigen in 10 μL of serum samples in a total volume of 16 μL for 5 min and subsequently with rp17-β-D -galactosidase conjugate in a volume of 10 μL for 5 and 10 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene tube coated with affinity-purified (antihuman IgG γ-chain) IgG for 5 and 10 min in the same way as in the present immunoassay I. During the incubations, the styrol test tubes containing the polystyrene beads and reaction mixtures were shaken, and the polystyrene test tubes were rotated with shaking, so that the polystyrene beads were rotated randomly, and small drops (16 to 30 μL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations, though only small parts of the solid phase surfaces were contacted at one time. The intent was to continuously mix thin aqueous layers of the reaction mixtures covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays were called thin aqueous layer immunoassays.) β-D -Galactosidase activity bound to the polystyrene tubes was assayed by fluorometry for 30 and 60 min. The present immunoassays I and II, in which only 15 to 25 min were used for the immunoreactions, were as sensitive if not more so than the previous immune complex transfer enzyme immunoassay requiring 150 min for the immunoreactions. In these earlier immunoreactions, the immune complex comprising the three components formed by 30 min incubation was trapped onto two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 60 min, and was then transferred to two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (antihuman IgG γ-chain) IgG for 60 min in a total volume of 150 μL. Furthermore, the present (sequential) immunoassay II (and probably I) could become approximately 10 times more sensitive by assaying bound β-D -galactosidase activity for a longer period of time (10 h), since β-D -galactosidase activity, bound nonspecifically in the presence of serum samples from HIV-1 seronegative subjects, was considerably low. J. Clin. Lab. Anal. 12:179–189, 1998. © 1998 Wiley-Liss, Inc. No abstract.     

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

In the immune complex transfer enzyme immunoassay for HIV-1 p24 antigen, different preparations of anti-p24 Fab′-β-D -galactosidase conjugate, various periods of time for immunoreactions involved, and shaking for incubations with polystyrene beads were tested. On the basis of the results of these experiments, p24 antigen was measured as follows. The antigen was reacted simultaneously with 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab′ conjugate and highly polymerized monoclonal mouse anti-p24 Fab′-β-D -galactosidase conjugate at 37°C for 2 hr. The immune complex formed comprising the three components was trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 1.5 hr and was transferred to white polystyrene beads coated with streptavidin in the presence of ϵN-2,4-dinitrophenyl-L -lysine for 1.5 hr. The incubations with polystyrene beads were performed at room temperature with shaking. β-D -Galactosidase activity bound to the white polystyrene beads was assayed by fluorometry at 30°C for 2 hr. The detection limit of p24 antigen (0.1 amol/tube and 10 amol (0.24 pg)/ml of serum) was equal to that obtained when the formation, trapping, and transferring of the immune complex were performed for 4, 16, and 3 hr, respectively, by incubation without shaking. Namely, the period of time required for the immune complex transfer enzyme immunoassay of p24 antigen was markedly shortened (25.5–7 hr) without loss of the sensitivity. By the improved immune complex transfer enzyme immunoassay, p24 antigen was detected 12–20 days earlier than the detection of antibodies to HIV-1, i.e., seroconversion by the conventional ELISA. J. Clin. Lab. Anal. 12:115–120, 1998. © 1998 Wiley-Liss, Inc.     

Rapid and ultrasensitive enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for HIV-1 p24 antigen

The immune complex transfer enzyme immunoassay for HIV-1 p24 antigen was performed in three different ways (in the present immunoassays I, II, and III) within much shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, p24 antigen was incubated simultaneously with 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab′ conjugate and monoclonal mouse anti-p24 Fab′-β-D -galactosidase conjugate in a total volume of 19 μL for 15 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min to trap the immune complex. After washing, the polystyrene bead was incubated with 35 μL of ϵN-2,4-dinitrophenyl-L -lysine for 15 min to elute the immune complex (the first eluate) and, after removing the first eluate, with an additional 35 μL of ϵN-2,4-dinitrophenyl-L -lysine for 1 min (the second eluate). The first and second eluates were incubated with a polystyrene test tube (12 × 75 mm) coated with streptavidin for 15 min. In the present (sequential) immunoassay II, a polystyrene bead of 6.35 mm in diameter successively coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab′ conjugate was incubated with p24 antigen in a total volume of 20 μL for 5 min and subsequently with monoclonal mouse anti-p24 Fab′-β-D -galactosidase conjugate in a volume of 5 μL for 20 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene test tube coated with streptavidin as described above. In the present (sequential) immunoassay III, p24 antigen was incubated with monoclonal mouse anti-p24 Fab′-β-D -galactosidase conjugate in a total volume of 19 μL for 10 min and with a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin- affinity-purified rabbit anti-p24 Fab′ conjugate for 20 min. The immune complex formed on the polystyrene bead was transferred as described above. The incubations were performed at room temperature either by shaking the polystyrene beads (one/assay) and the reaction mixtures in styrol test tubes (13.3 × 54 mm and 2.1 g) so as to randomly rotate the polystyrene beads or by rotating the polystyrene test tubes (12 × 75 mm) containing the reaction mixtures, so that small drops (19 to 70 μL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations (although they contacted only small parts of the solid phase surfaces at a time) to continuously mix thin aqueous layers covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays are called thin aqueous layer immunoassays.) The detection limits of p24 antigen by 1 hr assay of bound β-D -galactosidase activity in the present immunoassays I, II, and III were 0.1, 0.2 and 0.1 amol/assay, respectively, and were slightly higher than or equal to that by the previously reported immune complex transfer enzyme immunoassay, in which the immune complex was formed for 4 hr, was trapped for 16 hr, and was transferred for 3 hr followed by 1-hr assay of bound β-D -galactosidase activity. By 20-hr assay of bound β-D -galactosidase activity, the detection limit of p24 antigen was further lowered to 10 zmol/assay in the present (simultaneous) immunoassay I and to 3 zmol/assay in the present (sequential) immunoassay III. However, the nonspecific reaction(s) with serum samples from HIV-1 seronegative subjects hampered the improvement of the detection limit by 20-hr assay of bound β-D -galactosidase activity. J. Clin. Lab. Anal. 12:205–212, 1998. © 1998 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.     

Ultrasensitive and more specific enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV-1 in serum using affinity-purified rabbit anti-p24 Fab′ and monoclonal mouse anti-p24 Fab′

Previously, an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for p24 antigen of HIV-1 was developed. The immune complex comprising 2,4-dinitrophenyl-biotinyl-bovine serum albumin-rabbit anti-p24 Fab′ conjugate, p24 antigen, and rabbit anti-p24 Fab′-β-D-galactosidase conjugate was trapped onto polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, was eluted with ϵN-2,4-dinitrophenyl-L-lysine, and was transferred to polystyrene beads coated with streptavidin. β-D-Galactosidase activity bound to the streptavidin-coated polystyrene beads was assayed by fluorometry. This assay was highly sensitive. However, bound β-D-galactosidase activity had to be assayed for a long time (20 h), and the nonspecific signal was observed in 5% serum samples from subjects with low risk of HIV infection. In the present study, the assay time for bound β-D-galactosidase activity was shortened to 2.5 h by using 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab′ conjugate and affinity-purified rabbit anti-p24 Fab′-β-D-galactosidase conjugate. Furthermore, the nonspecific signal was found to increase with increasing periods of time for storage of serum samples at -20°C, and this increase was prevented without prolongation of the assay time for bound β-D-galactosidase activity and without loss of the sensitivity by substituting monoclonal mouse anti-p24 Fab′-β-D-galactosidase conjugate for affinity-purified rabbit anti-p24 Fab′β-D-galactosidase conjugate. © 1996 Wiley-Liss, Inc.     

Sensitive enzyme immunoassay of antibodies to HIV-1 p17 antigen using indirectly immobilized recombinant p17 for diagnosis of HIV-1 infection

Recombinant p17 (rp17) antigen of HIV-1 and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized onto polystyrene beads in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption; biotinyl-rp17 and biotinyl-MBP-rp17 were immobilized indirectly onto streptavidin-coated polystyrene beads; and 2,4-dinitrophenyl (DNP)-MBP-rp17 was immobilized indirectly onto (anti-DNP) IgG-coated polystyrene beads. These directly and indirectly immobilized antigens were incubated with urine samples containing antibody IgG to p17 antigen and subsequently with rp17-β-D -galactosidase conjugate or (anti-human IgG γ-chain) Fab′-β-D -galactosidase conjugate. β-D -Galactosidase activity bound to the polystyrene beads was assayed by fluorometry. When rp17-β-D -galactosidase conjugate was used, signals (fluorescence intensities for bound β-D -galactosidase activity) were much higher with the indirectly immobilized antigens than those with the directly immobilized antigens. By experiments using (anti-human IgG γ-chain)Fab′-β-D -galactosidase conjugate, the binding of rp17-β-D -galactosidase conjugate to antibodies against p17 antigen bound to directly immobilized rp17 antigen was shown to be seriously limited as compared with that to antibodies against p17 antigen bound to indirectly immobilized DNP-MBP-rp17. When rp17-β-D -galactosidase conjugate and serum samples were used, serum interference was much less with indirectly immobilized DNP-MBP-rp17 than with directly immobilized rp17 antigen, and the sensitivity of enzyme immunoassay for antibody IgG to p17 antigen using indirectly immobilized DNP-MBP-rp17 was 1,000- to 3,000-fold higher than that of enzyme immunoassay using directly immobilized rp17 antigen and Western blotting for p17 band. This sensitive enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as conventional methods for antibodies to HIV-1 and earlier than Western blotting for p17 band. J. Clin. Lab. Anal. 12:343–350, 1998. © 1998 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)     

Use of indirectly immobilized recombinant p17 antigen for detection of antibodies to HIV-1 by enzyme immunoassay

Recombinant HIV-1 p17 antigen (rp17) and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption, and biotinyl-rp17, biotinyl-MBP-rp17, and 2,4-dinitrophenyl (DNP)-MBP-rp17 were immobilized indirectly onto polystyrene beads, which had been coated with streptavidin alone, with biotinyl-bovine serum albumin and streptavidin and with (anti-2,4-dinitrophenyl group) IgG. These immobilized antigens were tested by incubation with diluted serum from an HIV-1 seropositive subject in the absence and presence of serum from HIV-1 seronegative subjects and, after washing, with rp17 beta-D-galactosidase conjugate. Higher positive signals (fluorescence intensities for bound -beta-D-galactosidase activity) and less serum interference were obtained with indirectly immobilized antigens than with directly immobilized ones. Enzyme immunoassay using biotinyl-MBP-rp17 indirectly immobilized onto polystyrene beads, which had been coated sequentially with biotinyl-bovine serum albumin and streptavidin, was approximately 1,000-fold more sensitive than that using directly immobilized rp17 antigen and Western blotting for p17 band. This enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as or even earlier than conventional methods and considerably earlier than Western blotting for HIV-1 p17 band. In addition, the sensitivity was further improved approximately 10-fold by incubation with shaking for immunoreactions and by increase of both the number of polystyrene beads and the volume of serum samples used per assay.     

Immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen

The immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen is described. Serum samples containing antibody IgM to HIV-1 p17 antigen were incubated simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p17 (rp17) conjugate and rp17-β-D -galactosidase conjugate, and the immune complex formed comprising the three components was trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. Subsequently, the immune complex was transferred to white polystyrene beads coated with monoclonal mouse (antihuman IgM) IgG in the presence of excess of ϵN-2,4-dinitrophenyl-L -lysine. The signal for antibody IgM to p17 antigen was the fluorescence intensity by fluorometric assay of β-D -galactosidase activity bound to the white polystyrene beads. The periods of time required for the formation, trapping, and transferring of the immune complex comprising the three components were more than 4 hr, 2 hr, and 3 hr, respectively. The immunoassay developed was shown to be specific by inhibition of transferring the immune complex in the presence of excess of nonspecific IgM but not IgG. Signals for antibody IgM to p17 antigen with serum samples of HIV-1 seroconversion serum panels,—that is, with serum samples in early stages of the infection—tended to be higher than those with serum samples from HIV-1 asymptomatic carriers probably long after the infection and patients with ARC and AIDS. In contrast, signals for antibody IgG to p17 antigen with serum samples of HIV-1 seroconversion serum panels tended to be higher than signals for antibody IgM to p17 antigen but were much lower than signals for antibody IgG to p17 antigen with serum samples from HIV-1 asymptomatic carriers and patients with ARC and AIDS. J. Clin. Lab. Anal. 12:329–336, 1998. © 1998 Wiley-Liss, Inc.     

Detection of anti-ovalbumin IgG in serum by immune complex transfer enzyme immunoassay

An immune complex transfer enzyme immunoassay for anti-ovalbumin IgG in serum is described. Serum-specific antibody was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-ovalbumin conjugate and ovalbumin-peroxidase conjugate. The complex formed by the three components was trapped onto polystyrene balls coated with anti-2,4-dinitrophenyl group IgG, eluted with epsilonN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgG-gamma-chain. Bound peroxidase activity was determined by fluorometry. This enzyme immunoassay was 300- to 1,000-fold more sensitive and more reliable than the enzyme-linked immunosorbent assay (ELISA). Anti-ovalbumin IgG was detected in 100% of healthy subjects using this method while only 14% were detected by ELISA.     

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.     

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.     

Measurement of anti-thyroglobulin IgG in serum by novel and sensitive immune complex transfer enzyme immunoassay

Anti-thyroglobulin IgG in human serum was measured by a novel and sensitive immune complex transfer enzyme immunoassay. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between human anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. The polystyrene ball was washed to eliminate most nonspecific IgG in test serum, and the complex was eluted from the polystyrene ball, to which nonspecific IgG had been adsorbed, 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 gamma-chain) Fab'-horseradish peroxidase conjugate. The present enzyme immunoassay was 1,000 to 3,000-fold more sensitive than the conventional enzyme immunoassay, in which a human thyroglobulin-coated polystyrene ball was incubated with serum containing human anti-thyroglobulin IgG and, after washing, with rabbit (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-thyroglobulin IgG was demonstrated in about 10% of healthy subjects and in all patients with Graves' disease and chronic thyroiditis. The principle of the present method may make it possible to sensitively measure other autoantibodies including anti-thyroid peroxidase and anti-thyrotropin receptor antibodies to aid diagnosis of thyroid diseases.     

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

In the previous immune complex transfer enzyme immunoassay for antibody IgG to p17 of HIV-1, the immune complex comprising 2,4-dinitrophenyl-bovine serum albumin-recombinant p17 conjugate, anti-p17 IgG, and recombinant p17-β-D-galactosidase conjugate was trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by overnight incubation and was transferred to polystyrene beads coated with (antihuman IgG γ-chain) IgG by 3 hr incubation in the presence of excess of ϵN-2,4-dinitrophenyl-L -lysine. These processes were made efficient by incubation with shaking and by using solid phases with larger surface areas. In addition, the volume of serum samples used was increased from 10 μl to 100 μl. As a result, the sensitivity was improved 20–30-fold and was ˜100,000-fold higher than that of Western blotting for p17 band, even when both trapping and transferring of the immune complex were performed for only 30 min. Furthermore, testing many samples became easily possible with higher sensitivity using microplates and a fluororeader. J. Clin. Lab. Anal. 11:244–250, 1997. © 1997 Wiley-Liss, Inc. No abstract.     

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.