principle and applications of ultrasensitive enzyme immunoassay (immune complex transfer enzyme...

Journal of Clinical Laboratory Analysis 7:376-393 (1993)

Principle and Applications of Ultrasensitive Enzyme lmmunoassay (Immune Complex Transfer Enzyme Immunoassay) for

Antibodies in Body Fluids Eiji Ishikawa, Seiichi Hashida, Takeyuki Kohno, Kouichi Hirota, Kazuya Hashinaka,

and Setsuko Ishikawa Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki, Japan

The sensitivity and specificity of enzyme immunoassay for antibodies in body fluids have been improved considerably by trans- ferring 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-l 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. C 1993 Wiley-Liss, Inc.

K e y words: anti-thyroglobulin IgG, autoantibody, Graves’ disease, chronic thyroiditis, anti-HTLV-l laG. ATL. anti-HIV-1 IaG, AIDS

INTRODUCTION

For the last two decades, the conventional enzyme-linked immunosorbent assay (ELISA) for antibodies in body fluids has been used successfully to aid diagnosis of autoimmune diseases and infections. For example, ELISA is one of the most widely used methods to detect antibodies to human im- munodeficiency virus (HIV) and human T-cell leukemia virus (HTLV) for diagnosis of HIV and HTLV infections (1). However, the sensitivity of the conventional ELISA is not sufficiently high for some purposes. Relevant autoantibodies are not demonstrated in all patients with autoimmune diseases. Higher sensitivity is recommended to narrow a window pe- riod after HIV infection. Higher sensitivity is required to detect antibodies in body fluids such as urine and saliva containing much lower concentrations of immunoglobulins than sendp lasma . This article reviews the principle and applications of recently developed ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibodies in body fluids.

PRINCIPLE OF IMMUNE COMPLEX TRANSFER ENZYME IMMUNOASSAY

In the most widely used conventional ELISA, antigen- coated solid phase is reacted with antibodies in samples and, after washing, with anti-immunoglobulin antibody-enzyme conjugate (Fig. I ) . Enzyme activity bound to the solid phase is correlated to the amount of antibodies to be measured. The

0 1993 Wiley-Liss, Inc.

sensitivity of this assay is seriously limited by the nonspecific binding of the conjugate to the solid phase, which is probably caused by the nonspecific binding of nonspecific immunoglobulins and other SubStdnce(S) in the samples. This drawback has been overcome by a method to transfer the immune complex of labelled antigen and antibodies to be measured from one solid phase to another, eliminating nonspecific immunoglobulins and other interfering substance(s) (immune complex transfer enzyme immunoassay) ( 2 ) .

In the initially developed method (Method I, Fig. 2) ( 3 ) , antibodies to be measured are reacted with 2,4-dinitrophenyl- biotinyl-antigen, and the immune complex formed is trapped onto solid phase coated with (anti-2,4-dinitrophenyl group) IgG (the first solid phase). After washing, the immune complex is eluted from the first solid phase with excess of ~N-2,4-dinitrophenyl-L-lysine and transferred onto solid phase coated with avidin (the second solid phase). Nonspecific immunoglobulins and other interfering substance(s) are mini- mized by transfer of the immune complex from the first solid phase to the second solid phase. The immune complex on

Received May 26, 1993; accepted June 9. 1993

Address reprint requests to Eiji Ishikawa. M.D., Department of Biochemis- try. Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.

Abbreviations: Ab, antibody; Ag, antigen: DNP-Lys. ~N-2.4-dinitrnphenyl- Mysine; Enz. enzyme: Ig. immunoglobulin.

Enzyme lmmunoassay for Antibodies 377

the second solid phase is reacted with (anti-immunoglobulin) antibody Fab’-enzyme conjugate, and the enzyme activity bound to the second solid phase is correlated to the amount of antibodies to be measured. This original method has been modified in various ways (Methods 11-VIII).

In Method I1 (Fig. 3) (4j, antibodies to be measured are reacted with 2,4-dinitrophenyl-antigen, and the immune complex formed is trapped onto solid phase coated with (anti- 2,4-dinitrophenyl group) IgG. After washing, the immune complex is eluted from the solid phase with excess of EN-2 ,4-dinitrophenyI-L-lysine and transferred to solid phase coated with antibody IgG to the antigen. Antibodies in the immune complex on the last solid phase are reacted with anti- immunoglobulin antibody Fab’-enLyme conjugate.

In Method I11 (Fig. 4) (3, antibodies to be measured are reacted with 2,4-dinitrophenyl-antigen, and the immune complex formed is trapped onto solid phase coated with (anti-

Ag - Ab solid phase

Fig. 1. Conventional ELISA.

Anti - DNP - DNP. Ab solid phase Biotin - Ag

Ab Ant i -DNP- DNP - Ag solid Dhase

I 000 DNP-Lvs I Stieptavidin solid phase

Anti - lg - Enz l m

Anti - Ag ~ solid phase ID

Anti - lg - Enz I D

Fig. 2. DNP: 2,4-dinitrophenyl group.

Immune complex transfer enzyme immunoassay I (Method I). Fig. 3. DNP: 2,4-dinitrophenyl group.

Immune complex transfer enLyme immunoassay I1 (Method 11).

378 lshikawa et al.

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Fig. 5. DNP: 2.4-dinitrophenyl group.

Immune complex transfer enzyme immunoassay 1V (Method 1V).

Fig. 4. DNP: 2.4-dinitrophenyl group.

Immune complex transfer enLyrne immunoassay 111 (Method 111).

2,4-dinitrophenyl group) IgG. After washing, the immune complex is eluted from the solid phase with excess of EN-2 ,4-dinitrophenyl-L-lysine and transferred to solid phase coated with anti-immunoglobulin antibody IgG. The antigen in the immune complex on the last solid phase is reacted with enzyme-labelled antibody Fab’ to the antigen.

In Method IV (Fig. 5) (6,7), antibodies to be measured are reacted with 2,4-dinitrophenyl-antigen-enzyme conjugate, and the immune complex formed is trapped onto solid phase coated with (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex is eluted from the solid phase with excess of ~N-2,4-dinitrophenyl-L-lysine and transferred to solid phase coated with anti-immunoglobulin IgG.

In Method V (Fig. 6) (8), antibodies to be measured are reacted simultaneously with 2,4-dinitrophenyl-antigen and antigen-enzyme conjugate. The immune complcx formed, con- sisting of the three components. is trapped onto solid phase coated with (anti-2,4-dinitrophenyl group) IgG. After wash-

ing, the immune complex is eluted from the solid phase with excess of ~N-2,4-dinitrophcnyl-L-lysine and transferred to solid phase coated with anti-immunoglobulin IgG.

In Method VI (Fig. 7) (2,9), 2,4-dinitrophenyl-biotinyl- antigen and streptavidin-coated solid phase are substituted for 2,4-dinitrophenyl-antigen and anti-immunoglobulin IgC- coated solid phase, respectively, in Method V.

In Method VII (Fig. 8), antibodies to be measured are reacted with 2,4-dinitrophenyl-antigen-enzyme conjugate, and the immune complex formed is trapped onto solid phase coated with (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex is eluted from the solid phase with excess of ~N-2,4-dinitrophenyl-L-lysine and, after reaction with biocytin-S-S-anti-imniunoglobulin IgG, transferred onto streptavidin-coated solid phase. The immune complex is eluted from the second solid phase by reduction with 2-mercaptoethylamine and transferred onto anti-immunoglobulin IgC- coated solid phase.

In Method VIll (Fig. 9), antibodies to be measured are reacted simultaneously with 2,4-dinitrophenyl-antigen and antigen-enzyme conjugate. The immune complex formed, con- sisting of the three components, is trapped onto solid phase coated with (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex is eluted from the solid phase with excess of ~N-2,4-dinitrophenyl-L-lysine and, after reaction

Enzyme lmrnunoassay for Antibodies 379

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Immune complcx transfer enzyme immunoassay V (Method V).

with biocytin-S-S-anti-immunoglobulin IgG, transferred onto streptavidin-coated solid phase. The immune complex is eluted from the second solid phase by reduction with 2-mercaptoethylamine and transferred onto anti-immunoglobulin IgG- coated solid phase.

Obviously from the principles described above, Methods I-V, VII, and Vlll can discriminate the immunoglobulin classes of antibodies to be measured, and Method V1 can mea- sure antibodies regardless of the immunoglobulin classes.

SENSITIVITY OF IMMUNE COMPLEX TRANSFER ENZYME IMMUNOASSAY

Methods V-VIII are the most sensitive (2). In these methods, the immune complexes containing enzyme-labeled antigens are transferred from one solid phase to another to minimize the nonspecific binding of enzyme-labeled antigens. In Methods I-IV, however, enzyme-labelled reactants are reacted with the immune complexes on solid phase in the final step, and bound enzyme activities are assayed without transfer of the immune complexes. This is one of factors to limit the sensitivity of Methods I-1V.

The sensitivity of Methods V and VI depends on the detection limit of enzymes as label. The use of P-D-galactosidase from Escherichia coli, which can be detected with higher sensitivity than other enzymes (lo), improves the sensitivity of

Anti - DNP - DNP - Ab Ag - Enz solid phase Biotin - Ag

I 000 DNP-Lvs

Fig. 7. Immune complex transfer enzyme immunoassay VI (Method V1). DNP: 2,4-dinitrophenyl group.

Methods V and VI - 10-fold as compared with that of horseradish peroxidase (Fig. 10). Method V for anti-insulin IgG and anti-thyroglobulin IgG using P-D-galactosidase from E . coli as label was 2,000- to 4,000-fold more sensitive than the corresponding conventional ELISA (8, I 1 ). The high sensitivity of Method V using P-D-galactosidase from E. coli as label was demonstrated in the measurement of anti-thymglobulin IgG in serum from healthy subjects and patients with autoimmune thyroid diseases. Anti-thyroglobulin IgG was detected not only in all patients with Graves’ disease and chronic thyroiditis but also in all healthy subjects (Fig. 11) (8,12), whereas anti-thyroglobulin antibodies were detected only in -60% of the patients and in a few percent of healthy subjects by the conventional ELISA and hemagglutination test (8,12). Dif- ference between the highest level of anti-thyroglobulin IgG in serum of the patients and the lowest level in serum of healthy subjects shown by Method V was 10,000,000-fold (Fig. 11). The sensitivity of Methods VII and VIII remains to be evaluated.

The sensitivity of the immune complex transfer enzyme immunoassay. as in other immunoassays with solid phase, is limited by the nonspecific binding of enzyme-labeled reactants to solid phase. Body fluids contain substances to en- hance the nonspecific binding of enzyme-labeled reactants, and most of them are unknown in nature except for nonspe-

380 lshikawa et al.

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Immune complex transfer enzyme immunoassay VII (Method W).

cific immunoglobulins and antibodies to enzymes as label. The sensitivity of Methods I and I1 is obviously limited by the nonspecific binding of nonspecific immunoglobulins, although nonspecific immunoglobulins are eliminated more completely than in the conventional ELISA. In Methods V and VlII (Figs. 6, 9), antibodies to enzymes as label in body fluids are bound specifically to antigen-enzyme conjugates. The complexes formed are bound nonspecifically to (anti- 2,4-dinitrophenyl group) IgG-coated solid phase, released in part and bound specifically to anti-immunoglobulin IgG-coated solid phase in the final step. In Method VII (Fig. 8), antibodies to enzymes as label in body fluids are bound specifically not only to antigen-enzyme conjugates but also to (anti-2,4-dinitrophenyl group) IgG-coated solid phase and anti- immunoglobulin IgG-coated solid phase in the final step. An- tibody 1gG to P-D-galactosidase from B. coli is known to be present in body fluids of a significant part of healthy sub-

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Immune complex transfer enzyme immunoassay VTII (Method

jects (Figs. 12, 13) (9) and to limit the sensitivity of Method V (12,13). The adverse effect of anti-P-D-galactosidase IgG can be eliminated by using inactive P-D-galactosidase with an amino acid sequence different from that of the native enzyme, which is commercially available. When sera were tested by Method V for antithyroglobulin IgG (12) and anti-HTLV-I IgG (1 3 ) , the signals were widely distributed in the presence of excess of thyroglobulin or HTIN-I antigen alone, but were uniformly lowered in the presence of excess of both thyroglobulin or HTLV-I antigen and inactive P-D-galactosidase (Fig. 14) (12,13). Thus the useof inactive 6-D-galactosidase improves the sensitivity and specificity of Method V using P-D-galactosidase as label. It remains to be investigated whether antibodies to other enzymes limit the sensitivity of Method V in a similar manner.


sensitive than conventional methods such as gelatin particle agglutination test using HTLV-I as antigen, the conventional ELISA using HTLV-I as antigen and alkaline phosphatase from calf intestine as label, and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I as antigens (Fig. 16) (15). Test results for sera of HTLV-I camers by this method were superior to those by other methods (Fig. 17) (15). Se- rum samples used had been tested by gelatin particle agglutination using HTLV-I as antigen and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I as antigens. Serum samples, for which the maximal dilution of samples with the diluent included in the kit to cause gelatin particle agglutination was high (128 to 16,384-fold), were all positive, except for some samples, which were negative by Western blotting. Serum samples, for which the maximal dilution to cause the agglutination was low (8- to 64-fold) but the positivity was confirmed by Western blotting, were also all positive. Most of serum samplcs, for which the maximal dilution to cause the agglutination was low (8- to 64-fold) and the positivity was denied by Western blotting, were negative. Namely, these samples were False-positive by gelatin particle agglutination. Serum samples, which were negative by gelatin particle agglutination, were all negative. However, some of serum samples, for which the maximal dilution to cause the agglutination was low (8- to 64-fold) and the positivity was indeterminate or denied by Western blotting, were positive by Method V using the recombinant gag-env hybrid protein as antigen. Namely, a most widely used confirmatory test, Western blotting, was suggested not completely to discriminate truly positive samples from truly negative ones. In addition, the difference between the lowest signal for positive samples and the highest signal for negative samples was much larger than that by the conventional ELISA using HTLV-I as antigen and alkaline phosphatase from calf intestine as label (Fig. 1) as shown in Figures 17 and 18, making it possible to discriminate positive and negative samples more unequivocally.

Although Method V using recombinant gag p24( 14- 139)- env gp46(217-315) hybrid protein as antigen was superior in the sensitivity to conventional methods such as gelatin particle agglutination test using HTLV-I as antigen, the conventional ELISA using HTLV-I as antigen, and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I as antigens as described above, there were some drawbacks. Since the recombinant gag-env hybrid protein was soluble only in the presence of sodium dodecyl sulfate, horseradish peroxidase had to be used as label rather than P-D-galactosidase from E . coli, which can be detected with higher sensitivity than horseradish peroxidase, and the specificity of the method could not be tested by preincubation of serum samples with excess of the recombinant gag-env hybrid protein. Further- more, there was no confirmatory test to examine whether truly positive and negative samples are discriminated, because West- ern blotting, which has been used as the most reliable confir-

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Dilution of Anti-HIV-l Serum with Normal Serum

Fig. 10. Dilution curves of anti-HIV-1 serum by various methods. Anti- HIV-I serum was serially diluted with serum from a seronegative subject and tested by various methods. Closed circles and triangles indicate dilution curves by Methods V and VI, respectively, using recombinant p24 of HIV-I as antigen and P-D-galactosidase from E . coli as label. Open circles and triangles indicate dilution curves by Methods V and 1’1, respectively, using recombinant p24 of HIV-1 as antigen and honeradish peroxidase a5 label. Open squares indicate a dilution curve by dot blotting with recombinant p24 of HIV- 1 as antigen. Test results by gelatin particle agglutination using HIV-1 as antigen are also shown.

APPLICATION OF IMMUNE COMPLEX TRANSFER ENZYME IMMUNOASSAY FOR ANTIBODIES IN SERUM

Method V has been applied to the measurement of antithyroglobulin IgG and anti-HTLV-I IgG in serum.

As described above, antithyroglobulin IgG was demonstrated, by Method V using P-D-galactosidase from E . cali as label, in serum of not only all patients with Graves’ disease and chronic thyroiditis but also all healthy subjects, although at lower concentrations than in the patients (2,s). The presence of antithyroglobulin IgG at low concentrations in healthy subjects was confirmed by preincubation of sera with excess of thyroglobulin in the presence of excess of inactive P-D-galactosidase (Fig. 15) (12,14).

Method V for anti-HTLV-I IgG using recombinant gag p24( 14- 139)-envgp46(217-315) hybridproteinas antigenand horseradish peroxidase as label was 100- to 3,000-fold more

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matory test, is not sufficiently sensitive as described above. In order to overcome these drawbacks, synthetic peptides and a recombinant protein, which are soluble in an appropriate buffer in the absence of detergents and can be labeled with P-D-galactosidase from E. coli, have been substituted for the recombinant gug-env hybrid protein.

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The synthetic peptides and recombinant protein used as antigens were Cys-gag p19( 100-130) (16). Cys-env gp46- (188-224) (17), Ala-Cys-envgp46(237-262) (18), andrecom- binant gag p24(14-214) (19). Cys was bound at or near the C-terminus of the peptides to react with maleimide groups introduced into P-D-galactosidase molecules for conjugation. The specificity was tested by preincubation with excess of the synthetic peptides and recombinant gag p24( 14-214) (16-19). The sensitivity of Method V using these synthetic and recombinant antigens and P-D-galactosidase from E . coli as label was 30 to 3,000-fold higher than that of conventional methods such as gelatin particle agglutination test using HTLV-I as antigen, the conventional ELISA using HTLV-I as antigen and Western blotting using four core proteins (p19, p24, p28, and p53) as antigens (Fig. 19). With Cys-env gp46(188-224), there were no false- negative samples, although some false-positive results were observed (Fig. 20). With the other antigens, however, some false-positive results as well as some false-negative ones were observed (Figs. 21-23). By Method V using Cys-gag p19( 100- 130) as antigen, a significant number of serum samples, for which the maximal dilution to cause the agglutination was low (8- to 64-fold) and the positivity was denied by Western blotting, were positive from bound P-D-galactosidase activity alone (Fig. 23), and the false-positivity of these samples was indicated only by no significant decrease in the sig-

Fluorescence Intensity for Bound Peroxidase Activity by Method V

nal when serum samples were preincubated with excess of Cys-gag P 19 ( 100- 1 30) ( 16). Therefore, Cys-env gp46 ( 1 88-224) could be used for screening. The other antigens could not be used for screening hut could be used as an aid for confirming the positivity and negativity (Tables 1 3 2 ) . Se- rum samples for which the maximal dilution to cause gelatin

Fig. 12. Presence of antibodies to p-D-galactosidasc from E . coli in se- m ~ n of healthy subjects. Anti-(J-0-galactosidase antibodies in of 107 healthy subjects were measured by Methods V and VI using horseradish peroxidase as label.


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Fig. 13. Confirmation of the presence of antibodies to P-D-galactosidase from E. coli in serum of healthy subjects. Sera from 107 healthy subjects were tested by Methods V and VI for antibodies to P-D-galactosidase from E . coli before and afterpreincubation with excess of inactive P-D-galactosidase.

particle agglutination was high (128 to 16,384-fold) (Group 1 ), and serum samples, for which the maximal dilution to cause the agglutination was low (8 to 64-fold) but the positivity was confirmed by Western blotting (Group 2-l), were all positive with at least three of the four antigens. Most of serum samples, for which the maximal dilution to cause the agglutination was low (8- to 64-fold) and the positivity was denied by Western blotting (Group 2-2-2), were negative with at least three of the four antigens. Namely, these samples were

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false-positive by gelatin particle agglutination. Serum samples, which were negative by gelatin particle agglutination (Group 3), were all negative with at least three of the four antigens, except for some samples (2.6%), which were positive with two of the four antigens, remained to be examined by other methods. These results were consistent with those by conventional methods. However, some of serum samples, for which the maximal dilution to cause the agglutination was low (8- to 64-fold) and the positivity was indeterminate or

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Fig. 14. Elimination of interference by antibody IgG to P-D-galactosidase from E . coli using inactive P-D-galactosidase in Method V for antithyroglobulin IgG in serum. Sera from 119 healthy males and 95 healthy females

were tested by Method V for anti-thyroglobulin IgG using P-D-galactosidase from E . coli as label before and aftcr preincubation with excess of inactive P-D-galactosidase in the presence of excess of thyroglobulin.

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nitithyroglobulin IgG using P-D-galactosidase from E. coli as label before and after preincubation with excess of thyroglobulin in the presence of excess of inactive p-D-galacrosidase.

Dilution of Anti-HTLV-l Serum with Normal Seturn (-fold)

Fig. 16. Dilution curves of anti-HTLV-I serum by Method V using recombinant gag-em hybrid protein as antigen and the conventional ELISA using HTLV-I as antigen. Sera from four HTLV-I-infected subjects were serially diluted with serum from a seronegative subject and tested by Method V using recombinant gag p24( 14-139)-em gp46(217-315) hybrid protein as antigen and horseradish peroxidase as label (open symbols) and the conventional ELTSA using HTLV-I as antigen and alkaline phosphatase as label (Eitest-ATL; Eisai Co., Tokyo, Japan) (closed symbols). Test results by gelatin particle agglutination using HTLV-I as antigen (SERODIA-ATLA; Fujirebio, Tokyo, Japan) and Western blotting using four core proteins (p19, p24, p28 and p53) of HTLV-I as antigens (Fujirebio) are also shown.

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Fig. 17. Comparison of test results for anti-HTLV-T TgG or antibodies in serum by Method V using recombinant gag-env hybrid protein of HTLV-I as antigen, gelatin particle agglutination using HTLV-I as antigen, and West- em blotting using four core proteins of HTLV-I as antigens. Sera from HTLV- I-infected sub,jects were tested by Method V using recombinant gag p24(14-l39)-env gp46(217-315) hybrid prntein as antigen and horseradish peroxidase as label, gelatin particle agglutination test using HTLV-I as antigen (SERODTA-ATLA; Fujirebio. Tokyo, Japan) and Western blotting using four core proteins (p19, p24, p28. and p53) of HTLV-I as antigens (Fujirehio). Open and closed triangles indicate sera that were negative and positive, respectively, by Western blotting. Open squares indicate sera that were negative by Western blotting but positive by Method V.


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Fig. 18. Comparison of test results for anti-HTLV-I IgG or antibodies in serum by the conventional ELISA, gelatin particle agglutination, and West- em blotting. Sem from HTLV-I-infected subjects were tested by the conventional ELISA using HTLV-I as antigen and alkaline phosphatase as label (Eitest-ATL; Eisai Co., Tokyo. Japan), gelatin particle agglutination test using HTLV-I as antigen (SERODIA-ATLA; Fujirebio. Tokyo, Japan) and West- cm blotting using four core proteins (p19, p24; p28, and p53) of HTLV-I as antigens (Fujirebio). Open and closed triangles indicate sera that were negative and positive, respectively, by Westem blotting. Open squares indicate sera that were negative by Western blotting but positive by the convcn- tional ELISA.

denied by Western blotting (Group 2-2-I), were positive with at least three of the four antigens. This indicated that there were some serum samples, which were indeterminate or negative by Western blotting but could be shown to be truly positive with high probability by Method V using the four antigens. Reliability of test results must be improved by demonstrating antibodies to increasing numbers of different antigens, and more useful antigens remain to be developed.

Fig. 19. Dilution curves of anti-HTLV-I serum by Method V using a synthetic peptide, Cys-env gp46( 188-224), as antigen and the conventional ELISA using HTLV-I as antigen. Sera from four HTLV-I-infected subjects were serially diluted with serum from a seronegative subject and tested by Method V using a synthetic peptide, Cys-env gp46(188-224), as antigen and 6-D-galactosidase from E . coli as label (open symbols) and the conventional ELISA using HTLV-I as antigen and alkaline phosphatase as label (Eitest- ATL; Eisai Co., Tokyo, Japan) (closed symbols). Test results by gelatin particle agglutination using HTLV-I as antigen (SERODIA-ATLA; Fujirebio, Tokyo, Japan) and Westem blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I as antigens (Fujirebio) are also shown.

By Method V using 6-D-galactosidase from E . coli as label, antithyroglobulin IgC was measurable in urine of most of patients with Graves' disease and chronic thyroiditis (Fig. 24) (14). Those levels were well correlated to serum levels, whether the values were corrected by the concentration of creatinine or IgG in urine or IgG in both urine and serum (Fig. 25) (14). This indicated that antithyroglobulin IgCi in urine derived from that in blood. By the conventional ELISA. however, urinary antithyroglobulin IgG was measurable in only 10% of the patients (Fig. 26) and, therefore, only high levels of antithyroglobulin TgG in urine were correlated to those in serum (Fig. 27) (14).

APPLlCATlON OF IMMUNE COMPLEX TRANSFER ENZYME IMMUNOASSAY FOR ANTIBODIES IN URINE AND SALIVA

Urine and saliva can be collected more easily with no in- vasive procedure, less expenses, and less possibility of infections than serum, plasma, or blood. However, the concentration of immunoglobulins is extremely low in urine and saliva (Table 3 ) . The concentrations of IgA in saliva and urine of healthy subjects are - 20-fold and 2,000-fold, respectively, lower than that in serum. The concentrations of IgG in saliva and urine are - 7.50-fold and 4,.500-fold, respectively, lower than that in serum. Therefore, a high sensitivity is required for detecting antibodies in saliva and urine samples, and Method V has been used for detecting antithyroglohulin IgG, anti-HTLV-I IgG and anti-HIV-1 TgG in urine.

Levels of anti-HTLV-1 IgG in urine were also well correlated to those in serum, when measured by Method V using a synthetic peptide, Cys-env gp46( 188-224) as antigen and P-D-galactosidase from E . coli as label (Fig. 28). This to- gether with other results will he described in detail elsewhere.

Method V was used for detecting anti-HIV-1 reverse transcriptase (RT) TgG using recombinant RT as antigen and horseradish peroxidase as label and anti-HIV-1 p17 IgG using recombinant p17 as antigen and P-D-galactosidase from E . coli as label. These methods were at least 10-fold more sensitive than conventional methods such as the conventional ELISA using five recombinant proteins of HIV-1 (gp120,

386 lshikawa et al.

0 m b -

A P


Gelatin Particle Agglutination

Fig. 20. Cornpaison of test results for anti-HTLV-I IgG or antibodies in serum by Method V using a synthetic peptide, Cys-env gp46(188-224). as antigen, gelatin particle agglutination using HTLV-I as antigen and Wcstcrn blotting using four core proteins of HTLV-I as antigens. Sera from HTLV-I- infected subjects were tested by Method V using a synthctic peptide. Cys- e m gp46( 188-224), as antigen and P-D-galactosidase from E . culi as label, gelalin particle agglutination test using HTLV-I as antigen (SERODIA-ATLA: Fujirebio. Tokyo. Japan) and Wcstern blotting using four core proteins (p19, p24. p28. and p53) of HTLV-I as antigens (Fujirebio). Open and closed triangles indicate sera that were negative and positive, respectively. by West- ern blotting. Open squares indicate sera that were negative by Western blotting but positive by Method V using Cys-env gp46( 188-224) as antigen.

gp4 1, p24, p 17, and p 15) as antigens and horseradish peroxidase as label and gelatin particle agglutination test using HIV-1 as antigen (Fig. 29). When unconcentrated urine samples from 100 seronegative subjects and 70 seropositive subjects were tested, the sensitivity and specificity were both 100%. The positivity and negativity with one of the two antigens could be confirmed with the other antigen. In addition. the positivity with low signals could be confirmed with - 10-fold con- centrated urine samples. The 10-fold concentration of urine samples was possible only by 15-20 min centrifugation in a microconcentrator. These results, described in detail elsewhere, indicated that diagnosis of HIV infection be possible by detecting anti-HIV IgG in unconcentrated urine, although more useful antigens remain to be developed. In contrast, the sensitivity and specificity by conventional methods were lower. The sensitivity and specificity by the conventional ELISA using five recombinant proteins (gp120, gp4 1 , p24, p 17, and p 15) of HIV- 1 as antigens and horseradish peroxidase as label (ABBO'IT HTLV-I11 EIA. Abbott Laboratories, North Chi- cago, TL) were 91% and 99%, respectively. The sensitivity

10' lo2 lo3 lo4

Negative Maximal Dilution of Serum with Buffer to Cause Gelatin Particle Agglutination ( -fold )

Fig. 21. Comparison of test results for anti-HTLV-I IgG or antibodies in serum by Method V using a synthetic peptide, Ala-Cys-env gp46(237-262). as antigen, gelatin particle agglutination using HTLV-I as antigen. and West- em blotting using four core proteins of HTLV-I as antigens. Sera froin HTLV- I-infected subjects were tested by Method V using a synthetic peptide. Ala-Cys-em gp46(237-262). as antigen and p-D-galactosidase from E . coli as label, gelatin particle agglutination test using HTLV-I as antigcn (SERODIA- ATLA; Fujirebio. Tokyo, Japan), and Western blotting using four core proteins (p19, p24, p28 and p53) of HTLV-I as antigens (Fujirebio). Open and closed triangles indicate sera that were negative and positive. respectively. by Western blotting. Open squares indicate sera that were negative by West- ern blotting but positive by Method V using Ala-Cys-env gp46(237-262) as antigen.

and specificity by gelatin particle agglutination test using HIV- 1 as antigen (SERODIA-HIV, Fujirebio, Tokyo, Japan) were 82% and 97%, respectively.

Since the concentration of IgG in saliva is -5-fold higher than that in urine (Table 3), the measurement of antibodies in saliva would be easier than in urine, and diagnosis of infections with HTLV and HIV by detecting antibodies in saliva would be more reliable than in urine.

DIFFICULTIES TO OVERCOME

The sensitivity of Methods V and VI depends upon the nonspecific binding of antigcn-enayme conjugates to solid phase. In order to minimize the nonspecific binding of the conjugates, the molar ratio of antigen to enzyme in the conjugate molecules should be within a narrow range. This is possible only after many trials under different conditions, re- quiring a significant amount of purified antigen. An optimal condition is different for each antigcn. The yield of the conjugates is often low. The procedure for the preparation of antigen conjugates is rather complex, as described for the preparation of HIV- 1 p24 conjugates in the Appendix. Anti-


0

! o o 3 3 Q y o

10' lo2 lo3 lo4 1 O U L l m d ' " " l l l i ' ' l l " l l i ' " 8 8 " ' i '

Negative Maximal Dilution of Serum with Buffer lo Cause Gelatin Particle Agglutination ( -fold)

Fig. 22. Comparison of test results for anti-HTLV-I IgG or antibodies in serum by Method V using recombinant gag p24 as antigen, gelatin particle agglutination using HTLV-I as antigen and Western blotting using four core proteins of HTLV-I as antigens. Sera from HTLV-I-infected subjects were tested by Method V using recombinant giig p24(14-214) as antigen and P-D-galactosidase from E . coli as label. gelatin particle agglutination test using HTLV-I as antigen (SERODIA-ATLA; Fujirebio, Tokyo, Japan) and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I as antigens (Fujirebio). Open and closed triangles indicate sera that were neptive and positive. respectively, by Western blotting. Open squares indicate sera that were negative by Western blotting but positive by Method V using recombinant gag p24(14-214) as antigen.

gen conjugates are prepared usually by random use of amino groups of antigen molecules. This may limit the reactivity of some antigens with antibodies.

P-D-Galactosidase from E . coli used as label has been assayed by fluorometry using 4-methylumbelliferyl-~-D- galactoside as substrate, and the sensitivity is the highest among those of enzymes used as label in enzyme immunoassay (10). However, this fluorometric assay is no more sufficiently sensitive in Methods V-VIII, in which the nonspecific binding of antigen-enzyme conjugates is markedly lowered.

APPENDIX Preparation of 2,4-Dinitrophenyl-Bovine Serum Albumin-Recombinant p24 Conjugate

1 . aN-Maleimidohexanoyl-E"2,4-dinitrophenyl-Llysine. An aliquot (0.15 ml) of 100 mmoliL eN-2,il-dinitrophenyl- L-lysine-HC1 in 50% (viv) N,N-dimethylformamide was incubated with 0.75 ml of 0.1 moliL sodium phosphate buffer, pH 7.0 (buffer A) and 0.1 ml of 100 mmoliL N-succinimidyl- 6-maleimidohexanoate in N,N-dimethylformamide at 30°C for 60 rnin.

ci n 3

$ a, S ... - a,

C a,

u) e

F 1 o5

loll 1 o3 A

A

Negative Maximal Dilution of Serum with Buffer to Cause of Gelatin Particle Agglutination ( -fold )

Fig. 23. Comparison of test results for anti-HTLV-I IgG or antibodieq in serum by Method V using a synthctic peptide. Cys-gag p19(100-130), as antigen, gelatin particlc agautination using HTLV-I as antigen, and Western blotting using four core proteins of HTLV-I as antigens. Sera from HTLV-I- infected subjects were tested by Method V using a synthetic peptide. Cys- gag p19(100-130), as antigen and P-D-galactosidase from E . coli as label, gelatin particle agglutination test using HTLV-I as antigen (SERODIA-ATLA: Fujirebio, Tokyo, Japan) and Western blotting using four core proteins ( ~ 1 9 , p24, p28. and pS3) of HTLV-I as antigens (Fujirebio). Open and closed triangles indicate sera that were negative and positive. respectively. by West- ern blotting.

2.2,4-Dinitrophenyl-bovine serum albumin. Bovine serum albumin (12 mg, 180 nmol) in 0.45 ml of buffer A was incubated with 50 p,l of 40 mmoliL N-succinimidyl-S-acetylmercaptoacetate in N,N-dimethylformamide at 30°C for 30 min. The reaction mixture was further incubated with 50 pl of 1 mol/L glycine-NaOH, pH 7.0,30 pl of 0.1 mol/L EDTA, pH 7.0, and 60 ~1 of 1 moliL hydroxylamine, pH 7.0, at 30°C for 10 rnin and with I .0 ml of the aN-maleimidohexanoyl- EN-2 ,4-dinitrophenyl-L-lysine solution at 30°C for 30 min. The reaction mixture was subjected to gel filtration on a column ( I .(I X 30 cm) of Sephadex G-25 using buffer A. The amount of bovine serum albumin was calculated from the absorbance at 280 nm, and the average number of 2,4-dinitrophenyl groups introduced per bovine serum albumin molecule was 5.6, which was calculated from the absorbance at 280 nm and 360 nm (14).

3. 6-Maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin. 2,4-Dinitrophenyl-bovine qerum albumin (3.7 mg) in 1 .O ml of buffer A was incubated with 0.1 ml of 3.3 mmoVL N-succinimidyl-6-maleimidohexanoate in N,N-dimethylformamide at 30°C for 30 min. The reaction mixture was sub-

388 lshikawa et al.

TABLE 1. Test Results of Sera for Anti-HTLV-I Antihodies by Various Methods (I)a

Gclatin Cutoff particle index for aggluti- the optical Western blottingusing4 nation density at core proteins of HTLV-I using 405 nm by Cutoff index lor the fluorescence intensity of bound

p-D-galactosidase activity by immune complex transfer enzyme immunoassay'

Number HTLV-I ELlSA Group of using no. sera I' 1Ib HTLV-I p19 p24 p28 p53 gp46(188-224) gp46(237-262) p19 (100-130) p24 (14- 214)

1 84 + + + + 3.2;4.6;5.1; NT 123-481,056 13; 14; 36; 21; 25; 29: 51: 1.7; 23; 25; 26 (90 sera) 5.5-23 80-149,093 81-77.897 43-58, 140

3 + + + + 3.1-15 NT 9,510-23,776 - 32-1, 531 5.8-1 84 I + + + + 5 . 0 NT 102 31 4.154

I .240 I + + + - 1 + + + - t - - - - 163

-

- ? - - - - -

- -

2- I 8 + + + 2.9-20 + + + + 1,109-101,990 1.5; 13-4,633 5.9; 20-4,577 18-1,155 (29sera) 13 + + 1.8-24 + + + + S2-20,204 2.5; 24-7,903 23-2. 376 1.2; 2.4: 8.6;

58-1,061 3 + + 2.8-4.7 + + + + 761-35,570 3.4-360 1 . 3 4 5 17-459 2 + + 7.5i2.9 + + + + 498:21 - 11; 25 40; 36 3 + + 2.0-4.3 + + + + 87-7,919 1.9-6.064 6.2-84 - ; (3.8)

2-2- 1 1 + + + 8 . 8 + + t I 3,916 2,383 44 94 1 (6 sera) 1 + + 2.1 + & * i 3,265 125 268 3.4

1 + + 1.5 ? ? - - 5,670 1,164 7.6 53 1 + + + 2.1 + - - - 1,781 41 0 35 I + + - + - - - 363 18 6.3 I + -

- -

1.6 - - 1s 2.3 - - _ -

"Plus symbols ( + or + + j indicate that the maximal dilution of serum with the diluent included in the kit to cause agglutination was 16 to 64-fold or 128 to 16,384-fold. Minus syrnhol ( - j indicates sera that showed cutoff indexes (ratios of signals to the cutoff value) below 1 , 'Gelatin particle agglutination I and I1 indicate SERODIA-ATLA, Fujirebio, and SERODIAHTLV-I, Fujimbio, respectively. 'Cutoff indexes in parenthese\ are those lor sera ha t showed inhibition degrees < 75% by excess of the peptides or pZ4. and those without parenthesis are for sera that showed inhibition degrees j 7.5% (> 80% for 99% of the sera).

jected to gel filtration using Sephadex G-25 and 0.1 moliL sodium phosphate buffer, pH 5.0, containing 5 mmoliL EDTA (buffer B). The average number of maleimide groups introduced per 2,4-dinitrophenyl bovine serum albumin molecule was 3.1 (20).

4. Mercaptoacetyl-recombinant p24. Recombinant p24 (0.7 mg) in 2 .O ml of buffer A was incubated with 0.2 ml of 8.25 mmoliL N-succinimidyl-S-acetylmercaptoacetate in N,N- dimethylformamide at 30°C for 30 min. The reaction mixture was incubated with 0.1 ml of 0.1 moUL EDTA, pH 7.0, 0.2 ml of 1 moliL glycine-NaOH, pH 7.0, and 0.28 ml of 1 moliL hydroxylamine.HC1, pH 7.0, at 30°C for 15 min, and subsequently subjccted to gel filtration using Sephadex G-25 and buffer B. The concentration of recombinant p24 was determined by a commercial protein assay kit using bovine serum albumin as standard. The average number of thiol groups introduced per recombinant p24 molecule was 1.5 (20).

5. 2,4-Dinitrophenyl-bovine serum albumin-recombinant p24 conjugate. Mercaptoacetyl-recombinant p24 (0.22 mg, 9.2 nmol) in 50 pl of buffer B was incubated with 6-maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin (0.15 mg, 2 . 3 nmol) in 10 pl of buffer B at 4°C for 20 h. After incubation, the reaction mixture was incubated with 10 pl of 10 mmoliL 2-mercaptoethylamine in buffer B at 30°C for 15 min and subsequcntly with 20 p1 of 10 mmoliL N-ethyl-

maleimide in buffer B at 30°C for 15 min. The reaction mixture was subjected to gel filtration on a column (1.5 X 45 cm) of Ultrogel AcA 44 using 10 mmol/L sodium phosphate buffer, pH 7.0, containing 0.1 moliL NaCl and 0.1 giL bovine serum albumin (buffer C). The average number of recombinant p24 molecules conjugated per 2,4-dinitrophenyl-bovine serum albumin molecule was 2.5, which was calculated from the concentration of 2,4-dinitrophenyl-bovine serum albumin and the total protein concentration determined by a commercial protein assay kit as described above. The amount of 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate was calculated as described for 2,4-dinitrophenyl-bovine serum albumin.

Preparation of 2,4-Dinitrophenyl-Biotinyl-Bovine Serum Albumin-Recombinant p24 Conjugate

1 . 6-Maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin. 2,4-Dinitrophenyl-bovine serum albumin (1.8 mg) in 0.5 ml of buffer A was incubated with 50 p1 of 16.5 mmoliL N-succinimidyl-6-maleimidohexanoate in N,N-dimethylformamide at 30°C for 30 min. The reaction mixture was subjected to gel filtration using Sephadex G-25 and buffer B . The average number of maleimide groups introduced per 2,4-dInitrophenyl-bovine serum albumin inolecule was 10.5 (20).


TABLE 2. Test Results of Sera for Anti-HTLV-I by Various Methods (IT)" Gelatin Cutoff particle index for aggluti- the optical Western blotting using 4 nation density at core proleins of HTLV-I using 405 nm by Cutoff index for the fluorescence intensity of bound

P-D-galactosidase activity by immune complex transfer enzyme immunoassay

Number HTLV-I ELISA Group of using no. scra I 11 HTLV-I p19 p24 p28 p53 gp46(188-224) gp46(237-262) p19 (100-130) p24(14-214)

- - 2-2-2 1 + + - ~ 841 16 (64 sera) 2 + -,+ -

I + - - - 4.2 (29) I + + - 2 + - - - 16; 12 (1.3); - I + - -

1 + - 1.3 4 + - -

1 + - 1.8 33 t - - - -;(1.2-49) - 3 + - - - - i(1.5; 11) -

- - -

- - - - 22;55 - - -

- - - - - 57

3.7 (4.6)

- - _ _ - - -

_ - - _ - - - _ _ _ - -

- - - 3.2 - - _ - - - - 1.7-383 - (1.9-151 -

- 14 + - - - ; ( l . l ) I .6-28 _ - - _ - - (6.5) - - - _ - -

- - - - -

- - _ _ _ - - - - - - - NT 3 89

(l16sera) 2 - 1.1; 1.6 NT NT 1 NT - (1 5 6 . 3 ) 5

- - - - - I .4-2.1 - NT 5 NT 1.2-17 - - (0.26-1.7) 9 NT 6.4 1 NT I

2 NT 24 8.2 1

- - - - -

- (2 .5) - - - - -

- - - - -

- - - - - (1.8) - - -

26 - - - NT I .3; 1.4 - 1.3; 1.7 -

(1.6)

- - - - - -

- - - -

"See Table 1 for symbols

TABLE 3. Concentration of IgA and IgG in Serum, Saliva, and I Jrine

Number Body of Concentration"

fluid Sex Age 5amples TgA 'gG

Yr gil g/l Serum Male 7-85 49 3 I ? 1 3 15 2 6 2

(3 8 - 36) k m a l e 16-87 56 3 2 2 1 8 15 2 6 4

(6 1 - 32) Male + Female 7-87 105 3 2 k 1 5 1 5 ? 6 3

( I 1 - 7 1)

( 1 I - 8 7)

(1 1 - 8 7) (3 8 - 36)

mgil mg/l Saliva Male 26-39 8 160 '-e 95 20 k 20

(49 - 260) (1 I - 53) Femdle 27-37 4 135 2 44 20 2 7 3

(I0 - 27) Male + Female 26-39 12 151 2 80 20 -t- 17

( 1 1 - 53) Urine Mdle 7-85 49 1 4 + . 2 1 2 9 + 2 1

( 0 6 - 15) Female 16-87 56 1 9 i - 2 3 3 7 ? 2 4

Mdle + Female 7-87 105 1 6 ? 2 3 3 3 i - 2 3 (0 6 - 41)

(90 - 193)

(49 - 260)

(0 14 - 7 1 1

(0 23 - 16) 10 8 - 41)

(0 14 - 16)

"Concentrdtion of IgA and 1gG nas expressed as mean + SD Values for the concentration of IgA and IgG in urine were calculated by transforming edLh vdlue to natural logarithm

2. N-Biotinyl-2-mercaptoethylamine. An aliquot (0.1 ml) of 44 mmoUL biotin-N-hydroxysuccinimide in N,N-dimethylformamide was incubated with 1.0 ml of 4.4 mmoliL 2-mercaptoethylamine in 0.1 moliL sodium phosphate buffer, pH 7.0, containing 5 mmoliL EDTA at 30°C for 30 min. Af- ter incubation, 0.1 ml of 1 moliL glycine-NaOH, pH 7.0, was added to the reaction mixture to eliminate remaining biotin-N-hydroxysuccinimide. 3.6-Pvlaleimidohexano~1-2,4-dinitrophen~1-biotiny1-bovine

serum albumin. 6-Maleimidohexanoyl-2,4-dinitrophenyl- bovine serum albumin (1.2 mg, 18 nmol) in 0 5 ml of buffer B was incubated with 40 ~1 of the N-biotinyl-2-mercaptoethylamine solution at 30°C for 30 min. After incubation, the reaction mixture was subjected to gel filtration using Sephadex G-25 and buffer B. The average number of biotin residues introduced per 2,4-dinitrophenyl-bovine serum albumin molecule was 5. l , which was calculated from the decrease in the number of maleimide groups (20).

4. 2,4-Dinitrophenyl-biotinyl-bovine serum albumin-recombinant p24 conjugate. Mercaptoacetyl-recombinant p24 (0.29 mg, 12 nmol) in 0.32 ml of buffer B was incubated with 6-maleimidohexanoyl-2,4-dinitrophenyl-biotinyl-bovine serum albumin (0.2 mg, 3.0 nmol) in 0.1 ml of buffer B at 4°C for 20 h. After incubation, the reaction mixture was incubated with 10 pl of 0.1 moliL 2-mercaptoethylamine in

390 lshikawa et al.

A 0 B

1041 1 o3

l o 5 -

104 -

lo3 -

lo2 - 0

1 0 ' 1

1 o'

10'

10' loo 10' lo2

1- -I-- - - - - - - L loo 10' l o2

0

Fluorescence Intensity for Bound 8-D-Galactosidase Activity without Preincubation with Thyroglobulin

Fig. 24. Measurement of antithyroglobulin IgG in urine from 45 healthy subjects (A,C) and 31 patients with autoimmune thyroid diseases (16 patients with Graves' disease and 15 patients with chronic thyroiditis) (B,D) by Method V using P-D-galactosidase from E . c d i as label. Circlcs and squares indicate males and females, respectively. The dotted lines indicate tentative cut-off values for the fluorescence intensity of bound p-D-galactosidase activity and the decrease in the fluorescence intensity of bound p-11-galactosidase activity by preincubation with excess of thyroglobnlin.

buffer B at 30°C for 15 min and subsequently with 20 (1.1 of 0.1 moliL N-ethylmaleimide in buffer B at 30°C for 15 min. The reaction mixture was subjected to gel filtration on a column (1 .5 x 45 cm) of Ultrogel AcA 44 using buffer C. The average number of recombinant p24 molecule conjugated per 2,4-dinitrophenyl-biotinyl-bovine serum albumin molecule was 4.3, which was calculated in the same way as that of 2,4-dinitrophenyl-bovine serum albumin-recombinant p24 conjugate. The amount of 2,4-dinitrophenyl-biotinyl-bovine serum albumin-recombinant p24 conjugate was calculated as described for 2,4-dinitrophenyl-bovine serum albumin.

lo2 lo3 lo4 105 lo6 10' l o2 103 10' lo5 10' 10'

Fluorescence lntensiiy for Bound O-D-Galactosidase Activity Observed with Serum

Fig. 25. Correlation of antithyroglobulin levels in urine to those in serum measured by Method V using P-D-galactosidase from E . coli as label. Antithyroglobulin IpG in urine and serum from 31 patients with autoimmune thyroid diseases (16 patients with Graves' disease and 15 patients with chronic thyroiditis) was measured. Circles and squares indicate males and females. respectively. A: No correction. B: Corrected by the concentration of creatinine in urine. C: Corrected by the concentration of IgC in urine. D: Corrected by the concentrations of IgC in urine and serum.

mount of peroxidase was calculated from the absorbance at 403 nm (20). The reaction mixture was subjected to gel filtration using Sephadex G-25 and buffer B. The number of maleimide groups introduced per peroxidase molecule was - 1 .0 (20).

2. Recombinant p24-peroxidase conjugate. Mercaptoacetyl- recombinant p24 (0.24 mg. 10 nmol) in 55 pl of buffer B was incubated with 6-maleimidohexanoyl-peroxidase (0.1 mg, 2.5 nmol) in 3.0 IJ.I of buffer B at 4°C for 20 h. After incubation, the reaction mixture was incubated with 10 ~1 of 10 mmoliL 2-mercaptoethylamine in buffer B at 30°C for 15 min and subsequently with 20 ~1 of 10 mmoliL N-ethylmaleimide in buffer B at 30°C for 15 min. The reaction mixture was

Preparation of Recombinant p24-Peroxidase Conjugate

1. 6-Maleimidohexanoyl-peroxidase. Horseradish peroxidase (2.0 mg) in 0.3 ml of buffer A was incubated with 30 ~l of 30 mmoliL N-succinimidyl-6-maleimidohexanoate in N,N-dimethylformamide at 30°C for 30 min. The a-

subjected to gel filtration on a column ( I .5 x 45 cm) of Ultrogel AcA 44 using buffer C. The average number of recombinant p24 molecules conjugated per peroxidase molecule was 1 . I , which was calculated from the concentration of peroxidase


1~ A

0.1

0.01 r

0.002 -

07. CmDO 0

I

,!

Y I 1

x D

l l 0

0

0

Absoi'oance for Bound Peroxidase Activity without Preincubation with Thyroglobulin

Fig. 26. Measurement of antithyroglobulin IgG in urine from 31 patients with autoimmune thyroid diseases (16 patients with Graves' disease and 15 patienth with chronic thyroiditis) by the conventional ELISA using horseradish peroxidase as label (MESACUP Anti-TG Test, Medical and Biologi- cal Lahoratories Co., Nagoya, Japan). Bound peroxidase activity was assayed for 15 min (A,C) or for 90 min (B,D). Circles and squares indicate males and females, respectively.

I l o

0

0

0

8

0

p

a O.Ool? 0 0.01 0 1 1 0 01 0.1 1

Absorbance for Bound Peroxidase Activity Observed with Serum

Fig, 27. Correlation of antithyroglobulin levels in urine to those in serum measured by the conventional ELISA using horseradish peroxidase as label (MESACUP Anti-TG Test. Medical ,and Biological Laboratories Co.. Nagoya, Japan). Antithyroglobulin IgG in urine and serum from 31 patients with autoimmune thyroid diseases (16 patients with Graves' disease and 15 patients with chronic thyroiditis) was measured. Bound peroxidase activity with urine samples was assayed for 15 min (A) and 90 min (B). Circles and squares indicate males and females, respectively.

.- . x ' 03F

a, i ._ c

8 - s ._ c 1 0 4

0 . . . * *

*. * .

I o3 I o4 I o5 1 o6 Fluorescence Intensity for Bound B-D-Galactosidase

Activity Observed with Serum

Fig. 28. Correlation of anti-HTLV-1 IgG levels in urine to those in serum measured by Method V using a synthetic peptide, Cys-env gp46 (188-224), as antigen and P-0-galactosidase fromE. roli as label.

(20) and the total protein concentration determined by a commercial protein assay kit as described above. The amount of recombinant p24-peroxidase conjugate was calculated from peroxidase activity (20).

Preparation of Recombinant p24-p-D-Galactosidase Conjugate

1. 6-Maleimidohexanoyl-recombinant p24. Recombinant p24 (0.5 mg) in 0.18 ml of buffer A was incubated with 18 p1 of 8.25 mmoliL N-succinimidyl-6-maleimidohexanoate in N,N-dimethylfonnamide at 30°C for 30 min. The reaction mixture was subjected to gel filtration using Sephadex G-25 and buffer B. The average number of maleimide groups introduced per recombinant p24 molecule was 1.3 (20).

2. Recombinant p24-P-D-galactosiddse conjugate. &Male- imido-hexanoyl-recombinant p24 (0.1 1 mg, 4.6 nmol) in 0.12 ml of buffer B was incubated with P-D-galactosidase (EC 3.2.1.23)fromE. coli(0.3mg, 0.56nmoljin0.1 mlofbuffer B at 4°C for 20 h. After incubation, the reaction mixture was incubated with 10 pl of 0. I moliL N-ethylmaleimide in buffer B at 30°C for 15 min and subsequently with 20 p1 of 0.1 moliL 2-mercaptoethylamine in buffer B at 30°C for 15 min, The reaction mixture was subjected to gel filtration on a column (1.5 X 45 cmj of Ultrogel AcA 22 using buffer C containing 1 .O mmoliL MgCI2 and 1 .O g/L NaN3. The amount of recombinant p24-P-D-galactosidase conjugate was calculated from p-D-galactosidase activity (20).

392 lshikawa et al.

Dilution al Unne from Seropositive Subjects with Unne from a Seronegative Subject (-lold)

Fig. 29. Dilution curves of anti-HIV-1 IgG in urine by various methods. Urine samples from seropositive subjects were serially diluted with urine from a seronegative subject and tested by Method V (open symbols) using recombinant RT as antigen and horseradish peroxidase as label (A) and recombinant p17 as antigen and P-D-galactosidase from E. coli as label (B), the conventional ELISA using five recombinant proteins of HIV-I (gpl20.

gp41, p24. p17, and PIS) as antigens and horseradish peroxidase as label ( A B B O T HILV-I11 EIA, Abbott Laboratories, North Chicago. IL) (closed symhols with broken lines) and gelatin panicle agglutination test using HIV-I as antigen (SERODIA-HIV. Fujirebio. Tokyo. Japan) (closed symbols with solid lines).

REFERENCES

1.

2.

3 .

4.

5 .

6 .

7.

8.

9.

10.

1 1 .

Constantine NT: Serologic tests for the retroviruses: Approaching a de- cade ofevolution. AfDS7:1-13, 1993. Ishikawa E, Kohno T: Development and applications of sensitive enzyme immunoassay for antibodies: A review. JClin LubAnul3:252-265, 1989. Kohno T, Ishikawa E: A novel eruyme imniunoassay of anli-insulin IgC in guineapig serum. BiochemBiophysRes Commun 147:644-649, 1987. Kohno T, Mitsukawa T. Matsukura S , lshikawa E: Novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for antithyroglobulin IgG in human serum. J Clin Lab Anal 2:209-214, 1988. Kohno T. Mitsukawa T, Matsukura S, Ishikawa E: Novel and sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-thyroglohulin IgG in human sernm. Clin Chem E n q m Cornrns 1:89-96, 1988. Kohno T, lshikawa E: Novel enzyme immuno transfer enzyme immunoassay) lor anti-insulin IgG in guinea pig serum. AnulLerr21:1019-1031, 1988. Kohno T, Mitsukawa T, Matsukura S, Ishikawa E: Sensitive time-resolved fluorimetric inmune-complex-transkr immunoassay for antithyroglobulin IgG in serum. J Clin Lab Anal 4224-230, 1990. Kohno T, Mitsukawa T, Matsukura S. Tsunetoshi Y. Ishikawa E: More sensitive and simpler immune complex transfer enzyme ininmunoassay for antithyroglohulin 1gG in serum. J Clin Lab Anal 3: 163-168, 1989. Kohno T, Hirota K , Ishikawa E: Presence of antibodies against P-D-galactosidase from Escherichia coli in serum of healthy subjects examined by immune complex transfer enzyme immunoassay. Clin Chem Enzyrn Con~rns 439-49, 199 I . Ishikawa E Development and clinical application of sensitive enzyme immunoassay for macromolecular antigen: A review. Clin Biochem 20:375-385, 1987. lshikawa E? Hashida S, Kohno T: Development of ultrasensitive enzyme immunoassay reviewed with emphasis on factors which limit the senaitivity. Mol Cell Prob 5:81-95, 1991.

12. Kohno T, Katsumaru H , Nakamoto H, Yasuda T, Mitsukawa T, Matsukura S, Tsunetoshi Y, IshikawaE: Use of inactive P-D-galactosidase forelim- ination of interference hy anti-P-D-galactosidase antibodies in immune complex transfer enzyme immunoassay for antithyroglohulin IgG in serum using P-D-galactosidase from E . coli as label. J Clin Lib Anal 5: 197-205, 1991.

13. Kohno T. Sakoda I , Ishikdwa E: Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) lgG in serum using a synthetic peptide, env gp46(188-209), as antigen. J Clin Lab Anal 5:25-37, 1991.

14. Yogi Y, Hirota K, Kohno 'I. Toshimori H. Matsukura S , Setoguchi T. Ishikawa E: Measurement of antithyroglohulin IgG in urine of patients with autoimmune thyroid diseases by sensitive enzyme immunoassay (immune complex transfer enzymt: immunoassay). J C h i Lab Anal 7:70-79, 1993.

15. Kohno H, Kohno T, Sakoda I, Ishikawa E: Sensitive detection of anti- human T-cell leukemia virus type I IgG in human serum by a novel enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant gag-env hybrid protein as antigen. .I Virol Merh- ods31:77-92, 1991.

16. Kohno T, Sakoda 1, Suzuki M, lzumi A, Ishikawa E: Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, cys-gug p19(100-130), a h

antigen. J Clin Lab Anal 5 :307- 3 16 199 1. 17. Kohno T, Sakoda I, Ishikawa E: Sensitive enzyme immunoassay (im-

mune 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. JCIinLubAnal6:105-112, 1992.

18. Kohno T, Sakoda I , lshikawa E: Scnsitive en7yme immunoassay (immune complex transfer enzyme immunoassay) for (antihuman T-cel I leukemia virus type I) ImmunogIobulin G in semm using a synthetic peptide, Ala-Cys-Env gp46(237-262). as antigen. J Clin Lab Anal 6:162-169, 1992.

19. Kohno T, Hirota K, Sakoda I , Yaniasaki M. Yokoo Y, Ishikawa E: Sen- sitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgC in serum


using recombinant gag p24(14-214) as antigen. J Clin Lab Anal 6:302-310, 1992.

20. Ishjkawa E, Imagawa M, Hashida S, Yoshitake S, Hamaguchi Y. Ueno

T: Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassay 4: 209-327, 1983.

principle and applications of ultrasensitive enzyme immunoassay (immune complex transfer enzyme...

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