[57]
B I O T I N Y L A T E D P R O T E I N A IN I M M U N O A S S A Y
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[57] B i o t i n y l a t e d P r o t e i n A in I m m u n o a s s a y B y IKUO TAKASHIMA, N O B U O HASHIMOTO, a n d H S H I - C H I C H A N G
Enzyme-linked immunoassay (ELISA) has been used extensively in serological diagnostic tests. In order to apply ELISA for serodiagnosis of zoonotic viral diseases such as Japanese encephalitis (JE), the conjugates must be prepared specifically to individual animal species involved in the transmission cycle of the virus. Since Guesdon et al.1 introduced avidinbiotin into ELISA, this system has been used widely to detect or quantify immunoglobulins 2,3 or antigens. 4,5 Protein A is known to bind IgG in several species of animals via the Fc portion of the IgG molecule. 6 In this chapter, we describe the use of biotin-labeled protein A ELISA for simultaneous detection of JE antibody in the sera of humans, swine, and several other vertebrates using the same reagents. Materials and Reagents
Antigen: purified JE virus JaGAr-01 strain vaccine (total N 96.6 /zg/ml) from Biken (Osaka, Japan) 7 Sera: swine sera were collected from endemic and nonendemic areas of Japan; human, cattle, horse, monkey, dog, and pigeon sera were collected from an endemic area; and rabbit, rat, and mice sera were obtained from individuals experimentally infected with the JE virus Protein A: salt-free lyophilized powder (E-Y Laboratories, San Mateo, CA) Horseradish peroxidase-labeled avidin (HRP-avidin): commercially available product of E-Y Laboratories Substrate: 0.2 mM 2,2'-azinodi(3-ethylbenzthiazolinesulfonic acid) (ABTS; Sigma Chemical Co., St. Louis, MO) and 0.004% H202 in 50 mM citrate buffer (pH 4.0) l j. L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 2 S. Jackson, J. A. Sogn, and T. T. Kindt, J. lmmunol. Methods 48, 229 (1982). 3 D. V. Subba Rao, N. L. McCartney-Francis, and D. D. Metcalfe, J. Immunol. Methods 57, 71 (1983). 4 C. Kendall, I. lonescu-Mediu, and G. R. Dreesman, J. Imrnunol. Methods 56, 329 (1983). 5 R. H. Yolken, F. J. Leister, L. S. Whitcomb, and M. Santosham, J. lmmunol. Methods 56, 319 (1983). 6 A. Forsgren and J. Sjoquist, J. Immunol. 97, 882 (1966). 7 K. Takaku, T. Yamashita, T. Osanai, I. Yoshida, M. Kato, H. Goda, M. Takagi, T. Hirota, T. Amano, K. Fukai, N. Kunita, K. Inoue, K. Shoji, A. Igarashi, and I. Ito, Biken J. 11, 25 (1968).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
498
APPLICATIONS
[5 7]
Coating buffer: 50 m M sodium carbonate-bicarbonate buffer (pH 9.6) Rinsing buffer: phosphate-buffered saline (PBS, pH 7.4) containing 0.05% Tween 20 (Tween 20-PBS) Serum or biotin-protein A diluent buffer: PBS containing 1% Tween 80 (Tween 80-PBS) HRP-avidin diluent buffer: 50 mM phosphate buffer (pH 8.0) containing 0.5 M NaCI and 0.1% Tween 20 (Tween 20-0.5 M NaC1 PB)
Biotin Labeling of Protein A The procedure is essentially the same as that described by Guesdon et Protein A (1 mg/ml) is dialyzed against 0.1 M NaHCO3 at 4 °. After dialysis, 1 ml of the protein A solution is mixed with 60/xl of biotin-Nhydroxysuccinimide ester (E-Y Laboratories; 1 mg/ml in dimethyl sulfoxide). The solution is kept at room temperature for 4 hr and dialyzed overnight against PBS at 4 ° with several changes of PBS. al. l
Procedure for ELISA The procedure followed is that previously described. 8 Briefly, the optimal dilutions of the reagents are determined by checkerboard titration. JE antigen is diluted with coating buffer to a concentration of 8 units. Onehundred microliters of the antigen is delivered into U-shaped polystyrene microtiter plates (96 U, PS, SH, Nunc, Denmark). The plates are incubated at 37° for 3 hr to fix the antigen onto the wells. The solution is then discarded, and the wells are rinsed once with Tween 20-PBS. After tapping the plates, serum samples (diluted serially at 2-fold with Tween 80PBS) are delivered to each well at a volume of 50 ~1. The plates are kept at 37° for 40 min and then rinsed 3 times with Tween 20-PBS. Biotinprotein A in Tween 80-PBS (50 tzl containing 4 units) is delivered into each well, and the plates are incubated at 37° for 40 min. The solution is again discarded, and the wells are washed 3 times with Tween 20-0.5 M NaCI PB. The HRP-avidin in Tween 20-0.5 M NaC1 PB (50/zl at 8 units) is delivered into each well, and the plates are incubated at 37° for 40 min. Next, the contents of the plates are discarded, and the wells are washed 3 times with Tween 20-0.5 M NaCI PB. Finally, 100 ~1 of freshly prepared ABTS substrate solution is added to each well. g H.-C. Chang, I. Takashima, J. Arikawa, and N. Hashimoto, J. Virol. M e t h o d s 9, 143 (1984).
[57]
BIOTINYLATED PROTEIN A IN IMMUNOASSAY
499
Following an incubation for 1 hr at 37°, the degree of color development is measured by a microplate photometer (Corona MTP-12, Corona Electric, Katsuda, Japan) at a wavelength of 405 nm. The cutoff point is set at an average of the OD values of control wells (without serum) plus an additional factor of 0.1. End-point titers of serum are expressed as a reciprocal of the highest serum dilution at which the OD value is above the cutoff point. The E L I S A titers are compared with the titers of the hemagglutination inhibition (HI) test described by Clarke and Casals. 9
Application E L I S A antibody titers to JE virus were determined in swine sera collected from nonendemic (Hokkaido) and endemic (Shizuoka) areas of Japan (Fig. 1A). E L I S A titers of 106 swine sera from Hokkaido were lower than 1 : 5. HI titers of all these sera from Hokkaido were negative (
B I O T I N Y L A T E D P R O T E I N A IN I M M U N O A S S A Y
497
[57] B i o t i n y l a t e d P r o t e i n A in I m m u n o a s s a y B y IKUO TAKASHIMA, N O B U O HASHIMOTO, a n d H S H I - C H I C H A N G
Enzyme-linked immunoassay (ELISA) has been used extensively in serological diagnostic tests. In order to apply ELISA for serodiagnosis of zoonotic viral diseases such as Japanese encephalitis (JE), the conjugates must be prepared specifically to individual animal species involved in the transmission cycle of the virus. Since Guesdon et al.1 introduced avidinbiotin into ELISA, this system has been used widely to detect or quantify immunoglobulins 2,3 or antigens. 4,5 Protein A is known to bind IgG in several species of animals via the Fc portion of the IgG molecule. 6 In this chapter, we describe the use of biotin-labeled protein A ELISA for simultaneous detection of JE antibody in the sera of humans, swine, and several other vertebrates using the same reagents. Materials and Reagents
Antigen: purified JE virus JaGAr-01 strain vaccine (total N 96.6 /zg/ml) from Biken (Osaka, Japan) 7 Sera: swine sera were collected from endemic and nonendemic areas of Japan; human, cattle, horse, monkey, dog, and pigeon sera were collected from an endemic area; and rabbit, rat, and mice sera were obtained from individuals experimentally infected with the JE virus Protein A: salt-free lyophilized powder (E-Y Laboratories, San Mateo, CA) Horseradish peroxidase-labeled avidin (HRP-avidin): commercially available product of E-Y Laboratories Substrate: 0.2 mM 2,2'-azinodi(3-ethylbenzthiazolinesulfonic acid) (ABTS; Sigma Chemical Co., St. Louis, MO) and 0.004% H202 in 50 mM citrate buffer (pH 4.0) l j. L. Guesdon, T. Ternynck, and S. Avrameas, J. Histochem. Cytochem. 27, 1131 (1979). 2 S. Jackson, J. A. Sogn, and T. T. Kindt, J. lmmunol. Methods 48, 229 (1982). 3 D. V. Subba Rao, N. L. McCartney-Francis, and D. D. Metcalfe, J. Immunol. Methods 57, 71 (1983). 4 C. Kendall, I. lonescu-Mediu, and G. R. Dreesman, J. Imrnunol. Methods 56, 329 (1983). 5 R. H. Yolken, F. J. Leister, L. S. Whitcomb, and M. Santosham, J. lmmunol. Methods 56, 319 (1983). 6 A. Forsgren and J. Sjoquist, J. Immunol. 97, 882 (1966). 7 K. Takaku, T. Yamashita, T. Osanai, I. Yoshida, M. Kato, H. Goda, M. Takagi, T. Hirota, T. Amano, K. Fukai, N. Kunita, K. Inoue, K. Shoji, A. Igarashi, and I. Ito, Biken J. 11, 25 (1968).
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.
498
APPLICATIONS
[5 7]
Coating buffer: 50 m M sodium carbonate-bicarbonate buffer (pH 9.6) Rinsing buffer: phosphate-buffered saline (PBS, pH 7.4) containing 0.05% Tween 20 (Tween 20-PBS) Serum or biotin-protein A diluent buffer: PBS containing 1% Tween 80 (Tween 80-PBS) HRP-avidin diluent buffer: 50 mM phosphate buffer (pH 8.0) containing 0.5 M NaCI and 0.1% Tween 20 (Tween 20-0.5 M NaC1 PB)
Biotin Labeling of Protein A The procedure is essentially the same as that described by Guesdon et Protein A (1 mg/ml) is dialyzed against 0.1 M NaHCO3 at 4 °. After dialysis, 1 ml of the protein A solution is mixed with 60/xl of biotin-Nhydroxysuccinimide ester (E-Y Laboratories; 1 mg/ml in dimethyl sulfoxide). The solution is kept at room temperature for 4 hr and dialyzed overnight against PBS at 4 ° with several changes of PBS. al. l
Procedure for ELISA The procedure followed is that previously described. 8 Briefly, the optimal dilutions of the reagents are determined by checkerboard titration. JE antigen is diluted with coating buffer to a concentration of 8 units. Onehundred microliters of the antigen is delivered into U-shaped polystyrene microtiter plates (96 U, PS, SH, Nunc, Denmark). The plates are incubated at 37° for 3 hr to fix the antigen onto the wells. The solution is then discarded, and the wells are rinsed once with Tween 20-PBS. After tapping the plates, serum samples (diluted serially at 2-fold with Tween 80PBS) are delivered to each well at a volume of 50 ~1. The plates are kept at 37° for 40 min and then rinsed 3 times with Tween 20-PBS. Biotinprotein A in Tween 80-PBS (50 tzl containing 4 units) is delivered into each well, and the plates are incubated at 37° for 40 min. The solution is again discarded, and the wells are washed 3 times with Tween 20-0.5 M NaCI PB. The HRP-avidin in Tween 20-0.5 M NaC1 PB (50/zl at 8 units) is delivered into each well, and the plates are incubated at 37° for 40 min. Next, the contents of the plates are discarded, and the wells are washed 3 times with Tween 20-0.5 M NaCI PB. Finally, 100 ~1 of freshly prepared ABTS substrate solution is added to each well. g H.-C. Chang, I. Takashima, J. Arikawa, and N. Hashimoto, J. Virol. M e t h o d s 9, 143 (1984).
[57]
BIOTINYLATED PROTEIN A IN IMMUNOASSAY
499
Following an incubation for 1 hr at 37°, the degree of color development is measured by a microplate photometer (Corona MTP-12, Corona Electric, Katsuda, Japan) at a wavelength of 405 nm. The cutoff point is set at an average of the OD values of control wells (without serum) plus an additional factor of 0.1. End-point titers of serum are expressed as a reciprocal of the highest serum dilution at which the OD value is above the cutoff point. The E L I S A titers are compared with the titers of the hemagglutination inhibition (HI) test described by Clarke and Casals. 9
Application E L I S A antibody titers to JE virus were determined in swine sera collected from nonendemic (Hokkaido) and endemic (Shizuoka) areas of Japan (Fig. 1A). E L I S A titers of 106 swine sera from Hokkaido were lower than 1 : 5. HI titers of all these sera from Hokkaido were negative (
![[125I]protein A: a tracer for general use in immunoassay.](/assets/img/hold.png)
