J. Biochem., 82, 261-266 (1977)
Use of Rabbit Antibody IgG Bound onto Plain and Aminoalkylsilyl Glass Surface for the Enzyme-Linked Sandwich Immunoassay Kanefusa KATO, Yoshitaka HAMAGUCHI, Sachiyo OKAWA, Eiji ISHIKAWA, Keiko KOBAYASHI,* and Nobuhiko KATLTNUMA* Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki-gun, Miyazaki 889-16, and *Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770 Received for publication, February 12, 1977
Rabbit antibody IgG was bound onto aminoalkylsilyl or plain glass rods by simple adsorption. For comparison, rabbit antibody IgG was also bound onto glutaraldehyde-activated aminoalkylsilyl glass rods. These antibody-glass rods were tested by the sandwich procedure using Fab' fragments of rabbit antibody conjugated with /3-D-gaIactosidase from Escherichia coli. The glutaraldehyde-activated aminoalkylsilyl glass showed the largest capacity to bind antigen and the plain glass showed the smallest. However, the antibody-glass rods prepared by simple adsorption were as useful for the sandwich immunoassay of macromolecular antigens as those prepared with glutaraldehyde. With all the antibody-glass rods prepared, 0.1 to 10 fmol of ornithine 3-aminotransferase from rat liver and 2,4-dinitrophenyl human IgG were measurable. More than 10 fmol of the antigens may be measurable with larger amounts of the antibody^-D-galactosidase complexes, although the non-specific binding of the complexes to the solid phase increases to limit the sensitivity of the immunoassay.
Engvall and Perlmann have developed the enzymelinked immunoassay of antibodies ( 7 ) . Subsequently, the sandwich technique has been developed for the enzyme-linked immunoassay of macromolecular antigens. Enzymes used for labelling antibodies are alkaline phosphatase [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] (1, 2), peroxidase [donor: hydrogen. . „ Abbreviations: IgG, immunoglobulin G; Fab, mono, . , .' , . . T -, T, ~ valent fragments derived from pepsin-treated IgG; Buffer A, 0.01 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl, 1 mM MgCI,, 0.1% NaN,, and 0.1% bovine serum albumin; OAT, ornithine 3-aminotransferase from rat liver. Vol. 82, No. 1, 1977
peroxide oxidoreductase, EC 1.11.1.7] (j), glucose oxidase [/9-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4] (4), and ^-D-galactosidase [/3-r>galactoside galactohydrolase, EC 3.2.1.23] (5). Antibodies have been labelled with enzymes using glutaraldehyde ( 2 - 4 ) or N, N'-ophenylenedimaleimide (5). The solid phases used are polystyrene tubes (2, 3), cellulose (4), and Sepharose 4B (5). We have developed a novel method for the conjugation of Fab' fragments of rabbit IgG with . , ., . „ , . ,. ,. fl-D-galactosidase from Escherichia coli and have r °~ demonstrated that 0.3 fmol of human IgG is measurable using Fab' fragments of purified rabbit anti(human IgG) antibody conjugated with yS-Dgalactosidase and rabbit anti(human IgG) IgG
261
262 K. KATO, Y. HAMAGUCHI, S. OKAWA, E. ISHIKAWA, K. KOBAYASHI, and N. KATUNUMA coupled to Sepharose 4B (6). We have recently used rabbit antibody bound onto aminoalkylsilyl glass treated with glutaraldehyde and have shown that the assay with the glass is more sensitive, simpler and more reproducible than that with Sepharose 4B (7). In subsequent studies, however, we have found that antibody can be bound onto glass surface by simple adsorption for the immunoassay. The present paper shows that a highly sensitive sandwich immunoassay of macromolecular antigens is possible with rabbit antibody IgG bound onto aminoalkylsilyl or plain glass by simple adsorption as well as with the antibody bound onto glutaraldehyde-activated aminoalkylsilyl glass.
Biological Laboratories Ltd. (Nagoya) and from Miles Laboratories, Inc. (Kankakee), respectively. The IgG fractions from these antisera and normal rabbit serum were prepared using NatSO4 (14) and DEAE-cellulose (11). Binding of Rabbit IgG onto Class Rods— Rabbit IgG was bound onto the glass surface in four different manners. Binding with glutaraldehyde was performed by the method of Robinson et al. (15) as described previously (7). Pyrex glass rods (3 mm in diameter and 5 mm in length) were aminoalkylated with 3-aminopropyltriethoxysilane (Nakarai Chemicals Ltd., Kyoto). The aminoalkylsilyl glass rods were activated with 1 % glutaraldehyde (Nakarai Chemicals Ltd., Kyoto), immersed in 0.25 M sodium phosphate buffer (pH 7.5) containing MATERIALS AND METHODS 0.1 mg/ml of IgG fraction from either rabbit antiAntigens and Antibodies—Crystalline ornithine sera or normal serum for 30 min, and stored at 4°C d-aminotransferase (OAT) [L-ornithine: 2-oxoacid overnight. The rabbit IgG-glass rods were aminotransferase, EC 2.6.1.13] from rat liver and washed with 0.01 M sodium phosphate buffer (pH rabbit antisera against the crystalline enzyme were 7.0) containing 0.1M NaCl, 1 mM MgCl,, 0.1% prepared as described previously (8). The crystal- NaN,, and 0.1 % bovine serum albumin (Buffer A) line enzyme preparation was homogenous on both and stored in Buffer A at 4°C until use. The glass ultracentrifugation and electrophoresis and could rods were washed several times during the storage be stored in 0.1 M sodium phosphate buffer (pH before use. For the binding by simple adsorption, 7.0) at —20°C without loss of activity for at least plain or aminoalkylsilyl glass rods were treated 6 months. Amounts in fig of the crystalline with rabbit IgG fractions in the same manner except enzyme were determined with Folin-Ciocalteu re- that the activation with glutaraldehyde was omitted. agent (9) using bovine serum albumin (Fraction V, As a control, plain glass rods were also treated with Armour Pharmaceutical Co., Chicago) as a stand- glutaraldehyde and then with rabbit IgG fractions ard, and amounts in fmoles were calculated by as described above. taking its molecular weight as 170,000 (8). The Rabbit Antibody (Fab')-f3-D-Galactosidase IgG fraction from the antiserum against the crystal- Complex—The rabbit antibody (Fab>/9-r>galactoline enzyme was obtained by fractionation with sidase complexes were prepared as described pre(NH4),SO4 (10) followed by passage through a viously (6). The Fab' fragments of rabbit antibody column of DEAE-cellulose (DE-52, Whatman Bio- containing sulfhydryl groups were treated with chemicals Ltd., Kent) (11). Human IgG was excess N, N'-o-phenylenedimaleimide (Aldrich obtained from Miles Laboratories, Inc. (Kankakee) Chemicals Company, Inc., Milwaukee) to introduce and its amounts were determined from the absor- maleimide residues. The maleimide residues of bance at 280 nm using the value of £ ^ = 1 5 (12). the dimaleimide-treated Fab' fragments were allow2, 4-Dinitrophenyl human IgG was prepared using ed to react with the sulfhydryl groups of fi-D2,4-dinitrophenyl sulfonic acid (sodium salt) galactosidase from Escherichia coli (Boehringer (Tokyo Kasei Kogyo Co., Ltd., Tokyo) by the Mannheim, Mannheim) to give the complexes. method of Eisen et al. (13). The number of 2, 4- Amounts of the complexes are expressed as units dinitrophenyl residues coupled to human IgG of /3-D-galactosidase activity, one unit being demolecules and the amount of 2, 4-dinitrophenyl fined as the amount that hydrolyzes one fimole of human IgG were calculated from the absorbance 4-methylumbelliferyI-/?-D-galactoside (Sigma Chemat 280 and 360 nm (12,13). The rabbit antisera ical Co., St. Louis) per min under the conditions against human IgG and 2, 4-dinitrophenyl bovine described previously (7). serum albumin were obtained from Medical and Sandwich Procedure—Each of the IgG-glass /. Biochem.
IgG BOUND ON GLASS FOR IMMUNOASSAY rods was incubated in a volume of 0.15 ml of Buffer A, containing various amounts of antigen, with shaking at 37°C for 4 h and the incubation mixtures were allowed to stand at 4°C overnight. Each rod was washed twice with 1 ml of Buffer A, and was incubated with the antibody-/3-D-galactosidase complex in 0.15 ml of Buffer A with shaking at 37°C for 6 h. (The binding to the antibody-glass rods of the antigens and the complexes used were almost maximal within 4 and 6 h, respectively.) Each rod was then washed twice with 1 ml of Buffer A and was transferred to another test tube which was free of ^-D-galactosidase. /S-D-Galactosidase activity bound to each rod was assayed using 4methylumbelliferyl-/3-D-galactoside as substrate (16) as described previously (7). The amounts of the complexes used for the assay of OAT and 2, 4-dinitrophenyl human IgG were 1,650 and 2,050 punits, respectively. These amounts of the complexes were chosen so that the ratios of j9-r>galactosidase activity bound to the solid phase in the presence of the antigens (10 fmol) to that in their absence were maximal. Temperature of Experiments—Unless otherwise specified, experiments were carried out at 20 to 25°C throughout.
263
4. 0 0.01
100
(fmol/assay tube) RAT ORNITHINE J-AMINOTRANSFERASE
Fig. 1. Sandwich immunoassay of crystalline OAT (A). Closed circles, triangles, and squares indicate the first assays with glutaraldehyde-activated aminoalkylsilyl, non-activated aminoalkylsilyl, and plain glass rods, respectively. Open symbols indicate the corresponding second assays, all of which were performed with glutarRESULTS aldehyde-activated aminoalkylsilyl glass rods. All glass Amounts of Antigen Adsorbed on the Antibody- rods used were loaded with anti-OAT.
Glass Rods—The antibody-glass rods were prepared in three different manners. Rabbit IgG from anti-OAT serum was bound onto the aminoalkylsilyl or plain glass rods by simple adsorption. For comparison, the same IgG was bound onto the aminoalkylsilyl glass rods activated with glutaraldehyde. These glass rods were tested by the sandwich procedure with the Fab' fragments of anti-OAT antibody conjugated with ^-D-galactosidase. The activity of /3-D-galactosidase bound to the glutaraldehyde-activated aminoalkylsilyl glass rods was the highest and the activity bound to the plain glass rods was the lowest (Fig. 1). These findings suggest that different amounts of IgG were bound onto the glass rods prepared in different manners. However, amounts of IgG bound onto the glass rods were unknown, since the change in the concentration of IgG before and after the treatment of glass rods was too small to determine precisely from the absorbance at 280 nm.
Vol. 82, No. 1, 1977
The amount of IgG bound onto the glass rods must be related to the amount of antigen that can be adsorbed. So, the amount of antigen that can be adsorbed on each of the glass rods was determined in the following manner. Various amounts (0.1 to 100 fmol) of crystalline OAT were subjected to the sandwich immunoassay with anti-OAT-glass rods prepared in different manners (the first assay) and the incubation media used for the first assay or portions of them were again subjected, after removing the glass rods used for the first assay, to the sandwich immunoassay using anti-OAT-aminoalkylsilyl glass rods prepared with glutaraldehyde (the second assay). The results are shown in Fig. 1 and Table I. £-r>Galactosidase activities bound in the second assay did not increase very much with any glass rod tested when less than 10 fmol of the antigen was subjected to the first assay (Fig. 1). This indicates
264 K. KATO, Y. HAMAGUCHI, S. OKAWA, E. ISHIKAWA, K. KOBAYASHI, and N. KATUNUMA TABLE I. Amounts of OAT bound to anti-OAT- glass rods. The experimental conditions were the same as in Fig. 1 except that one tenth volume of the medium for the first assay was used for the second assay. Values are the means of duplicate experiments. AAS: aminoalkylsilyl. OAT (fmol/assay tube)
fl-o-Galactosidase activity bound in the second assay (/iunits/rod)»
Free after the first assay
Antibody IgG-glass Glutaraldehyde-activated AAS Non-activated AAS Plain
68 220 385
31 71
93 69 29
Normal IgG-glass Glutaraldehyde-activated AAS Non-activated AAS Plain
443 430 440
110 98 108
0 2 0
First assay of 100 fmol of OAT with
7.0
Bound to the first glass
*• These values were used to calculate the amount of free OAT after the first assay from the corresponding curve in Fig. 1.
that less than 10 fmol of the antigen was almost completely bound to the glass rods used in the first assay. When 100 fmol of the antigen was used, one tenth volume of the incubation media used for the first assay was subjected to the second assay. From j9-r>galactosidase activities bound in the second assays and the corresponding curve in Fig. 1, the amounts of the antigen remaining free in the media after the first assays were determined and those values were subtracted from 100 fmol to give the amounts of the antigen bound in the first assays: 93, 69, and 29 fmol for the glutaraldehydeactivated aminoalkylsilyl, non-activated aminoalkylsilyl, and plain glass rods, respectively (Table I). Control experiments were performed in the same manner as described above except that the normal rabbit IgG-glass rods prepared in the three different manners described above were used for the first assays. The results are shown in Fig. 2. /9-D-GaIactosidase activity bound in the first assays did not significantly increase with increase in the amount of the antigen added, indicating the specificity of the present immunoassay. The activities bound in the second assays were similar to those in the first assay with anti-OAT-aminoalkylsiryl glass rods prepared with glutaraldehyde. These findings indicate that the antigen was not adsorbed non-specifically on the surface of test tubes and/or
600k 400
0.01
10
100
(fmol/assay tube) RAT ORNITHINE J-AMINOTRANSFERASE
Fig. 2. Sandwich immunoassay of crystalline OAT (B). The experimental conditions and symbols are the same as in Fig. 1 except that glass rods used for the first assays were loaded with normal rabbit IgG.
/ . Biochem.
IgG BOUND ON GLASS FOR IMMUNOASSAY
265
200
DISCUSSION Rabbit antibody IgG adsorbed non-specifically onto the aminoalkylsilyl or plain glass surface was shown to be practically useful for the sandwich immunoassay of macromolecular antigens. The antibody-glass can be readily prepared, easily washed and provide high sensitivities of the sandwich immunoassay. They can be re-used after treatment with NaOH and KiCr,O,-H,SO4. Experiments in this laboratory have shown that these glass rods are also useful for the competitive binding immunoassay of antigens and haptens (to be published). The inner surface of conventional glass test tubes may be used as solid phase. Rabbit antibody IgG bound on the glutaralml uL 0 6.01 0.1 1 10 100 dehyde-activated aminoalkylsilyl glass may be 2,4-DINITROPHENYL HUMAN IgG (fmol/assay tube) bound in part covalently but in part non-covalently. Fig. 3. Sandwich immunoassay of 2,4-dinitrophenyl Rabbit antibody IgG covalently bound must also human IgG. All glass rods used were loaded with anti serve for the immunoassay. However, rabbit anti(human IgG) and the anti(2,4-dinitrophenyl>/3-t>-galac- body IgG molecules bound covalently and nontosidase complex was used throughout. Closed circles, covalently may possess slightly different abilities glutaraldehyde-treated aminoalkylsilyl; open circles, to serve for immunoassay. As shown in Fig. 1, aminoalkylsilyl; closed triangles, glutaraldehyde-treated less than 10 fmol of OAT were almost completely plain; open triangles, plain. adsorbed, that is the same amount of the antigen was adsorbed on the antibody-glass rods prepared in different manners and, therefore, the amount of glass rods in the first assays and was fully recovered the complex bound should be the same. However, /3-D-galactosidase activities bound to these glass rods in the second assays and, therefore, validate the were slightly different. So, the antigen molecules above estimation of the amount of the antigen adsorbed on these glass rods may possess different adsorbed onto the glass rods. Applicability of the Antibody-Glass Rods to the abilities to bind the complex or the complex bound Sandwich Immunoassay—As shown in Fig. 1, 0.1 to may have different enzyme activities. Otherwise, 10 fmol of crystalline OAT were measurable with the antibody molecules bound with the antigen may all the glass rods prepared. The applicability of be desorbed to some extent during the washing the antibody-glass rods was further tested by using and/or the incubation with the complex. These a model antigen, 2,4-dinitrophenyl human IgG, possibilities remain open to further investigation. which contained 8.3 2, 4-dinitrophenyl residues per More than 10 fmol of the antigens tested were molecule. The model antigen was assayed by not measurable as shown in Figs. 1 and 3. The using the anti(human IgG>glass rods and the anti maximal amount of antigen that can be assayed is (2, 4-dinitrophenyl)-j9-r>-galactosidase complex. limited by two factors: the capacity of the solid As shown in Fig. 3, 0.1 to 10 fmol of the antigen phase to bind antigens and the amount of the comwas measurable with all the glass rods tested. Less plexes used. When 100 fmol of OAT was subthan 0.1 fmol of both antigens may be measurable jected to the sandwich immunoassay, 93, 69, and by careful assay. These findings indicate that 29 fmol were bound to the glutaraldehyde-activated rabbit antibody IgG bound onto glass surface by aminoalkylsilyl, non-activated aminoalkylsilyl, and simple adsorption can be generally used for the plain glass rods, respectively (Table I). The sandwich immunoassay of macromolecular anti- amount of anti-OAT-/3-r>galactosidase complex used was 1,650 ^units, that is, 31 fmol. (Assumgens. Vol. 82, No. 1, 1977
266 K. KATO, Y. HAMAGUCHI, S. OKAWA, E. ISHIKAWA, K. KOBAYASHI, and N. KATUNUMA
ing that /3-D-galactosidase of the complex bound to the antigen on the solid phase showed the same activity as that in the original preparation, 53 //units of the complex corresponds to 1 fmol). However, the maximal amount of the complex that can be bound appears to be about 14.6 fmol (775 punits), which was obtained when 100 fmol was subjected to the sandwich procedure (Fig. 1). This suggests that only 47% of the complex molecules prepared contained the Fab' fragments specific for the antigen. (The Fab' fragments specific for human IgG were present in 55% of the rabbit anti (human IgG) Fab'-/3-D-galactosidase complex molecules prepared in the same manner (6)). So, the maximal amount of the antigen that could be assayed was limited by the amount of the complex used. Experiments in this laboratory have shown that larger amounts of the antigens are measurable by increasing the amount of the complexes used. However, the non-specific binding of the complexes increases with increase of the amounts of the complexes used, limiting the sensitivity of the sandwich immunoassay. If the complex is prepared from purified mono-specific antibody, sensitivity may be improved and the maximal amount of antigen that can be assayed may be increased by using the same amount of the complex. 2,4-Dinitrophenyl human IgG was assayed with the anti(human IgG)-glass rods, since much less activity of ^-D-galactosidase was bound when the anti (2, 4-dinitrophenyl)-glass rods were used. Even with the anti(human IgG)-gIass rods, /9-Dgalactosidase activities bound were much lower than those in the assay of OAT. This suggests that only a small proportion of the 2, 4-dinitrophenyl residues coupled to human IgG may be available for binding with the antibody, although 2, 4-dinitrophenyl human IgG used contained 8.3 2, 4-dinitrophenyl residues per molecule. This is supported by the following calculation. Assuming that 53 //units of the complex corresponds to 1 fmol, as mentioned above, the numbers of the anti (2, 4-dinitrophenyl)-/3-D-gaIactosidase complex molecules bound are calculated to be 1.1, 0.43, and 0.18 per molecule of 2,4-dinitrophenyl human IgG added for the assay, when 0.1, 1.0, and 10 fmol respectively of the antigen were subjected to the sandwich immunoassay using the glutaraldehydeactivated glass (Fig. 3). These numbers are too small, since less than 1 fmol of 2, 4-dinitrophenyl
human IgG was almost completely bound to the solid phase, which was shown in the same manner as for OAT (Fig. 1). One reason for this may be that the distances between 2,4-dinitrophenyl residues and the amino groups of human IgG to which 2, 4-dinitrophenyl groups are coupled are too small for the binding with antibody (17). An appropriate spacer between 2,4-dinitrophenyl residues and amino groups of human IgG may be helpful to overcome this difficulty. Preliminary experiments in this laboratory have shown that more 2, 4-dinitrophenyl residues bind with antibody when N-2, 4-dinitrophenyl-e-aminocaproic acid is coupled to human IgG than when 2,4-dinitrophenyl sulfonic acid is coupled. We thank Ms. Setsuko Tahara and Ms. Yoko Sueishi for their secretarial and technical assistance. REFERENCES 1. Engvall, E. & Perlmann, P. (1972) / . Immunol. 109, 129-135 2. Belanger, L., Sylvestre, C, & Dufour, D. (1973) Clin. Chim. Acta 48, 15-18 3. Stimson, W.H. & Sinclair, J.M. (1974) FEBS Lett. 47, 190-192 4. Maiolini, R. & Masseyeff, R. (1975) /. Immunol Method. 8, 223-234 5. Kato, K., Hamaguchi, Y., Fukui, H., & Ishikawa, E. (1976) Eur. J. Biochem. 62, 285-292 6. Kato, K., Fukui, H., Hamaguchi, Y., & Ishikawa, E. (1976) / . Immunol. 116, 1554-1560 7. Hamaguchi, Y., Kato, K., Fukui, H., Shirakawa, I., Ishikawa, E., Kobayashi, K., & Katunuma, N. (1976) J. Biochem. 80, 895-898 8. Matsuzawa, T., Katsunuma, T., & Katunuma, N. (1968) Biochem. Biophys. Res. Commun. 32, 161-166 9. Layne, E. (1957) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 3, pp. 447-454, Academic Press, New York 10. Arnon, T. & Shapira, E. (1967) Biochemistry 6, 3942-3950 11. Levy, H.B. & Sober, H.A. (1960) Proc. Soc. Exp. Biol. Med. 103, 250-252 12. Palmer, J.L. & Nisonoff, A. (1974) Biochemistry 3, 863-869 13. Eisen, H.N., Carsten, M.E., & Belman, S. (1954) / . Immunol. 73, 296-308 14. Kekwick, R.A. (1940) Biochem. J. 34, 1248-1257 15. Robinson, P.J., Dunnill, P., & Lilly, M.D. (1971) Biochim. Biophys. Acta 242, 659-661 16. Woolen, J.W. & Walker, P.O. (1965) Clin. Chim. Acta 12, 647-658 17. Wilder, R.L., Green, G., & Schumaker, V.N. (1975) Immunochemistry 12, 49-54
/ . Biochem.
Use of Rabbit Antibody IgG Bound onto Plain and Aminoalkylsilyl Glass Surface for the Enzyme-Linked Sandwich Immunoassay Kanefusa KATO, Yoshitaka HAMAGUCHI, Sachiyo OKAWA, Eiji ISHIKAWA, Keiko KOBAYASHI,* and Nobuhiko KATLTNUMA* Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki-gun, Miyazaki 889-16, and *Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, Tokushima University, Tokushima, Tokushima 770 Received for publication, February 12, 1977
Rabbit antibody IgG was bound onto aminoalkylsilyl or plain glass rods by simple adsorption. For comparison, rabbit antibody IgG was also bound onto glutaraldehyde-activated aminoalkylsilyl glass rods. These antibody-glass rods were tested by the sandwich procedure using Fab' fragments of rabbit antibody conjugated with /3-D-gaIactosidase from Escherichia coli. The glutaraldehyde-activated aminoalkylsilyl glass showed the largest capacity to bind antigen and the plain glass showed the smallest. However, the antibody-glass rods prepared by simple adsorption were as useful for the sandwich immunoassay of macromolecular antigens as those prepared with glutaraldehyde. With all the antibody-glass rods prepared, 0.1 to 10 fmol of ornithine 3-aminotransferase from rat liver and 2,4-dinitrophenyl human IgG were measurable. More than 10 fmol of the antigens may be measurable with larger amounts of the antibody^-D-galactosidase complexes, although the non-specific binding of the complexes to the solid phase increases to limit the sensitivity of the immunoassay.
Engvall and Perlmann have developed the enzymelinked immunoassay of antibodies ( 7 ) . Subsequently, the sandwich technique has been developed for the enzyme-linked immunoassay of macromolecular antigens. Enzymes used for labelling antibodies are alkaline phosphatase [orthophosphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] (1, 2), peroxidase [donor: hydrogen. . „ Abbreviations: IgG, immunoglobulin G; Fab, mono, . , .' , . . T -, T, ~ valent fragments derived from pepsin-treated IgG; Buffer A, 0.01 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl, 1 mM MgCI,, 0.1% NaN,, and 0.1% bovine serum albumin; OAT, ornithine 3-aminotransferase from rat liver. Vol. 82, No. 1, 1977
peroxide oxidoreductase, EC 1.11.1.7] (j), glucose oxidase [/9-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4] (4), and ^-D-galactosidase [/3-r>galactoside galactohydrolase, EC 3.2.1.23] (5). Antibodies have been labelled with enzymes using glutaraldehyde ( 2 - 4 ) or N, N'-ophenylenedimaleimide (5). The solid phases used are polystyrene tubes (2, 3), cellulose (4), and Sepharose 4B (5). We have developed a novel method for the conjugation of Fab' fragments of rabbit IgG with . , ., . „ , . ,. ,. fl-D-galactosidase from Escherichia coli and have r °~ demonstrated that 0.3 fmol of human IgG is measurable using Fab' fragments of purified rabbit anti(human IgG) antibody conjugated with yS-Dgalactosidase and rabbit anti(human IgG) IgG
261
262 K. KATO, Y. HAMAGUCHI, S. OKAWA, E. ISHIKAWA, K. KOBAYASHI, and N. KATUNUMA coupled to Sepharose 4B (6). We have recently used rabbit antibody bound onto aminoalkylsilyl glass treated with glutaraldehyde and have shown that the assay with the glass is more sensitive, simpler and more reproducible than that with Sepharose 4B (7). In subsequent studies, however, we have found that antibody can be bound onto glass surface by simple adsorption for the immunoassay. The present paper shows that a highly sensitive sandwich immunoassay of macromolecular antigens is possible with rabbit antibody IgG bound onto aminoalkylsilyl or plain glass by simple adsorption as well as with the antibody bound onto glutaraldehyde-activated aminoalkylsilyl glass.
Biological Laboratories Ltd. (Nagoya) and from Miles Laboratories, Inc. (Kankakee), respectively. The IgG fractions from these antisera and normal rabbit serum were prepared using NatSO4 (14) and DEAE-cellulose (11). Binding of Rabbit IgG onto Class Rods— Rabbit IgG was bound onto the glass surface in four different manners. Binding with glutaraldehyde was performed by the method of Robinson et al. (15) as described previously (7). Pyrex glass rods (3 mm in diameter and 5 mm in length) were aminoalkylated with 3-aminopropyltriethoxysilane (Nakarai Chemicals Ltd., Kyoto). The aminoalkylsilyl glass rods were activated with 1 % glutaraldehyde (Nakarai Chemicals Ltd., Kyoto), immersed in 0.25 M sodium phosphate buffer (pH 7.5) containing MATERIALS AND METHODS 0.1 mg/ml of IgG fraction from either rabbit antiAntigens and Antibodies—Crystalline ornithine sera or normal serum for 30 min, and stored at 4°C d-aminotransferase (OAT) [L-ornithine: 2-oxoacid overnight. The rabbit IgG-glass rods were aminotransferase, EC 2.6.1.13] from rat liver and washed with 0.01 M sodium phosphate buffer (pH rabbit antisera against the crystalline enzyme were 7.0) containing 0.1M NaCl, 1 mM MgCl,, 0.1% prepared as described previously (8). The crystal- NaN,, and 0.1 % bovine serum albumin (Buffer A) line enzyme preparation was homogenous on both and stored in Buffer A at 4°C until use. The glass ultracentrifugation and electrophoresis and could rods were washed several times during the storage be stored in 0.1 M sodium phosphate buffer (pH before use. For the binding by simple adsorption, 7.0) at —20°C without loss of activity for at least plain or aminoalkylsilyl glass rods were treated 6 months. Amounts in fig of the crystalline with rabbit IgG fractions in the same manner except enzyme were determined with Folin-Ciocalteu re- that the activation with glutaraldehyde was omitted. agent (9) using bovine serum albumin (Fraction V, As a control, plain glass rods were also treated with Armour Pharmaceutical Co., Chicago) as a stand- glutaraldehyde and then with rabbit IgG fractions ard, and amounts in fmoles were calculated by as described above. taking its molecular weight as 170,000 (8). The Rabbit Antibody (Fab')-f3-D-Galactosidase IgG fraction from the antiserum against the crystal- Complex—The rabbit antibody (Fab>/9-r>galactoline enzyme was obtained by fractionation with sidase complexes were prepared as described pre(NH4),SO4 (10) followed by passage through a viously (6). The Fab' fragments of rabbit antibody column of DEAE-cellulose (DE-52, Whatman Bio- containing sulfhydryl groups were treated with chemicals Ltd., Kent) (11). Human IgG was excess N, N'-o-phenylenedimaleimide (Aldrich obtained from Miles Laboratories, Inc. (Kankakee) Chemicals Company, Inc., Milwaukee) to introduce and its amounts were determined from the absor- maleimide residues. The maleimide residues of bance at 280 nm using the value of £ ^ = 1 5 (12). the dimaleimide-treated Fab' fragments were allow2, 4-Dinitrophenyl human IgG was prepared using ed to react with the sulfhydryl groups of fi-D2,4-dinitrophenyl sulfonic acid (sodium salt) galactosidase from Escherichia coli (Boehringer (Tokyo Kasei Kogyo Co., Ltd., Tokyo) by the Mannheim, Mannheim) to give the complexes. method of Eisen et al. (13). The number of 2, 4- Amounts of the complexes are expressed as units dinitrophenyl residues coupled to human IgG of /3-D-galactosidase activity, one unit being demolecules and the amount of 2, 4-dinitrophenyl fined as the amount that hydrolyzes one fimole of human IgG were calculated from the absorbance 4-methylumbelliferyI-/?-D-galactoside (Sigma Chemat 280 and 360 nm (12,13). The rabbit antisera ical Co., St. Louis) per min under the conditions against human IgG and 2, 4-dinitrophenyl bovine described previously (7). serum albumin were obtained from Medical and Sandwich Procedure—Each of the IgG-glass /. Biochem.
IgG BOUND ON GLASS FOR IMMUNOASSAY rods was incubated in a volume of 0.15 ml of Buffer A, containing various amounts of antigen, with shaking at 37°C for 4 h and the incubation mixtures were allowed to stand at 4°C overnight. Each rod was washed twice with 1 ml of Buffer A, and was incubated with the antibody-/3-D-galactosidase complex in 0.15 ml of Buffer A with shaking at 37°C for 6 h. (The binding to the antibody-glass rods of the antigens and the complexes used were almost maximal within 4 and 6 h, respectively.) Each rod was then washed twice with 1 ml of Buffer A and was transferred to another test tube which was free of ^-D-galactosidase. /S-D-Galactosidase activity bound to each rod was assayed using 4methylumbelliferyl-/3-D-galactoside as substrate (16) as described previously (7). The amounts of the complexes used for the assay of OAT and 2, 4-dinitrophenyl human IgG were 1,650 and 2,050 punits, respectively. These amounts of the complexes were chosen so that the ratios of j9-r>galactosidase activity bound to the solid phase in the presence of the antigens (10 fmol) to that in their absence were maximal. Temperature of Experiments—Unless otherwise specified, experiments were carried out at 20 to 25°C throughout.
263
4. 0 0.01
100
(fmol/assay tube) RAT ORNITHINE J-AMINOTRANSFERASE
Fig. 1. Sandwich immunoassay of crystalline OAT (A). Closed circles, triangles, and squares indicate the first assays with glutaraldehyde-activated aminoalkylsilyl, non-activated aminoalkylsilyl, and plain glass rods, respectively. Open symbols indicate the corresponding second assays, all of which were performed with glutarRESULTS aldehyde-activated aminoalkylsilyl glass rods. All glass Amounts of Antigen Adsorbed on the Antibody- rods used were loaded with anti-OAT.
Glass Rods—The antibody-glass rods were prepared in three different manners. Rabbit IgG from anti-OAT serum was bound onto the aminoalkylsilyl or plain glass rods by simple adsorption. For comparison, the same IgG was bound onto the aminoalkylsilyl glass rods activated with glutaraldehyde. These glass rods were tested by the sandwich procedure with the Fab' fragments of anti-OAT antibody conjugated with ^-D-galactosidase. The activity of /3-D-galactosidase bound to the glutaraldehyde-activated aminoalkylsilyl glass rods was the highest and the activity bound to the plain glass rods was the lowest (Fig. 1). These findings suggest that different amounts of IgG were bound onto the glass rods prepared in different manners. However, amounts of IgG bound onto the glass rods were unknown, since the change in the concentration of IgG before and after the treatment of glass rods was too small to determine precisely from the absorbance at 280 nm.
Vol. 82, No. 1, 1977
The amount of IgG bound onto the glass rods must be related to the amount of antigen that can be adsorbed. So, the amount of antigen that can be adsorbed on each of the glass rods was determined in the following manner. Various amounts (0.1 to 100 fmol) of crystalline OAT were subjected to the sandwich immunoassay with anti-OAT-glass rods prepared in different manners (the first assay) and the incubation media used for the first assay or portions of them were again subjected, after removing the glass rods used for the first assay, to the sandwich immunoassay using anti-OAT-aminoalkylsilyl glass rods prepared with glutaraldehyde (the second assay). The results are shown in Fig. 1 and Table I. £-r>Galactosidase activities bound in the second assay did not increase very much with any glass rod tested when less than 10 fmol of the antigen was subjected to the first assay (Fig. 1). This indicates
264 K. KATO, Y. HAMAGUCHI, S. OKAWA, E. ISHIKAWA, K. KOBAYASHI, and N. KATUNUMA TABLE I. Amounts of OAT bound to anti-OAT- glass rods. The experimental conditions were the same as in Fig. 1 except that one tenth volume of the medium for the first assay was used for the second assay. Values are the means of duplicate experiments. AAS: aminoalkylsilyl. OAT (fmol/assay tube)
fl-o-Galactosidase activity bound in the second assay (/iunits/rod)»
Free after the first assay
Antibody IgG-glass Glutaraldehyde-activated AAS Non-activated AAS Plain
68 220 385
31 71
93 69 29
Normal IgG-glass Glutaraldehyde-activated AAS Non-activated AAS Plain
443 430 440
110 98 108
0 2 0
First assay of 100 fmol of OAT with
7.0
Bound to the first glass
*• These values were used to calculate the amount of free OAT after the first assay from the corresponding curve in Fig. 1.
that less than 10 fmol of the antigen was almost completely bound to the glass rods used in the first assay. When 100 fmol of the antigen was used, one tenth volume of the incubation media used for the first assay was subjected to the second assay. From j9-r>galactosidase activities bound in the second assays and the corresponding curve in Fig. 1, the amounts of the antigen remaining free in the media after the first assays were determined and those values were subtracted from 100 fmol to give the amounts of the antigen bound in the first assays: 93, 69, and 29 fmol for the glutaraldehydeactivated aminoalkylsilyl, non-activated aminoalkylsilyl, and plain glass rods, respectively (Table I). Control experiments were performed in the same manner as described above except that the normal rabbit IgG-glass rods prepared in the three different manners described above were used for the first assays. The results are shown in Fig. 2. /9-D-GaIactosidase activity bound in the first assays did not significantly increase with increase in the amount of the antigen added, indicating the specificity of the present immunoassay. The activities bound in the second assays were similar to those in the first assay with anti-OAT-aminoalkylsiryl glass rods prepared with glutaraldehyde. These findings indicate that the antigen was not adsorbed non-specifically on the surface of test tubes and/or
600k 400
0.01
10
100
(fmol/assay tube) RAT ORNITHINE J-AMINOTRANSFERASE
Fig. 2. Sandwich immunoassay of crystalline OAT (B). The experimental conditions and symbols are the same as in Fig. 1 except that glass rods used for the first assays were loaded with normal rabbit IgG.
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IgG BOUND ON GLASS FOR IMMUNOASSAY
265
200
DISCUSSION Rabbit antibody IgG adsorbed non-specifically onto the aminoalkylsilyl or plain glass surface was shown to be practically useful for the sandwich immunoassay of macromolecular antigens. The antibody-glass can be readily prepared, easily washed and provide high sensitivities of the sandwich immunoassay. They can be re-used after treatment with NaOH and KiCr,O,-H,SO4. Experiments in this laboratory have shown that these glass rods are also useful for the competitive binding immunoassay of antigens and haptens (to be published). The inner surface of conventional glass test tubes may be used as solid phase. Rabbit antibody IgG bound on the glutaralml uL 0 6.01 0.1 1 10 100 dehyde-activated aminoalkylsilyl glass may be 2,4-DINITROPHENYL HUMAN IgG (fmol/assay tube) bound in part covalently but in part non-covalently. Fig. 3. Sandwich immunoassay of 2,4-dinitrophenyl Rabbit antibody IgG covalently bound must also human IgG. All glass rods used were loaded with anti serve for the immunoassay. However, rabbit anti(human IgG) and the anti(2,4-dinitrophenyl>/3-t>-galac- body IgG molecules bound covalently and nontosidase complex was used throughout. Closed circles, covalently may possess slightly different abilities glutaraldehyde-treated aminoalkylsilyl; open circles, to serve for immunoassay. As shown in Fig. 1, aminoalkylsilyl; closed triangles, glutaraldehyde-treated less than 10 fmol of OAT were almost completely plain; open triangles, plain. adsorbed, that is the same amount of the antigen was adsorbed on the antibody-glass rods prepared in different manners and, therefore, the amount of glass rods in the first assays and was fully recovered the complex bound should be the same. However, /3-D-galactosidase activities bound to these glass rods in the second assays and, therefore, validate the were slightly different. So, the antigen molecules above estimation of the amount of the antigen adsorbed on these glass rods may possess different adsorbed onto the glass rods. Applicability of the Antibody-Glass Rods to the abilities to bind the complex or the complex bound Sandwich Immunoassay—As shown in Fig. 1, 0.1 to may have different enzyme activities. Otherwise, 10 fmol of crystalline OAT were measurable with the antibody molecules bound with the antigen may all the glass rods prepared. The applicability of be desorbed to some extent during the washing the antibody-glass rods was further tested by using and/or the incubation with the complex. These a model antigen, 2,4-dinitrophenyl human IgG, possibilities remain open to further investigation. which contained 8.3 2, 4-dinitrophenyl residues per More than 10 fmol of the antigens tested were molecule. The model antigen was assayed by not measurable as shown in Figs. 1 and 3. The using the anti(human IgG>glass rods and the anti maximal amount of antigen that can be assayed is (2, 4-dinitrophenyl)-j9-r>-galactosidase complex. limited by two factors: the capacity of the solid As shown in Fig. 3, 0.1 to 10 fmol of the antigen phase to bind antigens and the amount of the comwas measurable with all the glass rods tested. Less plexes used. When 100 fmol of OAT was subthan 0.1 fmol of both antigens may be measurable jected to the sandwich immunoassay, 93, 69, and by careful assay. These findings indicate that 29 fmol were bound to the glutaraldehyde-activated rabbit antibody IgG bound onto glass surface by aminoalkylsilyl, non-activated aminoalkylsilyl, and simple adsorption can be generally used for the plain glass rods, respectively (Table I). The sandwich immunoassay of macromolecular anti- amount of anti-OAT-/3-r>galactosidase complex used was 1,650 ^units, that is, 31 fmol. (Assumgens. Vol. 82, No. 1, 1977
266 K. KATO, Y. HAMAGUCHI, S. OKAWA, E. ISHIKAWA, K. KOBAYASHI, and N. KATUNUMA
ing that /3-D-galactosidase of the complex bound to the antigen on the solid phase showed the same activity as that in the original preparation, 53 //units of the complex corresponds to 1 fmol). However, the maximal amount of the complex that can be bound appears to be about 14.6 fmol (775 punits), which was obtained when 100 fmol was subjected to the sandwich procedure (Fig. 1). This suggests that only 47% of the complex molecules prepared contained the Fab' fragments specific for the antigen. (The Fab' fragments specific for human IgG were present in 55% of the rabbit anti (human IgG) Fab'-/3-D-galactosidase complex molecules prepared in the same manner (6)). So, the maximal amount of the antigen that could be assayed was limited by the amount of the complex used. Experiments in this laboratory have shown that larger amounts of the antigens are measurable by increasing the amount of the complexes used. However, the non-specific binding of the complexes increases with increase of the amounts of the complexes used, limiting the sensitivity of the sandwich immunoassay. If the complex is prepared from purified mono-specific antibody, sensitivity may be improved and the maximal amount of antigen that can be assayed may be increased by using the same amount of the complex. 2,4-Dinitrophenyl human IgG was assayed with the anti(human IgG)-glass rods, since much less activity of ^-D-galactosidase was bound when the anti (2, 4-dinitrophenyl)-glass rods were used. Even with the anti(human IgG)-gIass rods, /9-Dgalactosidase activities bound were much lower than those in the assay of OAT. This suggests that only a small proportion of the 2, 4-dinitrophenyl residues coupled to human IgG may be available for binding with the antibody, although 2, 4-dinitrophenyl human IgG used contained 8.3 2, 4-dinitrophenyl residues per molecule. This is supported by the following calculation. Assuming that 53 //units of the complex corresponds to 1 fmol, as mentioned above, the numbers of the anti (2, 4-dinitrophenyl)-/3-D-gaIactosidase complex molecules bound are calculated to be 1.1, 0.43, and 0.18 per molecule of 2,4-dinitrophenyl human IgG added for the assay, when 0.1, 1.0, and 10 fmol respectively of the antigen were subjected to the sandwich immunoassay using the glutaraldehydeactivated glass (Fig. 3). These numbers are too small, since less than 1 fmol of 2, 4-dinitrophenyl
human IgG was almost completely bound to the solid phase, which was shown in the same manner as for OAT (Fig. 1). One reason for this may be that the distances between 2,4-dinitrophenyl residues and the amino groups of human IgG to which 2, 4-dinitrophenyl groups are coupled are too small for the binding with antibody (17). An appropriate spacer between 2,4-dinitrophenyl residues and amino groups of human IgG may be helpful to overcome this difficulty. Preliminary experiments in this laboratory have shown that more 2, 4-dinitrophenyl residues bind with antibody when N-2, 4-dinitrophenyl-e-aminocaproic acid is coupled to human IgG than when 2,4-dinitrophenyl sulfonic acid is coupled. We thank Ms. Setsuko Tahara and Ms. Yoko Sueishi for their secretarial and technical assistance. REFERENCES 1. Engvall, E. & Perlmann, P. (1972) / . Immunol. 109, 129-135 2. Belanger, L., Sylvestre, C, & Dufour, D. (1973) Clin. Chim. Acta 48, 15-18 3. Stimson, W.H. & Sinclair, J.M. (1974) FEBS Lett. 47, 190-192 4. Maiolini, R. & Masseyeff, R. (1975) /. Immunol Method. 8, 223-234 5. Kato, K., Hamaguchi, Y., Fukui, H., & Ishikawa, E. (1976) Eur. J. Biochem. 62, 285-292 6. Kato, K., Fukui, H., Hamaguchi, Y., & Ishikawa, E. (1976) / . Immunol. 116, 1554-1560 7. Hamaguchi, Y., Kato, K., Fukui, H., Shirakawa, I., Ishikawa, E., Kobayashi, K., & Katunuma, N. (1976) J. Biochem. 80, 895-898 8. Matsuzawa, T., Katsunuma, T., & Katunuma, N. (1968) Biochem. Biophys. Res. Commun. 32, 161-166 9. Layne, E. (1957) in Methods in Enzymology (Colowick, S.P. & Kaplan, N.O., eds.) Vol. 3, pp. 447-454, Academic Press, New York 10. Arnon, T. & Shapira, E. (1967) Biochemistry 6, 3942-3950 11. Levy, H.B. & Sober, H.A. (1960) Proc. Soc. Exp. Biol. Med. 103, 250-252 12. Palmer, J.L. & Nisonoff, A. (1974) Biochemistry 3, 863-869 13. Eisen, H.N., Carsten, M.E., & Belman, S. (1954) / . Immunol. 73, 296-308 14. Kekwick, R.A. (1940) Biochem. J. 34, 1248-1257 15. Robinson, P.J., Dunnill, P., & Lilly, M.D. (1971) Biochim. Biophys. Acta 242, 659-661 16. Woolen, J.W. & Walker, P.O. (1965) Clin. Chim. Acta 12, 647-658 17. Wilder, R.L., Green, G., & Schumaker, V.N. (1975) Immunochemistry 12, 49-54
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