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Enhancement of immunoblot staining using a mixed chromogenic substrate
Journal of Immunological Methods, 121 (1989) 295-296
295
Elsevier JIM05266
L e t t e r to the editors
Enhancement of immunoblot staining using a mixed chromogenic substrate Paul R. Y o u n g Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. (Received 24 November 1988, revised received 13 March 1989, accepted 20 April 1989)
Dear Editors, Many modifications to the basic technique of immunoblotting have been described since it was first introduced by Towbin et al. in 1979. These have primarily focused on the immunological detection stage and generally involve the use of amplification steps employing reagents such as peroxidase-anti-peroxidase (PAP) conjugates, the biotin-avidin/streptavidin complex and colloidal gold with silver enhancement (Glass II et al., 1981; Brada and Roth, 1983; Wilchek and Bayer, 1984). Regardless of which method is employed, final image development is dependent in most cases on the generation of an insoluble coloured product from the reaction of an enzyme conjugate (usually horseradish peroxidase, HRP) with a suitable substrate. The two most frequently used substrates for H R P are 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 4-chloro-l-naphthol (CN). The former provides reasonable detection sensitivity, but poor photographic reproduction owing to a low contrast brown reaction product, while the latter generates a blue product of high contrast ideal for reproduction but of relatively low sensitivity. Many authors have commented on increases in detection sensitivity obtained with alkaline phosphatase (AP) when compared to H R P and have often attributed this finding to an improvement in the solubility of the substrates. While
Correspondence to: P.R. Young, Sir Albert Sakzewski Virus Research Laboratory, The Royal Children's Hospital, Herston Road, Brisbane 4029, Australia.
the solubility of DAB in aqueous solutions is indeed poor, often requiring pre-filtration before use, dissolving this substrate completely in methanol prior to addition to the reaction buffer overcomes this problem and results in detection levels approaching those of AP substrates. The simplistic assumption that mixing the two H R P substrates, DAB and CN, might achieve both high sensitivity and improved contrast has been investigated in experiments which involved the use of these chromogenic compounds either alone or in combination (CND) to develop immunoblots reacted with both polyclonal and monoclonal antibodies and appropriate peroxidase-conjugated probes. The C N D substrate reaction mix comprised: 10 mg DAB dissolved in 5 ml methanol (requiring stirring for 10-15 min) and 30 mg CN dissolved in a separate 5 ml methanol. Both solutions were then mixed with 40 ml PBS together with 10/~1 30% H202 just prior to use. Not only was the product of the C N D reaction found to be of higher contrast (a strong black precipitate was formed) but an apparent synergistic effect between the two substrates resulted in greatly improved sensitivity levels. In many cases, most notably with the monoclonal antibodies, a greater than ten-fold improvement in detection sensitivity was found when compared with blots developed with either substrate alone. The increased detection sensitivities obtained with C N D on immunoblots involves no additional 'enhancement' steps in the procedure, the substrates are inexpensive and the product of the reaction is black, making it ideal for photographic reproduction. Furthermore, its use is not restricted
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
296
solely to immunoblots, being equally applicable to immunocytochemical procedures.
References Brada, D. and Roth, J. (1983) 'Golden blot' - Detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes. Anal. Biochem. 142, 79-83.
Glass II, W.F., Briggs, R.C. and Hnmilica, L.S. (1981) Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels. Science 211, 70-72. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. Wilchek, M. and Bayer, E.A. (1984) The avidin-biotin complex in immunology. Immunol. Today 5, 39-43.
295
Elsevier JIM05266
L e t t e r to the editors
Enhancement of immunoblot staining using a mixed chromogenic substrate Paul R. Y o u n g Department of Medical Microbiology, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, U.K. (Received 24 November 1988, revised received 13 March 1989, accepted 20 April 1989)
Dear Editors, Many modifications to the basic technique of immunoblotting have been described since it was first introduced by Towbin et al. in 1979. These have primarily focused on the immunological detection stage and generally involve the use of amplification steps employing reagents such as peroxidase-anti-peroxidase (PAP) conjugates, the biotin-avidin/streptavidin complex and colloidal gold with silver enhancement (Glass II et al., 1981; Brada and Roth, 1983; Wilchek and Bayer, 1984). Regardless of which method is employed, final image development is dependent in most cases on the generation of an insoluble coloured product from the reaction of an enzyme conjugate (usually horseradish peroxidase, HRP) with a suitable substrate. The two most frequently used substrates for H R P are 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 4-chloro-l-naphthol (CN). The former provides reasonable detection sensitivity, but poor photographic reproduction owing to a low contrast brown reaction product, while the latter generates a blue product of high contrast ideal for reproduction but of relatively low sensitivity. Many authors have commented on increases in detection sensitivity obtained with alkaline phosphatase (AP) when compared to H R P and have often attributed this finding to an improvement in the solubility of the substrates. While
Correspondence to: P.R. Young, Sir Albert Sakzewski Virus Research Laboratory, The Royal Children's Hospital, Herston Road, Brisbane 4029, Australia.
the solubility of DAB in aqueous solutions is indeed poor, often requiring pre-filtration before use, dissolving this substrate completely in methanol prior to addition to the reaction buffer overcomes this problem and results in detection levels approaching those of AP substrates. The simplistic assumption that mixing the two H R P substrates, DAB and CN, might achieve both high sensitivity and improved contrast has been investigated in experiments which involved the use of these chromogenic compounds either alone or in combination (CND) to develop immunoblots reacted with both polyclonal and monoclonal antibodies and appropriate peroxidase-conjugated probes. The C N D substrate reaction mix comprised: 10 mg DAB dissolved in 5 ml methanol (requiring stirring for 10-15 min) and 30 mg CN dissolved in a separate 5 ml methanol. Both solutions were then mixed with 40 ml PBS together with 10/~1 30% H202 just prior to use. Not only was the product of the C N D reaction found to be of higher contrast (a strong black precipitate was formed) but an apparent synergistic effect between the two substrates resulted in greatly improved sensitivity levels. In many cases, most notably with the monoclonal antibodies, a greater than ten-fold improvement in detection sensitivity was found when compared with blots developed with either substrate alone. The increased detection sensitivities obtained with C N D on immunoblots involves no additional 'enhancement' steps in the procedure, the substrates are inexpensive and the product of the reaction is black, making it ideal for photographic reproduction. Furthermore, its use is not restricted
0022-1759/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
296
solely to immunoblots, being equally applicable to immunocytochemical procedures.
References Brada, D. and Roth, J. (1983) 'Golden blot' - Detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein A-gold complexes. Anal. Biochem. 142, 79-83.
Glass II, W.F., Briggs, R.C. and Hnmilica, L.S. (1981) Identification of tissue-specific nuclear antigens transferred to nitrocellulose from polyacrylamide gels. Science 211, 70-72. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. Wilchek, M. and Bayer, E.A. (1984) The avidin-biotin complex in immunology. Immunol. Today 5, 39-43.