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Enhanced enzyme-linked immunosorbant assay on membranes for the identification of mutants and pathogens
Life Sciences, Vol. 49, pp. 865-868 Printed in the U.S.A.
Pergamon Press
ENHANCED ENZYME-LINKED IMMUNOSORBANT ASSAY ON MEMBRANES FOR THE IDENTIFICATION OF MUTANTS AND PATHOGENS Matthew E. May and Carl W. Vermeulen* Department of Biology, The College of William and Mary, Williamsburg, Virginia 23185 USA (Received in final form July 16, 1991) Summa~
With variations in the concentrations of antibodies and blocking agents and reduction in incubation times, qualitative enzyme-linked immunosorbant assay can be performed in a matter of two hours, for more rapid identification of mutants and antigens. The usual enzyme-linked immunosorbant assay techniques, ELISA, as done on membranes (1) are too slow for many biochemical research laboratories. For example, after some variety of electrophoresis such as detergent augmented polyacrylamide gel electrophoresis, SDS-Page (2,3,4) or agarose electrophoresis of surface antigens (5), there are several electroblotting techniques (6,7) that could use highly specific immunochemical visualization, in lieu of less specific staining of gels with dyes such as 'silver stain' (8,9). Similarly, in the clinical field the potential for highly specific immunochemical analysis would prove to be an invaluable diagnostic tool for the identification of pathogens such as S a l m o n e l l a , or for E. coli, of which a few strains cause a great deal of diarrheal disease. In order to speed up the procedure we modified concentrations and reaction times of the prevailing membrane ELISA procedure, and have tested these modifications on both the ability to identify colonies, as well as to identify bacterial smears. In general we did the following: concentrations of blocking agents were increased by 30% such that saturation of non-specific protein binding sites was more rapidly attained. Similarly, when the concentration of primary antibody was increased 5-fold, binding of antibody to the protein complex is also accelerated. We found that bacterial smears that had been cultivated on nutrient agar for only 1 hour at 37.5c gave positive tests. Finally, by eliminating the overnight, secondary, conjugated antibody binding step in the prevailing procedure, as well as reductions of all remaining steps by at least half, brought the total time for ELISA to under two hours. Thus, ELISA identification of bacterial surface antigens has been shortened from approximately 30 hours to about 2 hours. The benefits of shortening this powerful biochemical tool should be obvious, especially since it has been found that even sectors of colonies are identifiable (10), and that several different secondary conjugated antibodies might be used to give a variety of colors (11) for simultaneous multitests. Malerials and Methods Bacteriology. Various concentrations of clinically derived E s c h e r i c h i a c o l i were used to analyze the sensitivity of the shortcnded procedure. E. c o l i E412 (R:KI:H?) (12,13), E. coli 'Bort' (O18ac:K1:H7) (14), and various clinical strains sensitive to Kl-specific phage eK1E (15) were plated and incubated for one hour at 37.5c. E. coli E457 (O6:K5:H?) (12,13) was used as the negative control. All axenic cultures were 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
866
Rapid Identification of Mutants & Pathogens
Vol. 49, No. 12, 1991
grown on nutrient agar, or in L-broth (0.3% glucose, 0.7% tryptone) at 37.5c. For mixed cultures, human fecal samples, doped with Kl-encapsulated E. ¢oli E412, were also tested. Sampling. Four types of samplings were made. (a) In the first, we were interested in noting the lower limit of detection using the 'dip test.' A nitrocellulose, NC, strip was dipped into broth cultures of known bacterial concentration. (b) To ascertain the earliest that colonies could be identified, serial dilutions were plated and incubated at 37.5c. At one hour intervals, narrow strips of NC membrane were then laid across the surface of the agar upon which the colonies were growing. (c) With regard to fecal samples, a 10-fold dilution was swabbed onto half of an agar plate and incubated at 37.5c. Hourly, NC strips were laid across the agar bridging both the smeared and the unsmeared sectors. In all cases, the NC strips were then dried in the incubator for 5 minutes or more at 37.5c before being subjected to ELISA. ELISA. The bacteria-treated NC strips were then placed in 2.5 ml of washing buffer (10% skim milk, the blocking agent, and 90% distilled water) at 18c. We then added 1.0 ml more cold skim milk and allowed the solution to gently shake at 65 rpm for 30 minutes to saturate protein binding sites on the membranes. This liquid was then decanted. Then 2.5 ml of washing buffer (18c) was added and immediately decanted to rid the membrane of excess blocking agent. This step was repeated once again. We then added 2.5 ml of washing buffer (18c) containing 5 I.tl of ascites fluid at 4c (murine m o n o c l o n a l - I g M against m c n i n g o c o c c a l B p o l y sacch ar i d e, which is identical to E_...coli K1 capsule (16,17,18,19)), as the primary antibody, and shaken for 15 minutes. The solution was then refrigerated for an additional 15 minutes at 65 rpm to allow primary antibody to attach to K1 of the relevant bacterial surface antigens. This solution was then replaced with 2.5 ml of 18c washing buffer containing 1 I.tl of HRP::secondary antibody (goat/anti-mouse IgM antibody conjugated with horse-radish peroxidase, from Kirkegaard and Perry, Inc., Gaithersburg, MD) at 5c, and swirled this for 30 minutes at 65 rpm. The membrane was then twice washed with 2.5 ml of 18c washing buffer and twice placed on the shaker for 1 minute at 65 rpm. The NC was then placed into 2.5 ml acetate buffer (50 mM sodium acetate, pH 5.0) at room temperature to rid the membrane of excess washing buffer, and allowed to shake for 1 minute at 65 rpm. We then developed the membrane, again at room temperature, with the peroxidase/membrane ELISA reagents according to methods and products of Kirkegaard and Perry, Inc. In this method the membrane is placed in enough 10c developer (equal volumes of 4-chloro-l-naphthol (1.4 g/l in an organic base) and H 2 0 2 (0.02% in citric acid buffer) to cover the membrane and swirled at 75 rpm until purple bands appeared. Development was stopped by decanting developer and adding 22c distilled water. The membranes were then dried and stored at room temperature in the dark as the pigment producted is photolabilc. Resuhs In the dip test, using serial dilutions of an overnight culture adjusted to I x l 0 9 cfu/ml, we found that positive results were directly obtainable to as low as 3x103 to lxl04cfu/ml. In the case where a dilute suspension of bacteria was spread upon agar, colonies of K l - p o s s e s s i n g E__~.coli strains clearly were discernible from those which did not possess KI capsules. Fecal bacteria possessing the proper antigen swabbing a 100-fold dilution of stool sample. Concomitantly with the attachment of the should remain in the for at least 30 minutes.
were detectable within
one hour of
the above, we found Ihat there is a minimum time period for primary antibody to lhe substrate antigen. The membrane solution containing the primary antibody and washing buffer The solution should bc swirled for 15 minutes and then be
Vol. 49, No. 12, 1991
Rapid Identification of Mutants & Pathogens
placed at 4c for another 15 minutes. The procedure done without both of these will give erratic results with very low concentrations of bacteria. Incubation for the bacteria was also reduced from overnight to 1 hour since by then there sufficient growth to give a positive reaction on the membrane. Moreover, we found that this new procedure, used with other polyclonal antibody preparations as a Bort-O18 rabbit antibody preparation, is just as effective under the parameters.
867
steps time was also such same
Dis¢~lssi0n The arsenals of biochemists and clinicians must be stocked with the greatest variety of methods available in order that a right one might be applied to the problem at hand. In the overlapping realms of biochemistry, molecular biology and molecular genetics, selection of successfully transfcctcd mutants that are producing important biological components is critical. Detection of the desired mutant colony from among backgrounds of millions of unwanted colonies is often a major hurdle that requires a great deal of ingenuity. While an accelerated ELISA method may not be absolutely necessary for this pursuit of the rare mutant or variant, we know of no scientist who would willingly choose a 20 to 30-hour procedure over an equally discriminating 2-hour method. In the clinical and veterinarian arenas, while time is often of the critical essence, it is even more important that the clinician be able to discern whether or not a specific pathogen is present. Here for the sake of choosing a model of growing medical interest, we have chosen diarrhea (5,20,21). Currently no American clinical laboratory routinely assays the serotype make-up of the E . ¢ o l i population in stool samples. This disease is especially critical in infants who become rapidly dehydrated. This lack of testing is presumably because the prevailing method for identification of E . ¢ o l i is a long and tedious process. Our method can detect almost immediately any strain or mixture of strains (using a mix of relevant primary antibodies). Of particular interest in the assay for bacteruria, which is noted as 'positive' when the bacterial count is greater than l x l 0 4 cfu/ml, the sensitivity of our dip test falls within this range. Since only a few pathogenic serotypes are very common, such ELISA testing now holds promise of more rapid diagnosis. Alluded to earlier, antibody mixes could be used so that different conjugated secondary antibodies could produce different colors simultaneously depending on which of several antigens were present (11). Moreover, as more and more monoclonal antibodies are prepared in time, this procedure will become increasingly more useful. Acknowl¢dgmcnlS This work was partially supported by NIH (NIAID) 1-R15-A127775-01 (CWV), and by a Minor Research Grant from the College of William and Mary in Virginia (MEM). References 1. A. H. SHUURS, and B. K. VAN WEEMER, Clinica, Chimica Acta 81 1-40 (1977). 2. R. C. GOLDMAN, and L. LEIVE, Eur. J. Biochem. 107 145-153 (1980). 3. I. M. HELANDER, in Enterobacterial Surface Antigens: Methods for Molecular Characterisation (T. K. KORHONEN, E. A. DAWES, and P. H. M,a,KEL.~., Eds.) Elsevier, Amersterdam. 263-274 (1985). 4. U. K. LAEMMLI, Nature 227 680-685 (1970). 5. F. ORSKOV, V. SHARMA, and I. ORSKOV, J. Hyg. Camb. 95 551-575 (1984). 6. M. BITTNER, K. KUPFERER, and C. F. MORRIS, Anal. Biochem. 102 459-471 (1980). 7. H. TOWBIN, T. STAEHELIN, and J. GORDON, Proc. Natl. Acad. Sci. USA 76 4350-4354 (1979). 8. J. MORRISSEY, Anal. Biochem. 117 307-310 (1981). 9. C. M. TSAI, C. E. FRASCH, Anal. Biochem. 119 115-119 (1982).
868
Rapid Identification of Mutants & Pathogens
Vol. 49, No. 12, 1991
10. H. SCHNEIDER, C. A. HAMMACK, M. A. APICELLA, and J. M. GRIFFIS, Infect. Immun. 56 942-946 (1988). 11. D. Y. MASON, and R. SAMMOUS, J. Clin. Pathol. 31 454-460 (1978). 12. A. CROSS, P. GEMSKI, J. SADOFF, F. ORSKOV and I. ORSKOV, J. Infect. Dis. 149 184-193 (1984). 13. P. GEMSKI, A. CROSS, and J. C. SADOFF, FEMS Microbiol. Letters 9 193-197 (1980). 14. R. BORTOLUSSI, P. FERR1ERI, and P. QUIE, Infect. Immun. 39 1136-1141 (1983). 15. P. H. M,~KELA, in Enterobacterial Surface Antigens: Methods for Molecular Characterisation (T. K. KORHONEN, E. A. DAWES, and P. H. M)iKEL,~, Eds.) Elsevier, Amersterdam. 155-177 (1985). 16. W. C. BOGARD, Jr, D. L. DUNN, K. ABERNATHY, C. KILGARRIFF, and P. C. KUNG, Infect. Immun. 55 899-908 (1987). 17. B. M. KAUFMAN, A. S. CROSS, S. L. FUTROFSKY, H. F. SIDBERRY, and J. C. SADOFF, Infect. Immun. 52 617-619 (1986). 18. C. W. VERMEULEN, A. S. CROSS, W. R. BYRNE, and W. ZOLLINGER, Infect. Immun. 56 2723-2730 (1988). 19. W. D. ZOLLINGER, and R. E. MANDRELL, Infect. Immun. 40 257-264 (1983). 20. F. ORSKOV, and I. ORSKOV, in Eschcrichia ¢oli Infections in Domestic Animals (H. WlLLINGER, and A. WEBER, Eds.), Verlag Paul Parey, Berlin, 7-14 (1979). 21. World Health. Diarrheal Diseases and Nutrition. W.H.O. Geneva (1988).
Pergamon Press
ENHANCED ENZYME-LINKED IMMUNOSORBANT ASSAY ON MEMBRANES FOR THE IDENTIFICATION OF MUTANTS AND PATHOGENS Matthew E. May and Carl W. Vermeulen* Department of Biology, The College of William and Mary, Williamsburg, Virginia 23185 USA (Received in final form July 16, 1991) Summa~
With variations in the concentrations of antibodies and blocking agents and reduction in incubation times, qualitative enzyme-linked immunosorbant assay can be performed in a matter of two hours, for more rapid identification of mutants and antigens. The usual enzyme-linked immunosorbant assay techniques, ELISA, as done on membranes (1) are too slow for many biochemical research laboratories. For example, after some variety of electrophoresis such as detergent augmented polyacrylamide gel electrophoresis, SDS-Page (2,3,4) or agarose electrophoresis of surface antigens (5), there are several electroblotting techniques (6,7) that could use highly specific immunochemical visualization, in lieu of less specific staining of gels with dyes such as 'silver stain' (8,9). Similarly, in the clinical field the potential for highly specific immunochemical analysis would prove to be an invaluable diagnostic tool for the identification of pathogens such as S a l m o n e l l a , or for E. coli, of which a few strains cause a great deal of diarrheal disease. In order to speed up the procedure we modified concentrations and reaction times of the prevailing membrane ELISA procedure, and have tested these modifications on both the ability to identify colonies, as well as to identify bacterial smears. In general we did the following: concentrations of blocking agents were increased by 30% such that saturation of non-specific protein binding sites was more rapidly attained. Similarly, when the concentration of primary antibody was increased 5-fold, binding of antibody to the protein complex is also accelerated. We found that bacterial smears that had been cultivated on nutrient agar for only 1 hour at 37.5c gave positive tests. Finally, by eliminating the overnight, secondary, conjugated antibody binding step in the prevailing procedure, as well as reductions of all remaining steps by at least half, brought the total time for ELISA to under two hours. Thus, ELISA identification of bacterial surface antigens has been shortened from approximately 30 hours to about 2 hours. The benefits of shortening this powerful biochemical tool should be obvious, especially since it has been found that even sectors of colonies are identifiable (10), and that several different secondary conjugated antibodies might be used to give a variety of colors (11) for simultaneous multitests. Malerials and Methods Bacteriology. Various concentrations of clinically derived E s c h e r i c h i a c o l i were used to analyze the sensitivity of the shortcnded procedure. E. c o l i E412 (R:KI:H?) (12,13), E. coli 'Bort' (O18ac:K1:H7) (14), and various clinical strains sensitive to Kl-specific phage eK1E (15) were plated and incubated for one hour at 37.5c. E. coli E457 (O6:K5:H?) (12,13) was used as the negative control. All axenic cultures were 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
866
Rapid Identification of Mutants & Pathogens
Vol. 49, No. 12, 1991
grown on nutrient agar, or in L-broth (0.3% glucose, 0.7% tryptone) at 37.5c. For mixed cultures, human fecal samples, doped with Kl-encapsulated E. ¢oli E412, were also tested. Sampling. Four types of samplings were made. (a) In the first, we were interested in noting the lower limit of detection using the 'dip test.' A nitrocellulose, NC, strip was dipped into broth cultures of known bacterial concentration. (b) To ascertain the earliest that colonies could be identified, serial dilutions were plated and incubated at 37.5c. At one hour intervals, narrow strips of NC membrane were then laid across the surface of the agar upon which the colonies were growing. (c) With regard to fecal samples, a 10-fold dilution was swabbed onto half of an agar plate and incubated at 37.5c. Hourly, NC strips were laid across the agar bridging both the smeared and the unsmeared sectors. In all cases, the NC strips were then dried in the incubator for 5 minutes or more at 37.5c before being subjected to ELISA. ELISA. The bacteria-treated NC strips were then placed in 2.5 ml of washing buffer (10% skim milk, the blocking agent, and 90% distilled water) at 18c. We then added 1.0 ml more cold skim milk and allowed the solution to gently shake at 65 rpm for 30 minutes to saturate protein binding sites on the membranes. This liquid was then decanted. Then 2.5 ml of washing buffer (18c) was added and immediately decanted to rid the membrane of excess blocking agent. This step was repeated once again. We then added 2.5 ml of washing buffer (18c) containing 5 I.tl of ascites fluid at 4c (murine m o n o c l o n a l - I g M against m c n i n g o c o c c a l B p o l y sacch ar i d e, which is identical to E_...coli K1 capsule (16,17,18,19)), as the primary antibody, and shaken for 15 minutes. The solution was then refrigerated for an additional 15 minutes at 65 rpm to allow primary antibody to attach to K1 of the relevant bacterial surface antigens. This solution was then replaced with 2.5 ml of 18c washing buffer containing 1 I.tl of HRP::secondary antibody (goat/anti-mouse IgM antibody conjugated with horse-radish peroxidase, from Kirkegaard and Perry, Inc., Gaithersburg, MD) at 5c, and swirled this for 30 minutes at 65 rpm. The membrane was then twice washed with 2.5 ml of 18c washing buffer and twice placed on the shaker for 1 minute at 65 rpm. The NC was then placed into 2.5 ml acetate buffer (50 mM sodium acetate, pH 5.0) at room temperature to rid the membrane of excess washing buffer, and allowed to shake for 1 minute at 65 rpm. We then developed the membrane, again at room temperature, with the peroxidase/membrane ELISA reagents according to methods and products of Kirkegaard and Perry, Inc. In this method the membrane is placed in enough 10c developer (equal volumes of 4-chloro-l-naphthol (1.4 g/l in an organic base) and H 2 0 2 (0.02% in citric acid buffer) to cover the membrane and swirled at 75 rpm until purple bands appeared. Development was stopped by decanting developer and adding 22c distilled water. The membranes were then dried and stored at room temperature in the dark as the pigment producted is photolabilc. Resuhs In the dip test, using serial dilutions of an overnight culture adjusted to I x l 0 9 cfu/ml, we found that positive results were directly obtainable to as low as 3x103 to lxl04cfu/ml. In the case where a dilute suspension of bacteria was spread upon agar, colonies of K l - p o s s e s s i n g E__~.coli strains clearly were discernible from those which did not possess KI capsules. Fecal bacteria possessing the proper antigen swabbing a 100-fold dilution of stool sample. Concomitantly with the attachment of the should remain in the for at least 30 minutes.
were detectable within
one hour of
the above, we found Ihat there is a minimum time period for primary antibody to lhe substrate antigen. The membrane solution containing the primary antibody and washing buffer The solution should bc swirled for 15 minutes and then be
Vol. 49, No. 12, 1991
Rapid Identification of Mutants & Pathogens
placed at 4c for another 15 minutes. The procedure done without both of these will give erratic results with very low concentrations of bacteria. Incubation for the bacteria was also reduced from overnight to 1 hour since by then there sufficient growth to give a positive reaction on the membrane. Moreover, we found that this new procedure, used with other polyclonal antibody preparations as a Bort-O18 rabbit antibody preparation, is just as effective under the parameters.
867
steps time was also such same
Dis¢~lssi0n The arsenals of biochemists and clinicians must be stocked with the greatest variety of methods available in order that a right one might be applied to the problem at hand. In the overlapping realms of biochemistry, molecular biology and molecular genetics, selection of successfully transfcctcd mutants that are producing important biological components is critical. Detection of the desired mutant colony from among backgrounds of millions of unwanted colonies is often a major hurdle that requires a great deal of ingenuity. While an accelerated ELISA method may not be absolutely necessary for this pursuit of the rare mutant or variant, we know of no scientist who would willingly choose a 20 to 30-hour procedure over an equally discriminating 2-hour method. In the clinical and veterinarian arenas, while time is often of the critical essence, it is even more important that the clinician be able to discern whether or not a specific pathogen is present. Here for the sake of choosing a model of growing medical interest, we have chosen diarrhea (5,20,21). Currently no American clinical laboratory routinely assays the serotype make-up of the E . ¢ o l i population in stool samples. This disease is especially critical in infants who become rapidly dehydrated. This lack of testing is presumably because the prevailing method for identification of E . ¢ o l i is a long and tedious process. Our method can detect almost immediately any strain or mixture of strains (using a mix of relevant primary antibodies). Of particular interest in the assay for bacteruria, which is noted as 'positive' when the bacterial count is greater than l x l 0 4 cfu/ml, the sensitivity of our dip test falls within this range. Since only a few pathogenic serotypes are very common, such ELISA testing now holds promise of more rapid diagnosis. Alluded to earlier, antibody mixes could be used so that different conjugated secondary antibodies could produce different colors simultaneously depending on which of several antigens were present (11). Moreover, as more and more monoclonal antibodies are prepared in time, this procedure will become increasingly more useful. Acknowl¢dgmcnlS This work was partially supported by NIH (NIAID) 1-R15-A127775-01 (CWV), and by a Minor Research Grant from the College of William and Mary in Virginia (MEM). References 1. A. H. SHUURS, and B. K. VAN WEEMER, Clinica, Chimica Acta 81 1-40 (1977). 2. R. C. GOLDMAN, and L. LEIVE, Eur. J. Biochem. 107 145-153 (1980). 3. I. M. HELANDER, in Enterobacterial Surface Antigens: Methods for Molecular Characterisation (T. K. KORHONEN, E. A. DAWES, and P. H. M,a,KEL.~., Eds.) Elsevier, Amersterdam. 263-274 (1985). 4. U. K. LAEMMLI, Nature 227 680-685 (1970). 5. F. ORSKOV, V. SHARMA, and I. ORSKOV, J. Hyg. Camb. 95 551-575 (1984). 6. M. BITTNER, K. KUPFERER, and C. F. MORRIS, Anal. Biochem. 102 459-471 (1980). 7. H. TOWBIN, T. STAEHELIN, and J. GORDON, Proc. Natl. Acad. Sci. USA 76 4350-4354 (1979). 8. J. MORRISSEY, Anal. Biochem. 117 307-310 (1981). 9. C. M. TSAI, C. E. FRASCH, Anal. Biochem. 119 115-119 (1982).
868
Rapid Identification of Mutants & Pathogens
Vol. 49, No. 12, 1991
10. H. SCHNEIDER, C. A. HAMMACK, M. A. APICELLA, and J. M. GRIFFIS, Infect. Immun. 56 942-946 (1988). 11. D. Y. MASON, and R. SAMMOUS, J. Clin. Pathol. 31 454-460 (1978). 12. A. CROSS, P. GEMSKI, J. SADOFF, F. ORSKOV and I. ORSKOV, J. Infect. Dis. 149 184-193 (1984). 13. P. GEMSKI, A. CROSS, and J. C. SADOFF, FEMS Microbiol. Letters 9 193-197 (1980). 14. R. BORTOLUSSI, P. FERR1ERI, and P. QUIE, Infect. Immun. 39 1136-1141 (1983). 15. P. H. M,~KELA, in Enterobacterial Surface Antigens: Methods for Molecular Characterisation (T. K. KORHONEN, E. A. DAWES, and P. H. M)iKEL,~, Eds.) Elsevier, Amersterdam. 155-177 (1985). 16. W. C. BOGARD, Jr, D. L. DUNN, K. ABERNATHY, C. KILGARRIFF, and P. C. KUNG, Infect. Immun. 55 899-908 (1987). 17. B. M. KAUFMAN, A. S. CROSS, S. L. FUTROFSKY, H. F. SIDBERRY, and J. C. SADOFF, Infect. Immun. 52 617-619 (1986). 18. C. W. VERMEULEN, A. S. CROSS, W. R. BYRNE, and W. ZOLLINGER, Infect. Immun. 56 2723-2730 (1988). 19. W. D. ZOLLINGER, and R. E. MANDRELL, Infect. Immun. 40 257-264 (1983). 20. F. ORSKOV, and I. ORSKOV, in Eschcrichia ¢oli Infections in Domestic Animals (H. WlLLINGER, and A. WEBER, Eds.), Verlag Paul Parey, Berlin, 7-14 (1979). 21. World Health. Diarrheal Diseases and Nutrition. W.H.O. Geneva (1988).