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An immunochemical film-badge for detecting organic contaminants in water
Environment International Vol. 2, pp. 67-.68, Pergamon Press Ltd. 1979. Printed in Great Britain.
An immunochemical Film-Badge for Detecting Organic Contaminants in Water* H.R. Lukens and C.B. Williams IRT Corporation, 7650 Convoy Court, San Diego, CA 92138, U.S.A.
An immunochemical method for the determination of specific organic environmental contaminants has been developed. The technique involves the use of a glass backed 10/zm indium film on which is deposited a layer of antibody toward the organic compound of interest. Subsequent exposure of the film to the compound results in a diminution of the film's optical transmittance, and the effect is visually discernable at the exposure boundary when part of the film is not exposed. Experiments with BSA and anti-BSA were used to define correct conditions for test-strip preparation, and the method was then evaluated by using it to detect 2-amin0benzimidazole (2ABZI), a degradation product of several carbamate fungicides. At its present stage of development the method will detect 0.25 ppm of 2ABZI in water.
A means of depositing a monolayer of antibody on indium film in such a manner that it can then bind its antigen has been accomplished with the objective of developing a system to detect organic contaminants in water. This order is the reverse of the arrangement described by Giaever, wherein bovine serum albumin (BSA), the antigen, was first deposited on metal film and then anti-BSA, the antibody was deposited on the BSA (Giaever, 73). The conditions used by Giaever were such that the reverse order could not work because the antibody preferentially binds to the metal with the sites that would otherwise bind the antigen (Giaever, 76). Hence, a deposition of anti-BSA on the indium would produce a layer incapable of binding BSA. In the present work, conditions have been developed that will allow the antibody to be deposited on a glass substrate in such a fashion that it still retains its reactivity with its antigen or hapten. Reagents used were BSA and anti-BSA, provided by W.B. Dandliker of Scripps Clinic and Research Foundation, 2-aminobenzimidazole (2ABZI), and anti2ABZI developed in previous work (Lukens, 75). Indium films approximately 10 /~m thick were deposited by tungsten filament evaporation of indium metal from a tantalum boat onto clean glass microscope slides invacuum (10-5 torr). The glass slides had been cleaned sequentially with detergent, distilled water, reagent grades acetone and ethanol, dried, and then finally cleaned with argon plasma for 15 min under vacuum. The thickness of the indium films was measured with a Sloan thickness monitor. Deposition of protein onto indium film was carried *This work was carried out under EPA contract 68-02-2202. 67
out by either partially dipping the slide in a solution of the protein or by placing drops of solution on the slide. In both cases, the slides were then thoroughly rinsed with distilled water and allowed to dry in air. Subsequent deposition of reagent onto the film was carried out in a similar manner and incubation periods of 10 min were used. A McBeth-Ansco Optical Transmission Densitometer was used to measure the optical transmittance in order to determine whether deposition of reagent had occurred. However, deposition was usually apparent by simple visual inspection, since the difference in transmission between a deposition area and the adjacent, nonexposed area was easily discerned. As a preliminary test of the procedures a 10/~m indium film was first coated with BSA from a neutral saline solution containing 294 ppm BSA and then coated with anti-BSA from a neutral saline solution containing 294 ppm of anti-BSA. The results were in accord with Giaever's findings. The measured transmissions after incubation with BSA and after incubation with anti-BSA were 0.310 and 0.270, respectively. Results from the reversal of the order of exposure were also in accord with Giaever's findings, as shown in Table 1. The antigen-sensitive end of the antibody molecule Table 1. Attempt to react BSA with film of anti-BSA Experimental condition Indium film only After incubation of indium with an anti-BSA After subsequent incubation with BSA
Measured transmittance 0.385 0.285 0.280
68
H.R. Lukens and C.B. Williams
(the Fab end) has a predominance of reactive amino groups, while reactive carboxyl groups predominate at the other end (Fc end). Therefore, in order to decrease the reactivity of the former and increase the reactivity of the latter, three aliquots o f the 294 ppm anti-BSA solution were made 0.0008 M, 0.004 M and 0.03 M in KOH, respectively. The anti-BSA was deposited on 20 /~m indium films from the alkaline solutions, and BSA was then successfully bound to the antibody films in each case as shown in Table 2. Anti-2ABZI was deposited on 10 ~um in. film from slightly alkaline solution and thoroughly rinsed. The films were incubated with neutral solutions of 2ABZI at concentrations of 2.5 ppm, 0.25 ppm and 0.025 ppm. The experiments were carried out in triplicate, and the results are given in Table 3. It can be seen that the method is capable of detecting 0.25 ppm 2ABZI in water; and, in fact, the deposition from the 0.25 ppm o f solution of 2-ABZI was visually apparent. The compound was not detected at 0.025 ppm. Table 2. Experiment with anti-BSA deposited on 20/am indium from solutions of specified KOH molarity Experimental Conditions In film alone After incubation with anti-BSA After subsequent incubation with BSA
0.0008 M KOH 0.004 M KOH
0.03 M KOH
0.078
0.076
0.079
0.072
0.071
0.063
0.060
0.060
0.055
Table 3.2ABZI vs anti-2ABZI on indium film Experimental conditions
After incubation with anti-2ABZl After subsequent incubation with 2ABZI
Table 4. Specifity of the method. Transmittance before and after incubation of anti-ABZI films with several reagents at 2.5 ppm concentration Experimental conditions Reagent Benzimidazole 5 Chlorobenzimidazole2ABZl Before incubation 0.315 0.295 0.345 After incubation 0.310 0.290 0.320 Change in transmittance 0.005 0.005 0.025
In conclusion, a method has been developed for the detection of organic contaminants in water. The method utilizes an indium film deposited on glass on which a film of antibody toward the contaminant o f interest is bound. In its present version the method is a film-badge system capable o f visually detecting sub-ppm levels o f contaminant. It is anticipated that a competitive binding version of the method, wherein fluorescein-labeled contaminant is incubated with the antibody, film after the film has been incubated with the water sample in question, will enable even greater sensitivity to be obtained. References
Transmittance with varying concentrations of 2ABZI 2.5 ppm 0.25 ppm 0.337 +0.004 0.333 + 0.006 0.318 +0.002
The specificity of the method was tested by comparing the change in transmittance effected by incubating films of anti-2ABZI on indium with solutions containing 2.5 ppm of benzimidazole, 5chlorobenzimidazole, and 2ABZI. The results, given in Table 4, shows that the 2ABZI is significantly more reactive with its antibody than the other two reagents. It was found that 2ABZI would bind to indium film, but it would not bind to a film of BSA on indium.
0.320 +0.005
Giaever, I. (1973) The antibody antigen reaction - - a visual observation, J. Im m un. 110, 1424-1426. Giaever, I. (1976)Private communication. Lukens, H.R., Williams, C.B., Levison, S.A., Dandliker W.B and Muryama, D. (1975) A fluorescence immunoassay technique for detecting organic environmental contaminants, EPA-650/I-75-004, U.S. Environmental Protection Agency, Washington, D.C.
An immunochemical Film-Badge for Detecting Organic Contaminants in Water* H.R. Lukens and C.B. Williams IRT Corporation, 7650 Convoy Court, San Diego, CA 92138, U.S.A.
An immunochemical method for the determination of specific organic environmental contaminants has been developed. The technique involves the use of a glass backed 10/zm indium film on which is deposited a layer of antibody toward the organic compound of interest. Subsequent exposure of the film to the compound results in a diminution of the film's optical transmittance, and the effect is visually discernable at the exposure boundary when part of the film is not exposed. Experiments with BSA and anti-BSA were used to define correct conditions for test-strip preparation, and the method was then evaluated by using it to detect 2-amin0benzimidazole (2ABZI), a degradation product of several carbamate fungicides. At its present stage of development the method will detect 0.25 ppm of 2ABZI in water.
A means of depositing a monolayer of antibody on indium film in such a manner that it can then bind its antigen has been accomplished with the objective of developing a system to detect organic contaminants in water. This order is the reverse of the arrangement described by Giaever, wherein bovine serum albumin (BSA), the antigen, was first deposited on metal film and then anti-BSA, the antibody was deposited on the BSA (Giaever, 73). The conditions used by Giaever were such that the reverse order could not work because the antibody preferentially binds to the metal with the sites that would otherwise bind the antigen (Giaever, 76). Hence, a deposition of anti-BSA on the indium would produce a layer incapable of binding BSA. In the present work, conditions have been developed that will allow the antibody to be deposited on a glass substrate in such a fashion that it still retains its reactivity with its antigen or hapten. Reagents used were BSA and anti-BSA, provided by W.B. Dandliker of Scripps Clinic and Research Foundation, 2-aminobenzimidazole (2ABZI), and anti2ABZI developed in previous work (Lukens, 75). Indium films approximately 10 /~m thick were deposited by tungsten filament evaporation of indium metal from a tantalum boat onto clean glass microscope slides invacuum (10-5 torr). The glass slides had been cleaned sequentially with detergent, distilled water, reagent grades acetone and ethanol, dried, and then finally cleaned with argon plasma for 15 min under vacuum. The thickness of the indium films was measured with a Sloan thickness monitor. Deposition of protein onto indium film was carried *This work was carried out under EPA contract 68-02-2202. 67
out by either partially dipping the slide in a solution of the protein or by placing drops of solution on the slide. In both cases, the slides were then thoroughly rinsed with distilled water and allowed to dry in air. Subsequent deposition of reagent onto the film was carried out in a similar manner and incubation periods of 10 min were used. A McBeth-Ansco Optical Transmission Densitometer was used to measure the optical transmittance in order to determine whether deposition of reagent had occurred. However, deposition was usually apparent by simple visual inspection, since the difference in transmission between a deposition area and the adjacent, nonexposed area was easily discerned. As a preliminary test of the procedures a 10/~m indium film was first coated with BSA from a neutral saline solution containing 294 ppm BSA and then coated with anti-BSA from a neutral saline solution containing 294 ppm of anti-BSA. The results were in accord with Giaever's findings. The measured transmissions after incubation with BSA and after incubation with anti-BSA were 0.310 and 0.270, respectively. Results from the reversal of the order of exposure were also in accord with Giaever's findings, as shown in Table 1. The antigen-sensitive end of the antibody molecule Table 1. Attempt to react BSA with film of anti-BSA Experimental condition Indium film only After incubation of indium with an anti-BSA After subsequent incubation with BSA
Measured transmittance 0.385 0.285 0.280
68
H.R. Lukens and C.B. Williams
(the Fab end) has a predominance of reactive amino groups, while reactive carboxyl groups predominate at the other end (Fc end). Therefore, in order to decrease the reactivity of the former and increase the reactivity of the latter, three aliquots o f the 294 ppm anti-BSA solution were made 0.0008 M, 0.004 M and 0.03 M in KOH, respectively. The anti-BSA was deposited on 20 /~m indium films from the alkaline solutions, and BSA was then successfully bound to the antibody films in each case as shown in Table 2. Anti-2ABZI was deposited on 10 ~um in. film from slightly alkaline solution and thoroughly rinsed. The films were incubated with neutral solutions of 2ABZI at concentrations of 2.5 ppm, 0.25 ppm and 0.025 ppm. The experiments were carried out in triplicate, and the results are given in Table 3. It can be seen that the method is capable of detecting 0.25 ppm 2ABZI in water; and, in fact, the deposition from the 0.25 ppm o f solution of 2-ABZI was visually apparent. The compound was not detected at 0.025 ppm. Table 2. Experiment with anti-BSA deposited on 20/am indium from solutions of specified KOH molarity Experimental Conditions In film alone After incubation with anti-BSA After subsequent incubation with BSA
0.0008 M KOH 0.004 M KOH
0.03 M KOH
0.078
0.076
0.079
0.072
0.071
0.063
0.060
0.060
0.055
Table 3.2ABZI vs anti-2ABZI on indium film Experimental conditions
After incubation with anti-2ABZl After subsequent incubation with 2ABZI
Table 4. Specifity of the method. Transmittance before and after incubation of anti-ABZI films with several reagents at 2.5 ppm concentration Experimental conditions Reagent Benzimidazole 5 Chlorobenzimidazole2ABZl Before incubation 0.315 0.295 0.345 After incubation 0.310 0.290 0.320 Change in transmittance 0.005 0.005 0.025
In conclusion, a method has been developed for the detection of organic contaminants in water. The method utilizes an indium film deposited on glass on which a film of antibody toward the contaminant o f interest is bound. In its present version the method is a film-badge system capable o f visually detecting sub-ppm levels o f contaminant. It is anticipated that a competitive binding version of the method, wherein fluorescein-labeled contaminant is incubated with the antibody, film after the film has been incubated with the water sample in question, will enable even greater sensitivity to be obtained. References
Transmittance with varying concentrations of 2ABZI 2.5 ppm 0.25 ppm 0.337 +0.004 0.333 + 0.006 0.318 +0.002
The specificity of the method was tested by comparing the change in transmittance effected by incubating films of anti-2ABZI on indium with solutions containing 2.5 ppm of benzimidazole, 5chlorobenzimidazole, and 2ABZI. The results, given in Table 4, shows that the 2ABZI is significantly more reactive with its antibody than the other two reagents. It was found that 2ABZI would bind to indium film, but it would not bind to a film of BSA on indium.
0.320 +0.005
Giaever, I. (1973) The antibody antigen reaction - - a visual observation, J. Im m un. 110, 1424-1426. Giaever, I. (1976)Private communication. Lukens, H.R., Williams, C.B., Levison, S.A., Dandliker W.B and Muryama, D. (1975) A fluorescence immunoassay technique for detecting organic environmental contaminants, EPA-650/I-75-004, U.S. Environmental Protection Agency, Washington, D.C.