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Quantification of antibody immobilization using peroxidase enzyme–substrate reaction
Microchemical Journal 65 Ž2000. 105᎐111
Quantification of antibody immobilization using peroxidase enzyme᎐substrate reaction Chandra S. Theegala, Ahmad A. SuleimanU Department of Chemistry, Southern Uni¨ ersity and A & M College, Baton Rouge, LA 70813, USA Received 22 February 2000; received in revised form 24 April 2000; accepted 25 April 2000
Abstract Due to the multitude of antigens and antibodies that are currently being studied by immunoscientists and researchers, numerous and diverse antibody immobilization techniques were developed. Newer and improved methods of detection and immobilization are constantly being developed. As a result, especially when no prior research exists, scientists and researchers are faced with the challenge of choosing the best immobilization technique. Assessment of antibody immobilization based on the final antibody᎐antigen reaction may not always be accurate, as certain unknown processes or reactions may account for a part Žor whole. of the transducer response. The present paper describes an alternative method for scientific quantification of antibody attachment on the biosensing surface or transducer using the peroxidase enzyme᎐substrate ŽTMB. colorimetric reaction. The proposed method can be used to pre-screen various immobilization techniques or pre-coatings, or can be used as a secondary quantification method. Four different pre-coatings on piezoelectric crystals and three affinity membranes were tested to evaluate the degree of antibody attachment. Results indicate that polystyrene-coated crystals had an average immobilization of 10.55% and the Immobilon membrane had 18.22% immobilization. Although the precision of the proposed technique varied significantly between different transducer surfaces ŽCVs 1.7᎐21.7%., results clearly demonstrate that polystyrene coating on crystal is approximately 450% superior to glutaraldehyde coating. Similarly, the
U
Corresponding author. Tel. q1-225-771-3990; fax: q1-225-771-3992. E-mail address: [email protected] ŽA.A. Suleiman.. 0026-265Xr00r$ - see front matter 䊚 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 0 . 0 0 0 3 7 - 0
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C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
Immobilon membrane had 720% higher antibody immobilization than Ultrabind membrane. The sensitivity of the overall technique is so high that each test requires only 3 l of diluted, labeled antibody solution Ž1 grml.. 䊚 2000 Elsevier Science B.V. All rights reserved. Keywords: Antibody; Immobilization; Piezocrystal; Immunosensor; Affinity membrane
1. Introduction Immobilization of antibodies on a biosensing surface or transducer can be done using various methods such as passive adsorption, covalent attachment, polymer or gel entrapment, crosslinking, electropolymerization, and photo-immobilization w1᎐3x. For piezocrystals, the innumerable list of actual coatings range from a simple Protein A or thin saline layer w4x to ophenylenediamine w2x, polyŽethylene oxide.polyŽpropylene oxide. triblock copolymers w5x, alkanethiol and amino-alkoxysilane monolayers, polyethylenimine-glutaraldehyde and plasma coatings w6x, copolymer coating of hydroxy-ethyl methacrylate and methylmethacrylate w7x, Alcian Blue w8x and ethylenediamine plasma-polymerized film matrix w9x. Each of these methods has its own advantages and disadvantages w1x; therefore, no single universally acceptable immobilization method exists. New antibody immobilization methods such as electropolymerization and the use of plant and animal tissues are constantly being developed. Currently, researchers and scientists working in this area have to identify an appropriate immobilization technique that best suits their application. However, with such numerous and diverse immobilization techniques that are currently available, scientific assessment of the antibody attachment with different methods is a tedious task. The present paper describes a simple scientific method for the quantification of antibody attachment on a biosensing or transducer surface using the peroxidase enzyme᎐substrate wtetramethylbenzidine ŽTMB.x colorimetric reaction. Furthermore, in certain biosensing applications, assessment of antibody immobilization based on the final antibody᎐antigen reaction response may not be accurate as certain unknown processes or reactions may account for a part Žor whole. of the transducer response. In such in-
stances, a secondary antibody quantification technique, such as the one proposed here, may prove to be very helpful.
2. Experimental 2.1. Materials Peroxidase labeled E. coli antibody solution Ž100 grml. was prepared by adding 1 ml of 0.1 M phosphate buffer ŽPBS. solution ŽpH 7.0. to a vial containing lymphosized antibody powder ŽKirkegaard & Perry Laboratories, Inc.. This antibody solution was further diluted 100-fold with PBS buffer to yield a 1-grml antibody solution. The TMB substrate and TMB stop solution were used as received ŽKirkegaard & Perry Laboratories, Inc.. Clip-mounted, AT-cut, 10 MHz piezoelectric crystals with gold electrodes ŽBliley Electric Co., Erie, PA. were used. Three affinity membranes, Ultrabind ŽGelman Science, Inc.., Protran ŽSchleicher & Schuell. and Immobilon ŽMillipore Corp.. were used as received. All other chemicals used were of analytical grade. 2.2. Apparatus A spectrophotometer ŽHitachi, Model U-2001. with a 1-cm cuvette with a 1-ml sample volume was used and all measurements were made at 450 nm. Two micro-syringes Ž5 and 10 l. and two variable volume pipettors with disposable dispensers Ž0᎐2 ml. were used for measuring and transferring antibody, TMB substrate and stop solutions. All washing and color development reactions were performed in 5-ml glass beakers placed on an orbital shaker ŽBig Bill, Model M49125, with 3r4 inch drive. set at 100 rev.rmin.
C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
2.3. Immobilization of antibody 2.3.1. Piezocrystals The crystals were first immersed in 1 N NaOH for 10 min, rinsed twice with distilled water ŽDI. and air-dried. A similar treatment was performed with 1 N HCl. Finally, 50 l of conc. HCl were placed on the gold electrode Žone side only. for 2 min and which was rinsed two times with DI and air dried. Following this surface preparation, the antibody was immobilized using one of the following methods. To permit statistical assessment three crystals were used for each method. 2.3.2. Direct adsorption The surface prepared crystals were kept horizontally on a flat surface and 3 l of peroxidaselabeled E. coli antibody Ž1 grml. was spread on the gold electrode on one side of the piezocrystal. The surface was allowed to air dry and immediately transferred to 100% humid chambers and kept overnight at 4⬚C. On the following day, the crystals were first washed in PBS buffer, followed by two distilled water washings. 2.3.3. Protein A Using a microsyringe, 3 l of protein A was placed on the cleaned gold electrode of the crystal and gently spread Žon the gold electrode only. using a glass rod. The crystals were allowed to air dry. The crystals were then washed for 10 min in PBS buffer, followed by two distilled water washings. The crystals were again air dried and 3 l of peroxidase labeled E. coli antibody Ž1 grml. was spread on the gold electrode and washed as described in the direct adsorption method. 2.3.4. Glutaraldehyde Two microliters of 1% glutaraldehyde, 2 l of BSA Ž4 mgrml. and 3 l of peroxidase-labeled E. coli antibody Ž1 grml. was mixed and evenly spread onto the gold electrode of the crystals. The crystals were air dried and washed with PBS, followed by two washes with distilled water. 2.3.5. Polystyrene Solid polystyrene was crushed to small pieces and 4 mg of the crushed pieces was dissolved in 1
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ml of toluene. On one side of each crystal Žon gold electrode., 2 l of polystyrene solution was coated and allowed to air dry. The crystals were washed with PBS, followed by two washings with distilled water. The crystals were again air dried and 3 l of peroxidase-labeled E. coli antibody Ž1 grml. was spread onto the electrodes and washed as described earlier. 2.4. Membranes The three affinity membranes were cut to 1-cm2 pieces and 3 l of HRP-labeled E. coli antibody Ž1 grml. was placed at the center of the membrane. Due to the inherent absorbent property of the membranes, there was no need to spread the antibody on the surface. The membranes were incubated overnight in 100% humid chambers at 4⬚C. The membranes were placed in 5-ml beakers with PBS, which were placed on orbital shaker Ž100 rev.rmin, 3r4 inch drive. and washed for 10 min. The PBS wash was followed by two similar distilled water washings. All antibody immobilization Žon the membranes. quantification was performed in triplicate. 2.5. Color de¨ elopment Each of the antibody coated and washed crystalrmembrane was placed in a 5-ml beaker, with the coated surface facing up. To this beaker, 0.5 ml of TMB substrate was added and the beaker was immediately placed on an orbital shaker Ž100 rev.rmin.. The peroxidase enzyme, in presence of H2 O2 , catalyses the oxidation of colorless TMB substrate to a blue colored product. After a specific reaction time Ž0᎐25 min., 1.5 ml of TMB STOP reagent is added to terminate the reaction. In addition to terminating the reaction, the TMB STOP solution changes the color of the blue substrate to yellow and enhances the absorbance signal four to five fold. Exactly 10 min after stopping the reaction, the absorbance of the solution was measured at 450 nm. A 1:3 ratio of TMB:STOP was chosen to yield high color stability during the spectrophotometric measurements. Based on the absorbance value of the sample and
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predetermined calibration curves, the percent-immobilized antibody was determined.
3. Results and discussion 3.1. Effect of HRP enzyme-TMB substrate reaction time on absorbance The HRP enzyme induced TMB substrate oxidation reaction is limited by either unreacted substrate or hydrogen peroxide Žor other strong oxidizing agents.. For this study, 3 l of labeled antibody Ž1 grml. was coated on one side of the piezocrystals and the crystals were air-dried. The crystals were placed in 5-ml beakers and 0.5 ml of TMB substrate was added. The crystals were not washed with either PBS or distilled water. The beaker was immediately placed on an orbital shaker Ž100 rev.rmin. and the reaction was terminated after 2, 4, 6, 10, 15 and 25 min Žwith 1.5 ml TMB STOP solution.. This study was conducted to determine the optimum reaction time needed for the quantification of antibody attachment. Results from this study ŽFig. 1. indicate that the absorbance of the substrate Žafter the stop reagent was added. increased with increase in reaction time. However, after 10᎐12 min, the reaction appears to slow down, possibly due to the exhaustion of unreacted substrate or oxidizing agent ŽH2 O2 .. The calibration curves, developed with 6-, 10- and 20-min reaction times were linear within the range of 0᎐3 ng of labeled antibody. However, a reaction time of 10 min was chosen for all further studies, as it was important to have unreacted labeled antibody at the time of termination of the reaction.
Fig. 1. Effect of reaction time on the absorbance of TMB substrate.
9, 12, 15, 20, 30, 40, 90, 120, 180, 240 and 1440 min.. After the drying process, the crystals were not washed with PBS or DI water. To serve as a control, three crystals were coated with labeled antibody and kept in 100% humid chamber at 4⬚C for 1440 min Ž1 day.. The antibody remaining Žor immobilized. was estimated based on the colorimetric procedure described earlier ŽSection 2.5.. As mentioned earlier, a 10-min reaction time was chosen for these studies. Results indicate ŽFig. 2. that the enzymerantibody on the humid controls did not disintegrate significantly Ž95% confidence limits. during the 1440 min of incubation; thereby, assuring that the concept is a statistically acceptable procedure if all incubations are done in humid chambers. However, the air drying process seemed to drastically affect the absorbance; thereby, limiting this technique to humid incubation.
3.2. Effect of air drying on absorbance This study was conducted to assess the degree of disintegration of the enzyme andror antibody during the air drying process and to account for absorbance loss due to the drying process itself. The antibody-coated crystals Ž3 l of HRP labeled antibody, 1 grml. were allowed to air dry Žnot in humid chamber. for different durations Ž0, 3, 6,
Fig. 2. Effect of air drying of labeled antibody on the absorbance of TMB substrate after stopping the reaction after 10 min.
C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
Fig. 3. Calibration curve for HRP enzyme-TMB substrate reaction at 6 min reaction time. Reaction terminated after 6 min with TMB stop agent.
3.3. Calibration cur¨ es Three different calibration curves were developed for three different reaction times, namely 6, 10 and 20 min. For the calibration curves, different quantities of labeled antibody Ž0᎐3 ng. was added to 0.5 ml substrate in 5-ml beakers and the beakers were placed immediately on an orbital shaker Ž100 rev.rmin.. After specific reaction times Ž6, 10 and 20 min., the reaction was terminated and the absorbance of the solution was measured 10 min later. The reaction was directly performed in a beaker so as to facilitate its applicability to multiple biosensor surfaces Žsuch as piezocrystals, membranes, etc... Results ŽFigs. 3, Fig. 4 and Fig. 5. indicate that all the calibration curves are linear within the instrument’s absorbance range Ž0᎐4 absorbance .. Each individual point on these calibration curves is an average of
Fig. 4. Calibration curve for HRP enzyme᎐TMB substrate reaction at 10-min reaction time. Reaction terminated after 10 min with TMB stop agent.
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Fig. 5. Calibration curve for HRP enzyme᎐TMB substrate reaction at 20-min reaction time. Reaction terminated after 20 min with TMB stop agent.
three absorbance values. As anticipated, the longer reaction times lowered the range due to higher intensity of the end product. 3.4. Antibody immobilization quantification The antibody coated and washed crystals and the membranes Žin triplicate. were placed in 5-ml beakers and the TMB substrate was added, then the absorbance of the colored substrate was measured as mentioned in the methodology section. To serve as controls, a set of piezocrystals and affinity membranes were coated with 3 l of antibody and the color was developed immediately without any washing. For the controls, there was no significant variation in absorbance between different piezocoatings and different membranes; therefore, an average value was used as an indication of 100% antibody response. For each of the piezocrystal and affinity membrane, the amount of antibody immobilized after the washing procedure was determined as described in the color development section. Results of the coated piezocrystals ŽFig. 6. indicated that the polystyrene coated crystals had the highest degree of immobilization of 10.55% Žof what was applied., while the glutaraldehyde coated crystals not only had the lowest immobilization Ž2.36%. but also had the least precision Ž21.7% CV.. However, it should be noted that, unlike other crystal coatings, the glutaraldehyde layer has a depth factor associated with it and the color development may not be a true indication of the degree of immobilization. This is
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membrane has approximately 720 or 650% higher immobilization than the other.
4. Conclusion
Fig. 6. Antibody immobilization expressed as a percent of antibody applied on the various piezocrystal coatings. Polystyrene coated crystals had the highest attachment Ž10.55%. and the direct adsorption had the least coefficient of variation.
due to the fact that a part Žor whole. of the antibody could be embedded deeply in the glutaraldehyderBSA layer or the HRP-labeled site may not be accessible for the TMB substrate. However, antithesis to this potential drawback, if the HRP enzyme is attached at the same location as the antigen᎐antibody attachment site, this proposed technique could be extremely useful for the determination of the optimum glutaraldehyder BSArantibody proportions. With respect to affinity membranes, the Immobilon membrane was unquestionably superior with respect to antibody immobilization ŽFig. 7.. The antibody attachment was approximately 720% and 650% better compared to Ultrabind and Protran membranes, respectively. The Immobilon membrane also exhibited the best precision Ž2.5% CV., while Ultrabind and Protran had higher coefficients of variation Ž11.9% and 8.2%, respectively.. When compared with the variation with piezocrystals, with the exception of glutaraldehyde coatings, the average variability associated with the membranes was higher. This variation was most likely introduced during the washing procedure and standardizing the washing procedure might lower the variability. In statistical terms, although a CV of 11.9% may appear to be a high number, it does not undermine the importance or applicability of the overall procedure, especially when the results suggests that one
Currently, there is a great need for developing newer and improved methods of immobilization of antibodies on biosensorrtransducer surfaces. However, scientific selection of the right immobilization technique is a very difficult task. Assessment of antibody immobilization using the final antibody᎐antigen reaction may not necessarily be accurate, as certain known or unknown processes may account for a part or whole of the transducer response. The proposed paper describes a technique for scientific quantification of antibody immobilization on biosensor surfaces. The degree of antibody immobilization using four different pre-coatings on piezocrystals ŽDirect Adsorption, Protein A, Glutaraldehyde, Polystyrene. and three affinity membranes ŽUltrabind, Protran and Im m obilon . was assessed. Polystyrene-coated piezocrystals and the Immobilon membrane had the highest degree of immobilization Ž10.55% and 18.22%, respectively.. Results indicate that the proposed technique can be effectively applied to quantify the degree of immobilization on transducer or biosensor surfaces. Researchers who are screening several immobilization techniques w4,10,11x can utilize this technique as a secondary quantification tool or as
Fig. 7. Antibody immobilization expressed as a percent of antibody applied on the various affinity membranes. Immobilon had the highest attachment and lowest coefficient of variation.
C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
a pre-screening tool to select the optimum immobilization techniqueŽs.. This quantification of the degree of immobilization will not only help in screening for the best biosensorrtransducer surface, but also will facilitate maximization of antibody immobilization on a specific membrane or coating. For example, one could maximize the immobilization rate by varying the proportions of glutaraldehyde, BSA and antibody. Despite promising results, the technique has a varying degree of variability Ždepending on membrane or coating type. as evidenced by both very low and very high coefficient of variation Ž1.7 and 21.7%.. The variability associated with the attachment and washing procedure may be responsible for this variation. Standardizing the attachment and washing procedure may lower this variation. Irrespective of this variability, the importance or applicability of the overall procedure is not impaired significantly, especially when the results suggest that one crystal coating is 450% superior than another or one membrane has 650᎐720% higher immobilization than another. Furthermore, the overall technique is very simple, requiring a few immunosupplies and a visible spectrophotometer. The sensitivity of the overall technique is so high that each test requires only 3 l of diluted labeled antibody solution Ž1 grml..
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Acknowledgements This work was supported by a research center in Minority Institutions, Grant RR-09104, from the National Center for Research Resources, National Institutes of Health. References w1x O.A. Sadik, J.M. Van Emon, Designing Immunosensors for Environmental Monitoring, Chemtech, 1997, pp. 38᎐46. w2x S.H. Si, J.H. Chen, F.J. He, L.H. Nie, S.Z. Yao, Chin. J. Chem. 14 Ž3. Ž1996. 222᎐227. w3x H. Gao, R. Luginbuhl, H. Sigrist, Sensors Actuators B Chem. 38 Ž1᎐3. Ž1997. 38᎐41. w4x B. Konig, M. Gratzel, Anal. Lett. 26 Ž8. Ž1993. 1567᎐1585. w5x J.T. Li, J. Carlsson, J.N. Lin, K.D. Caldwell, Bioconjugate Chem. 7 Ž5. Ž1996. 592᎐599. w6x H. Ghourchian, N. Kamon, Anal. Chim. Acta 300 Ž1-3. Ž1995. 99᎐105. w7x X. Chu, Z.H. Lin, G.L. Shen, R.Q. Yu, Analyst 120 Ž12. Ž1995. 2829᎐2832. w8x J. Lagace, S. Arsenault, E.A. Cohen, J. Immunol. Methods 175 Ž1. Ž1994. 131᎐135. w9x K. Nakanishi, H. Muguruma, I. Karube, Anal. Chem. 68 Ž1996. 1695᎐1700. w10x B.S. Attili, A.A. Suleiman, Anal. Lett. 28 Ž12. Ž1995. 2149. w11x F. Caruso, E. Rodda, D.N. Furlong, J. Colloid Interface Sci. 178 Ž1996. 104᎐115.
Quantification of antibody immobilization using peroxidase enzyme᎐substrate reaction Chandra S. Theegala, Ahmad A. SuleimanU Department of Chemistry, Southern Uni¨ ersity and A & M College, Baton Rouge, LA 70813, USA Received 22 February 2000; received in revised form 24 April 2000; accepted 25 April 2000
Abstract Due to the multitude of antigens and antibodies that are currently being studied by immunoscientists and researchers, numerous and diverse antibody immobilization techniques were developed. Newer and improved methods of detection and immobilization are constantly being developed. As a result, especially when no prior research exists, scientists and researchers are faced with the challenge of choosing the best immobilization technique. Assessment of antibody immobilization based on the final antibody᎐antigen reaction may not always be accurate, as certain unknown processes or reactions may account for a part Žor whole. of the transducer response. The present paper describes an alternative method for scientific quantification of antibody attachment on the biosensing surface or transducer using the peroxidase enzyme᎐substrate ŽTMB. colorimetric reaction. The proposed method can be used to pre-screen various immobilization techniques or pre-coatings, or can be used as a secondary quantification method. Four different pre-coatings on piezoelectric crystals and three affinity membranes were tested to evaluate the degree of antibody attachment. Results indicate that polystyrene-coated crystals had an average immobilization of 10.55% and the Immobilon membrane had 18.22% immobilization. Although the precision of the proposed technique varied significantly between different transducer surfaces ŽCVs 1.7᎐21.7%., results clearly demonstrate that polystyrene coating on crystal is approximately 450% superior to glutaraldehyde coating. Similarly, the
U
Corresponding author. Tel. q1-225-771-3990; fax: q1-225-771-3992. E-mail address: [email protected] ŽA.A. Suleiman.. 0026-265Xr00r$ - see front matter 䊚 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 6 - 2 6 5 X Ž 0 0 . 0 0 0 3 7 - 0
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C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
Immobilon membrane had 720% higher antibody immobilization than Ultrabind membrane. The sensitivity of the overall technique is so high that each test requires only 3 l of diluted, labeled antibody solution Ž1 grml.. 䊚 2000 Elsevier Science B.V. All rights reserved. Keywords: Antibody; Immobilization; Piezocrystal; Immunosensor; Affinity membrane
1. Introduction Immobilization of antibodies on a biosensing surface or transducer can be done using various methods such as passive adsorption, covalent attachment, polymer or gel entrapment, crosslinking, electropolymerization, and photo-immobilization w1᎐3x. For piezocrystals, the innumerable list of actual coatings range from a simple Protein A or thin saline layer w4x to ophenylenediamine w2x, polyŽethylene oxide.polyŽpropylene oxide. triblock copolymers w5x, alkanethiol and amino-alkoxysilane monolayers, polyethylenimine-glutaraldehyde and plasma coatings w6x, copolymer coating of hydroxy-ethyl methacrylate and methylmethacrylate w7x, Alcian Blue w8x and ethylenediamine plasma-polymerized film matrix w9x. Each of these methods has its own advantages and disadvantages w1x; therefore, no single universally acceptable immobilization method exists. New antibody immobilization methods such as electropolymerization and the use of plant and animal tissues are constantly being developed. Currently, researchers and scientists working in this area have to identify an appropriate immobilization technique that best suits their application. However, with such numerous and diverse immobilization techniques that are currently available, scientific assessment of the antibody attachment with different methods is a tedious task. The present paper describes a simple scientific method for the quantification of antibody attachment on a biosensing or transducer surface using the peroxidase enzyme᎐substrate wtetramethylbenzidine ŽTMB.x colorimetric reaction. Furthermore, in certain biosensing applications, assessment of antibody immobilization based on the final antibody᎐antigen reaction response may not be accurate as certain unknown processes or reactions may account for a part Žor whole. of the transducer response. In such in-
stances, a secondary antibody quantification technique, such as the one proposed here, may prove to be very helpful.
2. Experimental 2.1. Materials Peroxidase labeled E. coli antibody solution Ž100 grml. was prepared by adding 1 ml of 0.1 M phosphate buffer ŽPBS. solution ŽpH 7.0. to a vial containing lymphosized antibody powder ŽKirkegaard & Perry Laboratories, Inc.. This antibody solution was further diluted 100-fold with PBS buffer to yield a 1-grml antibody solution. The TMB substrate and TMB stop solution were used as received ŽKirkegaard & Perry Laboratories, Inc.. Clip-mounted, AT-cut, 10 MHz piezoelectric crystals with gold electrodes ŽBliley Electric Co., Erie, PA. were used. Three affinity membranes, Ultrabind ŽGelman Science, Inc.., Protran ŽSchleicher & Schuell. and Immobilon ŽMillipore Corp.. were used as received. All other chemicals used were of analytical grade. 2.2. Apparatus A spectrophotometer ŽHitachi, Model U-2001. with a 1-cm cuvette with a 1-ml sample volume was used and all measurements were made at 450 nm. Two micro-syringes Ž5 and 10 l. and two variable volume pipettors with disposable dispensers Ž0᎐2 ml. were used for measuring and transferring antibody, TMB substrate and stop solutions. All washing and color development reactions were performed in 5-ml glass beakers placed on an orbital shaker ŽBig Bill, Model M49125, with 3r4 inch drive. set at 100 rev.rmin.
C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
2.3. Immobilization of antibody 2.3.1. Piezocrystals The crystals were first immersed in 1 N NaOH for 10 min, rinsed twice with distilled water ŽDI. and air-dried. A similar treatment was performed with 1 N HCl. Finally, 50 l of conc. HCl were placed on the gold electrode Žone side only. for 2 min and which was rinsed two times with DI and air dried. Following this surface preparation, the antibody was immobilized using one of the following methods. To permit statistical assessment three crystals were used for each method. 2.3.2. Direct adsorption The surface prepared crystals were kept horizontally on a flat surface and 3 l of peroxidaselabeled E. coli antibody Ž1 grml. was spread on the gold electrode on one side of the piezocrystal. The surface was allowed to air dry and immediately transferred to 100% humid chambers and kept overnight at 4⬚C. On the following day, the crystals were first washed in PBS buffer, followed by two distilled water washings. 2.3.3. Protein A Using a microsyringe, 3 l of protein A was placed on the cleaned gold electrode of the crystal and gently spread Žon the gold electrode only. using a glass rod. The crystals were allowed to air dry. The crystals were then washed for 10 min in PBS buffer, followed by two distilled water washings. The crystals were again air dried and 3 l of peroxidase labeled E. coli antibody Ž1 grml. was spread on the gold electrode and washed as described in the direct adsorption method. 2.3.4. Glutaraldehyde Two microliters of 1% glutaraldehyde, 2 l of BSA Ž4 mgrml. and 3 l of peroxidase-labeled E. coli antibody Ž1 grml. was mixed and evenly spread onto the gold electrode of the crystals. The crystals were air dried and washed with PBS, followed by two washes with distilled water. 2.3.5. Polystyrene Solid polystyrene was crushed to small pieces and 4 mg of the crushed pieces was dissolved in 1
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ml of toluene. On one side of each crystal Žon gold electrode., 2 l of polystyrene solution was coated and allowed to air dry. The crystals were washed with PBS, followed by two washings with distilled water. The crystals were again air dried and 3 l of peroxidase-labeled E. coli antibody Ž1 grml. was spread onto the electrodes and washed as described earlier. 2.4. Membranes The three affinity membranes were cut to 1-cm2 pieces and 3 l of HRP-labeled E. coli antibody Ž1 grml. was placed at the center of the membrane. Due to the inherent absorbent property of the membranes, there was no need to spread the antibody on the surface. The membranes were incubated overnight in 100% humid chambers at 4⬚C. The membranes were placed in 5-ml beakers with PBS, which were placed on orbital shaker Ž100 rev.rmin, 3r4 inch drive. and washed for 10 min. The PBS wash was followed by two similar distilled water washings. All antibody immobilization Žon the membranes. quantification was performed in triplicate. 2.5. Color de¨ elopment Each of the antibody coated and washed crystalrmembrane was placed in a 5-ml beaker, with the coated surface facing up. To this beaker, 0.5 ml of TMB substrate was added and the beaker was immediately placed on an orbital shaker Ž100 rev.rmin.. The peroxidase enzyme, in presence of H2 O2 , catalyses the oxidation of colorless TMB substrate to a blue colored product. After a specific reaction time Ž0᎐25 min., 1.5 ml of TMB STOP reagent is added to terminate the reaction. In addition to terminating the reaction, the TMB STOP solution changes the color of the blue substrate to yellow and enhances the absorbance signal four to five fold. Exactly 10 min after stopping the reaction, the absorbance of the solution was measured at 450 nm. A 1:3 ratio of TMB:STOP was chosen to yield high color stability during the spectrophotometric measurements. Based on the absorbance value of the sample and
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predetermined calibration curves, the percent-immobilized antibody was determined.
3. Results and discussion 3.1. Effect of HRP enzyme-TMB substrate reaction time on absorbance The HRP enzyme induced TMB substrate oxidation reaction is limited by either unreacted substrate or hydrogen peroxide Žor other strong oxidizing agents.. For this study, 3 l of labeled antibody Ž1 grml. was coated on one side of the piezocrystals and the crystals were air-dried. The crystals were placed in 5-ml beakers and 0.5 ml of TMB substrate was added. The crystals were not washed with either PBS or distilled water. The beaker was immediately placed on an orbital shaker Ž100 rev.rmin. and the reaction was terminated after 2, 4, 6, 10, 15 and 25 min Žwith 1.5 ml TMB STOP solution.. This study was conducted to determine the optimum reaction time needed for the quantification of antibody attachment. Results from this study ŽFig. 1. indicate that the absorbance of the substrate Žafter the stop reagent was added. increased with increase in reaction time. However, after 10᎐12 min, the reaction appears to slow down, possibly due to the exhaustion of unreacted substrate or oxidizing agent ŽH2 O2 .. The calibration curves, developed with 6-, 10- and 20-min reaction times were linear within the range of 0᎐3 ng of labeled antibody. However, a reaction time of 10 min was chosen for all further studies, as it was important to have unreacted labeled antibody at the time of termination of the reaction.
Fig. 1. Effect of reaction time on the absorbance of TMB substrate.
9, 12, 15, 20, 30, 40, 90, 120, 180, 240 and 1440 min.. After the drying process, the crystals were not washed with PBS or DI water. To serve as a control, three crystals were coated with labeled antibody and kept in 100% humid chamber at 4⬚C for 1440 min Ž1 day.. The antibody remaining Žor immobilized. was estimated based on the colorimetric procedure described earlier ŽSection 2.5.. As mentioned earlier, a 10-min reaction time was chosen for these studies. Results indicate ŽFig. 2. that the enzymerantibody on the humid controls did not disintegrate significantly Ž95% confidence limits. during the 1440 min of incubation; thereby, assuring that the concept is a statistically acceptable procedure if all incubations are done in humid chambers. However, the air drying process seemed to drastically affect the absorbance; thereby, limiting this technique to humid incubation.
3.2. Effect of air drying on absorbance This study was conducted to assess the degree of disintegration of the enzyme andror antibody during the air drying process and to account for absorbance loss due to the drying process itself. The antibody-coated crystals Ž3 l of HRP labeled antibody, 1 grml. were allowed to air dry Žnot in humid chamber. for different durations Ž0, 3, 6,
Fig. 2. Effect of air drying of labeled antibody on the absorbance of TMB substrate after stopping the reaction after 10 min.
C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
Fig. 3. Calibration curve for HRP enzyme-TMB substrate reaction at 6 min reaction time. Reaction terminated after 6 min with TMB stop agent.
3.3. Calibration cur¨ es Three different calibration curves were developed for three different reaction times, namely 6, 10 and 20 min. For the calibration curves, different quantities of labeled antibody Ž0᎐3 ng. was added to 0.5 ml substrate in 5-ml beakers and the beakers were placed immediately on an orbital shaker Ž100 rev.rmin.. After specific reaction times Ž6, 10 and 20 min., the reaction was terminated and the absorbance of the solution was measured 10 min later. The reaction was directly performed in a beaker so as to facilitate its applicability to multiple biosensor surfaces Žsuch as piezocrystals, membranes, etc... Results ŽFigs. 3, Fig. 4 and Fig. 5. indicate that all the calibration curves are linear within the instrument’s absorbance range Ž0᎐4 absorbance .. Each individual point on these calibration curves is an average of
Fig. 4. Calibration curve for HRP enzyme᎐TMB substrate reaction at 10-min reaction time. Reaction terminated after 10 min with TMB stop agent.
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Fig. 5. Calibration curve for HRP enzyme᎐TMB substrate reaction at 20-min reaction time. Reaction terminated after 20 min with TMB stop agent.
three absorbance values. As anticipated, the longer reaction times lowered the range due to higher intensity of the end product. 3.4. Antibody immobilization quantification The antibody coated and washed crystals and the membranes Žin triplicate. were placed in 5-ml beakers and the TMB substrate was added, then the absorbance of the colored substrate was measured as mentioned in the methodology section. To serve as controls, a set of piezocrystals and affinity membranes were coated with 3 l of antibody and the color was developed immediately without any washing. For the controls, there was no significant variation in absorbance between different piezocoatings and different membranes; therefore, an average value was used as an indication of 100% antibody response. For each of the piezocrystal and affinity membrane, the amount of antibody immobilized after the washing procedure was determined as described in the color development section. Results of the coated piezocrystals ŽFig. 6. indicated that the polystyrene coated crystals had the highest degree of immobilization of 10.55% Žof what was applied., while the glutaraldehyde coated crystals not only had the lowest immobilization Ž2.36%. but also had the least precision Ž21.7% CV.. However, it should be noted that, unlike other crystal coatings, the glutaraldehyde layer has a depth factor associated with it and the color development may not be a true indication of the degree of immobilization. This is
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membrane has approximately 720 or 650% higher immobilization than the other.
4. Conclusion
Fig. 6. Antibody immobilization expressed as a percent of antibody applied on the various piezocrystal coatings. Polystyrene coated crystals had the highest attachment Ž10.55%. and the direct adsorption had the least coefficient of variation.
due to the fact that a part Žor whole. of the antibody could be embedded deeply in the glutaraldehyderBSA layer or the HRP-labeled site may not be accessible for the TMB substrate. However, antithesis to this potential drawback, if the HRP enzyme is attached at the same location as the antigen᎐antibody attachment site, this proposed technique could be extremely useful for the determination of the optimum glutaraldehyder BSArantibody proportions. With respect to affinity membranes, the Immobilon membrane was unquestionably superior with respect to antibody immobilization ŽFig. 7.. The antibody attachment was approximately 720% and 650% better compared to Ultrabind and Protran membranes, respectively. The Immobilon membrane also exhibited the best precision Ž2.5% CV., while Ultrabind and Protran had higher coefficients of variation Ž11.9% and 8.2%, respectively.. When compared with the variation with piezocrystals, with the exception of glutaraldehyde coatings, the average variability associated with the membranes was higher. This variation was most likely introduced during the washing procedure and standardizing the washing procedure might lower the variability. In statistical terms, although a CV of 11.9% may appear to be a high number, it does not undermine the importance or applicability of the overall procedure, especially when the results suggests that one
Currently, there is a great need for developing newer and improved methods of immobilization of antibodies on biosensorrtransducer surfaces. However, scientific selection of the right immobilization technique is a very difficult task. Assessment of antibody immobilization using the final antibody᎐antigen reaction may not necessarily be accurate, as certain known or unknown processes may account for a part or whole of the transducer response. The proposed paper describes a technique for scientific quantification of antibody immobilization on biosensor surfaces. The degree of antibody immobilization using four different pre-coatings on piezocrystals ŽDirect Adsorption, Protein A, Glutaraldehyde, Polystyrene. and three affinity membranes ŽUltrabind, Protran and Im m obilon . was assessed. Polystyrene-coated piezocrystals and the Immobilon membrane had the highest degree of immobilization Ž10.55% and 18.22%, respectively.. Results indicate that the proposed technique can be effectively applied to quantify the degree of immobilization on transducer or biosensor surfaces. Researchers who are screening several immobilization techniques w4,10,11x can utilize this technique as a secondary quantification tool or as
Fig. 7. Antibody immobilization expressed as a percent of antibody applied on the various affinity membranes. Immobilon had the highest attachment and lowest coefficient of variation.
C.S. Theegala, A.A. Suleiman r Microchemical Journal 65 (2000) 105᎐111
a pre-screening tool to select the optimum immobilization techniqueŽs.. This quantification of the degree of immobilization will not only help in screening for the best biosensorrtransducer surface, but also will facilitate maximization of antibody immobilization on a specific membrane or coating. For example, one could maximize the immobilization rate by varying the proportions of glutaraldehyde, BSA and antibody. Despite promising results, the technique has a varying degree of variability Ždepending on membrane or coating type. as evidenced by both very low and very high coefficient of variation Ž1.7 and 21.7%.. The variability associated with the attachment and washing procedure may be responsible for this variation. Standardizing the attachment and washing procedure may lower this variation. Irrespective of this variability, the importance or applicability of the overall procedure is not impaired significantly, especially when the results suggest that one crystal coating is 450% superior than another or one membrane has 650᎐720% higher immobilization than another. Furthermore, the overall technique is very simple, requiring a few immunosupplies and a visible spectrophotometer. The sensitivity of the overall technique is so high that each test requires only 3 l of diluted labeled antibody solution Ž1 grml..
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Acknowledgements This work was supported by a research center in Minority Institutions, Grant RR-09104, from the National Center for Research Resources, National Institutes of Health. References w1x O.A. Sadik, J.M. Van Emon, Designing Immunosensors for Environmental Monitoring, Chemtech, 1997, pp. 38᎐46. w2x S.H. Si, J.H. Chen, F.J. He, L.H. Nie, S.Z. Yao, Chin. J. Chem. 14 Ž3. Ž1996. 222᎐227. w3x H. Gao, R. Luginbuhl, H. Sigrist, Sensors Actuators B Chem. 38 Ž1᎐3. Ž1997. 38᎐41. w4x B. Konig, M. Gratzel, Anal. Lett. 26 Ž8. Ž1993. 1567᎐1585. w5x J.T. Li, J. Carlsson, J.N. Lin, K.D. Caldwell, Bioconjugate Chem. 7 Ž5. Ž1996. 592᎐599. w6x H. Ghourchian, N. Kamon, Anal. Chim. Acta 300 Ž1-3. Ž1995. 99᎐105. w7x X. Chu, Z.H. Lin, G.L. Shen, R.Q. Yu, Analyst 120 Ž12. Ž1995. 2829᎐2832. w8x J. Lagace, S. Arsenault, E.A. Cohen, J. Immunol. Methods 175 Ž1. Ž1994. 131᎐135. w9x K. Nakanishi, H. Muguruma, I. Karube, Anal. Chem. 68 Ž1996. 1695᎐1700. w10x B.S. Attili, A.A. Suleiman, Anal. Lett. 28 Ž12. Ž1995. 2149. w11x F. Caruso, E. Rodda, D.N. Furlong, J. Colloid Interface Sci. 178 Ž1996. 104᎐115.