Immunochemical Approaches for Purification and Detection of TNT

A highly sensitive immunochemical method for immunoaffinity purification (IAP) ..... In one study, ELISA was employed for detection of TNT traces on t...
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Anal. Chem. 2001, 73, 2461-2467

Immunochemical Approaches for Purification and Detection of TNT Traces by Antibodies Entrapped in a Sol-Gel Matrix Miriam Altstein,*,†,‡ Alisa Bronshtein,‡ Baruch Glattstein,§ Arie Zeichner,§ Tsippy Tamiri,§ and Josef Almog§

Institute of Plant Protection, The Volcani Center, Bet Dagan 50250, Israel, and Division of Identification and Forensic Science, Israel National Police Headquarters, Jerusalem 91906, Israel

A highly sensitive immunochemical method for immunoaffinity purification (IAP) and detection of trace amounts of TNT was developed on the basis of antibodies (Abs) in a ceramic matrix (sol-gel). The study resulted in: (i) a highly sensitive and reproducible TNT ELISA (I50 and I20 values of 0.4 ( 0.09 ppb and 0.12 ( 0.03 ppb, respectively; n ) 12), which is highly specific to TNT; and (ii) successful entrapment of the Abs that bound free analyte from solution. Binding was found to be highly reproducible, dose dependent, and only slightly (1.2-1.8-fold) lower than that in solution. The entrapped Abs did not leach from the matrix and were tolerant of absolute ethanol, acetone, and acetonitrile. Bound analytes could be easily eluted from the sol-gel matrix at high recoveries. The sol-gel-based IAP method described above introduces a simple one-step procedure that has a high potential to serve as a suitable and convenient immunochromatographic device for cleanup and concentration of TNT from “real field” samples in a manner that complies with both chemical and immunochemical residue analysis methods. In recent years, there has been an increasing need to have the ability to identify trace amounts of explosives such as 2,4,6trinitrotoluene (TNT) in two major fields: (i) in environmental monitoring of contaminants in soil and water near sites of buried explosives,1 and (ii) in post-explosion analysis, which usually includes traces of the unreacted material.2 Currently, the common analytical methods for TNT detection include high-pressure liquid chromatography (HPLC) with ultraviolet (UV) absorption,3,4 electrochemical5,6 or mass spectrometric † Present address: Department of Cell Biology and Neurosciences, Boyce Hall, Room 5419, University of California Riverside, Riverside, California 925210146. Phone: 909-536-3812. ‡ The Volcani Center. § Israel National Police Headquarters. (1) Wallenberg, S. R.; Bailey, C. G. Anal. Chem. 2000, 72, 1872-1878. (2) Beveridge, A. Forensic Investigation of Explosions; Tailor and Francis Publishers: London, 1998; pp 231, 314. (3) Weisberg, C. A.; Ellickson, M. L. Am. Lab. 1998, 32N-32V. (4) Ko ¨hne, A. P.; Dornberger, U.; Welsch, T. Chromatographia 1998, 48, 9-16. (5) Bratin, K.; Kissinger, P. T.; Briner, R. C.; Bruntlett, C. S. Anal. Chim. Acta 1981, 130, 295-311. (6) Lewin, U.; Efer, J.; Engewald, W. J. Chromatogr. A, 1996, 730, 161-167.

10.1021/ac001376y CCC: $20.00 Published on Web 04/28/2001

© 2001 American Chemical Society

detection,7 or gas chromatography (GC) with chemiluminescence8 or mass spectrometric (MS) detection. In many cases, these methods are susceptible to interferences by the sample matrix because of the presence of irrelevant compounds such as plasticizers, oils, and paints.9 In this study, we introduce two immunochemical approaches, based on the specific ability of antibodies (Abs) to bind an antigen (target analyte), to improve the detectability of trace amounts of TNT explosives: immunoaffinity purification (IAP) and enzyme immunoassays (EIA). Immunochemical methods, such as IAP and EIA, have been in use for many years in the pharmaceutical and biomedical fields for purification, isolation, and identification of a variety of medically important compounds.10,11 In recent years, EIA has been integrated into the agrochemical, environmental, and forensic fields for the determination of pesticide residues, xenobiotics, environmental contaminants, and explosives.12-16 Commercial kits for ∼50 compounds are available, and >200 assays have been described in the literature. Among the environmental contaminants that have been studied in the past decade, TNT is of major concern because of its biological persistence, toxicity, and mutagenicity,17,18 and more than dozen EIA and immunosensors have been developed for its detection at the sites of former ammunition plants or military installations and in groundwater.19-26 TNT is also of high interest in forensic contexts, and its monitoring by (7) Beveridge, A. Forensic Investigation of Explosions; Tailor and Francis Publishers: London, 1998; pp 283, 306. (8) Beveridge, A. Forensic Investigation of Explosions; Tailor and Francis Publishers: London, 1998; pp 234, 237. (9) Kolla, P. J. Forensic Sci. 1991, 36, 1342-1359. (10) Wilchek, M.; Miron, T.; Kohn, J. Methods Enzym. 1984, 104, 3-55. (11) Yalow, R. S. Annu. Rev. Biophys. Bioeng. 1980, 9, 327-345. (12) Van Emon, J. M., Geriach, C. L., Johnson, J. C., Eds. Environmental Immunochemical Methods: Perspectives and Applications; ACS Symposium Series 646; American Chemical Society: Washington, D.C.; 1996. (13) Sherry, J. P. Chemosphere 1997, 34, 1011-1025. (14) Meulenberg, E. P.; Mulder, W. H.; Stoks, P. G. Environ. Sci. Technol. 1995, 29, 553-561. (15) Hock, B. Acta Hydrochim. Hydrobiol. 1993, 21, 71-83 (16) Fetterolf, D. D.; Mudd, J. L.; Teten, K. J. Forensic Sci. 1991, 36, 343-349. (17) Levine, B. S.; Furedi, E. M.; Gordon, D. E.; Barkely, J. J.; Lish, P. M. Fundam. Appl. Toxicol. 1990, 15, 373-380. (18) Spanggord, R. J.; Mortelmans, K. E.; Griffin, A. F.; Simon, V. F. Environ. Mutagen. 1982, 4, 163-179. (19) Heiss, C.; Weller, M. G.; Niessner, R. Anal. Chim. Acta. 1999, 396, 309316. (20) Schuetz, A. J.; Winklmair, M.; Weller, M. G.; Niessner, R. Fresenius J. Anal. Chem. 1999, 363, 625-631.

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enzyme-linked immunosorbent assay (ELISA) on exposed body parts has been reported.16 Unlike EIAs, which are becoming quite common, implementation of IAP is still very limited. IAP, which uses the capability of Abs to bind to specific analytes and separate them from a complex matrix, provides a promising approach to analyte purification and concentration. The method is simple, cost-effective, highly specific to the target analyte, and most important, is fully compatible with EIAs as well as with chemical analytical methods, such as HPLC, GC, and GC combined with MS. Successful application of IAP requires, first and foremost, immobilization of the Abs, which is in many cases, a lengthy, multistep process. Consequently, there is a definite need for simplified methods of Ab immobilization and the introduction of new, simple, chemically stable, and nonreactive matrixes to exploit the full analytical potential of this approach. Sol-gel technology,27 which enables the incorporation of bioactive molecules into ceramics, glasses, and other inorganic materials,27-30 offers promising solutions for all of the above requirements. The sol-gel process is a method for preparation of inorganic oxide matrixes of metals and semimetals by direct hydrolysis and polycondensation of active monomeric precursors. The resulting matrixes have a large surface area; high porosity, inertness, and stability to chemical and physical agents; and visible and UV optical clarity. The reactions are performed at room temperature (RT), thus enabling organic and bioorganic molecules to be entrapped within the forming silica network. The biomolecules, which are strongly encapsulated within the matrix and cannot diffuse out, generally retain their activity, gain higher stability, and can react with ligands that diffuse into the highly porous matrix. The enhanced stability of the entrapped biomolecules and the physical and chemical properties of the matrix are among the reasons for the attractiveness of the sol-gel approach to immobilization in general and to that of proteins in particular. As a result, the sol-gel chemical route to materials has been studied quite intensively in recent years, resulting in many biomaterials having diverse applications.28-47 (21) Pfortner, P.; Weller, M. G.; Niessner, R. Fresenius J. Anal. Chem. 1998, 360, 781-783. (22) Kra¨mer, P. Anal. Chim. Acta, 1998, 376, 3-11. (23) Narang, U.; Gauger, P. R.; Ligler, F. S. Anal. Chem. 1997, 69, 1961-1964. (24) Bart, J. C.; Judd, L. L.; Hoffman, K. E.; Willkins, A. M.; Kusterbeck, A. W. Environ. Sci. Technol. 1997, 31, 1505-1511. (25) Keuchel, C.; Niessner, R. Fresenius J. Anal. Chem. 1994, 350, 538-543. (26) Whelan, J. P.; Kusterbeck, A. W.; Wemhoff, G. A.; Bredehorst, R.; Ligler, F. S. Anal. Chem. 1993, 65, 3561-3565. (27) Brinker, C. J., Scherer, G. W., Eds. Sol-Gel science: the Physics and Chemistry of Sol-Gel Processing; Academic Press: Boston, 1990. (28) Avnir, D., Braun, S., Eds. Biochemical Aspects of Sol-Gel Science and Technology. Kluwer Academic Publishers: Boston, 1996. (29) Livage, R. C. R. Acad. Sci. Paris, II. 1996, 322, 417-427. (30) Avnir, D.; Braun, S.; Lev, O.; Ottolenghi, M. Chem. Mater. 1994, 6, 16051614. (31) Gill, I.; Ballesteros, A. J. Am. Chem. Soc. 1998, 120, 8587-8598. (32) Gill, I.; Ballesteros, A. Trends Biotechnol. 2000, 18, 282-296. (33) Tess, M. E.; Cox, J. A. J. Pharm. Biomed. Anal. 1999, 19, 55-68. (34) Wang, J.; Pamidi, P. V. A.; Rogers, K. R. Anal. Chem. 1998, 70, 11711175. (35) Pope, E. J. A.; Braun, K.; Peterson, C. M. J. Sol-Gel Sci. Tech. 1997, 8, 635-639. (36) Cichna, M.; Knopp, D.; Niessner, R. Anal. Chim. Acta 1997, 339, 241250. (37) Cichna, M.; Mark, P.; Knopp, D.; Niessner, R. Chem. Mater. 1997, 9, 26402646.

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The successful application of the sol-gel doping methodology to a wide variety of proteins and enzymes and the above-mentioned long list of potential advantages of the sol-gel technique prompted us to extend the range of these bioactive materials to include additional enzymes and Abs for agricultural and environmental immunosensing applications. In previous studies, we successfully entrapped enzymes and Abs in the sol-gel matrix.48-52 In the present study, we have extended the scope of the technique by the entrapment of TNT Abs for further characterization of the sol-gel-entrapped Abs and their development as an IAP device for clean-up and concentration of these compounds for environmental and forensic purposes. MATERIALS AND METHODS Antibodies. Monoclonal anti-TNT immunoglobulins (IgGs) (IgG concentration 10.6 mg/mL >99% purity; Strategic BioSolutions; DE) was used throughout the study. Immunochemical Methods. Preparation of Trinitrophenyl (TNP)-Ovalbumin (OV)-Coating Antigen. The coating antigen was prepared according to Habeeb.53 Briefly, 1 mL of 0.001 mg 2,4,6-trinitrobenzenesulfonic acid (TNBS, Sigma) was added to 1 mg of OV (Sigma) that was dissolved in 2 mL of 4% NaHCO3, pH 8.5 (0.2:1 molar ratio of hapten:carrier). The reaction was allowed to react at 40 °C for 2 h, then 1 mL of 10% sodium dodecyl sulfate (SDS, 99%; Sigma) was added to the mixture, followed by the addition of 0.5 mL of 1 N HCl. The reaction mixture was dialyzed against 5 L of 10 mM sodium phosphate buffer containing 28 mM NaCl, pH 7.2. The product was used as the coating antigen for the ELISA described below. TNT Two-Step Competitive ELISA. The assay that was developed was the two-step competitive ELISA, in which tested compounds or TNT standard (kindly provided by the Israel National Police) in solution competed with an antigen-carrier conjugate immobilized on a 96-well microtiter plate for binding to anti-TNT IgG. The assay served to determine: (i) crossreactivity of anti-TNT IgGs, (ii) the amount of free TNT that did not bind to the sol-gel-entrapped IgG or to the IgGs in solution, and (iii) the amount of eluted TNT from the sol-gel immunoaffinity purification (IAP) columns. (38) Armon. R.; Dosoretz, C.; Starosvetsky, J.; Orshansky, F.; Saadi, I. J. Biotechnol. 1996, 51, 279-285. (39) Livage, J.; Roux, C.; Da Costa, J. M.; Desportes, I.; Quinson, J. F. J. Sol-Gel Sci. Technol. 1996, 7, 45-48. (40) Jordan, J. D.; Dunbar, R. A.; Bright, F. V. Anal. Chim. Acta, 1996, 332, 83-91. (41) Kawakami, K. Biotech. Tech. 1996, 10, 491-494. (42) Sampath, S.; Lev, O. Anal. Chem. 1996, 68, 2015-2021. (43) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 6, 1120A-1127A. (44) Wang, R.; Narang, U.; Prassad, P. N.; Bright, F. V. Anal. Chem. 1993, 65, 2671-2675. (45) Audebert, P.; Demaille, C. Chem. Mater. 1993, 5, 911-913. (46) Glezer, V.; Lev, O. J. Am. Chem. Soc., 1993, 115, 2533-2534. (47) Lan, E. H.; Dunn, B.; Zink, J. L. Chem. Mater. 2000, 12, 1874-1878. (48) Altstein, M.; Aharonson, N.; Segev, G.; Turniansky, A.; Avnir, D.; Bronshtein, A. Ital. J. Food Sci. 2000, 12, 191-206. (49) Altstein, M.; Segev, G.; Aharonson, N.; Ben Aziz, O.; Turniansky, A.; Avnir, D. J. Agric. Food Chem. 1998, 46, 3318-3324. (50) Bronshtein, A.; Aharonson, N.; Avnir, D.; Turniansky, A.; Altstein, M. Chem. Mater. 1997, 9, 2632-2639. (51) Bronshtein, A.; Aharonson, N.; Turniansky, A.; Altstein, M. Chem. Mater. 2000, 12, 2050-2058. (52) Turniansky, A.; Avnir, D.; Bronshtein, A.; Aharonson, N.; Altstein, M. J. SolGel Sci. Tech. 1996, 7, 135-143. (53) Habeeb, A. F. S. A. Anal. Biochem. 1966, 14, 328-336.

Wells of microtiter plates (NUNC Maxisorp; Roskilde, Denmark) were coated with 200 µL of TNP-OV conjugate diluted 1:250 in 0.5 M carbonate buffer (CB), pH 9.6. After an overnight (ON) incubation at 4 °C, the wells were washed three times with phosphate-buffered saline (140 mM NaCl in 50 mM sodium phosphate, pH 7.2; PBS) containing 0.1% (v/v) Tween-20 (PBST), and 100 µL of test (unknown) samples or standard (12 serial dilutions of TNT ranging from 0.001 to 2 ng/well) were added to the wells, together with 100 µL anti-TNT IgG (diluted 1:80 000 in PBST). Plates were incubated ON at 4 °C and washed as above with PBST, and 200 µL of a secondary Ab (goat anti-mouse horseradish peroxidase GAM-HRP, Sigma), diluted 1:3000 in PBST, was added to the plates. The plates were incubated for 2 h at RT, rinsed with PBST, and tested for HRP activity by the addition of 200 µL of substrate solution containing 96 µg/mL tetramethylbenzidine (TMB) and 0.004% H2O2 in 0.1 M sodium acetate buffer, pH 5.5. The reaction was stopped after 10-20 min by the addition of 100 µL of 4 M sulfuric acid, and the absorbance was measured using a Labsystems Multiscan Multisoft ELISA reader at 450 nm. Content of TNT in unknown samples was determined by comparison with a TNT calibration curve. Each sample was tested in duplicate at five dilutions. Only samples that paralleled the calibration curve were considered. Cross reactivity was determined similarly by comparing the ability of various diand trinitroaromatic compounds to compete with the immobilized TNP-OV conjugate on the anti-TNT IgG. Determination of Ab Titers by ELISA. The procedure was essentially similar to that of the two-step competitive ELISA, except that after an ON adsorption of the TNP-OV conjugate onto the plate and the washing, 200 µL of the tested Ab sample (undiluted or diluted 1:2, 1:4, 1:8, 1:16 in PBST) or anti-TNT IgGs (diluted 1:80 000 to 1:2 560 000 in PBST) were added to the wells and incubated ON at 4 °C. Plates were washed as above with PBST, 200 µL of GAM-HRP diluted 1:3000 in PBST, was added to the wells, and the plates were processed as described above for the TNT ELISA. Binding of TNT to IgGs in Solution. Anti-TNT purified IgGs (2.5 µL corresponding to 26.5 µg) were incubated with 30-1000 ng of TNT in a total volume of 400 µL of PBS, and the mixture was incubated for 15 min at RT on a slow shaker. Nonspecific binding was determined in the presence of an identical amount of normal mouse serum (NMS) IgG (NMS-IgG, Sigma) under similar conditions. At the end of the incubation, bound TNT was separated from the unbound compound by means of Centricon30 tubes (Millipore, Bedford, MA). The bound complex was washed twice with 400 µL of PBS, and the amount of unbound material was determined by a two-step competitive ELISA as described above. Sol-Gel Entrapment of IgGs. Sol-Gel Entrapment of AntiTNT IgGs. The entrapment was carried out by a two-step procedure in which hydrolysis was followed by polymerization of tetramethoxysilane (TMOS, ABCR, 99%, Karlsruhe, Germany). An acidic silica sol solution was obtained by mixing TMOS with 2.5 mM HCl in double-distilled water (DDW) at a molar ratio of 1:8 in the presence of 10% polyethylene glycol (PEG-400; Merck, Germany; average molecular weight, 400 g/mol, corresponding to approximately seven methylene units in the chain). The mixture was stirred for 1 min until a clear solution was obtained and was

then sonicated for 30 min in an ELMA ultrasonicator bath (model T-460/H, 285 W, 2.75 L; Singen-Hohentwiel, Germany). The reaction was carried out in a well-ventilated fume hood. Anti-TNT IgGs (2.5 µL, corresponding to 26.5 µg, unless otherwise indicated) and similar protein amounts of NMS-IgG were premixed with 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, 99.99%; Sigma), pH 7.5, to a final volume of 0.5 mL and were added to an equivolume amount of prehydrolyzed TMOS. The solution was quickly mixed for 5 s, and gelation occurred within 1-2 min. After 10 min, the gels (total volume, 1 mL) were washed with 2 mL of HEPES buffer, pH 7.5, and were kept wet (with 2 mL of HEPES buffer, pH 7.5, on top) at 4 °C until use. Best results were obtained using gels that were stored ON at 4 °C and used on the second day after preparation. Gels exhibited high stability and could be used for over 2 months after preparation. Binding of TNT to Sol-Gel-Entrapped Anti-TNT IgGs. Wet gels were thoroughly crushed, transferred into inverted 5-mL plastic syringes, and packed in 1-mL columns. The sol-gel columns were washed with 50 mL of PBS prior to sample application. For optimal binding, columns were kept under buffer at all times during the experiment. TNT (20 ng unless otherwise indicated) was applied in a volume of 1 mL of PBS to the sol-gel column. Unbound TNT was washed out using 20 mL of PBS (termed the wash-through fraction) and was tested for TNT content by the two-step competitive ELISA, as described above. Binding experiments were performed uisng sets of three solgel columns: (A) an experimental column containing anti-TNT IgG, (B) a control column (for the determination of nonspecific binding, e.g., adsorption onto all sites other than the active site of the Abs) containing NMS-IgG (NMS-IgG was used for the determination of nonspecific binding because the Ab was generated in mice), and (C) an empty control column with no protein. The protein concentration in all of the columns was the same, regardless of the nature of the entrapped protein. The extent of binding to column A is referred to below as total binding, and that to columns B and C, as nonspecific binding. Specific binding (i.e., binding to the active site of the Ab) was defined as the difference between the total binding and the nonspecific binding. Binding in all cases was highly reproducible, and the variations between experiments did not exceed 5%. Elution of TNT from Sol-Gel-Entrapped Abs. Elution of TNT was performed with sol-gel columns doped with 2.5 µL of antiTNT IgG to which 10-300 ng TNT was applied. After the regular binding procedure, columns were washed with 20 mL of PBS, and elution was performed using 2 mL of absolute ethanol (99.8%), acetone (99.8%), or acetonitrile (99.9%). The TNT content of the eluate was determined by ELISA as described above, after diluting the samples (to reduce the concentration of the organic solvent, which interferes with the ELISA) to a final concentration of 10%. Leaching of Abs from the Sol-Gel Matrix. Anti-TNT (10 µL, corresponding to 106 µg of IgG) was entrapped in the sol-gel matrix as described above. Columns were washed with 50 mL of PBS, followed by an additional rinse with 10 mL of eluting solvent (absolute ethanol) or PBS. The sample was concentrated using Centiplus YM-30 (Millipore; Bedford, MA) and brought up to 2 mL of PBS. Ab titers were determined by ELISA (see above) Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

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Table 1. Binding Capacity of Sol-Gel-entrapped Anti-TNT IgGsa TNT applied on column, ng

Figure 1. Dose response curve of TNT binding to sol-gels doped with anti-TNT IgGs. A constant amount (20 ng) of TNT was applied on sol-gel columns doped either with 0.3-10 µL (3.2-10.6 µg) of anti-TNT IgG or with equivalent amounts of NMS-IgG (to determine nonspecific binding).

against a calibration curve of anti-TNT IgGs that underwent a similar concentration. RESULTS AND DISCUSSION Development of a TNT ELISA. The first part of the study involved the development of a sensitive TNT ELISA for the analysis of pre- and post-IAP fractions. The development of the TNT ELISA involved two sets of experiments: The first was intended to determine the optimal concentration of the coating conjugate (TNP-OV), IgGs, and secondary Ab. The second experiment was intended to determine the I50, limit of detection (I20), and cross reactivity of the Ab. The first set of experiments revealed that a 1:250 dilution of the TNP-OV conjugate and a 1:160 000 dilution (final) of the anti-TNT IgG resulted in high binding and a low background. The second set of experiments revealed that the I50 and the I20 values for TNT were 0.4 ( 0.09 ppb and 0.12 ( 0.03 ppb, respectively; n ) 12. No cross reactivity was observed under the tested conditions with dinitro-(2,4dinitrophenol, 2,4 -dinitrophenylhydrazine, m-dinitrobenzene, 2,6dinitrotoluene, 2,4-dinitroaniline, 2,6-dinitroaniline, and 2,4dinitrotoluene) or trinitro-(2,4,6-trinitroaniline, 2,4,6-trinitrophenol, and 1,3,5-trinitrobenzene) compounds (data not shown). Ab Entrapment in Sol-Gel and TNT Binding. Preliminary requirements in the development of an IAP are the determination of optimal conditions for Ab immobilization and the assessment of optimal binding conditions of the analyte. Our preliminary experiments with a variety of biomolecules [antiatrazine monoclonal antibodies (Mabs) and IgGs, anti-DNP polyclonal Abs and IgGs, and enzymes of the cholinesterase group] indicated that the best sol-gel format for protein entrapment was a wet gel prepared by the two-step procedure at a silane:water ratio (r) of 1:8, containing 10% PEG.49-52 On the basis of the above results, we chose the same procedure and the same sol-gel composition for the entrapment of the TNT Mabs. All of the experiments in this part were performed using a standard TNT compound prepared from stock solutions of 1 mg/mL dissolved in acetone. The first experiments in this part were intended to determine the binding of a constant amount of TNT (20 ng) to varying amounts of encapsulated anti-TNT IgGs. The data in Figure 1 indicate that the entrapped Abs bind free TNT in a saturable manner and that the binding exhibited dose dependency with 2464 Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

2.5 µL IgG 10 30 100 300 10 µL IgG 100 300 1000 3000

nonspecific binding, ng

specific binding, ng

specific binding, %

0 6 0 0

10 26 38 90

100 87 38 30

9 9 0 4

99 213 278 640

99 71 28 21

a Binding was performed using 26.5 and 106 µg of sol-gel-entrapped anti-TNT IgG. Nonspecific binding was monitored using equivalent amounts of NMS-IgG. Binding is calculated as the difference between the total amount of TNT applied on the column and the unbound TNT that was detected (by ELISA) in the wash-through fraction.

respect to the amount of entrapped Abs. Specific binding ranged from a minimum of 5% at 0.3 µL IgGs to a maximum of 80% at 10 µL. In most of the experiments, the extent of nonspecific binding ranged between 20 and 28% of the applied analyte (Figure 1). Binding to nondoped sol-gel columns (that contained no protein) was in the same range. Nonspecific binding was not affected by the protein load or by the amount of analyte applied on the solgel column (see below). Characterization of Sol-Gel-Entrapped Abs. Determination of Binding Capacity. The binding capacity with respect to TNT load on the column was determined using constant amounts of entrapped Abs (2.5 and 10 µL) and amounts of TNT analyte ranging from 10 to 300 ng and from 100 to 3000 ng, respectively. The data in Table 1 reveal dose dependency and maximal capacities of 90 and 640 ng TNT, for 2.5 and 10 µL of IgG, respectively; namely, a 4-fold increase in the amount of entrapped Abs resulted in a 7-fold increase in binding capacity, hinting that the binding ratio of analyte:Ab in the sol-gel (1.75:1) might not differ significantly from that in solution (2:1) (see below). The nonspecific binding did not increase with the increase in the applied dose and remained low (10-15% interfered with the binding of analytes. Examination of TNT binding to sol-gel-entrapped anti-TNT IgGs in the presence of organic solvents (acetone, ethanol, and acetonitrile) revealed that Ab-TNT interactions were not affected, up to a concentration of 20% (data not shown). The enhanced stability of the entrapped Abs to the organic solvents, as indicated by the above sets of experimental findings can be attributed, most probably, mainly to the protective nature of the matrix, which reduces the freedom of peptide chain refolding and causes the denaturation and inactivation of biomolecules. The ability of the cage silanols to bind the protein at several of its sites is another factor contributing to the enhanced stability of the entrapped biomolecules. Previous studies performed in several laboratories, including ours, revealed that sol-gel entrapment facilitates long-term storage of Abs and enzymes at room temperature40,48,49 and protects against damage caused by drastic thermal and pH changes.30,55,56 The stability the sol-gel imparts to entrapped biomolecules introduces a major advantage, because it enables elution of target analytes from sol-gel IAP columns under harsh conditions (e.g., extreme pH buffers or organic solvents), which are necessary in cases of a strong Ab-antigen interaction, and it also facilitates the prolonged storage and ensures long shelf life of ready-to-use prepacked IAP columns. Leaching of Sol-Gel Entrapped Abs. Another important aspect of the development of an IAP method is the ability to maintain the Abs firmly attached to the supporting matrix. Theoretically, leaching can be a much more severe problem in sol-gel than in other methods because of the high porosity of (54) Zu ¨ hlke, J.; Knopp, D.; Niessner, R. Fresenius, J. Anal. Chem. 1995, 352, 654-659. (55) Chen, Q.; Kenausis, G. L.; Heller, A. J. Am. Chem. Soc. 1998, 120, 45824585. (56) Shtelzer, S.; Rappoport, S.; Avnir, D.; Ottolenghi, M.; Braun, S. Biotech. Appl. Biochem. 1992, 15, 227-235.

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Table 3. Elution of Bound TNT from Sol-Gel Anti-TNT-Doped Columns with Various Organic Solventsa eluting solvent acetone ethanol acetonitrile

bound TNT ng

eluted TNT ng

elution recovery %

18 17 18

22 18 18

122 106 100

a Binding was performed using 2.5 µL of sol-gel-entrapped antiTNT IgG and 20 ng of TNT. Bound TNT represents the difference between the total amount of TNT applied on the column and the unbound TNT that was detected (by ELISA) in the wash-through fraction. Elution was performed using 2 mL of solvent. Eluted TNT was determined by ELISA after diluting the sample to a final concentration of 10% solvent.

the matrix and the fact that Abs are not covalently bound to it. In a previous study we found that extensive washing of sol-gelentrapped biomolecules (anti-atrazine Abs, anti-DNP antiserum, and cholinesterases) with PBS or an eluting solvent (glycine-HCl buffer, pH 3.5) resulted in negligible leaching that was, in most cases, below the detection limit of the assay.49-51 In the present study we further tested the effect of an eluting solvent (ethanol) on leaching. The data revealed that no leaching occurred and that the amount of Abs found in the wash-through was below the limit of detection of the assay. Evaluation of this limit was determined from an Ab standard curve that covered a dilution range of 1:40 000 to 1:1 280 000. We found that the amount of the Ab that was present in 200 µL of PBS and ethanol wash-through (out of a total volume of 2 mL) corresponded to