Environ. Sci. Technol. 2007, 41, 2888-2893
Development of a Luminex Based Competitive Immunoassay for 2,4,6-Trinitrotoluene (TNT) GEORGE P. ANDERSON,* JACQUELINE D. LAMAR, AND PAUL T. CHARLES Center for Bio/Molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, D.C.
Previously, a displacement immunoassay for 2,4,6trinitrotoluene (TNT) was demonstrated using the Luminex 100. The work presented utilized this same specialized flow cytometer to demonstrate a highly sensitive and rapid competitive immunoassay for TNT. This required a TNT analog to be attached to the microsphere surface. Various linkers were evaluated; bovine serum albumin provided over 3 times more binding sites in comparison to various shorter diamine linkers. For this assay TNB-coated microspheres were added to samples; then biotinylated antiTNT antibody and the reporter molecule, Streptavidin-RPhycoerythrin, were added. In the absence of TNT, a highly fluorescent complex was formed on the surface of the microsphere. The presence of TNT resulted in dose-dependent decreased fluorescence. Various anti-TNT antibodies were evaluated; Mab 30-1 gave the strongest response, yielding the lowest limit of detection (10 000 >10 000
100 1413 14.9 1.2 2.5 nd nd
a
nd: No data.
TABLE 2. Analysis of Acetone Extracts of TNT Contaminated Soil FIGURE 1. Competitive assay comparing various Bt-anti-TNT antibodies. Bt-Mab 30-1 binding to the TNB-BSA-coated microspheres generated the largest signal. The Bt-Mab-anti-RDX (negative control) generated negligible signal response.
soil sample extract (TNT conc. µg/L) dilution factor
G18L1
G55XAa
7.47 6.41 6.42 6.07 4.42 3.59 2.92
33.31 22.10 16.67 14.91 18.73
92.03 58.34 57.22 51.27 42.21 31.83 13.69
5.38 6.2
18.01 16.2
52.26 238
G18L3
G51L1
G16L2
100 200 400 800 1600 3200 6400
1.01 1.23 1.16 7.98
1.85 2.14 1.78 1.46 2.58 8.14 5.42
averageb HPLC
1.13 1.7
1.77 1.5
a G55XA was diluted an additional factor of 10. b The average value of the TNT concentration (µg/L) was obtained by averaging the values obtained from the responsive portion of the standard curve, values shown in bold. For the HPLC tests, single samples were analyzed using EPA method 8330-50:50 MeOH/H20 at a flow rate of 1.4 mL/min with 25-cm C18 Altech Altima column. Samples were diluted 1:10 with 50:50 MeOH/H20 prior to injection. The four samples with less TNT were concentrated 10-fold by evaporation of the acetone with a N2 stream prior to HPLC analysis.
FIGURE 2. The percent displacement for TNT and various nitrocompounds is plotted. These values were determined by performing a displacement assay similar to those shown in Figure 1 for each compound. The curves shown were used to calculate the concentration of each compound at 50% inhibition and the percent cross reactivity in comparison to TNT, shown in Table 1.
and bovine serum albumin (BSA) were obtained from SigmaAldrich (St. Louis, MO). Mouse monoclonal antibodies specific for ricin and SEB (Mab Ric-07-A-G1, and Mab SEB03b2a) were the kind gifts of Dr. Robert Bull (Naval Medical Research Center, Silver Spring, MD). Mab 3D11 anticholera toxin was purchased from Biodesign International, Saco, MA. Cholera toxin was from Calbiochem (San Diego, CA), ricin from Vector (Burlingame, CA), and SEB from Toxin Technology Inc. (Sarasota, FL). Biotinylation of Antibodies. The antibodies were biotinylated using NHS-LC-Biotin (Pierce) dissolved in dimethyl sulfoxide (1.4 g/L). Before the addition of the biotin, the pH of the antibody solution was increased by the addition of half-volume of 0.1 M sodium tetraborate/0.1 M NaCl, pH 9.1. The antibodies were reacted with a 5:1 molar excess of the NHS-LC-Biotin for 1 h at room temperature, followed by separation from free biotin by gel filtration on a Bio-gel P10 column (Bio-Rad, Hercules, CA). Biotinylated (Bt) antibody concentrations were determined by UV absorbance at 280 nm. Preparation of the TNB-Coated Microspheres. Microspheres were activated using the standard protocol for two
FIGURE 3. TNT competitive assays in various matrices. TNT spiked into samples prepared in either 90% fresh water, 30% seawater, and 90% seawater, with the remainder being PBSTB, were tested. These samples all responded to the addition of TNT in a manner similar to using the standard conditions (100% PBSTB). step carbodiimide coupling chemisty provided by the manufacturer. After the final wash step of the activation protocol, the microspheres were resuspended in 0.1 mL solutions of three diamine compounds: ethylenediamine, 1, 5-diaminopentane, and 1, 8-diaminooctane, at final concentration of 0.1 M in 0.1 M sodium tetraborate/0.1 M sodium chloride. Additional activated microspheres were VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Percent control response profiles for multiplex competitive immunoassays on the Luminex 100. A is the TNT assay; B is the SEB assay; C is the ricin assay; D is the cholera toxin assay. Four sets of microspheres were mixed, Lx-BSA-TNB, Lx-SEB, Lx-ricin, and Lx-CTX, and then added to all the samples that contained various concentrations of TNT or one of the toxins. Then a mixture containing Bt-Mab 30-1, Bt-Mab SEB03B2A, Bt-Mab Ric07AG1, and Bt-Mab 3D11 (each at 0.5 µg/mL and SA-RPE at 8 µg/mL) was added to each well. After incubating for 40 min the samples were evaluated sequentially by the Luminex 100. The median fluorescence intensity was adjusted to a percent of the control response to permit display of all four assays on the same scale. Each assay showed concentration-dependent inhibition by the respective antigen concentrations, with little to no-response to nontarget antigens. resuspended in 0.1 mL of the borate buffer containing 10 mg/mL BSA. All sets were vortexed to resuspend and then stored in the dark overnight. Microspheres were then washed twice using 0.1 M carbonate buffer, pH 8.0, by centrifugation and then resuspended in carbonate buffer containing 2,4,6trinitrobenzene sulfonic acid (0.1 M). After a 2 h incubation period, the TNB coated microspheres were washed twice with PBS containing 0.05% Tween 20 and 1 mg/mL BSA (PBSTB), then resuspended in 0.5 mL of PBSTB and stored in the dark at 4 °C until use. A volume of 0.5 µL of resuspended microspheres provided over 100 microspheres per assay. Toxin and BSA-coated microspheres were prepared using the same standard protocol, coating 100 ul of various bead sets (34, 54, 58, and 77) with at least 100 µg protein (cholera toxin, BSA, ricin, and SEB respectively) at g1 mg/mL. Luminex-Based Standard TNT Competitive Assay. For the TNT standard competitive assay, each well in the columns to be utilized was filled with 60 µL of PBSTB. Then in the top row, 6 µL of 100 µg/mL TNT was added and mixed. Ten-fold serial dilutions were then prepared down the column, leaving the eighth row as PBSTB only. Microspheres were diluted with PBSTB so that 5 µL could be added to each well. The Bt-anti-TNT antibody (5 µg/mL), Bt-Mab 30-1 for most assays, was premixed with SA-RPE (40 µg/mL) for at least 5 min. Then 6 µL of the antibody-SA-RPE mixture was added to each well. The entire plate was shaken briefly to mix and then held in the dark for at least 30 min prior to measuring on the Luminex 100. Mixed Competitive Assays. The standard protocol was utilized except that the Luminex bead mixture consisted of microspheres coated with TNB-BSA, ricin, SEB, and cholera toxin. A Bt-antibody mixture was utilized which contained 5 µg/mL Bt-Mab 30-1, Bt-Mab SEB03b2A, Bt-Mab-Ric-07AG1, and Bt-Mab 3D11 anti-Cholera toxin. To this mixture was added SA-RPE (50 µg/mL). The assay was then completed as described above. 2890
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Results Several initial experiments, not shown, were performed to determine the amount of Bt-anti-TNT antibody and SA-RPE required to achieve optimal assay results. In addition, TNB attached to microspheres via linkers of different lengths were compared for their responsiveness in the standard TNT competitive assay. This test was completed using two of the available Bt-anti-TNT antibodies: Bt-Mab 30-1 and Bt-Mab A1.1.1 (Figure S1). Lx-BSA-TNB microspheres produced 3-4 times more fluorescence as compared to any other bead set, indicating that the anti-TNT antibodies bound more effectively to their surface. Thus, the Lx-BSA-TNB microspheres were used in subsequent experiments. Next, each available anti-TNT antibody was tested in the competitive assay (Figure 1); Bt-Mab 30-1 produced the largest response. Bt-Mab 30-1 and A1.1.1 appeared most sensitive responding to 1 ng/mL TNT, Bt-Mab 5-1 and 2E3 responded at 10 ng/mL, and while Bt-ScFv TNB 12H also responded at 10 ng/mL, it exhibited a very small total change; limits of detection based on a change >3 SD from background. To better evaluate these antibodies, their equilibrium binding to the TNB-coated microspheres was determined at a series of concentrations and each antibody’s dissociation constant (Kd) determined as described previously, see Figure S2 (27, 28). The resultant Kd was lowest for Bt-Mab 30-1 and Bt-A1.1.1, as one would have expected from their relative sensitivities. Once an optimal TNB coated microsphere and an anti-TNT antibody was selected, we examined how this antibody in this competitive assay cross-reacted to other nitroamines and nitroaromatics. Using the same assay format as the standard TNT competitive assay, cross-reactants were tested to determine their inhibition of antibody binding. Figure 2 shows the results of this assay, and Table 1 shows the calculated percent cross reactivities. TNB proved more effective than TNT in competing for the binding of Mab 30-
1, while tetryl, 2,4-DNT, 2A-2,4-DNT, and 2,6-DNT were progressively less effective than TNT. RDX showed virtually no cross reactivity, displacing no more than the acetonitrile control (not shown). Analysis of acetone extracts from soil samples was also performed. Soil samples were obtained from archived material collected at Umatilla Army Depot Activity (26); acetone extracts were prepared as described previously (23). All extracts were diluted by a factor of 100 or more to prepare the first tested dilution; then subsequent 1:1 dilutions were prepared. The value obtained for each dilution tested was fit to the standard curve, Figure S3. The dilutions that gave a response fit by the standard curve are shown in Table 2. The values that fit to the more accurate portion of the curve (1 ng/mL and 100 ng/mL) were averaged (yellow shaded values in Table 2). The averages compared very closely to the values determined by HPLC (23) for the four lower concentration samples; the high concentration sample also tested very high, but was a factor of 4 lower than measured by HPLC. Effectiveness of the competitive assay for analysis in complex matrices was also evaluated. Samples prepared in either 90% fresh water, 30% seawater, and 90% seawater, with the remainder being PBSTB, were tested. These samples all responded to the addition of TNT similar to the standard conditions (100% PBSTB), as shown in Figure 3. Results did suggest various matrices may slightly alter signal levels, thus for quantitative results, the standard curve should be performed in the matrix being evaluated. The multiplexing capabilities of Luminex 100 with inclusion of a TNT competitive assay was demonstrated by combining the TNT assay with competitive assays for three protein toxins: ricin, cholera toxin (CTX), and staphylococcal enterotoxin B (SEB) (Figure 4). Panels A, B, C, and D show the response to increasing concentrations of TNT, SEB, ricin, and CTX, respectively. In each case microspheres coated with competing antigen rapidly lost signal as antigen concentration in the sample was increased. These results indicated that competitive assays are easily multiplexed. However, limits of detection may be negatively impacted, as signal response from microsphere sets with irrelevant antigen appeared to vary by approximately 10%. The final experiment was to evaluate the on-rate of the competitive assay. Figure 5 shows that in the absence of free antigen, TNT, antibody binds very quickly, reaching a median fluorescence intensity of 1481 units in only 1 min, while 10 ng/mL TNT greatly depressed the binding rate, allowing only 422 units over the same time period. This equates to approximately a 70% reduction in signal.
Discussion The Luminex 100 has proven to be a highly versatile platform for multiplexed immunoassays (6-10). The work presented here evaluated a competitive immunoassay for TNT. Initial efforts explored a displacement assay, using TNB-coated microspheres saturated with Bt-anti-TNT antibody and SARPE prior to sample addition. This method yielded minimal displacement of bound antibody even at high TNT concentrations (not shown), thus was abandoned in favor of a standard competitive immunoassay format. To create a microsphere coated with an effective TNT analog the carboxyl microspheres, provided by Luminex, required conversion to free amino groups to facilitate reaction with TNB sulfonic acid. A series of diamine linkers as well as BSA were tested as the intermediary. Unexpectedly, we found the microspheres prepared using the BSA linker superior to the short diamino-alkane linkers (Figure S1). This result suggests a number of possibilities, from ineffective coupling of the diamino-alkanes, to an increased number of amines provided by BSA, or an increased affinity of the anti-TNT antibodies to TNB attached to surface via BSA.
FIGURE 5. On-rate analysis. The binding of the Bt-Mab 30-1(0.5 µg/mL) SA-RPE (4 µg/mL) to TNB-BSA microspheres was monitored as a function of time and free TNT concentration. Panel A shows the response during the first 4 min (240 s), and panel B shows the response over a 40 min time period (2400 s). Distinct differences in binding were observed within 1 min between 0 and 10 ng/mL TNT. Perhaps, BSA proved best, since the antibodies were developed by immunizing animals with TNT analogs attached to proteins. We next evaluated the available anti-TNT antibodies for use in the competitive assay; Bt-Mab 30-1 generated the largest signal. Results shown in Figure 1 compared to those by Charles et al. (26) showed a remarkable correspondence. The estimated limits of detection for the various antibodies were also similar to the standard ELISA competitive assay (26) and the Luminex displacement immunoassay (23). Zeck et al. (22) also evaluated Mab A1.1.1 in a competitive assay; utilizing immobilized antibody and a fluorescent analog of TNT. They estimated their detection limit to be 0.06 ng/mL; however, they used a very small amount of analog and the response range topped out at 1 ng/mL. Their results do imply that Luminex competitive assay could be reformulated for extreme sensitivity; an area for future investigation. Kds were also determined for the anti-TNT antibodies (Figure S2). Mab A1.1.1 and Mab 30-1 had the lowest Kds (∼10 nM) while Mab 5-1 and Mab 2E3 were both about twice that value, while the measured Kd for single chain antibody was very high, >300 nM. Mab A1.1.1 is the only antibody for which a Kd had been reported (∼0.8 nM) (22). Several explanations are possible for this much lower Kd, their method was different, and in actuality, measured the antibody’s affinity for a fluorescent analog, as opposed to TNT, just as our measurement actually measures the antibody’s affinity for BSA-TNB. Mab 30-1’s observed cross reactivity to nitro-compounds was similar to that found by the competitive ELISA (26). This VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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work found TNB to be 1413 vs 514%, tetryl to be 14.9 vs 8%, and 2,4-DNT to be 2.5 vs 20% determined via ELISA. While far from exact matches, the correspondence was much better than that observed by the Luminex displacement assay that found tetryl to be more effective than TNT (187%) and TNB to be less effective (80%). Since displacement utilized a different TNT analog than the competitive assays, it was not surprising that its cross reactivity pattern had less in common than the two competitive assays, which both used TNB-BSA on the capture surface. The competitive assay on the Luminex platform was also evaluated for its ability to quantify acetone extracts of soil samples (Table 2). The concentrations determined for the four samples containing low to medium concentrations of TNT were nearly identical to the values obtained by HPLC. Sample G55XA, which contained an extremely high concentration TNT, was a factor of 4 lower; part of this variance might result from the dilution (1000-fold+) necessary for analysis by this assay, while the HPLC tested that sample with minimal dilution. To evaluate the Luminex TNT competitive assay’s utility for use in complex matrices, experiments were conducted using 90% fresh water from Lake Michigan and 30% and 90% seawater from the Gulf of Mexico. TNT samples tested in these matrices gave responses similar to the TNT standard curve in PBST (Figure 3). The previously utilized displacement assay had required dilution to 30% to avoid loss of the microspheres (32); however, no dilution was required for the competitive assay. These results suggest this assay will produce equivalent results in fresh or marine waters without interferences from salts. The competitive assay could also be multiplexed. In addition to the TNB microspheres, three sets of toxin-coated microspheres were tested. While small molecule assays would have been preferred, the toxin assays were available and served to showcase the systems multianalyte capability. The final test, perhaps the most important in terms of transitioning the Luminex competitive assay to surveillance applications due to the necessity to obtain rapid high throughput answers, was the on-rate evaluation. While the standard protocol required an excess of 30 min, the on-rate binding experiment showed that an automated detection system could perform rapid analyses (1-2 min), while still maintaining a low limit of detection (e10 ng/mL). In summation, the Luminex based TNT competitive assay was shown to be highly sensitive (e1 ng/mL) using our standard protocol, but by monitoring inhibition of binding the measurement could potentially be performed rapidly (1-2 min). The undemanding requirements, having an available antibody and attaching an analyte analog to the microsphere surface, make this assay easy to replicate for numerous targets. This format should allow rapid automated screening for numerous compounds simultaneously. These features make Luminex an attractive choice for the simultaneous screening for compounds such as explosives like TNT, RDX, and PETN, or other environmental hazards.
Acknowledgments J.D.L. was supported by the National Science Foundation Science and Engineering Apprentice Program. This research was supported by the Office of Naval Research. We also thank BAE Systems for providing use of the Luminex 100. The views expressed here are those of the authors and do not represent the opinions of the U.S. Navy, the U.S. Department of Defense, or the U.S. Government.
Supporting Information Available Figure S1 shows the comparison of using various linkers to attach TNB to microspheres; Figure S2 shows the equilibrium 2892
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binding curves of the various anti-TNT antibodies and their calculated Kds; Figure S3 shows the TNT standard curve used for calculating the values shown in Table 2. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Kaplan, D. L.; Kaplan, A. M. 2,4,6-trinitrotoluene surfactant complexes-decomposition, mutagenicity, and soil leaching studies. Environ. Sci. Technol. 1982, 16, 566-571. (2) Won, W. D.; Disalvo, L. H.; Ng, J. Toxicity and mutagenicity of 2,4,6-trinitrotoluene and its microbial metabolites. Appl. Environ. Microbiol. 1976, 31, 576-580. (3) Roberts, W. C.; Commons, B. J.; Bausum, H. T.; Abermathy, C. O.; Murphy, J. J.; Khanna, K; Ohanian, E. V. Overview of the Health Effects of Selected Munitions Chemicals; EPA 822-R-93022; U.S. Environmental Protection Agency: Washington, DC, 1993. (4) Shiver-Lake, L. C.; Patterson, C. H.; van Bergan, S. K. New horizons: Explosive detection in soil extracts with a fiber-optic biosensor. Field Anal. Chem. Technol. 2000, 4, 239-245. (5) Charles, P. T.; Gauger, P. R.; Patterson, C. H.; Jr.; Kusterbeck, A. W. On-site immunoanalysis of nitrate and nitroaromatic compounds in groundwater. Environ. Sci. Technol. 2000, 34, 4641-4650. (6) Biagini, R. E.; Smith, J. P.; Sammons, D. L.; MacKenzie, B. A.; Striley, C. A. F.; Robertson, S. K.; Snawder, J. E. Simultaneous measurement of specific serum IgG responses to five select agents. Anal. Bioanal. Chem. 2004, 379, 368-374. (7) Dunbar, S. A.; Vander Zee, C. A.; Oliver, K. G.; Karem, K. L.; Jacobson, J. W. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP (TM) system. J. Microbiol. Methods 2003, 53, 245-252. (8) Jia, X. C.; Raya, R.; Zhang, L.; Foord, O.; Walker, W. L.; Gallo, M. L.; Haak-Frendscho, M.; Green, L. L.; Davis, C. G. A novel method of Multiplexed Competitive Antibody Binning for the characterization of monoclonal antibodies. J. Immunol. Methods 2004, 288, 91-98. (9) Kellar, K. L.; Douglass, J. P. Multiplexed microsphere-based flow cytometric immunoassays for human cytokines. J. Immunol. Methods 2003, 279, 277-285. (10) McBride, M. T.; Gammon, S.; Pitesky, M.; O’Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents. Anal. Chem. 2003, 75, 19241930. (11) Bromberg, A.; Mathies, R. A. Multichannel homogeneous immunoassay for detection of 2, 4, 6-trinitrotoluene (TNT) using a microfabricated capillary array electrophoresis chip. Electrophoresis 2004, 25, 1895-1900. (12) Buttner, W. J.; Findlay, M.; Vickers, W.; Davis, W. M.; Cespedes, E. R.; Cooper, S.; Adams, J. W. In situ detection of trinitrotoluene and other nitrated explosives in soils. Anal. Chim. Acta. 1997, 341, 63-71. (13) Goldman, E. R.; Cohill, T. J.; Patterson, C. H.; Anderson, G. P.; Kusterbeck, A. W.; Mauro, J. M. Detection of 2,4,6-trinitrotoluene in environmental samples using a homogeneous fluoroimmunoassay. Environ. Sci. Technol. 2003, 37, 4733-4736. (14) Goldman, E. R.; Hayhurst, A.; Lingerfelt, B. M.; Iverson, B. L.; Georgiou, G.; Anderson, G. P. 2,4,6-Trinitrotoluene detection using recombinant antibodies. J. Environ. Monit. 2003, 5, 380383. (15) Ju ¨ licher, P.; Mussenbrock, E.; Renneberg, R.; Cammann, K. Broadening the antibody specificity by hapten design for an enzyme-linked immunoassay as an improved screening method for the determination of nitroaromatic residues in soils. Anal. Chim. Acta. 1995, 315, 279-287. (16) Medintz, I. L.; Goldman, E. R.; Lassman, M. E.; Hayhurst, A.; Kusterbeck, A. W. K.; Deschamps, J. R. Self-assembled TNT biosensor based on modular multifunctional surface-tethered components. Anal. Chem. 2005, 77, 365-372. (17) Psillakis, E.; Naxakis, G.; Kalogerakis, G. Detection of TNTcontamination in soil samples using SPME and GC/MS. Global Nest 2000, 2, 227-236. (18) Shankaran, D. R.; Gobi, K. V.; Sakai, T.; Matsumoto, K.; Imato, T.; Toko, K.; Miura, N. A novel surface plasmon resonance immunosensor for 2,4,6-trinitrotoluene (TNT) based on indirect competitive immunoreaction: A promising approach for onsite landmine detection. IEEE Sensors J. 2005, 5, 616-621. (19) Wang, J.; Hocevar, S. B.; Ogorevc, B. Carbon nanotube-modified glassy carbon electrode for adsorptive stripping voltammetric
(20) (21)
(22)
(23)
(24)
detection of ultratrace levels of 2,4,6-trinitrotoluene. Electrochem. Commun. 2004, 6, 176-179. Wilson, R.; Clavering, C.; Hutchinson, A. Electrochemiluminescence enzyme immunoassays for TNT and pentaerythritol tetranitrate. Anal. Chem. 2003, 75, 4244-4249. Xu, X.; van de Craats, A. M.; de Bruyn, P. C. Highly sensitive screening method for nitroaromatic, nitramine and nitrate ester explosives by high performance liquid chromatographyatmospheric pressure ionization-mass spectrometry (HPLCAPI-MS) in forensic applications. J. Forensic Sci. 2004, 49, 11711180. Zeck, A.; Weller, M. G.; Niessner, R. Characterization of a monoclonal TNT-antibody by measurement of the crossreactivities of nitroaromatic compounds. Fresenius´ J. Anal. Chem. 1999, 364, 113-120. Anderson, G. P.; Moreira, S. C.; Charles, P. T.; Medintz, I. L.; Goldman, E. R.; Zeinali, M.; Taitt, C. R. TNT detection using multiplexed liquid array displacement immunoassays. Anal. Chem. 2006, 78, 2279. Bakaltcheva, I. B.; Shriver-Lake, L. C.; Ligler, F. S. A fiber optic biosensor for multianalyte detection: importance of preventing fluorophore aggregation. Sens. Actuators, B 1998, 51, 46-51.
(25) Danilova, N. P. ELISA screening of monoclonal-antibodies to haptens-influence of the chemical-structure of hapten-protein conjugates. J. Immunol. Methods 1994, 173, 111-117. (26) Charles, P. T.; Shriver-Lake, L. C.; Francesconi, S. C.; Churilla, A. M.; Rangassammy, J. G.; Patterson, C. H.; Deschamps, J. R.; Kusterbeck, A. W. Characterization and performance evaluation of in vivo and in vitro produced monoclonal anti-TNT antibodies for the detection of TNT. J. Immunol. Methods 2004, 284, 1526. (27) Seideman, J.; Peritt, D. A novel monoclonal antibody screening method using the Luminex-100 (TM) microsphere system. J. Immunol. Methods 2002, 267, 165-171. (28) Waterboer, T.; Sehr, P.; Michael, K. M.; Franceschi, S.; Nieland, J. D.; Joos, T. O.; Templin, M. F.; Pawlita, M. Multiplex human papillomavirus serology based on in situ-purified glutathione S-transferase fusion proteins. Clin. Chem. 2005, 51, 1845-1853.
Received for review September 29, 2006. Revised manuscript received February 6, 2007. Accepted February 8, 2007. ES062333N
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