Immunochemical Technology for Environmental Applications

Chapter 13

Optimization of Electrochemiluminescence Immunoassay for Sensitive Bacteria Detection

Downloaded by STANFORD UNIV GREEN LIBR on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch013

Hao Yu Calspan Systems Research Laboratories Corporation, Building E3549, Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD 21010

Electrochemiluminescence Immunoassay(ECLIA) offers high sensitivity for detection of antigens in solution. However, conventional ECLIAs developed for bacteria detection are not optimal and many factors which may affect the ECLIA results still remain unclear. In this report, ECLIA kinetics, antibody biotinylation and non-specific binding were investigated. Results of these studies showed that the system optimization in ECLIA could substantially increase detection limit by at least 10-fold compared to conventional ECLIA.

Electrogenerated chemiluminescence (ECL) as a highly sensitive detection technology has received considerable attention in chemical analysis and clinical diagnostics (1-4). Applications of E C L immuno- and nucleic acid-based assays for biological and environmental sample analysis were reported (5-7). The principle of ORIGEN ECL (IGEN, Gaithersburg, MD) has been described previously(4). Briefly, an ECL employs a metal chelate, ruthenium (Il)-trisbipyridyl, (Ru(bpy) ), in redox reaction which are conducted on the surface of an anode in the presence of electron carriers, tripropylamine (TPA). Both (Ru(bpy) ) and TPA are oxidized to (Ru(bpy) and TPA+, respectively. Deprotonated TPA+ spontaneously becomes TPA-, which creates a high energy state (Ru(bpy) *). Relaxation of (Ru(bpy) *) emits photons at 622 nm on the surface of the electrode. In the E C L Immunoassay (ECLIA), antibody-coated magnetic particles are used as primary capturing antibody and (Ru(bpy) )-conjugated antibodies, (Ru-Ab), as Tag-label to generate the E C L signal. The quantity of captured antigens is determined by measuring the ECL intensity at 622 nm. Sample media does not contribute any ECL signals. Unlike the fluorescence and chemiluminescence assays, the ECLIA have higher signal to noise (S/N) ratios and better reproducibilities (4, 6). That is because the noise contributed by natural fluorescence from biological samples remains at a minimum in ECLIA. Furthermore, unlike conventional chemiluminescence assays, the ECLIA reproducibilities are under well-controlled electric potentials and does not depend upon the substrate concentration or reaction time. Additionally, unlike Radio Immunoassay (RIA), the ECLIA is an non-radio immunoassay and is safe to use. 2+

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© 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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We have demonstrated that the ECLIA can be used for sensitive bacteria detection. Detection of biotoxoids at femtogram level. Detection of 100 spores/mL in buffer, and 2000 cells/mL of E. coli in environmental and food samples have been reported (5-7). This report focuses on the study of assay kinetics, non-specific binding, and varying molar incorporation ratios (biotin to protein (b/p)) in order to optimize ECLIA for the detection of biological agents, such as B. subtilis var. niger, Salmonella typhimurium, E. coli 0157:H7 and Yersinia pestis.


Materials and Methods Immunochemical Reagents. Goat anti-5. subtilis var. niger polyclonal antibody was obtained from the United States Naval Medical Research Institute. Polyclonal goat antiSalmonella sp. and anti-£. coli 0157 antibodies were obtained from the Kirkegaard Perry Lab.(KPL; Gaithersburg, MD). Mouse mti-Yersinia pestis ( F l positive) monoclonal antibody was obtained from BioDesign International (Kennebunk, ME). A l l antibodies were affinity purified. Biotin-DA/P-hydroxysuccinimide (NHS) (spacer arm 2.24 nm, Molecular Devices, Sunnyvale, CA), was used for antibody biotinylation. The initial b/p ratios used for conjugation ranged from 5 to 25. The final b/p molar ratios obtained ranged from 1.2 to 5.8 as determined using the absorbance measurement of each conjugate at 280 nm and 362 nm according to manufacturer's protocol (Molecule Device, Sunnyvale, CA). Ru(bpy) -NHS,(Ru-NHS) was purchased from IGEN for Ru(bpy) -antibody label. Molar ratios of Ru-antibody (Ru-Ab) conjugates were determined by absorbance at 280 and 455 nm. A final molar ratio of 4 was obtained. Irradiated B. subtilis var. niger spores and anti-K Pestis Fl-antigen (control #C092) were obtained from the United States Army Medical Research Institute for Infectious Diseases (Ft. Detrick, MD). Irradiated E. coli 0157:H7 and heat-killed Salmonella typhimurium were obtained from Dr. Jerry Crawford (United States Department of Agriculture) and KPL, respectively. A l l cells were centrifuged at 12,000 x(g) and resuspended in phosphate buffered saline (PBS) before use. Cell counts were determined by a hemacytometer. Ten-fold cell dilutions were made from a stock solution of 5xl0 cells/mL. Only 10 mL from each dilution was used in ECLIA. The final volume for the ECLIA was 280 mL as described (5). 2+

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Magnetic Particles. Streptavidin (SA)-coated polystyrene magnetic particles with 2.8 and 4.8 mm diameter (M-280 and M-450) were obtained from Dynal (Lake Success, NY). Super paramagnetic microbeads (#481-01) coated with SA were obtained from Miltenyi Biotec (Sunnyvale, CA). Organic polymer based, SA-coated BioMag paramagnetic particles (8-4680, size around 1 mm in diameter) were purchased from Advanced Magnetics (Cambridge, MS). SA-coated Magnetic Pore Glass (MPG) paramagnetic glass particles (MSTR0502) were given as a gift by M P G (Lincoln Park, NJ). ECLIA, and Fluorescence Microscopy. All immunoassays described in this report used sandwich immunoassay format. Biotin-Ab and Ru-Ab from the same animal species were used for primary and secondary antigen capture, respectively. The magnetic particles along with captured antigen-antibody complex were collected on the surface of electrode by a permanent magnet. There was no wash step involved during the E C L reaction. Finally, the E C L signal (at 622 nm) was measured by a photomultiplier tube. The E C L signal intensities were proportional to the antigen concentrations. The conventional ECLIA protocols used in this report were described previously (5, 7).

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS


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A fluorescence microscopic study was performed on an Olympus BH-2 microscope for examination of the particle natural fluorescence. The excitation/emission filters (490/525 nm) were selected. Results The ECLIA results using different b/p molar ratios are shown in Figure 1. Results indicated that the ECLIA intensities increased as the b/p molar ratios increased. Saturation of ECLIA intensities occurred when the b/p molar ratios increased up to 4 or greater in these experiments. B. thuringiensis spores at 10 cells/mL were used as negative control (-). Negative control also increased as the b/p molar ratio increased. Three types of SA-coated magnetic particles were examined by fluorescence microscopy. Some features of these magnetic particles were summarized (data not shown). The comparative results revealed that both SA-Dynabeads and SA-BioMag beads have minimal non-specific binding compared to S A - M P G beads under the same experimental conditions. Furthermore, fluorescence microscopy results revealed that Dynabeads (M-2.8 and M-4.5) emit broader auto-fluorescence at 520 nm. Within the visible wavelength range (such as 490 to 520 nm), the use of Dynabeads will interfere with the Fluorescein Isothiocyanate (FITC) based assays. SA-BioMag particles and SA-MPG beads, on the other hand, are good candidates for fluorescent assays because there are no natural fluorescence emitted from these beads. Dynabeads have stronger magnetization and spherical shape compared to other types of magnetic beads. Therefore, in an ECLIA, Dynabeads are good candidates for rapid capturing and rapid wash-off from the surface of the electrode. Natural fluorescence from magnetic particles is not a concern in ECLIA, therefore, Dynabeads are the best choice over others in ECLIA application. Polyclonal anti-£. coli and anti-Salmonella sp., and monoclonal anti-Yersinia pestis antibodies were selected for ECLIA kinetic studies. Goat anti-Salmonella sp. (1000 and 2000 cells/mL), anti-E. coli (500 and 1000 cells/mL) were used. Negative controls (-) (without antigens in both sandwich immunoassays) were used. Results of kinetic measurement in Figure 2a shows that the ECLIA intensities are proportional to the reaction time when polyclonal Ru-Ab is added into pre-incubated Biotin-Ab and antigen complex. However, when primary and secondary antibodies were monoclonal, the ECLIA intensities were saturated after 30 minutes reaction time (Figure 2b). The increase in reaction time also slightly increased the intensities of the negative control (-) in experiments using either polyclonal or monoclonal antibodies. When using polyclonal antibodies, kinetic results suggest that the longer the reaction time between Ru-Ab and Biotin-antibody-antigen complex is, the better the S/N ratios will be. The optimal reaction time for the ECLIA for Y. pestis using monoclonal antibodies is about 30 minutes. In Figures 3 and 4, the conventional ECLIA and optimized ECLIA are compared. Unlike the 2% serum used in conventional protocol, SuperBlock Blocking Buffer (Pierce, Rockford, IL) was used in optimized ECLIA for magnetic bead dilutions. An extended biotin spacer (2.24 nm) was used in current biotinylation with the b/p ratio of 4 compared to 1.35 nm biotin-Ab spacer used in the conventional assay. Reaction between the antigens and primary antibody was carried in ice for 5 minutes and incubation time with Ru-Ab was about 50 minutes using polyclonal and up to 30 minutes using monoclonal antibodies. These incubation time is significantly different compared to the time in conventional assays. A l l reactions were carried in polystyrene test tubes instead of glass tubes. Results of optimized ECLIA show at least 10-fold intensity increase (compared to signal to noise > 3) for B. subtilis var. niger and E. coli detection compared to the conventional assays previously reported.


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12 -•— Sal.1,000 cells/mL Sal.2,000 cells/mL 10 -A- E.c. 500 cells/mL

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Figure 2a. ECLIA kinetic studies using polyclonal anti-E. coli 0157:H7 (E. c.) and anti-Salmonella sp. (Sal.)antibodies.

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Figure 2b. ECLIA kinetic studies using monoclonal anti-Y. pestis ( F l positive) antibody.


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Figure 4. Results of conventional and optimized ECLIA for E. coli cell detection.


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Discussion In a sandwich immunoassay, antibodies from different species are used for antigen capturing (9). This allows the antibodies to interact with different epitopes of the target antigens. However, in this investigation the same antibodies (either goat IgG polyclonal or mouse IgG monoclonal antibodies) were used as primary and secondary antibody capture. In current ECLIA for micron-sized spores and intact bacterial cells capturing, Biotin-Ab coated on the beads are allowed to interact with antigens prior to adding the Ru-Ab. A competitive reaction between Ru-Ab and Biotin-Ab on the same antigen binding sites will begin after adding Ru-Ab. In addition to the competitive reaction, polyclonal Biotin- and Ru-Ab could also bind to different binding sites (multiple sites or epitopes) on the surface of those antigens. ECLIA results have demonstrated that the current sandwich immunoassay format can be used for bacteria cell capturing, including either using anti-5. subtilis spores and anti-Salmonella (polyclonal) or anti-K pestis (monoclonal) antibodies. In addition, the binding sites are potentially available for both Biotin-Ab and RuAb since no covalent bonds are formed between the antibody and antigen. The on/off rate of the free antigens to antibodies (Biotin-Abs and Ru-Abs) depends upon their equilibrium-constant. This constant could vary if the antibodies were modified by probe labels (in the current case, such as biotin and Ru(bpy) )(70). The b/p molar ratio seems to play an important role on the detection limit of the assay. Gretch and coworkers (77) reported that molar ratios of biotin to antibody which were equal to or greater than 8 had the maximum antigen recovery potential on SA-conjugated agarose matrix. Even higher b/p molar ratios were reported in the application of flow cytometry by labelling growth factor (72). Our ECLIA results show that b/p molar ratios between 4 and 6 were optimal in our experimental conditions. The b/p molar ratios greater than 6 did not significantly improve the ECLIA detection limit. Experiments also suggested the optimal b/p ratios have to be determined in each experiment. However, the high molar ratios of Biotin-NHS to antibody could potentially disrupt the antibody-binding capability to antigens (72). Reaction times in ECLIA are critical. The longer the reaction time is, the higher the intensities are. However, over an extensive incubation, the Ru-Ab potentially could adhere to the magnetic particles. This may explain why the negative controls (-) of ECLIA results increased slightly during the longer incubation. In our lab. 2.5% to 5% serum in PBS, 3% BSA and 10% skim milk as blocking solutions were investigated (data not shown). Five percent goat serum seemed to perform well when goat polyclonal antibodies were used in ECLIA. The use of SuperBlock Blocking Buffer provides the best blocking solution, to preventing protein non-specific binding in the presence of magnetic carriers. Optimization of ECLIA showed the enhancement of assay sensitivity (linear range) and detection limits for bacteria detection. About 10-fold increase in detection limit in optimized assay (approximately 5000 cells/mL with S/N ratio of 4.1) compared to the conventional assay (50000 cells/mL with S/N ratio of 4.9). The detection limit is approximately 10 cells/mL with S/N ratio of 3.28 in optimized assay compared to the conventional assay at 100 cells/mL (S/N ratio: 3). ECLIA time is about 1 hour including the extended 50 minutes incubation time. In addition, the reaction between SA-beads and Biotin-Ab is almost instant because of the high affinity constant (K = 10 M" ) between SA and biotin. There is no need to extend this pre-incubation time in ECLIA. It is interesting to note that the ECLIA kinetics of polyclonal and monoclonal antibodies are different. In the polyclonal antibody case, the ECLIA responses were directly proportional to the incubation time up to 5 hours. This suggests that a competition process gradually occurred between the Biotin-Ab and Ru-Ab on the available multiple antigen binding sites. At the same time, the Ru-Ab could be adhered 2+

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to magnetic beads even in the presence of blocking solution. Therefore, proportional ECLIA responses versus reaction time are expected. In using monoclonal antibody, the ECLIA responses reached a plateau around 30 minutes. Result suggest that a strong and specific antibody-antigen binding process occurred and that the mono-binding sites are occupied by either Biotin-Ab or Ru-Ab. Fifteen to 30 minutes reaction time is optimal under current ECLIA condition when monoclonal antibodies are employed. The ECLIA is a relative new technology. Besides the major advantage of no natural fluorescence involved in detection. Some disadvantages also should be taken into account. If samples contain some chemicals, such as proline, oxalate, gentamicin, streptomycin and N A D H , the ECLIA could cause false positive readings upon internal electron transfer from these chemicals (13). ECLIA technique has been successfully used as an alternative method in clinical and environmental applications for biological agent detection. It is believed that the ECLIA technique also could be applied in environmental monitoring for sensitive bacteria detection.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Norffsinger, J.B.; Danielson, N.D. Anal. Chem. 1987, 59, 865-868. Danielson, N.D.; He, L.; Norffsinger, J.B.; Trelli, L.; J. Pharm. Biomed. 1989, 7, 1281-1285. Uchikura, K.; Kirisawa, M . ; Anal. Sci. 1991, 7, 803-804. Blackburn, G.F.; Shah, H.P.; Kenten, J.H.; Leland, J.; Kamin, R.A.; Link, J.; Peterman, J.; Powell, M.J.; Shah, A.; Talley, D.B.; Tyagi, S.K.; Wilkins, E.; Wu, T.; Massey, R.J.; Clinical Chem. 1991, 37, 1534-15. Gatto-Menking, D.L.; Yu, H.; Bruno, J.G.; Goode, M.T.; Miller, M . ; Zulich, A.W.; Biosensors and Bioelectronics 1995, 10, 501-507. Yu, H.; Bruno, J.G.; Cheng, T.; Calomiris, J.J.; Goode, M.T.; Gatto-Menking, D.L.; J. of Biolum. Chemilum. 1995, 10, 239-245. Yu, H.; Bruno, J.G.; Applied and Environmental Microbiology 1996, 62, 587-592. Leland, J.K.; Powell, M.J.; J. of Electrochem. Soc. 1990, 137, 3127-3131. Immunological Reagents for Research catlog, Jackson ImmunoResearch Laboratories, Inc. West Grove, Pennsylvania 1993, 6. Bredehorst, R.; Wemhoff, G.A.; Kusterbeck, A.W.; Charles, P.T.; Thompson, R.B.; Ligler, F.S.; Vogel, C.W.; Anal. Biochem. 1991, 193, 272-279. Gretch, D.R.; Suter, M . ; Stinski, M.F.; Anal. Biochem. 1987, 163, 270-277. Jong, Marg O.D.; Rozemuller, H.; Bauman, Jan G.J.; Visser, Jan W . M . ; J. of Immunol. Methods 1995, 184, 101-112. Savage, M.D; Mattson, G.; Desai, S.; Nielander, G.W.; Morgensen, S.; Conklin, E. J.; Avidin-Biotin Chemistry: A Handbook, Chapter 2, Pierce Chemical Company, 1992. Lee, W.Y.; Neiman, T.A.; Anal. Chem. 1995, 67, 1789-1796.


Immunochemical Technology for Environmental Applications

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