Application of an Immunomagnetic Assay System for Detection of

Chapter 25

Application of an Immnnomagnetic Assay System for Detection of Virulent Bacteria in Biological Samples 1

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Downloaded by UNIV OF GUELPH LIBRARY on October 8, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch025

Hao Yu and Peter J. Stopa 1

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Calspan SRL Corporation and SCBRD-RTE, Building E 3549, U.S. Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD 21010 Virulent pathogenic bacteria could pose a serious health threat in contaminated food and water resources. Traditional bacterial culture methods or enzyme linked immunosorbent assay (ELISA) for identification of the bacteria are time consuming and labor intensive. Some new technologies are very sensitive but analysis time can be lengthy. For example, the polymerase chain reaction (PCR) can be used to amplify small quantities of genetic material to determine the presence of bacteria. This is a sensitive method that requires pure samples and considerable laboratory time. Alternative methods should be quicker and retain sensitivity. The magnetic separation technique appears promising for rapid bacterial isolation from the media prior to the detection. In this work, immunomagnetic assay system (IMAS) has been coupled to an electrochemiluminescent (ECL) technology for rapid and sensitive bacterial detection of biological samples within an hour. The sensitivity of the IMAS-ECL for Bacillus anthrax spores (sterne), Escherichia coli O157:H7 and Salmonella typhimurium detection is about 1000 cells/mL in biological samples. In addition, IMAS can also be coupled to a flow cytometer or any analytical instruments for target agent monitoring. Results of this study strongly suggest that IMAS methodology is useful for rapid and sensitive detection. Rapid and sensitive screening methods for Escherichia coli 0 1 5 7 Ή 7 , Salmonella typhimurium and other virulent bacteria such as Bacillus anthracis in contaminated food, water and other biological samples are important to prevent the spread of bacteria. Traditional methods for bacterial identification and detection are time consuming (e.g., membrane filtration onto eosin-methylene blue agar or culture takes 24-48 hours). The immunoblotting technique is very sensitive and the detection levels of 1-10 colony 0097-6156/96/0646-O297$15.00/0 © 1996 American Chemical Society

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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forming unit/g (CFU/g) for E. coli 0157 can be obtained by using capture on hydrophobic grid membranes (1,2, 3), or by overnight culture. The polymerase chain reaction and restriction fragment mapping identification techniques can be definitive and extremely sensitive, but require hours of processing and expertise in molecular biology. Enzyme linked immunosorbent assay (ELISA) can be rapid (less than a few hours), but it is labor intensive and multiple pipetting is required. An immuno-latex bead based agglutination assay has been used to detect E. coli 0157:H7 (4), but the assay is involved a pre-enrichment culture procedure which limits the application for rapid screening. An immunomagnetic assay system (IMAS) has been developed for effective magnetic particle capture followed by rapid detection techniques. This approach can speed up the assay time to less than one hour and also increase the sensitivity by significantly reducing biological sample interference. The IMAS includes a magnetic separator for capturing the antigen and an electrochemiluminescent (ECL) detector for detection. Detection sensitivities of as low as picogram or attomogram levels can be achieved for bacteria and toxoid, as well as ds-DNA (5, 6, 7) using ECL. Alternatively, in IMAS configuration, afluorescencemicroscope can be used for bacterial identification or a continuous fluorimeter or aflowcytometer can be coupled to IMAS for routine positive antigen screening. The current work illustrates the utility of easy, rapid, and sensitive detection for B. anthrax spores, E. coli 0157:H7 and Salmonella sp. in biological samples by use of IMAS. The extension of current methodologies could apply to environmental and clinical needs by isolating specific molecules and soluble antigens in biological samples.

Experimental procedures 1. Bacteria, antibodies and magnetic particles Heat-killed Κ coli 0157:H7, Salmonella typhimurium and anti-£. coli 0157,

-Salmonella sp. antibodies were obtained from Kirkegaard Perry Labs.(KPL; Gaithersburg, MD). Irradiated E. coli 0157:H7 and Salmonella sp. and ATCC-11775 (E. coli) were obtainedfromUSDA (Philadelphia, PA). Nonpathogenic E. coli 0111:B4 strain and B. anthrax spores (Sterne strain) were obtainedfromSigma Chemical Co.(St. Louis, MO) and USAMRIID (Ft. Derrick, MD), respectively. AntiSalmonella sp. antibody is broadly reactive with all sero group D of Salmonella sp. Goat anti-5. anthracis GT-576, -577 and -578 antibodies were obtained from Antibodies Inc. (Davis, CA). These antibodies were very specific to surface antigens of the spore coating rather than vegetative cells. Bacterial cell counts were performed with a hemacytometer and the stock bacterial suspensions in phosphate buffered saline (PBS, 10 mM phosphate, pH 7.4) contained 10 cells/mL. Streptavidin-coated 9


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Immunomagnetic Assay System & Virulent Bacteria299

magnetic beads, Dyna M-280 and MACS microbeads were obtainedfromDynal, Inc. (Lake Success, NY.) and Miltenyi Biotec Inc. (Sunnyvale, CA.), respectively. 2+

2. Biotin, fluorescein and Ru(bpy) -antibody conjugations N-hydroxy-succinimide (NHS)-biotin and fluorescein-NHS ester reagents from Molecular Device Corp. were used to label polyclonal anti-2?. anthracis, E. coll and Salmonella antibodies. Ru(bpy) - NHS ester was obtainedfromIGEN Corp.(Gaithersburg, MD) and used for antibody-conjugation (8). Both fluoresceinand biotin-antibody conjugation assays were performed for one hour. Unreacted labels were removed by using a G-25 Sephadex (PD-10) size exclusion chromatographic column (Pharmacia, Sweden). All protein concentrations were determined by the Bradford Protein Assay (BioRad Corp., Hercules, CA). A sandwich immunoassay, with biotinylated antibody as the primary capture antibody and fluorescein- or Ru(bpy) -labeled antibodies as Tag-antibodies, was used in these studies. 3

2+


3

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3. Sample processing Environmental water samples including bay water, pond water, stream water and tap water were collected along the Chesapeake Bay area in Maryland. Fresh ground beef and poultry samples were purchasedfromretail grocery stores. Liquid samples and minced solid samples (5 gram with their liquids) were placed in 15 mL polypropylene tubes and agitated for 30 minutes. One mL of supernatantfromthese samples was inoculated with varying amounts of target bacteria. Undiluted juice and water samples were similarly inoculated with bacteria. 4. IMAS and immunoassay A diagram of the IMAS is shown in Figure 1. One mL of biological samples mixed with 100 (100 ng) biotinylated antibody plus 100 //L (100 μg) streptavidincoated beads were processed by IMAS at a rate of 2 minutes/sample. Following the IMAS procedure, 100 μL of sample were collected for further measurement. An ORIGEN® analyzerfromIGEN was used for ECL assay. The principle of ECL has been previously described (5, 6). Previously collected samples were incubated for 30 minutes with 100 mL of Tag-antibody (200 ng) prior to the ECL assay. Following the sampling process, an optical orfluorescencemicroscope can be employed for cell identification or other analytical instruments can be used for more definitive analysis such as a flow cytometer. The IMAS was built in house. It works with four subsystem procedures which sequentially mix, magnetically capture,rinseand collect particlesfromthe sample though aflowcell. Downstream from the flow cell, a peristaltic pump (ColeParmer, Chicago, IL) is used at aflowrate of 2 mL/minute. Bacteria in artificially inoculated food supernatant fluids, environmental water and biological samples were subjected to the separator prior to the ECL assay. IMAS



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F i g u r e 1. A diagram of the IMAS. Samples are i n i t i a l l y processed by the IMAS, then analyzed by an ECL, a f l u o r i m e t e r and a flow cytometer. Samples a l s o can be c o l l e c t e d on a m i c r o n - s i z e d membrane f i l t e r f o r i d e n t i f i c a t i o n by a f l u o r e s c e n c e microscope.

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F i g u r e 2. ECL d e t e c t i o n f o r E. coli 0157:H7 and Salmonella sp. assays i n b u f f e r . R e s u l t s i n d i c a t e the d e t e c t i o n l i m i t s approximately 100 to 1000 c e l l s / m L . ECL v a l u e s r e p r e s e n t the mean and standard d e v i a t i o n of three independent measurements. Values i n amount of antigens i n d i c a t e the l o g of cells/mL used i n each assay.



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was accomplished by adding 100 yiL (200 ng) of biotinylated antibody to 100 to 200 μg of streptavidin-coated magnetic beads. This was followed by adding the antibodycoated magnetic beads to various concentrations of bacteria in 1 mL of sample supernatant for 15 minutes with continuous shaking. Samples were collected after magnetic separation. Magnetic particles were resuspended in PBS and washed again followed by resuspension in 50 μL of PBS. The E C L analyzer was operated at a photomultiplier tube (PMT) setting of 650V (or lOOOx gains) and a carousel vortexing speed of 100 rpm. In thefluorescencesandwich immunoassay, biotinylated antibody at 5 ng/^L was used for antigen (100 μL) capture and 100 μL offluorescein-conjugatedantibody at 5 ng/mL were used to enable detection. One hundred μL samples with or without bacteria were flowed through a Jasco-920fluorometer(Jasco, Inc., Japan). The BioRad Bryte HS® (Microscience Ltd., England) was used for flow cytometry studies. Forward light scattering andfluorescentdata were obtained from the flow cytometer. The data were collected under the 1.1 μL/minute flow rate and 0.7 Bar pressure conditions.

Results This experiment was designed to determine internal standards for bacterial detection. In Figure 2 the detection limits of E C L results for E. coll and Salmonella were approximately between 100 and 1000 cells/mL in buffer. Both results showed a dynamic range over four orders of magnitude. E. coll O l l l :B4 and ATCC-11775 at 10 cells/mL were selected as negative control antigens. E. coll and Salmonella ψ. were inoculated in various food and water samples. The final concentration of bacteria in these samples was 2000 cells/mL. Figure 3 shows the results of E C L assays with and without E. coll. The E C L intensities with different samples should be compared to the intensity in PBS buffer condition. The signal to noise ratios of biological samples that spiked with 2000 bacteria cells/mL in E C L determinations were between 5 and 10. In Figure 4, the E C L results of Salmonella detection in different water and food samples are illustrated. Even though some interference can be seen in fish, beef and juice samples, the signal to noise ratios were still significant. Detection for B. anthrax spores in biological sample is shown in Figure 5. In flow cytometry assays, a negative control assay was performed by injection of immunoparticles into the flow cytometer without bacteria present. The results of the negative control assays showed that there were no fluorescence (FL) or forward light scattering (LS) peaks in both cases (Figures 6a and 6b). Miltenyi M A C magnetic particles used in these assays were too small to be detected in the current assay scale. On the other hand, there were distinguishable peaks revealed in the LS result (Figure 6c) and the FL (Figure 6d) when E. coll antigen at 10 cells/mL was introduced into 6

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F i g u r e 3. IMAS-ECL assay of E. coli 0157:H7(2000 cells/mL) i n v a r i o u s b i o l o g i c a l samples. ECL values r e p r e s e n t the mean and standard d e v i a t i o n of three independent measurements.

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Samples with (+)/without

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F i g u r e 4. IMAS-ECL d e t e c t i o n of Salmonella sp.(2000 c e l l s / m L ) i n v a r i o u s food supernatant f l u i d s and environmental samples. ECL values represent the mean and standard d e v i a t i o n of three independent measurements.



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F i g u r e 5. IMAS-ECL d e t e c t i o n of B. anthracis (500 c e l l s / m L ) i n v a r i o u s b i o l o g i c a l samples. ECL v a l u e s r e p r e s e n t the mean and standard d e v i a t i o n of three independent measurements. A l l ECL v a l u e s should be compared to the ECL v a l u e a t b u f f e r c o n d i t i o n . N e g a t i v e c o n t r o l i s PBS without B. anthrax antigen.

the flow system. The shape and the location of the peaks in both histograms indicated that the particle size distribution of the bacteria was approximately a few micrometers in diameter.

The peak height in both LS and F L (Figures 6b and 6d) windows

corresponds to the quantity of the bacterial cells detected by flow cytometry. In addition, pathogenic bacterial antigens from various samples were further examined by other techniques. Results from fluorescence microscopy showed that about 60-80% of the bacterial cells were captured by immunoparticles (data not shown).

5

A distinguishable fluorescent signal was detected at >10 cells/mL by

fluorimetric measurement (data not shown) in the fluorescence assay.

Discussion IMAS provides a rapid, sensitive and facile technique for virulent bacterial detection that is sensitive to at least >1000 cells/mL in biological samples. Total IMAS assay procedure is less than one hour. The main advantages using magnetic separation prior



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F i g u r e 6. Forward l i g h t s c a t t e r i n g (LS) and p a r t i c l e f l u o r e s c e n c e (FL) histograms of IMAS-flow cytometry showed d i s t i n g u i s h a b l e peaks (b and d) , r e s p e c t i v e l y , i n the presence of E. coli 0157:H7 a n t i g e n s (10 cells/mL) compared to the c o n t r o l s (without E. coli 0157:H7 antigens) as a and c. 6

to the E C L or other assays are in reducing the direct interference from the biological samples; concentrating the target antigens from large volume to small volume; and avoiding the operator's direct contact with the samples which contain the pathogenetic bacteria. IMAS in conjunction with E C L is a powerful approach to perform very sensitive and rapid assays. The IMAS technique is capable of broader applications to any type of prokaryotic and eukaryotic cell assay, as well as soluble antigens, and nucleic acids(5,6, 7). The advantages of non-radioisotope labels, nonintrinsic fluorescence background and controlled electric potential make E C L technology more sensitive and effective than EI A, chemiluminescence and fluorescence in clinical and biological applications. In the E. coll and Salmonella sp. immunoassays, both E C L responses were not linear over a broad range. This is probably related to the immunologic "Bell Effect" (9) which is apparent beyond 10 ,10 cells/mL or a simple physical over loading of the ECL flow cell. In the latter hypothesis, increased amounts of captured bacteria do not lead to proportionately higher levels of E C L signal because bacterial absorption of light emitted by the E C L labels diminishes the E C L signal intensity reaching the PMT. Results of the IMAS-ECL assay for B. anthracis, E. coli and Salmonella ψ. showed that detection limits were about >100 cells/mL in PBS and >1000 cells/mL in most biological samples. These results may not be as sensitive as the 5

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immunoblotting technique; however, this is sensitive enough to detect the presence of bacteria in biological samples. More importantly, the early detection (within an hour) of this rapid assay could prevent these virulent bacteria from spreading and could save lives. Both heat-killed and irradiated bacterial E. coli cells were tested in my lab. It is believed that the heating process can cause bacterial cell membrane break-down. Therefore, cell surface antigens may not be in a condition for recognition by the antibody which was produced against normal surface antigen. Our results indicated that there were no difference in E C L assays whether heat-killed or irradiated bacterial cells were used. Results from other researchers (10, 11) using the same polyclonal antibodies from K P L (not necessary the same lot) demonstrated a similar detection sensitivity for both live and heat-killed E. coli 0157:H7 and Salmonella sp. Bacterial detection in biological samples constitutes authentic sample detection. In the case of solid meat samples, only the interference from their fluids was considered. The efficiency of bead collection from solid meat remains unknown. In order to perform quantitative E C L assays, positive detections in buffer were done as standard procedure prior to assays of real biological samples. Decreased sensitivity of E C L detection in biological samples was expected because of the sample interference. It was also anticipated that the competition between the primary and secondary antibodies could reduce the ability to detect bacteria. However, this was apparently not a significant problem in this protocol. The limitation of the E C L assay like any immunoassays is largely dependent upon the antibody affinity to the antigens; however, the advantage using IMAS-ECL instead of ELISA and culture-based methods is that IMAS-ECL is more sensitive than ELISA or fluorescence (6) and the total assay time is only one hour. Many hand-held test kits and commercial products, including Dynal antibodybased test kits (Dynal, Inc.) have been developed for E. coli 0157 and Salmonella sp. detection. These testing kits could be very rapid; however, they are not suitable for quantitative detection. In the present work, the IMAS method is useful for both sensitive detection and relative quantitation. The IMAS-ECL assay has shown the most sensitive detection for these bacteria, however, the results of IMAS-fluorimetry and IMAS-flow cytometry assays currently are not promising. Improvement of these assays could potentially increase sensitivity and make the IMAS more useful in biological detection as well as environmental monitoring.

Acknowledgement: This work was funded under U.S. Air Force HSC scholarship.

Literature cited 1.

Doyle, M.P. and Schoeni, J.L. Isolation of Escherichia coliO157fromretail fresh meats and poultry. Appl. Environ. Microbiol. 53, 2394-2396, (1987). In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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3.

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6.

7. 8.

9.

10. 11.

ENVIRONMENTAL IMMUNOCHEMICAL METHODS Todd, E.C.D., Szabo, R.A., Peterkin, P., Sharpe, A.N., Parrington, L., Bundle, D., Gidney, M.A.J. and Peny, M.B. Rapid hydrophobic grid membranefilter-enzyme-labeledantibody procedure for identification and enumeration of Escherichia coli 0157 in foods. Appl. Environ. Microbiol. 54, 2536-2540, (1988). Szabo, R., Todd, E., Mackenzie, J., Parrington, L. and Armstrong, A. Increased sensitivity of the rapid grid membrane filter enzyme-labeled antibody procedure for Escherichia coliO157detection in foods and bovine feces. Appl. Environ. Microbiol. 56, 3546-3549, (1990). Notermans, S., Wernars, K., Soentoro, P.S., Dufrenne, J. and Jansen, W. DNA- hybridization and latex agglutination for detection of heat-labile and shiga-like toxin-producing Escherichia coli in meat. Int. J. Food Microbiol. 13, 31-40, (1991). Gatto-Menking, D.L., Yu, H., Bruno, J.G., Goode, M.T., Miller, M. and Zulich, A.W. Sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor.Biosensors Bioelectronics, 10:501-50, (1995). Yu, H., Bruno, J.G., Cheng, T., Calomiris, J.J., Goode, M.T. and GattoMenking, D.L. A comparative study of PCR product detection and quantitation by electrochemiluminescence and fluorescence. J. Biolum. Chemilum., 10, 239-245, (1995). Yu, H. Enhancing Immunoelectrochemluminescence for Sensitive Bacterial Detection, J. Immunol. Methods, in press, (1996). Blackburn, G.F., Shah, H.P., Kenten, J.H., Leland, J., Kamin, R.A., Link, J., Peterman, J., Powell, M.J., Shah, Α., Talley, D.B., Tyagi, S.K., Wilkins, E., Wu, T.. and Massey, R.J. Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clinical Chem. 37, 1534-15, (1991). Ugelstad, J., Kilaas, L., Aune, Ο., Bjorgum, J., Heije, R., Schmid, R., Stenstad, P. and Berge, A. Monodisperse polymer particles: Preparation and new biochemical and biomedical applications. In: M . Uhlen, E. Homes and O. Olsvik (Eds.) Advances in Biomagnetic Separation. Eaton Publishing, Natick, Mass., p. 6-7, (1994). Tison, D.L. Culture confirmation of Escherichia coli serotypeO157:H7by direct immuno-fluorescence, J.Clin. Microbiolgy 28, 3546-3549, (1990). Park, C.H., Hixon,D.L., Morrison, W.L. and Cook, C.B. Rapid diagnosis of enterohemorrhagic Escherichia coli O157:H7 dirctlyfromfecal specimens using immuno-fluorescence stain. Am. J. Clin. Pathol. 101, 91-94, (1994).


Application of an Immunomagnetic Assay System for Detection of

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