Immunomagnetic Separation of Pathogenic Organisms From Environmental Matrices

Gary P. Yakub and Kathleen L. Stadterman-Knauer

1. Introduction

One of the most difficult challenges in the analysis of environmental samples is to separate the organism of interest from a sample that is high in background debris. Immunomagnetic separation (IMS) is one technique that has been developed to accomplish this in a rapid and reliable assay.

Immunomagnetic separation (or biomagnetic separation) involves a superparamag-netic, monodispersed, polystyrene microsphere that is coated with a specific ligand. When added to a heterogeneous target suspension, the microspheres bind to the desired target. Using a powerful magnet, the microsphere-target complex is then removed from the suspension (1). Many different targets of interest can be isolated with this technique, including fungal/bacterial cells or spores, protozoan parasites, cellular and subcellular material, proteins, and nucleic acid products. This wide range of application makes IMS one of the most versatile techniques available for the purification of target products from heterogeneous sample matrices.

1.1. Molecular Separation

Molecular separations involve the binding of a superparamagnetic microsphere to a nucleic acid product such as a segment of cDNA or tRNA. This technique is useful for the purification of nucleic acid products prior to downstream applications such as PCR and hybridization (2-6).

1.2. Clinical Separation

Clinical separations involve the isolation of a target from a sample of human tissue, body fluid, or waste product. Some of the targets that have been isolated include proteins (7,8), specific cell types such as cancer cells (9,10), peripheral blood mononuclear cells such as monocytes, T-cells, B-cells, and granulocytes (11-13), and a variety of pathogenic organisms such as Shigella spp. (14), Escherichia coli 0157 strains (15), Mycobacterium spp. (16,17), and Bordetella pertussis (18).

From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ

1.3. Environmental Separation

Environmental separations involve the isolation of a target organism from an environmental matrix such as soil, surface or ground water, raw and treated wastewaters, animal tissue, fluid, feces, and food or food byproducts. In the environmental field, IMS is most often used to isolate organisms that are pathogenic to humans. Organisms such as Cryptosporidium parvum (19-21), E. coli O157:H7 (22-27) Mycobacterium (30,31), Helicobacter pylori (32,33), Vibrio spp. (34,35), Microsporidia (36), Yersinia enterocolitica (37), Bacillus spp. (38,39), Coxiella (40), and hepatitus A virus (41) have all been successfully isolated with the IMS technique. Additionally, fungal plant pathogens (42), equine pathogens (43), and sulfate-reducing bacteria of importance to the offshore oil industry (44) have also been isolated from environmental matrices by the IMS procedure.

The basic IMS procedure is fairly straightforward. A sample or sample concentrate is obtained. One or more buffers are added to optimize the separation environment. The proper immunomagnetic beads are added and the sample is mixed for a period of time to allow the beads to associate with the target organism. A powerful magnet is used to separate the bead-target complex from the remaining debris. The beads are then dissociated from the target organisms and removed from the suspension. What remains are the purified target organisms. The specific methodology can be somewhat variable owing to the inherent variability of the different target organisms, and an exhaustive presentation of each individual organism's technique would be beyond the scope of this volume. A representative environmental IMS methodology will be presented in detail to allow the reader to become familiar with the basic techniques. This methodology can then be easily adapted to other target organisms of interest.

1.4. IMS of Cryptosporidium parvum

As a model, the remaining sections of this chapter will deal with the immunomag-netic separation of the human intestinal parasite, C. parvum, from environmental matrices. C. parvum is a coccidian protozoan pathogen of the mammalian digestive tract. It infects a variety of domestic animals and is highly prevalent in cattle. It forms an environmentally stable oocyst, 4-6 ^m in diameter. The main infection mode is the fecal-oral route. Oocysts can enter the potable water supply through fecal contamination (45). The resulting disease in humans, cryptosporidiosis, causes gastrointestinal upset, profuse watery diarrhea, cramps, and nausea. This disease is self-limiting in healthy immunocompetent hosts but can be fatal to immunosuppressed individuals as well as the very old and the very young (46). The infectious dose for 50% of immunocompetent individuals (ID50) is 132 to as few as 30 oocysts (47). For the immunosup-pressed, the infectious dose has been estimated to be as low as one oocyst (48). The mortality rates of C. parvum among these individuals vary from 52 to 68% (49). The largest documented outbreak of cryptosporidiosis occurred in the spring of 1993 in Milwaukee. Oocysts passed through the drinking water treatment plant and infected the general population. An estimated 403,000 people were affected, and several people died as a result of the disease (50). Because of the risk to the immunocompromised and the potential for oocysts to pass through drinking water treatment systems,

Cryptosporidium has become one of the most important contaminants found in drinking water today (49).

Current methods to enumerate oocysts from environmental and drinking waters include IMS. The United States Environmental Protection Agency (EPA) method 1623 (51) for water samples includes an initial concentration step using a cartridge filter. IMS is then used to isolate and purify the oocysts from the sample concentrate, followed by an immunofluorescent microscopic assay. Researchers have reported average oocyst recoveries in deionized water ranging from 68 to 83% using the Dynal kit described below (52). At turbidity levels up to 500 NTU, oocyst recoveries were similar to the recoveries obtained in deionized water. At a turbidity of 5000 NTU, oocyst recoveries greater than 35% were obtained (52). Other researchers have reported average recoveries of oocysts ranging from 62 to 100% in seeded environmental water concentrates with turbidities ranging from 210 NTU to 11,480 NTU (53).

2. Materials

1. Flat-sided sample tubes, 16 x 125 mm Leighton type with 60 x 10 mm flat-sided magnetic capture area (Dynal, Lake Success, NY, L10, cat. no. 740.03 or equivalent) (see Note 1).

2. Sample mixer: Dynal cat. no. 947.01 or equivalent (see Note 2).

3. Magnetic particle concentrator for 10 mL and test tubes (Dynal MPC-1®, cat. no. 120.01 or equivalent).

4. Magnetic particle concentrator for microcentrifuge tubes (Dynal MPC-M®, cat. no. 120.09 or equivalent (see Note 3).

5. Merifluor Direct Immunofluorescence Assay for Cryptosporidium/Giardia (Meridian Diagnostics, Cincinnati, OH, cat. no. 250050).

6. Dynabeads® anti-Cryptosporidium kit (Dynal cat. no. 730.01 or equivalent) (see Notes 4-6). Each kit contains (54):

a. Anti-Cryptosporidium immunomagnetic beads, coated with purified antibodies against Cryptosporidium that are covalently bonded to the bead surface. The beads are supplied as a suspension in phosphate-buffered saline (PBS), pH 7.4, with 0.1% bovine serum albumin (BSA).

b. 10X SL buffer A (clear, colorless solution).

c. 10X SL buffer B (magenta solution).

7. Hydrochloric acid, 0.1 N, ACS grade or equivalent.

8. Sodium hydroxide, 1.0 N, ACS grade or equivalent.

1. Begin with a water sample or water sample concentrate of approx 10 mL contained in a Leighton tube (see Notes 7-10).

2. Prepare a 1X dilution of SL buffer A (clear, colorless solution) in reagent water from the 10X solution provided with the Dynal kit. A volume of approx 1.5 mL 1X SL buffer A will be needed for each sample analyzed.

3. To each Leighton tube containing a sample, add 1 mL of the 10X SL buffer A (not the diluted 1X SL buffer A).

4. To each Leighton tube from step 3, add 1 mL. 10X SL buffer B (magenta solution).

5. To each Leighton tube from step 4, add 100 ^L of the anti-Cryptosporidium bead suspension (see Note 11). Tighten the screw cap on the Leighton tube.

6. Affix the sample tube to the Dynal sample mixer and rotate at approx 18 rpm for 1 h at room temperature.

7. After rotating 1 h, remove the sample tube from the mixer and place it in the MPC-1 concentrator with the flat side toward the magnet.

8. Gently rock the tube through 90°, tilting cap end and base end up and down in turn, for 2 min with approx one tilt/s (see Notes 12-15).

9. After tilting, rapidly remove the cap and pour the liquid into a suitable container. Do not remove the Leighton tube from the MPC-1 concentrator during this step (see Notes 16 and 17).

10. Return the Leighton tube to the upright position and remove the tube from the MPC-1 concentrator. Resuspend the sample in 1 mL of the 1X SL buffer A (prepared in step 2). Mix gently to resuspend all material (see Note 18). Do not vortex.

11. Quantitatively transfer all liquid to a labeled 1.5-mL microcentrifuge tube (see Note 19).

12. Place the microcentrifuge tube into the MPC-M concentrator with the magnetic strip in place.

13. Gently rock the MPC-M concentrator through 180° by hand for 1 min with approx one rock/s (see Note 20).

14. Immediately aspirate the remaining liquid from the microcentrifuge tube and cap (see Notes 21 and 22). If you are processing more than one sample, conduct three 90° rolling actions between the processing of each tube. Do not remove the tubes from the MPC-M concentrator during this step (see Note 23).

15. Remove the magnetic strip from the MPC-M concentrator (see Note 24).

16. Add 50 ^L of 0.1 N hydrochloric acid, cap the microcentrifuge tube, and vortex vigorously for 10-15 s (see Notes 25 and 26).

17. Place the tube in the MPC-M concentrator without the magnetic strip and allow to stand in a vertical position for at least 10 min at room temperature.

18. Vortex vigorously for 5-10 s (see Note 26).

19. Replace the magnetic strip in the MPC-M concentrator and allow to stand undisturbed for approx 10 s.

20. Prepare a well slide from the Merifluor kit by adding 5 ^L of 1.0 N sodium hydroxide to each sample well (see Note 27). Prepare one well for each processed Leighton tube.

21. Without removing the microcentrifuge tube from the MPC-M concentrator, transfer all the liquid from the tube and cap onto the prepared well slide (see Notes 28 and 29).

22. Allow the slide to sit undisturbed overnight to air dry (see Note 30). Proceed with an appropriate immunofluorescent assay to identify the Cryptosporidium oocysts (see Note 31).

4. Notes

1. Our laboratory utilizes Dynal (Oslo, Norway) as our sole supplier for all IMS reagents and equipment. Dynal equipment is an integral part of US EPA method 1623 (51) and has been shown by other researchers to provide the best recoveries for Cryptosporidium (52,53).

2. The sample mixer holds up to twelve 10-mL Leighton tubes. If you plan to process batches of more than 12 samples frequently, it would be advisable to purchase two mixers.

3. It is advisable to purchase one MPC-1 and one MPC-M concentrator for each analyst who will be working at the same time, to minimize sample processing time.

4. If you are testing for both Cryptosporidium and Giardia, Dynal makes a combination kit for the simultaneous recovery of both organisms (cat. no. 730.02).

5. Store the Dynal kits at 4-8°C and use prior to the expiration date listed on the carton.

6. One test kit contains enough reagent for 10 tests.

7. Because some organisms are found rather infrequently in nature, a general procedure must be developed to concentrate a large water sample down to a 10-mL. volume. For Cryptosporidium oocysts, much research is currently being conducted to determine which methods of sample concentration are best suited to a particular matrix. Processes such as centrifugation, density gradient floatation, and microfiltration are currently being investigated for utility in specific environmental matrices. For samples in which bacteria are the targets, a pre-enrichment step may be necessary.

8. It is recommended that the water concentrate packed pellet volume not exceed a 0.5 mL vol. If this is the case, vortex sample for 2 min, subdivide into two or more Leighton tubes, and bring the volume of each tube back to 10 mL. Pool all results for the final answer.

9. Water concentrates high in some chemical or physical compounds may exhibit an inhibitory effect on the IMS protocol. Two compounds known to cause interference are turbidity (52) and dissolved iron (55). Analyzing quality control recoveries of the intended target organisms can help to characterize the matrix in terms of interference as well as to monitor the separation process.

10. Allow all Dynal kit reagents to come to room temperature before use.

11. Be sure to vortex the bead suspension for a minimum of 10 s to resuspend the beads. After vortexing, invert the tube and inspect for signs of beads still sitting on the bottom of the tube. (Beads appear as reddish brown grains in the Cryptosporidium kit.) Vortex as long as needed to resuspend all the beads.

12. It is possible during the tilting process for the Leighton tube to fall out of the MPC-1 concentrator. Be sure to support the tube during the tilting process. Do not rely on the clip to hold the tube in place.

13. Use a mechanical countdown timer to time all steps accurately in the procedure.

14. Ensure that the tilting procedure is not interrupted during this step to avoid binding of magnetizable material in the sample.

15. The 90° vertical agitation used with the Dynal magnetic particle concentrator ensures that particulate matter and sample debris remain suspended in the solution in the Leighton tube while the bead-oocyst complexes are captured by the magnet (53).

16. Because of the possible presence of unrecovered pathogens, pour the liquid into an auto-clavable container and autoclave prior to disposal.

17. Researchers have reported that 3.1-4.6% of the oocysts were not captured in the initial separation (53).

18. During mixing, be sure to wash all the material off the flat side of the tube. This is best accomplished by adding the 1X SL buffer A near the top of the tube with a micropipet and allowing it to wash down the inside square surface.

19. Transfer the bead suspension either by direct pouring or the use of a micropipet. There will not be much room to add rinsate to the microcentrifuge tube, so restrict rinsing of the Leighton tube to three 200-^L vol of reagent water. Do not overfill the microcentrifuge tube.

20. At the end of this step, the beads should appear as a distinct brown dot at the back of the tube.

21. For this step, we recommend using a disposable glass Pastuer pipet and bulb. The long thin tip is less likely to disturb the bead pellet. Lower the tip into the solution along the front of the tube wall, gently aspirate out the solution, and remove the tip following the front wall of the microcentrifuge tube.

22. Dispose of this liquid into the same container used in step 9 (see Note 15).

23. Researchers have reported that 0.6-3% of the oocysts remained in the microcentrifuge tube (53).

24. This is the beginning of the dissociation step, whereby the superparamagnetic beads are removed from the target oocysts. The procedure outlined here is followed by slide fixing and microscopic examination of the captured oocysts. If the captured oocysts are to be subjected to viability studies or further molecular examination (i.e., PCR), other researchers have outlined dissociation protocols that will not interfere with these downstream activities (53,56).

25. Researchers have reported that 2-8% of the oocysts remain attached to the paramagnetic beads following the acid dissociation step (53).

26. Do not vortex so vigorously as to force the suspension into the underside of the microcentrifuge tube cap.

27. The Merifluor kit contains positive and negative control suspensions. Place a drop of each suspension onto separate wells of a treated well slide and process with the samples.

28. A micropipetor is adequate for this step. We use a 10-100-^L adjustable micropipetor set for approx 80 ^L in order to ensure that all the liquid is collected. The transfer may be accomplished in several passes.

29. Place the solution from the microcentrifuge tube directly into the bead of sodium hydroxide on the slide well. This will ensure adequate acid neutralization.

30. We cover the slides with an inverted plastic bowl to prevent disturbance or contamination.

31. Our laboratory currently utilizes the Merifluor kit listed in Subheading 2. (Materials), following the procedure outlined in Section 14 of USEPA Method 1623 (51).


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