WO1991002816A1

WO1991002816A1 - Isolating cells by elution from cationically derivatized cellulosic material

Info

Publication number: WO1991002816A1
Application number: PCT/US1990/004704
Authority: WO
Inventors: Calvin P. H. Vary
Original assignee: Idexx Corporation
Priority date: 1989-08-22
Filing date: 1990-08-20
Publication date: 1991-03-07

Abstract

A method of isolating cells from an impure mixture by: applying the impure mixture to a cationically derivatized cellulosic material under conditions allowing the cells to bind to the material; separating unbound mixture from the bound cells; and then applying an eluting solution to remove cells from the cellulosic material in a purified fraction. Useful cellulosic material includes cationically derivatized natural or synthetic 1-4-β-D-linked polymers of β-D glucopyranose, as well as derivatives thereof well known to those in the art.

Description

ISOLATING CELLS BY ELUTION FROM CATIONICALLY DERIVATIZED CELLULOSIC MATERIAL

Background of the Invention This invention relates to methods of isolating and purifying cells from impure mixtures.

Various methods for the recovery and evaluation of cells or their constituents from biological and other complex samples or mixtures have been reported in the literature or are available in commercial form. Wood et al., British Patent No. 1,441,022 discloses (1:84 et seq.) a method for evaluating the microorganism content of a material by various methods, including

"bringing a known quantity of the material into contact with an ion exchange resin, washing any remaining material from the resin while leaving the microorganisms retained on the resin, eluting the microorganisms from the resin in a suitable medium and thereafter concentrating and determining the quantity of the eluted microorganisms....suitable ion exchange resins are for example, anion exchange resin composed of quartenary ammonium exchange groups attached to a styrene divinylbenzene polymer lattice, anion exchange resin composed of polyalkylamine functional groups attached to a styrene divinylbenzene polymer lattice and cation exchange resin composed of nuclear sulfonic acid exchange groups attached to a styrene' divinylbenzene polymer lattice."

MacDonald (1986) Soil Biology and Biochemistry

18:399 describes a method to separate soil from soil microfloras in which soil is dispersed in an aqueous suspension of the sodium salt of Dowex Al at neutral pH and low electrolyte concentration,

"and microorganisms released by converting cation exchange groups in soil and on cell surfaces to the sodium form with the Na⁺ salt of the iminodiacetate resin Dowex Al."

Ferenci (1983) Applied and Environmental

Microbiology 45:384 reveals a method for the affinity immobilization of Escherichia coli on starch-Sepharose.

Immobilized cells are useful in biotechnology applications, e.g., as catalysts.

"E. coli cells were efficiently retained by [starch-Sepharose] columns loaded with over 10⁹ bacteria per ml of matrix. More than 95% of applied bacteria were bound at these densities. As previously demonstrated, this retention is starch dependent. At higher applied cell densities, even higher numbers were retained, although not necessarily in a starch-dependent manner... bacteria bound to starch-Sepharose in a receptor-dependent manner are eluted by soluble ligands of the lambda" receptor... The stability of interaction between E. coli and starch- Sepharose was investigated by subjecting columns containing immobilized bacteria to continuous elution. After the initial application of bacteria, when approximately 3 to 5% of bacteria passed through unabsorbed, the washout of bacteria was followed by continuing the elution with 200 column volumes of MMA under nongrowth conditions. The numbers of bacteria eluted per column volume of eluant settled to approximately

0.01% of bacteria present in the column. In total, 1.1% of retained bacteria were lost after elution with 200 column volumes of MMA." [Footnotes and references to tables omitted]

An ion exchange based device for the extraction of nucleic acids from cell lysates is available from Molecular Biosyste s, Inc., 10030 Barnes Canyon Road, San Diego, California 92121. The manual accompanying the product provides that. "The principles are as follows: lysates are applied to THE EXTRACTOR™ in a low ionic strength buffer; nucleic acids bind to the matrix while interfering proteins, mucopolysaccharides and pigments are washed away; nucleic acids are subsequently eluted using a high ionic strength buffered salt."

Summary of the Invention In general the invention features a method of isolating cells from an impure mixture by: applying the impure mixture to a cationically derivatized cellulosic material under conditions allowing the cells to bind to the material; separating unbound mixture from the bound cells; and then applying an eluting solution to remove cells from the cellulosic material in a purified fraction. Useful cellulosic material includes cationically derivatized natural or synthetic l-4-J-D-linked polymers of /3-D glucopyranose, as well as derivatives thereof well known to those in the art. In preferred embodiments the impure mixture is a crude biological sample comprising impurities that interfere with the detection of selective DNA hybridization, and the invention includes eluting cells from the cellulosic material in a fraction that is essentially free from impurities that interfere with detecting selective DNA hybridization and detection of such hybridization.

In preferred embodiments the impure mixture is applied to the cellulosic material under conditions such that: the cells bind to the cellulosic material; at least some contaminants do not bind to the cellulosic material; and other contaminants bind to the cellulosic material (e.g., colored material, including chlorophyll and its breakdown products, found in fecal matter binds to DEAE cellulose) and are not eluted at all, or at least not in the same fraction as the cells eluted from,the cellulosic material.

Other preferred embodiments include a column in which a cationically derived cellulosic material is mixed with or arranged in sequence with an anionically derived cellulosic material.

In preferred embodiments the anionically derivatized cellulose is derivatized with phosphoryl groups and the cationically derivatized cellulose is derivatized with diethylaminoethyl groups.

In other preferred embodiments elution is followed by detection of the eluted cells or detection of constituents of the eluted cells, e.g., by a selective nucleic and hybridization assay such as a polymerase chain reaction (PCR) assay or an enzymatic or other nonisotropic assay.

In preferred embodiments the cellulosic material is positioned within a compact disposable container.

In preferred embodiments the impure mixture is contacted with the cellulosic material at a pH 5.0 - 9.0 and an ionic strength of 10-200 mM; also, the eluting solution includes a polar organic solvent in a concentration effective to elute said cells from said cellulosic material and the eluting solution has a pH 7.8 - 8.0. In preferred embodiments the polar solvent is selected from the group consisting of: alcohols or polyalcohol of C₄ or less; ethers or polyethers of C₄ or less; aldehydes and ketones or C₄ or less; esters of C₄ or less. In particularly preferred embodiments the polar solvent is ethanol and in the most preferred embodiment it is about 40% ethanol volume/volume.

In preferred embodiments the eluting solution has an ionic strength sufficient to raise the ionic strength in the column to a level which elutes the cells from the cellulosic material.

When the cells are bound to the cationically derivatized cellulosic material any anion, e.g., -OH from NaOH, in sufficient concentration will elute the cells.

Thus in preferred embodiments the eluting solution is strongly basic. In particularly preferred embodiments the eluting solutions is 0.1 - 1.0 N NaOH.

In preferred embodiments the cells are suspended and loaded in a strongly basic solution and eluted with a strongly basic eluting solution. In particularly preferred embodiments both solutions are 0.1 - 1.0 N NaOH. Although the cells are loaded in a very high pH solution

(approximately pH 13) the buffering capacity of the column is such that the pH on the column is reduced during loading

(e.g., below pH 10) allowing the cells to bond to the cellulosic material.

Sufficient eluting solution, which is strongly basic, is applied to overcome the buffering capacity of the column. As the buffering capacity of the column is overcome a front of high pH moves down the column eluting the cells.

Contaminants that interfere with PCR are retained on the column.

The invention provides a broadly applicable, rapid, economic and relatively simple method for the purification and concentration of microorganisms from a variety of complex, high volume samples.

The invention avoids extensive labor-intensive manipulations involving complex reagent solutions sometimes required in previous known nucleic acid extractions.

Contaminants are separated from the cells during application, washing and elution. The intact cell itself is used as a protective isolation vehicle, and cell constituents are protected from sample components or separation reagents that would degrade or react with the cell constituents. Microorganisms are eluted in a narrow concentrated fraction with very few steps using stable, inexpensive, easy-to-prepare reagents. After elution cells may be lysed without further purification and cell constituents assayed directly.

Microorganisms can be recovered from extremely crude complex samples effectively free of impurities that interfere with, inhibit, or confuse the interpretation of a variety of isotopic and nonisotopic means of detection i.e., any such impurities remaining are present at levels too low to substantially interfere with detection. As an example, the invention can be used for non-isotopic assays which generally are particularly sensitive to contaminants found in many medical, agricultural, veterinary and industrial samples. The invention thus simplifies assays, of complex samples, particularly non-isotopic assays, which otherwise would require extensive and laborious purification-. Fecal samples, for example, which are easily obtained, can be used as a source for the determination of the presence or absence of a given microorganism in a number of organisms, notwithstanding the abundance of contaminants they contain, including a wide variety of colored and noncolored materials that obliterate readouts in colorimetric or other spectrophotometric analyses. Specifically, interference with florescence or luminescence based determinations (e.g. from bilirubin or porphyrins in the same) is controlled by the invention. The invention also provides a way to overcome the presence of significant levels of endogenous phosphatases or peroxidases which could otherwise prevent the use of detection systems based on those enzymes. Furthermore, the invention can remove substances that compete with or interfere with binding "hooks" that are critical to the detection system — e.g. avidin-biotin binding — can also be present.

The invention is particularly useful for assays that detect the presence of nucleic acids, whether isotopic or nonisotopic, where the sample is contaminated by high levels of nucleases, e.g., as is often found in fecal and other crude biological samples, or by DNA binding substances such as proteins, polyamines and mucopolysaccharides that interfere with detection by binding specifically or nonspecifically to probe, target DNA or substrate. The invention is also useful for polymerase chain reaction assays, which are sensitive, in addition to the above, to substances that inhibit or degrade DNA polymerase. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments We turn now to a description of preferred embodiments of the invention, after first briefly describing the drawing.

The Drawing Fig. 1 is a highly diagrammatic cross-sectional view of a cell purification column. Use of the Invention

Particularly preferred embodiments of the invention include the use of various anionically modified cellulosic materials, such as P-ll phosphocellulose (Watman) . Cationic cellulosic materials useful in the preferred embodiment include DE-52 (Watman) . These materials can be readily obtained by modifying commonly available cellulosic materials including poly-ø-D-glucopyranase, so as to add one or more of the above anionic or cationic functional groups. Typically the crude sample is suspended in buffer to provide the preferred cell-binding conditions described above; for example 10 mM Tris(pH 7.8) ImM EDTA can be used as a buffer. Typical elements for releasing the cells include aqueous/polar organic mixtures buffered to the above eluting conditions, for example, with 200 mM NaCl 0.05 M Tris (pH 7.8) 40% ethanol. Cells are released by a buffer with an ionic strength of 100 - 400 mM, preferably about 200 mM. Alternatively, the cells may be eluted with a high ionic strength eluting solution. In a one-step procedure the crude sample may be suspended in a basic solution e.g., 0.2 N NaOH and eluted with a basic solution e.g., 0.2N NaOH. The material is used to purify cells from any of a wide variety of samples, including milk, feces, semen, and uterine wash media. Typically the method is used to purify cells in preparation for an assay, such as an immunoassay, or most preferably, a nucleic acid hybridization assay. The standard sample may be food or it may be any biological fluid or sample, including milk, feces, semen or uterine wash media.

The invention is specifically illustrated below by a protocol for isolating Mycobacterium paratuberculosis from a fecal sample. This illustration is not intended to limit the invention.

Mycobacterium paratuberculosis is the causative agent of Johne's disease. Johne's disease is a chronic debilitating disorder of cattle and other ruminants. The disease is often associated with decreases in milk production and fertility and infected cattle may suffer diarrhea, weight loss and in some cases death. There is no cure for Johne's disease. It is of major economic concern to the cattle industry throughout the world. This organism has also been implicated in the etiology of Crohn's disease in humans.

The causative agent of Johne's disease is the bacterium Mycobacterium paratuberculosis. Control of Johne's disease has been hampered by several factors, most notably the lack of a rapid and sensitive assay for the causative agent and by the course of the disease. The problems arising from the lack of a convenient, rapid test for the presence of Mycobacterium paratuberculosis are complicated by the normal course of the disease. Johne's disease is typified by an extended initial phase of infection with few if any easily observed symptoms. Thus not only are resources wasted on infected asymptomatic cattle, but these animals act as reservoirs of infection, contaminating pasturage and other cattle.

Largely because of the ease of collection, fecal samples are an attractive source for the determination of whether or not an animal is infested with Mycobacterium paratuberculosis. At least two characteristics of^*fecal samples complicate their use as a diagnostic sample.

Animals in the initial phases of infection may not contain high levels of the causative agent. Furthermore, these samples are often extremely crude, containing feces contaminated with a variety of other microflora, soil, surface water, vegetation and insects. Thus an assay suitable for use under these conditions must be capable of accepting a large sample and of extracting the desired components from a sample that is extremely rich in substances that interfere with subsequent uses of the desired components.

The preferred embodiment employs a compact, high capacity single use cartridge or column. In Fig. 1, cartridge 10 consists of two 30 micrometer frits 12, 14, enclosing a two layer cellulosic separation system. The first layer 16, which may or may not closest to the sample inlet, consists of a layer of cationically derivatized cellulose, e.g. DE-52 (Watman), of volume of approximately 3.0 ml., e.g., P-ll (Watman), the second exchange layer 18 is comprised of phospho-cellulose occupying a packed volume of about 1 ml to give a total cartridge a packed volume of about 4 ml.

The sample is suspended in a minimal buffer of 10 mM Tris (pH 7.8) and 1 mM EDTA to a final concentration of about 10-11% by weight solids. Large particulate matter is removed by allowing the suspended sample to stand at room temperature for 10-20 minutes or by passage through cheese cloth or other 30μ or larger mesh. Up to 10 ml of sample is pushed or allowed to flow through the cartridge. The cartridge is then washed with 10 ml 50 M Tris (pH 7.8) followed by 10 to 20 ml of 50 mM Tris (pH 7.8) + 0.1M NaCl. The purified microorganisms are then eluted with 50 mM Tris (pH 7.8) 0.2M NaCl containing 40% ethyl alcohol. A brief spin is used to pellet microorganisms. The resulting pellet is resuspended in deionized 0.5ml water and heated to 120° C for 10 minutes.

Alternatively, the crude sample maybe suspended in 0.2N NaOH. The sample is vortexed, allowed to settle for 20 minutes and 0.1-1.5ml of suspension is loaded onto the cartridge. After allowing the sample to enter the cartridge bed the cells are eluted with 15ml of 0.2N NaOH. The eluate is pelleted and the pellet resuspended in a small amount of its supernatant. The resulting supernatant, from either method, contains ample nucleic acid, substantially free of inhibitory materials, for polymerase chain reaction (PCR) amplification or direct hybridization and analysis. For example, standard hybridization assays, such as those described by Falkow et al. in U.S. Patent 4,358,535, can be used. Polymerase chain reaction assays such as those described in Mullis et al. U.S. Patents 4,633,202 and 4,683,195 can be used. Each of the above U.S. patents is hereby incorporated by reference. Example 1

Preparation of Cellulose P-ll and DE-52 resins: Equilibration and removal of fines. 1. Weigh 40g P-ll and swell in total volume of 2L 10 mM Tris (pH 7.8) 1 mM EDTA by gentle inversion and let settle 1 hour.

2. Weigh 167g DE-52 and swell in total volume of 2L 10 mM Tris (pH 7.8) 1 mM EDTA by gentle inversion and let settle 1 hour.

3. Pour off supernatants above cellulose beds (1L for each) .

4. Resuspend by gentle inversion in 2L (add -1L 10 mM Tris pH 7.8) 1 mM EDTA) . 5. Let settle for 20 minutes.

6. Pour off supernatant which contains fines leaving behind cellulose bed.

7. Repeat steps 2-4 9X.

8. Upon final resuspension let cellulose settle for 1 hour and adjust volume of 10 mM Tris (pH 7.3) 1 mM

EDTA so that the ratio between 10 mM Tris (pH 7.8) 1 mM EDTA total volume and the cellulose bed is: 1:1 for the P-ll slurry; 1.67:1 for the DE-52 slurry. Example 2 Preparation of two-layer columns.

1. Set up columns in a column pouring template.

2. Add 3 ml P-ll slurry to each column.

3. Allow the P-ll slurry to settle for 10 minutes. 4. Add 3 ml of the DE-52 slurry to each column.

5. Seat upper Porex frit in mouth of column.

6. Push frit just to the column 4 ml mark.

7. Fill column with buffer to the 10 ml mark. 8. Press column cap securely into place.

Example 3

Fractionation of M. paratuberculosis from buffer and fecal extract.

10⁷ to 10⁸ microorganisms were spiked into TE buffer and separately into a TE buffered suspension of bovine fecal matter prepared at a ratio of a 5g wet weight solids/45 ml of buffer. The solutions were filtered through cheese cloth.. The samples were passed over a two-layer column as described above washed with 10 ml 50 mM Tris (pH 7.8) followed by 10 ml 50 mM Tris (pH 7.8) aliquots containing respectively and in order, 0.1M NaCl, 0.2M NaCl and 0.2M NaCl + 40% ETOH. Each 10 ml fraction was centrifuged to pellet any Mycobacterium paratuberculosis present." The pellets were resuspended in a minimal volume of their respective supernatants, transferred to 1.5 ml Eppendorf tubes and repelleted. Following the removal of residual supernatants, 0.5 ml of a 50% suspension of zirconium oxide beads in distilled deionized water was added and the microorganisms ruptured by mulling for 10 minutes in a bead beater mulling apparatus (BioSpec Products) and pelleted at 10K rpm for 30 seconds in a Brinkman microfuge. Alternatively the cells may be lysed by resuspending the pellet in 0.5 ml of distilled deionized water, heated at 120°C for 10 minutes and pelleted by centrifugation at 14K rpm for 30 seconds in a Brinkman microfuge. 10 μl of each extraction supernate was used as sample for the polymerase chain reaction. Primers were designed to be completely Mycobacterium paratuberculosis specific. Successful PCR gives a 229 BP diagnostic fragment, as visualized on agarose gels stained with ethidium bromide or by autoradiography of Southern blots following hybridization with a Myrobacterium paratuberculosis specific probe.

Table l

Fraction Tris Tris/ Tris/ Tris/ 0.2M 01.M NaCl 0.2M NaCl 0.2M NaCl/ 40% ETOM

Buffer Sample ++++

Fecal Sample ++

Example 4

M. paratuberculosis recovery as a fraction of sample size.

Constant amounts of M. paratuberculosis were spiked into, separately, 1, 5 and 10 ml of a fecal sample suspension prepared as in example 1. Samples were-applied to a two layer column of 3ml of DE-52 and 1 ml of P-ll. Columns were developed using successive elutions with 10 ml of 50 mM Tris (pH 7.8) ,50 mM Tris (pH 7.8) + 0.1M NaCl and 10 ml 50 mM Tris (pH 7.8) + 0.2M NaCl and 40% Ethanol. The latter two fractions were centrifuged and the resulting microbial pellet lysed for the PCR as described above.

M. paratuberculosis was found only in the ethanoic eluate and the level as detected by PCR was invariant between samples as judged by ethidium bromide stained DNA band intensity following agarose gel electrophoresis. These results are summarized in Table 2. Table 2 Tris/O.IM NaCl Tris/Q.2M NaCl/40% ETOH 1 ml sample — ++

0.25 ml sample — ++ 10 ml sample — ++

Example 5

An integrated test format for rapid field resolution of Mvcobacterium paratuberculosis.

Culture negative and positive fecal samples were obtained from the state veterinary testing laboratory in Madison, Wisconsin. Samples were prepared as in example 1 and the resulting final eluate fractions were assayed for Mvcobacterium paratuberculosis genomic DNA by PCR followed by Southern transfer and detection with a Mycobacterium paratuberculosis specific probe DNA and by slot blot analysis followed by detection with the same probe. A standard dilution series of Mycobacterium paratuberculosis spiked into a negative fecal sample provided a means for rough quantitation of the amount of Mycobacterium paratuberculosis recovered from the field isolates. The results are summarized in Table 3.

Table 3

Probe Probe

+ Culture + 6 0

Culture 2 4

Culture PCR

* determined negative by CMI test

Example 6

Loading and Elution under basic conditions. 1. Crude sample is suspended in 0.2N NaOH at a final concentration of approximately 10% by weight crude sample. 2. The sample is vortexed and allowed to settle for 20 minutes to remove large particulate matter.

3. 0.1-1.5ml of the sample suspension is pipetted onto the cartridge and allowed to flow into the cartridge bed. 4. The cells are eluted with 15mls of 0.2N NaOH.

The eluate is captured in a 15ml conical test tube. (Flow may be induced with a 20cc syringe. The flow rate should be approximately 1 drop/second) .

5. 15mls of eluate is collected and the tube is capped, inverted several times, and spun at 3000 RPM for 25 minutes in a Beckman TJ6 or other equivalent centrifuge.

6. The tubes are carefully removed, and all but about 200 μl of supernatant is pipetted off.

7. The pellet is resuspended by gentle suction and 200μl are transferred to a 1.5ml screw cap tube. 8. The aliquot of resuspended. pellet is heated at 120°C for 10 minutes, vortexed gently, then spun down into the base of the 1.5ml screw cap tube.

9. lμl of sample is added to 99μl of PCR reagent and PCR performed as described above.

Other embodiments are within the following claims. What is claimed is:

Claims

1. A method of isolating cells from an impure mixture by: applying said impure mixture to an cationically derivatized cellulosic material under conditions allowing said cells to bind to said material; separating unbound mixture from said bound cells; and then applying an eluting solution to remove cells from said cellulosic material in a purified fraction.

2. The method of claim 1 wherein said impure mixture is a crude biological sample comprising impurities that interfere with selective DNA hybridization or detection thereof, said method comprising eluting said cells from said cellulosic material in a fraction that is essentially free from said impurities.

3. The method of claim 1 wherein when said impure mixture is applied to the cellulosic material under conditions whereby some contaminants do not bind to said cellulosic material and other contaminants bind to the cellulosic material and remain bound under conditions in which said cells are eluted from said cellulosic material.

4. The method of claim 1 wherein said cationically derived cellulosic material is mixed with or arranged in sequence with an anionically derived cellulosic material.

5. The method of claim 1 or 4 wherein elution is followed by detection of the eluted cells or detection of constituents of said eluted cells.

6. The method of claim 5 wherein said cells or their constituents are detected by a selective hybridization assay.

7. The methods of claim 5 wherein said cells or their constituents are detected by non-isotopic means.

8. The method of claim 1 or 4 wherein the cellulosic material is positioned within a compact disposable container.

9. The method of claim 5 wherein said eluted cells are lysed to release cell constituents.

10. The method of claim 1 or claim 4 wherein said impure mixture is provided at a pH 5.0 - 9.0 and an ionic strength of 10-200 mM.

11. The method of claim 1 or claim 4 wherein said eluting solution comprises a polar organic solvent in a concentration effective to elute said cells from said cellulosic material.

12. The method of claim 10 wherein said eluting solution has a pH 7.8 - 8.0.

13. The method of claim 10 wherein said polar solvent is selected from the group consisting of: alcohols or polyalcohol of C₄ or less ethers or polyethers of C₄ or less; aldehydes and ketones or C₄ or less; esters of C₄ or less.

14. The method of claim 13 wherein said polar solvent is ethanol.

15. The method of claim 13 wherein the concentration of said ethanol in said eluting buffer is about 40% volume/volume.

16. The method of claim 1 or 4 wherein said eluting solution is of an ionic strength effective to elute said cells from said cellulosic material.

17. The method of claim 16 wherein said eluting solution is strongly basic.

18. The method of claim 17 wherein said eluting solution is 0.1 - 1.0 N NaOH.

19. The method of claim 17 wherein said impure mixture comprises crude sample resuspended in a strongly basic solution, said derivatized cellulose comprising a buffering substituant providing a localized column pH substantially below the pH of said strongly basic solution. and said cells are eluted by supplying base in said eluting solution sufficient to raise the localized column pH to a level which effects release of said cells from said derivatized cellulose.

20. The method of claim 19 wherein said strongly basic solution is 0.1 - 1.0 N NaOH and said eluting solution is 0.1 - 1.O N NaOH.