WO1994029721A1

WO1994029721A1 - A method of capturing particles

Info

Publication number: WO1994029721A1
Application number: PCT/SE1994/000519
Authority: WO
Inventors: Lahja SEVÉUS; Håkan DREVIN
Original assignee: Pharmacia Ab
Priority date: 1993-06-07
Filing date: 1994-06-01
Publication date: 1994-12-22
Also published as: AU6986594A; SE9301929D0

Abstract

A method of biospecifically capturing biological particles on a porous matrix, comprising bringing the particle suspended in an aqueous liquid into contact with the pore system of a porous matrix which is insoluble in water and the pore surfaces of which have bound thereon a bioaffinity counterpart to the particle to be captured, under conditions which enable desired particles to bind to the pore surfaces of the porous matrix via the bioaffinity counterpart. The method is characterized in that the matrix is elastically compressible, self-supporting and has capillaries of such pore dimensions as to enable the particles to pass through the matrix. The matrix is preferably brought into contact with the liquid in a compressed state and is allowed to relax after contact with the liquid. Alternatively, the matrix is subjected to cyclic compression-relaxation, preferably several times, during the incubation phase.

Description

A METHOD OP CAPTURING PARTICLES

TECHNICAL FIELD AND DESCRIPTION OF KNOWN PRIOR ART

Biospecific capturing of cells, cell organelles, virus and other particles of biological origin that can be suspended in aqueous media have earlier been used in cell culturing, immuno analysis, and other processes. The capturing stage has involved bringing an aqueous suspension (= sample) containing the particles into contact with a matrix, often a porous matrix, which is insoluble in the suspension medium. In order to make capturing of the particles selective, the matrix has exhibited a bioaffinity counterpart to a surface antigen on the particles.

The term bioaffinity counterpart relates to substance pairs which bind to one another via so-called biospecific affinity. The two components of such a pair are often referred to as the receptor and the ligand respectively. Examples in this respect include the pairs antibody-antigen/hapten, hormone- receptor, IgG(Fc)-protein A/protein G, lectin-carbohydrate structure, etc. The term also includes the fragments of the native forms and derivatives having a biospecific affinity which is the same as that of respective native forms. Examples in this respect include antibody active antibody fragments.

Earlier publications relating to the technical field covered by the present invention include:

EP-A2-437,287 which describes a porous solid-phase matrix for assaying methods which._ ιu*tilize reactions between ligand and receptor. The pores of the system include microspheres which are sufficiently large to be locked in the system. The surfaces of the microspheres present receptors which have a bioaffinity for the substance to be captured. In some of the embodiments described, the receptors are directed against specified viruses or bacteria. The matrix may be compressible, so as to enable sample liquid or reagent to be squeezed therethrough. Examples in this regard include filters and membranes. WO-A-9116116 relates particularly to the separation of selected particles (target particles) from mixtures which contain the selected particles and also other particles (non-target particles) . The matrix used is porous and is preferably composed of beads or pearls and shall always be placed in a column. The matrix presents receptors which are directed specifically to ligands which are unique to selected particles.

PROBLEMS SOLVED BY THE INVENTION

It has now been discovered that it is possible to increase the number of particles that are captured, by using a resilient, compressible and self-supporting matrix having pores of capillary dimensions. The capillary dimensions enable the matrix to draw in and retain the liquid/particle suspension under its own power. Because the matrix is self- supporting, it is not necessary to place the matrix in a peripheral vessel in order to prevent the collapse and drainage of the matrix. The resiliency of the matrix facilitates the absorption and drainage of the liquid. The matrix is particularly effective when compressed and relaxed in cycles, since this results in "agitation" of the liquid within the pores, which in turn results in shorter incubation times and also in more effective capture of the particles. The invention is based on the realization that suspended particles will settle instead of diffusing, and that the sediment thus produced will reduce the availability of the particles for reaction with the receptors of the matrix (bioaffinity counterpart to the particles) .

Because the invention increases the yield of the capture, it is possible to improve the sensitivity of any subsequent analysis. The invention provides potentially improved and simplified culturing and harvesting methods, when captured cells are to be cultivated. DISCLOSURE OF THE INVENTION

GENERAL DESCRIPTION

The invention provides an improvement to the method defined in the introduction for capturing suspended particles on a matrix. The inventive method is characterized in that the capturing matrix is elastically compressible, is self- supporting and presents capillaries of such pore dimensions as to enable the suspended particles to pass in and out of the matrix (when ignoring capture of the receptor that is bound to the matrix) . The matrix presents pores that are larger than the particles.

METHOD STAGES

According to the invention, the matrix is brought into contact with the liquid (= the sample) containing the particles to be captured. The matrix may initially be in either a relaxed state (i.e. not compressed) or in a compressed state. In the former case, the capillary forces draw liquid and suspended particles into the matrix. The same applies in the latter case, in which the matrix is in a compressed state, although the capillary forces are now amplified by allowing the matrix to relax in contact with the liquid. There is an endeavour for the particles to spread homogeneously in available pores in both instances. Spreading of liquid and particles throughout the matrix can be made more effective after a first contact with the liquid in both of said instances, by subjecting the matrix to at least one further compression-relaxation process, preferably two or more such processes. Compression-relaxation of the matrix during the capturing reaction (= incubation) will prevent the particles from settling (sedimentation draws the particles away from reaction with the bioaffinity counterpart) , which in turn makes the capturing reaction more effective and results in shorter incubation times. In this regard, the matrix may be compressed fully or only partially. The matrix may be placed in a vessel, so as to ensure that any liquid squeezed from the matrix will be reabsorbed by the matrix in the relaxing phase. When a matrix is used in a relaxed state, it is preferred that the matrix is not saturated with liquid when brought into contact with the sample.

Sedimentation can also be prevented by changing the position or attitude of the matrix during the incubation phase (by turning, twisting or reversing the matrix, or by turning it upside down, etc.).

THE MATRIX AND BIOAFFINITY COUNTERPART (RECEPTOR)

As a rule, the matrix is comprised of a base polymer, which is normally an organic polymer. For instance, the matrix may be based on a biopolymer, such as cellulose and collagen, or on a synthetic or semi-synthetic polymer, such as polyacrylamide, polyurethane, polyacrylate, derivatived cellulose, polyvinyl alcohol, etc. The polymer will prefer-a¬ bly exhibit hydrophilic groups, such as a plurality of amino and/or RO-groups, where R may be hydrogen, R (OCH2 ^CH2)n, etc. n is an integer, for instance an integer in the range 1-25. R may be hydrogen or a low molecular organic group, such that the group R¹-(OCH₂CH₂)n- will retain its general hydrophilic nature. Methyl and carboxymethyl are examples of R . The integer n need not necessarily be unitary on a matrix. For instance, if commercially available polyethylene oxide is used to introduce the group R - (OCH2CH2)n-, th^e integer n will vary in the final product

(since commercially available polyethylene oxide is comprised of a mixture in which n varies in respect of the individual components) . When the matrix base polymer is pronouncedly hydrophobic, its pore surfaces may be derivated to present the aforesaid hydrophilic groups. This can be achieved, for instance, by binding covalently or absorbing physically a polymer which exhibits the groups in question to the matrix pore surfaces. See, for instance, EP-A-211,046 and US-A- 4,535,

010. Stabilization of a matrix adsorbed polymer is usually achieved by cross-linking the polymer subsequent to its adsorption.

Generally speaking, there are two suitable main types of matrix:

(a) Compacted fibres (for instance fibres combined by compression or covalent bonds) ; and (b) continuous matrices (so-called monolithic matrices) .

The pores of the matrix shall have a size which will enable the suspended particles to penetrate the matrix and also to exit from the matrix, when the intention is not to capture the particles. Excessively small pores will result in clogging of the matrix, therewith rendering the matrix useless. Excessively large pores will result in low capillary forces and in the risk that the matrix will be unable to absorb and retain the aqueous liquid. In the case of embodiments at present preferred, the pore system is comprised of a three-dimensional network of pores which have a size that will enable the particles to move in three dimensions. In the case of those variants illustrated in the experimental part of this specification, the pore system includes through-penetrating pores, i.e. pores which extend between two sides of the matrix.

The magnitude of the capillary forces is determined by the hydrophilicity/hydrophobicity of the pore surfaces, in addition to being determined by the size of the pores. In relation to water pronouncedly hydrophobic surfaces produce weak capillary forces while pronouncedly hydrophilic surfaces produce strong capillary forces. As a rule of thumb, at least that part of the matrix which is filled with suspension will preferably provide a pore size which is at maximum in the range of 100 nm-100 μm. These pore dimensions will preferably be distributed uniformly in the body of the matrix. The water absorption capacity of the matrix will preferably lie in the range of 100-990 mg water/cm³ calculated on a dry matrix.

The capturing bioaffinity counterpart will preferably be distributed homogeneously in the matrix, at least in those parts that can be penetrated by the particles to be captured. The bioaffinity counterpart may be bound to the pore surfaces covalently, adsorptively or by biospecific affinity. In the case of this latter variant, there is used a bioaffinity reactant which is immobilized on the matrix and which has an affinity for the capturing counterpart. Relevant variants for binding the capturing counterpart by biospecific affinity are:

(A) The capturing counterpart is conjugated with an antigen/hapten or with biotin in combination with the matrix exhibiting the homologous antibody of the antigen/hapten and avidin/strepavidin respectively. The reverse is also possible theoretically. (B) A matrix bound anti-antibody that is biospecifically bound to an antibody that is specific to the particle to be captured.

On the date from which the convention priority of this application is claimed, it was preferred that the bioaffinity counterpart binds directly to a surface layer in the matrix pores. This surface layer also includes a coating which renders the surface hydrophilic.

PARTICLES

The particles are of a biological origin and are suspended in the aqueous liquid (= the sample) . The particles may consist in intact living or dead cells; particulate cell parts (cell organelles) , such as mitochondria and lysosomes; viruses; bacteriophages, etc. The surface structure (the ligand) used on the particles is often native, but may optionally be modified prior to bringing the particles into contact with the matrix.

Modification of the particles prior to capturing in the matrix may comprise, for instance, pre-incubating the particle suspension with antibodies directed against structures of interest. Such an antibody may be conjugated to an antigen/hapten or to biotin. When the antibody directed against the particles is not derivatived, particles can be captured via a matrix-bound anti-antibody. In the case of antibodies conjugated to an antigen/hapten/biotin, capture can be effective via a matrix-bound antibody directed against the antigen/hapten or matrix-bound avidin/strepavidin. Analogously with what has been said above with regard to antigen/hapten and biotin, the reverse can also be used, i.e. the matrix may present the antigen/hapten or may be biotinylated and the particle specific antibody may be conjugated to an antibody which is directed against the matrix-bound antigen/hapten or against avidin/strepavidin.

STAGES WHICH FOLLOW THE CAPTURING STAGE

The capturing stage is normally followed by one or more washing stages using an appropriate buffer to remove uncaptured particles and suspension medium. The matrix is preferably washed after each capturing reaction and each washing stage is terminated by compressing or sqeezing the matrix to drain washing liquid therefrom. Naturally, the matrix shall be allowed to relax when liquid is again applied, either before or during application of liquid.

FIELD OF USE OF THE INVENTION

The inventive method may be applied in a quantative or qualitative assay of the particle concerned. The assaying process may involve first eluating the particle with a buffer which breaks one of the bonds holding the particle to the matrix (the bond between the matrix and the bioaffinity counterpart or between the bioaffinity counterpart and the particle) . The types of bonds concerned can be broken by adding soluble, competitively binding substances (antigen, hapten, antibody, biotin, avidin/strepavidin) or by lowering or raising the pH to an acid or an alkaline pH-level respectively. Denaturizing conditions should be avoided if the particle is to remain intact. When necessary, the particle can be lysed prior to assaying the particle. Alternatively, the assay can be carried out directly on the matrix without preceding release/lysing.

Among the assaying methods concerned are those which utilize receptor-ligand interactions with a receptor marked or labeled with an analytically detectable group, such as enzyme, fluorophor, isotope, coenzyme, enzyme substrate, inert particle, etc. Particular mention can be made in this respect to immunological assaying methods which utilize reactions between antibody and antigen/hapten, often with the aid of an antibody which is directed against the particle (or a part thereof) and which may also be labeled or marked with an analytically detectable group or which can be shown in some other way. When the assay is carried out directly on the matrix, i.e. the captured particle is allowed to react with an appropriate reagent (possibly an antibody) , it is possible to achieve very short incubation times since the capillary pores are small, which in turn means that the reactions can be achieved quite independently of diffusion, or practically independently thereof. See for instance US-A-4,708,932. Other methodology may be used when the cells are eluated, such as conventional particle counts, flow cytometry, "fluorescence activated cell sorts (FACS)", immunocytochemistry, etc. When the particles are lysed and a released cell component is assayed, chromatographic and electrophoretic techniques, centrifugation, etc. , can be used to isolate or to enrich the component to be assayed.

After being captured, the cells may be released and cultured. One point of interest in this respect is that it has been observed that the cells grow on some of the matrices concerned and divide even after being caught. This enables cells captured on the matrix to be cultured without their intermediate release. This can afford advantages within immunocytochemical applications, among others.

The aforedescribed assaying and culturing methods can be applied for a diagnostic purpose when the sample (and the particles) derives from a multi-cell organism, preferably mammal, such as a human being. When applied in this way, the result of the assay is correlated to corresponding values of the organism in a healthy individual . When the values differ, this is taken as an indication that the organism from which the sample originates suffers from some form of disease or illness, the nature of which will depend on J he type of particle assayed, the bioaffinity counterpart that has been used in the capturing process and possibly also on what has been determined in respect of the captured particle.

The invention will now be described with reference to a number of examples. Specific embodiments of the invention are defined in the accompanying Claims.

MATERIALS AND METHODS

1. Cells

Target cells: (a) Surface immunoglobulin, IgM-positive B- lymphocytes from a patient with lymphoma. These cells have a diameter of 8-10 μm and carries as an identifier the CD19 surface marker. (b) Cultured C202 colon cancer cells with the surface antigen to C242 antibody (EP-A-521,842) . Control cells: (a) Monoclonal myeloblasts isolated from the blood of an acute leukemia patient. The cell size was 15-20 μ m. (b) Human erythrocytes. Size 10 μm. These control cells were selected because they were well characterized and have no markers binding to the capturing antibody (bio-affinity counterpart) . Cell characterization: Cells were characterized by using flow cytometry before application of the cells to the matrix.

2. The capturing bioaffinity counterpart (antibody and avidin)

(a) The capturing antibody for the surface IgM-positive B- lymphocytes was a rabbit anti-human IgM antibody, (b) The capturing antibody for C205 cancer cells was the C242 antibody (EP-A-521,842) . (c) Avidin was Neutralite® avidin with a specific biotin binding activity of 14 U/mg from Eurogentec S.A.

3. Matrices

Bioaffinity matrices were (a) cellulose viscose sponge in sheet form (Kettenbach AG, Germany) and (b) block polymerized sponge (Cellomeda®, a matrix based on cellulose from Cellomeda Oy, Turku, Finland) . The matrices were activated with cyanogen bromide (Thesis: The chemistry of the interaction of cyanogen bromide with polysaccharide resins. Joachim Kohn. Weiz ann Institute of Science, Rehovot,

Israel) , whereupon the capturing antibody or avidin was coupled to the matrices. Concentrations of the antibody were 100 or 1000 μg per cm . Finally, the matrices (0.2 cm³) were punched out from the sheet. Each piece of matrix has an absorption capacity of 50-60 μl of water.

Negative control matrices were Pharmacia CAP System matrices carrying mold allergen or anti-IgE (Kabi Pharmacia Diagnostics AB, Uppsala, Sweden) . The basic matrix was of the same type as those used for coupling antibody and avidin. Before use, each matrix was placed in a holder (the capsules used in Pharmacia CAP Systems (Kabi Pharmacia Diagnostics AB, Uppsala, Sweden) and soaked with buffer or culture medium. Note that since the experiment deals with living cells, detergents, such as Triton® and Tween®, and antimicrobials, such as Kathon®, were omitted in the soaking solutions and also in all other solution used in contact with the cells.

4. Sample application to the matrix and cell counting .1 Volume of cell suspension and number of cells: The cells were suspended in either a culture medium (RPM-1) or in a 0.1 M phosphate buffer at pH 7.2 containing 0.25% bovine serum albumin (BSA). Suspensions containing a single cell type and mixtures of cell types were prepared (equal number of each type) . The standard number of cells in the experiments was adjusted to 300,000 cells/50 ml suspension. Since the absorption capacity for the matrices used was 50 ml, the same volume was added to the matrix in each run. In order to find the maximum cell binding capacity, the number of cells was adjusted to 3,000,000 cells/50 ml in one experiment. In a number of experiments, the addition of cell suspension to the matrix and incubation of cells within the matrix was repeated two or three times, with new suspensions containing 300,000 cells/50 ml.

4.2 Application and cell capturing protocol: The matrix used in the experiment was first compressed with a glass rod to remove buffer solution therefrom. The matrix was then placed in a microtiter well containing 50 ml of the cell suspension without first drying the matrix. The cell suspension was absorbed instantaneously by the matrix and allowed to incubate for a period ranging from 15 minutes to an hour. After incubation, the cell suspension was removed from the matrix with the aid of a micropipette. The volume of the solution was measured. Finally, the matrix was washed by applying repeatedly 50 ml of buffer (5x) (0.1 M PBS pH 7.2. The matrix was rinsed, by pressing the matrix gently during every rinsing stage (5 minutes) . The removed incubation media and the used rins •_i_n_*<•g media were pooled and counted for cell content.

When an avidin matrix was used, the cell suspension, in a prestep prior to the addition to the matrix, was first incubated with an antibody reactive with its specific surface marker (anti-CD19 or anti-C242 mouse monoclonal antibody) and then with a biotinylated anti-mouse antibody (DAK0-CD19, clone HD37, isotype IgG-kappa; Dakopatts, Denmark) .

4.3 Counting and characterization of cells: The number of cells which were added to the matrix and those removed as uncaptured were counted in a Burker chamber. The different cell types were identified by their structural characteristics, mainly size. Flow cytometry and immunocytochemistry were used in the characterization process.

5. Special controls

In order to test the specificity of capturing the B- lymphocytes to the matrix, the specific IgM-binding sites were blocked by preincubating the cell suspension with the anti-IgM antibody before adding the cells to the matrix.

RESULTS Table 1 Target cells: B-lymphocytes. Buffer: Culture medium.

Control anti-IgM 300,000 cells CAP mixed with

B-cells in anti-IgM 300,000 67 mixed Cellomeda population

Control anti-IgM 300,000 cells Cellomeda

B-cells on anti-IgM 300,000 29 which IGM has been blocked with anti- IgM

These results clearly indicate a selective capture of target cells on the matrix used in the invention. In one experiment, a suspension containing 3,000,000 target cells was applied to the matrix. 560,000 of the cells were captured. This number of captured cells is extremely high, in comparison with the volume of the matrix and its water absorption capacity.

The effect of squeezing an elastic matrix during incubation

One of the above-mentioned matrices (0.2 cm 3) was placed in a transparent microtiter well equipped with a collecting vessel at the bottom. A suspension containing 300 plastic beads was pipetted onto the matrix. The number of particles appearing in the collecting vessel was determined with the aid of an inverted confocal mode microscope. The matrix was not squeezed (compressed) in run one. The cumulative number of particles that had passed through matrix after given time intervals was:

Time (min.) 2 5 7 10 15 20 30 washing 1x50 μl - 2 5 51 58 78 121 151

In a second run, the matrix was squeezed three times (compressed for about 10 seconds each time) followed by three washings/compressions (3x50 μl) . The cumulative numbers of particles that had passed through the matrix in each stage were:

1st compr. 2nd compr. 3rd compr. 1st wash 2nd wash 3rd wash 130 180 200 280 293 294

The results show compression of the matrix facilitates penetration of beads into the matrix.

Claims

1. A method of capturing biospecifically biological particles on a porous matrix in which particles suspended in an aqueous liquid are brought into contact with the pore system of the matrix, and in which the matrix is insoluble in said liquid and the pore surfaces of which have bound thereto a bioaffinity counterpart to the particle to be captured, under conditions which enable desired particles to bind to the pore surfaces of the porous matrix via the bioaffinity counterpart, characterized in that the matrix is elastically compressible, self-carrying and comprises capillaries of such pore dimensions as to enable the particles to pass through the matrix.

2. A method according to Claim 1, characterized in that the matrix is hydrophilic.

3. A method according to any one of Claims 1-2, charac- terized in that the matrix is brought into contact with the liquid in a compressed state and is then allowed to relax to its original state, wherein liquid and particles are sucked into the matrix and dispersed throughout the pore system of said matrix.

4. A method according to any one of Claims 1-2, charac¬ terized in that the matrix is brought into contact with the liquid in an uncompressed state and with the pore system empty of liquid.

5. A method according to any one of Claims 1-4, charac¬ terized by subjecting the matrix to at least one compression- relaxation phase, preferably several phases, subsequent to having brought the matrix into contact with said liquid.

6. A method according to Claim 5 , characterized by placing the matrix into a vessel which will ensure that liquid is reabsorbed during the matrix relaxing phase.

7. A method according to any one of Claims 1-6, charac¬ terized in that the matrix is comprised of compacted fibres.

8. A method according to any one of Claims 1-6, charac¬ terized in that the matrix is a continuous matrix.

9. A method according to any one of Claims 1-8, charac¬ terized in that the matrix is comprised of a polymer which preferably presents hydroxyl groups.

10. A method according to any one of Claims 1-9, charac¬ terized in that the bioaffinity counterpart bound to the pore surfaces of the matrix is an antibody which is directed against a surface antigen of the type of particles to be captured.

11. A method according to any one of Claims 1-10, charac¬ terized in that the bioaffinity counterpart binds to the pore surfaces via covalent bonds, physical adsorption or a bioaffinity bond, such as the bond between biotin and avidin or between hapten and antibody.

12. A method according to any one of Claims 1-9, charac- terized in that the bioaffinity counterpart bound to the matrix is biotin or avidin (including modified forms of biotin capable of binding to avidin or strepavidin and modified forms of avidin capable of binding to biotin) , preferably avidin; and in that the particle has been allowed to bond in a pre-stage to its avidin-modified or biotin- modified bioaffinity counterpart.

13. A method according to any one of Claims 1-12, charac¬ terized in that the particles are cells, viruses or cell organelles.

14. A method according to any one of Claims 1-13, charac¬ terized in that the captured particles are assayed qualita¬ tively or quantatively in a following stage.

15. A method according to Claim 14, characterized in that captured particles are cells or viruses and that they are eluated prior to being assayed, either intact or lysed.

16. A method according to any one of Claims 14, character- ized in that the captured particles are cells or viruses and that the particles are assayed on the matrix without preceding eluation.

17. A method according to any one of Claims 14-16, charac- terized by assaying captured particles immunochemically with the aid of an antibody directed against a cell component of the captured cell.

18. A method according to any one of Claims 14-17, charac- terized in that the particles originate from a biological sample taken from a multi-cell organism, preferably a mammal, such as a human being; and by correlating the result of the assay to a state of illness of the multicell organism.