EP0782704A1 - Separation cellulaire - Google Patents

Separation cellulaire

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Publication number
EP0782704A1
EP0782704A1 EP95930638A EP95930638A EP0782704A1 EP 0782704 A1 EP0782704 A1 EP 0782704A1 EP 95930638 A EP95930638 A EP 95930638A EP 95930638 A EP95930638 A EP 95930638A EP 0782704 A1 EP0782704 A1 EP 0782704A1
Authority
EP
European Patent Office
Prior art keywords
support
cells
target
specific binding
binding partner
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP95930638A
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German (de)
English (en)
Inventor
John Ugelstad
Per Stenstad
Lars Kilaas
Arvid Berge
Wenche Slettahjell Prestvik
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sinvent AS
Original Assignee
Sinvent AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sinvent AS filed Critical Sinvent AS
Publication of EP0782704A1 publication Critical patent/EP0782704A1/fr
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the present invention relates to a method for blocking the non-specific binding of cells in selective cell isolation procedures.
  • cell separation is a necessary step in the purging of bone marrow of malignant cells or to obtain a purified population of a particular cell type, for example for reconstitution or for subsequent investigation or manipulation, eg. by culture or by the introduction of genetic material.
  • Cell separation is also frequently used in the diagnosis of diseases and infections, for example by isolating pathogenic cells such as bacteria or protozoa or viruses from clinical samples, and in the monitoring of bacterial or other pathogenic contamination, for example in food or waste water. Cell separation thus has .implications in many important areas eg. the treatment and research of serious diseases, diagnosis, blood and tissue typing, food and environmental safety etc.
  • a problem with this form of separation is the non ⁇ specific binding of unwanted cells to the support resulting in the separation not only of the desired target cell which binds to the support by means of the target specific binding partner, but also other, undesired cells which bind non-specifically to the surface of the support.
  • the degree of non-specific binding may vary with the nature of the cells, support and affinity-binding system used, but generally speaking, this is a problem with all solid phase affinity capture based-systems, and particularly immunomagnetic separation. This can have potentially serious consequences, for example where it is important that only the desired cell is isolated eg. in bone marrow purging or in typing, and may lead, for example to confusing results in diagnostic systems.
  • BSA bovine serum albumin
  • casein has previously been used in the treatment of solid surfaces, eg. polystyrene to prevent physical adsorption of proteins.
  • Such utility has most notably found application in immunoassay procedures such as ELISAs, where plates are blocked by exposure to casein solutions (see for example Vogt et al. , J . Immunol. Methods. 101. 43-50, 1987), but other uses, for example in Western and Southern blotting, have also been described (see e.g. Johnson e_L al. , Gene Anal. Techn. 1, 3-8, 1984).
  • casein might be effective to prevent the non-specific adhesion of cells to solid supports.
  • the present invention thus provides the use of a milk protein to block the non-specific binding of cells to a solid phase selective cell-capture matrix.
  • the invention also provides a method of blocking non-specific binding of cells to a solid phase selective cell-capture matrix, said method comprising adding a milk protein to said matrix.
  • the solid phase selective capture matrix will be a solid support adapted for the selective separation of target cells by means of a target-specific binding partner, eg. the solid support may carry a target-specific binding partner.
  • the present invention thus provides a method of selectively isolating a target cell from a sample, comprising selectively binding said target cells to a solid support by means of a target-specific binding partner and separating said support-bound cells from the sample, characterised in that a milk protein is added to block non-specific binding of non-target cells.
  • cells is used herein to include all prokaryotic and eukaryotic cells and other viable entities such as viruses and mycoplasmas, and sub- cellular components such as organelles. Representative “cells” thus include all types of mammalian and non- mammalian animal cells, plant cells, protoplasts, bacteria, protozoa and viruses.
  • blocking includes reducing as well as preventing said non-specific binding.
  • the reduction in non ⁇ specific binding obtainable by the present invention is much higher than could have been predicted and significantly better than that obtained with BSA and other proteins. Indeed, treatment with milk protein of solid supports already pre-treated with BSA, leads to a marked reduction in non-specific binding compared to supports treated with BSA alone.
  • the milk protein may be any of the milk proteins known and described in the literature for blocking of non-specific adsorption in immunoassays, including for example, casein, whey protein concentrate, non fat dry milk and skim milk. Any of those milk proteins may be used, alone or in combination. Casein represents a convenient choice. Also included are derivatives of milk proteins such as partial hydrolysates or cross- linked proteins.
  • the solid support may be any of the well-known supports or matrices which are currently widely used or proposed for immobilisation, separation etc. These may take the form of particles, sheets, gels, filters, membranes, or microtitre strips, tubes or plates etc. and conveniently may be made of a polymeric material. Particulate materials eg. beads are generally preferred, due to their greater binding capacity, particularly polymeric beads, a wide range of which are known in the art. To aid manipulation and separation, magnetic beads are preferred.
  • the term "magnetic" as used herein, means that the support is capable of having a magnetic moment imparted to it when placed in a magnetic field, and thus is displaceable under the action of that field.
  • a support comprising magnetic particles may readily be removed by magnetic aggregation.
  • magnetic particles are superparamagnetic to avoid magnetic remanence and hence clumping, and advantageously are monodisperse to provide uniform kinetics and separation.
  • the preparation of superparamagnetic monodisperse particles is described by Sintef in EP-A-106873.
  • the monodisperse polymeric superparamagnetic beads sold as DY ABEADS by Dynal AS (Oslo, Norway) are exemplary of commercially available magnetic particles which may be used or modified for use according to the invention.
  • Non-specific binding of cells to such supports occurs by physical adsorption of the cells and has been observed with all types of solid surfaces which may be used in cell separation procedures, notably polymer supports, including both hydrophilic and hydrophobic surfaces and all such supports are included within the present invention.
  • polymer supports including both hydrophilic and hydrophobic surfaces and all such supports are included within the present invention.
  • hydrophobic supports are favoured for the coupling of specific binding partners such as antibodies (or at least that the surface be hydrophobic at the time of the coupling reaction) .
  • the reason for this is that the binding of antibodies to supports by chemical coupling in some cases is a slow reaction.
  • a spontaneous and high degree of adsorption of the antibody to the support surface results in a very large increase in the concentration of the antibody at the reaction site and thereby leads to a higher rate of chemical coupling.
  • the use of relatively hydrophobic surfaces therefore strengthens the need for effective blocking of non ⁇ specific adhesion of cells to the surface of the support.
  • milk protein in hindering non-specific binding of cells is clearly and unambiguously noticeable with any type of support surface, including rather hydrophilic surfaces but it is especially marked with hydrophobic surfaces, where casein is dramatically better than BSA when using optimal amounts of the latter.
  • the target-specific binding partner may be any grouping capable of recognising and binding to the target particle and conveniently may comprise any such binding partner as is conventionally used in separation and immobilisation techniques.
  • the binding partner may comprise a single entity capable both of binding selectively to the target cell and to the support, or a series of entities, comprising at one end a component capable of binding selectively to the target cell, and at the other end a component which binds to the support.
  • a series may comprise a primary binding partner recognising the target cell and a secondary binding partner, binding to the primary partner and to the support.
  • the binding partner will comprise an antibody, or antibody fragment, recognising an antigen on the surface of the cell, virus particle etc.
  • the antibody may be mono- or polyclonal or chimaeric and may be used in the form of a fragment which retains binding activity, eg. F(ab) 2 , Fab or Fv fragments (the Fv fragment is defined as the "variable" region of the antibody which comprises the antigen binding site) .
  • Alternative binding partners include proteins such as avidin, lectins, or ligands binding to receptors on the surface of the cells.
  • the binding partner may be chosen to recognise selectively surface epitopes specific to the particle, eg. surface antigens expressed only by a particular type of cell, or the binding partner may be of more general reactivity eg. capable of recognising a range of cells.
  • antibodies and their fragments are generally the preferred binding partner, particularly IgG and IgM antibodies.
  • Monoclonal antibodies can readily provide desired target specificity.
  • such antibodies or their fragments may be coupled directly to the support, for binding to the target cell, or they may be coupled indirectly, as mentioned above, as part of a binding partner series.
  • indirect coupling may be via a secondary antibody which is itself bound to the support and which binds to the target-specific, primary antibody at a site, eg. the Fc portion, which leaves the primary antibody free to bind the target.
  • a secondary antibody may be replaced by an immunoglobulin-binding protein eg. protein A.
  • the support may carry functional groups such as hydroxyl, carboxyl or amino groups which by well known methods may be activated to allow covalent binding of antibodies or of suitable ligands for attachment of antibodies.
  • suitable ligands are avidin or streptavidin which may be coupled directly to the support for subsequent binding with a biotinylated target-specific binding partner.
  • the support may also carry groups such as epoxy or aldehyde groups, in which case no activation is needed for establishment of covalent coupling of the antibodies or ligands to the support.
  • a large number of antibodies are available against specific markers expressed on cells of the haematopoietic system (see for example the range of antibodies against CD antigens available from Dako, Copenhagen) .
  • the anti-CD34 antibodies 12.8 and B1-3C5 useful for selecting early haematopoietic cells are available from Biosys S.A, France.
  • the literature also contains descriptions of numerous antibodies suitable for selecting infectious agents ' including bacteria, protozoa and viruses.
  • antibodies against the K88 (F4) fimbrial antigen of E.Coli are described by Lund et al in J.Clin.Microbiol. 2£.-2572-2575; Skjerve and Olsvik used a commercial polyclonal goat IgG in the immumagnetic separation of Salmonella (J.Fod Microbiol. 14.-H-18, 1989)
  • * sero-group specific monoclonal antibodies against Salmonella are described by Widjojoatmodo et al.. (Eur. J. Clin, Microbiol.Infect.Dis.10:935-938, 1991); Skjerve et al.
  • poly or monoclonal antibodies of the desired specificity may be obtained using standard techniques.
  • the binding partner may be chosen to bind wanted or unwanted particles ie. to achieve positive or negative selection, for example either to isolate a desired population of cells or to purge unwanted particles from a system.
  • the method of the invention has been found to be particularly useful in the positive selection of various desired cell populations, for example from blood, plasma or other body fluids or clinical samples, or from cell culture or other biological or artificial media etc.
  • binding partners such as antibodies and their fragments to supports to provide affinity capture matrices and are widely described in the literature.
  • the supports may be provided with a range of functional groups which may be activated to give a covalent bond between the support and the antibody by reaction of the activated group with amino or SH groups in the antibody.
  • the antibody so bound may, as mentioned above, be a target-specific antibody, or it may be a secondary antibody, binding the primary, target-specific antibody. Examples of the most common methods are: 1.
  • Coupling of antibodies to supports containing epoxy groups takes place without further activation as the epoxy groups react directly with amino groups and -SH groups on the antibody. Analogous methods may be used to couple other binding partner proteins.
  • the above-mentioned magnetic beads or other supports may be provided with a range of functional groups for attachment of the binding partner, (eg. hydroxyl, carboxyl, aldehyde, epoxy or amino groups) or they may be modified, eg. by surface coating, to introduce a desired functional group.
  • functional groups for attachment of the binding partner eg. hydroxyl, carboxyl, aldehyde, epoxy or amino groups
  • they may be modified, eg. by surface coating, to introduce a desired functional group.
  • US-A-4654267, 4336173 and 4459378 describe the introduction of many such surface coatings.
  • the binding of the target cells to the support may take place by a number of steps. For example all the reagents, including the specific binding partner may be bound stepwise to the support, which is then contacted with a sample containing the target cells. Alternatively the target cell population may be contacted, in a separate step, with the specific binding partner e.g. a monoclonal antibody, before exposure to an appropriate insoluble support.
  • the specific binding partner e.g. a monoclonal antibody
  • One preferred method is to bind an antibody or other target-specific binding partner to the support, prior to contacting with the target cells.
  • an antibody (as target-specific binding partner) may be bound to the target cells in a first step.
  • a secondary antibody capable of binding to the Fc region of the primary antibody is attached to the support, following which the cells and support are brought into contact.
  • a particularly advantageous coupling system for selectively binding a target cell to a solid support is described in our co-pending international patent application No. W094/20858. This is based on a hydroxyboryl/cis-diol linkage between a target-specific binding partner and the solid support.
  • the linkage may take the form of a binding partner system comprising a series of entities, and either the cell-recognising primary target-specific binding partner, or a secondary binding partner, binding the primary partner is attached to the support in this .
  • a primary or secondary antibody may be the subject of the hydroxyboryl/cis-diol linkage.
  • the hydroxyboryl-based system has been found to be particularly effective in the coupling, and subsequent release of IgM antibodies and the use of an IgM binding partner coupled to a support by means of a hydroxyboryl/ cis-diol linkage, represents a preferred aspect of the invention.
  • hydroxyboryl-based linkage a favourable orientation of the binding partner on the support is obtained, without detracting from its selective binding. This leads in turn to highly efficient and reliable binding of the target cell to the support.
  • an additional advantage of the hydroxyboryl-based system is that under certain conditions the binding of the hydroxyboryl/cis-diol residues may readily be reversed under mild conditions, thereby liberating the target cell in a simple and non ⁇ destructive manner.
  • the hydroxyboryl/cis-diol linking method is advantageous, it does seem to suffer particularly from the problem of non-specific cell binding, which appears to be especially marked with this method.
  • marked non-specific binding of B cells and monocytes has been observed, threatening severely to limit or complicate the use of the technique where such cells are present.
  • the use of milk protein according to this invention is particularly effective to block non-specific cell binding with solid supports provided with hydroxyboryl- based linkages and this represents one particularly preferred aspect of the invention.
  • Hydroxyboryl-based linkages may be broken simply and effectively by adding a competing cis-diol reagent as described in WO94/20858.
  • the time of addition of the milk protein, and the length of time the support is exposed or incubated with it will depend on the nature of the support, and linkage and also whether or not it is desired to liberate the target cells following separation. In all cases, the addition of the milk protein must be such as to permit a sufficient linkage of the binding partner to the support to take place,* in addition to preventing non-specific cell binding the milk protein will also have the effect of reducing binding of the binding partner to the surface. Generally speaking therefore, the milk protein is added after the binding partner is linked or bound to the support. Where the binding partner is a multi- component system, this will generally be after the support-binding component eg. the secondary antibody in the systems described above, is bound.
  • the support-binding component eg. the secondary antibody in the systems described above
  • Some milk protein may be added, if desired, during the binding partner attachment step, but generally speaking, the main portion is added after the binding partner (binding partner component) is bound.
  • the inclusion of additional protein during the binding partner attachment step may be advantageous in promoting binding of the binding partner in the correct orientation, and milk protein may be used to provide such additional protein.
  • proteinaceous binding partners such as antibodies tend to form hydrophobic interactions with hydrophobic surfaces which strengthen their linkage to the support. This may be advantageous in cases where subsequent detachment is undesired or where it is particularly desirable to avoid leakage of the binding partner from the support eg. in typing reactions. Thus, in such cases it may be desirable to avoid exposing the support to the milk protein for a longer period of time after the coupling reaction, to ensure that a stronger hydrophobic bond is formed. In such a situation the blocking step of incubating the support with milk protein may conveniently take place at any time sufficient for establishing a sufficiently strong linkage between the binding partner and the support, without any upper time limit.
  • the blocking agent may be added at any time after that the binding partner has been firmly linked to the support, which may vary from 15 minutes to 24 hours or longer.
  • the milk protein blocking agent may be added after 1 to 4 hours.
  • the support with binding partners attached may be stored for any length of time prior to use, especially if no detachment of the binding partner is desired.
  • the support For systems where at some stage release of the binding partner from the support may be desired, as for instance in the selective capture of cells, it may in some cases be preferable to use the support as soon as possible because of possible structural changes in the binding partner which may lead to non-reversible binding.
  • the presence of milk protein at the surface of the support will diminish the hydrophobic binding of the binding partner to the support, so that a sufficient release of the binding partner, eg. antibody, from the support may be achieved even after several weeks of storage at 4°C.
  • the support is stored in the presence of the blocking agent, and washed immediately prior to use. Washing is desirable, to remove any binding partner which may have leaked from the support.
  • Temperatures and other conditions during the blocking step are not especially critical and conventional laboratory procedures and conditions may be used. A temperature of 4°C during the blocking step has been found to be convenient.
  • concentration of milk protein used may also be varied according to circumstance, and choice. Concentrations of 0.01 to 0.1 % w/v are for example suitable, more particularly 0.05 to 0.1 % w/v.
  • the method of the invention may be applied to the isolation of any prokaryotic or eukaryotic cells, or other viable entities from biological or artificial media including whole blood, buffy coat and cell suspensions obtained by density gradient centrifugation.
  • a further aspect of the invention thus provides a kit for use in the method of the invention, comprising:
  • the cell separation method of the invention may have many uses, for example in bone marrow purging, depletion of normal T-cells in allografts, isolation of stem cells e.g. for reconstitution, isolation of pure cell sub-populations for functional studies, tissue typing, and diagnosis, for example detection of bacterial pathogens.
  • Non-specific cell binding may be a problem in such cell separation procedures as described for example in the situations below:
  • a direct method which in a simple but consistent way may be used to demonstrate the effect of blocking agents on the non-specific attachment of cells to the magnetic particles is based on measurements of cells which are so strongly attached to the magnetic beads that they are transported along with a large given number of the beads when these under given conditions were moved by a magnet.
  • This procedure was carried out as follows: A suspension of mononuclear cells obtained by gradient centrifugation of a buffycoat and containing 20 x 10 6 cells per ml was incubated with magnetic particles in an amount corresponding to 3 particles per cell. After 30 minutes of incubation at 4°C with gentle rotation, the tubes containing the particles and the cells were diluted 10 times with a PBS buffer, pH 7.4, and the particles with any cells attached to them were separated from the suspension with a magnet. The number of cells attached to the beads were estimated in a fluorescence microscope using a Burker haemocytometer after staining with acridine orange and ethidium bromide. The number of cells attached to the particles were expressed as % of total cells in the suspension.
  • Casein was also applied to improve particles which had previously been covered with BSA.
  • the casein was used as an aftertreatment of particles which were previously treated with BSA. The efficiency of this treatment was estimated as described above.
  • These particles which have been described in several papers (e.g. J. Ugelstad et al. in Progress of Polymer Science, 87-161, 1992) are magnetic particles covered with an epoxy resin and have a moderately hydrophilic surface ' . These particles were used for direct measurements of physical adsorption of cells, both naked and after treatment with BSA and casein. In short time experiments where the measurements of non-specific binding of cells were obtained after one hour attachment of protein, the measurements of cell addition may be considered as taking place on particles having the protein physically adsorbed. In cases when the particles where treated for a day or more with the proteins before measurements of cell adhesion the results may be considered as a case of adsorption of cells to particles covered with covalently bound proteins .
  • Dynabeads M450, 4.5 ⁇ m magnetisable beads (Dynal AS, Oslo, Norway) (10 g) carrying free epoxy groups were reacted with bis- (3-aminopropyl) a ine (100 g) for 5 hours at 70°C to yield surface -NH 2 groups on the beads. The beads were then washed seven times with 500 ml diglyme (diethyleneglycol dimethyl ether) . The beads (10 g) were then reacted with glycidyl methacrylate (200 g) at 70°C for 20 hours to yield surface vinyl groups.
  • Non-specific cell adhesion to these particles were measured as described above.
  • the non specific adhesion of cells was measured with various concentrations of proteins and with various incubation times for the pretreatment of the beads with protein solution.
  • Attachment of BSA or casein to the surface of the magnetic beads by help of glutaraldehyde was carried out as follows: M450 beads (100 mg) were dispersed in 2 ml PBS buffer, pH 7.4, containing either 0.1% BSA or 0.1% casein, and incubated for 20 hours at 20°C. Then the particles were isolated by a magnet and washed whereafter 2 ml of a glutaraldehyde solution (0.5 M) in 0.1 M phosphate buffer, pH 7.7, was added. This reaction took place at 20°C for 20 hours. After isolation and washing, 2 ml PBS buffer, pH 7.4, with either 0.1% BSA or 0.1% casein was added.
  • the values given are % of total cells attached to the beads, and are average values based upon more than 5 experiments.
  • Table I reveals that the advantage of casein as compared to BSA in decreasing the non-specific binding of cells is most pronounced for the case of particles with boronic acid coupled to the surface.
  • the particles used for measurements of non-specific cell contamination in target cells released after positive cell separation were particles with boronic acid covalently bound to the surface.
  • the preparation of these boronic acid particles for use in positive cell separation was similar to that described in Example 1 for boronic acid particles used for direct measurement of the non-specific adhesion of cells. Positive separation of cells applying these particles has so far been carried out for T4 cells, T8 cells B cells and monocytes.
  • the effect of casein as compared with BSA in preventing non-specific binding of cells and thereby contamination of non-target cells in the cell fraction obtained after removal of the particles from the isolated cells is described in detail for the case of T4 cells. Similar results were obtained by positive isolation of the other cells given above.
  • Bin-particles prepared as described were incubated with an IgGl, anti-CD4 monoclonal antibody, ST4 (Biosys, Compiegne, France) .
  • the particles were then isolated with a magnet and subsequently washed once with a PBS buffer. Then the particles were incubated for 1 hour at 4°C in a PBS buffer, pH 7.4, containing either 0.1% casein or 0.1% BSA and subsequently washed twice with the same PBS buffer at 4°C, immediately before use
  • PBMC Peripheral blood mononuclear cells
  • the PBMC suspension was then incubated with Dynabeads M450, coated with monoclonal antibodies against CD14 (5- 10 particles/target cell) (Dynal AS, Oslo, Norway) .
  • the particle concentration was at least 20 x 10 6 beads/ml.
  • the incubation took place in a PBS buffer, pH 7.4, containing 2% foetal calf serum, at 4°C for 30 minutes.
  • the magnetic beads, and thereby the monocytes were removed with a magnet and the PBMC depleted for monocytes was collected.
  • the boronic acid particles coated with anti-CD4 antibodies were incubated with PBMC depleted for monocytes.
  • the number of particles was adjusted to 10 - 20 particles per target cell, and in addition the particle concentration was kept at 25 x 10 6 beads/ml.
  • the incubation took place in a PBS buffer, pH 7.4, containing either 0.1 % BSA or 0.1 % casein at 4°C for 30 minutes. After incubation the suspension was diluted 10 times with the incubation buffer. Then the target cells with attached particles and excess particles were isolated by magnetic aggregation by help of an external magne .
  • the isolated cells were resuspended in PBS buffer, pH 7.4, with either 0.1 % BSA or 0.1 % casein at 4°C and again the cells with attached magnetic particles isolated by help of a magnet. This process of isolation and resuspension was repeated four times. Detachment of the beads.
  • the isolated cells were resuspended in a PBS buffer, pH 7.4, containing 50% foetal calf serum, 0.3% sodium citrate and 0.2 M sorbitol at 20°C.
  • the tubes with the cells were then placed on a Rock and Roller (Labinco, Breda, The Netherlands) for 2 hours. Then the beads were removed with a magnet. This process results in detachment of 80 to 90% of the cells from the beads.
  • the beads were resuspended in the detachment buffer and the suspension was pipetted 10 times. Again, the beads were removed by help of a magnet and the cells were collected and added to the cell suspension from the first step.
  • the number of cells in the different cell fractions was determined by use of a Coulter Multisiser II (Coulter Electronics Ltd., Luton, England) .
  • Fluorochrome conjugated antibodies against CD3, CD4, CD8, CD19 and CD56 were incubated with cells from the following three cell fractions: PBMC depleted for monocytes, the remaining cell fraction after T4 cell isolation and from the isolated cells depleted for magnetic beads. The cells were analyzed using a FACScan flow cytometer (Becton Dickinson) .
  • the efficiency of the blocking agents were determined by measurements of the contamination of non-target cells among the cells set free after isolation of cells and removal of the magnetic particles. Results: The number of target cells isolated from the cell suspension was practically equal with BSA and casein as blocking agents. With BSA as blocking agent the cell fraction released from the beads contained 85% of the target cells (T4 cells) . The contamination was mainly B cells which amounted to 13% of the isolated cells.
  • both the beads covered with BSA and the beads covered with casein were used for removing of T4 cells from the same type of cell suspension as described in Example 2. Both beads with BSA and beads with casein were capable of removing more than 95% of the target cells from the cell suspension.
  • the non-specific binding of cells was estimated from determination of non-target cells in the cell suspension before removal of T4 cells and after removal of the T4 cells by use of flow cytometry as described above. In this case also there is a clear difference between the beads with BSA and casein in non ⁇ specific binding of cells with a clear preference for the latter in giving less non-specific binding of non- target cells.
  • the IgG monoclonal antibody was coupled to the beads by means of excess aldehyde groups after which the system was treated with sodium borohydride in order to reduce the Schiff bases and the remaining aldehyde groups.
  • Both the particles with BSA and casein were effective in selective removal of target cells. Again the particles with casein tended to give less removal of non target cells along with the target cells.

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Abstract

La présente invention porte sur l'emploi d'une protéine du lait pour bloquer la fixation aspécifique de cellules à une matrice de capture cellulaire sélective en phase solide. D'une manière générale, la matrice de capture sélective en phase solide sera un support solide se prêtant à la séparation sélective de cellules cibles par l'entremise d'un partenaire de fixation ciblée, un anticorps, par exemple.
EP95930638A 1994-09-06 1995-09-05 Separation cellulaire Ceased EP0782704A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9417864A GB9417864D0 (en) 1994-09-06 1994-09-06 Cell separation
GB9417864 1994-09-06
PCT/GB1995/002094 WO1996007909A1 (fr) 1994-09-06 1995-09-05 Separation cellulaire

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Publication Number Publication Date
EP0782704A1 true EP0782704A1 (fr) 1997-07-09

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EP95930638A Ceased EP0782704A1 (fr) 1994-09-06 1995-09-05 Separation cellulaire

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EP (1) EP0782704A1 (fr)
JP (1) JPH10505423A (fr)
AU (1) AU3395595A (fr)
CA (1) CA2199020A1 (fr)
GB (1) GB9417864D0 (fr)
WO (1) WO1996007909A1 (fr)

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US8592219B2 (en) 2005-01-17 2013-11-26 Gyros Patent Ab Protecting agent
WO2006075964A1 (fr) * 2005-01-17 2006-07-20 Gyros Patent Ab Procede permettant de co-transporter un reactif avec une substance macromoleculaire amphiphile dans un conduit de transport microfluidique
US9656271B2 (en) * 2007-12-04 2017-05-23 Headway Technologies, Inc. Guided transport of magnetically labeled biological molecules and cells

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GB2189317B (en) * 1986-04-16 1990-10-24 London Polytech Method of detecting and enumerating sulphate-reducing bacteria
US4818686A (en) * 1986-09-15 1989-04-04 Coulter Corporation Chemical blocking agent against non-specific binding or staining of an antibody specific for terminal deoxynucleotidyl transferase in biological specimens during immunoassay
DE3850650T2 (de) * 1987-08-12 1995-03-02 Teijin Ltd Immuntestverfahren und reagenzsatz dazu.
US5047326A (en) * 1988-10-07 1991-09-10 Eastman Kodak Company Immunmological reagent composition and its use in the determination of chlamydial or gonococcal antigens
TW205096B (fr) * 1991-05-29 1993-05-01 Shionogi & Co
US5399484A (en) * 1991-10-08 1995-03-21 Eastman Kodak Company Use of blocking protein with high pH extraction in method to determine a microorganism associated with periodontal disease and kit useful therefor
GB9304979D0 (en) * 1993-03-11 1993-04-28 Sinvent As Imobilisation and separation of cells and other particles

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Also Published As

Publication number Publication date
GB9417864D0 (en) 1994-10-26
CA2199020A1 (fr) 1996-03-14
WO1996007909A1 (fr) 1996-03-14
JPH10505423A (ja) 1998-05-26
AU3395595A (en) 1996-03-27

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