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WO2009095665A1 - Analyse de coloration cellulaire et kit - Google Patents

Analyse de coloration cellulaire et kit Download PDF

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Publication number
WO2009095665A1
WO2009095665A1 PCT/GB2009/000243 GB2009000243W WO2009095665A1 WO 2009095665 A1 WO2009095665 A1 WO 2009095665A1 GB 2009000243 W GB2009000243 W GB 2009000243W WO 2009095665 A1 WO2009095665 A1 WO 2009095665A1
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Prior art keywords
antibody
tissue
cells
antibodies
fragment
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PCT/GB2009/000243
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English (en)
Inventor
Arthur Randall Bradwell
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Binding Site Ltd
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Binding Site Ltd
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Priority to EP09706451A priority Critical patent/EP2238456A1/fr
Publication of WO2009095665A1 publication Critical patent/WO2009095665A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • the invention relates to methods of staining cells in tissue or cell samples comprising antibodies which are specific for a single immunoglobulin heavy-chain/light chain combination, and to kits for such methods.
  • the reagents are especially useful for immunohistological, immunocytochemical and flow cytometry uses.
  • Immunohistology is the use of antibodies to directly or indirectly stain tissues so that they can be identified.
  • Immunocytochemistry is a technique for identifying cellular constituents (antigens) by means of antigen-antibody interactions, the site of antibody binding being identified either by direct labelling of the antibody or by use of a secondary labelling method.
  • Immunohistology and immunocytochemical techniques including methods of fixing tissue and visualising antigens in tissues using antigen-specific antibodies, or fragments of such antibodies, are generally known in the art. Reviews of the techniques and components used include:
  • Antibody molecules also known as immunoglobulins
  • the variable domains of the heavy and light chains combine to form an antigen-binding site, so that both chains contribute to the antigen-binding specificity of the antibody molecule.
  • the basic tetrameric structure of antibodies comprises two heavy chains covalently linked by a disulphide bond. Each heavy chain is in turn attached to a light chain, again via a disulphide bond. This produces a substantially "Y"- shaped molecule.
  • Heavy chains are the larger of the two types of chain found in antibodies, with typical molecular mass of 50,000-77,000 Da, compared with the smaller light chain (25,000 Da).
  • IgG is the major immunoglobulin of normal human serum, accounting for 70-75% of the total immunoglobulin pool. This is the major antibody of secondary immune responses. It forms a single tetramer of two heavy chains plus two light chains.
  • IgM accounts for approximately 10% of the immunoglobulin pool.
  • the individual heavy chains have a molecular weight of approximately 65,000Da and the whole molecule has a molecular weight of about 970,000Da.
  • IgM is largely confined to the intravascular pool and is the predominant early antibody.
  • IgA represents 15-20% of the human serum immunoglobulin pool. More than 80% of IgA occurs as a monomer. However, some of the IgA (secretory IgA) exists as a dimeric form.
  • IgD accounts for less than 1% of the total serum immunoglobulin.
  • IgE although scarce in normal serum, is found on the surface membrane of basophils and mast-cells. It is associated with allergic diseases such as asthma and hay-fever.
  • IgG In addition to the five main classes, there are four subclasses for IgG (IgGl, IgG2, IgG3 and IgG4). Additionally there are two subclasses for IgA (IgAl and Ig A2).
  • a plasma cell proliferates to form a monoclonal tumour of identical plasma cells. This results in production of large amounts of identical immunoglobulin molecules and is known as a monoclonal gammopathy.
  • Haematopathology has a long history of specialist practice in, for example, the United Kingdom, and other countries.
  • Haematopathology is a division of pathology which is concerned with diseases of the blood and of haemopoietic and lymphoid tissues.
  • the specialist nature of haematopathology has been enhanced by the advent of immunohistochemistry and molecular genetics, especially in the diagnosis of haematopoietic and lymphoid neoplasms.
  • Biopsies may be taken from, for example, lymph nodes.
  • Table 1 shows the immunohistochemistry of follicular lymphoma (based on Isaacson P.G. (2007) Supra.).
  • IgM and IgD play an important role in identifying the lymphoma.
  • the immunoglobulin light chains are monoclonal in nature.
  • the ability to identify whether a cell is producing IgM ⁇ or IgM ⁇ antibodies would assist in identifying the lymphoma.
  • Bone marrow may be biopsied. Different B cell lymphomas may be differentiated by expression of, for example, CD20, CD5, CDlO, CD21, CD23 and CyDl. Follicular lymphoma (FL) and lymphoplasmacytic lymphomas (LPC) tend to be IgM but not IgD positive. Chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL), mantle cells and splenic marginal zone lymphoma (SMZL) also express monoclonal light chains.
  • FL Follicular lymphoma
  • LPC lymphoplasmacytic lymphomas
  • CLL Chronic lymphocytic leukaemia/small lymphocytic lymphoma
  • SZL splenic marginal zone lymphoma
  • IgM, IgD and light chain monotype include classic Hodgkin's lymphoma, lymphocyte-rich nodular Hodgkin's lymphoma; nodular lymphocyte predominant Hodgkin's lymphoma; T-cell/histiocyte-rich diffuse large B cell lymphoma and angioimrnunoblastic T-cell lymphoma. This may be tested in conjunction with CD30, CDl 5, LMP-I, CD20, CD79a, Bcl-6 or CD45. A problem with such assays has been the reliable identification of Ig heavy chain class or Ig light chain.
  • the ability to identify the monoclonal expansion of, for example, ⁇ -lymphocytes in lymph nodes tissue by staining fixed or fresh tissue or cells on slides or in suspension (e.g. by using flow cytometry) has particular use.
  • a problem is distinguishing polyclonal expansion (such as due to infection or hyperplasia) from monoclonal (tumour) expansion.
  • the inventors have identified a particular type of antibody that is more specific than either total immunoglobulin or total light chain staining, which can both give high levels of background staining.
  • the Applicant has previously developed an assay for monitoring secreted immunoglobulins in plasma or serum by the heavy chain class and light chain type ratios.
  • WO 2006/079816 discloses a method monitoring a malignant plasma cell disease by monitoring the relative amounts of secreted immunoglobulins having a heavy chain class bound to lambda light chains, and immunoglobulins having the same heavy chain class but bound to kappa light chains. This exemplifies the detection of soluble immunoglobulins in plasma and serum samples. This is typically carried out using immunosorbent assays (such as ELISA), nephelometry, turbidimetry, or flow cytometry - type techniques determine the ratio in, for example, serum or plasma.
  • immunosorbent assays such as ELISA
  • nephelometry nephelometry
  • turbidimetry or flow cytometry - type techniques determine the ratio in, for example, serum or plasma.
  • the document indicates that the antibodies used may be separate antibodies specific for a particular heavy chain class or free light chain type.
  • the antibody may be specific for a specific heavy chain class, at the same time as having specificity for a light chain type.
  • the Applicant has since tried staining tissue samples with separate anti-Ig heavy chain specific antibodies and anti-light chain type antibodies. However, this was observed to produce problems with sensitivity. The Applicant tried to stain with anti-free light chain antibody. This failed to stain with high sensitivity and was observed to produce high levels of background staining. Monoclonal IgA ⁇ staining, for example, appeared to be hidden by background staining of IgG ⁇ .
  • tissue samples containing monoclonal proliferations of cells producing immunoglobulins can be successfully resolved using an adapted form of the types of antibodies shown in PCT/GB2006/078816. They have now used antibodies having specificity for a heavy chain class at the same time as having specificity for a light chain type to stain cells. Such antibodies produced good staining results.
  • the improved results may be due to the antibodies being specific for conformational epitopes spanning the heavy chain and light chains in the immunoglobulins. It is thought that the fixing or paraffin immobilisation methods used to prepare tissue samples may damage or denature some immunoglobulin molecules, but not these spanning regions to a significant extent. Free light chain specific antibodies, for example, appear to bind to previously hidden epitopes on the immunoglobulins leading to high background results.
  • the first aspect of the invention provides a method of staining one or more cells in a sample of tissue or cells, comprising contacting a sample of tissue of cells with an antibody, or a fragment thereof, the antibody having specificity for an immunoglobulin heavy chain class, at the same time as having specificity for a light chain type and detecting the binding of the antibody to the tissue or cells.
  • Having specificity for a heavy chain class at the same time as having specificity for a light chain type is intended to mean that the antibody, or a fragment thereof, is able to distinguish between different heavy chain classes and also able to distinguish between heavy chains of the same class, but which are bound to kappa or lambda light chains.
  • the antibody is capable of distinguishing between IgM ⁇ and IgM ⁇ , and is capable of distinguishing between IgM ⁇ and IgD ⁇ .
  • the method is particularly useful in immunohistological, immunocytochemical and flow cytometry uses.
  • the tissue or cells may be a fresh or fixed sample of tissue or cells.
  • the fixing of tissue and cells is discussed, for example, in the books by Polak and Van Noorden (1990, Supra.) and Bancroft and Stevens (1990, Supra), incorporated by reference herein in their entirety.
  • the fixing of cells and tissue sample is usually carried out to preserve the tissue or sample.
  • typical fixatives include formaldehyde, glutaraldehyde, parabenzoquinone, diethylpyrocarbonate, alcohols (such as methanol), acetone, formalin and mixtures of fixatives such as Carnoy's fluid, Bovin's and Zamboni's fixatives.
  • Tissue may also be freeze dried, frozen and/or embedded within paraffin or another resin, such as glycol methacrylate.
  • a resin such as glycol methacrylate.
  • the tissue or cell sample may be provided on, for example, a support such as a microscope slide.
  • a support such as a microscope slide.
  • Flow cytometry is known in the art for cell sorting. There are numerous text books describing flow cytometry, such as Practical Flow Cytometry, 3 rd Edit. (1994), H. Shapiro, Alan R. Liss, New York, and Flow Cytometry, First Principles, 2 nd Edit. (2001), A.L. Given, Wiley Liss.
  • the tissue samples or cells are fixed or freeze dried.
  • the tissue or cell sample is obtained from lymph node tissue or bone marrow tissue or lymphatic infiltrates in other tissues/organs.
  • Lymph node tissues are particularly useful for the characterisation and detection of lymphomas.
  • Antibodies having specificity for the following heavy chain and light chain combinations are particularly useful: IgD ⁇ , IgD ⁇ , IgM ⁇ and IgM ⁇ .
  • Tissue sample or cells may also be obtained from bone marrow. Bone marrow is of particular interest for the clearer identification or characterisation of monoclonal plasma cells in myeloma, plasmacytoma and AL amyloidosis.
  • Antibodies having specificity for IgG ⁇ , IgG ⁇ , IgA ⁇ , IgA ⁇ , IgD ⁇ and IgD ⁇ are especially preferred antibodies for such tissues.
  • Flow cytometry methods used in the current invention detect the number of positive cells to be counted, rather than the soluble concentration of soluble proteins exemplified in WO2006/079816. Counting the tumour cells is a direct measure of the tumour, whereas the concentration of soluble protein has no direct relationship to the tumour cell numbers. Large tumours may produce little or no soluble protein.
  • the cells are fixed to stabilize membrane bound proteins.
  • the method of staining the cells is carried out on a sample of tissue or cells on a support, such as a microscope slide.
  • a sample of tissue or cells on a support such as a microscope slide.
  • the cells are preferably in a cross-section of tissue. That is, stained cells are present, substantially attached to other cells from a cross-section of tissue obtained, for example, from a biopsy sample.
  • Aspirates of bone marrow or lymphoid tissue may also be used.
  • the cells from such aspirates are usually individual cells. These may be smeared across microscope slides before staining and viewing.
  • the antibody or fragment thereof is a polyclonal antibody.
  • Polyclonal antibodies allow some variability between different heavy chains of the same class to be detected because they are raised against a number of parts of the heavy chain. Methods of producing polyclonal antibodies having the desired specificity are disclosed in, for example, WO2006/07816. Using polyclonal antibodies having the desired specificity improves the labelling of the cells and tissue.
  • the antibody, or fragment thereof is specific for IgG ⁇ , IgG ⁇ , IgA ⁇ , IgA ⁇ , IgM ⁇ , IgM ⁇ , IgD ⁇ , IgD ⁇ , IgE ⁇ or IgE ⁇ .
  • the antibody may, in addition, be heavy chain subclass specific.
  • anti-IgA IgAl and Ig A2
  • anti-IgG such as IgGl, IgG2, IgG3 or IgG4 antibodies may be produced. This gives more detailed knowledge of the disease to be detected.
  • the antibody used may be a Fab or a F(ab') 2 fragment, whilst still retaining the ability to have specificity for an immunoglobulin heavy chain class, but at the same time having specificity for a light chain type.
  • the method of the invention may comprise contacting the tissue sample with two or more antibodies specific for a different heavy chain class but the same light chain type, or specific for the same heavy chain class but a different light chain type. This allows, for example, the ratio of cells having the same heavy chain class, but different light chain type, to be determined. Such a ratio might allow the detection of monoclonal diseases and the monitoring of the treatment of diseases to be carried out.
  • tissue or cell sample is contacted with antibodies or fragments specific for: IgG ⁇ and IgG ⁇ ; IgA ⁇ and IgA ⁇ ; IgM ⁇ and IgM ⁇ ; IgD ⁇ and IgD ⁇ ; and/or IgE ⁇ and IgE ⁇ .
  • Different labels may be used for different antibodies to allow the binding of different antibodies to be detected.
  • different coloured labels may be used to allow more than one antibody pair to be studied at the same time with, for example, flow cytometry.
  • the binding of the antibody to the cell may be determined because the antibody is directly labelled with a suitable label.
  • Methods of labelling antibodies are discussed in the books by Bancroft and Stevens, and Polak and Van Noorden, Supra.
  • the label attached to the antibody may be a visual label.
  • FITC fluorescein isothiocyanate
  • This may optionally be used with a fluorescent tissue counterstain such as Pontamine Sky Blue.
  • Other visual dyes include fluorescein, rhodamine, phycoerythrin, AMCA, Oregon GreenTM (Molecular Probes, Inc.) and CyDyesTM (Amersham Biosciences).
  • alkaline phosphatase may be used with a substrate, such as naphthol phosphate, to produce a coloured chromogen.
  • Horseradish peroxidase is generally know, in combination with a substrate such as 3,3'-diaminobenzidine (DAB) or other substrates generally known in the art, to produce a coloured precipitate.
  • DAB 3,3'-diaminobenzidine
  • Other enzymes that have been coupled to antibodies include ⁇ -D- galactosidase and glucose oxidase.
  • the antibodies may alternatively be labelled with particulate labels (such as colloidal gold) or indeed radioisotopes.
  • the antibody may be detected indirectly.
  • a second antibody raised against the immunoglobulin from the host species of the first antibody In indirect labelling the primary unconjugated antibody from a first animal type, or fragment thereof, is allowed to bind to the antigen on the cell.
  • Labels for the second tracer-conjugated antibody may be as defined above for direct visualisation techniques. That is, the second tracer-conjugated antibody may be attached to one or more of the labels described above.
  • the advantage of the indirect technique is that it is more sensitive than the direct technique and is still relatively rapid and inexpensive.
  • a further advantage of this method is that provided that the primary antibody host species remains the same, any number of tests can be performed using the common conjugated second antibody.
  • Antibodies may also be attached to, for example, avidin which is then used to attach labelled biotin to avidin to form an avidin/biotin complex.
  • the methods of the invention may be used in conjunction with one or more further antibodies, or fragments thereof, specific for antigens selected from: CD30, CD20, Bcl-2, Bcl-6, CDlO, CD5, CD21, CD23 and CyDl.
  • the method may additionally comprise the step of testing the sample with anti-lambda free light chain antibody or anti-kappa free light chain antibody to detect soluble free light chains in the sample.
  • anti-lambda free light chain antibody or anti-kappa free light chain antibody are sold by The Binding Site Limited, Birmingham, United Kingdom, under the trade mark "Freelite”.
  • the method includes the additional step of staining for free light chains to give an indication of the total amount of free light chains in the sample.
  • the invention also provides a tissue or cell staining kit for use in a method according to the invention, comprising at least one antibody or a fragment thereof, having specificity for an immunoglobulin heavy chain class, at the same time having specificity for a light chain type.
  • the kit may additionally comprise instructions for using the antibody in a method of the invention.
  • the antibody, or fragment thereof is a polyclonal antibody.
  • the antibody is preferably specific for IgG ⁇ , IgG ⁇ , IgA ⁇ , IgA ⁇ , IgM ⁇ , IgM ⁇ , IgD ⁇ , IgD ⁇ , IgE ⁇ or IgE ⁇ .
  • the antibody, or fragment thereof is heavy chain subclass specific.
  • the antibody may be an Fab or F(ab') 2 fragment.
  • the kit comprises two or more antibodies specific for different heavy chain classes, but the same light chain type, or specific for the same heavy chain class, but a different light chain type.
  • the antibodies may be especially specific for the same heavy chain class, but for different light chain types.
  • the kit comprises antibodies or fragments specific for: IgG ⁇ and IgG ⁇ ; IgA ⁇ and IgA ⁇ ; IgM ⁇ and IgM ⁇ ; IgD ⁇ and IgD ⁇ ; and/or IgE ⁇ and IgE ⁇ .
  • the antibody is preferably directly labelled, for example in the manner as defined above for a preceding aspect of the invention.
  • the kit comprises a second antibody for indirectly labelling the primary antibody used to bind the immunoglobulin in the cell.
  • a second antibody may be as defined above and labelled, for example, in the manner described above.
  • the indirect labelling may utilise an avidin biotin binding complex, as described above.
  • the kit comprises one or more antibodies, or fragments thereof, specific for an antigen selected from: CD30, CD20, Bcl-2, Bcl-6, CDlO, CD5, CD21, CD23 and CyDl.
  • the kit may additionally comprise one or more antibodies, or fragments thereof, specific for free lambda light chains or free kappa light chains, and/or total free light chains. Such antibodies may also be labelled directly or indirectly in the manner described above.
  • a further aspect of the invention provides a method of diagnosing or tracing the development or treatment of a haematopoietic or lymphoid disorder comprising the use of a method or a kit according to the invention.
  • the disorder is a lymphoma, leukaemia, myeloma, plasmacytoma or AL amyloidosis.
  • TSP Total Serum Protein
  • FLCK Free Light Chain kappa.
  • TSP Total Serum Protein.
  • IgG kappa multiple myeloma sample analysis using immunofixation probed with anti-IgG (IgG), anti-kappa (total) (K), anti lambda (total) ( ⁇ ), anti-IgG kappa (IgG) and anti-IgG lambda (IgG ⁇ ) antibodies.
  • IgG lambda multiple myeloma sample analysed using immunofixation probed with anti-IgG (IgG), anti-kappa (total) (K), anti lambda (total) ( ⁇ ), anti-IgG kappa (IgG) and anti-IgG lambda (IgG ⁇ ) antibodies.
  • IgM kappa multiple myeloma sample analysis using immunofixation probed with anti-IgM (IgM), anti-kappa (total) (K), anti-lambda (total) ( ⁇ ), anti-IgM kappa (IgM ⁇ ) and anti IgM lambda (IgM ⁇ ) antibodies.
  • IgM lambda multiple myeloma analysis using immunofixation probed with anti-IgM (IgM), anti-kappa (total) (K), anti-lambda (total) ( ⁇ ), anti-IgM kappa (IgM ⁇ ) and anti IgM lambda (IgM ⁇ ) antibodies.
  • FIGS 7 to 11 show lymphoid mode tissue stained with specific antiimmunoglobulin antibodies of the invention. Immunoglobulin producing cells are stained more darkly than the surrounding cells (4Ox magnification).
  • Figure 12 shows anti-IgM lambda stained lymphoid tissue at 1Ox magnification.
  • Figure 13 shows a schematic representation of an antibody (a) and a cross section through part of antibody (b). Heavy chains are shown as 10, light chains as 12 and the region that the antibodies used in the invention are believed to bind (heavy-light chain epitopes) as 14.
  • FCA Freund's Complete Adjuvant
  • HevyLiteTM antibodies (antibodies specific for a single Ig heavy-chain/light chain combination) are produced using a tolerisation, prime and boost regime. Simply, on day 0 tolerisation antigens are administered intravenously. After 4 days tolerisation antibody is injected intravenously, this is typically a sheep anti-toleragen antibody.
  • the sheep is primed with target antigen injected in Freund's Complete Adjuvant intradermally.
  • the sheep is then released, after 45 days the sheep is boosted with target antigen intradermally with Freund's Incomplete Adjuvant.
  • After 1 week the sheep is bled, taking a maximum of 40ml for analysis. Within 3 weeks of this time period the sheep is bled for production purpose (5x0.5L sheep / day). A minimum of 6 weeks after the production bleed further boosts may be administered using Freund's Incomplete Adjuvant.
  • Bleeds are taken in CPDA-I (Citrate Phosphate Dextrose Adenine) 500ml / bag, blood is then centrifuged at 3800rpm / lOmins. Plasma is decanted and respun at 3800 rpm / 20mins. The resultant supernatant is decanted and has 0.09% sodium azide, 2% sodium citrate added prior to shipping. Antibodies from the bleed are then purified using sequential antigen specific columns. Conjugation to Horseradish Peroxidase
  • Purified antibody is dialysed against 0.02M sodium bicarbonate buffer pH9.5 overnight. The antibody concentration is calculated using absorbance at 28OnM (antibody absorbance at 28OnM / 1.4).
  • Enzyme (horseradish peroxidase) is prepared at a ratio of 2:1 antibody: enzyme (w:w) and dissolved in distilled water to give a final concentration of 5mg/mL.
  • the peroxidase is activated using 20 ⁇ L 0.1 M sodium periodate solution / mg of peroxidase over a 20min time course.
  • the activated peroxidase enzyme solution is then dialysed against 0.001M sodium acetate buffer pH4.5 at 2-8C
  • Lymph node tissue was stained with antibodies specific for heavy chain/light chain pairs labelled with horseradish peroxidase.
  • B cells/plasma cells producing the relevant immunoglobulins were stained by the antibodies.
  • the staining with anti-IgA kappa, IgA lambda, IgG kappa, IgG lambda and IgM kappa is shown in Figures 7 to 11. Cells producing the relevant immunoglobulins are stained more darkly than the background cells.
  • tissue stained with the antibodies of the invention was compared with tissue stained using anti-lambda or anti-kappa antibodies, lower background staining was observed with the antibodies of the invention.

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Abstract

La présente invention concerne un procédé de coloration d'une ou plusieurs cellules au moyen d'un anticorps, ou d'un fragment de celui-ci, ayant une spécificité pour une classe de chaîne lourde d'immunoglobuline, tout en ayant une spécificité pour un type de chaîne légère, et de détection de la liaison de l'anticorps au tissu ou aux cellules. L'invention concerne en outre des kits de coloration de tissu ou de cellules comprenant de tels anticorps.
PCT/GB2009/000243 2008-01-29 2009-01-29 Analyse de coloration cellulaire et kit Ceased WO2009095665A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP09706451A EP2238456A1 (fr) 2008-01-29 2009-01-29 Analyse de coloration cellulaire et kit

Applications Claiming Priority (2)

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GB0801609A GB0801609D0 (en) 2008-01-29 2008-01-29 Hevylite diagnostic stain
GB0801609.9 2008-01-29

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US20130149792A1 (en) * 2010-02-05 2013-06-13 The Binding Site Group Limited Cancer prognosis assay
US10845362B2 (en) 2010-07-19 2020-11-24 The Binding Site Group Limited Competition assay

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130149792A1 (en) * 2010-02-05 2013-06-13 The Binding Site Group Limited Cancer prognosis assay
US10845362B2 (en) 2010-07-19 2020-11-24 The Binding Site Group Limited Competition assay

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