WO2009095665A1

WO2009095665A1 - Cell staining assay and kit

Info

Publication number: WO2009095665A1
Application number: PCT/GB2009/000243
Authority: WO
Inventors: Arthur Randall Bradwell
Original assignee: Binding Site Ltd
Current assignee: Binding Site Ltd
Priority date: 2008-01-29
Filing date: 2009-01-29
Publication date: 2009-08-06
Anticipated expiration: 2010-07-29
Also published as: EP2238456A1; GB0801609D0

Abstract

The application provides a method of staining one or more cells using an antibody, or a fragment thereof, 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. Tissue or cells staining kits comprising such antibodies are also provided.

Description

Cell Staining Assay and Kit

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:

(i) Introduction to Immunocytochemistry, 2^nd Edit. Polak J.M. and Van Noorden S., Bios.

Scientific Publishers (1990), see especially Chapters 3, 5, 6 and 7; and

(ii) Theory and Practice of Histological Techniques, 3^rd Edit. Bancroft J.D. and Stevens

A. (1990), pages 413-436 (authored by Robinson G., et al).

Antibody molecules (also known as immunoglobulins) have a twofold symmetry and are composed of two identical heavy chains and two identical light chains, each containing variable and constant domains. 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). There are five main classes of heavy chain which are γ, α, μ, δ and ε which are the constituents heavy chains for: IgG, IgA, IgM, IgD and IgE respectively. 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 molecules, together with J-chains, form a pentamer of five of the basic 4-chain structures. 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.

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).

There are two types of light chain: Lambda (λ) and Kappa (K). There are approximately twice as many K as λ molecules produced in humans, but this is quite different in some mammals. Each chain contains approximately 220 amino acids in a single polypeptide chain that is folded into one constant and one variable domain. Plasma cells produce one of the five heavy chain types together with either K or λ molecules. There is normally approximately 40% excess free light chain production over heavy chain synthesis. Where the light chain molecules are not bound to heavy chain molecules, they are known as "free light chain molecules". The free K light chains are usually found as monomers. The free λ light chains tend to form dimers. There are a number of proliferative diseases associated with antibody producing cells. These diseases are known as malignant plasma cell diseases. They are summarised in detail in the book "Serum-free Light Chain Analysis" A.R. Bradwell, available from The Binding Site Limited, Birmingham, UK (ISBN: 07044 24541).

In many such proliferative diseases 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.

However, despite improvements in the techniques available, a number of problems associated with immunohistochemical diagnosis of lymphoma have been highlighted in the review article by Isaacson P.G. (Histopathology (2007), 50, 821-834).

The production of specific immunoglobulins, or fragments of them, is often a very important marker in the identification of the specific type of cancer in a patient and, for example, to identify the primary source of secondary tumours found in a patient. This assists in identifying a course of treatment for the disease. Biopsies may be taken from, for example, lymph nodes.

The presence or absence of specific immunoglobulin heavy chain classes and immunoglobulin light chains in cells can also assist in the identification of other types of cancers. For example, Table 1 shows the immunohistochemistry of follicular lymphoma (based on Isaacson P.G. (2007) Supra.).

Table 1

* Follicular Dendritic Cell (FDC) framework not highlighted

This shows that IgM and IgD play an important role in identifying the lymphoma. Moreover, in the germinal centre and interfollicular cells the immunoglobulin light chains are monoclonal in nature. Hence, the ability to identify whether a cell is producing IgMλ or IgMκ 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.

Other diseases which may be differentiated with the assistance of 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. Indeed, Isaacson (2007 Supra.) states that "demonstration of Ig light chain restriction is beyond the capability of many immunohistochemistry laboratories". Clinical Immunology: A Practical Approach, Eds. Gooi & Chapel, IRL Press (1990), Chapt. 6, by A.C. Campbell, "Cellular investigations in the diagnosis of lymphoid malignancy", page 166 also states "Restriction of the light chain to a single type (Kappa or Lambda) provides formal evidence of the monoclonal origin and neoplastic nature of the tissue. Interpretation can be difficult, particularly with some high grade tumours, when there is high and unavoidable background staining with polyclonal IgG". There is therefore a need for improved techniques.

The ability to identify the monoclonal expansion of, for example, β-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. The document indicates that the antibodies used may be separate antibodies specific for a particular heavy chain class or free light chain type. Alternatively, the antibody may be specific for a specific heavy chain class, at the same time as having specificity for a light chain type. A schematic representation of the antibody binding site where such antibodies are believed to bind, is shown in Figure 12. The latter antibodies were produced by the tolerisation method generally disclosed in WO 97/17372, to produce antibodies having the required heavy chain class, light chain type specificity.

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 IgAκ staining, for example, appeared to be hidden by background staining of IgGκ.

However, they found that 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. For example, the antibody is capable of distinguishing between IgMλ and IgMκ, and is capable of distinguishing between IgMλ and IgDλ.

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. For example, 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. The selection of appropriate fixative methods and other preservation methods will vary depending on the tissue and whether circumstances allow the staining of fresh tissue. Such techniques are generally know in the art.

The tissue or cell sample may be provided on, for example, a support such as a microscope slide. However, it is also possible to suspend cells so that they can be counted, for example via flow cytometry. 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.

Preferably, the tissue samples or cells are fixed or freeze dried.

Preferably, 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: IgDκ, IgDλ, IgMκ and IgMλ. 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 IgGκ, IgGλ, IgAκ, IgAλ, IgDκ and IgDλ 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.

Preferably, where flow cytometry is used, the cells are fixed to stabilize membrane bound proteins.

Preferably, the method of staining the cells is carried out on a sample of tissue or cells on a support, such as a microscope slide. This is of interest where, for example, histopathology is used. 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.

Preferably, 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.

Preferably, the antibody, or fragment thereof, is specific for IgGλ, IgGκ, IgAλ, IgAκ, IgMκ, IgMλ, IgDλ, IgDκ, IgEλ or IgEκ. The antibody may, in addition, be heavy chain subclass specific. For example, anti-IgA (IgAl and Ig A2) and 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.

Preferably the tissue or cell sample is contacted with antibodies or fragments specific for: IgGλ and IgGκ; IgAλ and IgAκ; IgMλ and IgMκ; IgDλ and IgDκ; and/or IgEλ and IgEκ.

Different labels may be used for different antibodies to allow the binding of different antibodies to be detected. For example 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.

There are a number of different labels which are known in the art. The label attached to the antibody may be a visual label. For example, FITC (fluorescein isothiocyanate) is a well- known label. This may optionally be used with a fluorescent tissue counterstain such as Pontamine Sky Blue. Other visual dyes include fluorescein, rhodamine, phycoerythrin, AMCA, Oregon Green™ (Molecular Probes, Inc.) and CyDyes™ (Amersham Biosciences).

There are a number of enzyme labels generally known in the art. For example, 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. Other enzymes that have been coupled to antibodies include β-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. For example, 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. A second tracer-conjugated antibody, raised in another animal type and specific for the animal and immunoglobulin class of the primary antibody, is applied to the cell and allowed to bind to the primary antibody. The complex which forms can then be visualised. 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. Such antibodies are sold by The Binding Site Limited, Birmingham, United Kingdom, under the trade mark "Freelite".

Preferably 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.

Preferably, the antibody, or fragment thereof, is a polyclonal antibody. The antibody is preferably specific for IgGλ, IgGκ, IgAλ, IgAκ, IgMκ, IgMλ, IgDλ, IgDκ, IgEλ or IgEκ.

Preferably, the antibody, or fragment thereof, is heavy chain subclass specific. The antibody may be an Fab or F(ab')₂ fragment.

Preferably, 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.

Preferably, the kit comprises antibodies or fragments specific for: IgGλ and IgGκ; IgAλ and IgAκ; IgMλ and IgMκ; IgDλ and IgDκ; and/or IgEλ and IgEκ.

The antibody is preferably directly labelled, for example in the manner as defined above for a preceding aspect of the invention.

Preferably, the kit comprises a second antibody for indirectly labelling the primary antibody used to bind the immunoglobulin in the cell. Such a second antibody may be as defined above and labelled, for example, in the manner described above. Alternatively, the indirect labelling may utilise an avidin biotin binding complex, as described above.

Preferably, 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.

Preferably, the disorder is a lymphoma, leukaemia, myeloma, plasmacytoma or AL amyloidosis.

The invention will now be described by way of example only, with reference to the following figures:

Figure 1

IgA kappa multiple myeloma immunofixation analysis probed with anti-IgA (IgA), anti- kappa (total) (K), anti-lambda (total) (λ), anti IgA kappa (IgAκ) and anti-IgA lambda (IgAλ) antibodies. "TSP" is Total Serum Protein, FLCK is Free Light Chain kappa.

Figure 2

IgA lambda multiple myeloma sample analysis using immunofixation analysis probed with anti-IgA (IgA) , anti-kappa (total) (K), anti-lambda (total) (λ), anti IgA kappa (IgAκ) and anti- IgA lambda (IgAλ) antibodies. "TSP" is Total Serum Protein.

Figure 3

IgG kappa multiple myeloma sample analysis using immunofixation, probed with anti-IgG (IgG), anti-kappa (total) (K), anti lambda (total) (λ), anti-IgG kappa (IgG) and anti-IgG lambda (IgGλ) antibodies.

Figure 4

IgG lambda multiple myeloma sample analysed using immunofixation, probed with anti-IgG (IgG), anti-kappa (total) (K), anti lambda (total) (λ), anti-IgG kappa (IgG) and anti-IgG lambda (IgGλ) antibodies.

Figure 5

IgM kappa multiple myeloma sample analysis using immunofixation, probed with anti-IgM (IgM), anti-kappa (total) (K), anti-lambda (total) (λ), anti-IgM kappa (IgMκ) and anti IgM lambda (IgM λ) antibodies.

Figure 6

IgM lambda multiple myeloma analysis using immunofixation, probed with anti-IgM (IgM), anti-kappa (total) (K), anti-lambda (total) (λ), anti-IgM kappa (IgMκ) and anti IgM lambda (IgM λ) antibodies.

Figures 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 7 Anti IgA kappa

Figure 8 Anti IgA lambda

Figure 9 Anti IgG kappa

Figure 10 Anti IgG lambda

Figure 11 Anti IgM kappa

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.

Immunisation Protocol and Antibody Use

Buffers for HevyLite

Freund's Complete Adjuvant (FCA)

85% Marcol 52 Oil (Mineral Oil)

CAS Number: 8042-47-5

Supplier: ExxonMobil Lubricants & Specialities

Product Code: 2010B0201065, 407598, 750612-6

15% Mannide Monooleate CAS Number: 9049-98-3 Supplier: Sigma-Aldrich Product Code: M-8546

0.5mg/ml Mycobacterium butyricum (killed and dried)

CAS Number: N/A

Supplier Viogt Global (Becton Dickinson)

Freund's Incomplete Adjuvant (FIA)

85% Marcol 52 Oil (Mineral Oil)

CAS Number: 8042-47-5

Supplier: ExxonMobil Lubricants & Specialities

Product Code: 2010B0201065, 407598, 750612-6

1.0M Sodium Acetate

136.08g/L Sodium Acetate Supplier Fisher Scientific

IM Carbonate Buffer pH9.5

16.8g/L Sodium Hydrogen Carbonate 22.8g/L Disodium Carbonate Decahydrate Supplier Fisher Scientific Phosphate Buffered Saline (PBS)

85g/L Sodium Chloride

32g/L Disodium Hydrogen Phosphate Dihydrate 10.92g/L Sodium Dihydrogen Phosphate Dihydrate Supplier Fisher Scientific

CPDA (Blood collection bags)

Contains 63ml CPDA-I Anticoagulant Solution. Each 100ml solution contains:

327mg Acid. Citric. Monohydrate

2.63g Citrate

251mg Dihydrogenphosphate Dihydrate

3.19g Dextrose

27.5mg Adenine

Supplier Baxter

Tolerogen

HevyLite™ 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.

Immunisation

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

Antibody Preparation

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).

Horseradish Peroxidase Activation

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 μL 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

Antibody Labelling

The required amount of horseradish solution to antibody solution (2:1) is added and rotated at room temperature for 2h. The reaction is stopped by inactivating the sodium periodate coupling reagent using a solution of sodium borohydride (4mg/ml) using 200μL/10mg peroxidase, incubate for 2h at 2-8C in the dark. The labelled antibody solution is dialysed against 1 x PBS + 0.05% Proclin 300 (biocide). Finally antibody concentration is measured and Stabilzyme™ enzyme stabiliser, available from a number of companies, is added to give a final 10% concentration, BSA is added as a non-reactive protein buffer to lOmg/mL and the solution filter sterilised through 0.2μM filter.

Tissue Staining

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.

When 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.

Claims

1. A method of staining one or more cells in a sample of tissue or cells comprising contacting the sample of tissue or 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.

2. A method according to claim 1 wherein the tissue or cells are fixed tissue or cells, or fresh tissue or cells.

3. A method according to any preceding claim wherein the tissue or cells are mounted on a slide or are in suspension.

4. A method according to claim 3, wherein the cells are in suspension and detection of binding of the antibody or fragment is carried out via the use of flow cytometry.

5. Method according to claim 1, wherein the tissue or cell sample is from lymphoid tissue or bone marrow tissue.

6. A method according to any preceding claim wherein the antibody, or fragment thereof, is a polyclonal antibody.

7. A method according to claim 1 or claim 2, wherein the antibody, or fragment thereof, is specific for IgGλ, IgGκ, IgAλ, IgAκ, IgMx, IgMλ, IgDλ, IgDκ, IgEλ or IgEκ.

8. A method according to any preceding claim, wherein the antibody, or fragment thereof, is in addition, heavy chain subclass specific.

9. A method according to any preceding claim, wherein the antibody is a Fab or a F(ab')2 fragment.

10. A method according to any preceding claim comprising contacting the tissue sample with two or more antibodies, or fragments thereof, specific for different heavy chain class but the same light chain type or specific for the same heavy chain class, but a different light chain type.

11. A method according to claim 10, wherein the ratio of cells having the same heavy chain class, but different light chain type, is determined.

12. A method according to claim 10 or claim 11, wherein the tissue or cell sample is contacted with antibodies or fragments specific for: IgGλ and IgGκ; IgAλ and IgAκ; IgMλ and IgMκ; IgDλ and IgDκ; and/or IgEλ and IgEκ.

13. A method according to any preceding claim, wherein the antibody is directly labelled.

14. A method according to any preceding claim wherein the antibody is indirectly labelled.

15. A method according to any preceding claim wherein the tissue or cell sample is additionally contacted with 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.

16. A tissue or cell staining kit for use in a method according to any preceding claim comprising at least one antibody, or a fragment thereof, having specificity for an immunoglobulin heavy chain class, at the same time as having specificity for a light chain type.

17. A kit according to claim 16, additionally comprising instructions to use the antibody in a method according to any one of claims 1 to 15.

18. A kit according to claim 13 or claim 14 wherein the antibody, or fragment thereof, is a polyclonal antibody.

19. A kit according to claims 16 to 18, wherein the antibody, or fragment thereof, is specific for IgGλ, IgGκ, IgAλ, IgAκ, IgMκ, IgMλ, IgDλ, IgDκ, IgEλ or IgEκ.

20. A kit according to claims 16 to 19, wherein the antibody, or fragment thereof, is heavy chain subclass specific.

21. A kit according to claims 16 to 20, wherein the antibody is a Fab or a F(ab')₂ fragment.

22. A kit according to claims 16 to 21, comprising two or more antibodies, or fragments thereof, having specificity for an immunoglobulin heavy chain class, at the same time as having specificity for a light chain type.

23. A kit according to claim 22 comprising antibodies, or fragments thereof, wherein the ratio of cells having the same heavy chain class, but different light chain type, is determined.

24. A kit according to claims 16 to 23, wherein the antibody is labelled.

25. A kit according to claims 16 to 24, comprising an indirect label for the or each antibody.

26. A kit according to claims 16 to 25, additionally comprising 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.

27. A kit according to claims 16 to 26, additionally comprising an anti-free λ light chain or an anti-free K light chain antibody.

28. 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 any preceding claim.

29. A method according to claim 28, wherein the disorder is a lymphoma, leukaemia, myeloma, plasmacytoma or AL amyloidosis.