Stabilised calibrator or control

Abstract

A stabilised free light chain calibrator or control comprising mammalian serum and a predetermined amount of a free light chain, wherein the serum is depleted of one or more of alpha-1 antitrypsin (A1AT) and / or an immunoglobulin is claimed. Also claimed is an assay kit comprising the stabilised free light chain calibrator or control is also provided, as is a method of determining the presence of and / or concentration of a free light chain in a sample of serum using the stabilised free light chain calibrator or control or an assay kit comprising the same. A method of stabilising a free light chain calibrator or control comprising providing serum, depleting the serum of A1AT and / or an immunoglobulin an adding a predetermined amount of a free light chain, and a method of identifying the presence of, or progression of, a monoclonal gammopathy using the stabilised free light chain calibrator are also claimed.

Description

The invention relates to a stabilised free light chain calibrator or stabilised free light chain control comprising mammalian serum and a predetermined amount of a free light chain (FLC), wherein the serum is depleted of one or more of a-1 antitrypsin (A1AT) and / or an immunoglobulin. Antibody molecules (also known as immunoglobulins) have a twofold symmetry and are composed of two heavy chains and two 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 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 weight of 50,000-77,000 D, compared with the smaller light chain (25,000 D). There are five main classes of heavy chain which are y, a, p, 6 and e, which are the constituent 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 65,000 D and the whole molecule has a molecular weight of about 970,000 D. IgM is largely confined to the intravascular pool and is the dominant antibody produced in the primary immune response. IgA represents 15-20% of 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 is present on the plasma membranes of mature B-lymphocytes and accounts for less than 1% of the total immunoglobulin in the plasma. IgE is the least common serum immunoglobulin isotype, and IgE is most commonly found bound to Fc receptors on surface of mast cells and basophils. 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 IgA2). In healthy adult serum, IgGl, IgG2, IgG3 and IgG4 account for 60-70%, 14-20%, 4-8% and 2-6% of the total IgG pool, respectively. These percentages may be altered in certain disease types. There are two types of antibody light chain: Lambda (X) and Kappa (k). There are approximately twice as many k as A 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 A 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" (FLC). The k light chains are usually found as monomers. The A light chains tend to form dimers. There are a number of proliferative diseases associated with antibody producing cells. 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 immunoglobulins and is known as a monoclonal gammopathy. Diseases such as myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% respectively of cancer deaths in the United Kingdom. Multiple myeloma is the second-most common form of haematological malignancy after non-Hodgkin lymphoma. In Caucasian populations the incidence is approximately 40 per million per year. Conventionally, the diagnosis of multiple myeloma is based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine and related organ or tissue impairment such as hypercalcaemia, renal insufficiency, anaemia or bone lesions. Normal plasma cell content of the bone marrow is about 1% of nucleated cells, while in multiple myeloma the content is typically greater than 10%, frequently greater than 30%, but may be over 90%. AL amyloidosis is a protein conformation disorder characterised by the accumulation of monoclonal free light chain fragments as amyloid deposits. Typically, these patients present with heart or renal failure, but peripheral nerves and other organs may also be involved. There are a number of other diseases which can be identified by the presence of monoclonal immunoglobulins within the blood stream, or indeed urine, of a patient. These include plasmacytoma and extramedullary plasmacytoma, a plasma cell tumour that arises outside the bone marrow and can occur in any organ. When present, the monoclonal protein is typically IgA. Multiple solitary plasmacytomas may occur with or without evidence of multiple myeloma. Waldenstrom's macroglobulinaemia is a low-grade lymphoproliferative disorder that is associated with the production of monoclonal IgM. There are approximately 1,500 new cases per year in the USA and 300 in the UK. Serum IgM quantification is important for both diagnosis and monitoring. B-cell nonHodgkin lymphomas cause approximately 2.6% of all cancer deaths in the UK and monoclonal immunoglobulins have been identified in the serum of about 10-15% of patients using standard electrophoresis methods. Initial reports indicate that monoclonal free light chains can be detected in the urine of 60-70% of patients. In B-cell chronic lymphocytic leukaemia monoclonal proteins have been identified by free light chain immunoassay. Additionally, there are so-called monoclonal gammopathy of undetermined significance (MGUS) conditions. This term denotes the unexpected presence of a monoclonal intact immunoglobulin in individuals who have no evidence of multiple myeloma, AL amyloidosis, Waldenstrom's macroglobulinaemia, etc. MGUS may be found in 1% of the population over 50 years, 3% over 70 years and up to 10% over 80 years of age. Most of these are IgG- or IgM-related, although more rarely IgA-related or bi- clonal. Although most people with MGUS die from unrelated diseases, MGUS may transform into malignant monoclonal gammopathies. In at least some cases for the diseases highlighted above, the diseases present abnormal concentrations of monoclonal immunoglobulins or free light chains. Where a disease produces the abnormal replication of a plasma cell, this often results in the production of more immunoglobulins by that type of cell as that "monoclone" multiplies and appears in the blood. Immunofixation electrophoresis uses a precipitating antibody against the immunoglobulin molecules. Whilst this improves the sensitivity of the test it cannot be used to quantify monoclonal immunoglobulins because of the presence of the precipitating antibody. Immunofixation electrophoresis is also rather laborious to perform and interpretation may be difficult. Capillary zone electrophoresis is used in many clinical laboratories for serum protein separation and is able to detect most monoclonal immunoglobulins. However, when compared with immunofixation, capillary zone electrophoresis fails to detect monoclonal proteins in 5% of samples. These so-called "false negative" results encompass low-concentration monoclonal proteins. Total K and A assays, detecting both heavy chain bound and unbound light chains, have been produced. However, total k and total A assays are too insensitive for the detection of monoclonal immunoglobulin or free light chain. This is due to high background concentrations of polyclonal bound light chains which can interfere with such assays. A sensitive assay has been developed that can detect the free k light chains and separately, the free A light chains. This method uses a polyclonal antibody directed towards either the free k or the free A light chains. The possibility of raising such antibodies was also discussed as one of a number of different possible specificities, in WO 97 / 17372. This document discloses methods of tolerising an animal to allow it to produce desired antibodies that are more specific than prior art techniques could produce. The free light chain assay uses the antibodies to bind to free A or free k light chains. This assay is commercially produced by The Binding Site Group Ltd (now part of Thermo Fisher), Birmingham United Kingdom under the trademark "Freelite". The concentration of the free light chains is determined by nephelometry or turbidimetry. This involves the addition of the test sample to a solution containing the appropriate antibody in a reaction vessel or cuvette. A beam of light is passed through the cuvette and as the antigen-antibody reaction proceeds, the light passing through the cuvette is scattered increasingly as insoluble immune complexes are formed. In nephelometry, the light scatter is monitored by measuring the light intensity at an angle away from the incident light, whilst in turbidimetry light scatter is monitored by measuring the decrease in intensity of the incident beam of light. A series of calibrators of known antigen (i.e. free k or free A) concentration are assayed initially to produce a calibration curve of measured light scatter versus antigen concentration. This form of assay has been found to successfully detect free light chain concentrations. Furthermore, the sensitivity of the technique is very high. More recently mass spectrometry has been developed to detect and characterise immunoglobulins such as FLCs from serum and other samples. WO2015 / 154052 (Mayo Foundation) discloses methods of detecting immunoglobulin light chains, immunoglobulin heavy chains, or mixtures thereof, using mass spectrometry (MS). Samples comprising immunoglobulin light chains, heavy chains or mixtures thereof are immunopurified, reduced to separate light chains and heavy chains, and subjected to mass spectrometry to obtain a mass spectrum of the sample. This can be used to detect monoclonal proteins in samples from patients. It can also be used to fingerprint, isotype, and identify post-translational modifications such as disulphide bonds and glycosylation in monoclonal antibodies. Other variations and improvements have since been made to using MS to detect and quantify FLCs. WO 2015 / 131169 describes methods of monitoring conditions associated with abnormal antibody production. This uses enzymatic cleavage of target immunoglobulin and measuring one or more variable domain peptide fragments by quantitative mass spectrometry. The method is complex because it relies on the identification of variable domain peptide fragments unique to the specific target immunoglobulin associated with the disease and involves lengthy enzymatic cleavage. Other variations and improvements have since been made to using MS to detect and quantify FLCs. The characterisation of the amount or types of free-light chains (FLC), heavy chain or subclasses, or light chain-type bound to heavy chain class or subclass, is important in a wide range of diseases including B-cell diseases, such as multiple myeloma, and other immune-mediated diseases, such as nephropathy. Where free light chains are being detected by assays, it is desirable, if not essential, to have a calibrator or control for that free light chain included within the assay. This acts to calibrate the assay and to ensure that the assay is detecting the free light chain being assayed, or indeed is used to calibrate the assay to allow the concentration of the free light chain to be measured. Calibrators and controls containing free light chains are generally known in the art. They may typically be simply a known quantity of FLC within a buffer, or alternatively be used in combination with, for example, a serum albumin such as bovine serum albumin, or human serum albumin also in the buffer. Soiling K. (Scand.J.CIin.Lab.Inves. (1975) 407-412) discusses the production of radio immunoassays. These were calibrated using purified free light chains from urine. Brower J. et al (Clin.Chim.Acta (1985) 150, 267-274) looked at the estimation of FLCs in immunoglobulins by enzyme immunoassay. Purified kappa and lambda chains were utilised as calibrators, which has been purified from pooled urine of patients with multiple myeloma. Whilst standard serum was calibrated by adding known amounts of kappa or lambda, no stability issues were observed, or indeed, reported. Nelson, M. et al (B.J. Haematology (1992) 81, 223-230) also measured free kappa and lambda light chains in serum. Lambda and kappa light chains obtained from urine as Bence-Jones proteins were used for antigen standard curves and as a source of antigen with which to coat microtiter plates. No stability issues were reported. Abe M. et al (Clin.Exp.Immunol. (1998), 111, 457-462) looks at differences in kappa to lambda ratios in serum and urinary-free light chains. Monoclonal kappa and lambdatype Bence-Jones proteins (FLC) were obtained from patients with multiple myeloma or Waldenstrom's macroglobulinemia. Components were isolated from urine and serum respectively. These were used for ELISA assays. Similarly, Nakano et al (Journal of Immunoassay and Immunochemistry (2006), 27, 129-137 describes proposed reference material for human immunoglobulin light chain measurement. The paper proposed to use purified intact FLCs from urine. Some manufacturers use bovine serum albumin or human serum albumin in their controls or standards reference materials. This approach is ok if there is a direct link between higher order reference materials in a metrological calibration hierarchy. However, in circumstances where there is no internationally recognised primary reference material, clinical guidelines (ISO 17511:2020) often require the matrix of the assay calibrator to be as close as possible to that of the sample being tested. This is to reduce the likelihood of matrix-related noncommutability in the assignment process (Miller, GW et al. Clinical Chemistry (2023), 69, 228-238). Serum is the most commonly tested sample type for monoclonal gammopathies. Accordingly, ideally, the sample matrix should be serum-based. The gold standard for the detection of free light chains in serum is "Freelite™", from The Binding Site Limited, Birmingham, United Kingdom. This utilises pooled serum, into which a known quantity of kappa, lambda, or kappa and lambda free light chains is added. A preservative may also be included. The Applicant has reviewed different manufacturer's quality control release data for the detection of kappa, lambda and the kappa / lambda ratio compared with published data. They have now investigated and identified protein / protein interactions that may account for the discrepancies between quality control release and real-world data, thereby identifying an improvement step which would allow verification of the published ratio for the shelf life of the product. The Applicant unexpectedly identified that a-1 antitrypsin (A1AT) and immunoglobulins such as IgG, IgA and IgM affect the long-term stability of free light chain calibrators andcontrols. A1AT is a protease inhibitor and is produced primarily in the liver and is found in human serum typically between 1 to 1.9 g / L. It is an inhibitor of neutrophil elastase activity in the lung and hence can protect it from proteolytic damage. Whilst the interaction between A1AT and kappa free light chain is known, at no time has there been any suggestion that the stability of free light chain calibrators and controls should be investigated, and indeed, improved. Laurell C.B. and Thulin E. (Immunochemistry (1974), 11, 703-709 and Journal of Experimental Medicine (1975) 141, 453-465) report that complexes between kappachains and ai-antitrypsin were observed in plasma from myeloma patients secreting more than 1 g kappa-chains per litre of urine. This was also observed between A1AT and prealbumin and between IgA and A1AT. The absence of detectable lambda chain complexes with A1AT was thought to be due to lower reactivity of lambda-chain penultimate cysteinyl groups than that of the terminal kappa-chain thiol. The Authors suggested that kappa-chains can form disulphide links with proteins other than the heavy chains of immunoglobulins. They speculated that kappa chains in myeloma might cause complexation of intracellular enzymes containing unpaired thiols or form membrane complexes. The Authors did not, at any time, try and quantify kappa free light chains in body fluids nor did they suggest a long-term stability issue. Any interaction in the serum would have been expected to be equilibrated within the serum as the kappa and lambda light chains were already present within the serum. One reason for the review at this time is that the sensitivity of such assays has improved with the introduction of, for example, mass spectrometry with higher sensitivity. The Applicant unexpectedly identified that removal of A1AT provided an 80% improvement in stability. Moreover, the additional reduction of IgG, IgA and IgM resulted in a >95% improvement in the stability of a calibrator. The invention provides a stabilised free light chain calibrator or control comprising mammalian serum and a predetermined amount of a free light chain, where in the serum is depleted of one or more of A1AT and / or an immunoglobulin. For example, the serum may be depleted of A1AT and one or more of IgA, IgG and IgM. The serum may be depleted of A1AT and IgA, optionally the serum may also be depleted of IgG and / or IgM. The serum may be deleted of Al AT, IgA, IgG and IgM. The serum may be from a single source, but more typically is pooled from several different individuals, to provide a more accurate source of serum. Typically, the mammalian serum and free light chain are human. The free light chain may be selected from free kappa light chain, total free light chain (containing both kappa free light chain and lambda free light chain), and free lambda light chain. Most typically, the calibrator is free kappa light chain. The stabilised calibrator or control typically has a shelf life of 18 months, ideally at least 24 months, from manufacture to use by the customer when stored at 2-8 degrees. The stabilised calibrator or control may have a shelf life of about 6 months, ideally at least 4 months, from manufacture to use by the customer when stored at room temperature (about 16°C to about 25°C) or when stored at about 22°C. A calibrator is a material (in this case the free light chain) with known quantitative / qualitative characteristics (e.g., one or more of concentration, activity, intensity, and / or reactivity) that is used to calibrate, graduate, or adjust a measurement procedure. Calibrators may be provided as a dilution series provided separately, or as a single point calibrator that can be diluted in situ by the user or associated liquid handler. A control is a material (in this case the free light chain) with known quantitative / qualitative characteristics (e.g., one or more of concentration, activity, intensity, and / or reactivity) that is used to confirm that the assay calibration is performing to specification at point of use. The free light chain utilised in the free light chain calibrator or control is typically a mixture of polyclonal free light chains. The immunoglobulin that is depleted is typically selected from IgA, IgM and IgG, most typically all of IgA, IgM and IgG. Typically, the serum is depleted of A1AT and at least one immunoglobulin selected from IgA, IgM and IgG, and is more typically depleted of A1AT, IgG, IgM and IgA. It is not usually necessary to fully deplete the serum of these components. Concentrations of A1AT and / or an immunoglobulin simply need to be reduced below levels where they interfere with stability of the free light chain calibrator or control. The A1AT may be depleted by at least 80% of the original value to give a value less than 200 mg / L remaining in the calibrator or control. Each of the one or more depleted immunoglobulins (e.g., one or more of IgA, IgG and / or IgM) is typically depleted below 150mg / L in the calibrator or control. Typically, the predetermined amount of free light chain is between about 1 mg / L and about 200 mg / L or between about 1 mg / L and about 100 mg / L in the calibrator or control. Optionally, the predetermined amount of free light chain may be between about 0.1 mg / L and about 20 mg / L. The stabilised free light chain calibrator or control may additionally comprise up to about 0.3% w / v preservative. The preservative may, for example, be sodium azide, Epsilon-aminocaproic acid (EACA) or benzamidine, or a mixture thereof. Most typically, the concentrations utilised are about 0.099% w / v sodium azide, about 0.1% w / v EACA, and about 0.01% w / v benzamidine. Serum is the clear liquid part of the blood that remains after blood cells and clotting proteins have been removed. It contains a range or proteins, ions, lipid, hormones and sugars amongst other components. The stabilised free light chain calibrator or standard typically comprises at least the following components: pooled normal human serum that may have been clotted and de-lipidated, e.g., using established techniques. The depletion process is highly specific for the proteins that are removed (e.g., A1AT, and one or more of IgG, IgA and IgM), meaning that other proteins are not removed by the depletion process. All the other serum proteins (such as albumin, beta-globulins and complement proteins) and components are unchanged and should remain within the normal reference levels. To facilitate this the depleted-serum can be concentrated back to at least its original volume and the total protein levels measured and required to be between about 50 g / L and about 100 g / L (a normal range is about 60-80 g / L). The stabilised light chain calibrator or control may be obtainable by depleting the A1AT and / or immunoglobulin by affinity purification. Such affinity purification techniques are generally known in the art. They may be by immunoaffinity purification or alternatively using, for example Melon Gel to remove immunoglobulins. A further aspect of the invention provides an assay kit comprising an anti-free light chain specific control antibody, and an anti-free light chain specific antibody packaged with a stabilised free light chain calibrator or control according to the invention. The assay kit may be a nephelometric kit, a turbidimetric kit, a lateral flow kit or an ELISA assay kit. The kit may include instructions for performing the assay. The assay may be a direct assay or a competition or inhibition assay. Methods of determining the presence and / or concentration of free light chains in a sample of serum comprising the use of a stabilised free light chain calibrator or control, or kit, according to the invention are also provided. The methods may be, for example, capillary zone electrophoresis, an immunoassay or a mass spectrometry assay. Where the assay is an immunoassay, it may be nephelometric, turbidimetric, an ELISA assay or a lateral flow assay. The mass spectrometry assay may be, for example, LC-MS or MALDI-TOF. Methods of stabilising a free light chain calibrator or control comprising providing serum, depleting the serum of a-1 antitrypsin and / or an immunoglobulin and adding a predetermined amount of a free light chain to form the free light chain calibrator or control is also provided. The A1AT, immunoglobulin, serum, optional preservative and other components may be as described above. The invention also provides a method of identifying the presence of, or progression of, a monoclonal gammopathy, comprising the use of a stabilised free light chain calibrator, kit or method according to the invention. Monoclonal gammopathy may be, for example, myeloma, AL amyloidosis, a plasmacytoma, a plasma cell tumour, Waldenstrom's macroglobulinaemia, a B-cell non-Hodgkin lymphoma, a B-cell chronic lymphocytic leukaemia, smouldering multiple myeloma (SMM), or a monoclonal gammopathy of undetermined significance (MGUS). Brief Description of the Drawings The invention will now be described in detail, by way of example only, with reference to the figures. Figure 1 shows SDS-PAGE gels of the interactions between FLC Kappa and FLC Lambda with A1AT. Figure 2 shows Surface Plasmon Resonance (SPR) showing FLC Kappa antibodies have different binding kinetics to FLC Kappa—A1AT complex and FLC Kappa alone. Figure 3 shows stability of the different Kappa calibratorcurves using the measured on a turbidimetric analyser. Figure 4 shows spiking of A1AT into an artificial calibrator fluid and the effect on turbidimetric activity for Kappa and Lambda. Figure 5 shows impact on normal healthy sample values with unmodified or depleted calibrator measured on a turbidimetric analyser. Examples 1 Since 2001, Serum Free Light Chain measurements have become established as routine clinical laboratory tests. Katzmann JA et al., Clinical Chemistry (2002) 48, (9) 1437-1444 published a reference interval based on 282 normal samples establishing a kappa / lambda ratio reference range 0.26-1.65. The kappa / lambda ratio gives a strong indication of monoclonal gammopathy. In recent years, it has been suggested that the ratio may have changed. Here we present investigations identifying protein / protein interactions that may account for the discrepancies between QC release and real-world data; identifying an improvement step which would support the published ratio. The calibrators comprised kappa and pooled human sera with 0.099% w / v sodium azide, 0.1% w / v EACA and 0.01% w / v benzamidine. The calibrators were used with Freelite™ free light chain assay Optilite™ kits from The Binding Site Limited. The manufacturers QC release data was reviewed for kappa, lambda, and kappa / lambda ratio for all lots from 2018-2023 and compared to published data. To mimic field observation an accelerated stability study was performed on calibrators and protein / protein interactions identified using western blot. Subsequently, calibrators were depleted of alpha-1 antitrypsin (A1AT) and other serum proteins using specific affinity chromatography; accelerated stability was repeated. A calibrator of pure polyclonal free light chains (FLC) in human serum albumin was produced in the presence / absence of A1AT and additional interfering serum proteins. Analysis of FLC calibrators during an accelerated stability identified interactions between kappa FLC and ubiquitous serum proteins, including A1AT. The calibrator activity declined by 14.9±0.1% (mean±SEM) over a 4-month accelerated period @22°C, equivalent to 16 months real time. Calibrators depleted of A1AT and specific additional serum proteins showed significantly less decline in activity 1.4±1.4%, p<0.001. When blood donor sera were analysed using the calibrators following 4 months at 22°C, the results showed an increase in the reported kappa concentrations compared to those generated from frozen calibrators (19.0[15.4-22.5] to 21.3(17.0-26.0] mg / L, 12.5±3.8%, p<0.001). This observation was mirrored in kappa / lambda ratio (1.3(1.1-1.5] to 1.4(1.2-1.7] mg / L, 12.5±3.8%, p<0.001) and when clinical patient samples were assessed (30.1(14.0-47.1] to 34.3(15.5-56.9] mg / L, 13.8±1.4%, p<0.001). The depleted calibrator did not report an increase in sample results. Conclusion An unpredictable kappa / protein interaction was observed, particularly with A1AT in a time dependent fashion, which correlated to a reduction in calibrator activity. Removal of A1AT and other specific serum proteins remediated the issue, and the kappa / lambda ratio was verified irrespective of the age of the calibrator. Example 2 Pure proteins: Polyclonal FLC Kappa and Polyclonal FLC Lambda, were incubated with A1AT at three different molar concentration ratios for 0 and 7 days at 37°C. Control incubations of polyclonal FLC Kappa with HSA or with pure proteins in buffer were similarly set up (Table 1). At the end of the incubation the protein mixtures were analysed by reducing (Fig 1 A-C) and non-reducing (Fig 1 D-F) SDS-PAGE and stained with Coomassie brilliant blue. Incubation of polyclonal FLC kappa with A1AT for 7 days resulted in a decrease or disappearance in the FLC monomer band (25 kDa) and an electrophoretic mobility shift in the principal A1AT band from 60 to 50 kDa (Fig 1A and D). A weaker staining band of approximately 90 kDa was seen after 7 days. This later band was a complex of A1AT and FLC. This effect was greater at a Kappa: A1AT molar ratio of 2:1 and 4:1. In contrast, the interaction of polyclonal FLC lambda with A1AT only occurred with a molar ratio of 2:1. The changes to the SDS-PAGE mobility were absent when the polyclonal FLCs were incubated with buffer only. Incubation of A1AT in buffer for 7 days at 37°C resulted in a partial appearance of a 50 kDa band that was most likely due to autocatalysis (Fig IC and F). There is also evidence of proteolytical degradation of a portion of the FLC. Bands of 8 kDa were observed in Kappa: A1AT ratio of 2:1, 4:1 and Lambda ratio 2:1 lanes. This indicates that the interaction between polyclonal FLCs and A1AT is greater for kappa than lambda FLC and is concentration dependent. Monomeric FLC is preferred to dimeric. The interaction between A1AT and FLC is not a simple one. The relative intensity of the A1AT+FLC complex band (90 kDa) was less than expected compared to the disappearance of the more abundant FLC monomer band (25 kDa). The associated shift of the A1AT band (from 60 to 50 kDa) suggests either a degradation or a conformational re-arrangement occurs in the presence of FLC, which probably accounts for the change in mobility. Table 1: incubation mixtures of polyclonal FLCs and A1AT or HSA. Reaction Description Kappa (uM) Lambda (uM) Al AT (pM) HSA (pM) 1 Kappa: Al AT ratio - 2:1 25 0 12.5 0 2 Kappa: A1AT ratio - 4:1 25 0 6.25 0 3 Kappa: A1AT ratio - 8:1 25 0 3.13 0 4 Lambda: A1AT ratio - 2:1 0 25 12.5 0 5 Lambda: A1AT ratio - 4:1 0 25 6.25 0 6 Lambda: A1AT ratio - 8:1 0 25 3.13 0 7 Kappa: HSA ratio 2:1 25 0 0 12.5 8 Kappa: HSA ratio - 4:1 25 0 0 6.25 9 Kappa: HSA ratio - 8:1 25 0 0 3.13 10 Kappa PBS control 25 0 0 0 11 Lambda PBS control 0 25 0 0 12 A1AT PBS control 0 0 12.5 0 13 Albumin PBS control 0 0 0 12.5 Example 3 Surface Plasmon Resonance (SPR) was used to analyze the real-time kinetics between anti-FLC Kappa antibodies (Freelite™) and FLC kappa alone or FLC Kappa in a complex with A1AT. Polyclonal FLC Kappa was incubated with a ten-fold molar excess of pure A1AT. Control incubations of polyclonal FLC Kappa or A1AT alone were also set up. The sensograms produced when antibodies immobilized on the biosensor chip are reacted with different concentrations of pure proteins are shown in Figure 2 and the calculated binding constants are indicated in the table below each graph. The association (on-rate, ka) of the antibodies is higher against FLC Kappa alone than against the FLC Kappa+AIAT complex (Fig 2A and B). But the dissociation rate (kd) is more equivocal. This indicates that antibodies against FLC kappa have a lower affinity to the target analyte when it is complexed with A1AT. No binding of anti-FLC Kappa antibodies to A1AT alone was observed (Fig 2C). Example 4 Pooled human serum (clotted and de-lipidated) was depleted of alpha-l-antitrypsin (A1AT) and immunoglobulins by using a series of affinity chromatography steps. The following chromatography matrices (columns) were used: IgG depletion - Protein G Sepharose 4 Fast Flow (Cytiva), IgA depletion - CaptureSelect IgA-XL resin (Thermo Fisher Scientific, Inc.), IgM depletion - POROS CaptureSelect IgM-XL resin (Thermo Fisher Scientific, Inc.) and A1AT depletion - CaptureSelect AAT_XL resin (Thermo Fisher Scientific, Inc.). A single pass of the serum feedstock through these columns removed at least 80% of each target protein. The level of depletion was subsequently assessed by turbidimetric immunoassay after the volume had been corrected to match that of the feedstock. The total protein was adjusted to be at least as high as the original serum. (Table 2). Table 2 Analyte Analyte concentration at start g / L Analyte concentration at end g / L IgG 3.9 <0.166 IgA 0.98 <0.021 IgM 0.24 <0.102 Al AT 0.814 <0.34 Total Protein 55 84 Example 5 The unmodified (Fig 3A) Kappa FLC calibrator, the A1AT depleted (Fig 3B) and the A1AT and immunoglobulin depleted (Fig 3C) were prepared and either frozen at -20°C (• black line) or incubated at 22°C for 4 months (0 grey line). At the end of this period a Kappa FLC calibration curve was run for each calibrator fluid on the turbidimetric analyser (Optilite™). The unmodified calibrator demonstrated a loss in activity of about 15 to 20% during this time, compared to the frozen control (Fig 3A). This activity drop was substantially reduced when A1AT had been depleted from the calibrator fluid prior to incubation (Fig 3B). Depletion of both A1AT and immunoglobulins completely abolished this activity loss and both curves are indistinguishable from each other after 4 months (Fig 3C). Thus, the depletion of first A1AT and subsequently immunoglobulins served to stabilise the Kappa FLC calibrator. Example 6 A defined simple mixture of purified kappa FLC (A and B) or purified lambda FLC (C and D) and human serum albumin in buffer was produced and is stable when incubated for 21 days at 22°C compared to those at days 0-2 (Fig 4A and 4C). However, when physiological concentrations (0.5 g / l) of pure A1AT protein are spiked into this calibrator solution before storage the kappa calibrator loses activity after incubation for 21 days at 22°C (Fig 4B). The reduction in activity is similar in magnitude to that observed using the unmodified serum calibrator (Fig 3A). This reduction in activity is not seen for purified lambda FLC spiked with A1AT (Fig 4D). This supports the reasoning that A1AT is the main causative agent of the instability of serum-based FLC calibrators. Example 7 The unmodified (Fig 5A) Kappa FLC calibrator and the A1AT and IgG, IgA and IgM immunoglobulin depleted calibrator (Fig 5B) were prepared and either frozen at -20°C (solid line and lighter bar) or incubated at 22°C for 4 months (dashed line and darker bar). At the end of this period a Kappa FLC calibration curve was run for each calibrator fluid on the Optilite™ turbidimetric analyser. 51 normal healthy samples were run and read off each curve. A histogram visualization of the data shows a shift in the distribution of the samples read off the unmodified calibrator curve after 22°C for 4 months (Fig 5A dashed line) but not for the A1AT and immunoglobulin depleted calibrator (Fig 5B dashed line). The mean value was shifted + 13.5% for the former. This data shows that instability of the unmodified serum calibrator can affect not only the calibration curve but the reported (clinical) values of the FLC Kappa analyte.

Claims

1. A stabilised free light chain calibrator or control comprising mammalian serum and a predetermined amount of a free light chain, wherein the serum is depleted of one or more of alpha-1 antitrypsin (A1AT) and / or an immunoglobulin.

2. A stabilised free light chain calibrator or control according to claim 1, wherein the mammalian serum and free light chain are human.

3. A stabilised free light chain calibrator or control according to claims 1 or 2 wherein the free light chain is selected from (i) free kappa light chain, (ii) free lambda light chain, and (iii) free kappa light chain and free lambda light chain.

4. A stabilised free light chain calibrator or control according to claim 3, wherein the free light chain is free kappa light chain.

5. A stabilised free light chain calibrator or control according to claims 1 to 4, wherein the immunoglobulin is selected from one or more of IgA, IgM and IgG, most typically IgA and IgM and IgG.

6. A stabilised free light chain calibrator or control according to claims 1 to 5 wherein the serum is depleted of A1AT and at least one immunoglobulin.

7. A stabilised free light chain calibrator or control according to claim 6, wherein the serum is depleted of A1AT, IgG, IgM and IgA.

8. A stabilised free light chain calibrator or control according to claims 1 to 7 wherein the free light chain is polyclonal free light chain.

9. A stabilised free light chain calibrator or control according to claims 1 to 8, wherein the A1AT is depleted below 200 mg / L in the calibrator or control.

10. A stabilised free light chain calibrator or control according to claims 1 to 9, wherein when each depleted immunoglobulin is depleted below 150 mg / L in the calibrator or control.

11. A stabilised free light chain calibrator or control according to claims 1 to 10, where in the concentration of the predetermined amount of free light chain is between 1 mg / L and 200 mg / L, most typically between 1 mg / L and 100 mg / L.

12. A stabilised free light chain calibrator or control according to claims 1 to 11 wherein the calibrator additional contains up to 0.3 % w / v preservative.

13. A stabilised free light chain calibrator or control according to claims 1 to 12, wherein the serum comprises at least the following components: serum albumin, alpha globulin, beta globulin, gamma globulin.

14. A stabilised free light chain calibrator or control according to claims 1 to 13, obtainable by depleting the A1AT and / or immunoglobulin by affinity purification.

15. An assay kit comprising an anti-free light chain specific antibody packaged with a stabilised free light chain calibrator or control according to claims 1 to 14.

16. An assay kit according to claim 15, which is a mass spectrometry kit, a nephelometric kit, a turbidimetric kit, a lateral flow kit, or an ELISA assay kit.

17. A method of determining the presence of and / or concentration of a free light chain in a sample of serum comprising the use of a stabilised free light chain calibrator or control according to claims 1 to 14 or a kit according to claims 15 or 16.

18. A method according to claim 17 which is, capillary zone electrophoresis, an immunoassay or a mass spectrometry assay.

19. A method according to claim 18, wherein the immunoassay is nephelometric, turbidimetric, an ELISA assay or a lateral flow assay.

20. A method of stabilising a free light chain calibrator or control comprising providing serum, depleting the serum of alpha-1 antitrypsin (A1AT) and / or an immunoglobulin and adding a predetermined amount of a free light chain to form the free light chain calibrator or control.

21. A method of identifying the presence of, or progression of a monoclonal gammopathy comprising the use of a stabilised free light chain calibrator, kit or method according to any preceding claim.

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