Immunoassays

Abstract

The present invention relates to an immunoassay for identifying one or more free light chains. In particular, the present invention relates to a method for assaying free light chains (FLC) in a mammalian sample, comprising the use of at least one avian-derived antibody (IgY). The present invention also relates to a method for screening, diagnosing, monitoring or prognosing a disease in a patient, comprising carrying out the assay method described above. The present invention also relates to the use of avian-derived antibody (IgY) in an immunoassay for assaying FLC in a mammalian sample.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an immunoassay for identifying one or more free light chains.

[0002] 2. Background of the Invention Antibodies consist of two identical heavy chains and two identical light chains, each of which contains a variable domain and a constant domain. There are two types of light chains: kappa (κ) and lambda (λ). Each antibody expresses only one class of light chain. Normally, light chains are attached to heavy chains and are called bound light chains. However, there are usually some excess light chains that are not bound to heavy chains and are called "free light chains (FLC)". FLCs can be detected, for example, in the serum, plasma, or urine of an individual.

[0003] Serum free light chains (sFLCs) are important biomarkers for the diagnosis and management of immune-related diseases, such as smoldering multiple myeloma (SMM), multiple myeloma (MM), and other plasma cell disorders, such as monoclonal gammopathy of undetermined significance (MGUS), light chain amyloidosis (AL amyloidosis), and light chain deposition disease (LCDD). sFLCs are also markers for immune-stimulating diseases, such as multiple sclerosis, some liver diseases, and autoimmune diseases, such as systemic lupus erythematosus (SLE).

[0004] Both normal and abnormal plasma cells produce more light chains than heavy chains, and these excess light chains are released into the bloodstream. These FLCs are typically rapidly cleared and metabolized by the kidneys. Increased sFLCs can result from decreased renal clearance, increased polyclonal immunoglobulin production, or monoclonal gammopathy.

[0005] FLC can be detected by raising antibodies against a surface of FLC that is normally hidden in whole immunoglobulin molecules by binding of the light chain to the heavy chain. Methods for raising mammalian antibodies against FLC have been previously described (Bradwell et al, Clin. Chem., (2001), 47, 673-680).

[0006] Immunoassays are based on measuring the reaction of an antigen (e.g. a site on a protein) with an antibody specific for that antigen. Detection of free light chains can be performed by immunoassays in which an antibody is raised against at least one antigen in the region of FLC that is bound to the heavy chain in the intact antibody. Kits for the detection of κ or λ FLC are commercially available, for example "Freelite™" from The Binding Site Limited and "N Latex" from Siemens. These are based on polyclonal sheep antibody technology and multiple monoclonal antibodies, respectively.

[0007] A problem with current sFLC assays is that they are not interchangeable, because different assay methods may give different results for the same sample. One problem reported for FLC assays is that these molecules are known to be a heterogeneous population, exhibiting considerable diversity and often present in polymeric forms in blood, serum, plasma, and urine. These can be, for example, dimers, tetramers, or more highly polymerized forms. κFLC usually exists as a monomer, while λFLC tends to form dimers (Solling, Scand. J. Clin. Lab. Invest., (1976), 36, 447-452). Unless the anti-FLC antibody recognizes all molecular forms in equimolar amounts, sFLC assays will not give equivalent results in all samples. Caponi et al. (Clin. Chem. Lab. Med., (2016), 54, 1111-1113) highlight this inconsistency between existing FLC assays.

[0008] The monomer / dimer ratio of serum FLC may also differ between healthy individuals and patients with monoclonal gammopathy or other immune-stimulating diseases. For example, Kaplan et al. (Am. J. Hematol., (2014), 89, 882-888) showed that patients with AL and MM have abnormally increased dimerization of monoclonal FLC, with clonality values ​​of FLC dimers higher than those of monomers. These FLC pattern abnormalities were not observed in patients with MGUS, SMM, and AL amyloidosis as well as in healthy individuals. The importance of the polymeric diversity of FLC molecules was considered by the developers of the original sFLC assay (Bradwell et al, Clin. Chem., (2001), 47, 673-680). However, published data indicate that the antibodies generated bound the dimeric form with higher avidity compared to the monomeric form. Thus, the immunoassay generated using the mammalian-derived polyclonal antibodies described by Bradwell et al. is expected to preferentially recognize the dimeric form of FLC.

[0009] It has been shown that both the Freelite and N-Latex assays significantly overestimate the concentration of monoclonal FLC, especially in high titer samples, and polymerization of FLC has been shown to be the cause (de Kat Angelino CM et.al 2010. Clin Chem. 2010 Jul;56(7):1188-90., and Di Noto Get.al., Ann Clin Biochem. 2015 May;52(3):327-36).

[0010] Currently, there are no internationally accepted FLC reference materials or reference methods, and there are significant differences in the methodologies of currently available commercially available FLC assays and their responses to samples containing various multimeric forms of FLC.

[0011] Strategies have been proposed to facilitate quantification of multimeric FLC using mammalian-derived antibodies, including the use of reducing agents in the assay formulation (WO2017144896A1) for selective cleavage of inter-light chain (IL) disulfide bridges (Jerry and Kunkel, J Immunol November 1, 1972, 109 (5) 982-991). However, such strategies require additional steps and / or increase the complexity of the assay, and such methods have not yet been successfully applied.

[0012] It is important to be able to quantify FLC without being too dependent on the polymerization / multimerization state of the sample. It would also be advantageous to provide an assay that specifically detects λ or κ FLC without being too dependent on the multimeric form. FLC antibodies should only recognize "hidden" epitopes in intact immunoglobulins to avoid cross-reaction with light chains in whole immunoglobulins that would result in false elevation of FLC.

[0013] The present inventors have found that immunoassay reagents based on avian-derived antibodies (IgY) solve the problems associated with different multimeric forms of FLC.

[0014] Summary of the Invention In a first aspect, a method for assaying FLC in a mammalian sample is provided, comprising the use of at least one avian derived antibody (IgY).

[0015] In a second aspect, there is provided a method for screening, diagnosing, monitoring or prognosis of a disease in a patient, the method comprising carrying out an assay method according to the first aspect and comparing the result of the assay with at least one predetermined threshold value.

[0016] In a third aspect, there is provided the use of an avian derived antibody (IgY) in an immunoassay for assaying FLC in a mammalian sample.

[0017] In a fourth aspect, an assay kit is provided comprising at least one avian derived antibody for use in assaying for FLC in a mammalian sample.

[0018] Various embodiments of the invention are described herein and may be applied to any aspect of the invention, where technically feasible. Any embodiment described herein may be used alone or in combination with any other embodiment, where technically feasible.

[0019] In one embodiment, the avian-derived antibody is a chicken antibody.

[0020] In another embodiment, the sample is serum, plasma, or urine.

[0021] In another embodiment, the FLC is a λ FLC, a κ FLC, or a total FLC.

[0022] In another embodiment, the disease is selected from smoldering multiple myeloma, intact immunoglobulin myeloma, light chain myeloma, nonsecretory myeloma, monoclonal gammopathy of undetermined significance (MGUS), light chain amyloidosis (AL amyloidosis), Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, follicular center cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, pre-B cell leukemia, and acute lymphocytic leukemia.

[0023] In some embodiments, the avian derived antibody is attached to a support.

[0024] Further characteristics and advantages of the invention will become apparent from the following description, given by way of example only and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows a calibration curve for particles coated with anti-kappa IgY. [Diagram 2] FIG. 2 shows a calibration curve for particles coated with anti-lambda IgY. [Diagram 3]FIG. 3 shows the effect of reducing agents on kappa antigens using SDS-PAGE. [Figure 4] FIG. 4 shows the immunoturbidimetric response enhanced by chicken IgY anti-kappa particles and a comparison of the effect of monomeric and dimeric forms. [Diagram 5] FIG. 5 shows a comparison of the Freelite anti-kappa response and the effect of monomeric and dimeric forms. [Figure 6] FIG. 6 shows the effect of reducing agents on lambda antigen using SDS-PAGE. [Figure 7] FIG. 7 shows the immunoturbidimetric response enhanced by chicken IgY anti-lambda particles and a comparison of the effect of monomeric and dimeric forms. [Figure 8] FIG. 8 shows a comparison of the Freelite anti-lambda response and the effect of monomeric and dimeric forms.

[0026] Detailed Description The present invention relates to a method for assaying FLC in a mammalian sample, comprising the use of at least one avian-derived antibody (IgY). The avian-derived antibody can be an anti-FLC antibody, which can be an anti-kappa or anti-lambda FLC antibody. Measuring the amount of κ FLC, λ FLC, total FLC, and / or the ratio of κ FLC to λ FLC (κ FLC:λ FLC ratio or κ:λ ratio) can provide information about several different diseases or conditions in a patient. Normal plasma cells produce excess light chains, which are excreted in the serum. In healthy humans, about twice as many kappa light chains are produced as lambda light chains. In healthy individuals, these excess FLCs are reabsorbed and metabolized by the kidney. Individuals with certain diseases have elevated concentrations of FLC and / or altered ratios of κ FLC to λ FLC. Monoclonal gammopathy is a disorder caused by the abnormal proliferation of a single clone of plasma cells. Traditionally, one examines whether either λ FLC or κ FLC is increased. For example, multiple myeloma results from the monoclonal proliferation of malignant plasma cells, resulting in an increase in one type of cell producing one type of immunoglobulin, resulting in increased amounts of either lambda or kappa FLCs observed in an individual.

[0027] The inventors have surprisingly found that avian-derived antibodies can bind to FLC with stronger affinity than mammalian antibodies and / or can measure different multimeric forms more evenly. It would be particularly advantageous to be able to detect different multimeric forms with substantially equal affinity, since, for example, AL and MM patients have abnormally increased dimerization of monoclonal FLC (Kaplan et al, 2014). Existing sFLC assays use antibodies generated by immunizing laboratory mammals. Without wishing to be bound by any theory, it is believed that some of the problems with existing assays can be attributed to the close phylogenetic distance between humans and the mammals used to generate antibodies.

[0028] Methods for measuring the amount of a particular target molecule in a sample using binding molecules that specifically bind to the target are well known in the art. Any suitable immunoassay method can be used in appropriate embodiments of the present invention.

[0029] In some embodiments, the assay is an in vitro assay. In some embodiments, the assay is a nephelometric assay, a turbidimetric assay, a flow cytometry assay, a lateral flow assay, a radial flow assay, an immunofixation electrophoresis (IFE) assay, or an enzyme-linked immunosorbent assay (ELISA) assay. In some embodiments, the assay is a turbidimetric assay. In some embodiments, the assay is an ELISA assay.

[0030] In some embodiments, the assay includes the use of a biosensor comprising the avian-derived antibody and a physicochemical transducer or detector that converts one signal into another (e.g., optical, piezoelectric, electrochemical, electrochemiluminescent, etc.) for measurement and quantification. In particular, the biosensor can convert the binding event between the avian-derived antibody and its antigen (FLC) into a measurable signal (e.g., a change in optical properties), detect the signal, and process the detected signal into an assay result.

[0031] In some embodiments, the assay is a multiplex assay. Multiplexing allows for simultaneous quantification of multiple analytes in one sample. In some cases, the multiplex assay is a lateral flow device that includes multiple test lines. In some cases, the multiplex assay is a fluorescent assay. In some embodiments, the multiplex assay uses fluorescent beads. In some cases, the multiplex assay includes color-coded beads pre-coated with analyte-specific capture antibodies. In some cases, the multiplex assay includes color-coded beads pre-coated with analyte-specific capture antibodies, including, for example, Luminex xMAP assays.

[0032] In some embodiments, the assay is laboratory-based. Optionally, the assay is a point-of-care assay.

[0033] As used herein, an "assay" or "assay method" may be a qualitative or quantitative assay method. In the case of a qualitative assay, the assay may return a binary result (e.g., positive or negative) indicating whether the FLC concentration is above or below a certain threshold. Such an assay is particularly useful in initial screening to ascertain whether more detailed further analysis may be required. Quantitative assays generally return a numerical result, either on an absolute scale or on an arbitrary scale. Such assays are valuable for FLC measurement because changes in FLC concentration may indicate disease severity or progression, such as in the case of "smoldering" multiple myeloma, where periodic testing may be performed. In such a situation, a stable FLC assay value may indicate that the disease is stable and / or in remission. In contrast, a changing value may indicate that the disease is worsening. For example, an increase in total FLC concentration or an abnormal increase in the kappa:lambda ratio may indicate that the disease is progressing. Similarly, a decrease in total FLC or a kappa:lambda ratio moving closer to normal may indicate successful treatment and / or management.

[0034] In some embodiments, one or more of the IgYs used in the assay may be labeled (e.g., to allow detection of binding between the target molecule and the binding molecule). In some cases, the IgY is labeled by fluorescence, luminescence, radioactivity, isotopic labeling, or conjugation to an enzyme, binding agent, particle, or substrate. In some cases, the IgY is labeled with an enzyme capable of converting a substrate into a detectable analyte. Such enzymes include horseradish peroxidase, alkaline phosphatase, and other enzymes known in the art. In some cases, the IgY is conjugated to a specific antigen or one member of a binding pair, such as biotin.

[0035] In some embodiments, IgY is immobilized on a particle or surface. Immobilization allows processes such as separation of the immobilized IgY (and any bound components) from the fluid phase. Such particles or surfaces can be of any size and material that allows easy separation from the fluid phase (e.g., 10 nm to 10 cm or 100 nm to 1 cm), and can include surfaces or larger articles, such as areas of glass or plastic surfaces or wells of a microtiter plate (e.g., 96-well, 384-well, or 1536-well plates). Attachment to particles can also be used as a labeling method, since aggregation of small particles can increase scattering and thus turbidity of the sample.

[0036] In some cases, the IgY is coated onto nanoparticles having an average diameter of at least 40 nm (e.g., 40-300 nm), e.g., 50-260 nm, preferably 80-200 nm. Suitable materials for particles or surfaces to which the IgY can be attached include inorganic materials such as glass, ceramic, metal, or metal oxide (e.g., silica, titania, or zirconia); synthetic polymers such as thermoplastic or thermosetting polymers (e.g., polyolefins, polystyrene, polyesters, polyamides, polycarbonates, polyurethanes, epoxy resins, or phenolic resins); and / or natural polymers or modified (semi-synthetic) natural polymers (e.g., protein polymers such as latex rubber, cellulose, starch, or silk).

[0037] One particular example is the attachment of IgY to nanoparticles made of glass, silica, latex, metals (eg, gold), or polymeric materials (eg, polyethylene or polystyrene).

[0038] One particular example is the attachment of IgY to nanoparticles of latex particles (Ikerlat Polymers SL, Spain), the method comprising: Providing an immunoparticle according to any embodiment thereof; reacting the sample with the immunoparticles; detecting a change in reflectance, scattering, or transmittance of the sample, wherein the change in reflectance, scattering, or transmittance of the sample indicates the amount of FLC in the sample.

[0039] The sample is a sample selected from plasma and serum, the change in reflectance, scattering or transmittance is a change in transmittance, and the method is a turbidimetric method.

[0040] According to one embodiment of the above, the change is a change in reflectance or scattering, and the method is a nephelometric method.

[0041] The ability to perform the above methods as turbidimetric or nephelometric methods is a major advantage since such methods can be automated and performed on existing clinical analyzers that are in routine use in most clinical chemistry laboratories worldwide.

[0042] In certain embodiments, the IgY-specific binding agent may be capable of specifically binding to λ (lambda) FLC or κ (kappa) FLC. Specific binding in this context may be considered to have less than 5% (e.g., 0.001% to 5%), preferably less than 1% cross-reactivity. That is, lambda-specific IgY may have less than 5% or less than 1% cross-reactivity with kappa. Similarly, kappa-specific IgY may have less than 5% or less than 1% cross-reactivity with lambda.

[0043] Cross-reactivity is the relative signal generated in an assay method using a target antigen in an assay sample and a corresponding assay performed using another antigen in the same sample at the same concentration. In this case, the cross-reactivity of lambda-specific IgY to kappa is the signal generated in an assay method using a sample containing kappa FLC relative to the signal obtained in the same assay method performed with a sample containing lambda FLC. Similarly, the cross-reactivity of kappa-specific IgY to lambda is the signal generated in an assay method using a sample containing lambda FLC relative to the signal obtained in the same assay method performed with a sample containing kappa FLC.

[0044] In some embodiments, the FLC is selected from the group consisting of a kappa FLC, a lambda FLC, and a mixture thereof.

[0045] In some embodiments, the sample is a sample of blood, serum, plasma, saliva, urine, cerebrospinal fluid, or other biological fluid. Preferably, the sample is a serum, plasma, or urine sample. The test sample is taken from a mammal, preferably a human.

[0046] The avian-derived antibody (IgY) exhibits affinity for at least one constant domain of a mammalian immunoglobulin light chain. According to the embodiment, the IgY polyclonal antibody specifically binds to lambda or kappa FLC antigen with a KD of less than 20.0E-08M (e.g., 20.0E-08M to 1.0E-12M), preferably less than 10.0E-09M, and most preferably less than 2.0E-09M. One suitable way to measure the binding affinity of an antibody to its antigen is by surface plasmon resonance (SPR), for example using the Biacore™ assay platform and software (GE Healthcare).

[0047] In some embodiments, the avian antibody is an antibody from an avian species selected from chicken, duck, goose, turkey, and quail. In a preferred embodiment, the avian antibody in all aspects and embodiments described herein may be a chicken antibody. The chicken antibody may be a polyclonal antibody, a monoclonal antibody, or a recombinant monoclonal antibody. The monoclonal antibody or recombinant monoclonal antibody may be used individually or in combination (e.g., as a multiclonal preparation).

[0048] The antibodies of the present invention can be purified from eggs of immunized chickens immunized with natural monoclonal or polyclonal FLC or recombinant antigens specific for the constant regions of immunoglobulin light chains kappa and lambda.

[0049] The antibody of the present invention can be purified using well-known antibody purification techniques. Suitable examples of antibody purification techniques that can be used to purify the antibody of the present invention include precipitation (salting out) with ammonium sulfate or the like, ion exchange chromatography using diethylaminoester (DEAE) derivatives or carboxymethyl (CM) derivatives or the like, hydroxyapatite chromatography, gel filtration chromatography, and affinity chromatography using protein A or protein G, including binding to the antigen against which the antibody was raised, among others. It will be understood that a combination of the techniques listed above may be used to purify the antibody.

[0050] In some embodiments, IgY can specifically bind to λ or κ FLC. By "specifically bind" it is meant that the avian-derived antibody does not substantially bind to any other molecule than the desired FLC. For example, anti-lambda antibodies recognize kappa antigens or whole immunoglobulin light chains to a much lesser extent than lambda FLC. Such cross-reactivity can be used to generate a relative signal comparing the binding of the target antigen to another antigen. In some cases, the cross-reactivity is less than 1%.

[0051] In some embodiments, IgY has substantially equal reactivity with monomeric and dimeric forms of FLC, indicating that a given concentration of FLC in a sample will generate substantially equal immunoassay signals regardless of the polymeric nature of the FLC antigen. In some cases, IgY has substantially equal reactivity with all multimeric forms of FLC.

[0052] As used herein, the term "substantially equal" indicates that the difference between two values ​​is less than 20%, preferably less than 10%, and more preferably less than 5%.

[0053] In some embodiments, at least two IgYs with different specificities are used. In some cases, when at least two IgYs are present, one IgY has a higher affinity for either λ or κ FLC, and the other IgY has substantially the same affinity for λ and κ FLC.

[0054] In some embodiments, the κFLC:λFLC ratio in the sample is determined. In some cases, this κFLC:λFLC ratio is compared with the normal range of κFLC:λFLC ratio, and if there is a significant deviation, it is considered to indicate the presence of a pathology and / or the change in the activity or severity of the pathology. A certain ratio or deviation of this ratio above or below a threshold value may be attributed to a certain disease or group of diseases, and further testing can resolve the ambiguity. Also, the change in kappa to lambda ratio over time can indicate the change in the severity or progression of disease (e.g., indicating relapse or remission).

[0055] Normal ranges for the ratio of kappa to lambda light chains have been established (e.g., Rajkumar et al. Blood (2005) 106 (3): 812-817). These normal ranges were established using one manufacturer's assay (Freelite™ assay, Binding Site Ltd., Birmingham, UK) and may differ from ranges determined for other FLC assays.

[0056] In one embodiment, abnormal FLC ratio can be defined as kappa chain to lambda chain ratio less than 0.26 or greater than 1.65, for example, less than 0.10 or greater than 5.0.In certain embodiments, increased risk of disease progression (e.g., from precursor state such as MGUS, smoldering myeloma, or sporadic bone plasmacytoma to active myeloma) can be associated with increased deviation from normal kappa:lambda ratio.Similarly, worsening prognosis in certain proliferative conditions (e.g., multiple myeloma or chronic lymphocytic leukemia) can be associated with increased deviation from normal kappa:lambda ratio.

[0057] In some embodiments, the amount of total FLC concentration is measured. In some cases, the total FLC concentration is compared to a standard predefined value to determine whether the total amount of FLC is higher or lower than normal. The normal ranges for kappa and lambda chain concentrations in serum of healthy individuals have been defined as 3.3-19.4 mg / L for kappa chains and 5.7-26.6 mg / L for lambda chains [Katzmann et al. 2002, Clin Chem 48: 1437-1444]. These ranges were defined using the Freelite™ assay (Binding Site, Birmingham, UK) and may differ from ranges identified for other FLC assays. Calibration of currently used FLC assays is fraught with difficulties in standardization. Currently, there is no internationally accepted reference measurement system for sFLC (Tate et al. 2009. The Clinical biochemist. Reviews vol. 30,3: 131-40), and therefore results may vary significantly between different FLC assays (Schieferdecker et al. 2020. Blood cancer journal vol. 10,1 2. 9), and the average overestimation of FLC concentrations by the Freelite assay is estimated to be a 10-fold overestimation (Helden et al. 2019. Hematol Med Oncol 4:1-7). In this case, the actual normal range of kappa and lambda chain concentrations in serum of healthy individuals may be as low as 0.33-1.94 mg / L for kappa chains and 0.57-2.66 mg / L for lambda chains.

[0058] Appropriate cut-off values ​​for FLC concentrations higher than normal FLC concentrations may vary depending on the context of the assay. For example, a broad initial screening assay may use a lower FLC concentration as "normal" and accept a higher rate of false positives, which are discounted after further investigation. Assays used as a factor in deciding on aggressive or invasive testing or treatment may use higher values ​​for "normal". Thus, in certain embodiments, normal FLC concentrations for kappa FLC may be considered to be 0.2-50 mg / L, e.g., 0.2-50 mg / L, 0.33-1.94 mg / L, 2.0-50 mg / L, or 3.3-19.4 mg / L, etc. Significant deviations from the normal range may be considered, for example, higher than the normal range, or 10% higher than the upper limit of the range, or e.g., 20% higher than the upper limit of the range, depending on the clinical context.

[0059] Higher than normal sFLC concentration is associated with significantly increased likelihood of reduced survival.Total FLC concentration can also be monitored over time in subjects, where an increase in total FLC concentration indicates an increase in disease severity (e.g., recurrence), and a decrease in total FLC concentration indicates a decrease in disease severity (e.g., remission).

[0060] It is advantageous to be able to measure both the total sFLC concentration and the κ:λ ratio, because in patients with impaired renal clearance or polyclonal expansion of immunoglobulins, sFLC levels may be elevated but the sFLCκ:λ ratio is normal, whereas in patients with abnormal expansion of either kappa- or lambda-producing plasma cell clones, the sFLCκ:λ ratio is usually abnormal.

[0061] An abnormal free kappa / lambda ratio is associated with an increased risk of progression from precursor conditions such as MGUS, smoldering myeloma, and sporadic bone plasmacytoma to active myeloma, and portends a worse outcome in multiple myeloma and chronic lymphocytic leukemia, and thus the assay of the present invention can be used as an aid in determining the prognosis or appropriate treatment regimen in such conditions.

[0062] Thus, in an additional aspect, the present invention provides a method for distinguishing monoclonal plasma cell dyscrasias (such as monoclonal gammopathy and / or monoclonal myeloma) from renal disorders and / or polyclonal plasma cell dyscrasias, comprising measuring the κFLC:λFLC ratio in patients with elevated total FLC, wherein an abnormal ratio of κFLC:λFLC is indicative of a monoclonal plasma cell dyscrasia.

[0063] Also provided is a method for screening, diagnosing, monitoring, or prognosing a disease in a patient, comprising the use of at least one avian-derived antibody (IgY) for assaying FLC in a mammalian sample and comparing the assay result to at least one predetermined threshold value.

[0064] In some embodiments, the disease is a B-cell related disease. In some cases, the disease is selected from smoldering multiple myeloma, complete gammopathy, light chain myeloma, non-secretory myeloma, monoclonal gammopathy of undetermined significance (MGUS), light chain amyloidosis (AL amyloidosis), Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, follicular center cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, pre-B cell leukemia, and acute lymphocytic leukemia.

[0065] In some embodiments, after diagnosing the subject as having a disorder, the method further comprises administering to the subject a therapeutic agent (eg, a therapeutically effective amount) to treat the disorder.

[0066] In some embodiments, after diagnosing the subject as having a disorder, the method further comprises administering a treatment, such as plasma exchange or stem cell transplantation.

[0067] Also provided is the use of the avian derived antibody (IgY) in an immunoassay for assaying FLC in a mammalian sample.

[0068] There is also provided the use of at least one avian-derived antibody (IgY) in the manufacture of an assay kit for assaying FLC in a mammalian sample.

[0069] Also provided is an assay kit comprising at least one avian-derived antibody (IgY) for use in assaying for FLC in a mammalian sample.

[0070] In some embodiments, the assay kit further comprises at least one FLC reference sample.

[0071] In some embodiments, the IgY in the assay kit is attached to a substrate.

[0072] The assay kit can quantify the amount of λ FLC or κ FLC or the total amount of FLC in a sample.

[0073] Also provided is a method of distinguishing between at least two B cell related disorders in a mammalian subject.

[0074] In a further embodiment, the present invention relates to a method for assaying free light chain (FLC) in a mammalian sample, comprising the use of at least one avian-derived antibody (IgY), in which the FLC monomer:dimer ratio is determined. This determination can be achieved by comparing the method of the present invention with another method that responds differently to FLC monomer and FLC dimer. In particular, since the assay method of the present invention provides results that are relatively insensitive to the monomer:dimer ratio (see the examples below), and certain other known assay methods provide higher sensitivity to certain multimeric forms, comparing the results of two assay methods performed on the same sample may provide information about the multimeric composition of the FLC content of the sample. In this way, the monomer:dimer ratio can be determined by determining the deviation of the results between the assay method of the present invention and another assay method (such as the Freelite assay method or the N-latex assay method). This is particularly true when the other assay is based on mammalian (e.g., sheep, mouse, or rat) antibody binding.

[0075] Thus, in one embodiment, the method of the present invention may comprise additionally performing another FLC assay on the same sample using another assay method and comparing the results of the other assay method with the results of the assay method comprising the use of at least one avian-derived antibody (as described in any embodiment herein). Such another assay method may be, for example, an assay method based on the binding of a mammalian antibody, such as a sheep antibody. Such a mammalian antibody may be an anti-FLC antibody, and may be an anti-kappa FLC antibody or an anti-lambda FLC antibody. Such a mammalian antibody may be a monoclonal antibody (e.g., multiple monoclonal antibodies) or a polyclonal antibody.

[0076] In one embodiment, the other assay method may be a turbidimetric assay, a nephelometric assay, or an electrophoretic assay.

[0077] In one embodiment, both the assay method involving the use of at least one avian-derived antibody (as described in any embodiment herein) and another assay method (e.g., involving the use of a mammalian antibody) utilize the same detection method (such as turbidimetric, nephelometric, or fluorescent).

[0078] The invention will now be described with reference to the following non-limiting examples. EXAMPLES

[0079] Working Example Example 1: Preparation of polyclonal avian antibodies and their affinity purification a) Preparation of avian FLC antibodies For generation of polyclonal IgY antibodies against human FLC, the following immunization protocol can be used. 10-20 chickens were used for each immunization experiment. 10 mg of highly purified immunogen in phosphate buffer was emulsified with Freund's adjuvant and injected intramuscularly or subcutaneously into chickens. Injections were repeated every 2-4 weeks. Eggs were harvested 10-12 weeks after the start of injections. Yolks were isolated from the eggs and the IgY fraction from the yolk was defatted and then conventionally isolated by ammonium sulfate precipitation according to prior art egg antibody isolation methods (reviewed by Larsson A, et.al 1993 Poultry Science 72:1807-1812, 1993). The immunogens in the above examples are selected from purified Bence Jones protein, polyclonal free light chains purified from patients with acute renal failure, purified FLC produced by fractionation of whole molecule immunoglobulins, and recombinant peptides corresponding to the amino acid sequence of the constant region of immunoglobulin light chains.

[0080] b) Affinity purification of polyclonal antibody fractions Absorption process: 10 mg of purified IgG, IgA, and IgM proteins were immobilized on a GE Healthcare HITRAP NHS-activated HP column according to the method described in the column insert. The isolated IgY fraction was diluted to 4 mg / ml with phosphate buffered saline, and 200 to 300 ml of this IgY solution was passed through the column, and the non-adsorbed (flow-through) fraction was collected. This non-adsorbed fraction was then affinity purified against a mixture of the respective FLCs using the following method: Five milligrams of high-purity human free light chain was immobilized onto a GE Healthcare HITRAP NHS-activated HP column according to the method described in the column insert. 100 ml of the non-adsorbed fraction was passed through the column, followed by 25 ml of phosphate buffered saline. Antibodies with specific affinity to the immobilized FLC were eluted from the column with 15 ml of 0.1 molar citrate buffer, pH = 2.9. The eluted specific anti-FLC antibodies were dialyzed against phosphate buffered saline and concentrated to 3-5 mg / ml using Amicon Ultra centrifugal filtration devices with a molecular weight cut-off of 30.000 daltons.

[0081] Example 2: Turbidimetric assay of FLC (a) Preparation of nanoparticles coated with anti-FLC antibody An aliquot of anti-kappa or anti-lambda antibody prepared in Example 1 (e.g., 5 mg of IgY) was dialyzed against MOPS buffer at pH 8.4-8.6, followed by the addition of a 1 ml aliquot of 4% w / v chloromethyl-activated nanoparticles (available from Ikerlat Polymers, Spain or Thermo Fisher Scientific Inc., USA). After addition of nanoparticles to the purified anti-FLC antibody, the mixture is stirred for 24-72 h at 33 °C. An equal volume of glycine blocking buffer (pH 8.6) containing 10 mg / ml bovine serum albumin in borate buffer is added, and the mixture is incubated at 33 °C for 4-18 h. After this incubation, the particles are diluted to a total volume of 10 ml and dialyzed against a volume of 2000 ml of Tris-NaCl buffer R2, pH 8.8, containing 0.1% Tween® 20, 10 mg / ml egg albumin, and preservatives using a Float-A-Lyzer G2 dialysis device with a molecular weight cut-off of 1.000 kDaltons, and then adjusted to the final particle concentration in this storage buffer (R2) using dilution or centrifugal concentration.

[0082] (b) FLC assay using nanoparticles coated with anti-FLC antibodies. Calibrators for the assay were prepared using purified FLC and values ​​were assigned using the OD280 of purified FLC. The calibrator range was 0-1.6-3.1-6.3-12.5-25.0-50.0 mg / L. An assay buffer (R1) was prepared by mixing a soluble polymer such as polyethylene glycol, salts, and a pH buffer and optimized until the turbidimetric signal of the assay matched the Binding Site Freelite assay calibration. An example of this assay buffer is 25 mM TRIS, 180 mM NaCl, 0.1% Tween® 20, and 0.4% PEG 6000, pH 7.4. A latex-enhanced turbidimetric immunoassay was established on a clinical chemistry analyzer (ALCOR, Edif Instruments srl, Via Ardeatina 132-00179 Rome). A typical FLC assay used 3 μl sample, 180 μl R1 (assay buffer) and 60 μl R2 (antibody-coated nanoparticles). The calibration curves are shown in FIG. 1 for the anti-kappa nanoparticles and in FIG. 2 for the anti-lambda nanoparticles.

[0083] Example 3: Examination of the monomeric and dimeric structures of kappa FLC and lambda FLC Figure 3 shows an SDS-PAGE gel of native and reduced kappa FLC antigen. Purified kappa antigen was run on an SDS-PAGE (5-20%) gel. The lane labeled Native contains non-reduced kappa antigen clearly showing monomeric kappa FLC at approximately 24 kDa and dimeric kappa FLC at approximately 48 kDa. The band corresponding to the monomer is more intense than the dimer band. The lane labeled DTT is the same kappa antigen but reduced with 100 mM DTT before running on SDS_PAGE. The lane labeled L-cysteine ​​is the same kappa antigen reduced with 15 mM L-cysteine ​​before running on the SDS-PAGE gel. It is clear that these reducing agents (DTT and L-cysteine) efficiently convert the dimeric kappa FLC to the monomeric form of kappa FLC, as no band corresponding to the MW of the dimer is visible. Figure 6 shows an SDS-PAGE gel of native and reduced lambda FLC antigen. Purified lambda antigen was run on an SDS-PAGE (5-20%) gel. The lane labeled Native contains non-reduced kappa antigen clearly showing monomeric lambda FLC at approximately 24 kDa and dimeric lambda FLC at approximately 48 kDa. The band corresponding to the monomer is barely visible, whereas the dimer band is much stronger. The lane labeled DTT shows the same lambda antigen reduced with 100 mM DTT before running on SDS_PAGE. The lane labeled L-cysteine ​​shows the same lambda antigen reduced with 15 mM L-cysteine ​​before running on an SDS-PAGE gel. It is clear that these reducing agents (DTT and L-cysteine) efficiently convert the dimeric lambda FLC to the monomeric form of lambda FLC, as little or no band corresponding to the MW of the dimer is visible.

[0084] Example 4: Effect of polymer structure of FLC in turbidimetric assay (a) Conversion of light chain dimers to monomers Kappa FLC is thought to occur primarily as a monomer, whereas lambda FLC occurs primarily as a dimer. Non-reducing SDS-PAGE gels confirmed that, in the native state, the kappa antigen used for assay calibration consists primarily of monomers (Fig. 3), whereas the lambda antigen consists primarily of dimers (Fig. 6). Reducing agents such as glutathione, L-cysteine, tris(2-carboxyethyl)phosphine, B-mercaptoethanol, dithiothreitol (DTT), and dithioerythritol (DTE) reduce disulfide bonds between molecules, such as polymeric light chains. The efficiency of reduction of kappa dimers to kappa monomers by DTT and L-cysteine ​​is shown in Figure 3. The efficiency of reduction of lambda dimers to lambda monomers by DTT and L-cysteine ​​is shown in Figure 6.

[0085] (b) Effect of polymer structure of FLC on chicken antibodies based on turbidimetric assay of FLC Two separate calibrator series, kappa and lambda, were prepared as described above. To prepare the monomeric calibrator range, the first calibrator set was incubated with 15 mM L-cysteine ​​in assay buffer. The second set of calibrators (native calibrators containing dimeric and monomeric forms of FLC) was incubated in assay buffer without L-cysteine, followed by standard curve generation in an immunoturbidimetric assay. The ALCOR clinical chemistry assay was used as described in Example 2. The assay calibration curves for the native kappa FLC calibrators and the monomeric kappa FLC calibrators are shown in Figure 4. The assay calibration curves for the native lambda FLC calibrators and the monomeric lambda FLC calibrators are shown in Figure 7.

[0086] (c) Effect of polymer structure of FLC in Freelite Kappa Kit and Freelite Lambda Kit The Freelite Human Kappa Free kit (product code: LK016.CB) and the Freelite Human Lambda Free kit (product code: LK018.CB), both from The Binding Site, Birmingham, B15 1QT, UK, were adapted to run on an ALCOR clinical chemistry analyzer. One skilled in the art can optimize the parameter settings for these assays on any suitable automated clinical chemistry analyzer. Two separate calibrator series, kappa and lambda, were prepared as described above. To prepare the monomeric calibrator range, the first calibrator set was incubated with 15 mM L-cysteine ​​in assay buffer. The second set of calibrators (native calibrators containing dimeric and monomeric forms of FLC) was incubated in assay buffer without L-cysteine ​​before generating a standard curve in the Freelite assay. The assay calibration curves for the native kappa FLC calibrators and monomeric kappa FLC calibrators are shown in Figure 5. The assay calibration curves for the native lambda FLC calibrators and monomeric lambda FLC calibrators are shown in Figure 8.

Claims

1. A method for assaying free light chains (FLC) in a mammalian sample comprising the use of at least one avian-derived antibody (IgY).

2. The method of claim 1, wherein the IgY exhibits affinity for the constant domain of at least one mammalian immunoglobulin light chain.

3. The method of claim 1 , wherein the IgY is labeled.

4. 4. The method of claim 3, wherein the IgY is labeled by fluorescence, luminescence, radioactivity, isotope labeling, or by binding to an enzyme, particle, or substrate.

5. The method of claim 1 , wherein the IgY is immobilized on a particle or surface.

6. 6. The method of claim 5, comprising the use of a biosensor comprising immobilized IgY as a binder.

7. 10. The method of claim 1, wherein the assay is a nephelometric assay, a turbidimetric assay, a flow cytometry assay, a lateral flow assay, an immunofixation electrophoresis (IFE) assay, or an enzyme-linked immunosorbent assay (ELISA) assay.

8. The method of claim 1 , wherein the assay is a turbidimetric assay.

9. 2. The method of claim 1, wherein the IgY is coated onto nanoparticles having an average diameter of at least 40 nm, for example, 50-160 nm.

10. The method of claim 1 , wherein the assay is an ELISA assay.

11. The method of claim 1, wherein the IgY specifically binds (or is capable of specifically binding) to λ (lambda) FLC or κ (kappa) FLC.

12. 2. The method of claim 1, wherein the FLC is a λ (lambda) FLC, a κ (kappa) FLC, or a total FLC.

13. 2. The method of claim 1, comprising the use of at least two IgYs with different specificities, one IgY having a higher affinity for lambda FLC and another IgY having a higher affinity for kappa FLC.

14. 2. The method of claim 1, comprising the use of at least two IgYs with different specificities, one IgY having a higher affinity for either lambda FLC or kappa FLC, and the other IgY having substantially the same affinity for lambda FLC and kappa FLC.

15. 2. The method of claim 1, wherein the ratio of κ FLC to λ FLC (FLCκ:λ) in the sample is determined.

16. 16. The method of claim 15, wherein the FLCκ:λ ratio is compared to a normal range for FLCκ:λ and a significant deviation (e.g., >10% or >5%) from the normal range is calculated, wherein increasing deviation from the normal range is associated with an increased chance of disease progression and / or a worsening prognosis.

17. The method of claim 1, wherein the IgY has substantially equal reactivity with the monomeric and dimeric forms of FLC.

18. The method of claim 1, wherein the IgY has substantially equal reactivity to all multimeric forms of FLC.

19. 2. The method of claim 1, wherein the mammalian sample is a sample of blood, serum, plasma, saliva, urine, or cerebrospinal fluid, or other biological fluid, preferably serum, plasma, or urine.

20. 20. A method for screening, diagnosing, monitoring or prognosis of a disease in a patient, the method comprising carrying out an assay method according to any one of claims 1 to 19 and comparing the result of the assay with at least one predetermined threshold and / or calculating the deviation of the result of the assay from a normal range.

21. 21. The method of claim 20, wherein the disease is a B-cell related disease.

22. 22. The method of claim 21, wherein the disease is selected from smoldering multiple myeloma, complete immunoglobulin myeloma, light chain myeloma, non-secretory myeloma, monoclonal gammopathy of undetermined significance (MGUS), light chain amyloidosis (AL amyloidosis), Waldenstrom's macroglobulinemia, Hodgkin's lymphoma, follicular center cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, pre-B cell leukemia, and acute lymphocytic leukemia.

23. Use of avian-derived antibodies (IgY) in immunoassays to assay FLC in mammalian samples.

24. The use of claim 23 in a method according to any one of claims 1 to 19.

25. Use of at least one avian-derived antibody (IgY) in the manufacture of an assay kit for assaying FLC in a mammalian sample.

26. 20. An assay kit for use in the method of any one of claims 1 to 19.

27. An assay kit comprising at least one avian-derived antibody for use in assaying FLC in a mammalian sample.

28. 28. The assay kit of claim 27, further comprising at least one FLC reference sample.

29. 29. The assay kit of claim 27 or claim 28, wherein the IgY is attached to a substrate.

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