KAPPA LIGHT CHAIN CONSTANT (ckLC) MOUSE HYBRIDOMA, CLONE KC-31, PRODUCING MONOCLONAL ANTIBODIES TO CONSTANT REGION OF HUMAN KAPPA LIGHT CHAINS FOR DIAGNOSIS OF AL KAPPA AMYLOIDOSIS

Abstract

FIELD: biotechnology.SUBSTANCE: strain of the kappa light chain constant (kLC) hybridoma cell line, clone KC-31, deposited in the National Bioresource Center – Russian National Collection of Industrial Microorganisms (BRC VKPM) of the Kurchatov Institute Research Center under the registration number VKPM: H-243 dated September 9, 2025, to a producer of mouse monoclonal antibodies to the constant region of the human kappa light chain, suitable for the detection of AL kappa amyloidosis.EFFECT: development of a stable cell line producing highly specific monoclonal antibodies to a specific region of the constant region of the human kappa light chain, suitable for use in enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunofluorescence and Western blotting.1 cl, 5 dwg, 5 ex

Description

[0001] The invention relates to biotechnology and medicine, in particular to hybridoma technology, according to International Patent Classification C12N 5 / 12, and concerns a new stable cell line (strain) - a murine hybridoma kappa light chain constant (ckLC), clone KC-31, producing monoclonal antibodies to the constant region of the kappa light chains of human immunoglobulins. The resulting hybridoma is intended for the production of monoclonal antibodies applicable in immunohistochemistry, immunofluorescence, enzyme-linked immunosorbent assay, and Western blotting for the detection of kappa light chains, particularly for the diagnosis of AL kappa amyloidosis. The invention can find application both in research and in the biotechnological production of diagnostic reagents.

[0002] The hybrid cell line strain, clone KS-31, is deposited in the All-Russian Collection of Industrial Microorganisms and has the registration number VKPM: H-243 dated September 9, 2025.

[0003] AL amyloidosis (immunoglobulin light chain amyloidosis) is a disease belonging to the group of dysproteinoses characterized by the extracellular deposition of amyloid fibrils in tissues and organs, formed from abnormal immunoglobulin light chains. The pathogenetic basis of the disease is the clonal proliferation of bone marrow plasma cells secreting monoclonal immunoglobulin light chains (LC) or their fragments. Unlike multiple myeloma, the tumor clone is often small in size; however, the unique structural and functional properties of the LC produced determine their exceptional amyloidogenicity.

[0004] The key steps of amyloidogenesis in AL amyloidosis are: secretion of the precursor protein (a plasma cell produces a monoclonal light chain (kappa or lambda type), which acts as an amyloid precursor protein); proteolytic processing or proteolysis (in the systemic circulation or in the target extracellular matrix, whole-genome LC (~25 kDa) undergo limited proteolysis by macrophage or tissue proteases. This leads to cleavage of part of the variable (V) and / or constant (C) domains with the formation of fragments, mainly represented by the N-terminal regions of the V domain and fragments of the C domain); nucleation and polymerization (the resulting proteolytic fragments, which have an increased tendency toward β-sheet conformation, undergo partial unfolding.This exposes hydrophobic regions, which, under conditions of physicochemical stress, serve as the basis for irreversible aggregation, subsequently leading to the formation of insoluble fibrils rich in cross-β-folded structure; the formation of amyloid deposits (mature fibrils are deposited in the extracellular space of various organs (heart, kidneys, liver, nerves), mechanically and toxically disrupting their architecture and function) [1, 2].

[0005] Verification of AL amyloidosis and accurate typing of the amyloid protein (kappa vs. lambda) are critical for determining the patient's treatment strategy aimed at eradicating the plasma cell clone. Immunohistochemistry (IHC) using monoclonal and polyclonal antibodies to immunoglobulin light chains remains the gold standard for pathological diagnosis. However, the use of existing commercial antibodies to kappa and lambda light chains is associated with significant methodological problems and drawbacks, leading to false-negative and false-positive results. Antibodies to the constant domains and to the kappa light chain in general often demonstrate cross-linking with light chains present in circulating immunoglobulins, as well as with immunoglobulins fixed in the tissue stroma. This causes excessive background staining and makes typing of amyloidosis difficult [4].A large number of antibodies to the kappa light chain are commercially available, but they are often not tested immunohistochemically on tissue samples containing amyloid. Therefore, the use of antibodies that recognize a specific epitope of the kappa chain constant region, which is most frequently found among amyloidogenic kappa chain sequences, will reduce key limitations of immunohistochemical typing.

[0006] Previously, the inventors obtained rabbit polyclonal antibodies directed against a synthetic peptide corresponding to the amino acid sequence of the 116-130 region of the constant domain of the kappa light chain of immunoglobulin. It was found that this sequence occurs in all variants of the analyzed 112 amino acid sequences identified in patients with AL kappa amyloidosis. Immunization of rabbits with this peptide allowed us to obtain polyclonal antibodies AK3 and AK4, which demonstrated the ability to recognize both the peptide itself (by the dot blot method) and the native protein in an immunohistochemical study in 96-100% of cases [5]. Despite the high performance of polyclonal antibodies, the main problem with the use of polyclonal antibodies is the variability between batches from different animals, possible admixture of antibodies to third-party epitopes, which complicates the standardization of the production of the obtained antibodies.

[0007] The invention is illustrated by the following graphs and photographs.

[0008] Graph 1. Results of enzyme immunoassay of antibodies to the constant region of the kappa light chain, synthesized by the KC-31 hybridoma, from Example 2. The Y axis of the graph shows the optical densities obtained by solid-phase ELISA on the antigens: peptide of the constant region of kappa (116-130) conjugated with BSA (p116-130-BSA), peptide of the variable region of kappa (3-17) conjugated with BSA (p17-BSA), a mixture of immunoglobulins (the drug "Gammanorm"), free kappa chain isolated from the urine of a myeloma patient (kappa), bovine serum albumin (BSA) and isolated lambda AL-amyloid (AL-15) obtained according to the Westermark method [7]. The X axis shows the dilutions of the KC-31 supernatant. The antibodies bind to the kappa constant region peptide (ckLC) and weakly to the kappa free chain. No cross-reaction was detected with the kappa variable region, IgG, BSA, or AL-15. The working dilution of antibodies in the ELISA (titer) is 1:128, as indicated by the arrow on the graph.

[0009] Fig. 1. Illustration of Example 3. Immunohistochemical staining of the kidney of a patient with AL kappa amyloidosis with antibodies to the constant region of kappa light chains produced by hybridoma clone KC-31. A - Deposition of homogeneous acellular congophilic amyloid masses in the glomeruli, tubules and vessels of the kidney. B - Characteristic apple-green fluorescence of amyloid structures when examined under polarized light. C - Positive immunohistochemical staining of amyloid (brown color) with antibodies produced by hybridoma KC-31. Antibody from the supernatant, dilution 1:40.

[0010] Fig. 2. Illustration of immunohistochemical staining with antibodies to the KC-31 constant region, obtained using the KC-31 hybridoma supernatant. The absence of positive staining in amyloid with ATTR indicates the high specificity of the antibodies.

[0011] Assessment of the specificity of an antibody to the constant region of KC-31 on samples containing ATTR amyloid. A - Immunopositive staining of amyloid structures in the myocardium with a commercial antibody to transthyretin amyloid (anti-Prealbumin antibody, Abcam). B - Immunonegative staining of amyloid deposits with antibodies to the constant region of KC-31 in the myocardium of a patient with transthyretin amyloidosis. X100.

[0012] Fig. 3. Comparison of the results of immunohistochemical staining from Example 3 with antibodies produced by the KC-31 hybridoma and commercial antibodies to kappa light chain.

[0013] Specimen - Stomach of a patient with AL kappa amyloidosis (the amyloid type was determined by mass spectrometry). A - Massive acellular amyloid masses in the stomach. B - Characteristic apple-green fluorescence of amyloid structures when examined under polarized light. C - Positive immunohistochemical staining of amyloid (brown color) with antibodies produced by the KC-31 hybridoma. C - Negative immunohistochemical staining of amyloid with Rabbit anti-Kappa light chain antibody, clone SP148 (Abcam, USA).

[0014] Fig. 4. Illustration of immunofluorescence staining from Example 4. A - Positive (green) immunofluorescence of amyloid structures in the glomerulus of the kidney of a patient with AL kappa amyloidosis. B - Positive fluorescence of amyloid in the wall of the myocardial vessels of a patient with AL kappa amyloidosis.

[0015] Fig. 5. Western blot results using KC-31 antibodies produced by the kappa light chain constant (ckLC) hybridoma to the constant region of the kappa chain from Example 5. The working dilution of the antibodies was 1:30. The following were used as samples applied to a 12% gel under reducing conditions: the kappa variable region peptide (3-17) conjugated with BSA (p17-BSA), the kappa constant region peptide (116-130) conjugated with BSA (p116-130-BSA), a mixture of immunoglobulins (the drug "Gammanorm"), the free kappa chain isolated from the urine of a myeloma patient (kappa), and isolated lambda AL amyloid (AL15) obtained according to the Westermark method [7].As a result, antibodies to the constant region of human kappa light chains, clone KC-31, bind to the peptide of the constant region of kappa (p116-130), to the free chain of kappa, and very weakly to kappa in the composition of immunoglobulins (IgG) - the bands of binding of antibodies to antigens are indicated by arrows, but do not bind to the peptide of the variable region of kappa (p17) and lambda amyloid proteins (AL15).

[0016] The objective of the present invention was to develop a stable cell line producing highly specific monoclonal antibodies to a specific region of the constant region of the human kappa light chain, suitable for use in enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunofluorescence and Western blotting.

[0017] The problem is solved using the mouse hybridoma kappa light chain constant (ckLC), clone KC-31, which produces monoclonal antibodies to the constant region of human kappa light chains.

[0018] The technical result was achieved by obtaining a stable hybridoma line, deposited in the All-Russian Collection of Medical Products under number H-243 dated September 9, 2025, producing monoclonal antibodies to the most frequently occurring amino acid sequence in positions 116-130 of the constant domain of the kappa light chain, working in the methods of enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence and Western blotting, obtained by the method of hybridization of sensitized mouse lymphocytes with the myeloma line SP 2 / 0-Ag14.

[0019] The KC-31 hybridoma cell line produces antibodies to the constant region of the kappa light chain. Antibodies produced by the hybridoma bind to the constant region within amyloid fibrils and within the free kappa chain. To avoid false-negative staining during AL amyloidosis typing, antibodies produced by the KC-31 hybridoma can be used in combination with antibodies to the most common sequence within the kappa variable domain in AL kappa amyloidosis. Each hybridoma line producing monoclonal antibodies is unique because, as a descendant of a single B cell, it inherits a unique variable region of the antibody resulting from genetic recombination, hypermutations, and random nucleotide insertions. It also has a specific structure of variable domains and produces antibodies with unique specificity and affinity.

[0020] Steps of obtaining mouse hybridoma KS-31:

[0021] A synthetic peptide, a part of the conserved region of the constant domain of the human kappa light chain, corresponding to amino acid positions 116-130 - NH2-CFIFPPSDEQLKSGTA-CONH2, hereinafter referred to as "peptide 116-130" or "p116-130", was used as an antigen. The sequence was previously determined after analysis of 112 amino acid sequences of patients with AL-kappa amyloidosis, which showed that the constant region is the same in all samples studied [5].

[0022] Peptide 116-130 was conjugated to bovine serum albumin (BSA) using sulfosuccinimidyl 4-(N-maleimidophenyl) butyrate (Sulfo-SMPB, Thermo Scientific, USA), followed by gel filtration on G-25 sorbent.

[0023] Balb / c animals were immunized with a peptide antigen at a dose of approximately 50 μg per animal. The first immunization was administered as an emulsion with complete Freund's adjuvant, followed by incomplete adjuvant, with intervals between injections of approximately twenty days. Ten to fourteen days after the second immunization, specific antibody levels were determined using ELISA, and animals with elevated titers were given a booster dose of the antigen.

[0024] Approximately 72 hours after booster immunization, lymphocytes were isolated from the spleen and regional lymph nodes and fused with the SP2 / 0-Ag14 myeloma cell line based on the recommendations [6] but according to the accepted laboratory practice. Cell fusion was performed using a polyethylene glycol solution (50%, PEG 3000) prepared in RPMI-1640 medium with the addition of 10% dimethyl sulfoxide. The resulting cell suspension after fusion was distributed into the wells of a 96-well plate in RPMI-1640 medium containing 15% fetal bovine serum, and then incubated under standard conditions in a CO2 incubator.

[0025] Hybrid cell selection was performed by adding a selective mixture of HAT (hypoxanthine, aminopterin, thymidine, PanEco) to the medium. As the cultures grew, some of the medium was replaced with a medium containing only hypoxanthine and thymidine (HT, PanEco), and once stable growth was achieved (approximately by days 20-22), some of the medium was replaced with standard medium. Clonal formation was monitored microscopically.

[0026] Primary screening of supernatants of developing clones (culture fluid) using solid-phase ELISA was performed upon the appearance of visible colonies. The culture fluid was added to the wells of plates containing the immobilized antigen (p116-130-BSA). Antibody binding to the antigen was detected using an anti-mouse antibody conjugate with horseradish peroxidase (HRP) and tetramethylbenzidine (TMB), followed by stopping the reaction with sulfuric acid. Clones exhibiting specific binding were transferred to larger plates and further tested for the primary antigen (p116-130-BSA) and the BSA-conjugated peptide of the κ chain variable region to exclude cross-linking with either the variable region or the BSA carrier protein. The specificity of antibodies from selected clones was then tested by immunohistochemistry on sections with AL kappa amyloidosis samples.

[0027] Stable monoclonal hybridomas were obtained by two-fold cloning using limiting dilutions to one cell per well. At all stages of clone selection, supernatants were tested by ELISA and IHC to select clones with high productivity, growth rate, and antibody affinity. The selected lines were scaled up in culture flasks, then cryopreserved in the presence of fetal bovine serum and dimethyl sulfoxide and stored long-term in liquid nitrogen. Culture media for analysis were collected during the active growth phase of the hybridomas.

[0028] As a result, clone KC-31 was selected, producing monoclonal antibodies to the kappa constant region, applicable in ELISA, IHC, immunofluorescence and Western blotting (WB) methods.

[0029] Hybridoma KC-31 has the following characteristics.

[0030] Morphological features: the culture has the appearance of a suspension.

[0031] Cultivation conditions: 37°C, absolute humidity, 5% CO2 in RPMI-1640 medium with 15% fetal bovine serum (FBS), 2 mM L-glutamine, and 10 μg / ml gentamicin. For routine cultivation to produce antibodies, RPMI-1640 medium with FBS reduced to 3.75% is used. Freezing is carried out in 90% fetal bovine serum with 10% DMSO. Bacteria, fungi, yeast, and mycoplasma were not detected in the culture.

[0032] Isotype of monoclonal antibodies to the constant region of human kappa light chains (ckLC), clone KC-31, obtained from hybridoma KC-31: IgG2a. Activity (productivity) of the strain: when the cells reach the linear growth phase in the medium, the cell productivity is 6 μg / ml of antibodies. When testing the supernatant of the kappa light chain constant (ckLC) hybridoma, clone KC-31, the working dilution is 1:30-1:50, and by the immunofluorescence analysis 1:40. Antibody titer in ELISA is 1:128, in Western blotting 1:30.

[0033] Antibody hybridoma strain KC-31 is a stable cell line suitable for industrial-scale biotechnological production of antibodies, allowing the production of reproducible series of antibodies with high specificity.

[0034] The specificity of monoclonal antibodies of the IgG2a isotype obtained using the KC-31 hybridoma was tested by the following methods: enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunofluorescence and Western blotting (WB).

[0035] In ELISA it was shown that antibodies to the constant region of kappa of human light chains, clone KC-31, bind to the peptide of the constant region of kappa (116-130) - p116-130, but do not bind to the peptide of the variable region of kappa, weakly bind to the free chain of kappa isolated from the urine of a myeloma patient, do not bind to amyloid isolated from the liver of a patient with lambda AL amyloidosis (AL-15) and a mixture of IgG immunoglobulins (the drug "Gammanorm"). Western blotting (WB) showed that KC-31 antibodies react with the p116-130 peptide and the kappa free chain, but, in contrast to the ELISA results, weakly react with a mixture of immunoglobulins (the drug "Gammanorm"), but do not react with lambda amyloid proteins isolated from the liver of a patient with lambda AL amyloidosis (AL-15) Example 2, Graph 1.

[0036] The specificity and sensitivity of the KC-31 antibody clone, obtained by culturing the presented hybridoma using immunohistochemistry, were confirmed on 43 samples with different types of amyloidosis, and on an additional 10 samples without amyloid. Antibody validation was conducted in comparison with commercial antibodies to kappa light chains (Rabbit anti-Kappa light chain antibody, clone SP148 (Abcam, USA)). Validation results showed that KC-31 antibodies are more specific than commercial ones: in two samples containing AL kappa amyloid (the authenticity of the amyloid type was confirmed by mass spectrometry), the commercial antibody showed a false-negative reaction, while the developed KC-31 demonstrated positive immunostaining.

[0037] The invention is illustrated by the following examples.

[0038] Example 1. In vitro cultivation of the strain

[0039] KC-31 hybridoma clone cells are cultured in T-75 culture flasks without treatment (SPL, PanEco) in RPMI-1640 nutrient medium with 3.75-15% fetal bovine serum, 2 mM glutamine and 10 μg / ml gentamicin antibiotic at 37°C and 5% CO2. When seeding, the optimal cell density in a medium with 10% FBS is 0.3 * 10 6 cells / ml, after the cells reach a density above 10 6 cells / ml, serum-free medium is added to the cells, after which the hybridomas are cultured for another day.

[0040] The supernatant (culture fluid) is collected, filtered through a 0.22 µm filter, and supplemented with 10x Tris buffer solution (pH 7.4) and 0.09% sodium azide. The antibodies are aliquoted and frozen for long-term storage. If used within a month, they are stored at 2-8°C without loss of properties. The antibody titer upon binding to the antigen (p116-130, indicated by the arrow in Figure 1) is 1:128. Antibodies can be isolated from the cell fluid by affinity purification using a protein A sorbent.

[0041] Example 2. Conducting an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies synthesized by the KC-31 hybridoma

[0042] For solid-phase ELISA (Figure 1), antigens are individually adsorbed onto the wells of a 96-well plate at a concentration of 5 μg / ml using a carbonate-bicarbonate buffer with a pH of 9. 100 μl of the antigen solution is added to each well, and the plate is incubated overnight at 4°C. After incubation, the wells are washed three times with phosphate-buffered saline (PBS). To block non-specific binding, 100 μl of a 1% BSA solution in PBS is added to the wells and the plate is incubated for 1 hour at room temperature. After blocking, the plate is washed three times with PBS. Culture fluid containing KC-31 antibodies is applied to the immobilized antigens undiluted and in the form of successive serial dilutions. Incubation is carried out at room temperature for 1 hour on an orbital shaker. The sample is then washed four times with PBS-T (PBS containing 0.05% Tween-20). A conjugate of goat anti-mouse antibodies labeled with horseradish peroxidase (HRP) diluted in PBS-T is used for detection.The conjugate is added to the wells and incubated for 40 minutes at room temperature with gentle shaking. After incubation, the wells are washed six times with PBS-T and residual liquid is removed. To visualize antibody binding to antigens, 100 µl of a substrate-buffer solution containing TMB and hydrogen peroxide is added to each well, the wells are incubated for 10 minutes, and then the reaction is stopped by adding 50 µl of 20% sulfuric acid. Optical density is measured at a wavelength of 450 nm.

[0043] The following antigens were used: peptide of the variable region of the κ-chain (3-17) conjugated with BSA (p17-BSA); peptide of the constant region of the κ-chain (116-130) conjugated with BSA (p116-130-BSA); a mixture of immunoglobulins IgG (Gammanorm); free kappa chain isolated from the urine of a patient with myeloma; bovine serum albumin (negative control); lambda AL amyloid (AL-15) isolated according to the Westermark method [7].

[0044] According to the obtained data (Graph 1), the antibodies exhibit specific binding to the peptide of the constant region of the kappa chain (116-130) and to the free kappa chain (binding is present, but weaker). Reactions with other antigens are not observed.

[0045] Example 3. Conducting an immunohistochemical (IHC) study using monoclonal antibodies synthesized by hybridoma KC-31

[0046] Biopsy and autopsy specimens with AL kappa amyloidosis, as well as specimens with and without other types of amyloidosis, are used for immunohistochemical examination. They are fixed in 10% buffered formalin for no more than 48 hours. After histological processing, the specimens are embedded in paraffin as paraffin blocks, from which 3-4 µm-thick sections are cut. The sections are deparaffinized and rehydrated. Sections from each specimen are stained with hematoxylin and eosin and Congo red to confirm amyloidosis. To obtain immunohistochemical staining, thermal antigen retrieval is performed in Tris-EDTA buffer, pH 9, at 98°C for 20 minutes with preheating and subsequent cooling of the specimens for 20 minutes in the buffer. Endogenous peroxidase is blocked with a solution for blocking endogenous peroxidase. Incubation with antibodies in the form of supernatant (culture fluid) at a dilution of 1:40 takes 1 hour.A detection system is based on an enzymatic reaction with a polymer conjugate of horseradish peroxidase (poly-HRP) and DAB, followed by nuclei counterstaining with hematoxylin. Stained sections on glass slides are run through a series of alcohols and xylene and mounted under a coverslip using a xylene-based mounting medium. Positive staining is defined as the appearance of brown coloration on the section at the sites of antibody binding to the antigen being tested.

[0047] Forty-three tissue samples from patients with different types of amyloidosis were taken for immunohistochemical examination: 10 with AA amyloidosis, 9 with transthyretin amyloidosis (ATTR), 12 with AL lambda, 12 with AL kappa amyloidosis, and 10 blocks without amyloidosis (3 kidney samples, 4 myocardium, 3 stomach). Sections were stained with antibodies to the constant region of the kappa light chain obtained from the hybridoma kappa light chain constant (ckLC), clone KC-31 as a supernatant at a dilution of 1:40 and commercial antibodies to kappa light chains (Anti-Kappa light chain antibody [SP148] ab227654 (Abcam). It was shown that the KC-31 antibody reacted specifically with all samples containing AL kappa amyloid (Fig. 1) and showed no staining (as expected) for all cases not containing kappa amyloidosis - with AA and ATTR (Fig. 2).Compared with commercial antibodies, KC-31 reacted more specifically in some cases (1 stomach sample and 1 myocardium sample), while the commercial antibody showed a false negative reaction (Fig. 3).

[0048] Example 4. Conducting an immunofluorescence study using monoclonal antibodies synthesized by hybridoma KC-31

[0049] Biopsy and autopsy specimens with amyloidosis were used for immunofluorescence analysis. Sections of 3-4 μm thickness were used for the study, as were those used for immunohistochemistry. After deparaffinization and rehydration, thermal antigen retrieval was performed in Tris-EDTA buffer, pH 9, at 98°C for 20 minutes with preheating and subsequent cooling of the samples for 20 minutes in the buffer. Sections were coated with 150 μl of KC-31 supernatant antibodies at a 1:40 dilution and incubated for 1 hour in the dark. After washing, secondary anti-mouse antibodies conjugated to the fluorescent dye Alexa488 were used for detection. Antibodies were applied at 150 µl per section and incubated for an hour in the dark, then washed with wash buffer. To visualize nuclei, the intercalating dye DAPI was applied to the section and incubated for 10-15 minutes in the dark. Residual unbound reagents were then washed away with wash buffer.The slides were mounted under a coverslip in a water-based mounting medium for fluorescent slides. The slides were examined under a microscope with a reflected-light fluorescence module.

[0050] Staining of preparations containing AL kappa amyloidosis revealed visible specific fluorescence of amyloid structures in renal glomeruli and myocardial vessels. The KC-31 antibody was shown to retain specificity when used as a reagent for the immunofluorescence reaction (Fig. 4).

[0051] Example 5. Application of antibodies obtained using KC-31 hybridoma in Western blotting

[0052] Prior to electrophoretic separation, protein samples are incubated for 5 min at 95°C in Laemmli loading buffer containing 10% sodium dodecyl sulfate and 500 mM beta-mercaptoethanol to denature them. Up to 5 μg of protein (the amount is shown in Fig. 5) are loaded into the wells of a 12% polyacrylamide gel. Electrophoresis is carried out at a constant current of 30 W for each gel until the color band appears. Proteins are transferred to a 0.22 nm nitrocellulose membrane at a constant current of 300 mA for 30 min. The membrane is blocked with 1% BSA in PBS-T for 40 min and then incubated with KC-31 antibodies in the supernatant diluted 1:30 in 1% BSA on an orbital shaker overnight at 2-8°C. The membrane is then washed three times with PBS-T buffer and then incubated with secondary anti-mouse antibodies conjugated to HRP for 45 min. The membrane is washed three times with PBS-T for 7-10 min each. Antibody binding sites are visualized using the chromogen DAB.At the sites of antibody binding, brown stripes, clearly visible to the human eye, are formed on the membrane.

[0053] The following antigens were used to test the KC-31 antibody: the kappa variable region peptide (3-17) conjugated with BSA (p17-BSA), the kappa constant region peptide (116-130) conjugated with BSA (p116-130-BSA), a mixture of human IgG immunoglobulins (the drug "Gammanorm") in two concentrations, the free kappa chain isolated from the urine of a myeloma patient (kappa) and the lambda AL amyloid proteins (AL15) isolated according to the Westermark method [7]. The results are shown in Fig. 5. The antibodies bind well to the kappa constant region peptide and the free kappa chain. In contrast to the ELISA data, they bind weakly to IgG, which may indicate the presence of such an amino acid sequence in the composition of the drug.

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