Free light chain, quality control and uses thereof

Abstract

The application relates to a free light chain, a quality control product and application thereof, the free light chain comprising one or both of a free kappa light chain and a free lambda light chain; wherein the free kappa light chain has an amino acid sequence as shown in SEQ ID NO. 1 or an amino acid sequence with at least 90 percent identity thereof; and the free lambda light chain has an amino acid sequence as shown in SEQ ID NO. 2 or an amino acid sequence with at least 90 percent identity thereof. The quality control product prepared by using the free light chain of the application can be used for quality control of various detection methods such as immunofixation electrophoresis, immunoturbidimetry and mass spectrometry detection, so as to guarantee the accuracy of clinical related disease diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of free light chain detection, in particular to a free light chain, a quality control product and uses thereof. BACKGROUND

[0002] Free light chain (sFLC) refers to the monomer or dimer form of immunoglobulin light chain existing in a sample, which is not combined with heavy chain to form a complete immunoglobulin molecule, and is divided into two types of κ chain and λ chain.

[0003] During the synthesis and secretion of immunoglobulin by plasma cells, a small amount of light chain fails to combine with heavy chain, and these uncombined light chains are released to the outside of the cell in a free form, enter the blood circulation, become free light chains, including free κ light chain (κ-FLC) and free λ light chain (λ-FLC). In addition, in some pathological states, such as plasma cell abnormal proliferation diseases, plasma cells will synthesize and secrete a large amount of monoclonal light chains, resulting in a significant increase in the level of free light chains in serum. Once the processing capacity of the kidney is exceeded, free light chains will appear in urine, and the dimer form is also called Benchoi protein, which is a type of overflow proteinuria.

[0004] Free light chain (sFLC) detection has a variety of applications in clinical practice, mainly including:

[0005] 1. Diagnosis and monitoring of multiple myeloma (MM):

[0006] Diagnostic indicators: Under normal circumstances, the κ and λ light chains in the human body are in a relatively stable ratio. In multiple myeloma patients, due to the monoclonal proliferation of plasma cells, a large amount of free light chains of a single type will be produced, resulting in abnormal κ / λ ratio. When the κ / λ ratio in serum exceeds the normal range (0.26-1.65), accompanied by other related symptoms and abnormal examination, it is highly suggestive of multiple myeloma.

[0007] Disease monitoring: During treatment, the level of serum free light chain can be detected regularly to directly understand the activity of myeloma cells. If the treatment is effective, the level of serum free light chain will gradually decrease, and the κ / λ ratio will tend to be normal; otherwise, if the level continues to rise or remains high, it may mean that the disease has relapsed or progressed.

[0008] 2. Diagnosis and evaluation of light chain amyloidosis (AL)

[0009] Diagnostic value: In patients with light chain amyloidosis, monoclonal free light chains will misfold and deposit in tissues and organs, leading to organ dysfunction. Serum free light chain detection is important for the early detection of asymptomatic light chain amyloidosis. Even if the patient has not yet developed obvious clinical symptoms, if the serum free light chain level is abnormally elevated and the κ / λ ratio is imbalanced, combined with relevant imaging examination and tissue biopsy, it can help early diagnosis.

[0010] Prognosis evaluation: The level of serum free light chain is closely related to the prognosis of patients with light chain amyloidosis. High levels of serum free light chains often indicate more severe disease, more severe organ involvement, and relatively poor prognosis for patients. For example, patients with serum free light chain levels above a certain threshold have significantly lower survival rates than those with lower levels.

[0011] 3. Auxiliary diagnosis of other plasma cell diseases

[0012] In diseases such as monoclonal gammopathy of unknown significance (MGUS) and smoldering multiple myeloma (SMM), serum free light chain detection also has important auxiliary diagnostic value. Although these diseases have no obvious symptoms in the early stages of onset, abnormal changes in serum free light chains can serve as an important indicator for early detection and monitoring of disease progression.

[0013] 4. Diagnosis and differential diagnosis of kidney diseases

[0014] Glomerular disease: In some primary glomerular diseases such as minimal change nephropathy, membranous nephropathy, etc., and secondary glomerular diseases such as diabetic nephropathy, lupus nephritis, etc., due to damage to the glomerular filtration membrane, serum free light chains may enter the urine through the damaged filtration membrane, leading to an increase in urinary free light chains. By detecting serum and urinary free light chains, combined with other renal function indicators and pathological examination results, it can help to make a definitive diagnosis and determine the severity of the disease.

[0015] Tubular disease: In renal tubular disease, the reabsorption dysfunction of the renal tubule to the filtered light chain also leads to increased urinary excretion of free light chains. At this time, the serum free light chain level may be normal or slightly elevated, but urinary light chain detection will be significantly abnormal. Through the detection of serum and urinary free light chains and ratio analysis, it can help to differentiate from glomerular disease.

[0016] 5. Monitoring immune status after organ transplantation

[0017] Rejection monitoring: After organ transplantation, the body's immune system may produce an immune rejection reaction to the transplanted organ. Changes in serum free light chain levels may be associated with the occurrence of immune rejection. When rejection occurs, the activation and proliferation of immune cells may cause serum free light chain levels to rise. By regularly monitoring serum free light chains, potential rejection can be detected early, and immunosuppressive treatment regimens can be adjusted in a timely manner.

[0018] Infection monitoring: Transplant patients are prone to infection due to long-term use of immunosuppressive agents and low immunity. When infected, the body's immune response may also cause fluctuations in serum free light chain levels. Therefore, monitoring serum free light chain levels helps to detect complications such as infection in a timely manner and assess the patient's immune status and disease progression.

[0019] 6. Application in other diseases

[0020] a) Cardiovascular disease: Studies have shown that sFLC levels are associated with the severity and prognosis of heart failure, and may serve as a biomarker for inflammation and myocardial injury.

[0021] b) Autoimmune diseases: sFLC levels are associated with disease activity in rheumatoid arthritis (RA), and may reflect synovial inflammation and immune complex deposition. Some patients have elevated sFLC, which may be related to kidney involvement (lupus nephritis) or disease activity.

[0022] c) Solid tumors: A small number of solid tumor patients may secrete light chains or stimulate polyclonal plasma cell proliferation, leading to elevated sFLC, but blood system diseases need to be ruled out in combination with other indicators.

[0023] In the diagnostic guidelines for multiple diseases, free light chain detection quality control products play an important role. For example, the "Guidelines for the Diagnosis and Treatment of Multiple Myeloma Renal Damage (2024 Edition)" and the "Chinese Guidelines for the Diagnosis and Treatment of Multiple Myeloma (2022 Revision)" recommend using serum free light chain detection to achieve early screening, diagnosis and monitoring of multiple myeloma and related diseases, which has promoted the widespread application of free light chain detection quality control products in clinical practice.

[0024] Free light chain detection is mainly used in the clinical laboratory, and also provides strong laboratory evidence for the diagnosis, treatment monitoring and efficacy evaluation of related diseases in departments such as hematology, nephrology, infectious diseases, and neurology.

[0025] Clinical laboratory quality control products are used daily to monitor the stability of the detection system, evaluate the performance of the detection method or instrument, and compare between laboratories to ensure consistency of results. However, the quality control products of different manufacturers currently have differences, which affect the consistency of results, and commercial quality control products are expensive, increasing the burden on laboratories. There is an urgent need to promote the standardization of free light chain detection to improve the consistency of results and reduce the cost of calibration and quality control products through mass production. SUMMARY

[0026] In view of the deficiencies of the prior art, the purpose of the present application is to provide a free light chain, a quality control product and uses thereof.

[0027] The purpose of the present application is achieved by the following technical solutions:

[0028] In a first aspect, the present application provides a free light chain, which comprises one or both of a free kappa light chain and a free lambda light chain.

[0029] The free kappa light chain has an amino acid sequence as shown in SEQ ID NO. 1, and the free lambda light chain has an amino acid sequence as shown in SEQ ID NO. 2.

[0030] Amino acid sequence of free kappa light chain:

[0031] DISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGIPERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSGGGGSDYKDDDDKHHHHHH (SEQ ID NO. 1).

[0032] Amino acid sequence of free lambda light chain:

[0033] DISVAPGETARISCGEKSLGSRAVQWYQHRAGQAPSLIIYNNQDRPSGIPERFSGSPDSPFGTTATLTITSVEAGDEADYYCHIWDSRVPTKWVFGGGTTLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSHHHHHH (SEQ ID No. 2).

[0034] In some embodiments, the present application also provides a variant of the free light chain as described herein, which comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID No. 1 or 2, and substantially retains the biological function of the free light chain it is derived from.

[0035] More specifically, the variant differs from the free light chain as described herein only by the conservative substitution of one or more (e.g., up to 20, up to 15, up to 10, up to 5, or up to 1 amino acid conservative substitution) amino acid residues.

[0036] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is a function of the number of matching positions shared by the sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently accomplished by use of the algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443-453, as implemented in the computer program ALIGN available from DNAstar, Inc., using, for example, a program parameter of an affine gap cost of 1 for the gap open penalty and 0.5 for the gap extension penalty. Percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6, as incorporated in the GCG software package (available at www.gcg.com).

[0037] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of an amino acid residue for an amino acid residue with similar side chains, e.g., substitutions that take place within a family of amino acid residues that are physicochemically or functionally similar, e.g., have similar size, shape, charge, chemical properties, including ability to form covalent or hydrogen bonds, etc. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution is one in which the replacement amino acid residue is of the same family as the original. Methods of identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0038] Polynucleotide

[0039] In another aspect, the present application also provides a polynucleotide encoding the above-mentioned free light chain.

[0040] The polynucleotide of the present application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0041] The nucleotide full-length sequence of the free light chain of the present application or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the length of the fragment is short. Generally, a long fragment of sequence can be obtained by first synthesizing a plurality of small fragments and then ligating them together.

[0042] Vector

[0043] In another aspect, the present application also provides a vector comprising a polynucleotide encoding the above-mentioned free light chain.

[0044] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of a protein encoded by the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by the vector are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain various elements to control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector can also contain a replication initiation site.

[0045] Cell

[0046] In another aspect, the present application also provides a cell comprising a vector as described herein, which is obtained by introducing the vector into a host cell.

[0047] As used herein, the term "host cell" refers to a cell that can be used for introducing a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or other human cells. The host cell can include a single cell or a population of cells.

[0048] The introduction of a vector into a host cell can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, such as E. coli, the transformation of the cell can be performed by using CaCl2treatment after the exponential growth phase, using steps well known in the art. Another method is to use MgCl2. If necessary, the transformation can also be performed by electroporation. When the host is a eukaryote, the DNA transfection method can be selected from the following methods: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0049] Use

[0050] The application also provides use of the free light chain in preparation of a quality control product.

[0051] Quality control

[0052] The application also provides a quality control product comprising the aforementioned free light chain.

[0053] The quality control product comprises recombinant humanized free kappa light chain at a concentration of 0.1-50.0 mg / mL and recombinant humanized free lambda light chain at a concentration of 0.1-50.0 mg / mL, based on the final volume of the quality control product.

[0054] The quality control product further comprises a normal level of serum protein control. The normal level is relative to a disease level.

[0055] The normal level of serum protein control comprises: a buffer, a beta-2 component peak mimic, other component peak mimics, a surfactant, an alkaline earth metal salt, a protease inhibitor; the component peak mimic refers to a purified or recombinant protein or derivative of human or animal origin, which can produce signals similar to each component of serum proteins in an electropherogram.

[0056] Preferably, the buffer is a Good's buffer with a pH of 6.0-9.0 and a concentration of 20-200 mmol / L.

[0057] Preferably, the beta-2 component peak mimic is one or more of a purified complement protein, a recombinant complement protein, a purified immunoglobulin, a recombinant immunoglobulin, specifically one or more of purified complement protein C1q, purified complement protein C2, purified complement protein C3, purified complement protein C4, purified complement protein C5, recombinant complement protein C1q, recombinant complement protein C2, recombinant complement protein C3, recombinant complement protein C4, recombinant complement protein C5a, human IgA, human IgM, recombinant humanized IgA antigen, recombinant humanized IgM antigen; 2.0-8.0 mg / mL is a relatively optimal addition concentration of the purified complement protein or the recombinant complement protein, and 4.0 mg / mL is the optimal addition concentration of the purified complement protein or the recombinant complement protein; 0.1-50.0 mg / mL is the addition concentration of the purified immunoglobulin or the recombinant immunoglobulin, 2.0-15.0 mg / mL is a relatively optimal addition concentration, and 4.0-6.0 mg / mL is the optimal addition concentration of the purified immunoglobulin or the recombinant immunoglobulin. The human or recombinant humanized IgA can be IgA kappa or IgA lambda. The human or recombinant humanized IgM can be IgM kappa or IgM lambda.

[0058] Preferably, the other component peak mimetic is one or more of an albumin component peak mimetic, an alpha-1 component peak mimetic, an alpha-2 component peak mimetic, a beta-1 component peak mimetic, a gamma component peak mimetic.

[0059] Preferably, the albumin component peak mimetic is one or more of human serum albumin, bovine serum albumin, porcine serum albumin, with a preferable addition concentration of 20.0-80.0 mg / mL, and an optimal addition concentration of 60.0 mg / mL.

[0060] Preferably, the alpha-1 component peak mimetic is one or both of alpha-1 antitrypsin, alpha-1 microglobulin, with a preferable addition concentration of 1.5-8.0 mg / mL, and an optimal addition concentration of 3.0 mg / mL.

[0061] Preferably, the alpha-2 component peak mimetic is one or both of alpha-2 macroglobulin, haptoglobin, with a preferable addition concentration of 2.0-10.0 mg / mL, and an optimal addition concentration of 4.0 mg / mL.

[0062] Preferably, the beta-1 component peak mimetic is transferrin, with a preferable addition concentration of 1.0-5.0 mg / mL, and an optimal addition concentration of 3.0 mg / mL.

[0063] Preferably, the gamma component peak mimetic is one or both of human IgG, recombinant human IgG protein, with an addition concentration of 0.1-50.0 mg / mL, a preferable addition concentration of 5.0-30.0 mg / mL, and an optimal addition concentration of 15.0 mg / mL. The human or recombinant human IgG is IgG kappa or IgG lambda.

[0064] Preferably, the surfactant is Tween-80, with a concentration of 1.0-5.0 g / L.

[0065] Preferably, the alkaline earth metal salt is magnesium chloride, with a concentration of 5.0-200.0 mmol / L.

[0066] Preferably, the protease inhibitor is AEBSF, with a concentration of 0.5-3.0 mmol / L.

[0067] The concentrations of the substances in the normal level serum protein control are calculated based on the final volume of the normal level serum protein control.

[0068] Preferably, the normal level serum protein control is the serum protein electrophoresis control disclosed in CN 117825476A, for example, the serum protein electrophoresis control described in any one of embodiments 1-9 thereof.

[0069] The quality control product is in the form of lyophilized powder.

[0070] The quality control product can be used in immunofixation electrophoresis of agarose electrophoresis system, immunotyping electrophoresis of capillary electrophoresis system or free light chain detection system of mass spectrometry technology platform.

[0071] The quality control product can also be used in free light chain detection system of immunoturbidimetry analysis system.

[0072] Diagnostic applications

[0073] The present application also relates to the use of the free light chain, polynucleotide, vector, cell or quality control product as described herein in the manufacture of a diagnostic kit or medicament for a single free light chain related disease.

[0074] Preferably, the free light chain related disease is multiple myeloma, light chain amyloidosis, monoclonal gammopathy, kidney disease, cardiovascular disease, autoimmune disease, and other plasma cell diseases, etc.

[0075] Compared with the prior art, the positive effect of the present application is that the free light chain and the quality control product containing the same can be used in quality control of immunofixation electrophoresis analysis system, immunoturbidimetry analysis system, mass spectrometry detection, etc., to ensure the accuracy of clinical related disease diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 Results of immunofixation electrophoresis of free kappa light chain are shown;

[0077] Figure 2 Results of immunofixation electrophoresis of free lambda light chain are shown;

[0078] Figure 3 Results of immunofixation electrophoresis of normal level of serum protein control are shown;

[0079] Figure 4 Results of immunofixation electrophoresis of quality control product 1 (right panel) and quality control product 2 (left panel) composed of free light chain and normal level of serum protein control are shown. DETAILED DESCRIPTION

[0080] M protein in serum protein is an abnormal monoclonal immunoglobulin or fragment thereof, which is usually produced by monoclonal malignant proliferation of plasma cells or B lymphocytes. M protein can be generally classified into 7 types, such as IgG, IgA, IgM, IgD, IgE and free light chain (kappa, lambda) and non-secretory type, which can present a characteristic band, i.e. M band, after electrophoresis. The principle of immunofixation electrophoresis for detecting monoclonal immunoglobulin is as follows:

[0081] The abnormal bands in the serum or urine protein profile are mainly located in the beta and gamma globulin zones, which are always suspected of being monoclonal proteins (M proteins, paraproteins, monoclonal immunoglobulins) and thus are a sign of a gammopathy. The use of immunofixation techniques is precisely to better identify these abnormal bands.

[0082] Immunofixation electrophoresis is a simple and practical technique that allows the in situ fixation of proteins after electrophoresis by forming insoluble complexes with antibodies. It is simple to perform and easy to interpret, and is divided into four steps:

[0083] 1. Electrophoresis of the agarose gel to separate the proteins.

[0084] 2. Immunofixation of the electrophoresis proteins (immunoprecipitation) - the addition of the various antisera in the corresponding electrophoresis lanes. The antisera diffuse in the gel and precipitate the corresponding antibodies present. The proteins in the reference lane are fixed with a fixative.

[0085] 3. By washing, the non-precipitated soluble proteins are removed. The precipitate of the antigen-antibody complex thus remains in the gel profile.

[0086] 4. The precipitated proteins can be observed by colouring.

[0087] To detect and identify the suspected monoclonal component, the sample is subjected to electrophoresis in 6 lanes simultaneously. After electrophoresis, one of the lanes is used as a reference of the electrophoresis profile of the proteins of the sample as a whole. The presence or absence of a monoclonal component reacting with the anti-gamma (Ig G), alpha (Ig A) and mu (Ig M) heavy chains and free and bound anti-kappa and lambda light chain antisera is determined by the characteristics of the other 5 lanes. The immunofixation bands are then compared with the suspected bands in the reference profile, the corresponding bands should have the same electrophoretic position.

[0088] For the purposes of facilitating the understanding of the present application, a more detailed description thereof will be given below in relation to the drawings and to specific examples. It should be noted that the present application is not limited to the specific methods, protocols, cell lines, constructs and reagents described herein, and can likewise be varied. Unless otherwise defined, all the technical and scientific terms used in the present description have the same meaning as understood by a person skilled in the art belonging to the technical field of the present application. The terms used in the present description in the specification of the present application are only for the purposes of describing the specific examples and are not intended to limit the present application.

[0089] In the following example, immunofixation electrophoresis was performed using the HYDRAGEL 9IF kit supplied by Sebia, item number 4309, following the instructions.

[0090] Example 1 Preparation of free light chains

[0091] 1. Gene synthesis

[0092] The free kappa light chain with the amino acid sequence as shown in SEQ ID NO. 1, the recombinant humanized free lambda light chain with the amino acid sequence as shown in SEQ ID NO. 1 are respectively codon optimized to adapt to CHO cell expression, and are respectively synthesized into pcDNA3.1 vector.

[0093] 2. 100 ug plasmid extraction

[0094] I. Transformation

[0095] ①TOP1 competent cells are taken out from -80℃, quickly inserted into ice, after 5 minutes, the bacterial mass is melted, about 10 ng of target plasmid DNA is added, and the EP tube bottom is lightly mixed by hand (avoiding using gun suction to beat), and is placed in ice for 25 minutes.

[0096] ②42℃ water bath heat shock for 45 seconds, quickly put back on ice and stand for 2 minutes.

[0097] ③Add 700 μl of sterile culture medium LB without antibiotics to the centrifuge tube, mix well, then recover at 37℃, 200 rpm for 60 minutes.

[0098] ④Centrifuge at 50,000 rpm for 1 minute to collect bacteria, take about 100 μl of supernatant, lightly blow and resuspend the bacterial mass, and plate on LB medium containing ampicillin antibiotic.

[0099] ⑤Place the plate in a 37℃ incubator overnight.

[0100] II. Plasmid extraction - endotoxin-free plasmid extraction kit DP108

[0101] ①Before use, add all the RNase A provided in the kit to P1, mix well, and store at 2-8℃.

[0102] ②Before use, add anhydrous ethanol to the rinse solution PWF and MRDE.

[0103] ③Column equilibration step: add 2 ml of equilibration solution BL to the adsorption column CP7 (the adsorption column is placed in a 15 ml collection tube), centrifuge at 5,000 rpm (~ 4,500 xg) for 2 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube (the column treated with the equilibration solution is preferably used immediately).

[0104] ④Take 20-50 ml (select the appropriate amount according to the concentration of the cultured bacterial mass, and 100 ml is recommended for low copy) of overnight cultured bacterial solution, add it to the centrifuge tube, centrifuge at room temperature 5,000 rpm (~ 4,500 xg) for 3 min to collect the bacteria, and try to aspirate the supernatant.

[0105] 5. Try to remove the supernatant. To ensure that all the supernatant is removed, use a clean paper towel to remove the drops of water from the side of the tube.

[0106] 6. Add 2.5 ml of solution P1 (check if RNase A has been added) to the centrifuge tube containing the bacterial pellet. Mix the bacterial cell pellet thoroughly using a pipette or vortex.

[0107] 7. Add 2.5 ml of solution P2 to the centrifuge tube. Immediately invert the tube 6-8 times gently and let it stand at room temperature for 5 min.

[0108] 8. Add 1.25 ml of solution E3 to the centrifuge tube. Immediately invert the tube 6-8 times gently and mix thoroughly until a white, dispersed flocculent precipitate appears. Then let it stand at room temperature for 2-3 min. Centrifuge at 5,000 rpm (~ 4,500 x g) for 10 min. The white precipitate will be at the bottom of the tube. Carefully pour the entire solution into the filter CS1 (avoid pouring in a large amount of precipitate that can block the filter). Slowly push the plunger to filter the solution. The filtrate will be collected in a clean 15 ml tube (provided).

[0109] 9. Add an equal volume of solution EBT to the filtrate. Immediately invert the tube 7-10 times and mix thoroughly.

[0110] 10. Transfer 4 ml of the mixture to the adsorption column CP7 (put the adsorption column in a 15 ml collection tube). Centrifuge at 5,000 rpm (~ 4,500 x g) for 3 min at room temperature. Discard the waste in the collection tube and put the adsorption column CP7 back in the collection tube. Repeat this step until all the mixture has passed through the adsorption column CP7.

[0111] Add 2 ml of rinse GDE to the adsorption column CP7. Centrifuge at 5,000 rpm (~ 4,500 x g) for 3 min. Discard the waste in the collection tube and put the adsorption column back in the collection tube.

[0112] Add 3 ml of rinse MRDE (check if anhydrous ethanol has been added) to the adsorption column CP7. Centrifuge at 5,000 rpm (~ 4,500 x g) for 3 min. Discard the waste in the collection tube and put the adsorption column back in the collection tube.

[0113] Add 3.5 ml of rinse PWF to the adsorption column CP7. Centrifuge at 5,000 rpm (~ 4,500 x g) for 3 min. Discard the waste in the collection tube and put the adsorption column back in the collection tube.

[0114] Repeat step 13.

[0115] 5,000 rpm (~ 4,500 xg) centrifugation for 10 min, in order to remove the residual rinse solution in the adsorption column.

[0116] The adsorption column CP7 was placed in a clean 15 ml collection tube, and 0.5-1 ml elution buffer TB was added to the middle of the adsorption membrane, and then placed at room temperature for 2-3 min, and then centrifuged at 5,000 rpm (~ 4,500 xg) for 5 min at room temperature. The eluate in the 15 ml centrifuge tube was all transferred to a clean 1.5 ml centrifuge tube for use or stored at -20°C.

[0117] The prepared monoclonal antibodies (free kappa light chain and free lambda light chain) were verified by immunofixation electrophoresis. The immunofixation electrophoresis was performed on a Sebia agarose electrophoresis instrument (HYDRASYS2 SCAN FOCUSING), using a matching immunofixation electrophoresis kit (HYDRAGEL 9IF, Ref 4309, batch number: 08074 / 01), and the immunofixation electrophoresis detection was completed according to the specified operation steps. The results are shown in Figure 1 and Figure 2 .

[0118] Preparation of normal level serum protein control

[0119] According to the method in the serum protein electrophoresis quality control invention patent CN 117825476A, the raw materials were added according to Table 1, and then were divided and freeze-dried to obtain a lyophilized powder dosage form.

[0120] Table 1

[0121]

[0122] The human immunoglobulin used in this example is Human immunoglobulin for intravenous injection.

[0123] The immunofixation electrophoresis results of the prepared normal level serum protein control are shown in Figure 3 The results show that each band is clear, and no obvious abnormal precipitation line is observed in each lane.

[0124] Example 3 Immunofixation electrophoresis of monoclonal antibody

[0125] After mixing the free kappa light chain prepared in Example 1 with the normal level control prepared in Example 2, the obtained control 1 (the final concentration of free kappa light chain is 2 mg / mL) was subjected to immunofixation electrophoresis. The results are shown in Figure 4As shown in the right panel of FIG. 1, the free kappa light chain can be clearly distinguished in the kappa lane, indicating that the control 1, which is a combination of the free kappa light chain and the normal level control, can be used as a new control for immunofixation electrophoresis.

[0126] After mixing the free lambda light chain prepared in Example 1 with the normal level control prepared in Example 2, the resulting control 2 (the final concentration of the free lambda light chain is 2 mg / mL) was subjected to immunofixation electrophoresis, and the results are shown in the left panel of FIG. 2. Figure 4 As shown in the left panel of FIG. 2, the free lambda light chain can be clearly distinguished in the lambda lane, indicating that the control 2, which is a combination of the free lambda light chain and the normal level control, can be used as a new control for immunofixation electrophoresis.

[0127] Example 4

[0128] The normal level control prepared in Example 2, the control 1 prepared in Example 3 and the control 2 were subjected to quantitative detection using the free kappa light chain detection kit (Optilie Freelite kappa free kit, immunoturbidimetry, batch number: 544492-1) and the free lambda light chain detection kit (Optilie Freelite kappa lambda kit, immunoturbidimetry, batch number: 540302-2) of the Optilie company, respectively. The testing instrument was a full-automatic protein analyzer (model Optilite Type 864).

[0129] The detection steps were carried out according to the kit instructions, and the detection results are shown in Table 2.

[0130] Table 2

[0131] Indicator (mg / L) Reference interval Normal level quality control Quality control 1 Quality control 2 FLC-κ (mg / L) 3.30-19.40 17.83 mg / mL 1751.56 mg / mL 16.68 mg / mL FLC-λ 5.71-26.30 16.83 mg / mL 12.63 mg / mL 432.84 mg / mL FLC ratio κ / λ 0.26-1.65 1.06 138.68 0.04

[0132] As shown in Table 2, the average values of the free kappa light chain and the free lambda light chain of the normal level control are within the normal range, and the free light chain kappa / lambda ratio is normal; the free kappa light chain of the control 1 is significantly abnormal, and the free light chain kappa / lambda ratio is abnormally high; the free lambda light chain of the control 2 is significantly abnormal, and the free light chain kappa / lambda ratio is abnormally low. This set of controls can cover normal and abnormal conditions, and the concentration of the free light chain in the control 1 and the control 2 can be adjusted according to specific needs, thereby adjusting the free light chain kappa / lambda ratio. Further indicating that the prepared control is suitable for immunoturbidimetry detection of free light chain control.

[0133] The raw materials used in the present application are commercially available and widely available, and can be produced on a large scale.

[0134] The above description of the embodiments is given for the purpose of completeness to provide one of ordinary skill in the art with a thorough understanding of the application and does not limit the application to any one embodiment or application. It will be apparent to those skilled in the art that various modifications and variations can be made to the present embodiments without departing from the scope or spirit of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A free light chain, characterized in that, The free light chain consists of one or both of a free kappa light chain and a free lambda light chain; The free kappa light chain has an amino acid sequence as shown in SEQ ID NO. 1; and the free lambda light chain has an amino acid sequence as shown in SEQ ID NO.

2.

2. A polynucleotide, comprising, The free light chain of claim 1.

3. A vector, characterized in that, The polynucleotide of claim 2.

4. A cell, characterized in that, The polynucleotide of claim 2 or the vector of claim 3.

5. Use of the free light chain of claim 1 in the preparation of a quality control product for detecting free light chain in an immunofixation electrophoresis system or an immunonephelometry system.

6. A quality control article, characterized in that, The quality control product comprises the free light chain of claim 1, and is used for quality control of free light chain detection in an immunofixation electrophoresis system or an immunonephelometry system.

7. The quality control of claim 6, wherein, The free kappa light chain has a concentration of 0.1-50.0 mg / mL, and / or the free lambda light chain has a concentration of 0.1-50.0 mg / mL, based on the final volume of the quality control product.

8. The quality control according to claim 6 or 7, characterized in that The quality control product further comprises a normal level serum protein control, which comprises the following components: a buffer, a β-2 component peak mimic, other component peak mimics, a surfactant, an alkaline earth metal salt, a protease inhibitor; the component peak mimic refers to a purified or recombinant protein or derivative of human or animal origin, which can produce a signal similar to each component of serum protein.

9. The quality control of claim 8, wherein, Any of the following features are also included: 1) the buffer is a buffer with a pH of 6.0-9.0 and a concentration of 20-200 mmol / L; 2) the β-2 component peak mimic is one or more of a purified complement protein, a recombinant complement protein, a purified immunoglobulin, and a recombinant immunoglobulin, with a concentration of 2.0-8.0 mg / mL; 3) the other component peak mimic is one or more of an albumin component peak mimic, an α-1 component peak mimic, an α-2 component peak mimic, a β-1 component peak mimic, and a γ component peak mimic.

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