A monoclonal immunoglobulin and its use

Abstract

The present application relates to the field of immunofixation electrophoresis, and in particular to a monoclonal immunoglobulin and its use, wherein the heavy chain amino acid sequence of the monoclonal immunoglobulin is shown in SEQ ID NO.1 or 2, and the light chain amino acid sequence is shown in SEQ ID NO.3. The monoclonal immunoglobulin described herein can be used to prepare immunofixation electrophoresis quality control products, and by adding different types of monoclonal immunoglobulins, immunofixation electrophoresis quality control products containing various types of monoclonal immunoglobulins can be prepared. The quality control products can be used for quality control of immunofixation electrophoresis analysis systems to ensure the accuracy of diagnosis of clinically relevant diseases.

Description

Technical Field

[0001] The present application relates to the field of immunofixation electrophoresis, and in particular to a monoclonal immunoglobulin and its use. Background Art

[0002] M protein in serum protein is an abnormal monoclonal immunoglobulin or its fragment, usually produced by the monoclonal malignant proliferation of plasma cells or B lymphocytes. The detection of M protein can be carried out through a variety of tests, including serum protein electrophoresis, immunofixation electrophoresis, serum Ig quantification and urine Bence Jones protein detection. The detection of M protein is of great significance for the diagnosis of diseases such as multiple myeloma and macroglobulinemia. Generally speaking, it can be divided into 7 types, such as IgG, IgA, IgM, IgD, IgE, free light chains (κ, λ) and non-secretory types. After electrophoresis, it can present a characteristic band, namely the M band.

[0003] Immunofixation electrophoresis (IFE) is an immunochemical analysis technique that combines zone electrophoresis and immunoprecipitation reaction. It is a qualitative detection technique for identifying the clonal properties of immunoglobulins. It is commonly used in clinical laboratories for typing and identification of monoclonal immunoglobulins (M proteins), cryoglobulins, and Bence Jones proteins. Alfonso reported this technique in 1964; in 1969, Alper and Johnson successively reported and practiced a detection technique that included two processes, agarose gel protein electrophoresis and immunoprecipitation test; in 1976, this technique was identified as an antibody detection method. Immunofixation electrophoresis combines the high resolution of protein electrophoresis with the specificity of antigen-antibody reaction. It has been used to detect and identify various abnormal proteins in serum, urine, cerebrospinal fluid, and other body fluid samples. It is the preferred method for typing and identification of M proteins, cryoglobulins, Bence Jones proteins, cerebrospinal fluid oligoclonal proteins, etc. in clinical laboratories, and has gradually replaced traditional immunoelectrophoresis techniques.

[0004] The principle of immunofixation electrophoresis is to first perform zone electrophoresis on proteins in a gel medium. Due to the different relative molecular weights, charge properties and quantities of various proteins, the electrophoretic migration directions and speeds are also different, thus separating different proteins; then the protein fixative and heavy chain (γ, α, μ, δ, ε) and light chain (κ, λ) antisera of different types of immunoglobulins (IgG, IgA, IgM, IgD, IgE) are added to different lanes on the surface of the gel; after incubation, the fixative and antiserum penetrate and diffuse in the gel; the antiserum can form immune complexes with the corresponding immunoglobulins in the gel and precipitate into the gel; the unbound free antibodies and other proteins are eluted, and the formed antigen-antibody complexes are retained in the gel; after rinsing and staining, the reference lane and the precipitation zone immune complexes are colored, and the monoclonal components are separated according to the different electrophoretic migration distances of various proteins. According to the type of antiserum corresponding to the lane forming the immune complex, different types of immunoglobulins and their light chains can be typed and identified.

[0005] Immunofixation electrophoresis has high sensitivity and resolution, and the results are easier to analyze. As the importance of immunofixation electrophoresis in clinical applications continues to be recognized, more and more laboratories have begun to carry out this test project.

[0006] However, the immunofixation electrophoresis detection steps are relatively cumbersome, including sample pretreatment, pre-dilution, gel laying, sample loading, electrophoresis, antiserum addition, adsorption and removal of excess antiserum, drying, rinsing, staining, decolorization, re-drying and cleaning of the reusable spotting comb, sample loading template, and reagent preparation. There are many manual operation steps, the instrument automation level is low, the working state of the electrophoresis analyzer, the quality of the gel slice, and the quality of the antiserum reagent will affect the results, and the interpretation of the results is greatly affected by the subjective factors of the technicians. Therefore, the quality assurance of the test results is very important. However, there is currently no commercial immunofixation electrophoresis quality control product for laboratory internal quality control and inter-laboratory quality evaluation. Summary of the invention

[0007] The purpose of the present application is to provide a monoclonal immunoglobulin, the heavy chain amino acid sequence of the monoclonal immunoglobulin is shown as SEQ ID NO.1 or 2, and the light chain amino acid sequence is shown as SEQ ID NO.3.

[0008] Heavy chain gamma sequence 1:

[0009] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGDGFYVYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.1).

[0010] Heavy chain gamma sequence 2: IgG

[0011] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGDGFYVYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.2).

[0012] Light chain lambda sequence 1:

[0013] DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGGGTKLDI KGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO.3).

[0014] The monoclonal immunoglobulin is M protein. The protein formed by heavy chain γ sequence 1 and light chain λ sequence 1 is named M protein 1, and the protein formed by heavy chain γ sequence 2 and light chain λ sequence 1 is named M protein 2.

[0015] In some embodiments, the present invention also provides a variant of a monoclonal immunoglobulin as described herein, which comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the amino acid sequence shown in SEQ ID No. 1, 2 or 3, and substantially retains the biological function of the monoclonal immunoglobulin from which it is derived.

[0016] More specifically, the variants differ from the monoclonal immunoglobulins as described herein only by conservative substitutions of one or more (e.g., at most 20, at most 15, at most 10, at most 5 or at most 1 amino acid residues).

[0017] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent 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)), which has been incorporated 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, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0018] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the 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 amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds 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, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid 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).

[0019] Polynucleotide

[0020] In another aspect, the present invention also provides a polynucleotide encoding the above monoclonal immunoglobulin.

[0021] More specifically, the polynucleotide has an amino acid sequence shown in SEQ ID No. 4, 5 or 6.

[0022] The polynucleotide of the present invention 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.

[0023] The term "polynucleotide encoding a polypeptide / protein / antibody" may include a polynucleotide encoding the polypeptide / protein / antibody, or may also include additional coding and / or non-coding sequences.

[0024] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identity between the two sequences, and the polypeptides / proteins / antibodies encoded by them have substantially the same functions and activities. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably 95%.

[0025] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0026] Carrier

[0027] In another aspect, the present invention also provides a vector comprising a polynucleotide encoding the above monoclonal immunoglobulin.

[0028] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries 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, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0029] cell

[0030] In another aspect, the present invention also provides a cell comprising the vector as described herein, wherein the cell is obtained by introducing the vector into a host cell.

[0031] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia 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 fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or other human cells. Host cells may include a single cell or a cell population.

[0032] The vector can be introduced into the host cell using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0033] use

[0034] The present invention also provides use of the monoclonal immunoglobulin in preparing immunofixation electrophoresis quality control products.

[0035] Immunofixation electrophoresis quality control

[0036] The present invention also provides an immunofixation electrophoresis quality control product, which includes the monoclonal immunoglobulin.

[0037] Based on the final volume of the immunofixation electrophoresis quality control product, the concentration of the monoclonal immunoglobulin is 0.1-30 mg / mL, preferably 2 mg / mL.

[0038] The immunofixation electrophoresis quality control product also includes a normal level of serum protein electrophoresis quality control product. The normal level is relative to the disease level.

[0039] The normal level serum protein electrophoresis quality control products include: buffer, β-2 component peak simulator, other component peak simulators, surfactants, alkaline earth metal salts, and protease inhibitors; the component peak simulator refers to purified or recombinant proteins or derivatives of human or animal origin, which can produce signals similar to those of the serum protein components in the electrophoresis graph.

[0040] Preferably, the buffer is Good's buffer with a pH of 6.0-9.0 and a concentration of 20-200 mmol / L.

[0041] Preferably, the β-2 component peak mimic is one or more of purified complement protein, recombinant complement protein, purified immunoglobulin, and recombinant immunoglobulin, specifically 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 One or more of the humanized IgM antigens in the group; 2.0-8.0 mg / mL is the preferred addition concentration of purified complement protein or recombinant complement protein, and 4.0 mg / mL is the optimal addition concentration of purified complement protein or recombinant complement protein; 0.1-50.0 mg / mL is the addition concentration of purified immunoglobulin or recombinant immunoglobulin, 2.0-15.0 mg / mL is the preferred addition concentration, and 4.0-6.0 mg / mL is the optimal addition concentration of purified immunoglobulin or recombinant immunoglobulin. The human or recombinant humanized IgA can be IgAκ or IgAλ. The human or recombinant humanized IgM can be IgMκ or IgMλ.

[0042] The other component peak simulators are one or more of an albumin component peak simulator, an α-1 component peak simulator, an α-2 component peak simulator, a β-1 component peak simulator, and a γ component peak simulator.

[0043] Preferably, the albumin component peak mimic is one or more of human serum albumin, bovine serum albumin, and porcine serum albumin, with 20.0-80.0 mg / mL being a preferred addition concentration, and 60.0 mg / mL being an optimal addition concentration.

[0044] Preferably, the α-1 component peak mimic is one or both of α-1 antitrypsin and α-1 microglobulin, 1.5-8.0 mg / mL is a better addition concentration, and 3.0 mg / mL is the optimal addition concentration.

[0045] Preferably, the α-2 component peak mimic is one or both of α-2 macroglobulin and haptoglobin, 2.0-10.0 mg / mL is a better addition concentration, and 4.0 mg / mL is the optimal addition concentration.

[0046] Preferably, the β-1 component peak mimic is transferrin, with 1.0-5.0 mg / mL being a better addition concentration, and 3.0 mg / mL being the optimal addition concentration.

[0047] Preferably, the γ component peak mimic is one or both of human IgG and recombinant human IgG proteins, with an addition concentration of 0.1-50.0 mg / mL, a preferred 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 IgGκ or IgGλ.

[0048] Preferably, the surfactant is Tween-80 with a concentration of 1.0-5.0 g / L.

[0049] Preferably, the alkaline earth metal salt is magnesium chloride with a concentration of 5.0-200.0 mmol / L.

[0050] Preferably, the protease inhibitor concentration is AEBSF, and the concentration is 0.5-3.0 mmol / L.

[0051] The concentration of each substance in the normal level serum protein electrophoresis quality control product is calculated based on the final volume of the normal level serum protein electrophoresis quality control product.

[0052] Preferably, the normal level serum protein electrophoresis quality control product is the serum protein electrophoresis quality control product in publication number CN 117825476A, for example, the serum protein electrophoresis quality control product described in any one of Examples 1 to 9 thereof.

[0053] The immunofixation electrophoresis quality control product is in the form of a lyophilized powder.

[0054] The immunofixation electrophoresis quality control product can be used for immunofixation electrophoresis in an agarose electrophoresis system, immunophenotyping electrophoresis in a capillary electrophoresis system, or an M protein detection system in a mass spectrometry technology platform.

[0055] Diagnostic Applications

[0056] The present invention also relates to the use of the monoclonal immunoglobulin, polynucleotide, vector, cell or immunofixation electrophoresis quality control product as described herein in the preparation of a diagnostic kit or drug for a disease associated with monoclonal immunoglobulin. The monoclonal immunoglobulin-related disease is, for example, multiple myeloma, multiple myeloma, heavy chain disease, malignant lymphoma, chronic lymphocytic leukemia, macroglobulinemia, non-lymphoreticular tumor, monocytic leukemia or cryoglobulinemia.

[0057] Compared with the prior art, the positive effect of the present invention is that the monoclonal immunoglobulin and quality control products of the present invention can be used for quality control of the immunofixation electrophoresis analysis system to ensure the accuracy of diagnosis of clinically relevant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Display the results of immunofixation electrophoresis with background;

[0059] Figure 2 The results of immunofixation electrophoresis of M protein 1 are shown;

[0060] Figure 3 The results of immunofixation electrophoresis of commercial IgM-κ type M protein (denoted as mk) are shown;

[0061] Figure 4 The results of immunofixation electrophoresis with background + mk are shown;

[0062] Figure 5 The results of immunofixation electrophoresis of background + mk + M protein 1 are shown;

[0063] Figure 6 The results of immunofixation electrophoresis of M protein 2 are shown;

[0064] Figure 7 The results of immunofixation electrophoresis of background + mk + M protein 2 are shown. DETAILED DESCRIPTION

[0065] Principle of immunofixation electrophoresis to detect monoclonal immunoglobulins:

[0066] Abnormal bands in serum or urine protein profiles are mainly located in the beta globulin and gamma globulin regions, which are always suspected to be monoclonal proteins (M proteins, paraproteins, monoclonal immunoglobulins), and are therefore signs of gammopathies. Immunofixation techniques are used to better identify these abnormal bands.

[0067] Immunofixation electrophoresis is a simple technique that can fix proteins in place after electrophoresis by forming insoluble complexes between them and antibodies. It is simple to perform and interpret and consists of 4 steps:

[0068] 1. Perform electrophoresis on agarose gel to achieve protein separation.

[0069] 2. Electrophoretic protein immunofixation (immunoprecipitation) - Various antisera are added to the corresponding electrophoresis lanes. The antisera diffuse in the film and precipitate the corresponding antibodies present. The proteins in the reference lanes are fixed with a fixative.

[0070] 3. Remove the unprecipitated soluble proteins by washing, so that the precipitate of the antigen-antibody complex will remain in the film image.

[0071] 4. The precipitated protein can be visualized by staining.

[0072] To detect and identify suspected monoclonal components, the specimen is electrophoresed simultaneously in 6 lanes. After electrophoresis, one lane is used as a reference for the electrophoretic pattern of the complete specimen protein. The characteristics of the other 5 lanes are used to confirm or exclude the monoclonal components that react or are absent with anti-γ (Ig G), α (Ig A) and μ (Ig M) heavy chains and free and bound anti-κ and λ light chain antisera. The immunofixation bands are then compared with the suspected bands in the reference pattern, and the corresponding bands should have the same electrophoretic position.

[0073] For ease of understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the present invention is not limited to the ad hoc methods, schemes, cell lines, constructs and reagents described herein, and can be changed equally. Unless otherwise defined, all technical and scientific terms used in this specification are identical with the meanings generally understood by those skilled in the art belonging to the technical field of the present invention. The terms used in this specification in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.

[0074] In the following examples, immunofixation electrophoresis was performed using the HYDRAGEL 9IF kit supplied by Sebia with a catalog number of 4309, and the experimental steps were performed according to the instructions.

[0075] Example 1 Preparation of monoclonal immunoglobulin

[0076] 1. Gene Synthesis

[0077] ① The heavy chain γ sequence 1, heavy chain γ sequence 2 and light chain λ amino acid sequence 1 were codon optimized to adapt to CHO cell expression and synthesized into pcDNA3.1 vector respectively.

[0078] 2.100ug plasmid extraction

[0079] I. Transformation

[0080] ① Take out the TOP1 competent cells from -80℃ and quickly put them in ice. After 5 minutes, wait for the bacterial block to melt, add about 10ng of the target plasmid DNA and gently mix it by hand at the bottom of the EP tube (avoid using a gun to suck it), and let it stand in ice for 25 minutes.

[0081] ②Heat shock in a 42℃ water bath for 45 seconds, quickly return to ice and let stand for 2 minutes.

[0082] ③ Add 700 μl of sterile culture medium LB without antibiotics into the centrifuge tube, mix well and resuscitate at 37°C, 200 rpm for 60 minutes.

[0083] ④ Centrifuge at 5000 rpm for 1 minute to collect the bacteria, take about 100 μl of the supernatant, gently blow to resuspend the bacteria and spread it on a plate containing LB culture medium containing ampicillin antibiotics.

[0084] ⑤ Place the plate upside down in a 37°C incubator for overnight culture.

[0085] II. Plasmid Extraction - Endotoxin-free Plasmid Medium Extraction Kit DP108

[0086] ① Before use, add all the RNaseA provided by the kit to P1, mix well, and store at 2-8℃.

[0087] ②Please add anhydrous ethanol to the rinse solution PWF and MRDE before use.

[0088] ③ Column balancing step: Add 2 ml of balancing 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 x g) for 2 minutes, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. (It is best to use the column treated with the balancing solution immediately)

[0089] ④ Take 20-50ml (select the appropriate amount according to the concentration of the cultured bacteria, 100ml is recommended for low copies) of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge at 5,000rpm (~4,500xg) at room temperature for 3 minutes to collect the bacteria and remove the supernatant as much as possible.

[0090] ⑤ Try to absorb the supernatant as much as possible. To ensure that all the supernatant is absorbed, please use clean absorbent paper to absorb the water droplets on the bottle wall.

[0091] ⑥ Add 2.5 ml of solution P1 (please check whether RNase A has been added) to the centrifuge tube containing the bacterial cell pellet, and use a pipette or vortex oscillator to thoroughly suspend the bacterial cell pellet.

[0092] ⑦ Add 2.5 ml of solution P2 to the centrifuge tube, gently turn it upside down 6-8 times, and leave it at room temperature for 5 minutes.

[0093] ⑧ Add 1.25ml of solution E3 to the centrifuge tube, and immediately gently turn it upside down 6-8 times to mix thoroughly until white dispersed flocculent precipitates appear in the solution. Then place it at room temperature for about 2-3 minutes. Centrifuge at 5,000rpm (~4,500xg) for 10 minutes to allow the white precipitate to settle to the bottom of the tube (the centrifugation time can be increased appropriately), and carefully pour all the solution into the filter CS1 (please avoid pouring a large amount of precipitate to block the filter), slowly push the push handle to filter, and collect the filtrate in a clean 15ml tube (self-prepared).

[0094] ⑨Add an equal volume of EBT solution to the filtrate and immediately invert it upside down 7-10 times to mix thoroughly.

[0095] ⑩ Transfer 4 ml of the mixed solution to the adsorption column CP7 (the adsorption column is placed in a 15 ml collection tube), centrifuge at room temperature at 5,000 rpm (~4,500 x g) for 3 min, pour out the waste liquid in the collection tube, and put the adsorption column CP7 back into the collection tube. Repeat this step until all the mixed solution passes through the adsorption column CP7.

[0096] Add 2 ml of rinsing solution GDE to the adsorption column CP7, centrifuge at 5,000 rpm (~4,500 x g) for 3 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0097] Add 3 ml of rinsing solution MRDE to the adsorption column CP7 (please check whether anhydrous ethanol has been added), centrifuge at 5,000 rpm (~4,500 x g) for 3 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0098] Add 3.5 ml of rinsing solution PWF to the adsorption column CP7, centrifuge at 5,000 rpm (~4,500 xg) for 3 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0099] Repeat step 13.

[0100] Centrifuge at 5,000 rpm (~4,500 x g) for 10 min to remove the remaining rinse solution in the adsorption column.

[0101] Place the adsorption column CP7 in a clean 15ml collection tube, add 0.5-1ml elution buffer TB to the middle of the adsorption membrane, let stand at room temperature for 2-3min, and then centrifuge at room temperature at 5,000rpm (~4,500xg) for 5min. Transfer all the eluate in the 15ml centrifuge tube to a clean 1.5ml centrifuge tube for later use or store at -20℃.

[0102] Example 2 Preparation of normal immunofixation electrophoresis quality control product, i.e. background

[0103] According to the method in the invention patent "CN 117825476A" of serum protein electrophoresis quality control product, the raw materials are added according to Table 1 and then packaged, and the lyophilized powder dosage form is prepared by freeze-drying.

[0104] Table 1

[0105]

[0106]

[0107] The human immunoglobulin used in this example is Intravenous human immunoglobulin.

[0108] The immunofixation electrophoresis results of the prepared immunofixation electrophoresis quality control products are shown in Figure 1 , where each band is clear, no obvious abnormal precipitation line is seen in the ELP lane, and no obvious abnormal precipitation line is seen in the antibody lane either.

[0109] Example 3M protein 1 immunofixation electrophoresis

[0110] The M protein 1 was subjected to immunofixation electrophoresis. Figure 2 As shown, the results showed that M protein 1 was an IgGλ-type M protein, which was consistent with the design.

[0111] Commercial IgM-κ type M protein (denoted as mk) (purchased from: molecular innocation, Human IgM from Myeloma Plasma Affinity Purified) was subjected to immunofixation electrophoresis in the same manner as a control. The results are shown in Figure 3 As shown, the results showed IgM-κ type M protein, which was consistent with the actual situation.

[0112] mk and the background prepared in Example 2 were mixed, and the final concentration of mk after mixing was 2 mg / mL. Immunofixation electrophoresis was then performed in the same manner as a control. The results are as follows: Figure 4 As shown, the results cannot clearly distinguish the protein type of mk.

[0113] mk was mixed with the background prepared in Example 2 and M protein 1. The final concentrations of mk and M protein 1 in the mixed sample were 2 mg / mL, respectively. Immunofixation electrophoresis was then performed in the same manner. The results are as follows: Figure 5 As shown, the type of M protein in the sample could not be distinguished.

[0114] Example 4M Protein 2 Immunofixation Electrophoresis

[0115] The steps are the same as in Example 3, and the results are as follows Figure 6 As shown, the results showed that M protein 2 was an IgGλ-type M protein, which was consistent with the design.

[0116] After mixing mk, the background prepared in Example 2 and M protein 2, the final concentrations of mk and M protein 2 in the sample were 2 mg / mL, and then immunofixation electrophoresis was performed in the same manner. The results are as follows: Figure 7 As shown, the types of M proteins can be clearly distinguished, indicating that the combination of M protein 2 and background can be used as a new quality control product for immunofixation electrophoresis.

[0117] The raw materials used in this application are all commercially available raw materials, which have a wide range of sources and can be produced on a large scale.

[0118] The above description of the embodiments is to facilitate those of ordinary skill in the art to understand and apply the present application. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to pay creative labor. Therefore, the present application is not limited to the embodiments herein, and improvements and modifications made by those skilled in the art based on the contents disclosed in the present application without departing from the scope and spirit of the present application are within the scope of the present application.

Claims

1. A monoclonal immunoglobulin, characterized in that The heavy chain amino acid sequence of the monoclonal immunoglobulin is shown in SEQ ID NO.2, and the light chain amino acid sequence is shown in SEQ ID NO.

3.

2. A polynucleotide, characterized in that Encoding the monoclonal immunoglobulin according to claim 1.

3. A carrier, characterized in that Comprising the polynucleotide of claim 2.

4. A cell, characterized in that Comprising the polynucleotide according to claim 2 or the vector according to claim 3.

5. Use of the monoclonal immunoglobulin according to claim 1 in preparing a monoclonal immunoglobulin immunofixation electrophoresis quality control product.

6. An immunofixation electrophoresis quality control product, characterized in that: The immunofixation electrophoresis quality control product includes the monoclonal immunoglobulin according to claim 1.

7. The immunofixation electrophoresis quality control product according to claim 6, characterized in that: Based on the final volume of the immunofixation electrophoresis quality control product, the concentration of the monoclonal immunoglobulin is 0.1-30 mg / mL.

8. The immunofixation electrophoresis quality control product according to claim 7, characterized in that: The concentration of the monoclonal immunoglobulin was 2 mg / mL.

9. The immunofixation electrophoresis quality control product according to claim 6, characterized in that: The immunofixation electrophoresis quality control product also includes a normal level serum protein electrophoresis quality control product; the normal level serum protein electrophoresis quality control product includes the following components: buffer, β-2 component peak simulator, other component peak simulators, surfactant, alkaline earth metal salt, protease inhibitor; the component peak simulator refers to a purified or recombinant protein or derivative of human or animal origin, which can produce signals similar to the components of serum proteins in immunofixation electrophoresis.

10. The immunofixation electrophoresis quality control product according to claim 9, characterized in that: Also includes any of the following features: 1) The buffer solution is a buffer solution 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 purified complement protein, recombinant complement protein, purified immunoglobulin, and recombinant immunoglobulin, with a concentration of 2.0-8.0 mg / mL; 3) The other component peak simulators are one or more of an albumin component peak simulator, an α-1 component peak simulator, an α-2 component peak simulator, a β-1 component peak simulator, and a γ component peak simulator.

11. The immunofixation electrophoresis quality control product according to claim 10, characterized in that: The albumin component peak mimic is one or more of human serum albumin, bovine serum albumin, and porcine serum albumin, and the concentration is 20.0-80.0 g / L.

12. The immunofixation electrophoresis quality control product according to claim 10, characterized in that: The α-1 component peak simulator is one or both of α-1 antitrypsin and α-1 microglobulin, with a concentration of 1.5-8.0 g / L; the α-2 component peak simulator is one or both of α-2 macroglobulin and haptoglobin, with a concentration of 2.0-10.0 g / L.

13. The immunofixation electrophoresis quality control product according to claim 10, characterized in that: The β-1 component peak analog is transferrin, and the concentration is 1.0-5.0 g / L; the γ component peak analog is one or both of human IgG and recombinant human IgG proteins, and the concentration is 5.0-30.0 g / L.

14. The immunofixation electrophoresis quality control product according to claim 13, characterized in that: The human or recombinant human IgG is IgGκ or IgGλ.

15. Use of the monoclonal immunoglobulin according to claim 1, the polynucleotide according to claim 2, the vector according to claim 3, the cell according to claim 4, or the immunofixation electrophoresis quality control product according to any one of claims 6 to 14 in the preparation of a monoclonal immunoglobulin typing and identification kit.

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