Anti-histone antibody, anti-histone antibody immunodetection quality control product as well as preparation method and application of anti-histone antibody immunodetection quality control product

Abstract

The invention discloses an anti-histone antibody, an anti-histone antibody immunodetection quality control product as well as a preparation method and application of the anti-histone antibody immunodetection quality control product. The anti-histone antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 4-6, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of which the amino acid sequences are respectively shown as SEQ ID NO: 1-3. The anti-histone antibody has high specificity and low cross reactivity, and low-cost large-scale preparation can be realized. The quality control product based on the antibody has the advantages of excellent stability, high recovery rate and low matrix effect through formula and process optimization, is highly consistent with clinical serum, and significantly improves the detection reliability.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to anti-histone antibody, anti-histone antibody immunodetection quality control and its preparation method and application. BACKGROUND

[0002] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that can involve multiple organs and tissues of the body, and various autoantibodies and immune complexes can involve the heart, kidney, liver and other organs. The etiology and pathogenesis of SLE is complex. The production of autoantibodies is a major feature of SLE. In clinical examination, it is found that there are many autoantibodies in the serum of SLE patients. Autoantibody detection provides an important diagnostic basis for correct diagnosis and effective treatment of SLE. In clinical practice, anti-double-stranded DNA antibody (anti-dsDNA), anti-Sm antibody (anti-Sm), anti-nucleosome antibody (anti-nucleosome, AnuA) and anti-histone antibody (anti-hisTone antibody, AHA) are commonly considered as specific antibodies for the diagnosis of SLE.

[0003] Anti-histone antibody (AHA) is also an autoantibody that can be specifically expressed in various autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), and is related to RA bone erosion and systemic lupus erythematosus disease activity. The positive expression of AHA in the body of patients with systemic lupus erythematosus is related to age, lupus nephritis, complement system damage, etc., and monitoring its positive expression is helpful for early diagnosis and intervention of the disease.

[0004] The common mainstream detection platform on the market includes chemiluminescence immunoassay, chemiluminescence method, direct chemiluminescence method, magnetic particle chemiluminescence immunoassay, enzyme immunoassay, etc. The detection system needs to use matching quality control products for quality control. At present, there are few third-party anti-histone antibody IgG quality control products. SUMMARY

[0005] Therefore, it is necessary to provide anti-histone antibody, anti-histone antibody immunodetection quality control and its preparation method and application.

[0006] The first aspect of the present application provides an anti-histone antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 with the amino acid sequences shown in SEQ ID NO: 4~6, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 with the amino acid sequences shown in SEQ ID NO: 1~3, respectively.

[0007] In some embodiments, the constant region of the anti-histone antibody is a constant region of human IgG;

[0008] Optionally, the amino acid sequence of the heavy chain variable region is as set forth in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is as set forth in SEQ ID NO: 7;

[0009] Further optionally, the amino acid sequence of the heavy chain constant region is as set forth in SEQ ID NO: 10, and the amino acid sequence of the light chain constant region is as set forth in SEQ ID NO: 11.

[0010] A second aspect of the present application provides a nucleic acid molecule comprising an anti-histone antibody gene expression cassette fragment, wherein the anti-histone antibody gene expression cassette fragment encodes the anti-histone antibody of the first aspect of the present application.

[0011] A third aspect of the present application provides an expression vector comprising the nucleic acid molecule of the second aspect of the present application.

[0012] Optionally, the expression vector comprises m copies of the anti-histone antibody gene expression cassette fragment, wherein m is an integer greater than or equal to 1.

[0013] Further optionally, the m is 3.

[0014] A fourth aspect of the present application provides a host cell comprising the expression vector of the third aspect of the present application.

[0015] Optionally, the host cell comprises Pichia pastoris.

[0016] Optionally, the host cell is further capable of expressing a helper protein.

[0017] Further optionally, the helper protein comprises one or more of Ssa4, Bmh2, and VHb.

[0018] Further optionally, the host cell further comprises a second expression vector, wherein the second expression vector comprises a gene encoding the helper protein.

[0019] A fifth aspect of the present application provides an anti-histone antibody immunoassay quality control product, wherein the quality control product comprises the anti-histone antibody of the first aspect of the present application.

[0020] In some embodiments, the quality control product further comprises a matrix solution.

[0021] Optionally, the matrix solution comprises one or more of a matrix, a buffer, a protein stabilizer, a lyoprotectant, an excipient, an adjuvant, and a preservative, wherein the matrix comprises matrix serum, the buffer comprises 2-morpholinoethanesulfonic acid buffer, the protein stabilizer comprises casein hydrolysate, the lyoprotectant comprises trehalose, the excipient comprises D-mannitol, and the preservative comprises Proclin 300;

[0022] Further optionally, the matrix solution comprises one or more of matrix serum, 0.95% (w / v)~1% (w / v) 2-(N-morpholino)ethanesulfonic acid or a hydrate thereof, 0.23% (w / v)~0.27% (w / v) casein hydrolysate, 3% (w / v)~5% (w / v) D-mannitol, 0.8% (w / v)~1.2% (w / v) trehalose, and 0.08% (w / v)~0.12% (w / v) Proclin 300.

[0023] The sixth aspect of the present application provides a method for preparing an anti-histone antibody immunodetection quality control product, comprising mixing the anti-histone antibody of the first aspect of the present application with a matrix solution and lyophilizing to obtain a dry quality control product.

[0024] Optionally, the matrix solution comprises one or more of matrix serum, 0.95% (w / v)~1% (w / v) 2-(N-morpholino)ethanesulfonic acid monohydrate, 0.23% (w / v)~0.27% (w / v) casein hydrolysate, 3% (w / v)~5% (w / v) D-mannitol, 0.8% (w / v)~1.2% (w / v) anhydrous trehalose, and 0.08% (w / v)~0.12% (w / v) Proclin 300.

[0025] In some embodiments, the lyophilizing step comprises pre-freezing and drying.

[0026] Optionally, the pre-freezing conditions comprise pre-freezing at a temperature of -8°C~-12°C for 30min~50min, pre-freezing at a temperature of -38°C~-42°C for 1.4h~1.6h, and pre-freezing at a temperature of -40°C~-50°C for 3.4h~3.6h.

[0027] Optionally, the drying comprises primary drying and secondary drying, and the primary drying conditions comprise a vacuum degree of 0.18mbar~0.22mbar, and a temperature of -28°C~-32°C for 21h~23h, and then warming to -8°C~-12°C for 20min~40min.

[0028] The secondary drying condition includes a vacuum degree of 0.18 mbar to 0.22 mbar, a temperature of 8°C to 12°C, and a duration of 6 h to 8 h.

[0029] The seventh aspect of the present application provides an application of the anti-histone antibody of the first aspect of the present application in preparing the anti-histone antibody immunoassay quality control product of the fifth aspect of the present application; optionally, the anti-histone antibody immunoassay quality control product is suitable for an acridinium ester chemiluminescence detection platform.

[0030] The eighth aspect of the present application provides an anti-histone antibody immunoassay kit, which comprises the anti-histone antibody immunoassay quality control product of the fifth aspect of the present application or the anti-histone antibody immunoassay quality control product obtained by the preparation method of the sixth aspect of the present application.

[0031] The ninth aspect of the present application provides an application of the anti-histone antibody immunoassay quality control product of the fifth aspect of the present application, or the anti-histone antibody immunoassay quality control product obtained by the preparation method of the sixth aspect of the present application, or the kit of the eighth aspect of the present application in detecting an anti-histone antibody.

[0032] Optionally, the detecting the anti-histone antibody comprises the following steps: respectively detecting optical signals in a to-be-detected antibody sample and the anti-histone antibody immunoassay quality control product by using a chemiluminescence method; comparing and analyzing detection results of the to-be-detected antibody sample and the anti-histone antibody immunoassay quality control product to obtain quality information or antibody content information of the to-be-detected antibody sample.

[0033] Optionally, the detecting the anti-histone antibody adopts an acridinium ester chemiluminescence method.

[0034] The foregoing anti-histone antibody greatly reduces the non-specific cross-reaction that may be caused in a human immune detection system, improves the specificity and accuracy of detection, and can be mass-produced with low raw material cost. Further, the foregoing chimeric antibody is provided with the quality control product prepared by using an optimized formula, the quality control product exhibits excellent stability and can maintain reliable activity under different storage conditions; at the same time, the quality control product has a high recovery rate, effectively reflects the antibody content in a real sample, has low matrix effect, has good consistency with a clinical serum sample, and significantly improves the reliability and comparability of a detection result. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments and examples of the present application, and more completely understand the present application and its beneficial effects, the accompanying drawings needed to be used in the description of the embodiments or examples will be briefly introduced. Obviously, the accompanying drawings in the following description only represent some of the embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0036] Figure 1 A schematic diagram for increasing the gene copy number of pAO815 vector and homotail enzyme method in an embodiment of the present application, wherein a is a schematic diagram of pAO815 vector structure, and b is a schematic diagram of increasing the AHA copy number by using the homotail enzyme method;

[0037] Figure 2 A plasmid map of pFUSE-CHIg-hG1 in an embodiment of the present application;

[0038] Figure 3 A plasmid map of pFUSE2-CLIg-hk in an embodiment of the present application;

[0039] Figure 4 A gel electrophoresis verification of AHA IgG protein in an embodiment of the present application;

[0040] Figure 5 A linear relationship diagram of detecting quality control product matrix liquid 1 and clinical serum sample in an embodiment of the present application;

[0041] Figure 6 A linear relationship diagram of detecting quality control product matrix liquid 2 and clinical serum sample in an embodiment of the present application;

[0042] Figure 7 A linear relationship diagram of detecting quality control product matrix liquid 3 and clinical serum sample in an embodiment of the present application. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings. The preferred embodiments of the present application are shown in the accompanying drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein only for the purpose of describing specific embodiments and is not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0045] The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or a combination of all related listed items.

[0046] In the present application, "multiple", "various", "multiple times", "multiple", etc. are used without specific limitation, which means more than two or equal to two in quantity. For example, "one or more" means one or more than two.

[0047] As used herein, "combination thereof", "any combination thereof", "any combination thereof", etc. include all suitable combinations of any two or more listed items.

[0048] In this article, "suitable combination", "suitable way", "any suitable way", etc. The "suitable" in the above-mentioned terms means that the technical solutions of the present application can be implemented, the technical problems of the present application can be solved, and the expected technical effects of the present application can be achieved.

[0049] In this article, "preferably", "better", "better", "preferably" only describes the better implementation or embodiment, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0050] In the present application, "further", "more further", "particularly" and the like are used for description purposes, indicating differences in content, but should not be understood as limiting the protection scope of the present application.

[0051] In the present application, "optionally", "optional", "optional" means optional, that is, selected from two parallel schemes of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified, there is no contradiction or mutual restriction relationship, and each "optional" is independent.

[0052] In the present application, the terms "first", "second", etc. in "first aspect", "second aspect", etc. are only used for description purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", etc. only serve the purpose of non-exhaustive enumeration description, and it should be understood that it does not constitute a closed limitation on the quantity.

[0053] In the present application, the technical features described in an open manner include both the closed technical solutions composed of the listed features and the open technical solutions containing the listed features.

[0054] In the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical end points (i.e. the minimum value and the maximum value) of the numerical range, and every number between the two numerical end points. When a numerical interval refers only to integers within the numerical interval, unless otherwise specified, the two end point integers of the numerical range, and every integer between the two end points, are considered to be directly enumerated in the present disclosure, such as when t is an integer selected from 1-10, meaning that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein are to be understood as including any and all sub-ranges subsumed therein.

[0055] In the present application, unless otherwise specified, the temperature parameter allows for both constant temperature treatment and fluctuations within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0056] In the present application, %(w / w) and wt% both refer to weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass volume percentage.

[0057] Currently, AHA is expressed using natural serum or prokaryotic expression systems such as E. coli, or prepared using murine monoclonal antibody technology. However, there are the following disadvantages: 1) Natural high-value serum is difficult to obtain in large quantities, requires additional storage costs, has relatively low purity, and the activity is difficult to meet the dilution ratio requirement, is contaminated with impurities and poses a biological safety risk, and high-value samples are difficult to collect; when expressed using a prokaryotic expression system, post-translational modification is lacking, and inclusion bodies are easily formed, making purification difficult, affecting protein function, and making it difficult to obtain raw materials. 2) The preparation steps of murine monoclonal antibodies are complicated, and the murine antibodies may have cross-reactivity with non-target proteins, resulting in binding of the murine antibodies to non-target antigens, which can cause false positives or false negatives in experimental results.

[0058] Currently, liquid or lyophilized anti-histone IgG antibody quality control products are prepared using traditional buffer formulations such as Tris and PBS buffer; or anti-histone IgG antibody quality control products are prepared by supplementing a single saccharide excipient to a negative serum matrix. However, the reconstitution or bottle-opening stability is poor.

[0059] Based on this, the embodiments of the present application at least provide an anti-histone antibody, an anti-histone antibody immunodetection quality control product, and a preparation method thereof.

[0060] In the present application, the term "light chain" refers to the smaller chain of an immunoglobulin molecule. The basic structure of an immunoglobulin molecule is a symmetrical structure composed of four polypeptide chains connected by disulfide bonds, called monomers, in the shape of a "Y" or "T".

[0061] In the present application, the term "heavy chain" refers to two identical long chains consisting of 450-550 amino acid residues, which are relatively large in molecular weight.

[0062] In the present application, the term "chimeric antibody" refers to an antibody having at least a portion of the heavy chain variable region and at least a portion of the light chain variable region derived from one species; and at least a portion of the constant region derived from another species. For example, a chimeric antibody can include a murine variable region and a human constant region. A "humanized antibody" refers to an antibody containing complementarity determining regions (CDRs) derived from a non-human antibody, and framework regions and constant regions derived from a human antibody.

[0063] In the present application, the term "complementarity determining region" or "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed" US Department of Health and Human Services, NIH, 1991, and subsequent editions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the case, the term "CDR" is used to refer to a region containing one or more or even all of the major amino acid residues that play a role in the binding affinity of the antibody to the antigen or epitope it recognizes. In another specific embodiment, CDR refers to the highly variable regions of the heavy and light chains of immunoglobulins defined by IMGT.

[0064] In the present application, the term "variable region" refers to the region of the immunoglobulin light and heavy chains near the N-terminal amino acid sequence, which varies greatly.

[0065] In the present application, the term "constant region" refers to the C-terminal amino acid, which is relatively stable and varies little.

[0066] In the first aspect of the present application, an anti-histone antibody is provided, which includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NO: 4~6, respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 1~3, respectively.

[0067] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7.

[0068] In some embodiments, the antihistone antibody is a chimeric antibody, the constant region of which is the constant region of human IgG; further, the constant region of human IgG includes the light chain constant region and the heavy chain constant region of human IgG; even further, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 10.

[0069] A second aspect of this application provides a nucleic acid molecule comprising an antihistone antibody gene expression cassette fragment encoding the aforementioned antihistone antibody.

[0070] In this application, the term "nucleic acid molecule" primarily refers to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffer solutions, or salts.

[0071] In this application, the term "gene expression cassette fragment" refers to a DNA fragment containing necessary regulatory elements and the coding sequence of the target gene, which can guide the transcription and translation of the target gene in the host cell.

[0072] A third aspect of this application provides an expression vector comprising the aforementioned nucleic acid molecule.

[0073] In some embodiments, the expression vector can be obtained by conventional methods in the art, for example, by linking the nucleic acid molecules described in this application to various expression vectors. The expression vector can accommodate the nucleic acid molecules described in this application; for example, the expression vector may include plasmids, granules, bacteriophages, or viral vectors.

[0074] In this application, the term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are efficiently linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), also serve equivalent functions.

[0075] In some embodiments, the expression vector includes m copies of an anti-histone antibody gene expression cassette fragment, where m is an integer greater than or equal to 1. Non-limitingly, m can be, but is not limited to, 1, 2, 3, or 4. Further, m is 3.

[0076] In a fourth aspect of this application, a host cell is provided that includes the expression vector described above.

[0077] In this application, "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; members of the Bacillaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. Suitable microorganisms include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0078] In some implementations, the host cell includes Pichia pastoris.

[0079] Pichia pastoris is a methanol-nutritional yeast, utilizing methanol as its sole carbon source. Pichia pastoris contains two alcohol oxidases: AOX1 and AOX2. The AOX1 gene encodes most alcohol oxidases. By transforming the target gene into the yeast genome and supplementing with an appropriate amount of methanol, the expression of the foreign gene can be induced in large quantities. Promoter, gene copy number, and co-expression of accessory proteins are key factors affecting the expression of foreign proteins in Pichia pastoris. Post-translational modification and secretion processes are another challenge in the expression level of heterologous proteins. Co-expression of accessory proteins with the target protein helps the foreign protein fold correctly, thereby improving the synthesis and secretion of the target protein. Accessory proteins promote the secretion of foreign proteins extracellularly by assisting in correct folding and reducing intracellular accumulation. For example, the cytoplasmic molecular chaperone Ssa4, a member of the Hsp70 family, is responsible for guiding nascent proteins to the endoplasmic reticulum membrane, and the 14-3-3 isoform chaperone protein Bmh2 plays an important role in exocytosis and vesicle transport.

[0080] In some implementations, the host cell may also express accessory proteins. Further, accessory proteins include one or more of Ssa4, Bmh2, and VHb.

[0081] In some embodiments, the host cell further includes a second expression vector, which includes the gene encoding the aforementioned accessory protein.

[0082] It should be noted that the embodiments of this application use molecular biology techniques to screen for suitable chimeric antibody AHA gene sequences, and promote their exogenous secretion expression using Pichia pastoris, so that the detection effect is as close as possible to that of human AHA protein. This method can improve molecular stability; the preparation of anti-histone antibody quality control products avoids the risks of difficult raw material collection and low potency of purchased raw materials; using Pichia pastoris for AHA expression can increase AHA yield and significantly reduce raw material costs.

[0083] In a fifth aspect of this application, an antihistone antibody immunoassay quality control product is provided, comprising the aforementioned antihistone antibody.

[0084] In some implementations, the quality control material for anti-histone antibody immunoassay also includes a matrix solution.

[0085] In some implementations, the quality control material for anti-histone antibody immunoassay is prepared by mixing anti-histone antibodies and matrix solution in a specific ratio, which is adjusted according to the concentration of the quality control material.

[0086] In some embodiments, the matrix solution includes one or more of a matrix, a buffer, a protein stabilizer, a lyophilization protectant, an excipient, an adjuvant, and a preservative, wherein the matrix includes matrix serum, the buffer includes 2-morpholine ethanesulfonic acid buffer, the protein stabilizer includes casein hydrolysate, the lyophilization protectant includes trehalose, the excipient includes D-mannitol, and the preservative includes Proclin 300. Further, the matrix solution includes one or more of the following: matrix serum, 0.95% (w / v) to 1% (w / v) of 2-(N-morpholino)ethanesulfonic acid or its hydrate, 0.23% (w / v) to 0.27% (w / v) of casein hydrolysate, 3% (w / v) to 5% (w / v) of D-mannitol, 0.8% (w / v) to 1.2% (w / v) of trehalose, and 0.08% (w / v) to 0.12% (w / v) of Proclin 300.

[0087] In some embodiments, the concentration of 2-(N-morpholino)ethanesulfonic acid in the matrix solution may be, but is not limited to, 0.95% (w / v), 0.96% (w / v), 0.97% (w / v), 0.98% (w / v), 0.99% (w / v), 1% (w / v), or any value or range between two of the above.

[0088] In some embodiments, the concentration of casein hydrolysate in the matrix solution may be, but is not limited to, 0.23% (w / v), 0.24% (w / v), 0.25% (w / v), 0.26% (w / v), 0.27% (w / v), or any value or range between two of the above.

[0089] In some embodiments, the concentration of D-mannitol in the matrix solution may be, but is not limited to, 3% (w / v), 4% (w / v), 5% (w / v), or any value or range between two of the above.

[0090] In some embodiments, the concentration of trehalose in the matrix solution may be, but is not limited to, 0.8% (w / v), 0.9% (w / v), 1.0% (w / v), 1.1% (w / v), 1.2% (w / v), or any value or range between two of the above.

[0091] In some embodiments, the concentration of Proclin 300 in the matrix solution may be, but is not limited to, 0.08% (w / v), 0.09% (w / v), 0.1% (w / v), 0.11% (w / v), 0.12% (w / v), or any value or range between two of the above.

[0092] The sixth aspect of this application provides a method for preparing an anti-histone antibody immunoassay quality control, comprising mixing the above-mentioned anti-histone antibody with the above-mentioned matrix solution to obtain the quality control.

[0093] In some embodiments, the above-mentioned quality control samples are freeze-dried to obtain dry quality control samples.

[0094] In some implementations, the freeze-drying steps include pre-freezing and drying;

[0095] Furthermore, the pre-freezing conditions include: pre-freezing at -8℃ to -12℃ for 30 min to 50 min; pre-freezing at -38℃ to -42℃ for 1.4 h to 1.6 h; and pre-freezing at -40℃ to -50℃ for 3.4 h to 3.6 h.

[0096] Furthermore, the drying process includes primary drying and secondary drying. The conditions for primary drying include: a vacuum of 0.18 mbar to 0.22 mbar, maintained at -28°C to -32°C for 21 to 23 hours; and then heated to -8°C to -12°C for 20 to 40 minutes.

[0097] The conditions for secondary drying include a vacuum of 0.18 mbar to 0.22 mbar and a temperature of 8°C to 12°C for 6 to 8 hours.

[0098] It should be noted that in the embodiments of this application, hormone-free serum is used as the matrix, and hydrolyzed casein, anhydrous trehalose, D-mannitol and defoamer are added as protein protectants and lyophilization excipients for AHA quality control products, providing a stable solution matrix and improving the stability of the quality control products; using the above-mentioned optimized lyophilization process, the recovery yield is significantly improved.

[0099] The seventh aspect of this application provides the use of the aforementioned antihistone antibody in the preparation of the aforementioned antihistone antibody immunoassay quality control product; further, the antihistone antibody immunoassay quality control product is suitable for an acridinium ester chemiluminescence detection platform.

[0100] The eighth aspect of this application provides an anti-histone antibody immunoassay kit, comprising the above-described anti-histone antibody immunoassay quality control or the anti-histone antibody immunoassay quality control obtained by the above-described preparation method.

[0101] The ninth aspect of this application provides the use of the above-described antihistone antibody immunoassay quality control, or the antihistone antibody immunoassay quality control obtained by the above-described preparation method, or the above-described kit in the detection of antihistone antibodies.

[0102] In some embodiments, detecting antihistone antibodies includes the following steps: using chemiluminescence to detect the optical signals in the antibody sample to be tested and the antihistone antibody immunoassay quality control, respectively; comparing and analyzing the detection results of the antibody sample to be tested and the antihistone antibody immunoassay quality control to obtain the quality information or antibody content information of the antibody sample to be tested.

[0103] In some implementations, the antibody sample to be tested includes one or more of serum and plasma.

[0104] In some implementations, the detection of antihistone antibodies is performed using acridinium ester chemiluminescence.

[0105] The following are some examples.

[0106] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0107] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0108] In the following examples, anhydrous trehalose and D-mannitol were purchased from Sinopharm Chemical Reagent Co., Ltd.; 2-(N-morpholino)ethanesulfonic acid monohydrate, Proclin 300, and casein hydrolysate were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0109] Unless otherwise specified, all test materials used in the following examples are commercially available products.

[0110] Example 1: Immunogen Preparation (Obtaining Human Histones)

[0111] Based on the human histone sequences published by NCBI, the following human histone gene sequence was synthesized by Nanjing GenScript:

[0112] Human histone H3 amino acid sequence (aa1-136), C-terminal fused 3×flag-tag

[0113] MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERADYKDDDDKGDYKDDDDKIDYKDDDDK (SEQ ID NO: 9)

[0114] Example 2: Preparation of mouse AHA monoclonal antibody

[0115] Animal immunization: The prepared histones were used as immunogens to immunize mice, and whole-mouse anti-histone IgG monoclonal antibodies were prepared. Histones were mixed with an equal volume of Freund's adjuvant and emulsified repeatedly using a medical three-way tube and syringe, generally until the emulsified mixture remained in a droplet form on the surface of water without rapidly spreading. After thorough emulsification, 25 μg was injected into six Balb / c mice each, for a total of three immunizations, with an interval of 15 days.

[0116] Potency confirmation: Blood was collected from the tail vein, and the serum was used as the primary antibody to perform indirect ELISA to determine the serum titer of mice. When the serum dilution factor was 12800, an OD value ≥1 was considered to meet the fusion criteria.

[0117] Cell fusion: (1) Resuspend mouse myeloma cells (SP2 / 0 cells) and immune mouse spleen cells in 1640 incomplete medium, count them separately using a hemocytometer, and mix them in a 50ml sterile centrifuge tube at a ratio of spleen cells:SP2 / 0 cells = 1:3. After thorough mixing, add 1640 incomplete medium to 40mL; (2) Centrifuge at 1500rpm for 5min. (At this time, prepare 37℃ warm water for incubation of the cell fusion centrifuge tube) (3) After centrifugation, discard the supernatant and the liquid on the tube wall (use sterile absorbent paper strips to dry), gently tap the SP2 / 0 cells and immune mouse cells mixture to loosen it, and immerse the bottom of the centrifuge tube in 37℃ warm water. (4) Take out 1ml of incubated fusion agent PEG1450 from the incubator and add it evenly to the cell mixture mixture within 60s (rotate the centrifuge tube while adding). (5) After incubating for 45 seconds, take out the 1640 incomplete culture medium preheated in the 37℃ incubator, take 1 mL, and add it evenly to the precipitate over 60 seconds to dilute the PEG fusion agent (while rotating the centrifuge tube). Take another 1 mL and add it evenly over 30 seconds. Then slowly add the remaining 45 mL of culture medium. Use HAT selective medium to select hybridoma cells on a 96-well cell culture plate. Under a microscope, the total fusion rate is >95%. The supernatant of the histone-coated monoclonal cell wells is tested. Cells in wells with OD450 >0.8 are selected for subcloning. Finally, cell clones with a histone reaction positivity rate >99% are obtained as hybridoma cell lines secreting anti-human AHA IgG monoclonal antibodies.

[0118] Cell cloning: The obtained positive cell lines were cloned using limiting dilution, and cloned three times to obtain five hybridoma cell lines that produced high-titer anti-human AHA IgG monoclonal antibodies. These cells were then expanded and cryopreserved.

[0119] Ascites preparation: Female Balb / c mice aged 6-8 weeks were treated with paraffin for 10 days, and AHA IgG hybridoma cells were collected and processed at 2×10⁻⁶ cells / mL. 6 One cell per mouse was injected intraperitoneally. Seven days later, ascites rich in AHA IgG antibodies was collected from the mouse peritoneum.

[0120] Example 3: Amplification of the variable region gene of mouse AHA IgG monoclonal antibody and the constant region gene of human IgG antibody to synthesize mouse-human light and heavy chain genes.

[0121] Take approximately 10 hybridoma cell lines in the logarithmic growth phase. 7 Total RNA was extracted from cells according to the instructions of the OMEGA RNA Extraction Kit, and then RT-PCR amplification was performed. The amplified products were recovered and purified for later use.

[0122] Using primers designed by Orlandi et al. (1989) for amplifying the variable region of mouse genes, the variable region of the antibody gene was amplified using the amplification product as a template. The heavy chain VH (approximately 360 bp) and light chain VL (approximately 330 bp) fragments were recovered. The plasmid pFUSE-CHIg-hG1 (plasmid diagram shown in Figure 1) containing the human IgG heavy chain constant region was then used. Figure 2 As shown, the plasmids were purchased from Invivoge (product number: pfuse-hchg1) and the human light chain constant region plasmid pFUSE2-CLIg-hk (plasmid image shown). Figure 3 As shown, using a template (purchased from Invivoge, catalog number: pfuse2-hclk), the human heavy chain constant region gene CH and the human light chain constant region gene CL of IgG were amplified to synthesize AHAIgG. Three samples were sent for sequencing. The obtained sequences were classified and analyzed for homology in the GeneBank nucleic acid database. The amino acid and gene sequences of the AHAIgG mouse-human light and heavy chain genes are as follows:

[0123] Light chain variable region amino acid sequence

[0124] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPLTFGAGTKLELK (SEQ ID NO: 7)

[0125] amino acid sequence of the light chain constant region

[0126] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10)

[0127] LCDR1: KASQDVGTAVA (SEQ ID NO: 1)

[0128] LCDR2: WASTRHT (SEQ ID NO: 2)

[0129] LCDR3: QQYSSYPLT (SEQ ID NO: 3)

[0130] Heavy chain variable region amino acid sequence

[0131] QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARRSNSPFAYWGQGTLVTVSA (SEQ ID NO: 8)

[0132] Heavy chain constant region amino acid sequence

[0133] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

[0134] HCDR1:SYWMH (SEQ ID NO: 4)

[0135] HCDR2: EINPSNGRTNYNEKFKS (SEQ ID NO: 5)

[0136] HCDR3:RSNSPFAY (SEQ ID NO: 6)

[0137] Example 4: Construction of AHA IgG expression vector for mouse-human light and heavy chain genes

[0138] Glycerol-containing bacteria (recombinant plasmids of heavy and light chain pFUSE) were inoculated onto LB+Amp solid plates using an inoculation loop. After single colonies grew, they were picked with a sterile toothpick and transferred to LB+Amp liquid medium. The plates were incubated overnight at 37°C with a shaker. Once the bacterial cells became turbid, the plasmids were extracted using the Omega plasmid extraction kit instructions. Using the plasmids as templates, primers containing the 5'EcoRI gene and the 5'NotRI gene were designed to synthesize both heavy and light chain AHA IgG. The complete AHA IgG gene sequence was obtained using overlap extension PCR via the GSSSS linker. Successful gene construction was indicated by verification of correct sequence.

[0139] The AHA IgG blunt-terminated gene fragment and the pAO815 vector were digested using EcoRI and NotI restriction endonucleases from Nanjing Novizan, respectively, yielding sticky-terminated AHA IgG gene fragments and pAO815 vector double-digested fragments. The gene and vector fragments were then ligated using a reaction system containing T4 ligase, and heat-transformed into *E. coli* BL21(DE3). The colonies were screened on LB agar plates containing ampicillin (Amp). Colony growth was verified by colony PCR using primers at both ends of the gene. After successful verification, the colonies were inoculated into LB+Amp liquid medium. Once the medium became turbid, plasmid extraction was performed using an Omega plasmid extraction kit. After successful PCR verification, the samples were sent to BGI Genomics for gene sequencing. Correct sequencing indicated successful construction of the pAO815-AHA IgG vector.

[0140] Example 5: Increasing gene copy number using the same-tailed enzyme method

[0141] In the pAO815 plasmid vector, Bgl II and BamHI are isoskeletal enzymes. After ligation, the original restriction sites disappear. Therefore, by selecting a reference enzyme site, a repeat can be "added" to the target gene expression fragment on the pAO815 vector. Figure 1 As shown. A portion of the PAO815-AHA IgG plasmid was digested with BamHI and SalI, and purified by gel extraction to obtain a vector fragment with the downstream sequence of the His4 gene removed. Another portion of the PAO815-AHA IgG plasmid was digested with BglII and SalI, and purified to obtain a gene expression cassette fragment containing the downstream sequence of the His4 gene. The structure of the pAO815 vector is shown below. Figure 1 As shown in (a), the double enzyme digestion process is as follows: Figure 1As shown in (b) above. The enzyme-digested and purified vector and the AHA IgG gene expression cassette fragment were ligated, transformed into E. coil Top10 competent cells, and plated on LB plates containing 100 μg / mL Amp. Positive clones were screened by colony PCR using His4-F / His4-R primers, and their plasmids were verified by enzyme digestion. The correct recombinant vector was identified as two "AHA IgG" gene copy vectors, named PAO815-2AHA IgG.

[0142] Similarly, the PAO815-nAHA IgG (n=1,2,3…) plasmid containing n copies of the gene expression cassette was digested with BamHI and SalI, and the fragment was purified by gel extraction to obtain a vector fragment with the downstream sequence of the His4 gene removed. The PAO815-mAHA IgG (m=1,2,3…) plasmid was digested with BglII and SalI, and the fragment containing the downstream sequence of the His4 gene and a fragment containing m copies of the AHA IgG gene expression cassette was purified. The two fragments were ligated to obtain the recombinant vector pAO815-(n+m)AHA IgG containing n+m copies of the AHA IgG gene expression cassette. Due to plasmid size limitations, a higher gene copy number is not always better. This application verified that the protein expression level of AHA IgG was highest when the copy number was 3.

[0143] Example 6: Screening of strains co-expressing accessory proteins

[0144] Since higher copy numbers can increase protein expression levels, they also increase endoplasmic reticulum stress. Co-expression of accessory proteins can reduce protein misfolding and further enhance AHA IgG expression. These include the cytoplasmic molecular chaperone Ssa4, responsible for transporting nascent proteins to the endoplasmic reticulum membrane; the 14-3-3 type protein Bmh2, involved in protein exiting the endoplasmic reticulum; and the VHb gene, which effectively improves oxygen utilization in Pichia pastoris fermentation.

[0145] Using the Pichia pastoris genome as a template, the designed primer pairs Ssa4-F / Ssa4-R, Bmh2-F / Bmh2-R, and VHb-F / VHb-R were used respectively (where the Ssa4-F primer contains the EcoRI site, the VHb-R primer contains the NotI restriction site, the Ssa4-R and Bmh2-R primers contain the reverse complementary sequence of the GSSSS Linker (repeated twice, GSSSSGSSSS), and the Bmh2-F and VHb-F primers contain the G4S Linker sequence).

[0146] Overlap extension PCR: The overlap extension PCR procedure is shown in Table 1. The Ssa4-Linker-Bmh2 fusion protein gene sequence was obtained through the first round of overlap extension PCR in Table 1. The second round of overlap extension PCR was performed using the fusion gene as a template, as shown in Table 2, to obtain the gene sequence of the Ssa4-Linker-Bmh2-Linker-VHb fusion protein, hereinafter referred to as Ssa4-Bmh2-VHb. At this time, the 5' end of Ssa4 contains the EcoRI site, and the 3' end of VHb contains the NotI restriction site. The fusion gene product and plasmid pPICZαA were recovered by PCR gel digestion and digested with EcoRI and NotI restriction endonucleases from Novizumab, respectively, to recover the gene fragment Ssa4-Bmh2-VHb and the vector linear fragment pPICZαA. The fragments were ligated using a ligation system containing T4 ligase, transformed into E. coli Top10 competent cells, and plated on LB agar plates containing 25 μg / mL bleomycin. Colonies were validated by PCR, plasmids were extracted, and sequenced. Successful validation indicated successful construction, and the vector was named pPICZαA-Ssa4-Bmh2-VHb gene expression vector.

[0147] Table 1

[0148]

[0149] Table 2

[0150]

[0151] The obtained PAO815-2 AHA IgG and pPICZαA-Ssa4-Bmh2-VHb gene expression vectors were linearized with SalⅠ and Sac Ⅰ, respectively, and integrated into the His4 and 5'AOX1 regions of Pichia pastoris GS115. Both integration methods can produce his+ and mut+ strains, that is, strains that can synthesize histidine and utilize methanol. To improve transformation efficiency, GS115 cells were transformed using a two-step electroporation process. The preparation of competent GS115 cells and the electroporation transformation steps were performed according to the *Pichia pastoris Expression Manual*. In the first step, the PAO815-2 AHA IgG gene plasmid was linearized using SalI, and GS115 competent cells were transformed by electroporation. Cells were screened using YPDS+ampicillin solid plates. After single colonies emerged, they were inoculated into YPD+ampicillin liquid medium, and yeast genome was extracted. The procedure followed the instructions for the Tiangeng yeast genome extraction kit, and verification was performed using His4-F / His4-R primers. In the second step, Sac... I. Linearize the pPICZαA-Ssa4-Bmh2-VHb gene expression vector, transform the yeast strain (which has been prepared into competent cells) that was verified in the first step of electroporation, screen it with YPD+bleomycin solid plates, inoculate it into a vial after single colonies have grown, extract the yeast genome, and verify it with Ssa4-F / VHb-R. If it is correct, it is a successfully constructed co-expression strain.

[0152] Example 7: Induction, purification, and validation of AHA IgG

[0153] The effects of temperature, pH, and methanol addition on yeast expression levels were explored. Using a controlled variable method, single variable conditions were changed each time, and the optimal expression levels under various conditions were compared. The optimal temperature for controlling AHA IgG expression in Pichia pastoris was determined to be 28.5℃, the pH of the culture medium was 5.3, and the methanol addition was 2.0%. The protein purity was roughly estimated using ImageJ.

[0154] Fermentation supernatant acquisition: After culturing Pichia pastoris containing the target gene for 5 days, the fermentation broth in the shake flask was centrifuged. The first centrifugation was performed at 4000-5000 rpm for 10 minutes to remove yeast cells and obtain the fermentation supernatant. The second centrifugation was performed at 12000 rpm for 30 minutes to remove large aggregates or broken cells and other impurities to prevent clogging of the filter column during filtration.

[0155] Protein purification: Take an appropriate amount of well-mixed 50% BeyoGold™ His-tag Purification Resin, centrifuge at 4°C (1000g × 10s), discard the stock solution, add one column volume of non-denaturing lysis buffer to the gel and mix well to equilibrate the gel, centrifuge at 4°C (1000g × 10s), discard the liquid, and repeat the equilibration process 1-2 times, discarding the liquid each time. Add 4 mL of fermentation supernatant to every 0.5 mL of gel (equivalent to 1 mL of 50% gel) (1:8 ratio), mix BeyoGold™ His-tag Purification Resin and bacterial lysis supernatant, and gently shake at 4°C on a side-shaking or horizontal shaking table for 60 min to ensure sufficient binding of the protein to nickel ions in the gel. Open the cap at the bottom of the purification column and allow the liquid inside the column to flow out under gravity, collecting approximately 20 μL of the flow-through for subsequent analysis. The column was washed 5 times, with 1-2 column volumes of non-denaturing wash buffer added each time. Approximately 20 μL of wash buffer was collected after each wash for subsequent analysis. For example, the column volume after packing 1 mL of well-mixed 50% BeyoGold™ His-tag Purification Resin is 0.5 mL, meaning the wash volume for each wash after packing 1 mL of well-mixed 50% BeyoGold™ His-tag Purification Resin is 0.5 mL. The content of each elution was analyzed using a nano-drop micro-analyzer. It was found that most of the protein was eluted by the second elution. A 150 mM imidazole eluent maximized the elution of AHA IgG protein.

[0156] Concentration calculation: A protein concentration standard curve was prepared using the Coomassie Brilliant Blue G-250 method, and the concentration of AHAIgG protein in the eluent was measured to be 2.0 mg / mL.

[0157] Electrophoretic analysis: The theoretical molecular weights of AHA and IgG are approximately [values ​​missing]. The denaturing electrophoresis results are as follows: Figure 4 As shown, the heavy chain and light chain separate into two bands, and the correct protein expression was confirmed by mass spectrometry analysis by Suzhou Putai.

[0158] Purity analysis: The electrophoresis image of the purified AHA IgG protein was analyzed using ImageJ software. It was found that the concentration of the purified protein could reach 90%, which basically met the test requirements. 1M phosphate buffer containing 150mM imidazole was used as the protein storage solution for AHA IgG.

[0159] Example 8: AHA IgG Activity Detection

[0160] Activity assays were performed using commercially available AHA IgG raw materials from Feipeng Biotechnology and purified AHA IgG raw materials. The two anti-histone antibody raw materials were diluted with commonly used Tris buffer at 10-fold, 20-fold, 40-fold, 80-fold, 160-fold, and 320-fold dilutions, respectively. The assays were performed using an anti-histone IgG antibody assay kit (chemiluminescence method) and chemiluminescence immunoassay analyzer manufactured by Shenzhen Yahuilong Biotechnology Co., Ltd. Each sample was tested three times. The results are shown in Table 3. The results indicate that the AHA IgG purified from Pichia pastoris had the highest backcalculation concentration, reaching 2000 Au / mL, reducing costs by approximately 50% or more, significantly lowering raw material costs.

[0161] Table 3

[0162]

[0163] Example 9: Screening and Preparation of Anti-Histone IgG Antibody Quality Control Product Formulation

[0164] Considering the matrix effect, matrix serum or animal serum was used as the base matrix. Lyophilization protectants such as mannitol, excipients such as anhydrous trehalose, and other protein stabilizers such as casein and bovine serum albumin were added to various base matrices. First, three-level quality control samples were prepared using matrix solutions with different component formulations. Then, the lyophilized morphology, recovery rate, matrix effect, inter-bottle homogeneity, accelerated aging stability, and reconstitution stability of each experimental group were analyzed to screen for the most suitable matrix formulation.

[0165] Prepare the matrix solution according to the following three formulations.

[0166] Matrix solution 1: Newborn calf serum + 0.1% Proclin 300 + 4% D-mannitol + 1% anhydrous trehalose.

[0167] Matrix solution 2: Universal matrix serum + 0.1% Proclin 300 + 4% D-mannitol + 1% anhydrous trehalose.

[0168] Matrix solution 3: Matrix serum + 0.97% 2-(N-morpholino)ethanesulfonic acid monohydrate + 0.25% casein hydrolysate + 4% D-mannitol + 1% anhydrous trehalose + 0.1% Proclin 300.

[0169] (1) Preparation of quality control materials

[0170] Anti-histone IgG chimeric antibodies were added to the above three formulations to prepare three concentrations of quality control samples: 8.8±0.8 AU / mL, 66±6 AU / mL, and 110±10 AU / mL. The samples were then lyophilized and vacuum-capped for sampling.

[0171] (2) Freeze-drying of quality control products

[0172] After preparing the quality control sample according to the above formula, the optimized freeze-drying procedure of this application (Table 4) is called to freeze-dry the quality control sample. After the freeze-drying is completed, the sample is capped and taken under vacuum.

[0173] Table 4

[0174]

[0175] Example 10: Validation of the appearance, recovery rate, matrix effect, homogeneity, and stability of anti-histone IgG antibody quality control samples.

[0176] (1) Appearance

[0177] Visual examination under natural light with corrected vision showed that all three groups met the standards for appearance: loose material, dissolved in purified water, with no sediment or suspended matter. (See Table 5)

[0178] Table 5

[0179]

[0180] (2) Recovery rate

[0181] Recovery rates were calculated by detecting differences before and after freeze-drying. Matrix solution 3 showed a higher recovery rate. (See Table 6)

[0182] Table 6

[0183]

[0184] (3) Comparison of freeze-drying processes

[0185] Two bottles of quality control samples were randomly selected from those prepared using the novel freeze-drying procedure and the conventional freeze-drying procedure. One bottle was placed at 2–8°C as a control sample, and the other at 37°C as an experimental sample. On day 10, after removing the experimental sample, it was kept at 2–8°C for at least 30 minutes. Then, both the experimental and control samples were reconstituted and equilibrated at room temperature for 10–20 minutes before testing. Each sample was tested three times, and the average value of the test results for each sample was calculated. The relative deviation between the average value of each experimental sample and the average value of the control sample was also calculated. The stability of the quality control samples at 37°C on day 10 should meet the requirement of a relative deviation within ±10%. The comparative results showed that the samples prepared using the novel freeze-drying procedure exhibited the smallest deviation in stability on day 10 compared to the conventional freeze-drying procedure. (See Table 7)

[0186] Table 7

[0187]

[0188] (4) Evaluation of matrix effect

[0189] Two base sample pools were prepared, one containing AHA and the other containing clinical serum samples. AHA was added in parallel to both the control matrix and the human samples to cover the measurement range, ensuring a uniform concentration. At least 10 samples were prepared for each pool. Calibration testing was performed using the anti-histone antibody IgG assay kit (chemiluminescence method) from Shenzhen Yahuilong Biotechnology Co., Ltd. Ten control matrix solutions with different concentrations of the analyte and ten parallel clinical serum samples were also tested. A linear regression plot was constructed using the concentration values ​​of the clinical serum samples as the X-axis and the concentration values ​​of the control matrix solutions as the Y-axis, and a standard linear regression analysis was performed. The results for all three matrix solutions met the acceptance criteria, with a correlation coefficient (r) ≥ 0.975 and a slope ≤ 1.1 (0.9 ≤ slope ≤ 1.1). This indicates that there was no significant matrix difference between the anti-histone antibody IgG control prepared with the three matrix solutions and the clinical serum samples. (See Table 8 and...) Figures 5-7 )

[0190] Table 8

[0191]

[0192]

[0193] (5) Uniformity

[0194] Ten quality control samples from the smallest packaging units of the same batch were randomly selected and randomly numbered 1 to 10. Each packaging unit was tested three times on the testing system. The order of the three measurements was: 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9. The test results were recorded, and F and s were calculated according to formulas (1) to (11). bb s r and CV 瓶间 .

[0195] ············(1)

[0196] ············(2)

[0197] ···········(3)

[0198] ···········(4)

[0199] ···········(5)

[0200] n 0 = 1 α − 1 [ ∑ i = 1 α n i − ∑ i = 1 α n i 2 ∑ i = 1 α n i ] ···········(6)

[0201] ···········(7)

[0202] ···········(8)

[0203] ···········(9)

[0204] ············(10)

[0205] ···········(11)

[0206] In the formula:

[0207] --variance;

[0208] —Specify the parameter's i-th measurement value or calculation result;

[0209] —Overall average;

[0210] —Number of repeated measurements for sample i;

[0211] —The j-th result of sample i;

[0212] —mean square;

[0213] — Degrees of freedom;

[0214] —F-test value;

[0215] —Number of valid measurements;

[0216] —The number of samples drawn;

[0217] —Total number of tests;

[0218] —Standard deviation between bottles;

[0219] —Intra-bottle standard deviation (repeatability standard deviation);

[0220] when When ≤1, with replace calculate The results should meet the requirements. ≤10.0%.

[0221] when ≤ At that time, the test results showed no significant difference in uniformity between the bottles, and the calculation results... The results should meet the requirements. ≤10.0%.

[0222] when > , When the value is ≤0.3δ, the test results show no significant difference in uniformity between bottles, and the calculation results are as follows. The results should meet the requirements. ≤10.0%.

[0223] when > , When the value is greater than 0.3δ, the uniformity between bottles is considered to be poor, and the result does not meet the requirements.

[0224] Note: δ is the target standard deviation.

[0225] The formulations of all three matrix solutions were below 10%, but the homogeneity of matrix solution 3 was relatively stable across the negative, positive 1, and positive 2 groups. All are less than 5%. (See Table 9)

[0226] Table 9

[0227]

[0228]

[0229]

[0230] (6) Accelerated aging stability

[0231] Two bottles of quality control sample were randomly selected. One bottle was placed at 2–8°C as a control sample, and the other at 37°C as an experimental sample. On day 10, after removing the experimental sample, it was kept at 2–8°C for at least 30 minutes. Then, both the experimental and control samples were reconstituted and equilibrated at room temperature for 10–20 minutes before testing. Each sample was tested three times, and the average value of the test results for each sample was calculated. The relative deviation between the average value of each experimental sample and the average value of the control sample was also calculated. The stability of the components in the quality control sample after accelerated testing at 37°C on day 10 should meet the requirement that the relative deviation is within ±10%. The comparative results showed that the optimized formulation exhibited the smallest deviation in stability after accelerated testing on day 10. (See Table 10)

[0232] Table 10

[0233]

[0234] (7) Reconstitution stability

[0235] Four bottles of quality control samples were randomly selected from each of the negative, positive 1 and positive 2 samples. One bottle was reconstituted on day 0 and placed at ≤-20℃ as experimental sample 1, one bottle was reconstituted on day 65 and placed at 2~8℃ as experimental sample 2, one bottle was reconstituted on day 92 and placed at room temperature (20~25℃) as experimental sample 3, and the unreconstituted quality control samples were placed at 2~8℃ as control samples.

[0236] On day 100, experimental and control samples were reconstituted and tested. Each sample was tested three times, and the average value of the test results for each sample was calculated. At the same time, the relative deviation between the average value of each experimental sample and the average value of the control sample was calculated.

[0237] Acceptance criteria: The reconstitution stability of the quality control product at 20~25℃ should meet the requirement that the relative deviation is within ±10% after 8 days of reconstitution; the reconstitution stability at 2~8℃ should meet the requirement that the relative deviation is within ±10% after 35 days of reconstitution; and the reconstitution stability at ≤-20℃ should meet the requirement that the relative deviation is within ±10% after 100 days of reconstitution.

[0238] The results show that matrix solution 3 exhibits better reconstitution stability. (See Table 11)

[0239] Table 11

[0240]

[0241]

[0242]

[0243] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0244] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. An anti-histone antibody, characterized in that, It includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes amino acid sequences HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 4~6, and the light chain variable region includes amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 1~3.

2. The anti-histone antibody as described in claim 1, characterized in that, The constant region of the antihistone antibody is the constant region of human IgG; Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7; Further optionally, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 11, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

10.

3. A nucleic acid molecule, characterized in that, It includes an antihistone antibody gene expression cassette fragment, said antihistone antibody gene expression cassette fragment encoding the antihistone antibody as described in claim 1 or 2.

4. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 3; Optionally, the expression vector includes m copies of an anti-histone antibody gene expression cassette fragment, where m is an integer greater than or equal to 1; Alternatively, m can be 3.

5. A host cell, characterized in that, It includes the expression vector as described in claim 4; Optionally, the host cell includes Pichia pastoris; Optionally, the host cell may also express accessory proteins; Further, optionally, the accessory protein includes one or more of Ssa4, Bmh2, and VHb; Further, optionally, the host cell further includes a second expression vector, the second expression vector including the gene encoding the accessory protein.

6. A quality control product for anti-histone antibody immunoassay, characterized in that, The quality control product includes the anti-histone antibody as described in claim 1 or 2; Optionally, the quality control material further includes a matrix solution; Further optionally, the matrix solution includes one or more of a matrix, a buffer, a protein stabilizer, a lyophilization protectant, an excipient, an adjuvant, and a preservative, wherein the matrix includes matrix serum, the buffer includes 2-morpholine ethanesulfonic acid buffer, the protein stabilizer includes casein hydrolysate, the lyophilization protectant includes trehalose, the excipient includes D-mannitol, and the preservative includes Proclin 300; Further optionally, the matrix solution comprises one or more of the following: matrix serum, 0.95% (w / v) to 1% (w / v) of 2-(N-morpholino)ethanesulfonic acid or its hydrate, 0.23% (w / v) to 0.27% (w / v) of casein hydrolysate, 3% (w / v) to 5% (w / v) of D-mannitol, 0.8% (w / v) to 1.2% (w / v) of trehalose, and 0.08% (w / v) to 0.12% (w / v) of Proclin 300.

7. A method for preparing an anti-histone antibody immunoassay quality control, characterized in that, This includes mixing the anti-histone antibody as described in claim 1 or 2 with a matrix solution and lyophilizing it to obtain a dry quality control product; Optionally, the matrix solution comprises one or more of the following: matrix serum, 0.95% (w / v) to 1% (w / v) of 2-(N-morpholino)ethanesulfonic acid monohydrate, 0.23% (w / v) to 0.27% (w / v) of casein hydrolysate, 3% (w / v) to 5% (w / v) of D-mannitol, 0.8% (w / v) to 1.2% (w / v) of anhydrous trehalose, and 0.08% (w / v) to 0.12% (w / v) of Proclin 300.

8. The method for preparing the anti-histone antibody immunoassay quality control product as described in claim 7, characterized in that, The freeze-drying process includes pre-freezing and drying; Optionally, the pre-freezing conditions include: pre-freezing at a temperature of -8℃ to -12℃ for 30 min to 50 min; pre-freezing at a temperature of -38℃ to -42℃ for 1.4 h to 1.6 h; and pre-freezing at a temperature of -40℃ to -50℃ for 3.4 h to 3.6 h. Optionally, the drying includes primary drying and secondary drying. The conditions for primary drying include: a vacuum of 0.18 mbar to 0.22 mbar, maintained at a temperature of -28°C to -32°C for 21 to 23 hours; and a temperature increase to -8°C to -12°C for 20 to 40 minutes. The secondary drying conditions include a vacuum of 0.18 mbar to 0.22 mbar and a temperature of 8°C to 12°C for 6 to 8 hours.

9. An anti-histone antibody immunoassay kit, characterized in that, The kit includes the anti-histone antibody immunoassay quality control product as described in claim 6 or the anti-histone antibody immunoassay quality control product obtained by the preparation method described in claim 7 or 8.

10. The use of the anti-histone antibody immunoassay quality control product as described in claim 6, or the anti-histone antibody immunoassay quality control product obtained by the preparation method described in claim 7 or 8, or the kit described in claim 9 in the detection of anti-histone antibodies; Optionally, the detection of antihistone antibodies includes the following steps: using chemiluminescence to detect the optical signals in the antibody sample to be tested and the antihistone antibody immunoassay quality control, respectively; comparing and analyzing the detection results of the antibody sample to be tested and the antihistone antibody immunoassay quality control to obtain the quality information or antibody content information of the antibody sample to be tested; Optionally, the detection of antihistone antibodies is performed using acridinium ester chemiluminescence method.

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