Anti-PM-Scl monoclonal antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an anti-PM-Scl monoclonal antibody and application thereof. According to the invention, a PM-Scl recombinant protein is used as an antigen, and a phage display technology is used for screening to obtain the monoclonal antibody of the human PM-Scl. The monoclonal antibody is used as a raw material of a PM-Scl-IgG detection reagent quality control product, is good in universality and stability, can replace a clinical positive sample and be used as a quality control product of a third-party detection laboratory, saves the detection cost, is a human source, is closer to a natural sample in property compared with a human-mouse chimeric antibody, and has a good application prospect. Some matrix effect problems can be avoided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to monoclonal antibodies against PM-Scl and their applications. Background Art

[0002] In 1977, Wolfe et al. first reported the anti-PM-1 antibody in the sera of patients with overlap of myositis and systemic sclerosis. However, later studies found that this antibody was more commonly seen in patients with overlap of polymyositis (PM) and scleroderma (Scl), so it was also called the anti-PM-Scl antibody. Currently, the detection of PM-Scl antibodies mainly relies on the detection in medical laboratories. The quality control of medical laboratories aims to detect and control the precision of routine work in the laboratory and detect changes in its accuracy, so as to improve the consistency of specimen detection in routine work, thereby ensuring the accurate issuance of test reports.

[0003] The conventional method for preparing PM-Scl antibody quality control products is mainly to collect autoimmune positive sera. This method has huge defects. First, the positive rate of PM-Scl antibodies is relatively low, it is difficult to obtain clinical positive sera, the cost is very high, and the supply is often insufficient. Second, the difference between batches is large and the quantity is small, making it difficult to prepare on a large scale and industrialize. Third, it is very difficult to effectively ensure consistency as a conventional quality control product. Fourth, there is a lack of industry-standard reference materials for autoimmune detection kits, the compliance rate of detection by kits from different enterprises is relatively low, and the reactivity of positive sera in kits from different manufacturers varies, which is not conducive to the quality control of test results in different laboratories.

[0004] Therefore, the research and development of a quality control product suitable for quality evaluation during specimen detection of the PM-Scl antibody project in medical laboratories such as clinical laboratories, third-party physical examination centers, and provincial and municipal clinical inspection centers is of great significance for ensuring the reliability and accuracy of the test results of the PM-Scl antibody project. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide monoclonal antibodies against PM-Scl and their applications.

[0006] The present invention provides monoclonal antibodies against PM-Scl,

[0007] The amino acid sequence of CDR1 in its light chain variable region is at least one of those shown in SEQ ID NO:2 and / or SEQ ID NO:10;

[0008] The amino acid sequence of CDR2 in its light chain variable region is at least one of those shown in SEQ ID NO:3 and / or SEQ ID NO:11;

[0009] The amino acid sequence of CDR3 in its light chain variable region is at least one of those shown in SEQ ID NO:4 and / or SEQ ID NO:12;

[0010] The amino acid sequence of CDR1 in its heavy chain variable region is at least one of those shown in SEQ ID NO:6 and / or SEQ ID NO:14;

[0011] The amino acid sequence of CDR2 in its heavy chain variable region is at least one of those shown in SEQ ID NO:7 and / or SEQ ID NO:15;

[0012] The amino acid sequence of CDR3 in its heavy chain variable region is at least one of those shown in SEQ ID NO:8 and / or SEQ ID NO:16.

[0013] Furthermore, for the monoclonal antibody against PM-Scl of the present invention,

[0014] The light chain variable region includes CDR1 with the amino acid sequence shown in SEQ ID NO:2, CDR2 with the amino acid sequence shown in SEQ ID NO:3, and CDR3 with the amino acid sequence shown in SEQ ID NO:4; and / or

[0015] The light chain variable region includes CDR1 with the amino acid sequence shown in SEQ ID NO:10, CDR2 with the amino acid sequence shown in SEQ ID NO:11, and CDR3 with the amino acid sequence shown in SEQ ID NO:12;

[0016] The heavy chain variable region includes CDR1 with the amino acid sequence shown in SEQ ID NO:6, CDR2 with the amino acid sequence shown in SEQ ID NO:7, and CDR3 with the amino acid sequence shown in SEQ ID NO:8;

[0017] The heavy chain variable region includes CDR1 with the amino acid sequence shown in SEQ ID NO:14, CDR2 with the amino acid sequence shown in SEQ ID NO:15, and CDR3 with the amino acid sequence shown in SEQ ID NO:16.

[0018] Even further, for the monoclonal antibody against PM-Scl of the present invention,

[0019] The amino acid sequence of the light chain variable region is at least one of those shown in SEQ ID NO:1 and / or SEQ ID NO:9;

[0020] The amino acid sequence of the heavy chain variable region is at least one or more of those shown in SEQ ID NO:5 and / or SEQ ID NO:13.

[0021] In a specific embodiment of the present invention, the anti-PM-Scl monoclonal antibody includes the anti-PM-Scl monoclonal antibodies numbered PM12 and PM18;

[0022] The amino acid sequence of CDR1 of the light chain variable region of PM12 is shown in SEQ ID NO:2;

[0023] The amino acid sequence of CDR2 of the light chain variable region of PM12 is shown in SEQ ID NO:3;

[0024] The amino acid sequence of CDR3 of the light chain variable region of PM12 is shown in SEQ ID NO:4;

[0025] The amino acid sequence of CDR1 of the heavy chain variable region of PM12 is shown in SEQ ID NO:6;

[0026] The amino acid sequence of CDR2 of the heavy chain variable region of PM12 is shown in SEQ ID NO:7;

[0027] The amino acid sequence of CDR3 of the heavy chain variable region of PM12 is shown in SEQ ID NO:8;

[0028] The amino acid sequence of the light chain variable region of PM12 is shown in SEQ ID NO:1;

[0029] The amino acid sequence of the heavy chain variable region of PM12 is shown in SEQ ID NO:5;

[0030] The amino acid sequence of CDR1 of the light chain variable region of PM18 is shown in SEQ ID NO:10;

[0031] The amino acid sequence of CDR2 of the light chain variable region of PM18 is shown in SEQ ID NO:11;

[0032] The amino acid sequence of CDR3 of the light chain variable region of PM18 is shown in SEQ ID NO:12;

[0033] The amino acid sequence of CDR1 of the heavy chain variable region of PM18 is shown in SEQ ID NO:14;

[0034] The amino acid sequence of CDR2 of the heavy chain variable region of PM18 is shown in SEQ ID NO:15;

[0035] The amino acid sequence of CDR3 of the heavy chain variable region of PM18 is shown in SEQ ID NO:16;

[0036] The amino acid sequence of the light chain variable region of PM18 is as shown in SEQ ID NO:9;

[0037] The amino acid sequence of the heavy chain variable region of PM18 is as shown in SEQ ID NO:13;

[0038] In the present invention, the monoclonal antibody against PM-Scl is obtained by screening; in the present invention, after screening the phage library to obtain excellent-performance monoclonal antibodies against PM-Scl numbered PM12, PM18, PM30, PM46, and PM64, further detection of universality and stability found that: among them, PM12, PM18, and PM46 have good universality; PM12 and PM18 have good stability; among them, PM12 has the best performance.

[0039] Using the monoclonal antibody of the present invention as the raw material for the quality control product of the PM-Scl-IgG detection reagent has good universality and good stability, can replace clinical positive samples, and be used as the quality control product of the third-party testing laboratory, saving the detection cost.

[0040] The monoclonal antibody against PM-Scl described in the present invention has an IgG subtype in its constant region.

[0041] The present invention provides a nucleic acid, which comprises a nucleic acid encoding the monoclonal antibody against PM-Scl described in the present invention.

[0042] The nucleic acid described in the present invention can be DNA, RNA, cDNA or PNA. In the embodiments of the present invention, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (such as promoters or transcription terminators). The nucleic acid can be linear or circular in topology. The nucleic acid can be a part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be directly obtained from natural sources, or can be prepared with the assistance of recombinant, enzymatic or chemical techniques. The RNA form is mRNA obtained by gene transcription, etc.

[0043] In the present invention, the nucleic acid can be optimized or unoptimized. The optimization includes but is not limited to: codon usage preference, eliminating secondary structures (such as hairpin structures) that are unfavorable for expression, changing GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splicing sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA unstable regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.

[0044] The present invention also provides a transcription unit, which comprises a transcription element and the nucleic acid of the present invention; the transcription element includes but is not limited to a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, an origin of replication, a nucleic acid restriction site, and a homologous recombination site, such as an enhancer of a promoter, a poly(A) signal, etc.

[0045] The present invention provides a recombinant vector containing the nucleic acid of the present invention.

[0046] Furthermore, the source of the vector of the present invention includes plants, animals, bacteria, fungi, phages, or viruses, and the present invention does not make any limitation thereto. The viral vectors include: adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, or lentivirus vectors, etc. The phage vectors include phagemids and helper vectors, and the phagemids include but are not limited to pBluescript II-KS(+), pcomb3XSS, pCANTAB5E, or pKK233.3. The animals include mammals and non-mammals, and the mammalian expression vectors include but are not limited to pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vectors include but are not limited to pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vectors include but are not limited to pYES2, pYES3, pYES6, pAUR23, etc.

[0047] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of the operably linked coding gene in a specific host organism.

[0048] Once the recombinant vector is transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome by itself. In this specification, "plasmid" and "vector" can sometimes be used interchangeably because a plasmid is the most commonly used form of vector currently. However, the present invention intends to include such other forms of expression vectors that play an equivalent role and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or merely potential genomic inserts. In a specific embodiment of the present invention, the recombinant vector is pcomb3XSS.

[0049] The present invention provides host cells, which are characterized in that they are transfected or transformed with the recombinant vector as described in the present invention.

[0050] Furthermore, the transformation methods include: chemical transformation and electrotransformation; the transfection methods include calcium phosphate co-precipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0051] The host cells provided by the present invention can be derived from plants, animals, bacteria, fungi, phages or viruses, and the present invention does not limit this.

[0052] Further, the hosts include phages and / or mammalian cells. The model animals include commonly used experimental animals in the biomedical field such as mice, rats, rabbits, etc., and the present invention does not limit this. The mammalian cells include at least one of CHO cells, BHK cells, Sp2 / 0, HEK-293, HEK293T, Hep G2, HELA, CHO-K1, COS-1, COS-7, NIH3T3, A204, A549, D-407, CHO, HCS-2, HT-29, U87, Sf9, or FD-CHOS.

[0053] The present invention provides a reagent containing a monoclonal antibody against PM-Scl, which is characterized in that it includes at least one of the monoclonal antibody against PM-Scl as described in the present invention or the monoclonal antibody against PM-Scl obtained by culturing the host cells as described in the present invention and excipients.

[0054] The present invention provides the use of at least one of the following I) - VI) in the detection of PM-Scl:

[0055] I), the monoclonal antibody against PM-Scl as described in the present invention;

[0056] II), the nucleic acid as described in the present invention;

[0057] III), the recombinant vector as described in the present invention;

[0058] IV), the host cells as described in the present invention;

[0059] V), the monoclonal antibody against PM-Scl obtained by culturing the host cells as described in the present invention;

[0060] VI), the reagent as described in the present invention.

[0061] The present invention provides a PM-Scl detection kit, which is characterized in that it includes a PM-Scl immunoassay reagent and at least one of the following a) - c):

[0062] a), the anti-PM-Scl monoclonal antibody of the present invention;

[0063] b), the anti-PM-Scl monoclonal antibody obtained by culturing the host cell as described in the present invention;

[0064] c), the reagent of the present invention.

[0065] The present invention uses the PM-Scl recombinant protein as an antigen, and screens the monoclonal antibody against human PM-Scl by phage display technology. The monoclonal antibody against human PM-Scl is closer to the natural sample in nature than the human-mouse chimeric antibody, and can avoid some matrix effect problems. At the same time, the antibody described in the present invention is used as a quality control product for PM-Scl-IgG detection, has the characteristics of strong generality and high stability, can make the difference between PM-Scl-IgG detections controllable, and has wide application and popularization value. Description of the Drawings

[0066] Figure 1 Show the SDS-PAGE analysis results of the PM-Scl recombinant antibody. Detailed Embodiments

[0067] The present invention provides an anti-PM-Scl monoclonal antibody and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0068] The term "at least one (kind)" means one or more (kinds), and "a plurality (kinds)" means two (kinds) or more than two (kinds). "At least one (individual)" or its similar expression means any combination of these items, including any combination of single items (individual or kind) or plural items (individual or kind). For example, "at least one (individual) of a, b, or c", or, "at least one (kind) of a, b, and c" can all represent: a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single items (individual or kind) respectively, or multiple items (individual or kind).

[0069] The singular forms of "a", "the", and "said" used in the embodiments of the present application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0070] The terms "comprising", "having", or "including", including the use of their grammatical synonyms, should generally be understood as open-ended and non-limiting, e.g., not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context otherwise.

[0071] The use of any and all examples or exemplary language such as "for example" or "including" in this document is merely intended to better illustrate the invention and does not limit the scope of the invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0072] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values, and percentages used in this disclosure are modified by the term "about". Herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0073] Amino acid sequence of the light chain variable region of PM12: DIQMTQSPSSLSASVGDRVTVTCRASQTISNYLNWYQQKPGKAPKLLIYGASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLTFGPGTKVDIK (SEQ ID NO:1);

[0074] CDR1 of the light chain variable region of PM12: RASQTISNYLN (SEQ ID NO:2);

[0075] CDR2 of the light chain variable region of PM12: GASSLHS (SEQ ID NO:3);

[0076] CDR3 of the light chain variable region of PM12: QQSYNTPLT (SEQ ID NO:4);

[0077] Amino acid sequence of the heavy chain variable region of PM12: QVQLVQSGTEVRRPGASVRVSCKTSGYTFTSYGVQWVRQAPGKGLEWMGWINTGVGTTKYSQKFQDRVTISADTSAATVYLEVSSLTSEDTAVYFCARDRSLSMIIQIIKGEMDVWGQGTTVTVSS (SEQ ID NO:5);

[0078] CDR1 of the heavy chain variable region of PM12: SYGVQ (SEQ ID NO:6);

[0079] CDR2 of the heavy chain variable region of PM12: WINTGVGTTKYSQKFQD (SEQ ID NO:7);

[0080] CDR3 of the heavy chain variable region of PM12: DRSLSMIIQIIKGEMDV (SEQ ID NO:8);

[0081] Amino acid sequence of the light chain variable region of PM18: DIQMTQSPSTLSASVGDTVSLSCRASHSIDSWLAWYQQKPGRAPKLLIYKTSNLESGVPSRFRGSGFGTEFTLTISSLQPDDSATYYCQQYKSFSSWIFGQGTKLEIK (SEQ IDNO:9);

[0082] CDR1 of the light chain variable region of PM18: RASHSIDSWLA (SEQ ID NO:10);

[0083] CDR2 of the light chain variable region of PM18: KTSNLES (SEQ ID NO:11);

[0084] CDR3 of the light chain variable region of PM18: QQYKSFSSWI (SEQ ID NO:12);

[0085] Amino acid sequence of the heavy chain variable region of PM18: EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREYCSSTSCYLYGMDVWGQGTTVTVSS (SEQ ID NO:13);

[0086] CDR1 of the heavy chain variable region of PM18: SYYMH (SEQ ID NO:14);

[0087] CDR2 of the heavy chain variable region of PM18: IINPSGGSTSYAQKFQG (SEQ ID NO:15);

[0088] CDR3 of the heavy chain variable region of PM18: EYCSSTSCYLYGMDV (SEQ ID NO:16);

[0089] The antigen used in the present invention is the PM-Scl recombinant protein (ProSpec, catalog number PRO-444) as the antigen, which is used to prepare a monoclonal antibody specific for humanized PM-Scl;

[0090] All the materials used in the present invention are common commercially available products and can be purchased on the market. The present invention will be further described below in conjunction with the examples:

[0091] Example 1 Construction of a phage human library

[0092] Purchase autoimmune and allergic PBMC (D001201116) from Rubai Biotechnology. Use TRIzol reagent to extract total RNA from 5 × 10 6 PBMC according to the reagent instructions, and reverse transcribe to synthesize cDNA. Use conserved region primers to PCR amplify the light chain variable region and heavy chain variable region of the antibody respectively. The PCR reaction program is shown in Table 1: The PCR products are recovered by 1% agarose gel. The amplified light chain variable region and heavy chain variable region are spliced into scFv using the overlap-PCR method, and the products are recovered by 1% agarose gel and stored at -20 °C.

[0093] Table 1. PCR amplification program

[0094]

[0095] The phagemid vector pcomb3XSS and the purified and recovered ScFv fragment are digested with SfiI to construct a recombinant plasmid. The recombinant plasmid is electrotransformed into TG1 competent cells to construct an immune single-chain antibody library, and a primary phage single-chain antibody library is prepared.

[0096] Example 2 Panning of the phage human library

[0097] Coat the immunization tube with the PM-Scl antigen, block it with PBS containing 4% skim milk, add 1×10 12 of the phage library, bind for 1 h, wash, and first elute with 0.15 mol / L Arg-HAC (arginine acetate) with a pH of 3, 1.6 g / L peptone, and 1 g / L yeast extract, and then elute with 50 mmol / L Tris containing 20 mmol / L trypsin. In addition to measuring the titer of the eluted phage, the rest are all infected with 5 mL of TG1. After standing for 0.5 h, add 20 ml of 2YT-AG medium to an OD600 value of about 0.5, and add the pre-helper phage M13K07 at a ratio of 1:20, and amplify and culture overnight, and precipitate to collect the phage. Complete the first round of screening.

[0098] The second and third rounds of screening were completed in sequence to enrich phage antibodies with high affinity. After the phages eluted in the third round of panning infected TG1, they were spread on plates and cultured overnight. Single colonies were picked and inoculated into 3 pieces of 96-well 2 mL deep-well plates for shaking culture. When the OD600 value of the bacterial solution was about 0.5, the bacterial solution was preserved. Then, helper phage M13K07 was added, and the mixture was allowed to stand at 37 °C for 0.5 h and then induced overnight at 28 °C and 220 rpm to produce phages.

[0099] The ELISA plate was coated with PM-Scl antigen. After blocking with PBS containing 4% skim milk, 50 μL of phages was added. After washing, diluted anti-M13-HRP antibody was added. TMB was used for color development. The phages showing strong positive color development in ELISA were sequenced to obtain the antibody gene sequences, and the antibodies in the positive phages were numbered as PM12, PM18, PM30, PM46, and PM64.

[0100] Example 3 Expression and purification of PM-Scl recombinant antibody

[0101] The heavy-chain antibody gene and the light-chain antibody gene were respectively homologously recombined with the RGFc-PCMV3 and RCL-PCMV3 vectors digested with KasI / BamHI by PCR to obtain the expression vectors of the human full antibody sequences. After the recombinant full antibody plasmids were transferred into competent DH5α cells, positive clones were selected for sequencing and plasmid extraction. The extracted plasmids were transfected into HEK293 cells. After ELISA detection showed antibody expression 48 h after transfection, feeding was added, and the cell supernatant was collected 7 d after transfection to obtain the supernatant of the target protein.

[0102] The cell supernatant was purified with SPA to obtain the PM-Scl recombinant antibody. The antibody was stored in 10 mM PBS pH 7.2 buffer, and the concentration was adjusted to 2 mg / ml. Then, SDS-PAGE detection was performed. As Figure 1 shown, the purity of the purified antibody exceeded 92%.

[0103] Example 4 Evaluation of the universality of monoclonal antibodies against PM-Scl

[0104] The monoclonal antibodies against PM-Scl (PM12, PM18, PM30, PM46, and PM64) were diluted 100-fold, 500-fold, 1000-fold, and 5000-fold with negative serum (containing 0.1% P300 preservative), and then detected on the mainstream anti-PM-Scl antibody IgG assay kits on the market (the kits were respectively: the anti-PM-Scl antibody IgG assay kit of Yahui Long; the 18-item autoantibody spectrum detection kit of Haooubo; AESKULISA PM-Scl of AESKU; the anti-PM-Scl antibody IgG assay kit of Antu). The results are shown in Table 2.

[0105] Table 2. Reactivity of monoclonal antibody PM12 against PM-Scl on kits from different manufacturers

[0106]

[0107] Table 3. Reactivity of monoclonal antibody PM18 against PM-Scl on kits from different manufacturers

[0108]

[0109] Table 4. Reactivity of monoclonal antibody PM30 against PM-Scl on kits from different manufacturers

[0110]

[0111] Table 5. Reactivity of monoclonal antibody PM46 against PM-Scl on kits from different manufacturers

[0112]

[0113] Table 6. Reactivity of monoclonal antibody PM64 against PM-Scl on kits from different manufacturers

[0114]

[0115] Among them, 3 antibodies (PM12, PM18, PM46) can react on the mainstream commercial kits, and the reactivity of 2 antibodies (PM30, PM64) varies significantly on kits from different manufacturers, which may be related to the different antigens used in the kits from different manufacturers.

[0116] Example 5 Evaluation of the stability of monoclonal antibodies against PM-Scl

[0117] The antibodies were diluted 500-fold and 1000-fold respectively with negative serum (containing 0.1% P300 preservative), and the accelerated stability at 37°C and the freeze-thaw stability at -20°C were evaluated respectively, with the corresponding antibodies stored at 4°C - 8°C as the control. Detection was performed using the monoclonal antibody IgG assay kit for anti-PM-Scl from Antu, and the results are shown in Tables 7, 8, and 9 below.

[0118] Table 7. Stability test of monoclonal antibody PM12 against PM-Scl

[0119]

[0120] Table 8. Stability test of monoclonal antibody PM18 against PM-Scl

[0121]

[0122] Table 9. Stability test of monoclonal antibody PM46 against PM-Scl

[0123]

[0124] The freeze-thaw stability of 2 antibodies (PM12, PM18) was good. After 10 cycles of freeze-thaw at -20°C, the titer decrease was less than 10%. For antibody PM12, the signal value decrease was less than 10% after 7 days of acceleration at 37°C, and for antibody PM18, the signal value decrease was less than 20% after 10 days of acceleration at 37°C, showing good stability. For 1 antibody (PM46), the signal value decrease was greater than 20% after 10 days of acceleration at 37°C, indicating poor stability.

[0125] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A monoclonal antibody against PM-Scl, characterized in that: The amino acid sequence of CDR1 of the light chain variable region is at least one of SEQ ID NO: 2 and / or SEQ ID NO: 10; The amino acid sequence of CDR2 of the light chain variable region is at least one of SEQ ID NO: 3 and / or SEQ ID NO: 11; The amino acid sequence of CDR3 of the light chain variable region is at least one of SEQ ID NO: 4 and / or SEQ ID NO: 12; The amino acid sequence of CDR1 of the heavy chain variable region is at least one of SEQ ID NO: 6 and / or SEQ ID NO: 14; The amino acid sequence of CDR2 of the heavy chain variable region is at least one of SEQ ID NO: 7 and / or SEQ ID NO: 15; The amino acid sequence of CDR3 of the heavy chain variable region is at least one of SEQ ID NO: 8 and / or SEQ ID NO:

16.

2. The monoclonal antibody according to claim 1, characterized in that The light chain variable region comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO: 2, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 3, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 4; and / or The light chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO: 10, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 11, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 12; The heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO: 6, a CDR2 with an amino acid sequence as shown in SEQ ID NO: 7, and a CDR3 with an amino acid sequence as shown in SEQ ID NO: 8; The heavy chain variable region includes a CDR1 with an amino acid sequence as shown in SEQ ID NO:14, a CDR2 with an amino acid sequence as shown in SEQ ID NO:15, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:

16.

3. The monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the light chain variable region is at least one of SEQ ID NO: 1 and / or SEQ ID NO: 9; The amino acid sequence of the heavy chain variable region is at least one or more of SEQ ID NO: 5 and / or SEQ ID NO:

13.

4. The monoclonal antibody according to claim 3, characterized in that Its constant region is of IgG subtype.

5. A nucleic acid, characterized in that Comprising a nucleic acid encoding the monoclonal antibody according to any one of claims 1 to 4.

6. A recombinant vector, characterized in that Containing the nucleic acid according to claim 5.

7. A host cell, characterized in that Transfect or transform the recombinant vector as described in claim 6.

8. A reagent containing a monoclonal antibody against PM-Scl, characterized in that: The method comprises at least one of the monoclonal antibodies according to any one of claims 1 to 4 or the monoclonal antibodies obtained by culturing the host cell according to claim 7 and auxiliary materials.

9. Application of at least one of the following I) to VI) in PM-Scl detection: I), the monoclonal antibody according to any one of claims 1 to 4; II), the nucleic acid according to claim 5; III), the recombinant vector according to claim 6; IV), the host cell according to claim 7; V), culturing the monoclonal antibody obtained by the host cell according to claim 7; VI), the reagent according to claim 8.

10. PM-Scl detection kit, characterized in that, It includes PM-Scl immunoassay reagent and at least one of the following a) to c): a), the monoclonal antibody according to any one of claims 1 to 4; b), culturing the monoclonal antibody obtained by the host cell according to claim 7; c) The reagent according to claim 8.

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