Preparation method and application of progesterone recombinant monoclonal antibody

Abstract

The present application relates to the technical field of antibodies, in particular to a preparation method and application of a progesterone recombinant monoclonal antibody, wherein an excellent progesterone recombinant rabbit monoclonal antibody is screened, the heavy chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region of the antibody has an amino acid sequence as shown in SEQ ID NO:2. Experimental results show that the progesterone recombinant rabbit monoclonal antibody protein has a large expression amount, high specificity and high sensitivity, and can be applied to early spontaneous abortion risk assessment, prediction of adverse pregnancy, determination of ovulation, evaluation of luteal function loss and other clinical diagnoses, meanwhile, the antibody is applied to detection of progesterone content in serum, and has high accuracy, good repeatability, strong stability and good clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody technology, in particular to a preparation method and application of a progesterone recombinant monoclonal antibody. BACKGROUND

[0002] Progesterone (English name: Progesterone) is a steroid hormone, a steroid involved in the female menstrual cycle, pregnancy and the influence on the embryo, and the first compound with biological activity generated in the process of steroid biosynthesis. It is mainly produced by the corpus luteum and placenta, and most of the progesterone in the blood is bound to plasma proteins. Only a small amount exists in free form, and the main binding proteins are steroid binding protein, albumin, and sex hormone binding protein. The combination with albumin and steroid binding protein accounts for the majority, and the free form of biologically active progesterone only accounts for 2.5-3% of the total progesterone.

[0003] Progesterone is involved in the female menstrual cycle, supports pregnancy and embryo formation, and is a key hormone in the reproductive process. Under normal circumstances, the progesterone content begins to rise at the 11th week of normal pregnancy and reaches a peak at the 35th week. The progesterone content in the serum of patients with ectopic pregnancy is lower than that in normal pregnancy, and combined with β-HCG detection, it can be applied to the prediction of ectopic pregnancy; progesterone detection is also used to confirm ovulation, evaluate luteal function deficiency, and test the effectiveness of the ovulation induction process. It is currently believed that the detection of progesterone in combination is a method that can correctly evaluate luteal function in addition to endometrial biopsy, and can even replace endometrial biopsy.

[0004] The clinical significance of progesterone detection is particularly important for women during pregnancy. However, progesterone is a small molecule of steroid with antigenicity and no immunogenicity, so it is very difficult to obtain a high specificity progesterone antibody. There are many steroid small molecules similar in structure to progesterone in blood samples, which interfere with the detection of progesterone. At the same time, small molecule substances are easily affected by steric hindrance and matrix effect during detection. In the immunodetection of small molecule substances, there are generally problems of low sensitivity, narrow linear range, and insufficient accuracy. Therefore, it is necessary to screen a monoclonal antibody with high specificity and sensitivity.

[0005] Phage display technology is a unique gene recombination expression technology, and also a simple and effective screening tool. The entire coding antibody gene sequence in the immune organ after immunization is integrated into the vector by molecular cloning technology to form an antibody library. Phage binds to antigen and is selected to be displayed on the surface of phage as a fusion protein. After washing the vector with acidic or alkaline solution, antigen-binding phage is obtained, and specific antibodies can be obtained through multiple rounds of panning. SUMMARY

[0006] Therefore, the present application aims to provide a preparation method and application of a progesterone recombinant monoclonal antibody.

[0007] The present application provides a progesterone monoclonal antibody, which is obtained by screening an antigen after immunizing a New Zealand white rabbit through a phage display technology.

[0008] Further, the antigen is prepared by crosslinking KLH protein after adding EDC and NHS dropwise into P-11-HS to form an active intermediate P-11-HS-NHS.

[0009] In the present application, the progesterone monoclonal antibody,

[0010] the amino acid sequence of the CDR region of the heavy chain comprises at least one of the sequences shown in SEQ ID NO: 5, 6 or 7;

[0011] the amino acid sequence of the CDR region of the light chain comprises at least one of the sequences shown in SEQ ID NO: 8 or 9 or SAS.

[0012] Further, the progesterone monoclonal antibody,

[0013] the amino acid sequence of the three CDR regions of the heavy chain is GLSLSSNE, IDRSANT and ARGGYDVAYAFNI in sequence;

[0014] the amino acid sequence of the three CDR regions of the light chain is QSVYNNNN, SAS and LGGYSTAAA in sequence.

[0015] Further, the heavy chain variable region of the progesterone monoclonal antibody has an amino acid sequence shown in SEQ ID NO: 1, the light chain variable region of the progesterone monoclonal antibody has an amino acid sequence shown in SEQ ID NO: 2; the constant region of the heavy chain of the progesterone monoclonal antibody is a rabbit IgG1 subtype; and the constant region of the light chain of the progesterone monoclonal antibody is a rabbit K1 type.

[0016] The present application utilizes a phage display technology to obtain the progesterone recombinant rabbit monoclonal antibody, which has high specificity and high detection sensitivity.

[0017] The present application provides a nucleic acid encoding the progesterone monoclonal antibody.

[0018] Further, the nucleic acid comprises:

[0019] A nucleic acid encoding the heavy chain variable region of the progesterone monoclonal antibody, which has a sequence as shown in SEQ ID NO: 3;

[0020] A nucleic acid encoding the light chain variable region of the progesterone monoclonal antibody, which has a sequence as shown in SEQ ID NO: 4.

[0021] Further, the nucleic acid encoding the heavy chain variable region of the progesterone monoclonal antibody includes any one or more of the following:

[0022] A protein having at least 80% homology with the nucleic acid as shown in SEQ ID NO: 3 and encoding the same or similar function as the nucleic acid of SEQ ID NO: 3;

[0023] A nucleic acid in which one or more bases of the nucleic acid as shown in SEQ ID NO: 3 are modified, substituted, deleted or added;

[0024] A nucleic acid complementary or partially complementary to the nucleic acid as shown in SEQ ID NO: 3.

[0025] Further, the nucleic acid encoding the light chain variable region of the progesterone monoclonal antibody includes any one or more of the following:

[0026] A protein having at least 80% homology with the nucleic acid as shown in SEQ ID NO: 4 and encoding the same or similar function as the nucleic acid of SEQ ID NO: 4;

[0027] A nucleic acid in which one or more bases of the nucleic acid as shown in SEQ ID NO: 4 are modified, substituted, deleted or added;

[0028] A nucleic acid complementary or partially complementary to the nucleic acid as shown in SEQ ID NO: 4.

[0029] The present application provides an expression module, which includes a promoter, a terminator and the nucleic acid of the present application.

[0030] Further, the expression module further includes a single or multiple nucleic acids of the present application combined in series, fusion expression or other feasible ways to form an expression module, which is not limited by the present application.

[0031] The present application also provides a transcription unit, which refers to a DNA sequence from the start of the promoter to the end of the terminator. The promoter and the terminator can further include regulatory fragments on both sides or between them, which can include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, a transmembrane signal peptide and a homologous recombination site, such as an enhancer of the promoter, an ITR sequence, a polyA, a MIS signal peptide, etc., operably linked to the nucleic acid sequence.

[0032] The present application provides a recombinant vector, which comprises a vector backbone and the nucleic acid of the present application.

[0033] Further, the source of the vector backbone of the present application includes plants, animals, bacteria, fungi, bacteriophages, or viruses, which are not limited by the present application. The bacteriophage vector includes phagemids and helper vectors, and the phagemids include but are not limited to pBluescript II-KS(+), pcomb3XSS, pCANTAB5E or pKK233.3. The mammalian expression vector includes but is not limited to pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vector includes but is not limited to pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vector includes but is not limited to pYES2, pYES3, pYES6, pAUR23, etc.

[0034] In some embodiments of the present application, the nucleic acid encoding the progesterone monoclonal antibody is integrated with the vector backbone pCMV3 to construct a recombinant vector capable of replication and expression in host cells.

[0035] The recombinant vector of the present application refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism. The nucleic acid sequences or elements necessary for the expression in viruses, microorganisms or mammalian cells include promoters, ribosome binding sites and possibly other sequences. It is known that eukaryotic cells use promoters, enhancers and terminators. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or, in some cases, integrate into the genome. In this specification, "plasmid" and "vector" are sometimes used interchangeably, as the plasmid is the most commonly used form of vector currently. However, the present application is intended to include such other forms of expression vectors which function the same way, which are known in the art or will become known, including but not limited to: plasmids, phage particles, viral vectors and / or only potential genomic inserts. In specific embodiments, the nucleic acid encoding the progesterone monoclonal antibody provided by the present application can be constructed in various expression vectors.

[0036] The present application provides a host cell, which comprises at least one of the following I)~III):

[0037] I) secreting the progesterone monoclonal antibody as described in the present application;

[0038] II) genome-integrating the nucleic acid as described in the present application;

[0039] III) transfecting or transforming the recombinant vector as described in the present application.

[0040] In the present application, the transformation method includes chemical transformation and electroporation; the transfection method includes calcium phosphate co-precipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc. In some embodiments of the present application, the adeno-associated virus is used to transfect the host.

[0041] The host cell provided by the present application is derived from plants, animals, microorganisms or viruses, which are not limited in the present application. The host cell is transformed or transfected by the vector constructed by the recombinant DNA technology, so that the transformed host cell has the ability to replicate the vector encoding the protein or express the desired protein.

[0042] Further, in some specific embodiments, the host cell of the present application is a mammalian cell, specifically at least one selected from 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. In some specific embodiments, the host cell of the present application is a HEK-293 cell, which is used for expression of the progesterone monoclonal antibody. The results show that the progesterone monoclonal antibody of the present application has high expression amount, high purity and high concentration, which can meet the production demand and has a good application prospect in clinical diagnostic reagents.

[0043] The present application provides a preparation method of the progesterone monoclonal antibody, which comprises culturing the host as described in the present application to obtain the progesterone monoclonal antibody.

[0044] The present application provides a labeled antibody, which comprises a label and:

[0045] The progesterone monoclonal antibody of the present application;

[0046] Or the culture of the progesterone monoclonal antibody prepared by the preparation method of the present application.

[0047] Further, the label includes a chemical label and a biological label; the chemical label is an isotope and / or a chemical drug; the biological label includes biotin, avidin or enzyme labeling, and the enzyme labeling is preferably horseradish peroxidase or alkaline phosphatase.

[0048] The conjugate comprises a conjugation medium and:

[0049] The progesterone monoclonal antibody according to the present application;

[0050] or a culture of the progesterone monoclonal antibody prepared by the preparation method according to the present application.

[0051] Further, the conjugation medium is a solid medium or a semi-solid medium.

[0052] Still further, the conjugation medium is selected from colloidal gold, polystyrene flat plate or beads.

[0053] The present application provides the use of any one of a) ~ f) below in a product for detecting the level of progesterone in serum:

[0054] a), the progesterone monoclonal antibody according to the present application;

[0055] b), the nucleic acid according to the present application;

[0056] c), the recombinant vector according to the present application;

[0057] d), the host cell according to the present application;

[0058] e), the culture containing the progesterone monoclonal antibody prepared by the preparation method according to the present application;

[0059] f), the labeled antibody according to the present application;

[0060] g), the conjugate according to the present application.

[0061] The present application provides a product for detecting the level of progesterone in serum, which comprises any one of A) ~ D) below:

[0062] A), the progesterone monoclonal antibody according to the present application;

[0063] B), the culture containing the progesterone monoclonal antibody prepared by the preparation method according to the present application;

[0064] C), the labeled antibody according to the present application;

[0065] D), the conjugate according to the present application.

[0066] Further, the product according to the present application can be a test strip, which is coated with the progesterone monoclonal antibody according to the present application.

[0067] Still further, the product according to the present application can be a kit. The kit further comprises a coating buffer, a washing solution, a blocking solution and / or a color developing solution.

[0068] In some embodiments, the kit is suitable for the magnetic microparticle chemiluminescence of progesterone.

[0069] In some specific embodiments of the present application, the recombinant rabbit monoclonal antibody of progesterone is used as a detection antibody for the detection of progesterone, has good accuracy and sensitivity, and good repeatability.

[0070] The method for detecting serum progesterone is to detect progesterone by using the product of the present application.

[0071] The recombinant monoclonal antibody of progesterone provided by the present application is suitable for immunological detection of progesterone.

[0072] The present application uses screening to obtain a progesterone recombinant rabbit monoclonal antibody with excellent performance, the heavy chain variable region of which has an amino acid sequence as shown in SEQ ID NO: 1; and the light chain variable region of which has an amino acid sequence as shown in SEQ ID NO: 2. Experimental results show that the progesterone recombinant rabbit monoclonal antibody protein of the present application has large expression amount, strong specificity, high sensitivity, and can be applied to early spontaneous abortion risk assessment, prediction of adverse pregnancy, determination of ovulation, evaluation of luteal function loss and other clinical diagnoses. Meanwhile, the antibody is applied to detection of progesterone content in serum, has high accuracy, good repeatability, strong stability, and good clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 Preparation of immunogen;

[0074] Figure 2 Agarose gel electrophoresis graph for extraction of spleen RNA;

[0075] Figure 3 Agarose gel electrophoresis graph of VL gene PCR product;

[0076] Figure 4 Agarose gel electrophoresis graph of VH gene PCR product;

[0077] Figure 5 Agarose gel electrophoresis graph of scFv gene PCR product;

[0078] Figure 6 Agarose gel electrophoresis graph of PCR identification of recombinant bacteria liquid of antibody library;

[0079] Figure 7 SDS-PAGE electrophoresis graph of purified recombinant antibody;

[0080] Figure 8 Clinical detection correlation analysis of antibody 2 of the present application and a reference manufacturer;

[0081] Figure 9 Clinical detection correlation analysis of antibody 6 of the present application and a reference manufacturer;

[0082] Figure 10 Deviation of the test results of the antibody 2 of the present application from the theoretical concentration;

[0083] Figure 11 Deviation of the test results of the antibody 6 of the present application from the theoretical concentration. DETAILED DESCRIPTION

[0084] The present application provides a method for preparing and using a recombinant monoclonal antibody of progesterone. Those skilled in the art can refer to the content of the present application and make appropriate improvements to the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously make changes or appropriate changes and combinations to the method and application of the present application without departing from the content, spirit and scope of the present application, to realize and apply the technology of the present application.

[0085] 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. For the purposes of the present application, the definitions and terms of the art are specifically incorporated by reference from Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 common L-amino acids.

[0086] "Antibody" refers to a protein consisting of one or more polypeptides that specifically bind an antigen. One form of an antibody comprises the basic structural unit of an antibody. This form is a tetramer that consists of two identical pairs of antibody chains, each pair having one light chain and one heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains associate to contribute to the binding of the antigen, while the constant regions are responsible for the effector functions of the antibody.

[0087] The "variable region" of an antibody heavy or light chain is the N-terminal mature region of that chain. The currently known antibody types include kappa and lambda light chains, and alpha, gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon, and mu heavy chains or other types of equivalents thereof. A full-length immunoglobulin "light chain" (about 25 kDa or about 214 amino acids) comprises a variable region formed by about 110 amino acids at the NH2-terminus, and a kappa or lambda constant region at the COOH-terminus. A full-length immunoglobulin "heavy chain" (about 50 kDa or about 446 amino acids) also comprises a variable region (about 116 amino acids), and one of the heavy chain constant regions, such as gamma (about 330 amino acids).

[0088] An "antibody" includes any isotype of antibody or immunoglobulin, or an antibody fragment that retains the ability to specifically bind to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be labeled and detected, for example, by labeling with a radioisotope, an enzyme that generates a detectable substance, a fluorescent protein, biotin, and the like, and detected. Antibodies can also be bound to a solid phase carrier, including but not limited to polystyrene plates or beads, and the like.

[0089] The general procedure for preparing a natural antibody phage library is to extract total RNA from the immune antibody cells, which are usually obtained from immune organs and / or peripheral blood.

[0090] The amino acid sequence of the heavy chain variable region of antibody 2 is QSLEESGGGLVKPGGSLTLTCTVSGLSLSSNEIIWVRQAPGNGLEWVGGIDRSANTYYASWAKGRSTVTRNTNLNTVTLKMTSLTAADTATYFCARGGYDVAYAFNIWGPGTLVTVSS (SEQ ID NO: 1).

[0091] The amino acid sequence of the light chain variable region of antibody 2 is ALVMTQTPSPVSAAVGGTVTINCQASQSVYNNNNLSWYQQKPGQPPKLLIYSASILASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCLGGYSTAAAFGGGTKLEIK (SEQ ID NO: 2).

[0092] The nucleotide sequence of the heavy chain variable region of antibody 2 is: cagtcgctggaggagtccgggggaggcctggtcaagcctggaggatccctgacactcacctgcacagtctctggattatccctcagtagcaatgaaataatctgggtccgccaggctccagggaacgggctggaatgggtcggaggcattgataggagtgcgaacacatactacgcgagctgggcgaaaggccgatccaccgtcaccagaaacaccaacctgaacacggtgactctgaaaatgaccagtctgacagccgcggacacggccacttatttctgtgcgagaggtggttatgatgttgcttatgcttttaatatttggggcccaggcaccctggtcaccgtctcctca (SEQ ID NO: 3).

[0093] The nucleotide sequence of the light chain variable region of antibody 2 is: gcccttgtgatgacccagactccatcccctgtgtctgcagctgtgggaggcacagtcaccatcaactgccaggccagtcagagtgtttataataacaacaacttatcctggtatcagcagaaaccagggcagcctcccaaactcctgatctattctgcatccattctggcatctggggtcccatcgcggttcaaaggcagtggatctgggacacagttcactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtctaggaggatatagtactgctgcggctttcggcggaggaaccaagctggagatcaaa (SEQ ID NO: 4).

[0094] The CDR1 amino acid sequence of the heavy chain variable region is: GLSLSSNE (SEQ ID NO: 5).

[0095] The CDR2 amino acid sequence of the heavy chain variable region is: IDRSANT (SEQ ID NO: 6).

[0096] The CDR3 amino acid sequence of the heavy chain variable region is: ARGGYDVAYAFNI (SEQ ID NO: 7).

[0097] The CDR1 amino acid sequence of the light chain variable region is QSVYNNNN (SEQ ID NO: 8).

[0098] The CDR2 amino acid sequence of the light chain variable region is SAS.

[0099] The CDR3 amino acid sequence of the light chain variable region is LGGYSTAAA (SEQ ID NO: 9).

[0100] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market.

[0101] The present application is further described below in combination with examples:

[0102] Example 1 The phage platform screening preparation provided by the present application:

[0103] 1. Preparation of immunogen as shown in Figure 1

[0104] EDC (1.53 mg, 0.153 mL) and NHS (3.82 mg, 0.153 mL) were mixed, and the mixture was slowly added to a DMSO solution of P-11-HS (1.72 mg, 0.344 mL) at room temperature, and the active intermediate P-11-HS-NHS was formed by shaking the reaction at room temperature. Then, the active intermediate was slowly added to a solution of cross-linked protein KLH (20 mg, 2 mL) at room temperature, and the reaction was shaken at room temperature after addition. After the reaction was completed, the mixture was dialyzed in a neutral phosphate buffer, and then transferred to a 4-mL glass bottle and the volume was recorded.

[0105] 2. Animal immunization:

[0106] The prepared antigen P-11-HS-KLH was used to immunize New Zealand white rabbits for more than 3 times, with an interval of 30 days. The first immunization dose was 0.5 mg. The immunization antigen was emulsified with an equal volume of Freund's complete adjuvant, and the animal was immunized by subcutaneous injection at multiple points on the back. The antigen dose was halved for the second and subsequent immunizations. Ten days after the third immunization, the ear vein blood was collected and placed at 37°C for 1 h. Low-speed centrifugation was performed at low temperature for 10 min, and the antiserum was collected for titer evaluation.

[0107] 3. Antiserum titer detection

[0108] ​Serum titer is a semi-quantitative indicator commonly used to express the relative content of specific antibodies in antiserum, that is, the dilution of antiserum that binds to a certain amount of antigen under given conditions. The detection scheme of the present invention is as follows: ① Add mouse anti-rabbit antibody with a final concentration of 4μg / mL to the required volume of 0.05mol / L CB (pH=9.6) coating buffer, mix well and coat the chemiluminescent plate; ② Use unrelated protein to block at 37℃ for 2h to block the exposure site; ③ Dilute the test serum (444#, 445#, which are the rabbit serum numbers with relatively high titer and antiserum in animal immunization) from the initial 1 / 500 ratio and add 50μl / well, and set the benchmark manufacturer's antibody as a positive control; ④ The antibody is bound to the solid phase, and a specific enzyme-labeled antigen is added to detect the signal. The test results are shown in Table 1. When the titer reaches 1 / 32k or above, the immune effect is considered to be good, and the final booster immunization is carried out. The animal is sacrificed three days later and the spleen is retained for future use. The total RNA of spleen tissue is routinely extracted by Trizol method (such as Figure 2 The cDNA was synthesized by reverse transcription.

[0109] Table 1. Antiserum titer evaluation

[0110]

[0111] Example 2 scFv gene splicing and phage screening construction

[0112] scFv gene splicing: Using the cDNA obtained in Example 1 as a template, PCR amplify the light chain variable region and heavy chain variable region of the antibody respectively; the PCR reaction program is as follows:

[0113] Table 2. PCR amplification reaction program

[0114]

[0115] The PCR products were recovered from 1% agarose gel, and the light chain variable region was recovered as Figure 3 , heavy chain variable region recovery as Figure 4 The light chain variable region and heavy chain variable region amplified by PCR were spliced ​​into scFv using the overlap-PCR method, as shown in FIG. Figure 5 The target fragment scFv was ligated with the phagemid vector by enzyme digestion and electroporated into electrocompetent cells TG1 to construct a rabbit antibody gene library. The recombination rate of the antibody gene library was identified by bacterial liquid PCR. The agarose gel electrophoresis diagram is shown in the figure below. Figure 6The antibody gene library is prepared into a phage antibody library with the aid of helper phage. After 3 rounds of screening, 3 rounds of monoclonal strains are picked out from the library, and monoclonal phage antibodies are prepared for monoclonal phage ELISA evaluation. Some dominant clones are selected to construct antibodies 1 to 6. The diagnostic performance evaluation results of some sequences and samples are shown in Table 3. According to the results, the effect of antibody 2 is the best, and the effect of antibody 6 is the second. Two antibodies are selected for subsequent experiments. Antibody 2: the amino acid sequence of the heavy chain variable region is SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is SEQ ID NO: 2.

[0116] Table 3. Recombinant antibody clinical gradient evaluation

[0117]

[0118] Example 4. Expression and antibody purification of the recombinant monoclonal antibody of the application

[0119] 1. Recombinant antibody expression

[0120] The positive strain scFv sequence is used as a template to design two pairs of V H and V L primers, respectively, and PCR amplification is performed to obtain V H (the amino acid sequence is shown in SEQ ID NO: 1; the nucleotide sequence is shown in SEQ ID NO: 3) and V L (the amino acid sequence is shown in SEQ ID NO: 2; the nucleotide sequence is shown in SEQ ID NO: 4) target fragments. The heavy chain expression vector Sig-RGFc-PCMV3 and the light chain expression vector Sig-RLFc-PCMV3 constructed in the laboratory are digested with HindIII and BamHI, and then homologously recombined with the corresponding VH or VL, respectively. The recombinant plasmid is transformed into competent cells to construct a heavy and light chain co-expression vector.

[0121] The constructed plasmid is shaken and cultured, and a sufficient amount of plasmid containing the heavy chain and the light chain is extracted according to the conventional operation steps. After preparation, it is transfected into HEK293 cells in good condition, cultured for 3 days, and then supplemented with a feed (the feed is a commercially available product, and the addition volume is 3.5% of the cell culture supernatant). After seven days, the cell survival rate is not less than 65%, and the cell supernatant is harvested.

[0122] 2. Antibody SPA purification and SDS-PAGE identification column equilibration

[0123] The application uses A protein filler to purify the collected cell supernatant. First, the SPA column is filled with 5 ml and the flow rate is set to 8 mL / min. The chromatographic column is washed with 0.02 mol / L PBS pH=7.2~7.4 as the equilibrium buffer. The sample is loaded with a flow rate of 4 mL / min. When the UV peak graph rises, the flow-through collection starts. The equilibrium liquid is used to balance the chromatographic column. The flow rate is set to 8 mL / min. The chromatographic column is washed again with the equilibrium buffer until the UV peak graph drops to the baseline. The flow-through collection is stopped. 0.2 mol / L Gly+0.15 mol / L NaCl pH2.7 is used as the dissociation liquid. When the UV peak graph starts to rise, the target protein is collected. When the UV peak graph drops to the baseline, the collection is stopped. 1 mol / L Tris pH 8.5 is added to the collection tube to neutralize to pH 7. The collected proteins are combined and detected by SDS-PAGE, as shown in Figure 7 . It is shown that the purified and concentrated protein has high purity and high concentration, and can be used as a diagnostic reagent.

[0124] Example 5 Application of the prepared progesterone recombinant rabbit monoclonal antibody kit of the application

[0125] The prepared progesterone recombinant rabbit monoclonal antibody of the application is used as a magnetic bead coated antibody in a kit, and is used together with other components to determine the antigen in a sample. The specific performance evaluation is as follows:

[0126] 1. Accuracy determination:

[0127] 86 clinical samples are detected by using an existing mainstream commercial kit for value setting. The clinical samples need to be centrifuged before loading to remove the interference of the sample state. According to the system back calculation of the sample progesterone concentration, the correlation analysis is performed with the reference manufacturer. According to the detection results, the linear equation of antibody 2 is y = 1.0147x + 0.014, the correlation coefficient R 2 =0.9932, and the results are shown in Figure 8 . The linear equation of antibody 6 is y = 0.9849x + 0.757, the correlation coefficient R 2 =0.9902, and the results are shown in Figure 9 . According to the above results, the clinical accuracy of the two antibodies meets the requirements of in vitro diagnostic reagents.

[0128] 2. Precision evaluation:

[0129] The clinical high value, medium value and low value samples were detected using the same batch of reagents, each sample was repeated 20 times, the average value and standard deviation (SD) were calculated, and the coefficient of variation CV% was calculated to evaluate the within-run precision. The within-run coefficient of variation (CV) of the three levels of samples should be less than 5.0%, and the results are shown in Table 4, which shows that the repeatability of the kit is good and can be used for the development of diagnostic kits.

[0130] Table 4. Evaluation results of two antibodies

[0131]

[0132] 3. Sensitivity performance evaluation:

[0133] Select 5 clinical samples close to 0 value, each sample is repeated 3 times, and continuously detected for 4 days. The 60 data obtained by detection are verified according to the method of CLSI EP17-A. The LoB values of the three batches of kits prepared by the two antibodies of the application are less than 0.05 ng / mL, which are within the blank limit, indicating that the antibody sensitivity is high and meets the requirements.

[0134] 4. Linear analysis:

[0135] Select 3 clinical high value samples with progesterone concentration close to the upper limit of the expected linear range of about 130%, and 8 known clinical low value samples to mix in different proportions to obtain 9 linear samples with different concentrations, and calculate the theoretical concentration value after dilution. Finally, the deviation between the test results and the theoretical concentration is calculated. The linear regression coefficient R of antibody 2 is 0.9969, the results are shown in Table 6, the linear regression dilution R of antibody 6 is 0.9967, the results are shown in Table 7. 2 Figure 10 2 Figure 11 . The deviation of the diluted linear samples is less than 15%, which meets the requirements.

[0136] 5. Recovery performance evaluation:

[0137] Select 3 high value samples, dilute the high value samples with low value clinical serum according to a specific proportion, and the volume ratio of the high value clinical sample is less than 10% to prepare the recovery sample. Each recovery sample is repeated 3 times to calculate the recovery rate. The sample recovery rates of antibody 2 are 97.33%, 96.98% and 97.87% respectively, and the sample recovery rates of antibody 6 are 103.82%, 103.23% and 102.5% respectively. The detection deviation is less than 10%, and the recovery accuracy of the two antibodies meets the requirements.

[0138] 6. Stability evaluation:

[0139] ​​​​The reagent was placed at 37℃ for 7 days, and at the same time, the same batch of reagent was placed at 2-8℃ as a control group, and the antibody stability performance evaluation test was carried out, and the specific results are shown in Table 5. The performance indicators of the reagent after heat treatment can all meet the requirements, and the test results have no deviation.

[0140] Table 5. Accelerated heat stability results of progesterone magnetic particle detection reagent

[0141]

[0142] 7. Matrix effect analysis

[0143] 20 clinical samples in the detection linear range were selected, the sample state was normal, there was no hemolysis, there was no obvious precipitate, and the state was clear. According to the needs, the matrix was selected as mixed serum, quality control matrix, etc., and the commercial quality control sample was prepared. The concentration range of the prepared sample should be in the normal linear range of the kit. Generally, it should be avoided to be diluted to the vicinity of the detection limit to avoid dilution error. According to the evaluation results, the detection values of the two antibodies in the mixed serum and quality control matrix were within their 95% confidence intervals, meeting the requirements.

[0144] From the above results, it can be seen that antibody 2 is slightly better than antibody 6, and antibody 2 has reached a high level of consistency with the detection results of the current market mainstream manufacturers, has good stability, high sensitivity, and is used as a specific antibody in the in vitro detection kit. According to the above evaluation results, the detection performance meets the requirements of the kit, and plays an important role in the later application and clinical diagnosis.

[0145] The above is only a preferred embodiment of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A progesterone monoclonal antibody, characterized in that The amino acid sequences of the three CDR regions of its heavy chain are GLSLSSNE, IDRSANT, and ARGGYDVAYAFNI; The amino acid sequences of the three CDR regions of its light chain are QSVYNNNN, SAS and LGGYSTAAA, respectively.

2. The progesterone monoclonal antibody according to claim 1, characterized in that The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; The light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:

2.

3. The progesterone monoclonal antibody according to claim 1 or 2, characterized in that The constant region of the heavy chain is of rabbit IgG1 subtype; the constant region of the light chain is of rabbit K1 type.

4. A nucleic acid encoding the progesterone monoclonal antibody according to any one of claims 1 to 3.

5. The nucleic acid according to claim 4, characterized in that The nucleic acid encoding the heavy chain variable region of the progesterone monoclonal antibody is shown in SEQ ID NO: 3; The nucleic acid encoding the light chain variable region of the progesterone monoclonal antibody is shown in SEQ ID NO:

4.

6. A recombinant vector, characterized in that It comprises a vector backbone and the nucleic acid according to claim 4 or 5.

7. The recombinant vector according to claim 6, characterized in that The vector backbone is pCMV3.

8. A host cell, characterized in that Including at least one of the following I) to III): 1), secreting the progesterone monoclonal antibody according to any one of claims 1 to 3; II), genome integration of the nucleic acid according to claim 4 or 5; III), transfecting or transforming the recombinant vector according to claim 6 or 7.

9. A method for preparing a progesterone monoclonal antibody, characterized in that: The method comprises culturing the host cell as claimed in claim 8 to obtain progesterone monoclonal antibodies.

10. A labeled antibody, characterized in that Includes markers and: The progesterone monoclonal antibody according to any one of claims 1 to 3; Or a culture of the progesterone monoclonal antibody prepared by the preparation method according to claim 9.

11. The labeled antibody according to claim 10, characterized in that The markers include chemical markers and biological markers; the biological markers include biotin, avidin or enzymes; the chemical markers include isotopes.

12. A conjugate, characterized in that It includes a coupling medium and: The progesterone monoclonal antibody according to any one of claims 1 to 3; Or a culture of the progesterone monoclonal antibody prepared by the preparation method according to claim 9.

13. Use of any one of the following a) to g) in the preparation of a product for detecting progesterone levels in serum: a), the progesterone monoclonal antibody according to any one of claims 1 to 3; b) the nucleic acid according to claim 4 or 5; c) The recombinant vector according to claim 6 or 7; d) The host cell according to claim 8; e) A culture containing progesterone monoclonal antibodies obtained by the preparation method according to claim 9; f), the labeled antibody according to claim 10 or 11; g) The conjugate according to claim 12.

14. A product for detecting progesterone levels in serum, characterized in that: Including any one of the following A) to D): A), the progesterone monoclonal antibody according to any one of claims 1 to 3; B) a culture containing a progesterone monoclonal antibody obtained by the preparation method according to claim 9; C), the labeled antibody according to claim 10 or 11; D) The conjugate according to claim 12.

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