C-reactive protein monoclonal antibody and its preparation method and application

Abstract

The present invention discloses a C-reactive protein monoclonal antibody, a preparation method thereof, and an application thereof. The amino acid sequence of the heavy chain CDR3 of the antibody is shown in SEQ ID NO.3 or SEQ ID NO.9; the amino acid sequence of the light chain CDR3 of the antibody is shown in SEQ ID NO.6 or SEQ ID NO.12. The C-reactive protein monoclonal antibody has good specificity and high affinity, meets the requirements of full-scale CRP detection, has no cross-reaction with other proteins, can be used as a capture antibody, can be effectively applied to the ELISA method, and has good detection linearity, precision, stability and accuracy, and has important application value in clinical detection.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a C-reactive protein monoclonal antibody and a preparation method and application thereof. Background Art

[0002] C-reactive protein (CRP) is a pentameric acute-onset protein, also known as Pentraxin 1, primarily synthesized in the liver. From a pathological perspective, CRP can activate complement and enhance phagocytosis by phagocytes, thereby playing a regulatory role in clearing invading pathogens and damaged, necrotic, or apoptotic tissue cells. It plays a crucial role in the body's innate immune system. When infected or with tissue damage, CRP levels in plasma increase dramatically. It is often used alongside inflammatory factors such as white blood cells, interleukins, and procalcitonin (PCT) to aid in the diagnosis of acute infectious diseases and tissue damage. CRP levels are significantly elevated 4–6 hours after acute infection, peaking at 36–48 hours. Its half-life in the blood is approximately 19 hours. A significant and sustained elevation of CRP serves as an early marker of inflammation in bacterial sepsis, and its measurement is highly valuable for assessing the efficacy of acute inflammatory treatments.

[0003] CN112034178A discloses a C-reactive protein detection kit, which includes a first reagent and a second reagent; wherein the first reagent includes microspheres capable of adsorbing C-reactive protein, and the second reagent includes CRP antibodies and albumin (Alb). The kit can detect CRP levels in both low-value samples and high-value samples, and has high sensitivity and accuracy. At the same time, the scheme can use anti-human C-reactive protein antiserum and a small amount of microspheres for conventional immunoturbidimetry, avoiding the tedious coupling steps in the preparation of latex turbidimetric reagents, and has certain cost advantages.

[0004] CN107907690A discloses a high-sensitivity C-reactive protein (HRP) detection kit and its use method. The HRP detection kit includes a calibrator, reagent R1, enzyme conjugate working solution R2, magnetic bead conjugate working solution M, a cleaning solution, and a chemiluminescent substrate. Reagent R1 contains a pyridine component that can rapidly eliminate blood cells from whole blood, effectively preventing blood cells from engulfing magnetic beads. The kit combines chemiluminescence technology with immunomagnetic microparticles to provide a near-homogeneous reaction system and employs a one-step reaction mode, significantly improving detection sensitivity and precision. Carboxyl magnetic beads are used as solid-phase carriers. Due to their uniform size and shape, they allow the target substance to be quickly and effectively bound to the magnetic beads. Their spherical structure also eliminates nonspecific binders associated with irregularly shaped particles, thereby improving the specificity of the kit product.

[0005] Currently, there are over 50 CRP testing reagents on the market, with varying degrees of quality. Establishing a scientific, rigorous, standardized, and regulated evaluation system for testing reagents requires the use of standard substances for quality control and testing of CRP testing reagents, thus necessitating the development of standard reference materials. To address the varying quality of existing CRP testing reagents, developing a CRP testing reagent and testing system with excellent specificity, reproducibility, high potency, and good correlation with clinical test results remains a research priority in this field. Summary of the Invention

[0006] In response to the deficiencies in the existing technology and actual needs, the present invention provides a C-reactive protein monoclonal antibody and its preparation method and application. The present invention screens and obtains a C-reactive protein monoclonal antibody with good specificity, and applies it to the ELISA detection of C-reactive protein, and has good detection linearity, precision, stability and accuracy.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a C-reactive protein monoclonal antibody, wherein the amino acid sequence of the heavy chain CDR3 of the antibody is shown as SEQ ID NO.3 or SEQ ID NO.9; the amino acid sequence of the light chain CDR3 of the antibody is shown as SEQ ID NO.6 or SEQ ID NO.12.

[0009] Preferably, the amino acid sequence of the heavy chain CDR1 of the antibody is shown as SEQ ID NO.1 or SEQ ID NO.7, and the amino acid sequence of the heavy chain CDR2 is shown as SEQ ID NO.2 or SEQ ID NO.8.

[0010] Preferably, the amino acid sequence of the light chain CDR1 of the antibody is shown as SEQ ID NO.4 or SEQ ID NO.10, and the amino acid sequence of the light chain CDR2 is shown as SEQ ID NO.5 or SEQ ID NO.11.

[0011] Preferably, the amino acid sequence of the variable region of the heavy chain of the antibody is as shown in SEQ ID NO.13 or SEQ ID NO.15, and the amino acid sequence of the variable region of the light chain of the antibody includes the sequence shown in SEQ ID NO.14 or SEQ ID NO.16;

[0012] Preferably, the amino acid sequence of the heavy chain of the antibody is as shown in SEQ ID NO.17 or SEQ ID NO.19, and the amino acid sequence of the light chain of the antibody is as shown in SEQ ID NO.18 or SEQ ID NO.20.

[0013] In the present invention, the amino acid sequence of the variable region of the heavy chain of monoclonal antibody 20# is shown in SEQ ID NO.13, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.14.

[0014] In the present invention, the amino acid sequence of the heavy chain variable region of monoclonal antibody 22# is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.16.

[0015] In the present invention, the amino acid sequence of the heavy chain of monoclonal antibody 20# is shown in SEQ ID NO.17, and the amino acid sequence of the light chain is shown in SEQ ID NO.18.

[0016] In the present invention, the amino acid sequence of the heavy chain of monoclonal antibody 22# is shown in SEQ ID NO. 19, and the amino acid sequence of the light chain is shown in SEQ ID NO. 20.

[0017] In the present invention, the CDR1, CDR2 and CDR3 of the C-reactive protein monoclonal antibody and their corresponding sequence numbers are shown in Table 1.

[0018] Table 1

[0019]

[0020] SEQ ID NO. 1 (heavy chain CDR1 of monoclonal antibody #20): GFSFNTYAMN.

[0021] SEQ ID NO.2 (heavy chain CDR2 of monoclonal antibody 20#):

[0022] LIRSKSNNYETNYADSVKD.

[0023] SEQ ID NO. 3 (heavy chain CDR3 of monoclonal antibody #20): HGAVVEGAWFPY.

[0024] SEQ ID NO. 4 (light chain CDR1 of monoclonal antibody #20): RASENIYSNLA.

[0025] SEQ ID NO. 5 (light chain CDR2 of monoclonal antibody #20): AATNLAD.

[0026] SEQ ID NO. 6 (light chain CDR3 of monoclonal antibody #20): QHFWGSPPT.

[0027] SEQ ID NO. 7 (heavy chain CDR1 of monoclonal antibody #22): GYTFTRYWMH.

[0028] SEQ ID NO. 8 (heavy chain CDR2 of monoclonal antibody #22): NINPSNGGTNYNEKFKN.

[0029] SEQ ID NO. 9 (heavy chain CDR3 of monoclonal antibody #22): RPGNYYGYGLYYFDY.

[0030] SEQ ID NO.10 (light chain CDR1 of monoclonal antibody #22): KASQSVSNVVA.

[0031] SEQ ID NO.11 (light chain CDR2 of monoclonal antibody #22): YASNRYT.

[0032] SEQ ID NO. 12 (light chain CDR3 of monoclonal antibody #22): QQDYSSPLT.

[0033] SEQ ID NO.13 (heavy chain variable region of monoclonal antibody 20#):

[0034] EVQLEESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVALIRSKSNN YETNYADSVKDRFSISRDDSENMLYLQMNNLKSEDSAMYYCVRHGAVVEGAWFPYWGQ GTLVTVSAASTTAPS.

[0035] SEQ ID NO.14 (light chain variable region of monoclonal antibody 20#):

[0036] DIVMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGNSPQLLVYAATNLADGV PSRFSGSVSGTQYSLKINSLQSEDFGIYYCQHFWGSPPTFGGGTKLEIKRTDAAPTVSIFPPSS EQLTSGGA.

[0037] SEQ ID NO.15 (heavy chain variable region of monoclonal antibody 22#):

[0038] QVQLQQSGTELVKPGASVKLSCKASGYTFTRYWMHWVKQRPGQGLEWIGNINPSNG GTNYNEKFKNKATLTTNKSSSTAYMQLSSLTSEDSAVYYCARRPGNYYGYGLYYFDYWGQ GTTVTVSSASTTA。

[0039] SEQ ID NO.16 (Light chain variable region of monoclonal antibody 22#):

[0040] DIVMTQTPGFLLVSAGDRVTITCKASQSVSNVVAWYQQKPGQSPKLLIYYASNRYTGV PDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPLTFGAGTKLELK。

[0041] SEQ ID NO.17 (Heavy chain of monoclonal antibody 20#):

[0042] EVQLEESGGGLVQPKGSLKLSCAASGFSFNTYAMNWVRQAPGKGLEWVALIRSKSNNYETNYADSVKDRFSISRDDSENMLYLQMNNLKSEDSAMYYCVRHGAVVEGAWFPYWGQGTLVTVSAASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK。

[0043] SEQ ID NO.18 (Light chain of monoclonal antibody 20#):

[0044] DIVMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGNSPQLLVYAATNLADGVPSRFSGSVSGTQYSLKINSLQSEDFGIYYCQHFWGSPPTFGGGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0045] SEQ ID NO.19 (Heavy chain of monoclonal antibody 22#):

[0046] QVQLQQSGTELVKPGASVKLSCKASGYTFTRYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKNKATLTTNKSSSTAYMQLSSLTSEDSAVYYCARRPGNYYGYGLYYFDYWGQGTTVTVSSASTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHRQNYDSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK。

[0047] SEQ ID NO.20 (Light chain of monoclonal antibody 22#):

[0048] DIVMTQTPGFLLVSAGDRVTITCKASQSVSNVVAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYSSPLTFGAGTKLELK RTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0049] In a second aspect, the present invention provides a nucleic acid molecule encoding the C-reactive protein monoclonal antibody according to the first aspect.

[0050] In a third aspect, the present invention provides a recombinant vector having the nucleic acid molecule of the second aspect inserted therein, wherein the vector can, after transfecting a host cell, cause the transfected host cell to express the C-reactive protein monoclonal antibody of the first aspect.

[0051] In a fourth aspect, the present invention provides a recombinant cell expressing a C-reactive protein monoclonal antibody, wherein the recombinant cell is obtained by transfecting a host cell with the recombinant vector described in the third aspect.

[0052] In a fifth aspect, the present invention provides a method for preparing a C-reactive protein monoclonal antibody as described in the first aspect, the preparation method comprising:

[0053] The nucleic acid molecule encoding the C-reactive protein monoclonal antibody described in the first aspect is inserted into an expression vector to obtain a recombinant vector, and the recombinant vector is introduced into a host cell for culture, protein expression and purification, and finally the C-reactive protein monoclonal antibody is obtained.

[0054] Preferably, the vector comprises a pcDNA3.1(+) vector.

[0055] Preferably, the host cell comprises an Expi293 cell.

[0056] In this study, single B cell antibody preparation technology combined with the Expi293 eukaryotic expression system was used to prepare CRP monoclonal antibodies, which were then screened using an indirect ELISA method to obtain C-reactive protein monoclonal antibodies with good specificity and high affinity. The resulting monoclonal antibodies 20# and 22# exhibited good specificity and high sensitivity for both recombinant CRP protein and CRP(+) serum, meeting the requirements for full-scale CRP detection. They also showed no cross-reactivity with other proteins (including unrelated proteins PCT and FBS), laying a solid foundation for the further development of immunoassay kits and the establishment of other detection methods.

[0057] In a sixth aspect, the present invention provides use of the C-reactive protein monoclonal antibody described in the first aspect in preparing a C-reactive protein detection product.

[0058] In the present invention, the C-reactive protein monoclonal antibody screened has good specificity and high affinity, meets the full CRP detection requirements, has no cross-reaction with other proteins, and has important application value in clinical detection.

[0059] In a seventh aspect, the present invention provides an ELISA reaction system for detecting C-reactive protein, wherein the reaction system comprises the C-reactive protein monoclonal antibody described in the first aspect.

[0060] In the present invention, the expressed and obtained C-reactive protein monoclonal antibody is used as a capture antibody and / or a detection antibody, which is coated on an enzyme-linked immunosorbent assay microplate to construct an ELISA reaction system, thereby enabling C-reactive protein detection.

[0061] In an eighth aspect, the present invention provides use of the C-reactive protein monoclonal antibody described in the first aspect in detecting C-reactive protein.

[0062] In a ninth aspect, the present invention provides a method for detecting C-reactive protein, comprising using the C-reactive protein monoclonal antibody described in the first aspect as a capture antibody and detecting C-reactive protein by ELISA.

[0063] The C-reactive protein monoclonal antibody screened by the present invention has good specificity and high affinity, can be used as a capture antibody, and can be effectively applied in ELISA methods, such as the ELISA double-antibody sandwich method. It also has good detection linearity, precision and accuracy, and has a good correlation with the actual clinical serum value, meeting the CRP detection requirements.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) The present invention utilizes single B cell antibody preparation technology to prepare CRP monoclonal antibodies, and uses indirect ELISA method to screen and obtain C-reactive protein monoclonal antibodies 20# and 22# with good specificity and high affinity. Monoclonal antibodies 20# and 22# have the advantages of strong specificity, high sensitivity, wide linear range, and good stability for both recombinant CRP protein and CRP(+) serum. Among them, the detection linear range is [0.2-350μg / mL], which is better than the requirements of ultrasensitive CRP determination and conventional CRP determination, and far higher than the requirements of the full CRP linear determination range in YY / T 1513-2017 "C-reactive protein determination kit". The detection stability is as long as 3 years, far exceeding the standard of 1-year validity period of ordinary in vitro diagnostic kits. At the same time, there is no cross-reaction with other proteins (irrelevant proteins PCT and FBS), which lays a good foundation for the further development of immunoassay kits and the establishment of other detection method systems.

[0066] (2) The present invention establishes an ELISA detection system for C-reactive protein, using the screened C-reactive protein monoclonal antibody with good specificity and high affinity as the capture antibody for detection. The ELISA detection system has good detection linearity, precision, stability and accuracy, and has a good correlation with the actual clinical serum value, meeting the CRP detection requirements and having important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 is the SDS-PAGE electrophoresis diagram of the purified recombinant CRP protein;

[0068] Figure 2 This is the SDS-PAGE electrophoresis diagram of the purified CRP monoclonal antibody;

[0069] Figure 3 Figure 1 is the specificity test result of monoclonal antibodies (20#, 21#, 22#, 8#);

[0070] Figure 4 Figure 1 is the specificity verification result of monoclonal antibodies (20#, 22#);

[0071] Figure 5 This is the linear range analysis result diagram of the ELISA detection system of CRP protein;

[0072] Figure 6 This is the result of correlation analysis between ELISA detection and clinical sample detection. DETAILED DESCRIPTION

[0073] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0074] In the following embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this field or the product instructions shall be followed. Reagents or instruments used without manufacturer specified are all conventional products that can be purchased through regular channels. Detailed Description of the Invention

[0076] The present invention uses genetic engineering to construct a eukaryotic expression plasmid from the CRP gene (NG_013007.1), and the expression plasmid is transfected into eukaryotic expression cells Expi293 cells. After expression in the cells, the CRP protein is purified to obtain a recombinant CRP protein with high expression level and high purity. The recombinant CRP protein is used as an immunogen to immunize mice, and single B cells expressing specific antibodies are isolated from the mice by flow cytometry fluorescence sorting technology. mRNA of the single B cells is extracted, and the light and heavy chain nucleic acid sequences encoding the CRP monoclonal antibody protein are amplified by PCR to construct a recombinant plasmid. The plasmid is transfected into Expi 293 cells to prepare CRP monoclonal antibodies, and two monoclonal antibodies 20# and 22# with good specificity and high affinity are screened by indirect ELISA.

[0077] The specificity of CRP monoclonal antibodies was evaluated using other samples such as recombinant PCT and FBS as detection sources. The results showed that both antibodies 20# and 22# had good specificity for CRP recombinant protein and did not react with unrelated proteins PCT and FBS. This shows that the CRP monoclonal antibodies prepared by the present invention have good reaction specificity and no cross-reaction.

[0078] To demonstrate the practicality of the CRP monoclonal antibody of the present invention, the present invention further paired the CRP monoclonal antibodies to establish an ELISA (i.e., double-antibody sandwich ELISA) reaction system capable of detecting CRP in clinical samples and conducted performance evaluation. The results showed that using the CRP monoclonal antibody 22# prepared by the present invention as the coating antibody and the CRP monoclonal antibody 20# as the detection antibody achieved good detection results, representing the optimal combination. The optimal working concentrations of the paired antibodies were determined by checkerboard titration, ultimately determining the optimal working conditions to be: a coating antibody concentration of 5 μg / mL and a detection antibody dilution of 1:3000.

[0079] The present invention pairs these two antibodies and establishes an ELISA double antibody sandwich detection method, which has good detection linearity (y=0.0104x+0.2146, R 2 =0.9982), the intra-assay coefficient of variation (CV) of precision analysis was 6.70-9.37%, the inter-assay coefficient of variation was 8.52-9.10%, and the average recovery was 102%, which had a good correlation with the true clinical serum value and met the full CRP detection requirements.

[0080] Finally, the double-antibody sandwich ELISA detection system of the present invention was used to detect 48 clinically derived sera with known CRP concentrations. After obtaining the measured values ​​and the source values, regression analysis was performed; the results showed that the detection results of the CRP polyclonal antibody of the present invention and the double-antibody sandwich ELISA quantitative detection thereof were well correlated with the clinical detection results.

[0081] The experimental materials and reagents in the following specific embodiments of the present invention include:

[0082] The vector pcDNA3.1(+), restriction endonucleases EcoR I and Xho I, and the Expi293 eukaryotic expression system were purchased from Thermo Fisher Scientific. BALB / c mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Endotoxin-free plasmid extraction kit, Ni-NTA affinity chromatography column, Protein A purification column, Western blot reagent, rapid silver staining kit, biotin, and HRP-streptavidin were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0083] ELISA plates were purchased from Corning, USA;

[0084] TMB single-component colorimetric solution was purchased from Beijing Solebow Technology Co., Ltd.;

[0085] The full-wavelength microplate reader was purchased from Thermo Fisher.

[0086] Example 1 Screening and identification of C-reactive protein monoclonal antibodies

[0087] (1) The full-length CRP gene coding sequence (NG_013007.1) provided by GenBank was analyzed, and the restriction sites EcoR I and Xho I and protective bases were added to both ends of the coding sequence for full gene synthesis. The double-enzyme digested vector pcDNA3.1(+) was then connected and transformed with the CRP gene fragment that had been digested with the same enzymes. The recombinant plasmid was constructed and sent to BGI for sequencing. The CRP recombinant plasmid with the correct sequencing results was transfected into the Expi 293 eukaryotic expression system for expression. Since the recombinant DNA of CRP was fused with a 6×His-tag amino acid sequence at the N-terminus, it was purified using a conventional Ni ion affinity chromatography column. The silver staining results of SDS-PAGE electrophoresis showed that the purity of the recombinant CRP protein was high. The SDS-PAGE electrophoresis results of the purified recombinant CRP protein are as follows: Figure 1 As shown, recombinant CRP protein was obtained.

[0088] (2) Mice were immunized with recombinant CRP protein. Antibody titers were preliminarily detected four weeks after immunization. Mice were killed six weeks after immunization. The spleens of mice were removed, the spleen tissues were minced with scissors, and the spleen tissues were digested with trypsin to separate into single cells.

[0089] (3) Use phosphate buffered saline (PBSD) to suspend the spleen cells to a concentration of 2 × 10 7 Cells / mL were collected by filtration using a FACS tube with a filter. The cells were resuspended in 200 μL PBSD and 0.625 μL VIVD staining solution was added to determine the living cells. Different fluorescently labeled antibodies, anti-CD3, anti-CD8, anti-F4 / 80 and anti-CD20, were used to stain different lymphocytes. At the same time, fluorescently labeled probes prepared using the antigen protein CRP were added to stain the target B lymphocytes. Single B cells (CD3 － / CD8 － / F4 / 80 － / CD20 + / IgM － / IgD － / IgG + / Probe + ), meet the sorting strategy criteria: lymphocytes / viable / CD3 - CD8 - f / 4 / 80 - CD20 + / IgM - IgD - / IgG + / eOD+ The specific staining and screening scheme is shown in Table 2:

[0090] Table 2

[0091] Antibody Fluorescent dyes filter CD3 PerCP Cy55 B710 CD4 PerCP Cy55 B710 CD8 PerCP Cy55 B710 F4 / 80 PerCP Cy55 B710 B220 PE-CF594 G610 IgG1 FITC B515 IgG2a FITC B515 IgG2b FITC B515 IgG3 FITC B515 Hu IgG A680 R710

[0092] (4) The mRNA of a single B cell was extracted and random RT-PCR was performed to synthesize cDNA of all the mRNA. Two PCRs were performed using the cDNA as a template and multiple sets of primers targeting the light and heavy chain sequences of the CRP-specific antibody to amplify the nucleic acid sequences encoding the light and heavy chains of the CRP antibody with specific endonuclease sites. Finally, the above coding sequences were respectively digested and inserted into the pcDNA3.1(+) vector to construct recombinant plasmids encoding the light and heavy chains of the CRP-specific antibody.

[0093] The primers used to clone the antibody light and heavy chain coding sequences are described in Table 3. In addition to the common bases, the nucleotide sequences of the primers in Table 3 also include:

[0094] R:Guanine / Adenine (purine), guanine / adenine (purine)

[0095] Y:Cytosine / Thymine (pyrimidine)

[0096] K:Guanine / Thymine, guanine / thymine

[0097] M:Adenine / Cytosine

[0098] S:Guanine / Cytosine, guanine / cytosine

[0099] W:Adenine / Thymine

[0100] B:Guanine / Thymine / Cytosine, Guanine / Thymine / Cytosine

[0101] D:Guanine / Adenine / Thymine

[0102] H:Adenine / Cytosine / Thymine

[0103] V:Guanine / Cytosine / Adenine, Guanine / Cytosine / Adenine

[0104] N:Adenine / Guanine / Cytosine / Thymine, adenine / guanine / cytosine / thymine.

[0105] Table 3

[0106]

[0107]

[0108] The reverse transcription system used to synthesize cDNA of all mRNA is shown in Table 4:

[0109] Table 4

[0110] Element μL / well 150 ng / μL random primers 3 NEB lab 10mM dNTP 2 Superscript III (reverse transcriptase) 1 total 6

[0111] The reverse transcription parameter settings are shown in Table 5.

[0112] Table 5

[0113] temperature Time (minutes) 42℃ 10 25℃ 10 50℃ 60 94℃ 5 4℃ Maintain temperature

[0114] The PCR reaction system was prepared according to the components shown in Table 6; the PCR reaction parameters were based on the conditions after optimization of different primers.

[0115] Table 6

[0116] Element 1×(μL) cDNA 5 10× buffer 5 dNTP (10mM) 0.5 <![CDATA[MgCl2(25mM)]]> 1 5' primer (25 μM) 1 3' primer (25 μM) 1 HS Taq Plus Polymerase 0.3 <![CDATA[ddH2O]]> 36.2 total 50

[0117] (5) The light chain plasmid and heavy chain plasmid of the same antibody were mixed in a mass ratio of 1:1 and then transfected into Expi 293 cells for expression and assembly of the light and heavy chains of the monoclonal antibody. The cell culture medium was collected and the monoclonal antibody was affinity purified using Protein A. The purity was detected by silver staining after SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis results of the purified monoclonal antibody are shown in Figure 2. Figure 2 As shown, after expression and purification, four pairs of clear light (L) and heavy (H) chain bands (numbered 20#, 21#, 22#, and 8#) were visible on SDS-PAGE electrophoresis gel after silver staining, and the purity was high.

[0118] (6) The recombinant CRP protein and CRP(+) serum 1 (Shenzhen Children's Hospital) and CRP(+) serum 2 (Shenzhen Children's Hospital) were coated on enzyme-linked immunosorbent assay microplates at a concentration of 1 μg / mL by indirect ELISA. The four purified antibodies were screened to identify monoclonal antibodies that could recognize the recombinant CRP protein and CRP components in serum. The specificity test results of monoclonal antibodies (20#, 21#, 22#, 8#) are shown in Figure 2. Figure 3As shown in Figure 2, two monoclonal antibodies 20# and 22# that can specifically immunize CRP protein were obtained. Recombinant CRP, recombinant CRP + fetal bovine serum FBS (purchased from Sangon Biotech (Shanghai) Co., Ltd.), recombinant procalcitonin (PCT) (purchased from Feipeng Biotech Co., Ltd.) and FBS were used as detection sources, respectively. The specificity of the screened antibodies for CRP recognition was further tested. The specificity verification results of the monoclonal antibodies (20#, 22#) are shown in Figure 2. Figure 4 As shown, the results of the indirect ELISA method showed that both antibodies 20# and 22# had good specificity for the recombinant CRP protein, and did not react with the irrelevant proteins PCT and FBS, indicating that two monoclonal antibodies with good specificity were screened and numbered 20# and 22#.

[0119] The amino acid sequence of the heavy chain variable region of monoclonal antibody 20# is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.14; the amino acid sequence of the heavy chain of monoclonal antibody 20# is shown in SEQ ID NO.17; and the amino acid sequence of the light chain of monoclonal antibody 20# is shown in SEQ ID NO.18.

[0120] The amino acid sequence of the heavy chain variable region of monoclonal antibody 22# is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.16; the amino acid sequence of the heavy chain of monoclonal antibody 22# is shown in SEQ ID NO.19; and the amino acid sequence of the light chain of monoclonal antibody 22# is shown in SEQ ID NO.20.

[0121] Example 2 ELISA screening of CRP antibodies

[0122] To screen the binding affinity between each batch of purified CRP monoclonal antibodies and a CRP standard protein and compare the functional differences between each batch of purified monoclonal antibodies and commercially available antibodies, this example used ELISA to screen the functionality of several CRP monoclonal antibodies. Based on the absorbance results, six of the 11 available antibodies with the best functionality were selected for subsequent functional testing.

[0123] In this example, enzyme-linked immunosorbent assay (ELISA) was used to screen CRP monoclonal antibodies. Eleven CRP monoclonal antibodies (eight homemade antibodies, 8#, 10#, 11#, 27#, 28#, 20#, 21#, and 22#, as well as CRP antibody 1 from Beijing Company A, CRP antibody 2 from Beijing Company B, and CRP antibody 3 from Beijing Company B) were coated using a double-antibody sandwich ELISA. CRP standards were then used for binding. A commercial recombinant protein coupled to HPR was then used as a secondary antibody for binding. The absorbance values ​​were measured under the same culture conditions, and six preferred antibodies were selected.

[0124] 1. The experimental materials and reagents used in this example are as follows:

[0125] (1) Sample name

[0126] 11 types of CRP monoclonal antibodies (including 8 self-made ones 8#, 10#, 11#, 27#, 28#, 20#, 21#, 22# and CRP antibody 1 from Beijing A company, CRP antibody 2 from Beijing B company, and CRP antibody 3 from Beijing B company).

[0127] (2) Sample source

[0128] The samples to be tested this time are CRP monoclonal antibodies with immunogenicity after screening by single B cell technology, including 8 types: 8#, 10#, 11#, 27#, 28#, 20#, 21#, and 22#.

[0129] The control samples were CRP antibody 1 from Beijing Company A, CRP antibody 2 from Beijing Company B, and CRP antibody 3 from Beijing Company B.

[0130] (3) Sample storage conditions

[0131] Store the samples at 2-8°C.

[0132] (4) Experimental instruments

[0133] Microplate washer, microplate reader, 96-well ELISA plate, Na2CO3, NaHCO3, Tris, NaCl, BSA, Tween 20, KCl, Na2HPO4, KH2PO4, H2SO4 and TMB.

[0134] (5) Solution preparation

[0135] a. 10× ELISA coating buffer: Na2CO3 150mM, NaHCO3 250mM, adjust pH to 9.6, store at 4°C;

[0136] b. 1× ELISA diluent: Tris 10 mM, NaCl 150 mM, 0.5% BSA, 0.05% Tween 20, adjust pH to 7.4, store at 4°C;

[0137] c. 20× ELISA wash buffer (PBST): NaCl 2.74 M, KCl 54 mM, Na2HPO4 200 mM, KH2PO4 40 mM, 1% Tween 20, pH 7.4, store at room temperature;

[0138] d. ELISA stop solution: 0.5M H2SO4, stored at room temperature.

[0139] 2. The test steps are as follows:

[0140] (A) Monoclonal antibody coating

[0141] a. Dilute 10× ELISA coating buffer to 1×. Dilute each CRP monoclonal antibody to the specified concentration (5 μg / mL) using 1× ELISA coating buffer. Add 100 μL of the diluted sample coating buffer to each well of a new ELISA plate.

[0142] b. Seal the ELISA plate with sealing film and coat at 4°C overnight.

[0143] c. Dilute 20× ELISA Wash Buffer (PBST) to 1× and wash the plate using a plate washer: Turn on the plate washer and rinse once with deionized water. After rinsing, transfer the wash buffer tubing to the 1× ELISA Wash Buffer bottle. Place the plate in the plate washer and start the ELISA 3 wash program. The procedure is as follows: Aspirate all wells, add 300 μL of ELISA Wash Buffer per well, soak for 1 minute, and repeat three times. After the program is complete, rinse the plate washer once with deionized water and turn off the machine.

[0144] d. Prepare ELISA blocking solution: 2% BSA, freshly prepared for use;

[0145] e. Add ELISA blocking solution to each well of the plate, 200 μL, and block at room temperature for 1 hour.

[0146] f. Repeat the plate washing steps. The blocked ELISA plate can be sealed with a film for later use or directly proceed to the next experiment.

[0147] (B) CRP standard (antigen) binding

[0148] a. Dilute the CRP standard in 1× ELISA diluent to a gradient concentration of 200 μg / mL and 50 μg / mL. Add 100 μL to each well of the ELISA plate and incubate at room temperature for 30 minutes.

[0149] b. Repeat the plate washing step.

[0150] (C) Detection of antibody binding

[0151] a. Use the horseradish peroxidase labeling kit (Cat. No.: HRP-L-500) from Yingchuang Bio to label 1 mg of CRP-Ab8# antibody from Feipeng Bio as a secondary antibody.

[0152] b. Dilute the HRP-labeled CRP-Ab8# secondary antibody 8000-fold with 1× ELISA enzyme-labeled diluent. Add the diluted secondary antibody to the ELISA plate at 100 μL per well and incubate at room temperature for 30 minutes.

[0153] c. Repeat the plate washing steps

[0154] (D) Color development

[0155] a. Add TMB substrate to the ELISA plate, 100 μL per well, and incubate at room temperature for 15 minutes.

[0156] b. Add 100 μL of ELISA stop solution to each well of the microplate to terminate the reaction;

[0157] c. Place the ELISA plate into an ELISA reader and measure the absorbance at a single wavelength of 450 nm. Analyze the absorbance data.

[0158] 3. Results and analysis

[0159] Under the same culture conditions, TMB color development was stopped by adding stop solution after 15 minutes, and the absorbance was read using a microplate reader within 10 minutes. The absorbance results are shown in Table 7.

[0160] Table 7

[0161]

[0162] Table 7 shows that the 11 antibodies exhibited varying performance. The absorbance at 200 μg / mL ranged from 1.651 to 3.411, and at 50 μg / mL, it ranged from 0.403 to 0.883. According to the principle of antigen-antibody binding, samples with the highest absorbance should exhibit the best binding ability. The results showed that two samples (20# and 22#) had an absorbance greater than 3 at 200 μg / mL, four samples (8#, 11#, 21#, and CRP-B-3) had an absorbance between 2.4 and 3, and five samples (10#, 28#, 27#, CRP-A-1, and CRP-B-2) had an absorbance less than 2.4. Six samples (8#, 11#, 20#, 21#, 22#, and CRP-B-3) had an absorbance greater than 0.6 at 50 μg / mL. Screening based on absorbance, samples 8#, 11#, and 21# had good binding to CRP-B-3; samples 20# and 22# had excellent binding, so the above six antibodies were selected for affinity determination.

[0163] Example 3: Detection of CRP Antibody Affinity by BLI Method

[0164] To evaluate the affinity between each batch of purified CRP monoclonal antibodies and the CRP standard protein, this example established a process for detecting the affinity between the CRP monoclonal antibodies and the standard protein using biomembrane interferometry (BLI).

[0165] This method utilizes biomembrane interferometry technology. Six CRP monoclonal antibodies optimized by ELISA (five homemade antibodies, 8#, 11#, 20#, 21#, and 22#, and one CRP antibody purchased from Beijing Company B, 3) are bound to the surface of an AMC sensor. The antibody-bound sensor is then placed in a gradient-diluted CRP standard protein solution for binding and dissociation kinetics. By processing the binding and dissociation signals between the CRP monoclonal antibodies and the CRP standard, the affinity (KD) of the corresponding monoclonal antibodies for the CRP standard protein can be calculated. Table 8 shows the amino acid sequences corresponding to the CDR regions of the CRP monoclonal antibodies.

[0166] Table 8

[0167]

[0168] 1. The experimental materials and reagents used in this example are as follows:

[0169] (1) Experimental instruments

[0170] Biomolecular interaction analyzer (Fortebio), small high-speed refrigerated centrifuge, electronic balance, pH meter, vortex shaker.

[0171] (2) Reagents and materials

[0172] 96-well black sample plate, six ELISA-optimized CRP monoclonal antibodies, CRP standard diluent, AMC sensor, Tween-20, 1× PBS, glycine, and concentrated hydrochloric acid.

[0173] (3) Solution preparation

[0174] Sample diluent (SD buffer, pH = 7.4): Take 180 mL of 1×PBS, add 100 μL of Tween-20, and adjust the volume to 200 mL. After thorough mixing, filter with a 0.22 μm filter membrane and store for later use.

[0175] Monoclonal antibody working solution: Dilute the monoclonal antibody to 25 μg / mL working solution using sample diluent.

[0176] CRP standard working solution: Dilute the CRP standard with sample diluent to a standard working solution of 200 μg / mL.

[0177] (1) Regeneration solution: Weigh 0.150134 g of glycine and dissolve it in 180 mL of ultrapure water. Adjust the pH to 1.7 with concentrated hydrochloric acid, make up to 200 mL, and store at 4°C until use.

[0178] (2) Blank solution: SD buffer.

[0179] (3) 2. The experimental steps of this embodiment are as follows:

[0180] Sample plate order: Add SD buffer to wells BH in columns 1 and 3 of a 96-well black sample plate. Add 25 μg / mL of Company B's CRP antibody working solutions (3, 22#, 11#, 8#, 20#, 21#, and 21#, respectively) to wells BH in column 2. Add 200 μg / mL of CRP standard solution to wells BG in column 4. Add blank solution to wells H in column 4. Add eluent to wells BH in column 11. Add SD buffer to wells BH in column 12 to neutralize the detector after glycine treatment. The sample volume is 250 μL / well. Place seven AMC sensors in wells BH in column 1 of the sensor rack and allow them to wet with SD buffer for at least 10 minutes.

[0181] (2) Select the kinetic detection module in the Data Acquisition 12 software and set the detection conditions as follows: the sample plate temperature in the instrument parameters is 30°C, and the data acquisition frequency is 2 Hz. The test conditions are shown in Table 9.

[0182] Table 9

[0183] Step number Step Name Time / s Sample column Sample plate shaking speed / rpm 1 Baseline 120 1 1000 2 Curing 80 2 1000 3 Baseline 2 80 3 1000 4 Combine 60 4 1000 5 dissociation 80 3 1000 6 regeneration 30 12 1000 7 regeneration 30 12 1000 7 regeneration 30 12 1000

[0184] (3) Data were analyzed using data processing software (Data Analysis 12.0). Background was subtracted using a blank well corresponding to the unbound sample as a blank control. Steady-state fitting was performed using concentration as the horizontal axis and the binding value of each concentration sample as the vertical axis to calculate the affinity (KD) between the CRP monoclonal antibody and the CRP standard. The data analysis parameters are shown in Table 10. After analysis, the analysis data was exported.

[0185] Table 10

[0186]

[0187] 3. Results and analysis

[0188] The KD values ​​of the six antibodies were obtained through software analysis. The KD value indicates the affinity between the sample and the receptor. The smaller the KD value, the greater the affinity between the sample and the receptor. The KD values ​​of the six antibodies are shown in Table 11.

[0189] Table 11

[0190] Antibody No. KD Company B CRP Antibody 3 2.824E-08 8# 1.058E-08 11# 5.558E-08 20# 1.310E-12 21# 8.231E-09 22# 9.071E-10

[0191] The results in Table 11 show that 20# and 22# have good affinity with the CRP standard.

[0192] Example 4 ELISA method to detect the pairing of C-reactive protein monoclonal antibodies

[0193] The experimental steps include: coating C-reactive protein monoclonal antibodies 22#, 21#, 20# and 8# to 96-well ELISA reaction wells at a concentration of 5.0 μg / mL, blocking the ELISA plate with a sealing film, and coating at 4°C overnight; washing the plate three times with PBST, blocking with 2% BSA at 37°C for 1 hour, discarding the remaining blocking solution, and then washing with PBST three times; diluting C-reactive protein with 1× ELISA enzyme diluent to a concentration of 200 μg / mL, 150 μg / mL, 50 μg / mL and 0 μg / mL, adding 100 μL to each well of the ELISA plate, and standing at room temperature. Incubate for 30 minutes; discard the liquid in the wells and wash the plate three times with PBST; add biotin-labeled diluted C-reactive protein monoclonal antibodies at a dilution of 1:3000, 100 μL per well, incubate at room temperature for 30 minutes, discard the liquid in the wells, and wash the plate three times with PBST; add TMB substrate to the ELISA plate, 100 μL per well, and incubate at room temperature for 15 minutes; add ELISA stop solution to the ELISA plate, 100 μL per well, to terminate the reaction; place the ELISA plate in a microplate reader and detect the absorbance at a single wavelength of 450 nm; the detection results of the antibody ELISA combination screening coated antibodies are shown in Table 12.

[0194] Table 12

[0195]

[0196] From Table 12, it can be seen that the combination test results using 22# as the capture antibody and 20# as the detection antibody are the best.

[0197] Example 5 Construction of an ELISA reaction system for detecting C-reactive protein

[0198] In this example, an ELISA reaction system for detecting C-reactive protein was constructed, and the steps were as follows: the purified specific monoclonal antibody 22# was diluted as a capture antibody to different concentrations of 10 μg / mL, 5 μg / mL, and 1 μg / mL, and coated on an enzyme-linked immunosorbent assay microplate overnight at 4°C. Another specific monoclonal antibody 20# prepared was biotin-labeled using conventional experimental methods and diluted with PBS in proportion to 1:2000, 1:3000, 1:4000, and 1:6000, respectively. The optimal working concentration was determined to be a strong positive OD value of around 1.0 and a negative OD value of <0.1. The optimal working concentration of the paired antibody pair was determined by checkerboard titration, and the optimal working conditions were ultimately determined to be: 5 μg / mL of coating antibody and a 1:3000 dilution of detection antibody.

[0199] Example 6 Performance Evaluation of ELISA System

[0200] This example evaluates the performance of the ELISA system in Example 5.

[0201] (1) Analysis range test of standard curve

[0202] The recombinant CRP protein with a mass concentration of 1000 μg / mL was used as the detection standard and diluted with PBS to 500 μg / mL, 350 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL and 0.20 μg / mL. The ELISA system was constructed for determination. The protein concentration was used as the horizontal axis, and the corresponding A 450nm The standard curve is drawn as the vertical axis, and the linear range analysis results of the ELISA detection system of CRP protein are as follows Figure 5 As shown in the figure, the regression analysis results showed that the detection linearity was good when the CRP concentration was between 0.5 and 350 μg / mL (y = 0.0104x + 0.2146, R 2 =0.9982)

[0203] (2) Precision analysis

[0204] The recombinant CRP protein was diluted to 150 μg / mL and 100 μg / mL with PBS as quality control samples, respectively. The samples were measured in four batches using the constructed ELISA system, with eight replicate wells in each batch. The intra-batch precision and inter-batch precision were calculated, and the results are shown in Table 13. The results showed that the intra-batch coefficient of variation CV (coefficient of variation CV = standard deviation SD / mean × 100%) was 6.7-9.37%, which was less than the standard value of 10%, and the inter-batch coefficient of variation was 8.52-9.10%, which was less than the standard value of 15%, indicating that the precision of CRP detection was good.

[0205] Table 13

[0206]

[0207] (3) Recovery experiment

[0208] Using fetal bovine serum as the matrix fluid, dilute the CRP recombinant protein standard to concentrations of 1250 μg / mL, 1000 μg / mL, 625 μg / mL, and 500 μg / mL. Prepare the experimental samples as follows:

[0209] 1) Matrix solution 0.9 mL + PBS 0.1 mL;

[0210] 2) Analytical sample 2: 0.9 mL matrix solution + 0.1 mL 2500 μg / mL standard;

[0211] 3) Analytical sample 2: 0.9 mL matrix solution + 0.1 mL 1250 μg / mL standard;

[0212] 4) Analytical sample 3: 0.9 mL of matrix solution + 0.1 mL of 1000 μg / mL standard;

[0213] 5) Analytical sample 4: 0.9 mL matrix solution + 0.1 mL 625 μg / mL standard;

[0214] 6) Analysis of sample 5: 0.9 mL of matrix solution + 0.1 mL of 500 μg / mL standard.

[0215] The above samples were measured using the constructed ELISA system. Eight replicate wells were set for each sample, and the recovery rate was calculated after taking the average value (recovery rate = detection value / theoretical value × 100%). The results are shown in Table 14. In the recovery rate experiment, the recovery rates of CRP detection at different concentrations ranged from 96.7% to 106.0%, with an average recovery rate of 102%, indicating good accuracy for CRP detection.

[0216] Table 14

[0217]

[0218] (4) Comparison with clinical samples

[0219] The ELISA detection system was used to detect 48 clinical serum samples (Shenzhen Children's Hospital). The ELISA test results were used as the horizontal axis and the clinical serum sample values ​​were used as the vertical axis to draw a scatter plot and perform correlation analysis. The results of the correlation analysis between ELISA detection and clinical sample detection are shown in the figure. Figure 6 As shown, the test results were subjected to linear regression analysis (y = 0.9507x + 0.9561, R 2 =0.9761), and the two test results have a good correlation.

[0220] Example 7 Stability test of ELISA system

[0221] In this example, the stability of the two monoclonal antibodies with good affinity mentioned above was determined. The first batch of monoclonal antibodies was used as a control, and the hydrodynamic radius rh, polydispersity index PDI and melting temperature Tm values ​​were detected using the Prometheus Panta multifunctional protein stability analyzer. rh is the hydrodynamic radius of the protein. Within a certain range, as the protein denatures, rh will gradually increase; PDI is the polymer dispersity index, which is used to describe the molecular weight distribution of the polymer. The larger the PDI, the wider the distribution of molecules of different particle sizes; the smaller the PDI, the more uniform the molecular distribution. As the protein denaturation increases, the PDI gradually increases; Tm is the temperature at which half of the protein unfolds, which is used to reflect the thermal stability of the protein. The above three indicators can reflect the structural denaturation and aggregation of the protein. The appearance and stability indicators rh, PDI and Tm values ​​of the monoclonal antibodies were detected under different storage times and repeated freeze-thaw conditions to analyze the stability of the monoclonal antibodies.

[0222] 1. Test method

[0223] (1) Experimental materials

[0224] Prometheus Panta Multifunctional Protein Stability Analyzer, water bath. CRP monoclonal antibodies CRP#20 and CRP#22. Absolute ethanol, PR Panta standard capillaries.

[0225] 2. Experimental content

[0226] (1) Stability test of samples at -20°C.

[0227] Aliquots of CRP20# and CRP22# monoclonal antibodies were packaged into 50 μL / liquots at a concentration of 1 mg / mL and placed in a -20°C constant temperature refrigerator. Every 6 months from the date of storage, one unopened aliquot of each antibody was taken and its rh, PDI, and Tm values ​​were measured using the Prometheus Panta Multifunctional Protein Stability Analyzer:

[0228] ① Sample preparation: melt the sample at 4℃, hold one end of the capillary for sample loading, and insert the other end into the sample liquid 1mm above the surface. The EP tube can be slightly tilted to use the capillary action to fill the entire capillary with the sample. Be careful not to allow the outer surface of the capillary to be contaminated with the sample.

[0229] ②On-machine testing: The capillaries filled with samples are placed in the sample tray one by one and tested using PR.Panta control software;

[0230] ③ Enter sample-related information (Sample ID, Concentration, Solvent) in the sample information table, run the Discovery Scan, and adjust the excitation power so that the fluorescence signal value is within the range of 2000 to 15000 counts;

[0231] ④ Select the Size Analysis test function, select the High Sensitivity mode, and measure the hydrodynamic radius rh and polydispersity index PDI of the sample at 25°C;

[0232] ⑤ Click “Start Discovery Scan” to perform sample pre-scan; adjust “Excitation Power” so that the sample fluorescence peak is within the range of 2000 to 15000 counts.

[0233] ⑥After the Size Analysis is completed, add the Thermo unfolding test in the software: set the heating rate to 1°C / min, the heating range to 25-95°C, and determine the Tm value of the sample.

[0234] (A) Appearance stability

[0235] The CRP monoclonal antibody should be a clear, transparent liquid with no precipitation, flocculent matter, or suspended matter. The results of the appearance stability test are shown in Table 15.

[0236] Table 15

[0237] Serial number Test time point Test results 1 6 months The antibodies are all clear and transparent liquids without precipitation, flocs or suspended matter. 2 12 months The antibodies are all clear and transparent liquids without precipitation, flocs or suspended matter. 3 18 months The antibodies are all clear and transparent liquids without precipitation, flocs or suspended matter. 4 24 months The antibodies are all clear and transparent liquids without precipitation, flocs or suspended matter. 5 30 months The antibodies are all clear and transparent liquids without precipitation, flocs or suspended matter. 6 36 months The antibodies are all clear and transparent liquids without precipitation, flocs or suspended matter.

[0238] (B) Accuracy

[0239] The rh, PDI, and Tm values ​​of CRP20# and CRP22# monoclonal antibodies were determined using a Prometheus Panta multifunctional protein stability analyzer. Each sample was tested three times, and the average value was calculated. The absolute value of the relative deviation between the rh and Tm test results and the original sample test results should be ≤5%, and the PDI value should be less than 0.15.

[0240] The 6-month stability results are shown in Table 16:

[0241] Table 16

[0242]

[0243]

[0244] The 12-month stability results are shown in Table 17:

[0245] Table 17

[0246]

[0247] The 18-month stability results are shown in Table 18:

[0248] Table 18

[0249]

[0250]

[0251] The 24-month stability results are shown in Table 19:

[0252] Table 19

[0253]

[0254] The 30-month stability results are shown in Table 20:

[0255] Table 20

[0256]

[0257]

[0258] The 36-month stability results are shown in Table 21:

[0259] Table 21

[0260]

[0261] Analysis of results: After storage at -20°C for 6, 12, 18, 24, 30, and 36 months, the changes in the rh and Tm values ​​of CRP20# and CRP22# monoclonal antibodies were all within the allowable deviation range, and the PDI were all less than 0.15, indicating that CRP20# and CRP22# monoclonal antibodies can still maintain good performance and strong stability after storage at -20°C for 3 years.

[0262] (2) Determination of sample stability under repeated freeze-thaw conditions.

[0263] (A) Appearance stability

[0264] The CRP monoclonal antibody is a clear and transparent liquid with no precipitation, flocs, or suspended matter. There should be no visible changes in appearance after repeated freezing and thawing. The appearance stability results are shown in Table 22.

[0265] Table 22

[0266]

[0267]

[0268] (B) Accuracy

[0269] After storage at -20°C, CRP monoclonal antibody samples 20# and 22# were frozen and thawed seven times in a 37°C water bath. The rh, PDI, and Tm values ​​were determined using the Prometheus Panta Multifunctional Protein Stability Analyzer. Each sample was tested three times, and the average value was calculated. The absolute value of the relative deviation between the rh and Tm values ​​and the original sample test results should be ≤5%, and the PDI value should be less than 0.15.

[0270] The results of one freeze-thaw cycle are shown in Table 23.

[0271] Table 23

[0272]

[0273] The results of freeze-thaw 2 times are shown in Table 24

[0274] Table 24

[0275]

[0276]

[0277] The results of freeze-thaw 3 times are shown in Table 25

[0278] Table 25

[0279]

[0280] The results of 4 freeze-thaw cycles are shown in Table 26

[0281] Table 26

[0282]

[0283]

[0284] The results of 5 freeze-thaw cycles are shown in Table 27

[0285] Table 27

[0286]

[0287] The results of 6 freeze-thaw cycles are shown in Table 28

[0288] Table 28

[0289]

[0290] The results of 7 freeze-thaw cycles are shown in Table 29.

[0291] Table 29

[0292]

[0293]

[0294] Result analysis: Samples stored at -20℃ for 30 months were frozen and thawed seven times. The changes in the rh and Tm values ​​of CRP20# and CRP22# monoclonal antibodies were within the tolerance range, and the PDI was less than 0.15, indicating that CRP20# and CRP22# monoclonal antibodies maintained good performance and strong stability after seven freeze-thaw cycles.

[0295] In summary, the C-reactive protein monoclonal antibody screened by the present invention has good specificity and high affinity, can be used as a capture antibody, and can be effectively applied to ELISA methods, such as the ELISA double-antibody sandwich detection method. It also has good detection linearity, precision and recovery rate, good stability and good correlation with the actual clinical serum value, meeting the requirements of CRP detection.

[0296] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A C-reactive protein monoclonal antibody, characterized in that The amino acid sequence of the heavy chain CDR1 of the antibody is shown in SEQ ID NO.1, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.3; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.6; Alternatively, the amino acid sequence of the heavy chain CDR1 of the antibody is shown in SEQ ID NO.7, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.9; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.10, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.

12.

2. The C-reactive protein monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO.14; Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO.

16.

3. The C-reactive protein monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO. 17, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO. 18; Alternatively, the amino acid sequence of the heavy chain of the antibody is shown in SEQ ID NO.19, and the amino acid sequence of the light chain of the antibody is shown in SEQ ID NO.

20.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the C-reactive protein monoclonal antibody according to any one of claims 1 to 3.

5. A recombinant vector, characterized in that The recombinant vector is inserted with the nucleic acid molecule according to claim 4, and after transfecting a host cell, the vector can cause the transfected host cell to express the C-reactive protein monoclonal antibody according to any one of claims 1 to 3.

6. A recombinant cell expressing a C-reactive protein monoclonal antibody, characterized in that: The recombinant cell is obtained by transfecting a host cell with the recombinant vector according to claim 5.

7. The method for preparing a C-reactive protein monoclonal antibody according to any one of claims 1 to 3, characterized in that: The preparation method comprises: The nucleic acid molecule encoding the C-reactive protein monoclonal antibody according to any one of claims 1 to 3 is inserted into an expression vector to obtain a recombinant vector, and the recombinant vector is introduced into a host cell for culture, protein expression and purification, and finally the C-reactive protein monoclonal antibody is obtained.

8. Use of the C-reactive protein monoclonal antibody according to any one of claims 1 to 3 in the preparation of a C-reactive protein detection product.

9. An ELISA reaction system for detecting C-reactive protein, characterized in that: The reaction system comprises the C-reactive protein monoclonal antibody according to any one of claims 1 to 3.

10. A method for detecting C-reactive protein for purposes other than disease diagnosis or treatment, characterized in that: The detection method comprises using the C-reactive protein monoclonal antibody according to any one of claims 1 to 3 as a capture antibody or a detection antibody, and detecting C-reactive protein by ELISA.

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