Anti-monkey C-reactive protein monoclonal antibody, enzyme-linked immunosorbent assay kit and its application

Abstract

The present invention belongs to the field of biological detection technology, and particularly relates to a monoclonal antibody against monkey C-reactive protein, an enzyme-linked immunosorbent assay kit and its application. Named CRP-13, CRP-13 comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region has three complementary determining regions CDR-H1 to CDR-H3, and the amino acid sequences are successively as shown in SEQ ID NO.6 - SEQ ID NO.8; the light chain variable region has three complementary determining regions CDR-L1 to CDR-L3, and the amino acid sequences are successively as shown in SEQ ID NO.9 - SEQ ID NO.11. The monoclonal antibody of the present invention can specifically bind to monkey C-reactive protein with high specificity. By using the above monoclonal antibody to screen for paired monoclonal antibodies, an enzyme-linked immunosorbent assay kit for monkey C-reactive protein is constructed based on the combination of this pair of monoclonal antibodies, which can detect the content of monkey C-reactive protein in monkey serum, plasma or tissue fluid.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to an anti-monkey C-reactive protein monoclonal antibody, an enzyme-linked immunosorbent assay kit and applications thereof. Background Art

[0002] Infectious diseases are extremely common in clinical practice, and laboratory tests are crucial for their diagnosis and treatment. The four most commonly used indicators are C-reactive protein (CRP), procalcitonin (PCT), serum amyloid A (SAA), and interleukin-6 (IL-6).

[0003] C-reactive protein (CRP) is a nonspecific inflammatory marker, an acute-phase protein synthesized by the liver to protect the body when pathogens invade. It is one of the most widely used diagnostic indicators for infection in clinical practice. CRP levels rise rapidly in response to various non-infectious factors, such as infections or tissue cell damage, and are typically detectable within 5-8 hours of the onset of an inflammatory response. In assessing the inflammatory response to infection, CRP not only serves as a predictor and prognostic indicator for sepsis, but is also commonly used to help differentiate between bacterial and viral infections. While CRP levels typically increase following bacterial infections, they are lower in most patients with viral infections. However, levels can be significantly elevated in some viral infections, particularly those with viral meningitis.

[0004] Laboratory animals are the primary subjects for preclinical drug evaluation, and monkeys are a crucial species in preclinical research. Laboratory animals are used throughout both preclinical and clinical research stages in new drug development, with preclinical research being particularly popular. Various experimental methods are used to evaluate drug pharmacology, study mechanisms of action, and observe toxic effects to demonstrate efficacy and safety. Monkeys are the closest relative to humans, sharing 75% to 98.5% genetic homology with humans, making them the most closely related species to humans. Therefore, they are ideal animals for experimental use.

[0005] Currently, there is no monkey C-reactive protein enzyme-linked immunosorbent assay (ELISA) kit that can detect the level of monkey C-reactive protein in monkey serum, plasma or tissue fluid. Summary of the Invention

[0006] In order to solve the above problems, one of the objectives of the present invention is to provide an anti-monkey C-reactive protein monoclonal antibody, an enzyme-linked immunosorbent assay kit and its use, which can specifically bind to monkey C-reactive protein, and use the above monoclonal antibody to screen for matching monoclonal antibodies, and further construct a monkey C-reactive protein enzyme-linked immunosorbent assay kit based on the combination of the monoclonal antibodies, which is used to detect the content of monkey C-reactive protein in monkey serum, plasma or tissue fluid.

[0007] In a first aspect of the present invention, an anti-monkey C-reactive protein monoclonal antibody is provided, named CRP-13, wherein the CRP-13 comprises a heavy chain variable region and a light chain variable region;

[0008] The heavy chain variable region has three complementarity determining regions, the amino acid sequence of CDR-H1 is shown in SEQ ID NO.6, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.8;

[0009] The light chain variable region has three complementarity determining regions, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.9, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.10, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.11.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of CRP-13 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region of CRP-13 is shown in SEQ ID NO.4.

[0011] Furthermore, the monoclonal antibody further comprises:

[0012] An antibody with the same function obtained by replacing, deleting and / or adding one or more amino acids in the amino acid sequence of the monoclonal antibody; or comprising a heavy chain variable region having an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence having at least 80% homology with the light chain variable region; or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0013] In other embodiments, V H and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence. H and V L V H and V L Antibodies to the regions can be obtained by mutagenizing (eg, site-directed mutagenesis or PCR-mediated mutagenesis) nucleic acid molecules encoding SEQ ID NOs. 6-11 and then testing the encoded altered antibodies for retained function using functional assays described herein.

[0014] The second aspect of the present invention provides a use of the anti-monkey C-reactive protein monoclonal antibody in the preparation of a monkey C-reactive protein detection reagent or kit.

[0015] The third aspect of the present invention provides a monkey C-reactive protein enzyme-linked immunosorbent assay kit, the kit comprising two anti-monkey C-reactive protein monoclonal antibodies, CRP-7 and CRP-13, wherein the monoclonal antibody CRP-7 is used as a coating antibody and the monoclonal antibody CRP-13 is used as a detection antibody;

[0016] The CRP-7 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-H1 is shown in SEQ ID NO.12, the amino acid sequence of its CDR-H2 is shown in SEQ ID NO.13, and the amino acid sequence of its CDR-H3 is shown in SEQ ID NO.14; the light chain variable region has three complementarity determining regions, the amino acid sequence of its CDR-L1 is shown in SEQ ID NO.15, the amino acid sequence of its CDR-L2 is shown in SEQ ID NO.16, and the amino acid sequence of its CDR-L3 is shown in SEQ ID NO.17.

[0017] Furthermore, the amino acid sequence of the CRP-7 heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the CRP-7 light chain variable region is shown in SEQ ID NO.2.

[0018] Furthermore, the monoclonal antibody further comprises:

[0019] An antibody with the same function obtained by replacing, deleting and / or adding one or more amino acids in the amino acid sequence of the monoclonal antibody; or comprising a heavy chain variable region having an amino acid sequence having at least 80% homology with the heavy chain variable region; and a light chain variable region having an amino acid sequence having at least 80% homology with the light chain variable region; or an antibody obtained by connecting a tag to the N-terminus and / or C-terminus of the monoclonal antibody.

[0020] In other embodiments, V H and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the above sequence. H and V L V H and V L Antibodies to the regions can be obtained by mutagenizing (eg, site-directed mutagenesis or PCR-mediated mutagenesis) nucleic acid molecules encoding SEQ ID NOs. 12-17 and then testing the encoded altered antibodies for retained function using the functional assays described herein.

[0021] Furthermore, the monoclonal antibodies CRP-7 and CRP-13 are both obtained by immunizing monkeys with recombinant C-reactive protein.

[0022] Furthermore, the monkey C-reactive protein recombinant protein is obtained by selecting and optimizing a monkey C-reactive protein polypeptide using bioinformatics software, and the segment is predicted to be able to be expressed in Escherichia coli through analysis.

[0023] The present invention provides a monkey C-reactive protein recombinant protein, which realizes the efficient expression of the monkey C-reactive protein in Escherichia coli by optimizing the antigen peptide sequence and expression vector.

[0024] Furthermore, the amino acid sequence of the monkey C-reactive protein recombinant protein is shown in SEQ ID NO.5.

[0025] The fourth aspect of the present invention provides a use of the monkey C-reactive protein enzyme-linked immunosorbent assay kit, wherein the kit is used to detect the content of monkey C-reactive protein in monkey serum, plasma or tissue fluid.

[0026] The beneficial effects of the present invention are:

[0027] (1) The monoclonal antibody of the present invention can specifically bind to monkey C-reactive protein with high specificity.

[0028] (2) The present invention screened out a monoclonal antibody that specifically binds to monkey C-reactive protein based on monkey C-reactive protein recombinant protein, and used the above monoclonal antibody to screen a pairing monoclonal antibody. Further, based on the combination of the monoclonal antibodies, a monkey C-reactive protein enzyme-linked immunosorbent assay kit was constructed to detect the content of monkey C-reactive protein in monkey serum, plasma or tissue fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Shown is the standard curve graph of the monkey C-reactive protein ELISA test of the present invention. DETAILED DESCRIPTION

[0030] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0031] Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making any creative work are within the scope of protection of the present invention.

[0032] The above technical solution is described in detail below in conjunction with specific embodiments.

[0033] Example 1

[0034] Preparation of recombinant antigens:

[0035] This embodiment provides a monkey C-reactive protein recombinant protein, the amino acid sequence of which is shown in SEQ ID No. 5:

[0036] MEKLLCFLVLTSLSHAFGQTDMSTKAFVFPKESDNSTLKARLTKPLKAFTVCLHFYTELSSTRGYSINYATKRQNNEILIFRKSLIGYSFTVGGSEVHICTSWESASGIVEFPQLGKPRARKSLKRGYTVGEDASIILGQEQDSFGGSFETSLVGDIGNVNMWDFVLSPDENYAYRGGTFSPSVLYGSALKYEVQGEVFIKPQLWS.

[0037] This embodiment provides a method for preparing monkey C-reactive protein recombinant protein, comprising the following steps:

[0038] (1) Plasmid construction: Computational software was used to predict and select the main antigenic epitopes of monkey C-reactive protein for expression. This segment of polypeptide can be expressed in the Escherichia coli prokaryotic expression system. The gene was cloned into the pET28a vector through the E. coli expression system to obtain the recombinant plasmid pET28a-CRP that expresses monkey C-reactive protein.

[0039] (2) Induced expression: The recombinant plasmid pET28a-CRP was transformed into BL21 (DE3) competent cells, and the transformed bacteria were spread on LB agar plates containing 50 μg / mL kanamycin and cultured at 37°C overnight. A single colony was picked and inoculated into 4 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 230 rpm with shaking overnight. 1% of the total volume of the culture medium was inoculated into LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm with shaking for about 3 hours until the OD600 reached 0.6. IPTG was added at a final concentration of 0.1 mM and the cells were collected after induction at 16°C and 180 rpm for 15 hours.

[0040] (3) Purification of recombinant protein: Since the expressed recombinant protein carries a histidine tag, it is initially crudely purified using a protein purifier and a chromatography column, and then purified using ion exchange chromatography to obtain the recombinant protein, which is monkey C-reactive protein recombinant protein. The amino acid sequence is shown in SEQ ID No. 5:

[0041] MEKLLCFLVLTSLSHAFGQTDMSTKAFVFPKESDNSTLKARLTKPLKAFTVCLHFYTELSSTRGYSINYATKRQNNEILIFRKSLIGYSFTVGGSEVHICTSWESASGIVEFPQLGKPRARKSLKRGYTVGEDASIILGQEQDSFGGSFETSLVGDIGNVNMWDFVLSPDENYAYRGGTFSPSVLYGSALKYEVQGEVFIKPQLWS.

[0042] (4) Identify the purity of the recombinant protein by SDS-PAGE electrophoresis: Protein sample pretreatment: add an equal volume of 2×SDS loading buffer to each sample, place in a boiling water bath for 10 minutes, centrifuge at 12000 rpm for 3 minutes, and take the supernatant and add it to the sample well.

[0043] Example 2

[0044] Monoclonal antibody screening:

[0045] This embodiment provides a method for obtaining monoclonal antibodies by screening based on the recombinant monkey C-reactive protein, and the specific operations are as follows:

[0046] S1) Mouse Immunization: 6-week-old female BALB / c mice were immunized subcutaneously at multiple sites with 50 μg of recombinant simian C-reactive protein (RCP) mixed with an equal volume of Freund's complete adjuvant (CFA) and emulsified. At week 0 and week 2, immunizations were repeated using the same method, using an equal proportion of Freund's incomplete adjuvant mixed with the recombinant protein. At week 6, sera were collected from the mice for antibody titer determination. Mice with the highest titer were selected for a tail vein booster immunization with 20 μg of recombinant simian C-reactive protein. Three days later, spleens were harvested for hybridoma cell production.

[0047] S2) Screening of hybridoma cell lines:

[0048] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and cultured in HAT medium for selection. When the fused cells reached half the bottom of the well, positive clones were screened by indirect ELISA for monkey C-reactive protein. After 2-3 consecutive limiting dilution cloning cycles to achieve 100% positive clones, the cell lines were expanded and cryopreserved. The indirect ELISA screening method for positive clones was as follows: Microplates were first coated with recombinant protein in carbonate buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L with pure water) at a concentration of 1 μg / mL, 50 μL / well, at 4°C overnight. The plates were then blocked with 3% sucrose and 1% BSA (150 μL per well) at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20) and patted dry. 50 μL of cell culture supernatant was added and incubated at 37°C for 30 minutes. Shake out the liquid in the wells, wash the plate 4 times with PBST solution, pat dry, add 50 μL / well of HRP-labeled goat anti-mouse (diluted 1:5000 in PBS), react at 37°C for 30 min, wash the plate 4 times, pat dry, add 50 μL / well of TMB colorimetric solution, develop at room temperature for 10 min, and finally add 0.5 M sulfuric acid as a terminator to terminate the reaction. Measure the OD450 nm value with a microplate reader.

[0049] S3) Preparation of monoclonal antibody ascites:

[0050] After expanding the culture of the selected monoclonal cell line, female BALB / c mice were intraperitoneally injected with 0.2 mL (containing 2.5×10⁶ cells) pretreated with Freund's incomplete adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the mid-layer was collected.

[0051] S4) Monoclonal Antibody Purification:

[0052] Centrifuge the ascites at 12,000 rpm for 5 minutes. Remove 5 mL of the supernatant and add 20 mL of acetate buffer (68 mmol / L, pH 4.5). Mix thoroughly, then slowly add 50 μL of octanoic acid with stirring. Continue stirring for 30 minutes after addition. Centrifuge at 12,000 rpm for 30 minutes at 2-8°C, and remove the supernatant. Filter the supernatant through absorbent cotton, add saturated ammonium sulfate to 50% of the final volume (v / v), stirring. Continue stirring for 30 minutes after addition, and allow to settle at 2-8°C for 4-5 hours. Centrifuge at 12,000 rpm for 30 minutes at 2-8°C, and remove the precipitate. Resuspend the precipitate in 3 ml of Tris-HCl (10 mmol / L, pH 9.0), place it in a dialysis bag (MW: 8000-14000), and dialyze it against 2 L of Tris-HCl (10 mmol / L, pH 9.0) at 2-8°C for 14 hours. Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12,000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody. Determine the concentration of the purified monoclonal antibody using an ultramicrospectrophotometer. Store at -20°C to avoid repeated freeze-thaw cycles.

[0053] S5) Characterization of monoclonal antibody binding activity:

[0054] The purified antibodies were coated with monkey C-reactive protein recombinant protein and control carrier protein (monkey PCT recombinant protein) by indirect ELISA method to identify the affinity of the monoclonal antibody. The antibody was diluted to 1 μg / ml. The results are shown in Table 1. "Ctrl" in Table 1 represents the negative control monkey PCT mouse monoclonal antibody.

[0055] Table 1 - Binding activity identification of monoclonal antibodies

[0056]

[0057] As can be seen from Table 1, the binding activity of the 25 screened monkey C-reactive protein monoclonal antibodies to monkey C-reactive protein and control carrier protein was detected by indirect ELISA. The data showed that the monoclonal antibodies could specifically bind to monkey C-reactive protein.

[0058] Example 3

[0059] Screening and application of paired monoclonal antibodies:

[0060] 1. HRP labeling of antibodies:

[0061] The screened monoclonal antibodies were labeled with HRP according to the classic sodium periodate method. Finally, the labeled antibodies were dialyzed overnight in 0.01M PBS, pH 7.4 buffer, and glycerol was added at a volume of 1:1. The antibodies were stored in aliquots at -20°C.

[0062] 2. Establishment of double antibody sandwich immunoassay method:

[0063] The purified monoclonal antibody was diluted to a concentration of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) and added to the ELISA plate at 50 μL / well for overnight coating at 4°C. The next day, the coating solution was discarded and the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 3% sucrose and 1% BSA at 150 μL / well. The plate was incubated at 37°C for 2 h, the blocking solution was discarded, and the plate was patted dry. The monkey C-reactive protein recombinant protein sample to be tested and the monkey PCT protein control sample were diluted with PBS at 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, and then added to the ELISA plate at 50 μL / well, incubated at 37°C for 35 minutes, washed four times with PBST washing solution, patted dry, and enzyme-labeled monoclonal antibody diluted to 500, 1000, and 2000 times with PBS was added at 50 μL / well, incubated at 37°C for 35 minutes, washed four times, patted dry, and TMB color development solution was added at 50 μL / well. The color was developed at room temperature for 10 minutes. Finally, 0.5 M sulfuric acid was added at 50 μL / well to terminate the reaction, and the OD450nm value was measured with a microplate reader.

[0064] Then, the most appropriate reaction conditions were determined by adjusting the dilution of the coating antibody and HRP antibody.

[0065] Specifically: monoclonal antibody was coated in a microplate (coating buffer carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water), with a coating concentration gradient of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 50 μL / well, and incubated at 4°C overnight; the plate was washed once with washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 3% sucrose and 1% BSA, 150 μL per well, at 37°C for 2 hours, patted dry, and stored dry for later use; monkey C-reactive protein recombinant protein was diluted with PBS to a concentration of 10 ng / ml, and 50 μL was added to the microplate coated with the monoclonal antibody. At the same time, PCT protein was diluted to 10 ng / ml as a negative control, and reacted at 37°C for 30 minutes. Pour liquid from the wells, wash the plate four times with PBST, and pat dry. Dilute the HRP-labeled monoclonal antibody in PBS to concentrations of 1 μg / mL, 2 μg / mL, and 4 μg / mL, add 50 μL / well, react at 37°C for 30 minutes, wash the plate four times, pat dry, add 50 μL / well of TMB colorimetric solution, and develop at room temperature for 10 minutes. Finally, add stop solution to terminate the reaction and measure the OD450nm value using a microplate reader. Select the coating conditions that provide the most obvious positive / negative difference and the optimal reaction conditions for the HRP-labeled antibody.

[0066] By coating and HRP-labeling the monoclonal antibodies separately as described above, the optimal pairing was selected based on the testing of positive and negative samples, with the most significant difference in positive and negative test values. The final concentration determined was: 1 μg / mL for the coated monoclonal antibody CRP-7 and 1.5 μg / mL for the HRP-labeled monoclonal antibody CRP-13.

[0067] 3. After sequencing:

[0068] CRP-7 monoclonal antibody, including heavy and light chains:

[0069] The amino acid sequence of the CRP-7 heavy chain variable region is shown in SEQ ID NO.1, specifically as follows: EVQLVESGGGLVQPGGSLRLSCAASGILFNRKGIHWVRQAPGKGLEWVARIYDYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCANSTGAGFYSRVYWGQGTLVTVSS, the complementary determining region CDR-H1 amino acid sequence GILFNRKG (SEQ ID NO.12), the complementary determining region CDR-H2 amino acid sequence IYDYGGST (SEQ ID NO.13), and the complementary determining region CDR-H3 amino acid sequence ANSTGAGFYSRVY (SEQ ID NO.14).

[0070] The amino acid sequence of the CRP-7 light chain variable region is shown in SEQ ID NO.2, specifically as follows: DIQMTQSPSSLSASVGDRVTITCRASQGSSNYVAWYQQKPGKAPKLLIYSTASLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQTRFSFGSSTFGQGTKVEIK, the complementary determining region CDR-L1 amino acid sequence is QGSSNY (SEQ ID NO.15), the complementary determining region CDR-L2 amino acid sequence is YSTAS (SEQ ID NO.16), and the complementary determining region CDR-L3 amino acid sequence is QTRFSFGSST (SEQ ID NO.17).

[0071] CRP-13 monoclonal antibody, including heavy and light chains:

[0072] The amino acid sequence of the CRP-13 heavy chain variable region is shown in SEQ ID NO.3, specifically as follows: EVQLVESGGGLVQPGGSLRLSCAASGSLFTGNDMSWVRQAPGKRLEWVAYINFGRNSKYFPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCADGRTKLNFLSYWGQGTLVTVSS, the complementary determining region CDR-H1 amino acid sequence GSLFTGND (SEQ ID NO.6), the complementary determining region CDR-H2 amino acid sequence INFGRNSK (SEQ ID NO.7), and the complementary determining region CDR-H3 amino acid sequence ADGRTKLNFLSY (SEQ ID NO.8).

[0073] The amino acid sequence of the light chain variable region of the CRP-13 is shown in SEQ ID NO.4, specifically as follows: DIQMTQSPSSLSASVGDRVTITCRTSGGNHINLTWYQQKPGKAPQLLIYNAKTLADGVPSRFSGSGSGTQFTLTISSLQPEDFANYYCQQGWLSPDTFGQGTKVEIK, the amino acid sequence of the complementarity determining region CDR-L1 is GGNHIN (SEQ ID NO.9), the amino acid sequence of the complementarity determining region CDR-L2 is YNAKT (SEQ ID NO.10), and the amino acid sequence of the complementarity determining region CDR-L3 is QQGWLSPDT (SEQ ID NO.11).

[0074] 4. Linear detection based on double antibody sandwich ELISA detection method:

[0075] Referring to the above detection steps, the monkey C-reactive protein standard protein was first diluted with PBS buffer solution in a gradient manner. The concentrations after dilution were 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.1 ng / mL, 1.6 ng / mL, and 0 ng / mL, respectively. 50 μL of the diluted standard protein of different concentrations was taken for detection. Three parallel experiments were performed for each concentration to prepare a standard curve. The standard curve is shown as follows: Figure 1 As shown: According to the four-parameter fitting results, the linearity reaches 0.999.

[0076] 5. Sensitivity detection based on double antibody sandwich ELISA detection method:

[0077] Referring to the detection steps of Example 4, the monkey C-reactive protein standard protein was first diluted with PBS buffer solution to a gradient dilution. The concentrations after dilution were 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.1 ng / mL, 1.6 ng / mL, and 0 ng / mL, respectively. 50 μL of the diluted standard protein of different concentrations and 20 PBS buffers were taken for detection. The sensitivity test was performed. The standard curve and PBS buffer values ​​are shown in Table 2. The sensitivity of the double antibody sandwich ELISA detection method was calculated to be 0.78 ng / mL according to the formula "mean + 2SD".

[0078] Table 2: Sensitivity test data of the present invention

[0079]

[0080] 6. Identification of monkey serum samples based on double antibody sandwich ELISA detection method:

[0081] Referring to the above detection steps, 16 uninfected monkey sera and 16 infected monkey sera were measured and identified based on the double antibody sandwich ELISA detection method, and the measured OD values ​​are shown in Table 3.

[0082] Table 3 Results of double antibody sandwich ELISA on 16 samples of non-infected monkey sera and 16 samples of infected monkey sera

[0083]

[0084] From the test results, it can be seen that there are significant differences between infected and uninfected people.

[0085] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. An anti-monkey C-reactive protein monoclonal antibody, characterized in that: Named as CRP-13, the CRP-13 includes a heavy chain variable region and a light chain variable region; The heavy chain variable region has three complementary determining regions, the amino acid sequence of CDR-H1 is shown in SEQ ID NO.6, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.8; The light chain variable region has three complementary determining regions, the amino acid sequence of CDR-L1 is shown in SEQ ID NO.9, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.10, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

11.

2. The anti-monkey C-reactive protein monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the CRP-13 heavy chain variable region is shown in SEQ ID NO.3, and the amino acid sequence of the CRP-13 light chain variable region is shown in SEQ ID NO.

4.

3. Use of the anti-monkey C-reactive protein monoclonal antibody according to claim 1 or 2 in the preparation of a monkey C-reactive protein detection reagent.

4. A monkey C-reaction enzyme-linked immunosorbent assay kit, characterized in that: The kit comprises two anti-monkey C-reactive protein monoclonal antibodies, namely CRP-7 and the anti-monkey C-reactive protein monoclonal antibody CRP-13 according to claim 1 or 2, wherein the monoclonal antibody CRP-7 is used as a coating antibody and the monoclonal antibody CRP-13 is used as a detection antibody; The CRP-7 includes a heavy chain variable region and a light chain variable region, the heavy chain variable region has three complementary determining regions, the amino acid sequence of its CDR-H1 is shown in SEQ ID NO.12, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.13, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.14; the light chain variable region has three complementary determining regions, the amino acid sequence of its CDR-L1 is shown in SEQ ID NO.15, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.16, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

17.

5. The monkey C-reaction ELISA kit according to claim 4, characterized in that: The amino acid sequence of the CRP-7 heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the CRP-7 light chain variable region is shown in SEQ ID NO.

2.

6. The monkey C-reaction ELISA kit according to claim 4, characterized in that: The monoclonal antibodies CRP-7 and CRP-13 are both obtained by immunizing monkey C-reactive protein recombinant protein.

7. The monkey C-reaction ELISA kit according to claim 6, characterized in that: The amino acid sequence of the monkey C-reactive protein recombinant protein is shown in SEQ ID NO.5.

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