A combination of anti-calprotectin monoclonal antibodies and use thereof

Abstract

The application provides an anti-calprotectin monoclonal antibody combination and application thereof. The anti-calprotectin monoclonal antibody combination comprises a first antibody and a second antibody, wherein the first antibody comprises a first light chain variable region and a first heavy chain variable region, and the second antibody comprises a second light chain variable region and a second heavy chain variable region; the amino acid sequence of the first light chain variable region comprises VL-CDR1-3 shown in SEQ ID NO:1-3; the amino acid sequence of the first heavy chain variable region comprises VH-CDR1-3 shown in SEQ ID NO:4-6; the amino acid sequence of the second light chain variable region comprises VL-CDR1-3 shown in SEQ ID NO:7-9; and the amino acid sequence of the second heavy chain variable region comprises VH-CDR1-3 shown in SEQ ID NO:10-12. The anti-calprotectin monoclonal antibody combination can improve the sensitivity and accuracy of in-vitro detection.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a combination of anti-calprotectin monoclonal antibodies and their applications. Background Technology

[0002] Calprotectin, a member of the S100 protein family, is primarily secreted by neutrophils and macrophages. With a molecular weight of approximately 36 kDa, it is a heterodimer composed of S100A8 (calprotectin A) and S100A9 (calprotectin B), widely distributed in bodily fluids such as blood, cerebrospinal fluid, and synovial fluid. Under normal physiological conditions, its level remains low. However, when the body enters a pathological state, such as inflammation, infection, or tumors, the expression and secretion of calprotectin significantly increase. In the feces of patients with inflammatory bowel disease (IBD), calprotectin levels can be tens or even hundreds of times higher than in healthy individuals. Therefore, calprotectin is considered an important biomarker for diagnosing IBD, assessing disease activity, and monitoring treatment effectiveness. Furthermore, calprotectin levels are also closely related to the occurrence and development of various diseases, including rheumatoid arthritis, psoriasis, lung cancer, and colon cancer.

[0003] Monoclonal antibodies possess advantages such as high specificity, high affinity, and good homogeneity, playing a crucial role in biomarker detection and disease treatment. Currently, some studies have reported on monoclonal antibodies targeting calprotectin, but existing antibodies still have some shortcomings. For example, some antibodies have low specificity and are prone to cross-reaction with other members of the S100 protein family; some antibodies have low affinity, resulting in insufficient detection sensitivity and difficulty in accurately detecting low concentrations of calprotectin; furthermore, existing methods for preparing anti-calprotectin monoclonal antibodies are mostly cumbersome, costly, and have low yields, making it difficult to meet the needs of large-scale production and clinical applications. Therefore, developing an anti-calprotectin monoclonal antibody with high specificity, strong affinity, and a simple and efficient preparation method has significant practical significance and application value.

[0004] Immunoassay techniques are widely used in pathogen detection due to their high specificity and ease of operation. Existing technologies such as immunochromatography, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and chemiluminescence immunoassay can all be used for antigen detection. However, the core of these methods lies in using high-quality antibodies as recognition elements.

[0005] Therefore, there is an urgent need to provide a monoclonal antibody against calprotectin with high affinity and high specificity to construct a high-performance in vitro immunodiagnostic tool, thereby meeting the need for accurate detection of calprotectin. Summary of the Invention

[0006] This application provides a combination of anti-calprotectin monoclonal antibodies and their applications, aiming to solve the technical problems of low detection sensitivity, poor specificity, easy cross-reactivity, poor batch stability, and cumbersome preparation methods in the in vitro diagnosis of calprotectin infection. It is applicable to a variety of immunoassay methods and can be used to improve the early detection rate and diagnostic accuracy of calprotectin infection.

[0007] In a first aspect, this application provides an anti-calprotectin monoclonal antibody combination, comprising a first antibody and a second antibody, wherein the first antibody includes a first light chain variable region and a first heavy chain variable region, and the second antibody includes a second light chain variable region and a second heavy chain variable region, wherein...

[0008] The amino acid sequence of the first light chain variable region includes VL-CDR1 shown in SEQ ID NO:1, VL-CDR2 shown in SEQ ID NO:2 and VL-CDR3 shown in SEQ ID NO:3;

[0009] The amino acid sequence of the first heavy chain variable region includes VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5, and VH-CDR3 shown in SEQ ID NO:6;

[0010] The amino acid sequence of the second light chain variable region includes VL-CDR1 shown in SEQ ID NO:7, VL-CDR2 shown in SEQ ID NO:8, and VL-CDR3 shown in SEQ ID NO:9;

[0011] The amino acid sequence of the variable region of the second heavy chain includes VH-CDR1 shown in SEQ ID NO:10, VH-CDR2 shown in SEQ ID NO:11, and VH-CDR3 shown in SEQ ID NO:12.

[0012] According to this application, the monoclonal antibody combination contains two rabbit-derived antibodies that can recognize two epitopes of calprotectin, thereby achieving higher binding affinity and stronger specificity, effectively reducing the risk of false negatives and cross-reactions, and improving the sensitivity and accuracy of in vitro detection.

[0013] Specifically, the first and second antibodies each possess independent light and heavy chain variable regions, and their respective CDR1, CDR2, and CDR3 sequences have been validated through animal immunization, screening, and cloning, demonstrating stability and specific recognition capabilities. By clearly defining the six complementarity-determining regions (CDRs) of the two antibodies at the structural level, the precise localization and reproducibility of the antigen recognition sites are ensured, contributing to maintaining the consistency and stability of antibody function during subsequent recombinant expression.

[0014] Furthermore, the first and second antibodies can work synergistically when binding to the calprotectin antigen, resulting in a significant enhancement of the antibody combination provided in this application compared to a single antibody in terms of antigen capture, detection signal intensity, and anti-interference ability, thereby effectively improving the sensitivity and accuracy of in vitro detection of calprotectin.

[0015] In some embodiments, the amino acid sequence of the first light chain variable region is shown in SEQ ID NO:13; and the amino acid sequence of the first heavy chain variable region is shown in SEQ ID NO:14.

[0016] In some embodiments, the amino acid sequence of the second light chain variable region is shown in SEQ ID NO:15; and the amino acid sequence of the second heavy chain variable region is shown in SEQ ID NO:16.

[0017] In some of the above embodiments, by defining the full-length variable region sequence, rather than just the complementarity-determining region, the natural combination relationship between the frame region (FR) and the CDR can be fully preserved. The frame region plays an important role in maintaining the spatial conformation of the antibody variable region, stabilizing the correct folding of the CDR, and supporting the formation of the antigen-binding surface. Therefore, further defining the full-length variable region sequence based on the CDR sequence can ensure that the antibody maintains a stable tertiary structure and a high level of specific binding ability in recombinant expression and practical applications. Compared with the scheme that relies solely on the CDR sequence definition, this implementation can reduce structural perturbations or affinity reductions caused by frame region differences, ensure batch-to-batch consistency and reproducibility of antibody function, and thus exhibit higher stability and reliability in practical detection applications.

[0018] In some embodiments, the nucleotide sequence of the first light chain variable region is shown in SEQ ID NO:17; and the nucleotide sequence of the first heavy chain variable region is shown in SEQ ID NO:18.

[0019] In some embodiments, the nucleotide sequence of the second light chain variable region is shown in SEQ ID NO:19; and the nucleotide sequence of the second heavy chain variable region is shown in SEQ ID NO:20.

[0020] In some of the above embodiments, by explicitly defining the nucleotide sequence of the antibody's variable region, it can be ensured that the antibody can be stably expressed in different recombinant expression systems and maintain the correct amino acid sequence. Nucleotide-level definition not only covers the translation products of the amino acid sequence but also ensures the consistency between transcription and translation, thereby avoiding amino acid drift caused by codon differences or potential mutations. Compared to defining only at the amino acid level, nucleotide sequence definition provides a more direct technical basis for subsequent plasmid construction, vector assembly, and expression optimization, improving process controllability and production stability.

[0021] Secondly, this application provides the use of the anti-calprotectin monoclonal antibody combination according to any embodiment of the first aspect in the preparation of tools for detecting calprotectin.

[0022] According to this application, by applying the above-mentioned antibody combination to in vitro diagnostic tools, a highly sensitive and specific calprotectin detection system can be constructed. The antibody combination can simultaneously identify different binding sites of calprotectin antigens during the detection process, achieving multiple capture and signal enhancement, thereby significantly improving the sensitivity and accuracy of the detection. Compared with single antibodies, this combination has significant advantages in reducing false negatives, reducing cross-reactivity, and improving the reliability of detection results.

[0023] In some embodiments, the tool for detecting calprotectin is used to detect calprotectin in an in vitro sample, said in vitro sample including at least one of feces, serum, plasma, or whole blood.

[0024] In some of the above embodiments, by specifying the sample type, a variety of common clinical body fluid sources can be covered, which helps to achieve rapid and accurate detection in different clinical scenarios; serum, plasma, and whole blood samples are convenient for routine hematological testing, while stool is suitable for the diagnosis of gastrointestinal diseases. Compared with testing tools limited to a single type of sample, the tool described in this embodiment has wider applicability in clinical application.

[0025] In some embodiments, the anti-calprotectin monoclonal antibody is used in an immunoassay method for detecting calprotectin in an in vitro sample.

[0026] In some of the above embodiments, by introducing the antibody combination provided in this application into the immune detection system, the presence of calprotectin antigen in an in vitro sample can be directly detected by utilizing the principle of specific binding between antigen and antibody.

[0027] In some embodiments, the immunoassay method includes at least one of enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, chemiluminescence immunoassay, immunomicrosphere assay, and immunogold assay.

[0028] In some of the above embodiments, different immunoassay methods can utilize the high specificity recognition performance of the antibody combination of this application; enzyme-linked immunosorbent assay (ELISA) is suitable for high-throughput detection in laboratories; immunofluorescence and chemiluminescence methods have higher sensitivity and quantitative detection capabilities; immunomicrosphere methods facilitate multiplexing; and immunogold methods are suitable for rapid and convenient point-of-care testing. Therefore, with the antibody combination of this application, different detection platforms can be flexibly combined to meet the diverse needs of laboratory research and point-of-care testing.

[0029] In some embodiments, the tool for detecting calprotectin includes at least one of reagents, kits, test strips, and antibody chips.

[0030] In some of the above embodiments, the antibody combinations provided in this application can be used to prepare different forms of detection products. The reagents or kits are suitable for standardized laboratory testing, the test strips facilitate rapid screening and point-of-care testing, and the antibody chips can achieve high-throughput, automated parallel analysis. Through the expansion of various tool forms, this embodiment can cover a variety of different application scenarios, greatly improving the flexibility and promotional value of testing.

[0031] Compared with the prior art, the beneficial effects of this application are at least as follows:

[0032] 1. The anti-calprotectin monoclonal antibody combination provided in this application contains two antibodies obtained through animal immunization and selection. By clearly defining the CDR sequences of their respective light chain and heavy chain variable regions, it achieves efficient recognition of calprotectin and can obtain higher affinity and specificity in in vitro detection.

[0033] 2. Using two antibodies together in the same detection system can create a synergistic effect during antigen capture. Compared with single antibody detection, this can effectively reduce the risk of false negatives and cross-reactions, thereby improving the sensitivity and accuracy of the test results.

[0034] 3. By further defining the amino acid and nucleotide sequences of the full-length variable region, the structural consistency and functional stability of the antibody in recombinant expression and mass production can be ensured, thereby improving the reproducibility and reliability of the detection tool in clinical and industrial applications. Attached Figure Description

[0035] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0036] Figure 1 This is an SDS-PAGE protein electrophoresis image of monoclonal antibodies Ab2# and Ab3# in one embodiment of this application. Detailed Implementation

[0037] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0038] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0039] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0040] In this specification, unless otherwise specified, "parts" refers to "parts by weight".

[0041] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0042] I. Experimental Materials:

[0043] Calprotectin: In-house synthesized. The nucleotide sequence was commercially synthesized by Nanjing Genscript Biotech Co., Ltd., based on data on calprotectin published in the NCBI database (NCBI sequence numbers, NM_001160271.3 and NM_001177906.1).

[0044] Nickel ion affinity chromatography column, Protein G affinity chromatography column: purchased from GE Healthcare;

[0045] Freund's adjuvant: purchased from SIGMA;

[0046] New Zealand White Rabbit: Purchased from the China National Institutes for Food and Drug Control (Daxing);

[0047] II. Experimental Methods:

[0048] (1) Preparation of calprotectin:

[0049] A: Screening of bacterial strains: The constructed human calprotectin recombinant protein expression vector was transformed into Escherichia coli BL21(DE3) strain. Single colonies were picked from ampicillin-resistant (ampicillin concentration of 0.1 mg / mL) LB plates and inoculated into ampicillin-resistant LB medium. The culture was carried out overnight at 37°C and 220 rpm with shaking to obtain bacterial culture.

[0050] B: Induction of expression: The bacterial culture was diluted at a ratio of 1:100 and inoculated into fresh LB medium. It was cultured at 37°C and 220 rpm with shaking until the OD600nm value reached 0.6-0.8. Then, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 1 mmol / L, and expression was induced at 30°C and 180 rpm for 6 hours.

[0051] C: Protein Purification: 1) After induction of expression, centrifuge the bacterial culture (8000 rpm, 10 minutes, 4℃), collect the bacterial cells, resuspend the cells in lysis buffer (0.02 mol / L PBS, 0.5 mol / L NaCl, 10 mmol / L imidazole, pH 7.4), and disrupt the cells using an ultrasonic disruptor (300W power, 3 seconds working time, 5-second interval, total time 30 minutes); 2) Centrifuge the disrupted bacterial culture (12000 rpm, 20 minutes, 4℃) and collect the supernatant. Load the supernatant into a nickel ion affinity chromatography column pre-equilibrated with lysis buffer. After loading, wash with wash buffer (0.02 mol / L PBS, 0.5 mol / L NaCl, 10 mmol / L imidazole, pH 7.4). The chromatography column was washed with NaCl, 20 mmol / L imidazole, pH 7.4 until the UV absorbance returned to baseline. The target protein was then eluted with elution buffer (0.02 mol / L PBS, 0.5 mol / L NaCl, 250 mmol / L imidazole, pH 7.4), and the elution peak was collected. The collected elution peak target protein was dialyzed overnight in 0.02 mol / L PBS (pH 7.4) using a dialysis bag (molecular weight cutoff of 10 kDa). The dialysate was changed 3 times. After dialysis, the protein concentration was measured, and the protein purity was detected by SDS-PAGE electrophoresis.

[0052] (2) Animal immunization:

[0053] A: Mix the self-made immunogen with Freund's adjuvant in equal volumes to a suitable volume and emulsify it completely. Immunize the New Zealand white rabbits by subcutaneous injection at multiple points. Each New Zealand white rabbit is injected with 1000 μL of immunogen, once every two weeks, for a total of 10 New Zealand white rabbits.

[0054] B: After 10 immunizations, one week after the last immunization, blood was collected from the ear vein of the New Zealand white rabbits. The blood was collected, the serum was separated, and the antibody titer in the serum was tested using the ELISA method (enzyme-labeled plate coated with calprotectin). The OD values ​​of different dilutions of serum from different New Zealand white rabbits are shown in Table 1.

[0055] Table 1

[0056]

[0057] According to Table 1, rabbits #2, #3, and #5 had low antibody titers and were therefore eliminated; among rabbits #1 and #4, rabbit #1 had the best antibody titer and was selected for monoclonal cell preparation.

[0058] (3) Preparation of monoclonal antibodies:

[0059] A: Screening of hybridoma cells

[0060] a) The spleens of the New Zealand white rabbits obtained through screening were processed, and the isolated spleen cells were fused with myeloma cells. The fused cells were screened and cultured in plates using the limiting dilution method.

[0061] b) Screening and culturing monoclonal cell wells, and detecting the supernatant after cell culture by ELISA. The four monoclonal cell wells with the highest OD values ​​in the cell culture plate were selected as target hybridoma cells (1#, 2#, 3#, 4#) for culture and amplification, which were used for cryopreservation of cell seeds and purification and verification of monoclonal antibodies.

[0062] B: Preparation of monoclonal antibodies

[0063] a) Isolation of antibody variable region genes from hybridoma cells using RT-PCR: After homogenizing the optimal hybridoma cells, add cell lysis buffer for RNA extraction, precipitate the RNA from the aqueous phase with isopropanol, wash the precipitated RNA after centrifugation to remove impurities, resuspend and reverse transcribe to obtain cDNA.

[0064] b) Using existing New Zealand white rabbit-specific primers (synthesized by GenScript Biotech Co., Ltd.), PCR gene amplification was performed. Hybridoma cell cDNA was used as a template to amplify the variable regions of the heavy and light chains of the antibody. A 50 μL system contained 5 μL cDNA, HotStar Taq Plus enzyme, dNTPs, and 0.5 μM specific primers. PCR amplification was performed under the following conditions: pre-denaturation, 94℃, 5 min; amplification, 94℃, 30 s, 55℃, 30 s, 72℃, 50 s, 35 cycles; annealing, 72℃, 7 min. The obtained PCR product was identified by 1% agarose gel electrophoresis, the target fragment was recovered, and the sample was sent for sequencing to obtain the antibody gene sequence.

[0065] c) Constructing expression vectors for monoclonal antibodies:

[0066] Based on the antibody gene sequence obtained from step b, an expression vector for monoclonal antibodies was constructed. Homologous recombination arms were added to both ends of the variable region gene of the antibody heavy chain and the variable region gene of the light chain using homologous recombination primers. The expression plasmid containing the constant regions of the rabbit antibody heavy and light chains IgG1 was linearized using a double enzyme to generate homologous recombination arms. The variable region gene fragment with added homologous recombination arms and the linearized plasmid were ligated together by homologous recombination to form a complete expression vector, pCDNA3.4. The recombination product was transformed into TOP10 E. coli competent cells to amplify the plasmid. Through the above experimental operations, paired pCDNA3.4-antibody heavy chain plasmid and pCDNA3.4-antibody light chain plasmid were obtained.

[0067] d) Expression and purification of monoclonal antibodies:

[0068] The paired monoclonal antibody heavy and light chain expression plasmids obtained in step c were added to the culture medium at a 1:1 ratio. After thorough mixing, PEI transfection reagent (4 times the mass of DNA) was added. After mixing, the mixture was incubated at room temperature in the dark for 10 minutes. Then, the plasmids were added to 293T cells while shaking the cell culture flask. The cells were then incubated in a 5% CO2 incubator at 37°C with shaking for 5 days. The cell culture supernatant was then collected and purified using affinity purification (Protein A) to obtain the monoclonal antibody. The specific steps are as follows:

[0069] (1) Centrifuge the supernatant of the expressed antibody at 10000×g at room temperature for 30 min to remove the precipitate;

[0070] (2) Wash the affinity purification column containing Protein A thoroughly with 10 volumes of binding buffer;

[0071] (3) Pass the expression supernatant through the purification column at a flow rate of 5 mL / min;

[0072] (4) Wash the purification column thoroughly with 20 times the column volume of binding buffer;

[0073] (5) Elute the purification column with 0.1M citrate buffer (pH=3.0-3.5) until the elution peak drops to equilibrium, and adjust the pH to 7.0 with 1M Tris-HCl buffer (pH=9.0).

[0074] (6) The purified monoclonal antibody was concentrated using a concentrated centrifuge column, PBS was used as the antibody preservation buffer, and finally the concentration of the concentrated antibody was determined using an ultra-micro UV spectrophotometer.

[0075] Monoclonal antibodies Ab1#, Ab2#, Ab3#, and Ab4# were thus obtained.

[0076] III. Testing Section

[0077] 1. Screening of monoclonal antibodies:

[0078] a) ELISA indirect method verification: Calcavirin was coated onto an ELISA plate (0.1 μg / mL). Four antibody strains (Ab1#, Ab2#, Ab3#, Ab4#) were diluted to concentrations of 100, 10, 1, and 0 ng / mL, respectively. The diluted solutions were added to the ELISA plate, and the plate was incubated at 37°C for 1 h. After washing the plate, the ELISA-labeled secondary antibody dilution solution (5000-fold dilution) was added, and the plate was incubated at 37°C for 0.5 h. TMB was added for color development for 15 min, and the process was terminated. The absorbance (OD) was measured at wavelengths of 450 / 620 nm. The results are shown in Table 2.

[0079] Table 2

[0080]

[0081] Based on the analysis of Table 2, it can be seen that monoclonal antibodies Ab2#, Ab3#, and Ab4# have good recognition effects on calprotectin.

[0082] b) Confirmation of antibody pairing combinations

[0083] Thirty clinically positive and thirty negative samples were collected from relevant hospitals. Alternative monoclonal antibodies were combined using a sandwich method (forming a coated antibody-antigen-detection antibody complex). Fluorescent immunochromatography (coating conditions: 0.02M PBS buffer, coating concentration 1.8 mg / mL, drying temperature 53℃, drying time 13h; labeling conditions: 0.02M PBS buffer, antibody to fluorescent microspheres (particle size approximately 300 nm) mass ratio 1.2:1, labeling 12h; sample loading volume 100 μL, results read 15 min after loading) was used to detect the above 60 samples and the critical sample (calprotectin diluted with purified water to a concentration of 6 ng / mL). The ratio (I) of the sample T / C value to the critical sample T / C value was calculated, and this parameter was used as the critical value (I≥1 for positive, I<1 for negative). Antibody combinations with high positive-negative concordance rates and large sample gradients were screened. The results are shown in Table 3.

[0084] Table 3

[0085]

[0086] As shown in Table 3, the positive and negative concordance rates of samples were best when Ab2# antibody coating was used for Ab3# antibody detection and Ab4# antibody coating was used for Ab3# antibody detection.

[0087] c) Verification of cross-reactivity of monoclonal antibodies:

[0088] Two pairs of candidate monoclonal antibodies were combined using a sandwich method (forming a coated antibody-antigen-detection antibody complex). Different concentrations of hemoglobin, total IgG antibody, and lactoferrin were detected by fluorescence immunochromatography to observe the anti-interference ability of different antibodies against these easily cross-reactive substances. The cross-reactivity is shown in Table 4.

[0089] Table 4

[0090]

[0091] As shown in Table 4, the results indicate that the obtained monoclonal antibody showed the best detection effect on Ab2# antibody-coated + Ab3# antibody, with no cross-reactivity; therefore, monoclonal antibodies Ab2# and Ab3# were selected for use with calprotectin.

[0092] 2. Characterization of monoclonal antibodies Ab2# and Ab3#

[0093] a) Monoclonal antibody Ab2#:

[0094] Light chain:

[0095] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.17:

[0096] GACCCAATGCTGACCCAGACGGCCTCTCCTGTGAGCGCCGCCGTGGGGAGCACAGTTACCATCTCCTGTCAGGCCAGCCAGGATATCTCCAACTGCCTGGCCTGGTACCAGCAGAAGCCAGGCCAGCCCCCTAAGCTGTTGATCTACGATGCCAGCAACCT GGAGTCCGGCGTGCCCTCTAGATTCTCTGGCTCCGGAAGCGGAACCGAGTACACACTGACAATCAGCGGCGTCCAGTGCGACGACGCAGCCACCTACTACTGCCAGGCCTACGAGTCTCTGCCCAGATCCTTCGGCGGAGGCACGAAACTGGAGATCAAG.

[0097] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.13: DPMLTQTASPVSAAVGSTVTISCQASQDISNCLAWYQQKPGQPPKLLIYDASNLESGVPSRFSGSGSGTEYTLTISGVQCDDAATYYCQAYESLPRSFGGGTKLEIK.

[0098] The amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:1: QASQDISNCLA; the amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:2: DASNLES; and the amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:3: QAYESLPRS.

[0099] Heavy chain:

[0100] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.18:

[0101] CAGTCCGTCAAAGAGAGCGAGGGAGGTCTGTTCAAGCCAACCGACACACTCACCCTGACCTGCACCGTGTCCGGCGGCTCCATCTCCTCCAGCAGCTATAATATTGGCTGGGTGAGACAGGCCCCAGGAAGCGGACTGGAGTGGATCGGAAGCATCTACTACAGTGGGTCCACCTACTA CAACAGCTGGGCCAAGTCTAGGTCCACCATCACCAGGAACACCAACGAGAACACCGTCACCCTGAAAATGACCTCCCTGACCGCCGCCGACACAGCCACCTACTTCTGCGCCAGGCAGGTGCCTTACAACCTGGGCTTCGACCCATGGGGCCAGGGCACCCTGGTGACCGTGTCTTCC.

[0102] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.14:

[0103] QSVKESEGGLFKPTDTLTLTCTVSGGSISSSSYNIGWVRQAPGSGLEWIGSIYYSGSTYYNSWAKSRSTITRNTNENTVTLKMTSLTAADTATYFCARQVPYNLGFDPWGQGTLVTVSS

[0104] The amino acid sequence of the complementarity-determining region VH-CDR1 of the heavy chain variable region is shown in SEQ ID NO:4: SYNIG; the amino acid sequence of the complementarity-determining region VH-CDR2 of the heavy chain variable region is shown in SEQ ID NO:5: SIYYSGSTYYNSWAKS; and the amino acid sequence of the complementarity-determining region VH-CDR3 of the heavy chain variable region is shown in SEQ ID NO:6: QVPYNLGFDP.

[0105] b) Monoclonal antibody Ab3#:

[0106] Light chain:

[0107] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.19:

[0108] GATGTGGTGATGACCCAGACACCCAGTCCTGTGAGTGCCGCCGTGGGCGATACCGTGACCATCAAATGTCAGGCTTCCCAGTCCGTGTCCTCCAACCTCGCCTGGTACCAGCAGAAGCCCGCCAGGCCCCCCGGCTGCTGATCTACGGAGCCTCTAATCTGGA GTCCGGAGTGCCCTCCCGCTTCAGAGGCTCTGGCTCGGGGACCCAGTTCACCCTGACCATTTCCGGCATGAAAGCTGAGGACGTGGCCACCTATTATTGCCAGCAGTACAACAATTGGCCCCACTGTTCACCTTCGGCGGCGGGACTAAGCTGGAGATTAAG.

[0109] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.15:

[0110] DVVMTQTPSPVSAAVGDTVTIKCQASQSVSSNLAWYQQKPGQAPRLLIYGASNLESGVPSRFRGSGSGTQFTLTISGMKAEDVATYYCQQYNNWPPLFTFGGGTKLEIK.

[0111] The amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:7: QASQSVSSNLA; the amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:8: GASNLES; and the amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:9: QQYNNWPPLFT.

[0112] Heavy chain:

[0113] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.20:

[0114] CAGAAGCAGCTGTGGAGAGTGGCGGCGGACTGGATCAGCCCGCCGGCAGCCTGAAACTGTCCTGTAAGGACAGCGGCTACAATTTCACCCGCCTGGGCATGTGTTGGGTGCACCAGGCCCCAGGCAAGGGCCTGGAGTGGATTGCCTGCATCTCGGGGTACAACGGGATCACCAACTACGTCAGCAGA GTGAACGGCAGGTTCACCATCAGCAGCGATAACACCCAGAACATGGTGGACCTGGAGATGAATAGCCTGACCGCTGCAGACATGGCCGTGTACTTTTGCGCCACGAGACAGGGCGTGCAGTCCCCCAGATCCTGGTATTACTACGGACTGGATGTGTGGGGCCAGGGCACACTGGTGACCGTCTCCAGC.

[0115] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.16:

[0116] QKQLVESGGGLDQPAGSLKLSCKDSGYNFTRLGMCWVHQAPGKGLEWIACISGYNGITNYVSRVNGRFTISSDNTQNMVDLEMNSLTAADMAVYFCATRQGVQSPRSWYYYGLDVWGQGTLVTVSS.

[0117] The amino acid sequence of the complementarity-determining region VH-CDR1 of the heavy chain variable region is shown in SEQ ID NO:10: RLGMC; the amino acid sequence of the complementarity-determining region VH-CDR2 of the heavy chain variable region is shown in SEQ ID NO:11: ISGYNGITNYVSRVNG; the amino acid sequence of the complementarity-determining region VH-CDR3 of the heavy chain variable region is shown in SEQ ID NO:12: RQGVQSPRSWYYYGLDV.

[0118] c) Molecular weight determination of monoclonal antibodies Ab2# and Ab3#

[0119] Its SDS-PAGE protein electrophoresis chromatogram is as follows: Figure 1 As shown, according to Figure 1 Lane M is the marker, lane 1 is the monoclonal antibody Ab2#, and lane 2 is the monoclonal antibody Ab3#. Both monoclonal antibodies are reduced to two parts, one with a molecular weight of 50kDa and the other with a molecular weight of 25kDa.

[0120] 3. Development and validation of a calprotectin detection kit based on the obtained anti-calprotectin monoclonal antibody pairs (monoclonal antibody Ab2# and monoclonal antibody Ab3#).

[0121] Brief description of the reagent kit development process:

[0122] The Ab2# antibody was used as the coating antibody to coat the NC membrane, and the Ab3# antibody fluorescent microspheres were used as the detection antibody. A series of optimization studies were conducted on the coating conditions of the Ab2# antibody (coating buffer, coating concentration, drying temperature and time, etc.), the labeling conditions of the Ab3# antibody (antibody to fluorescent microsphere ratio, labeling buffer, labeling time, etc.), the sample loading volume, and the result reading time after loading. After the kit was finalized, the kit performance (sensitivity, repeatability, stability, etc.) was also studied.

[0123] The coating conditions were determined as follows: 0.02M PBS buffer, coating concentration 1.8 mg / mL, drying temperature 53℃, and drying time 13h.

[0124] Labeling conditions: 0.02M PBS buffer, antibody to fluorescent microspheres (particle size about 300 nm) mass ratio 1.2:1, labeling for 12 h; sample loading volume is 100 μL, and results are read 15 min after loading.

[0125] 1) Sensitivity:

[0126] Calprotectin was diluted to nine concentration levels: 2000, 1000, 500, 200, 100, 50, 10, 5, and 0 ng / mL. The analytical sensitivity was determined using a calprotectin detection kit (fluorescent immunochromatography). The concentration value was considered the analytical sensitivity when the T / C value of the sample was greater than 2.0 compared to the T / C value of 0 concentration. The results are shown in Table 5.

[0127] Table 5

[0128]

[0129] According to Table 5, the minimum concentration corresponding to a T / C value greater than 2.0 for the sample T / C value / 0 concentration is 5 ng / mL. Therefore, the present invention provides a calprotectin detection kit (fluorescent immunochromatography) with a sensitivity of 5 ng / mL for the detection of calprotectin.

[0130] (ii) Repeatability:

[0131] The concentrations of calprotectin in samples 1 (10 ng / mL), 2 (50 ng / mL), and 3 (500 ng / mL) were determined using a calprotectin assay kit (fluorescent immunochromatography). The results are shown in Table 6.

[0132] Table 6

[0133]

[0134] As shown in Table 6, the coefficient of variation (CV%) of the calprotectin detection kit (fluorescence immunochromatography) is less than 15%, indicating that the kit has good reproducibility.

[0135] (iii) Stability:

[0136] The calprotectin assay kit (fluorescent immunochromatography) was stored at 2–8°C. The kits were removed at 0, 3, 6, 9, and 12 months post-production, and the calprotectin concentrations in samples 1 (10 ng / mL), 2 (50 ng / mL), and 3 (500 ng / mL) were measured. The results are shown in Table 7.

[0137] Table 7

[0138]

[0139] As shown in Table 7, the deviation of the calprotectin detection kit (fluorescent immunochromatography) is less than 15%, indicating that the kit has good stability.

[0140] Finally, it should be noted that the above text has described the various embodiments of the present invention in detail. To avoid obscuring the concept of the present invention, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description. The above embodiments are only used to illustrate the technical solutions of this application, and not to limit them;

[0141] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A combination of anti-calprotectin monoclonal antibodies, characterized in that, The anti-calprotectin monoclonal antibody combination comprises a first antibody and a second antibody, the first antibody comprising a first light chain variable region and a first heavy chain variable region, the second antibody comprising a second light chain variable region and a second heavy chain variable region, wherein, the amino acid sequence of the first light chain variable region comprises a VL-CDR1 as shown in SEQ ID NO: 1, a VL-CDR2 as shown in SEQ ID NO: 2, and a VL-CDR3 as shown in SEQ ID NO: 3; the amino acid sequence of the first heavy chain variable region comprises a VH-CDR1 as shown in SEQ ID NO: 4, a VH-CDR2 as shown in SEQ ID NO: 5, and a VH-CDR3 as shown in SEQ ID NO: 6; the amino acid sequence of the second light chain variable region comprises a VL-CDR1 as shown in SEQ ID NO: 7, a VL-CDR2 as shown in SEQ ID NO: 8, and a VL-CDR3 as shown in SEQ ID NO: 9; the amino acid sequence of the second heavy chain variable region comprises a VH-CDR1 as shown in SEQ ID NO: 10, a VH-CDR2 as shown in SEQ ID NO: 11, and a VH-CDR3 as shown in SEQ ID NO:

12.

2. The anti-calprotectin monoclonal antibody combination according to claim 1, characterized in that, the amino acid sequence of the first light chain variable region is as shown in SEQ ID NO: 13; and the amino acid sequence of the first heavy chain variable region is as shown in SEQ ID NO:

14.

3. The anti-calprotectin monoclonal antibody combination according to claim 1, characterized in that, the amino acid sequence of the second light chain variable region is as shown in SEQ ID NO: 15; and the amino acid sequence of the second heavy chain variable region is as shown in SEQ ID NO:

16.

4. The anti-calprotectin monoclonal antibody combination according to claim 1, characterized in that, the nucleotide sequence of the first light chain variable region is as shown in SEQ ID NO: 17; and the nucleotide sequence of the first heavy chain variable region is as shown in SEQ ID NO:

18.

5. The anti-calprotectin monoclonal antibody combination according to claim 1, characterized in that, the nucleotide sequence of the second light chain variable region is as shown in SEQ ID NO: 19; and the nucleotide sequence of the second heavy chain variable region is as shown in SEQ ID NO:

20.

6. Use of the anti-calprotectin monoclonal antibody combination according to any one of claims 1-5 in the manufacture of a tool for detecting calprotectin.

7. Use according to claim 6, characterized in that, The tool for detecting calprotectin is used for detecting calprotectin in an in vitro sample, the in vitro sample comprising at least one of feces, serum, plasma, or whole blood.

8. Use according to claim 7, characterized in that, The anti-calprotectin monoclonal antibody combination is used in an immunoassay method for detecting calprotectin in an in vitro sample.

9. Use according to claim 8, characterized in that, The immunoassay method comprises at least one of an enzyme-linked immunoassay, an immunofluorescence assay, a chemiluminescence immunoassay, an immunomicrosphere assay, and an immunocolloidal gold assay.

10. The use according to any one of claims 6 to 9, characterized in that, The tool for detecting calprotectin comprises a reagent.

11. The use according to any one of claims 6 to 9, characterized in that, The tool for detecting calprotectin comprises a kit.

12. The use according to any one of claims 6 to 9, characterized in that, The tool for detecting calprotectin comprises a test strip.

13. The use according to any one of claims 6 to 9, wherein the compound is ###00003### 13 The tool for detecting calprotectin comprises an antibody chip.

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