Anti-ccrp antibodies or functional fragments thereof and uses thereof

Abstract

The present application relates to the field of biotechnology, in particular to anti-cCRP antibody or its functional fragment and application thereof. The present application not only provides a kind of monoclonal antibody (wild type) with high affinity to cCRP, but also carries out site-directed mutagenesis to the wild type, and verifies the affinity of each mutation, which provides a reference for the research in the field of immune detection of cCRP.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to anti-cCRP antibodies or their functional fragments and their applications. Background Technology

[0002] When the body is stimulated by trauma, infection, or other stimuli, an acute phase response occurs. Stimulated macrophages, fibroblasts, and endothelial cells release various pro-inflammatory factors, promoting the synthesis of acute phase reaction proteins in the liver. Common acute phase reaction proteins include C-reactive protein (CRP), serum amyloid protein, and haptoglobin. CRP is the main acute phase reaction protein in dogs, characterized by rapid synthesis and rapid decline. In healthy dogs, the level of canine CRP is <10 mg / L, rising within 4 to 6 hours after inflammatory stimulation, reaching its peak at 48 hours, at which point the serum concentration is approximately 100-1000 times higher than normal. After systemic inflammation subsides, it can decrease by 50% within 24 hours. Therefore, canine CRP is highly sensitive for early detection of infection. Furthermore, detecting CRP levels in canine serum can be used as an auxiliary diagnostic tool to differentiate between bacterial infections, viral infections, blood parasite infections, traumatic diseases, and tumors. Therefore, canine CRP (cCRP) is clinically used as a monitoring indicator for systemic inflammation and disease progression.

[0003] Currently, the detection of canine C-reactive protein in serum in clinical practice generally uses immunoassay methods such as single-path immunodiffusion, enzyme-linked immunosorbent assay (ELISA), and latex turbidimetry. The sensitivity and accuracy of immunoassay methods largely depend on the reactivity with antigens and antibodies, therefore, high-performance monoclonal antibodies are very important. Summary of the Invention

[0004] The purpose of this invention is to provide an anti-cCRP antibody or its functional fragment, which can achieve specific binding with canine C-reactive protein and ultimately be applied to an immunoassay product that can rapidly and accurately detect canine inflammatory responses.

[0005] Specifically as follows:

[0006] Anti-cCRP antibodies or their functional fragments possess the following complementarity-determining regions:

[0007] CDR-VH1: X1-Y-X2-VH, where X1 is T or N, and X2 is R or G;

[0008] CDR-VH2: X1-INPSSG-X2-TNYNQ-X3-FKH, where X1 is S or V, X2 is N or S, and X3 is I, K, or A;

[0009] CDR-VH3: SDY-X1-GSNIFD-X2, where X1 is Y or D, and X2 is Y;

[0010] CDR-VL1: RAS-X1-NIYYS-X2-A, where X1 is E or Q, and X2 is L or V;

[0011] CDR-VL2: X1-AN-X2-LED, where X1 is Y, W, or N; X2 is S;

[0012] CDR-VL3: KQ-X1-YD-X2-PPT, where X1 is A or Y, and X2 is C or V.

[0013] Furthermore, in the complementary decision region, X2 is R in CDR-VH1, X1 is S in CDR-VH2, X2 is Y in CDR-VH3, X1 is E in CDR-VL1, X2 is S in CDR-VL2, and X1 is A in CDR-VL3.

[0014] The CDR segmentation described in this invention uses the Kabat algorithm.

[0015] In this article, "CDR" refers to the "complementarity-determining region" within the variable sequence of the antibody. Each of the heavy and light chains has three CDRs, starting from the N-terminus of either the heavy or light chain.

[0016] Antigen binding sites may include six CDRs (CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, and CDR-VL3 in this invention). A polypeptide containing a single CDR (e.g., CDR-VH1, CDR-VH2, CDR-VH3, CDR-VL1, CDR-VL2, or CDR-VL3) can be termed a "molecular recognition unit." Crystallographic analysis of antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit may primarily be responsible for the specificity of the antigen binding site. Generally, CDR residues directly and substantially participate in influencing antigen binding.

[0017] The inventors of this invention have discovered that when the mutation sites in each complementarity-determining region are the aforementioned amino acid residues, the antibody has a better affinity for cCRP.

[0018] In an optional implementation, X1 in CDR-VH1 is T;

[0019] In an optional implementation, X1 in CDR-VH1 is N;

[0020] In an optional implementation, X2 in CDR-VH1 is R;

[0021] In an optional implementation, X2 in CDR-VH1 is G;

[0022] In an optional implementation, X1 in CDR-VH2 is S;

[0023] In an optional implementation, X1 in CDR-VH2 is V;

[0024] In an optional implementation, X2 in CDR-VH2 is N;

[0025] In an optional implementation, X2 in CDR-VH2 is S;

[0026] In an optional implementation, X3 in CDR-VH2 is I;

[0027] In an optional implementation, X3 in CDR-VH2 is K;

[0028] In an optional implementation, X3 in CDR-VH2 is A;

[0029] In an optional implementation, X1 in CDR-VH3 is Y;

[0030] In an optional implementation, X1 in CDR-VH3 is D;

[0031] In an optional implementation, X2 in CDR-VH3 is Y;

[0032] In an optional implementation, X1 in CDR-VL1 is E;

[0033] In an optional implementation, X1 in CDR-VL1 is Q;

[0034] In an optional implementation, X2 in CDR-VL1 is L;

[0035] In an optional implementation, X2 in CDR-VL1 is V;

[0036] In an optional implementation, X1 in CDR-VL2 is Y;

[0037] In an optional implementation, X1 in the CDR-VL2 is W;

[0038] In an optional implementation, X1 in CDR-VL2 is N;

[0039] In an optional implementation, X2 in CDR-VL2 is S;

[0040] In an optional implementation, X1 in CDR-VL3 is A:

[0041] In an optional implementation, X1 in CDR-VL3 is Y;

[0042] In an optional implementation, X2 in CDR-VL3 is C;

[0043] In an optional implementation, X2 in CDR-VL3 is V.

[0044] Preferably, each complementarity-determining region is selected from any one of the following mutation combinations:

[0045]

[0046]

[0047] Further, the antibody includes light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NO:1-4 in sequence; the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO:5-8 in sequence.

[0048] Furthermore, the antibody also contains a constant region.

[0049] Preferably, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD. Preferably, the species source of the constant region is cattle, horses, dairy cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, fighting cocks or humans.

[0050] Preferably, the functional fragment is selected from any one of the antibody's VHH, F(ab')2, Fab', Fab, Fv, and scFv.

[0051] Furthermore, the antibody is a labeled antibody.

[0052] The "labeled antibody" described in this invention is a marker that is labeled with a detectable marker.

[0053] The term "antibody" includes various forms of antibody structures, including but not limited to complete antibodies and antibody fragments. Antibodies according to the invention are preferably goat, sheep, mouse, rabbit, or rat antibodies, chimeric antibodies, or further genetically engineered antibodies, provided they retain the characteristic properties according to the invention. An "antibody fragment" comprises a portion of a full-length antibody, preferably its variable domains, or at least its antigen-binding site. Examples of antibody fragments include biantibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single-chain antibody molecules; scFv, sc(Fv)2; biantibodies; and multispecific antibodies formed from antibody fragments.

[0054] Preferably, the detectable markers include, but are not limited to, biotin, fluorescent dyes, enzymes that catalyze substrate color development, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers;

[0055] Preferably, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives, rhodamine dyes and their derivatives, Cy series dyes and their derivatives, and Alexa dyes. Series of dyes and their derivatives, ATTO TM Series dyes, TYE series dyes, quantum dot and protein dyes and their derivatives, etc.

[0056] The enzyme is one that catalyzes the color development of a substrate. Such enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase. Preferably, the enzyme catalyzing the color development of a substrate includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0057] Preferably, the radioactive isotopes include, but are not limited to, 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, and 18F;

[0058] Preferably, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, luciferin, fluorescein from crustaceans and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, rofenine and its derivatives, and peroxate and its derivatives.

[0059] Preferably, the nanoparticle-based markers include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles;

[0060] Preferably, the colloid includes, but is not limited to, colloidal metals, dispersed dyes, dye-labeled microspheres, and latex;

[0061] Preferably, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, and colloidal selenium.

[0062] On the other hand, the present invention discloses a detection kit containing the aforementioned anti-cCRP antibody or a functional fragment thereof.

[0063] Preferably, the detection kit is a fluorescence immunochromatographic detection kit.

[0064] Preferably, the samples tested by the kit are selected from bodily fluids such as whole blood, serum, plasma, urine, or saliva.

[0065] Beneficial effects:

[0066] This invention not only provides a monoclonal antibody (wild-type) with high affinity for cCRP, but also performs multiple active mutations on the wild-type antibody and verifies the affinity of each mutation, providing a reference for research in the field of immunoassay for cCRP. Attached image description:

[0067] Figure 1 Clinical correlation analysis between the reagent of this invention and the control reagent Detailed Implementation

[0068] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of formulations or unit doses herein. Unless otherwise stated, the techniques employed or considered herein are standard methods. Materials, methods, and examples are illustrative and not limiting in nature.

[0070] As used herein, the terms “comprising,” “including,” “having,” “may,” and their variations are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures.

[0071] Unless otherwise specified, the practice of this invention will employ conventional techniques of cell biology, molecular biology (including recombinant technologies), microbiology, biochemistry, and immunology, which are within the capabilities of those skilled in the art. Such techniques are well explained in the literature, such as *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Methods in Enzymology* (Academic Press, Inc.); and *Handbook of Experimental Immunology* (D.M. Weir and CC. Blackwyn). The references cited are: J.M. Miller and M.C. Salos (eds., 1987); F.M. Usubel et al. (eds., 1987); PCR: The Polymerase Chain Reaction (eds., Mullis et al., 1994); and J.E. C. Olgan et al. (eds., 1991), each of which is explicitly incorporated herein by reference.

[0072] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0073] Example 1: Preparation of Monoclonal Antibodies

[0074] 1. Mouse immunization and antibody detection

[0075] Ten 6-8 week old SPF-grade female BALB / c mice were selected. Freund's complete adjuvant was mixed with canine C-reactive protein at a concentration of 1 mg / ml in equal volumes and emulsified in an emulsifier. The emulsified antigen was then used to immunize the 6-8 week old SPF-grade female BALB / c mice, with each mouse receiving 50 μg of antigen protein via paw injection or subcutaneous injection in the back. Two weeks after the initial immunization, the antigen protein was mixed with Freund's incomplete adjuvant, emulsified, and again injected with 50 μg of antigen protein per mouse via paw injection or subcutaneous injection in the back. Two weeks later, blood was collected via tail vein, the supernatant was collected by centrifugation, and serum titer was determined using an indirect ELISA method. Mice with serum titers higher than 1:100,000 were selected for cell fusion.

[0076] 2. Cell fusion and positive hybridoma screening and subcloning

[0077] Cell fusion: SP2 / 0 myeloma cells and feeder cells in logarithmic growth phase were prepared. Mouse spleen cells or lymph node cells were mixed with SP2 / 0 cells at a 1:1 ratio and fused using an electrofusion apparatus to form hybridoma cells. After fusion, the hybridoma cells were transferred to serum-containing RPMI-1640 medium for 96-well plate culture, with 10n hybridoma cells per well. 5 Cell count, add feeder cells, at 10 per well 5 Supplementation was performed. After supplementation, the cell culture plate was placed in a cell culture incubator and cultured for 10 days. Positive hybridomas were screened after 10 days.

[0078] Screening and subcloning of positive hybridomas: Indirect ELISA was used to identify fusion cells in cell culture medium to determine whether specific antibodies were produced. Cells from positive wells were subcloned, and feeder cells were prepared on the same day as subcloning. Cells to be subcloned were gently pipetted to prepare a single-cell suspension, added to a counting chamber, and cell density was calculated. At a density of 100 cells per well, the required volume was taken, added to an appropriate amount of complete culture medium, mixed thoroughly, and then added to a 96-well plate. The monoclonal hybridoma cells with the highest affinity were selected.

[0079] 3. Production and purification of monoclonal antibodies

[0080] Two groups of 6-8 week old BALB / c mice were selected, and 500 μL of paraffin oil was injected intraperitoneally to suppress the immune response. One week after the paraffin injection, 0.5 ml of the aforementioned monoclonal hybridoma cells (approximately 1 × 10⁻⁶ cells) was injected intraperitoneally into one group of mice. 6 Quantity; Ascites fluid was collected two weeks later. The collected ascites fluid was purified by ammonium sulfate precipitation and affinity purification of protein A to obtain the target antibody.

[0081] Example 2: Validation of antibody performance

[0082] 1. ELISA Affinity Test

[0083] Goat anti-mouse antibody was diluted to 1 μg / mL using coating buffer (0.05 M pH 9.5 carbonate and bicarbonate buffer), and 100 μL was added to each well of a 96-well microplate. The plates were incubated overnight at 4°C. The plates were washed three times with PBS buffer containing 0.05% Tween-20. The antibody was then diluted to 1 μg / mL using dilution buffer (1% BSA, 0.1% PBST), and 100 μL was added to each well of a 96-well microplate. The plates were incubated at 37°C for 30 minutes. Dilute biotin-labeled canine C-reactive protein to 100 ng / mL with diluent. Remove the ELISA plate from its 37°C incubation and wash three times. Add 150 μL of the prepared canine C-reactive protein antibody dilution to the 96-well ELISA plate in row A. Add 100 μL of diluent to each well in rows B-H. Take 50 μL of the antibody dilution from row A and perform a 3-fold serial dilution from row B onwards. Incubate at 37°C for 30 minutes. Dilute horseradish peroxidase-labeled avidin 1:5000 with diluent. Remove the ELISA plate and wash three times. Add 100 μL of the diluted avidin to each well in a 96-well ELISA plate and incubate at 37°C for 30 minutes. Remove the ELISA plate and wash three times. Add the chromogenic buffer and incubate at room temperature for 3 minutes. Terminate the reaction by adding 0.5 M sulfuric acid and read the OD450 value using a plate reader.

[0084] The target antibody in Example 1 has a heavy chain variable region as shown in SEQ ID NO:9, wherein the amino acid sequences of each complementarity-determining region on the heavy chain variable region are as follows:

[0085] CDR-VH1: T(X1)-YG(X2)-VH;

[0086] CDR-VH2: V(X1)-INPSSGN(X2)-TNYNQK(X3)-FKH;

[0087] CDR-VH3: SDYD(X1)-GSNIFDY(X2);

[0088] Its light chain variable region is shown in SEQ ID NO:10, wherein the amino acid sequences of each complementarity-determining region on the light chain variable region are as follows:

[0089] CDR-VL1:RASQ(X1)-NIYYSL(X2)-A;

[0090] CDR-VL2: Y(X1)-ANS(X2)-LED;

[0091] CDR-VL3: KQY(X1)-YDV(X2)-PPT.

[0092] Based on the target antibody, mutations are made at sites related to antibody activity in the complementarity-determining region, where X1, X2, and X3 are mutation sites.

[0093] Table 1 Mutation sites related to antibody activity

[0094]

[0095] Table 2 Antibody activity analysis data

[0096]

[0097]

[0098] As can be seen from the table above, mutation 2 exhibits the best activity. Therefore, using mutation 2 as the backbone sequence, other mutation sites with good affinity were screened, and some results are shown below:

[0099] Table 3 Mutation sites related to antibody affinity

[0100]

[0101]

[0102] Canine C-reactive protein 100 ng / ml

[0103] The absorbance of OD450 for each mutation was measured, and the results are as follows:

[0104] Table 4. Affinity test for antibody mutations

[0105] Mutation 2-1 2.3508 Mutation 2-2 2.2889 Mutation 2-3 2.3147 Mutations 2-4 2.2727 Mutations 2-5 2.3744 Mutations 2-6 2.5072 Mutations 2-7 2.5854 Mutations 2-8 2.4133 Mutations 2-9 2.2015 Mutation 2-10 2.2834 Mutation 2-11 2.3120 Mutation 2-12 2.2201 Mutation 2-13 2.3672 Mutation 2-14 2.4220 Mutation 2-15 2.2897 Mutation 2-16 2.3017 Mutation 2-17 2.4115 Mutations 1-18 2.4637 Mutation 2-19 2.3609 Mutation 2-20 2.1844 Mutation 2-21 2.3514 Mutation 2-22 2.4038 Mutation 2-23 2.4122 Mutation 2-24 2.2289 Mutation 2-25 2.2547 Mutation 2-26 2.3308 Mutation 2-27 2.3087 Mutation 2-28 2.1984 Mutation 2-29 2.2003 Mutation 2-30 2.3069 Mutation 2-31 2.4408 Mutation 2-32 2.3502 Mutation 2-33 2.2144 Mutation 2-34 2.2564 Mutation 2-35 2.3368

[0106] 2. Stability test

[0107] The antibody concentration of this invention is 1 μg / mL, and the cCRP concentration is 100 ng / mL.

[0108] The antibody was subjected to accelerated thermal treatment at 37°C for 7 days in a predetermined buffer (PBS, 0.05% ProClin™ 300). The accelerated antibody was evaluated by SDS-PAGE and indirect ELISA methods, with a control group at 4°C, to assess the long-term stability of the antibody. Additionally, the antibody was subjected to five freeze-thaw cycles at -20°C, and the results are as follows:

[0109] Table 5 Stability Study

[0110]

[0111]

[0112] As can be seen from the above, mutations 2 to 2-35 can be stably stored at 4℃, and their properties remain stable even after being accelerated at 37℃ for 0.5 months (the slight decrease in reactivity does not affect the performance of the reagent), ensuring the performance of the reagent after opening the bottle, thereby ensuring the accuracy and stability of the test results.

[0113] Example 3: Application of Antibodies

[0114] This embodiment demonstrates the excellent performance of the antibody of the present invention in application by using a canine C-reactive protein monoclonal antibody in a fluorescent immunochromatographic assay.

[0115] A. Detection principle of the fluorescence immunochromatographic reagent kit

[0116] An immunofluorescence chromatography method based on the double-antibody sandwich method is employed. The canine C-reactive protein immunofluorescence quantitative reagent card includes a PVC base plate, a sample pad, a nitrocellulose membrane, and absorbent paper. The nitrocellulose membrane has a control C line coated with goat anti-mouse antibody, a detection T line coated with cCRP monoclonal antibody A, and a microsphere line coated with fluorescent microsphere-labeled cCRP monoclonal antibody B. During detection, cCRP in serum forms a complex with fluorescently labeled antibody B. Due to chromatographic action, the complex moves forward along the nitrocellulose membrane and is captured by antibody A at the detection T line, forming a "fluorescent microsphere-labeled B-cCRP-A" complex. This complex can be excited to produce 615 nm red light under ultraviolet light. The fluorescence intensity is proportional to the amount of reaction complex enriched on the test strip, allowing calculation of the antigen content in the sample.

[0117] B. Preparation of the fluorescence immunochromatographic reagent kit

[0118] The kit of the present invention comprises fluorescent microspheres labeled with 1 mg / mL antibody B, a nitrocellulose membrane coated with antibody A, antibody B and goat antimouse, and a reagent card assembly.

[0119] The fluorescent microspheres labeled with antibody B were prepared by activating the fluorescent microspheres with EDC for 0.5 h, mixing antibody B with the fluorescent microspheres at a mass ratio of 1:50, rotating and labeling at room temperature for 2.5 h, blocking with BSA blocking agent for 1 h, centrifuging at 12000 rpm to remove the supernatant after blocking, adjusting the concentration to 1 mg / mL with diluent, and sonicating in a water bath to obtain sufficiently dispersed fluorescent microspheres labeled with antibody B.

[0120] The nitrocellulose membrane was prepared by adjusting the concentration of antibody A and goat anti-mouse coating solution (0.05M pH=9.5 carbonate and bicarbonate buffer) to 1 mg / mL. The prepared antibody A was then applied to the detection line area of ​​the nitrocellulose membrane using a membrane scribing instrument, and the goat anti-mouse solution was applied to the control line area of ​​the nitrocellulose membrane. Fluorescent microspheres labeled with antibody B were then applied to the microsphere line area of ​​the nitrocellulose membrane. The nitrocellulose membrane was then dried in an oven at 45°C.

[0121] The reagent card assembly involves sequentially and tightly overlapping a sample pad, a nitrocellulose membrane, and an absorbent pad onto a plastic base plate. The assembled intermediates are then cut into 0.5cm wide strips using a cutting machine and placed into the plastic card housing. The plastic base plate, sample pad, absorbent pad, and plastic card housing are all common materials used in this field.

[0122] C. Detection method using a fluorescence immunochromatographic reagent kit

[0123] The Q3VET fluorescence instrument developed by Chongqing Zhongyuan Huiji Biotechnology Co., Ltd. was used for detection.

[0124] Standard curve plotting

[0125] cCRP was prepared into a series of concentration standards using serum: 0 mg / L, 3.125 mg / L, 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, and 400 mg / L. 5 μL of the standard was added to 995 μL of sample diluent and mixed thoroughly. 60 μL of the mixture was then added to the sample well of the reagent card. The card was inserted into a Q3VET fluorescence spectrometer, and after 3 minutes, the ratio of the signal intensity in the detection area to the control area was read, i.e., the T / C value (each control point was tested twice, and the average value was taken). The corresponding standard curve was then plotted.

[0126] Sample detection

[0127] Add 5 μL of serum / plasma to 995 μL of sample diluent and mix well; add 60 μL of the mixture to the sample well of the reagent card, insert the Q3VET fluorescence instrument, and read the ratio of the signal intensity of the detection area to the quality control area, i.e., T / C, after 3 minutes. The cCRP content in the sample can be obtained through the standard curve.

[0128] Clinical relevance of serum cCRP levels

[0129] The clinical relevance of the kit in this invention was verified by testing 120 canine serum samples with the same concentration gradient using the kit in this embodiment and the control kit.

[0130] The embodiments cannot exhaustively cover all possible schemes. In this embodiment, antibody A is selected to have mutations 2-13, and the heavy chain variable region of antibody B is shown in SEQ ID NO:11, and the light chain variable region is shown in SEQ ID NO:12.

[0131] The results are as follows Figure 1 As shown, the kit of this invention has a strong clinical correlation with the control kit, with an R² of 0.97. This indicates that the mutation 2-13 and antibody B of the invention, when applied to the fluorescent immunochromatographic kit, can achieve specific detection of cCRP.

[0132] D. Precision of serum cCRP detection

[0133] The fluorescence immunochromatographic kit of the present invention was used to detect low-value and high-value samples respectively. Each sample was tested 8 times, and the precision was calculated. The results are shown in Table 6. The precision is <10%.

[0134] Table 6 Precision of serum cCRP detection

[0135] test points Low value ug / g / Ml High value ug / Ml 1 35.6 103.1 2 33.7 106.0 3 34.6 100.8 4 36.3 98.6 5 31.9 93.9 6 33.2 90.5 7 34.1 95.5 8 32.6 91.1 Mean 34.0 97.4 SD 1.400 5.288 Cv 4％ 5％

[0136] The results show that mutations 2-13 and B in this invention, when applied to a fluorescent immunochromatographic kit, can achieve accurate detection of cCRP.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-cCRP antibody or a functional fragment thereof, characterized in that, It has the following complementary determinant regions: CDR-VH1: TYRVH; CDR-VH2: SINPSSGNTNYNQKFKH; CDR-VH3: SDYYGSNIFDY; CDR-VL1: RASENIYYSLA; CDR-VL2: YANSLED; CDR-VL3: KQAYDVPPT.

2. The anti-cCRP antibody or its functional fragment as described in claim 1, characterized in that, The antibody comprises light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L and heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H; the heavy chain backbone regions FR1-H, FR2-H, FR3-H and FR4-H are selected from SEQ ID NO:1-4 in sequence; the light chain backbone regions FR1-L, FR2-L, FR3-L and FR4-L are selected from SEQ ID NO:5-8 in sequence.

3. The anti-cCRP antibody or its functional fragment as described in claim 2, characterized in that, The antibody also contains a constant region.

4. The anti-cCRP antibody or its functional fragment as described in claim 3, characterized in that, The constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

5. The anti-cCRP antibody or its functional fragment as described in claim 4, characterized in that, The species source of the constant region is cattle, horses, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, geese, or humans.

6. A labeled antibody, characterized in that, The labeled antibody includes the anti-cCRP antibody as described in claim 5 or a functional fragment thereof.

7. The labeled antibody as described in claim 6, characterized in that, The labeling agents for the labeled antibodies include biotin, fluorescent dyes, enzymes that catalyze substrate color development, radioisotopes, chemiluminescent reagents, or nanoparticle-based labeling agents.

8. The labeled antibody as described in claim 7, characterized in that, The fluorescent dyes include fluorescein dyes, rhodamine dyes, Cy series dyes, Alexa Fluor series dyes, ATTO series dyes, or protein dyes; The enzymes that catalyze the color development of the substrate include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, or glucose-6-phosphate dehydrogenase. The radioactive isotopes include 212Bi, 131I, 111In, 90Y, 186Re, 211At, 125I, 188Re, 153Sm, 213Bi, 32P, 94mTc, 99mTc, 203Pb, 67Ga, 68Ga, 43Sc, 47Sc, 110mIn, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 121Sn, 161Tb, 166Ho, 105Rh, 177Lu, 172Lu, or 18F; The chemiluminescent reagents include luminol, luciferin, fluorescein from crustaceans, ruthenium bipyridine, acridine ester, dioxane, rofenol, or peroxyoxalate; The nanoparticle-based markers include colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, or rare earth complex nanoparticles.

9. The labeled antibody as described in claim 8, characterized in that, The colloid includes colloidal metals, dispersed dyes, dye-labeled microspheres, or dye-labeled latex.

10. The labeled antibody as described in claim 9, characterized in that, The colloidal metal includes colloidal gold or colloidal silver.

11. A test kit, characterized in that... The test kit contains the anti-cCRP antibody or its functional fragment as described in any one of claims 1-5.

12. The detection kit as described in claim 11, characterized in that, The detection kit is a fluorescence immunochromatographic detection kit.

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