Application of IGFBP4 and CCL14 protein antibodies and combination thereof in colorectal cancer diagnosis

Abstract

The invention provides an application of an IGFBP4 and CCL14 protein antibody and a combination thereof in colorectal cancer diagnosis, by optimizing a eukaryotic expression process of recombinant IGFBP4 and CCL14 antigens, the expression quantity and immunogenicity are greatly improved, a monoclonal antibody with high affinity and high specificity is screened, the optimal IGFBP4 and CCL14 antibodies are screened out, and the colorectal cancer diagnosis efficiency is improved. The antibody provided by the invention can effectively solve the problems of low affinity and specificity of the existing antibody, has a wider detection range, shows an extremely high synergistic diagnosis value during combined detection, remarkably improves the diagnosis efficiency of early colorectal cancer, and has an important clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody screening and disease diagnosis. Specifically, it relates to the application of antibodies against IGFBP4 and CCL14 proteins and their combination in the diagnosis of colorectal cancer. Background Art

[0002] IGFBP4 is one of the members of the insulin-like growth factor binding protein (IGFBP) family, consisting of 258 amino acids with a molecular weight of approximately 28 - 32 kDa. Its core function is to regulate the bioavailability and activity of IGF by binding insulin-like growth factors (IGF-1 and IGF-2) with high affinity. The IGF system plays a key role in cell proliferation, differentiation, and apoptosis, and IGFBP4 indirectly inhibits the IGF-mediated growth-promoting and anti-apoptotic signaling pathways by inhibiting the binding of IGF to its receptor. In addition, the activity of IGFBP4 can also be dynamically regulated by proteases (such as pregnancy-associated plasma protein-A, PAPP-A) that cleave its binding region to release IGF. The role of IGFBP4 in tumors is complex. On the one hand, it plays a tumor-suppressive role by inhibiting IGF signaling. For example, in colorectal cancer, high expression of IGFBP4 is associated with a longer survival time of patients, possibly due to the inhibition of IGF-1-driven tumor growth. On the other hand, overexpression of IGFBP4 in certain tumors (such as glioblastoma) may promote invasion through non-IGF-dependent pathways (such as integrin signaling).

[0003] CCL14 belongs to the C-C motif chemokine family and exists in an inactive precursor form (CCL14-1-74). After cleavage by proteases (such as neutrophil elastase), the active fragment CCL14-9-74 is released. It recruits immune cells such as monocytes and dendritic cells by binding to CCR1 and CCR3 receptors, regulating inflammatory responses and immune surveillance. The role of CCL14 in the tumor microenvironment is dual. In liver cancer, high expression of CCL14 is associated with an increase in the infiltration of cytotoxic T cells (CD8 + T cells) and an extended survival time of patients, suggesting its tumor-suppressive role by enhancing anti-tumor immunity. In contrast, in breast cancer, CCL14 may promote the metastasis of tumor cells by activating the CCR1 / ERK pathway. In addition, CCL14 can induce angiogenesis, and its pro-cancer or anti-cancer effects highly depend on the tumor type and the composition of immune cells in the microenvironment. As a prognostic marker, high expression of CCL14 in liver cancer and kidney cancer indicates a good prognosis, while in breast cancer, it is associated with a poor prognosis.

[0004] Diagnostic markers for colorectal cancer (CRC) include various types, covering serological, histological, fecal, and molecular markers, etc. Serological markers: carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and carbohydrate antigen 125 (CA125) are commonly used markers for CRC screening and postoperative monitoring, but they have the problem of low specificity (inflammation, smoking, etc. can also increase their levels); Fecal markers: fecal occult blood test (FOBT), but it is easily interfered by diet or gastrointestinal bleeding; Histological markers (biopsy or surgical specimens): pathological examination (gold standard), immunohistochemical markers (microsatellite instability (MSI) / mismatch repair (MMR)), but they are invasive and costly; Molecular markers: MSI / MMR, KRAS proto-oncogene (KRAS) / B-Raf proto-oncogene (BRAF) mutations, epigenetic markers (methylated genes), but they have low sensitivity and high costs.

[0005] Patent document CN115466794B discloses the diagnosis of CRC based on the RNA editing site combination of insulin-like growth factor-binding protein 7 (IGFBP7), apoptosis-inducing factor (BLCAP), and peptidylprolyl isomerase A (PPIA) genes, with relatively high sensitivity, but it relies on complex RNA editing detection techniques and has high costs; Patent CN118006789B discloses the diagnosis of CRC using protein combinations such as NADPH oxidase 1, etc., but it does not solve the problem of cross-reaction of markers in inflammation; Patent CN113398249A mainly focuses on the therapeutic applications of CCL14, such as viral vector delivery, and there are few patents for diagnostic reagents or detection methods directly used for early tumor screening, lacking specific detection probes or rapid screening tools. Existing products are kits based on the combined detection of CEA + CA19-9, but they have insufficient sensitivity (<50%) for early tumors (such as stage I).

[0006] Therefore, there is an urgent need to find antibodies that can detect IGFBP4 protein and CCL14 protein with high sensitivity and specificity, and have stable performance, can efficiently detect the levels of IGFBP4 and CCL14 in the test sample, and can use IGFBP4 and CCL14 proteins as combined markers for early diagnosis, efficacy evaluation, and prognostic intervention of colorectal cancer, etc. Summary of the Invention

[0007] In view of the problems existing in the prior art, the present invention provides an antibody against IGFBP4 and CCL14 proteins, their combination, and their application in the diagnosis of colorectal cancer. By optimizing the eukaryotic expression process of recombinant IGFBP4 and CCL14 antigens, the expression level and immunogenicity are significantly improved. As a positive control product in the kit of the present invention, it has excellent correlation, consistency, and stability. Dozens of antibodies targeting the epitopes of IGFBP4 and CCL14 are respectively screened by hybridoma technology, and a group of excellent paired antibodies are respectively selected from them. After verification, they can be applied to enzyme-linked immunosorbent assay and chemiluminescence immunoassay technology, providing a combination of colorectal cancer diagnostic markers with high sensitivity and high specificity, and solving the problem of insufficient accuracy in early tumor screening, typing, and efficacy monitoring in the prior art.

[0008] In the prior art solutions, when using traditional hybridoma technology or recombinant protein immunization of animals to prepare monoclonal or polyclonal antibodies against IGFBP4 protein or CCL14 protein, there are problems of insufficient antibody affinity and specificity due to unoptimized immunogen design. Moreover, when detecting IGFBP4, CCL14 proteins or gastrointestinal tumor markers by existing ELISA or chemiluminescence methods, the kit is not optimized for the combination of IGFBP4 and CCL14 antibodies, resulting in low detection sensitivity and specificity, and unable to achieve efficient diagnosis of gastrointestinal tumors (especially colorectal cancer). The present invention has developed brand-new highly specific antibodies against IGFBP4 and CCL14 and their combination, which can be used for the preparation of diagnostic kits to detect IGFBP4 and CCL14 in different samples to achieve clinical value.

[0009] On the one hand, the present invention provides an antibody against IGFBP4 protein, including a first antibody and / or a second antibody; the first antibody includes:

[0010] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.1, CDR2 consisting of the amino acid sequence of SEQ ID NO.2, and CDR3 consisting of the amino acid sequence of SEQ ID NO.3 in the heavy chain variable region, and

[0011] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.4, CDR2 consisting of the amino acid sequence of SEQ ID NO.5, and CDR3 consisting of the amino acid sequence of SEQ ID NO.6 in the light chain variable region;

[0012] The second antibody includes:

[0013] (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.7, CDR2 consisting of the amino acid sequence of SEQ ID NO.8, and CDR3 consisting of the amino acid sequence of SEQ ID NO.9 in the heavy chain variable region, and

[0014] (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.10, CDR2 consisting of the amino acid sequence of SEQ ID NO.11, and CDR3 consisting of the amino acid sequence of SEQ ID NO.12 in the light chain variable region.

[0015] The present invention optimizes the eukaryotic expression process of recombinant IGFBP4 antigen, expresses and purifies the recombinant protein in the eukaryotic expression system, and greatly improves the expression level and immunogenicity of IGFBP4. Using the purified recombinant protein as an immunogen to immunize mice or other appropriate animals to obtain immune spleen cells; adopting the hybridoma technology to fuse the immune spleen cells with myeloma cells to obtain a hybridoma cell line secreting anti-IGFBP4 monoclonal antibody and screening 10 monoclonal antibodies with high affinity and high specificity against IGFBP4 therefrom; cloning, expressing, and purifying the genes of the screened monoclonal antibodies to obtain purified antibody products. The antibodies obtained through three rounds of screening are further screened for excellent paired antibodies by the double-antibody sandwich ELISA test method. The screened IGFBP4 paired antibodies, either alone or in combination with other antibodies, have higher diagnostic value when diagnosing digestive tract tumors, such as colorectal cancer.

[0016] Furthermore, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.16.

[0017] On the other hand, the present invention provides a kit for detecting IGFBP4 protein, and the kit includes the antibody of IGFBP4 protein as described above.

[0018] It can be understood that the kit can be an ELISA detection kit, a chemiluminescent immunoassay detection kit, etc.

[0019] In some embodiments, the kit is a chemiluminescent immunoassay detection kit. By screening the optimal and appropriate paired IGFBP4 first antibody and second antibody, a double-antibody combination chemiluminescent immunoassay detection kit is constructed.

[0020] In another aspect, the present invention provides an antibody against CCL14 protein, including a first antibody and / or a second antibody; the first antibody includes:

[0021] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.17, CDR2 consisting of the amino acid sequence of SEQ ID NO.18, and CDR3 consisting of the amino acid sequence of SEQ ID NO.19 in the heavy chain variable region, and

[0022] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.20, CDR2 consisting of the amino acid sequence of SEQ ID NO.21, and CDR3 consisting of the amino acid sequence of SEQ ID NO.22 in the light chain variable region;

[0023] The second antibody includes:

[0024] (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.23, CDR2 consisting of the amino acid sequence of SEQ ID NO.24, and CDR3 consisting of the amino acid sequence of SEQ ID NO.25 in the heavy chain variable region, and

[0025] (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.26, CDR2 consisting of the amino acid sequence of SEQ ID NO.27, and CDR3 consisting of the amino acid sequence of SEQ ID NO.28 in the light chain variable region.

[0026] By optimizing the eukaryotic expression process of recombinant CCL14 antigen, the present invention expresses and purifies the recombinant protein in a eukaryotic expression system, greatly improving the expression level and immunogenicity of CCL14. Using the purified recombinant protein as an immunogen to immunize mice or other suitable animals to obtain immune spleen cells; adopting the hybridoma technology to fuse the immune spleen cells with myeloma cells to obtain a hybridoma cell line secreting anti-CCL14 monoclonal antibody and screening 10 monoclonal antibodies with high affinity and high specificity against CCL14 therefrom; cloning, expressing and purifying the genes of the screened monoclonal antibodies to obtain purified antibody products. The antibodies obtained through screening are further screened for excellent paired antibodies by the double antibody sandwich ELISA test method. The screened CCL14 paired antibodies, alone or in combination with other antibodies, have higher diagnostic value when diagnosing digestive tract tumors, such as colorectal cancer.

[0027] Further, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.30; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.32.

[0028] In another aspect, the present invention provides a kit for detecting CCL14 protein, and the kit includes the antibody against CCL14 protein as described above.

[0029] It can be understood that the kit can be an ELISA detection kit, a chemiluminescent immunoassay detection kit, etc.

[0030] In some embodiments, the kit is a chemiluminescent immunoassay detection kit. By screening the optimal and suitable paired first antibody and second antibody against CCL14, a dual-antibody combination chemiluminescent immunoassay detection kit is constructed.

[0031] In another aspect, the present invention provides a kit for detecting IGFBP4 protein and / or CCL14 protein, and the kit includes the antibody against IGFBP4 protein as described above, and / or the antibody against CCL14 protein as described above.

[0032] In some embodiments, combining the kit for detecting IGFBP4 protein and the kit for detecting CCL14 protein can be used to simultaneously detect IGFBP4 protein and CCL14 protein, and thus can be applied to the field of simultaneous detection of multiple markers, such as the field of gastrointestinal tumor diagnosis, to improve the diagnostic efficiency through combined detection.

[0033] In another aspect, the present invention provides the use of an antibody for preparing a reagent for predicting whether an individual has colorectal cancer, and the antibody includes the antibody against IGFBP4 protein as described above, and / or the antibody against CCL14 protein as described above.

[0034] Traditional single-target antibody drugs have many limitations themselves. In existing cancer diagnosis technologies, single biomarker (such as CCL14) detection has problems of low sensitivity and high false positive rate. Although IGFBP4 is related to the tumor microenvironment, its clinical application for single detection is not yet mature. The present invention uses IGFBP4 and CCL14 as combined markers, and solves the problem of insufficient diagnostic efficiency of single markers through the combined detection of antibodies targeting IGFBP4 and CCL14 proteins, improving the accuracy and specificity of cancer diagnosis.

[0035] Although IGFBP4 and CCL14 are known diagnostic markers for colorectal cancer, there are significant differences in the diagnostic efficacy when using different IGFBP4 antibodies and / or CCL14 antibodies to diagnose colorectal cancer. During the diagnosis of colorectal cancer, it is necessary to detect blood samples through IGFBP4 antibodies and / or CCL14 antibodies. There are complex interfering substances in the blood samples, the matrix has a great influence, cross-reactions are likely to occur, or there are interferences from some non-linear fragments. It is difficult to obtain ideal test results by directly using existing antibodies, which seriously affects the accuracy of the diagnostic results.

[0036] Through the design, expression of antigens, and screening of monoclonal antibodies, the antibodies of IGFBP4 and CCL14 proteins finally obtained in the present invention have better detection sensitivity and specificity, and the signals are more stable, with less cross-reaction. When used for the diagnosis of colorectal cancer, the diagnostic efficacy can be significantly improved.

[0037] Furthermore, it includes an antibody of IGFBP4 protein and an antibody of CCL14 protein.

[0038] Furthermore, the antibody of IGFBP4 protein includes a first antibody and a second antibody. The amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.16.

[0039] The antibody of CCL14 protein includes a first antibody and a second antibody. The amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID NO.29, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.30; the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO.31, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.32.

[0040] Furthermore, the reagent is used to detect the content of antigens that can bind to antibodies in body fluid samples; the body fluid samples include any one or more of blood, plasma, serum, lymph fluid, cerebrospinal fluid, synovial fluid, urine, saliva, and mucus.

[0041] In some embodiments, the reagent for predicting the recurrence risk of colorectal cancer is a detection reagent prepared with IGFBP4 and CCL14 proteins as detection targets. The present invention uses antibodies to detect IGFBP4 and CCL14 proteins.

[0042] Furthermore, the reagent is used to detect the presence, relative abundance, or concentration of biomarkers in body fluid samples.

[0043] The present invention screens for biomarkers (IGFBP4 and CCL14 proteins) for predicting the recurrence risk of colorectal cancer from blood. There are significant differences in the levels of these two biomarkers in the blood of colorectal cancer patients and healthy individuals. By collecting a blood sample, the likelihood of an individual having colorectal cancer can be predicted or diagnosed by detecting the levels of IGFBP4 and CCL14 proteins in the individual's blood. Alternatively, the levels of IGFBP4 and CCL14 proteins in the blood of a group can be detected, and then the group can be classified into colorectal cancer patients and healthy individuals.

[0044] In another aspect, the present invention provides a kit for predicting whether an individual has colorectal cancer, which kit comprises an antibody against the IGFBP4 protein as described above, and an antibody against the CCL14 protein as described above.

[0045] In another aspect, the present invention provides an antibody combination for predicting whether an individual has colorectal cancer, which antibody combination comprises an antibody against the IGFBP4 protein as described above, and an antibody against the CCL14 protein as described above.

[0046] Further, it comprises an antibody against the IGFBP4 protein and an antibody against the CCL14 protein.

[0047] Further, the antibody against the IGFBP4 protein comprises a first antibody and a second antibody. The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.16.

[0048] The antibody against the CCL14 protein comprises a first antibody and a second antibody. The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.30; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.32.

[0049] In another aspect, the present invention provides a system for predicting whether an individual has colorectal cancer, which system comprises a data analysis module for analyzing the detection value of an antigen, which antigen is detected by an antibody, and the antibody comprises an antibody against the IGFBP4 protein as described above, and / or an antibody against the CCL14 protein as described above.

[0050] Further, the data analysis module uses the detection values of biomarkers of known samples as the training set. According to the conditions of colorectal cancer patients after surgery, they are divided into a non-recurrence group, an in-situ recurrence group, and a distant metastasis group. The relationship between the detection values of the non-recurrence group, the in-situ recurrence group, and the distant metastasis group is analyzed to construct a model.

[0051] Further, the system further includes a data storage module, a data input interface, and a data output interface; the data storage module is used to store the detection values of biomarkers; the data input interface is used to input the detection values of biomarkers, and the data output interface is used to output the prediction results.

[0052] The beneficial effects of the present invention are as follows:

[0053] 1. Provide an efficient method for recombinant expression of IGFBP4 and CCL14 proteins. As immunogens for inoculating experimental animals and positive quality control products in kit products, the IGFBP4 and CCL14 proteins provided by the present invention have the characteristics of high yield, good stability, and strong immunogenicity;

[0054] 2. Provide a brand-new specific recognition IGFBP4 antibody sequence, which can be used to construct an IGFBP4 detection kit. Combined with the double antibody sandwich method design, the cross-reaction rate is extremely low (<2.0%), and the sensitivity reaches the ng / mL level, which can detect IGFBP4 protein with high precision;

[0055] 3. Provide a brand-new specific recognition CCL14 antibody sequence, which can be used to construct a CCL14 detection kit. Combined with the double antibody sandwich method design, the cross-reaction rate is extremely low (<3.0%), and the sensitivity reaches the ng / mL level, which can detect CCL14 protein with high precision;

[0056] 4. The diagnostic value of the kit used alone or in combination in digestive tract tumors, especially in colorectal cancer; the combined detection model (IGFBP4 + CCL14 logistic regression) significantly improves the discrimination ability (AUC≥0.92) compared with a single biomarker. Description of the Drawings

[0057] Figure 1 It is a process flow chart for recombinant expression of IGFBP4 protein and CCL14 protein.

[0058] Figure 2 It is the SDS-PAGE detection result after purification of recombinant IGFBP4 and CCL14 antigens.

[0059] Figure 3 It is a process flow chart for the preparation of monoclonal antibodies against IGFBP4 and CCL14.

[0060] Figure 4 It is a flow chart of animal immunization.

[0061] Figure 5 It is the 4 SDS-PAGE detection results of monoclonal antibodies of IGFBP4 and CCL14.

[0062] Figure 6 It is the purification process flow chart of monoclonal antibody of IGFBP4 and monoclonal antibody of CCL14

[0063] Figure 7 It is the regression curve of the IGFBP4 detection kit.

[0064] Figure 8 It is the regression curve of the CCL14 detection kit.

[0065] Figure 9 It is the ROC curve of the diagnostic test results of different markers for colorectal cancer. Specific implementation manners

[0066] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way. The reagents used in this embodiment are all known products and are obtained by purchasing commercially available products.

[0067] Example 1. Screening of IGFBP4 and CCL14 antibodies

[0068] 1. Expression of recombinant antigens IGFBP4 and CCL14 proteins

[0069] The specific implementation plan is shown in Figure 1 the recombinant protein expression and preparation process flow chart shown below. The steps are as follows:

[0070] (1) Construction of recombinant antigen expression vector: Through bioinformatics analysis, the nucleotide sequences of IGFBP4 (UniProt number: P22692) (SEQ ID NO.41) and CCL14 (UniProt number: Q16627) (SEQ ID NO.42) are obtained. Through gene synthesis method, the expression sequences of IGFBP4 and CCL14 (SEQ ID NO.43 and SEQ ID NO.44) are obtained. The nucleotide sequences of IGFBP4 and CCL14 are respectively cloned into the multiple cloning sites of the pTT5 expression vector by molecular cloning method. After screening positive clones and verifying them by sanger sequencing and double digestion without error, the vector is transformed into the host cell Escherichia coli TOP10, and finally glycerol bacteria are stored in frozen state. This part of the work is entrusted to Anhui General Biotechnology Co., Ltd.

[0071] (2) Expression of recombinant antigens

[0072] 1. Materials required: LB medium (solid / liquid), sodium ampicillin solution, protease inhibitor, Binding buffer, Elution buffer, Ni-NTA beads, 40% glycerol, 293 cells, KOP293 medium, KPM medium, TA-293, KE-293, KT-Feed, PBS.

[0073] 2. Experimental procedures

[0074] 1) Vector preparation

[0075] a. The expression vector synthesized by outsourcing (5 μg of vector dry powder) is dissolved in 100 μL of purified water, dissolved and mixed evenly to obtain a 50 ng / μL vector solution.

[0076] b. Take 2 μL of the vector solution and transform it into TOP10 competent cells; take 100 μL of the transformation suspension and spread it on an LB plate, and culture it overnight at 37 °C.

[0077] c. After culturing for 16 - 18 h, pick a single clone into 5 mL of liquid LB medium, and culture it on a shaker at 37 °C and 250 rpm for 8 h.

[0078] d. After culturing for 8 h, inoculate the seed bacterial solution at a ratio of 1:50 into 200 mL of liquid LB medium, and culture it on a shaker at 37 °C and 250 rpm overnight (16 - 18 h).

[0079] e. Extract the vector according to the instructions of the endotoxin-free vector extraction kit.

[0080] 2) Cell transfection

[0081] a. HEK-293 cells are placed in a 5% CO2 constant temperature shaker and cultured with constant shaking at 37 °C and 120 rpm.

[0082] b. Use cells with a viable cell ratio ≥ 97% and a viable cell density of 2 × 106 cells / mL for transfection, and prepare 200 mL of cells one day in advance.

[0083] c. Prepare the transfection complex: Prepare two 50 mL centrifuge tubes. In one tube, dilute the vector DNA (200 μg) in 10 mL of serum-free medium (Opti-MEM), and in the other tube, dilute 1.2 mL of the liposome transfection reagent (Lipofectamine 3000) in 10 mL of serum-free medium.

[0084] d. Gently mix the diluted vector and the transfection reagent, and let it stand at room temperature for 10 minutes to form a DNA-liposome complex.

[0085] e. Add the complex dropwise to the cell culture medium and gently mix evenly.

[0086] f. Add 4 mL of 293 cell protein expression enhancer (KE-293 MAX) and 4 mL of transient transfection nutrient additive (KT-Feed) 24 hours after transfection.

[0087] g. Collect the fermentation broth after continuous culture for 5 days.

[0088] (3) Recombinant antigen purification: The fermentation broth was purified by nickel affinity chromatography and desalted and buffer-exchanged with G25. After the purified sample passed the quality inspection, it was aliquoted and stored frozen. The SDS-PAGE detection results of the purified IGFBP4 and CCL14 recombinant antigens are shown respectively as Figure 2 shown. After sequencing verification, the amino acid sequences of the purified IGFBP4 and CCL14 recombinant antigens are SEQ ID NO.43 and SEQ ID NO.44.

[0089] II. Screening and preparation of monoclonal antibodies against IGFBP4 and CCL14

[0090] The specific implementation plan for monoclonal antibody screening is detailed in the Figure 3 process flow chart of monoclonal antibody preparation shown as follows. The specific implementation steps are as follows:

[0091] (1) Animal immunization

[0092] Immunize 5 white variant laboratory mice (BALB / c) with the above-obtained IGFBP4 and CCL14 recombinant antigens respectively, divided into primary immunization, second immunization, third immunization, fourth immunization and booster immunization. The injection time, injection adjuvant and injection method for each immunization are detailed in the Figure 4 immunization flow chart shown.

[0093] (2) Serum titer detection

[0094] After the immunization is completed, collect blood from the submandibular vein, and detect the antibody titer by indirect ELISA. Select mice with OD450 greater than 0.2 for spleen fusion. The operation process of detecting the antibody titer by indirect ELISA is as follows:

[0095] 1. Required materials: Coating buffer (pH 9.6 carbonate buffer), blocking solution (5% BSA), antigen (purified IGFBP4 protein and CCL14 protein), orbital blood, HRP-labeled secondary antibody (anti-mouse IgG), TMB (3,3',5,5'-tetramethylbenzidine) chromogenic solution, termination solution (2M H2SO4), microplate reader.

[0096] 2. Experimental steps:

[0097] ① Antigen coating:

[0098] a) Dilute the antigen to 0.5 ug / ml using carbonate buffer (pH 9.6);

[0099] b) Add the diluted antigen to the ELISA plate wells, 100 μL per well, and incubate at 37 °C for 2 h;

[0100] c) Discard the liquid, and wash 3 times with PBST (PBS + 0.05% Tween-20), 3 min each time;

[0101] ② Block non-specific binding:

[0102] Add 200 μL of blocking solution (5% BSA) to each well, and incubate at 37 °C for 1 h;

[0103] Discard the liquid, and wash 3 times with PBST;

[0104] ③ Add the sample to be tested and incubate:

[0105] a) Gradient dilute the sample to be tested (orbital blood) starting from a low concentration (1:1000, 1:4000, 1:16000, 1:64000, 1:256000);

[0106] b) Add 100 μL of the diluted sample to each well, and incubate at 37 °C for 1 - 2 h;

[0107] c) Wash 5 times with PBST, and pat dry (ensure that unbound antibodies are removed);

[0108] ④ Add the enzyme-labeled secondary antibody:

[0109] a) Dilute the secondary antibody with the diluent according to the recommended ratio, add 100 μL to each well, and incubate at 37 °C for 1 hour;

[0110] b) Wash 5 times with PBST, and pat dry (avoid background elevation caused by residual secondary antibody);

[0111] ⑤ Color development and termination of reaction:

[0112] a) Add 100 μL of TMB substrate solution to each well, and react at room temperature in the dark for 10 - 15 min (observe the blue color gradually deepening, and avoid over-color development);

[0113] b) Add 50 μL of termination solution (H2SO4) to each well, the solution turns from blue to yellow, and immediately measure the absorbance at 450 nm (OD450) with an enzyme-linked immunosorbent assay reader.

[0114] The detection results of the serum titers of IGFBP4 and CCL14 in mice are shown in Tables 1 and 2:

[0115] Table 1 Indirect ELISA for detecting the serum titer of IGFBP4 in mice

[0116]

[0117] Table 2 Indirect ELISA Detection of Serum Titer of CCL14 in Mice

[0118]

[0119] As can be seen from Table 1, the orbital blood tests of five mice KY0195, KY0196, KY0197, KY0198, and KY0199 in the IGFBP4 project all met the standards (OD450 value ≥ 0.2 at a dilution of 1:64000). The results in Table 2 show that the orbital blood tests of four mice KY0232, KY0233, KY0234, and KY0235 in the CCL14 project met the standards (OD450 value ≥ 0.2 at a dilution of 1:64000), and all can be used for subsequent fusion screening.

[0120] (3)Cell Fusion

[0121] Blood was taken from the eyeballs and reserved as a positive control. The spleens of the mice were isolated, ground, counted, and cell fusion was carried out according to the ratio of spleen cells: sp2 / 0 cells = 8:1. 1 mL of PEG1450 was added, and the whole process was carried out under the condition of a 37°C water bath. Preheated blank medium was added to terminate, and after centrifugation, HAT conditioned medium was added for pressure screening.

[0122] (4)Monoclonal Antibody Screening

[0123] Most of the fused cells are cells of multiple clones and need to continue with monoclonal plating. After three rounds of screening (ELISA, IgG), 10 hybridoma cells that highly secrete specific antibodies against IGFBP4 and CCL14 were obtained respectively.

[0124] After primary screening by indirect ELISA and SPR verification, 10 hybridoma cells secreting and expressing IGFBP4 antibodies (Code: IGFBP4-mAb-10-01~IGFBP4-mAb-10-10) were screened from 55 clones, and their sequences and performances are shown in Table 3. 10 hybridoma cells secreting and expressing CCL14 antibodies (Code: CCL14-mAb-15-01~CCL14-mAb-15-10) were screened from 42 clones, and their sequences and performances are shown in Table 4. They are sorted in descending order according to the binding titer (OD450 value).

[0125] Table 3 Hybridoma Cells Secreting and Expressing IGFBP4 Antibodies

[0126]

[0127] Table 4 Hybridoma Cells Secreting and Expressing CCL14 Antibodies

[0128]

[0129] As can be seen from Table 3 and Table 4, the affinities and specificities of the 10 antibodies against IGFBP4 and CCL14 obtained by screening are all excellent. Among them, the two optimal antibodies against IGFBP4 are IGFBP4-mAb-10-01 and IGFBP4-mAb-10-02 respectively, and their cross-reactivities with CCL14 are both <2.0%; the two optimal antibodies against CCL14 are CCL14-mAb-15-01 and CCL14-mAb-15-02 respectively, and their cross-reactivities with IGFBP4 are both <3.0%.

[0130] (5)Purification of monoclonal antibodies

[0131] Hybridoma cells were expanded and cultured, fermented to secrete antibodies, the supernatant of the fermentation broth was collected, and after Protein A affinity chromatography and G25 desalting and buffer exchange, the purified samples were qualified by quality inspection and then aliquoted and frozen. For the specific implementation plan, please refer to Figure 6 the process flow chart of monoclonal antibody purification shown

[0132] III. Screening of ligand antibodies

[0133] The 10 high-affinity antibodies against IGFBP4 and CCL14 obtained by the above screening were paired and detected according to the antibodies to obtain excellent antibody pairs. The paired antibody screening used the double-antibody sandwich ELISA test method to verify whether the two antibodies could bind to the antigen simultaneously to form a "capture antibody - antigen - detection antibody" complex. The specific method steps are as follows:

[0134] (1)Required materials: Coating buffer (pH 9.6 carbonate buffer), blocking solution (5% BSA), antigens (purified IGFBP4 protein and CCL14 protein), antibody pairs to be screened (antibodies described in Table 5 and Table 6), HRP-labeled secondary antibody (anti-mouse IgG), TMB (3,3',5,5'-tetramethylbenzidine) chromogenic solution, termination solution (2M H2SO4), microplate reader.

[0135] (2)Experimental steps:

[0136] ① Coating the capture antibody:

[0137] a) Dilute the capture antibody (unlabeled antibody) to 1 μg / mL, add it to the microplate wells (100 μL / well), and incubate at 37°C for 2 h.

[0138] b) Wash the plate (wash 3 times with PBST).

[0139] ② Blocking: Add 200 μL of blocking solution, block at 37°C for 1 h, and wash the plate.

[0140] ③ Antigen binding: Add antigen diluted in gradient (such as 0.05 μg / mL, 100 μL / well), incubate at 37 °C for 1 h, and wash the plate.

[0141] ④ Detection of antibody binding:

[0142] a) Add detection antibody (biotinylated modification), incubate at 37 °C for 1 h, and wash the plate.

[0143] b) Add secondary antibody labeled with horseradish peroxidase (HRP) (anti-mouse IgG-HRP), incubate and then wash the plate.

[0144] ⑤ Color development and reading:

[0145] a) Add TMB color development solution (100 μL / well), develop color in the dark for 10 - 15 min.

[0146] b) Add stop solution (50 μL / well), immediately measure the absorbance at 450 nm (OD value) with an enzyme-linked immunosorbent assay (ELISA) reader.

[0147] The screening results of IGFBP4 paired antibodies are shown in Table 5, and the screening results of CCL14 paired antibodies are shown in Table 6.

[0148] Table 5 Screening of IGFBP4 Paired Antibodies

[0149]

[0150] Table 6 Screening of CCL14 Paired Antibodies

[0151]

[0152] As shown in the results of Table 5, the optimal paired antibodies obtained by immunizing IGFBP4 are IGFBP4-mAb-10-05 (capture antibody) and IGFBP4-mAb-10-07 (labeling antibody), and the titer reaches 0.81. As shown in the results of Table 6, the optimal paired antibodies obtained by immunizing CCL14 are CCL14-mAb-15-01 (capture antibody) and CCL14-mAb-15-08 (labeling antibody), and the titer reaches 0.96. It can be found that when IGFBP4-mAb-10-07 is used as the capture antibody and IGFBP4-mAb-10-05 is used as the labeling antibody, the titer of the paired antibodies decreases significantly to 0.34; when CCL14-mAb-15-08 is used as the capture antibody and CCL14-mAb-15-01 is used as the labeling antibody, the binding titer of the paired antibodies also decreases significantly to 0.37. The reason may be that antibodies recognize different epitopes and there is steric hindrance. When the IGFBP4 and CCL14 antigens are immobilized by the IGFBP4-mAb-10-05 and CCL14-mAb-15-01 capture antibodies respectively, the other epitope may be more fully exposed, facilitating the binding of the detection antibody; conversely, if the positions are exchanged, the epitope may not be exposed enough, resulting in low binding efficiency. In addition, there are differences in the affinity of different antibodies. The capture antibody needs to bind to the antigen quickly and firmly (high affinity), while the detection antibody may rely more on the continuous binding ability of the antigen (high binding capacity). From the results of Table 3 and Table 4, it can be known that the binding titer of the IGFBP4-mAb-10-05 antibody is higher than that of the IGFBP4-mAb-10-07, and the binding titer of the CCL14-mAb-15-01 antibody is higher than that of the CCL14-mAb-15-08. The affinities of IGFBP4-mAb-10-05 and CCL14-mAb-15-01 are better and are more suitable as capture antibodies.

[0153] Name IGFBP4-mAb-10-05 (capture antibody) as the first IGFBP4 antibody and IGFBP4-mAb-10-07 (labeling antibody) as the second IGFBP4 antibody. Name CCL14-mAb-15-01 (capture antibody) as the first CCL14 antibody and CCL14-mAb-15-08 (labeling antibody) as the second CCL14 antibody.

[0154] Through DNA sequencing and amino acid sequence analysis, it was confirmed as follows: The CDR1 sequence of the heavy chain of the anti-IGFBP4 first antibody is SEQ ID NO.1, the CDR2 sequence is SEQ ID NO.2, the CDR3 sequence is SEQ ID NO.3, the CDR1 sequence of the light chain is SEQ ID NO.4, the CDR2 sequence is SEQ ID NO.5, and the CDR3 sequence is SEQ ID NO.6; The CDR1 sequence of the heavy chain of the anti-IGFBP4 second antibody is SEQ ID NO.7, the CDR2 sequence is SEQ ID NO.8, the CDR3 sequence is SEQ ID NO.9, the CDR1 sequence of the light chain is SEQ ID NO.10, the CDR2 sequence is SEQ ID NO.11, and the CDR3 sequence is SEQ ID NO.12.

[0155] The CDR1 sequence of the heavy chain of the anti-CCL14 first antibody is SEQ ID NO.17, the CDR2 sequence is SEQ ID NO.18, the CDR3 sequence is SEQ ID NO.19, the CDR1 sequence of the light chain is SEQ ID NO.20, the CDR2 sequence is SEQ ID NO.21, and the CDR3 sequence is SEQ ID NO.22; The CDR1 sequence of the heavy chain of the anti-CCL14 second antibody is SEQ ID NO.23, the CDR2 sequence is SEQ ID NO.24, the CDR3 sequence is SEQ ID NO.25, the CDR1 sequence of the light chain is SEQ ID NO.26, the CDR2 sequence is SEQ ID NO.27, and the CDR3 sequence is SEQ ID NO.28.

[0156] ⑥ Recombinant antibody vector construction: Total RNA of hybridoma cells was extracted, and the RNA was reverse transcribed into cDNA by RT-PCR reaction. The antibody light chain and heavy chain sequences were cloned, and the antibody light chain and heavy chain sequences were constructed onto the T vector, and then DNA sequencing analysis was performed to obtain the antibody gene sequence.

[0157] ⑦ Antibody production and purification preparation: The antibody gene sequence obtained in step ⑥ was transfected into CHO cells and expanded in culture. The antibody was purified by protein A / G affinity chromatography, and the SDS-PAGE detection of the purified antibody is as Figure 5 shown, and the purified antibody was stored in phosphate buffer (PBS) by dialysis method.

[0158] Meanwhile, in this embodiment, the thermal stability Tm value of the screened antibodies was determined by circular dichroism (CD) to evaluate the stability of the antibodies, and compared with existing antibodies. The existing IGFBP4 antibody was purchased from Abcam, catalog number ab205581, and the CCL14 antibody was purchased from Abcam, catalog number ab272383. The test results are shown in Tables 7 and 8. Here, Tm represents the midpoint temperature of protein thermal denaturation, that is, the temperature when 50% of the protein unfolds, reflecting the trend of protein conformational change during the temperature change process. The higher the Tm, the better the stability.

[0159] Table 7 Changes in the stability of IGFBP4 antibody

[0160]

[0161] Table 8 Changes in the stability of CCL14 antibody

[0162]

[0163] It can be seen from Tables 7 and 8 that the stability of the screened monoclonal antibodies of IGFBP4 and CCL14 is significantly better than that of the commercially available IGFBP4 and CCL14 antibodies.

[0164] Example 2. Construction of IGFBP4 and CCL14 antibody detection kits

[0165] In this embodiment, the first antibody and second antibody of IGFBP4 screened in Example 1; and the first antibody and second antibody of CCL14 were used to construct an IGFBP4 and CCL14 combined detection kit.

[0166] The kit includes:

[0167] Quality control products: IGFBP4 positive control (high concentration); IGFBP4 positive control (low concentration); CCL14 positive control (high concentration); CCL14 positive control (low concentration).

[0168] Coated antibodies: Biotin-labeled IGFBP4 first antibody; Biotin-labeled CCL14 first antibody; Magnetic bead particles coated with streptavidin; 100 mM phosphate buffer, pH 7.4; Preservative.

[0169] Labeled antibodies: Azabutyrate-labeled IGFBP4 second antibody; Azabutyrate-labeled CCL14 second antibody; Preservative; 100 mM phosphate buffer, pH 7.4; Preservative.

[0170] Standard products: IGFBP4 standard products (5 ng / ml, 25 ng / ml, 125 ng / ml, 250 ng / ml, 500 ng / ml).

[0171] Dilution buffer: 2× universal dilution buffer, 15 ml.

[0172] The IGFBP4 and CCL14 combined detection kit constructed in this example includes a detection kit for detecting IGFBP4 and a detection kit for detecting CCL14, which can respectively detect the concentrations of IGFBP4 and CCL14 in the sample to be tested, so as to obtain the detection values of IGFBP4 and CCL14 respectively. That is, it can be used to detect IGFBP4, or to detect CCL14, or to detect IGFBP4 and CCL14 simultaneously.

[0173] The method for detecting the concentration of the combined biomarker IGFBP4 and / or CCL14 in the sample to be tested using the combined detection kit provided in this example is as follows:

[0174] Among them, to detect the concentration of IGFBP4 in the sample to be tested:

[0175] 1) Coating: Dilute the first antibody of IGFBP4 with carbonate CBS coating solution to 0.1 - 10 μg / ml (according to the corresponding inspection specifications), add 50 μl per well to a 96-well plate, and incubate overnight at 4°C.

[0176] 2) Blocking: The next day, dry the 96-well plate by shaking it, add 200 μl of blocking solution to each well, block at room temperature (22 - 25°C, the same below) for 1 h, then wash the plate 3 times with a plate washer, dry it immediately for use, or store it at -20°C for later use.

[0177] 3) Adding the test sample: Set up control wells, 8 standard wells (for making a standard curve) and wells for the sample to be tested. Add blank dilution buffer to the control wells, add IGFBP4 working solutions with gradient concentrations (serially diluted 8-fold with the dilution buffer, and the gradient concentrations are 0 ng / L, 7.8125 ng / L, 15.625 ng / L, 31.25 ng / L, 62.5 ng / L, 125 ng / L, 250 ng / L, 500 ng / L, 1000 ng / L) to the 8 standard wells respectively, and add the sample to be tested to the wells for the sample to be tested. The added volume is 50 μl for each. Seal the enzyme-labeled plate with a sealing film, incubate at 37°C for 30 minutes, and set up duplicate wells. React with shaking at 220 rpm at room temperature for 1 h, wash the plate 3 times with a plate washer, and pat it dry.

[0178] 4) Adding the enzyme-labeled second antibody: Dilute the biotinylated second antibody of IGFBP4 to the working concentration with the dilution buffer. Add 50 μl to each well, react with shaking at 220 rpm at room temperature for 1 h, wash the plate 3 times with a plate washer, and pat it dry.

[0179] 5) Adding enzyme solution: Dilute the SA-HRP stock solution 10,000 times with the dilution solution to obtain the enzyme working solution. Add 50 μl to each well, react with shaking at 220 rpm at room temperature for 45 minutes to 1 hour, wash the plate 3 times with a plate washer, and pat dry.

[0180] 6) Adding chromogenic substrate: Add 50 μL of tetramethylbenzidine (TMB) with a concentration of 4 g / L and 5 μL of hydrogen peroxide with a mass fraction of 0.02% to each well, and incubate at 37 °C for 15 minutes.

[0181] 7) Adding stop solution: Add 50 μL of 1 M phosphoric acid to each well to terminate the reaction.

[0182] 8) Reading and calculation: Detect the absorbance (OD value) of each well at 450 nm.

[0183] 9) Fitting the standard curve to calculate the corresponding concentration: Use the concentration of the IGFBP4 working solution as the ordinate and its OD value as the abscissa to plot the standard curve, and obtain the regression curve as Figure 7 shown (y = 58.127x + 34111, R 2 = 0.9922). Substitute the OD value of the test sample obtained by detection into the regression equation to calculate the concentration of IGFBP4 in the test sample.

[0184] The specific method for detecting the CCL14 concentration in the test sample is the same as above, and the constructed regression curve is as Figure 8 shown (y = 213.33x + 146715, R 2 = 0.994).

[0185] III. Performance evaluation of the chemiluminescence kit

[0186] In this example, the performance of the IGFBP4 and CCL14 joint detection kit was also evaluated, mainly including the following aspects:

[0187] (1) Recovery rate

[0188] Add IGFBP4 protein with a final concentration of 500 ng / ml and CCL14 protein with a final concentration of 100 ng / ml to the certified serum (IGFBP4 concentration 543 ng / ml, CCL14 concentration 92 ng / ml determined by mass spectrometry) and plasma samples respectively, repeat the measurement and calculate their mean values, and examine their recovery rates (see Table 9). The recovery rate is the ratio of the measured value to the theoretical value.

[0189] Table 9 Determination of recovery rate

[0190]

[0191] As shown in Table 9, the average recovery rates of the IGFBP4 and CCL14 combined detection kit in serum, EDTA plasma, and heparin plasma samples all reached over 90%.

[0192] (2)Precision

[0193] IGFBP4 protein quality control solutions and CCL14 protein quality control solutions (prepared with negative human serum) containing 5, 10, 50, and 250 ng / mL were used respectively to examine their within - batch / between - batch precision. For within - batch precision: Use the same batch of kits to quantitatively detect the certified samples. Each sample was continuously measured 20 times, and the SD values and coefficient of variation (CV) of samples with different concentrations were calculated respectively. For between - batch precision: Select 3 different batches of kits to quantitatively detect the certified samples. Each sample was repeatedly measured 8 times using the same kit, and the SD values and coefficient of variation (CV) of samples with different concentrations were calculated respectively. The test results are shown in Table 10.

[0194] Table 10 Within - batch / between - batch precision

[0195]

[0196] It can be seen that the chemiluminescence immunoassay kit constructed in this example has a sensitivity of 5 ng / mL, and the within - batch / between - batch precision meets the detection requirements.

[0197] (3)Linearity

[0198] Appropriately add IGFBP4 protein (1000 ng / ml) and CCL14 protein (1000 ng / ml) to the certified serum and plasma samples. Dilute the test samples 10 - fold, 100 - fold, and 1000 - fold in a serial dilution manner. The linear range is the ratio of the measured value to the theoretical value of the IGFBP4 and CCL14 contents in the diluted samples. The results are shown in Table 11.

[0199] Table 11 Results of linear analysis

[0200]

[0201] As can be seen from Table 11, the linear detection range for IGFBP4 detection is 95 - 104%, and the linear detection range for CCL14 detection is 97 - 105%.

[0202] Example 3 Application of the IGFBP4 and CCL14 antibody detection kit in the diagnosis of colorectal cancer

[0203] 1. Diagnostic efficacy of using the IGFBP4 and CCL14 chemiluminescence immunoassay kits alone or in combination

[0204] In this example, the chemiluminescent immunoassay kits for IGFBP4 and CCL14 constructed in Example 2 were used. The ability of using one kit alone and combining two kits was investigated respectively. Serum IGFBP4 and CCL14 in healthy people (60 cases) and colorectal cancer patients (55 cases) were tested, and their ability to distinguish diseased populations was evaluated by SPSS 2.0 software respectively.

[0205] When the chemiluminescent immunoassay kit for IGFBP4 was used alone, the antibody combination used in the kit was the first antibody for IGFBP4 (the heavy chain amino acid sequence is SEQ ID NO.33, and the light chain amino acid sequence is SEQ ID NO.34) and the second antibody for IGFBP4 (the heavy chain amino acid sequence is SEQ ID NO.35, and the light chain amino acid sequence is SEQ ID NO.36) to construct the chemiluminescent immunoassay kit. The test results for distinguishing healthy people and colorectal cancer patients are shown in Table 12. The cutoff value of IGFBP4 content for distinguishing whether a patient has colorectal cancer is 757.4 ng / ml. If it is greater than or equal to 757.4 ng / mL, the patient is judged to have colorectal cancer; if it is less than 757.4 ng / mL, the patient is judged to be a healthy person (therefore, the detection accuracy of IGFBP4 for colorectal cancer screening needs to reach 0.1 ng / mL).

[0206] When the chemiluminescent immunoassay kit for CCL14 was used alone, the antibody combination used in the kit was the first antibody for CCL14 (the heavy chain amino acid sequence is SEQ ID NO.37, and the light chain amino acid sequence is SEQ ID NO.38) and the second antibody for CCL14 (the heavy chain amino acid sequence is SEQ ID NO.39, and the light chain amino acid sequence is SEQ ID NO.40) to construct the chemiluminescent immunoassay kit. The test results for distinguishing healthy people and colorectal cancer patients are shown in Table 12. The cutoff value of CCL14 content for distinguishing whether a patient has colorectal cancer is 125.6 ng / mL. If it is greater than or equal to 125.6 ng / mL, the patient is judged to have colorectal cancer; if it is less than 125.6 ng / mL, the patient is judged to be a healthy person (therefore, the detection accuracy of CCL14 for colorectal cancer screening needs to reach 0.1 ng / mL).

[0207] When the IGFBP4 and CCL14 chemiluminescence immunoassay kits are used in combination, a model is constructed through the SPSS 2.0 algorithm software. The constructed Logistic regression formula is: P = exp(-2.903 + 0.006X1 + 0.004X2) / [1 + exp(-2.903 + 0.006X1 + 0.004X2)] (X1 is the actual detected value of IGFBP4 (ng / mL); X2 is the actual detected value of CCL14 (ng / mL); when P > 0.487, it is judged as positive; when P ≤ 0.487, it is judged as negative). Compare its diagnostic performance with that of using the IGFBP4 or CCL14 chemiluminescence immunoassay kit alone. The results are shown in Table 12 and Figure 9 as follows.

[0208] Table 12 Diagnostic performance of the combined model

[0209]

[0210] It can be seen from Table 12 that compared with using the IGFBP4 or CCL14 chemiluminescence immunoassay kit alone, the constructed logistic regression model (IGFBP4 + CCL14) by combining the two indicators of IGFBP4 and CCL14 has better diagnostic performance, and there is obvious synergistic effect, which can better distinguish tumor patients from healthy people.

[0211] Meanwhile, 66 suspected colorectal cancer patients were detected using the IGFBP4-CCL14 combined detection kit of the present invention, and then the P value was calculated according to the above formula. The results showed that among the 66 tested samples, the P value of 52 cases was greater than 0.487, and 45 of them were diagnosed as colorectal cancer patients by histological pathological examination; the P value of 14 cases was less than 0.487, and 4 of them were histologically confirmed as colorectal cancer patients, and the remaining 10 cases were patients with other gastrointestinal diseases. The overall accuracy rate was 68%, the false negative rate was 6%, and the false positive rate was 10.6%. The overall diagnostic effect was good.

[0212] 2. Differences in the diagnostic efficacy of different IGFBP4 and CCL14 antibody combinations

[0213] In this embodiment, the following two antibody combinations are used respectively: The first one: the first antibody of IGFBP4, the second antibody of IGFBP4, combined with the first antibody of CCL14 and the second antibody of CCL14; The second one: the commercially available antibody combination. Chemiluminescent immunoassay kits are constructed respectively, and the combined diagnostic model (IGFBP4 + CCL14) is used for the diagnosis of colorectal cancer. The test samples include the sera of 200 healthy people and 360 colorectal cancer patients. They are randomly divided into a test group and a verification group, among which the test group includes the sera of 100 healthy people and 180 colorectal cancer patients, and the verification group includes the sera of 100 healthy people and 180 colorectal cancer patients.

[0214] The first antibody combination includes: the first antibody of IGFBP4 (the heavy chain amino acid sequence is SEQ ID NO.33, and the light chain amino acid sequence is SEQ ID NO.34) and the second antibody of IGFBP4 (the heavy chain amino acid sequence is SEQ ID NO.35, and the light chain amino acid sequence is SEQ ID NO.36), the first antibody of CCL14 (the heavy chain amino acid sequence is SEQ ID NO.37, and the light chain amino acid sequence is SEQ ID NO.38) and the second antibody of CCL14 (the heavy chain amino acid sequence is SEQ ID NO.39, and the light chain amino acid sequence is SEQ ID NO.40). A model is constructed by the SPSS 2.0 algorithm software, and the constructed Logistic regression formula is: P = exp (-2.872 + 0.008X1 + 0.005X2) / [1 + exp (-2.872 + 0.008X1 + 0.005X2)] (X1 is the actual detected value of IGFBP4 (ng / mL); X2 is the actual detected value of CCL14 (ng / mL); when P > 0.472, it is judged as positive; when P ≤ 0.472, it is judged as negative).

[0215] The second antibody combination includes: a commercially available first antibody against IGFBP4 (purchased from Abcam, catalog number ab205581) and a commercially available second antibody against IGFBP4 (purchased from Sino Biological, catalog number 10967-MM02), a commercially available first antibody against CCL14 (purchased from Abcam, catalog number ab272382) and a commercially available second antibody against CCL14 (purchased from Abcam, catalog number ab272383). A model was constructed using SPSS 2.0 algorithm software, and the constructed Logistic regression formula is: P = exp (-2.893 + 0.005X1 + 0.007X2) / [1 + exp (-2.893 + 0.005X1 + 0.007X2)] (X1 is the actual detected value of IGFBP4 (ng / mL); X2 is the actual detected value of CCL14 (ng / mL); when P > 0.545, it is judged as positive; when P ≤ 0.545, it is judged as negative).

[0216] The test results of the two antibody combinations for distinguishing healthy people and colorectal cancer patients are shown in Table 13.

[0217] Table 13 Diagnostic performance of different IGFBP4 and CCL14 antibody combinations

[0218]

[0219] It can be seen from Table 13 that although the detected markers are both IGFBP4 and CCL14 proteins and are based on the same combined diagnostic model, there are obvious differences in the diagnostic results when using different antibodies for detection. The reason may be that the existing antibodies not only have low detection sensitivity but also have obvious differences in cross-reactivity and stability. For example, the existing IGFBP4 antibody cross-reacts with other members of the IGFBP family or chemokine analogs. Therefore, using the preferred antibody combination after mutation provided by the present invention, including the first antibody and second antibody against IGFBP4 and the first antibody and second antibody against CCL14, to construct a chemiluminescent immunoassay kit can significantly improve the diagnostic efficacy of colorectal cancer.

[0220] Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

Claims

1. An antibody against IGFBP4 protein, characterized in that, Comprising a first antibody and / or a second antibody; the first antibody comprises: (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.1, CDR2 consisting of the amino acid sequence of SEQ ID NO.2, and CDR3 consisting of the amino acid sequence of SEQ ID NO.3 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.4, CDR2 consisting of the amino acid sequence of SEQ ID NO.5, and CDR3 consisting of the amino acid sequence of SEQ ID NO.6 in the light chain variable region; The second antibody comprises: (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.7, CDR2 consisting of the amino acid sequence of SEQ ID NO.8, and CDR3 consisting of the amino acid sequence of SEQ ID NO.9 in the heavy chain variable region, and (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.10, CDR2 consisting of the amino acid sequence of SEQ ID NO.11, and CDR3 consisting of the amino acid sequence of SEQ ID NO.12 in the light chain variable region.

2. The antibody against the IGFBP4 protein according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

16.

3. An antibody against CCL14 protein, characterized in that, Comprising a first antibody and / or a second antibody; the first antibody comprises: (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.17, CDR2 consisting of the amino acid sequence of SEQ ID NO.18, and CDR3 consisting of the amino acid sequence of SEQ ID NO.19 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.20, CDR2 consisting of the amino acid sequence of SEQ ID NO.21, and CDR3 consisting of the amino acid sequence of SEQ ID NO.22 in the light chain variable region; The second antibody comprises: (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.23, CDR2 consisting of the amino acid sequence of SEQ ID NO.24, and CDR3 consisting of the amino acid sequence of SEQ ID NO.25 in the heavy chain variable region, and (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.26, CDR2 consisting of the amino acid sequence of SEQ ID NO.27, and CDR3 consisting of the amino acid sequence of SEQ ID NO.28 in the light chain variable region.

4. The antibody against CCL14 protein according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.30; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

32.

5. A kit for detecting IGFBP4 protein and / or CCL14 protein, characterized in that, An antibody comprising the IGFBP4 protein as claimed in claim 1 or 2, and / or an antibody comprising the CCL14 protein as claimed in claim 3 or 4.

6. Use of an antibody for the preparation of a reagent for predicting whether an individual has colorectal cancer, characterized in that, The antibody comprises the IGFBP4 protein as claimed in claim 1 or 2, and / or the CCL14 protein as claimed in claim 3 or 4.

7. The use according to claim 6, wherein An antibody comprising the IGFBP4 protein and an antibody comprising the CCL14 protein; the antibody comprising the IGFBP4 protein comprises a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.14; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.16; The antibody comprising the CCL14 protein comprises a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.29, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.30; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

32.

8. A kit for predicting whether an individual has colorectal cancer, characterized in that, An antibody comprising the IGFBP4 protein as claimed in claim 1 or 2, and an antibody comprising the CCL14 protein as claimed in claim 3 or 4.

9. An antibody combination for predicting whether an individual has colorectal cancer, characterized in that, An antibody comprising the IGFBP4 protein as claimed in claim 1 or 2, and an antibody comprising the CCL14 protein as claimed in claim 3 or 4.

10. A system for predicting whether an individual has colorectal cancer, characterized in that, The system comprises a data analysis module for analyzing the detection value of an antigen, the antigen being detected by an antibody, the antibody comprising the IGFBP4 protein as claimed in claim 1 or 2, and / or the CCL14 protein as claimed in claim 3 or 4.

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