Application of antibodies and combinations of IGFBP4 and CCL14 proteins in the diagnosis of colorectal cancer

Abstract

The application provides application of an antibody of IGFBP4 and CCL14 protein and a combination thereof in diagnosis of colorectal cancer, and solves the problems of low affinity and specificity of existing antibodies by optimizing eukaryotic expression process of recombinant IGFBP4 and CCL14 antigens, greatly improving expression and immunogenicity, screening high-affinity and high-specificity monoclonal antibodies, and screening optimal IGFBP4 and CCL14 antibodies. The antibody has a wider detection range, and has extremely high synergistic diagnostic value when combined detection is performed, significantly improves the diagnostic efficiency of early colorectal cancer, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the technical field of antibody screening and disease diagnosis, and more specifically, to the application of antibodies and combinations thereof containing IGFBP4 and CCL14 proteins in the diagnosis of colorectal cancer. Background Technology

[0002] IGFBP4 is a member of the insulin-like growth factor binding protein (IGFBP) family, composed 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 to insulin-like growth factors (IGF-1 and IGF-2) with high affinity. The IGF system plays a crucial role in cell proliferation, differentiation, and apoptosis, and IGFBP4 indirectly inhibits IGF-mediated growth-promoting and anti-apoptotic signaling pathways by inhibiting the binding of IGF to its receptor. Furthermore, IGFBP4 activity can also be enhanced by the release of IGF through cleavage of its binding region by proteases (such as pregnancy-associated plasma protein-A, PAPP-A), dynamically regulating IGF signaling. IGFBP4 plays a complex role in tumors. On one hand, it exerts a tumor-suppressive effect by inhibiting IGF signaling. For example, in colorectal cancer, high IGFBP4 expression is associated with longer patient survival, possibly due to the inhibition of IGF-1-driven tumor growth. On the other hand, overexpression of IGFBP4 in some tumors (such as glioblastoma) may promote invasion through non-IGF-dependent pathways (such as integrin signaling).

[0003] CCL14 belongs to the CC motif chemokine family. It exists in an inactive precursor form (CCL14-1-74), which is cleaved by proteases (such as neutrophil elastase) to release the active fragment CCL14-9-74. It recruits immune cells such as monocytes and dendritic cells by binding to CCR1 and CCR3 receptors, regulating inflammatory responses and immune surveillance. CCL14 plays a dual role in the tumor microenvironment. In liver cancer, high CCL14 expression is associated with cytotoxic T cells (CD8+). + Increased T-cell infiltration is associated with prolonged patient survival, suggesting that CCL14 exerts its anti-tumor effect by enhancing anti-tumor immunity. Conversely, in breast cancer, CCL14 may promote tumor cell metastasis by activating the CCR1 / ERK pathway. Furthermore, CCL14 can induce angiogenesis, and its pro- or anti-tumor effects are highly dependent on tumor type and the composition of immune cells in the tumor microenvironment. As a prognostic marker, high expression of CCL14 in liver and kidney cancer indicates a favorable prognosis, while in breast cancer it is associated with a poor prognosis.

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

[0005] Patent CN115466794B discloses a combination of RNA editing sites based on insulin-like growth factor binding protein 7 (IGFBP7), apoptosis-inducing factor (BLCAP), and peptidyl prolyl isomerase A (PPIA) genes for CRC diagnosis, which has high sensitivity but relies on complex RNA editing detection technology, resulting in high costs. Patent CN118006789B discloses a combination of proteins such as NADPH oxidase 1 for CRC diagnosis, but does not address the issue of cross-reactivity of biomarkers in inflammation. Patent CN113398249A mainly focuses on the therapeutic applications of CCL14, such as viral vector delivery, while there are few patents on diagnostic reagents or detection methods directly used for early tumor screening, and there is a lack of specific detection probes or rapid screening tools. Existing products are kits based on the combined detection of CEA and CA19-9, but their sensitivity for early-stage tumors (such as stage I) is insufficient (<50%).

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

[0007] To address the problems existing in the prior art, this invention provides antibodies against IGFBP4 and CCL14 proteins, their combinations, 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 positive control samples in the kit of this invention, they exhibit excellent correlation, consistency, and stability. Dozens of antibodies targeting the IGFBP4 and CCL14 epitopes were obtained through hybridoma technology, and a set of excellent paired antibodies was selected from each. These antibodies have been verified to be applicable to enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay, providing a combination of highly sensitive and specific diagnostic biomarkers for colorectal cancer, thus solving the problem of insufficient accuracy in early tumor screening, subtyping, and efficacy monitoring in the prior art.

[0008] Existing technologies using traditional hybridoma techniques or recombinant protein immunization of animals to prepare monoclonal or polyclonal antibodies against IGFBP4 or CCL14 proteins suffer from insufficient antibody affinity and specificity due to unoptimized immunogen design. Furthermore, current ELISA or chemiluminescence methods for detecting IGFBP4, CCL14 proteins, or gastrointestinal tumor markers do not optimize kits for combinations of IGFBP4 and CCL14 antibodies, resulting in low detection sensitivity and specificity, hindering efficient diagnosis of gastrointestinal tumors (especially colorectal cancer). This invention develops novel, highly specific antibodies against IGFBP4 and CCL14, and their combinations, which can be used in the preparation of diagnostic kits to detect IGFBP4 and CCL14 in different samples, thereby achieving clinical value.

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

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

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

[0012] The second antibody includes:

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

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

[0015] This invention optimizes the eukaryotic expression process of recombinant IGFBP4 antigen, expressing and purifying the recombinant protein in a eukaryotic expression system, significantly increasing the expression level and immunogenicity of IGFBP4. Using the purified recombinant protein as an immunogen, mice or other suitable animals are immunized to obtain immune spleen cells. Hybridoma technology is used to fuse these immune spleen cells with myeloma cells, obtaining hybridoma cell lines that secrete anti-IGFBP4 monoclonal antibodies. Ten high-affinity, high-specificity monoclonal antibodies against IGFBP4 are then screened from these hybridoma cells. The selected monoclonal antibodies are then gene-cloned, expressed, and purified to obtain purified antibody products. The antibodies obtained after three rounds of screening are further screened using a double-antibody sandwich ELISA method to identify superior paired antibodies. The selected IGFBP4 paired antibodies, alone or in combination with other antibodies, have higher diagnostic value for gastrointestinal tumors, such as colorectal cancer.

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

[0017] On the other hand, the present invention provides a kit for detecting IGFBP4 protein, the kit comprising an antibody against IGFBP4 protein as described above.

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

[0019] In some embodiments, the kit is a chemiluminescent immunoassay kit. A dual-antibody chemiluminescent immunoassay kit is constructed by screening for optimal and appropriately paired IGFBP4 primary and secondary antibodies.

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

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

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

[0023] The second antibody includes:

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

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

[0026] This invention optimizes the eukaryotic expression process of recombinant CCL14 antigen, expressing and purifying the recombinant protein in a eukaryotic expression system, significantly increasing the expression level and immunogenicity of CCL14. Using the purified recombinant protein as an immunogen, mice or other suitable animals are immunized to obtain immune spleen cells. Hybridoma technology is used to fuse these immune spleen cells with myeloma cells, obtaining hybridoma cell lines that secrete anti-CCL14 monoclonal antibodies. Ten high-affinity, high-specificity monoclonal antibodies against CCL14 are then screened from these cells. The selected monoclonal antibodies are then gene-cloned, expressed, and purified to obtain purified antibody products. The selected antibodies are further screened using a double-antibody sandwich ELISA method to identify superior paired antibodies. The selected CCL14 paired antibodies, alone or in combination with other antibodies, have higher diagnostic value for gastrointestinal tumors, such as colorectal cancer.

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

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

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

[0030] In some embodiments, the kit is a chemiluminescent immunoassay kit. A dual-antibody chemiluminescent immunoassay kit is constructed by screening for optimal and appropriately paired CCL14 primary and secondary antibodies.

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

[0032] In some approaches, kits for detecting IGFBP4 protein and CCL14 protein are combined to simultaneously detect both proteins. This allows for applications in fields requiring the simultaneous detection of multiple biomarkers, such as gastrointestinal tumor diagnosis, where combined detection can improve diagnostic efficacy.

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

[0034] Traditional single-target antibody drugs have many limitations. In existing cancer diagnostic techniques, the detection of a single biomarker (such as CCL14) suffers from low sensitivity and high false-positive rates. Although IGFBP4 is associated with the tumor microenvironment, its clinical application as a standalone marker is not yet mature. This invention uses IGFBP4 and CCL14 as combined biomarkers, employing combined detection of antibodies targeting IGFBP4 and CCL14 proteins to address the insufficient diagnostic efficacy of single biomarkers and improve the accuracy and specificity of cancer diagnosis.

[0035] While IGFBP4 and CCL14 are known diagnostic markers for colorectal cancer, the diagnostic efficacy varies significantly when using different IGFBP4 and / or CCL14 antibodies. This is because colorectal cancer diagnosis requires testing blood samples with IGFBP4 and / or CCL14 antibodies. Blood samples contain complex interfering substances, significant matrix influence, and are prone to cross-reactivity or interference from nonlinear fragments. Directly using existing antibody detection methods often yields unsatisfactory results, severely impacting diagnostic accuracy.

[0036] Through the design, expression, and screening of antigens and monoclonal antibodies, the antibodies for IGFBP4 and CCL14 proteins obtained in this invention have better detection sensitivity and specificity, more stable signals, and less cross-reactivity, which can significantly improve diagnostic efficacy when used for colorectal cancer diagnosis.

[0037] Furthermore, this includes antibodies against IGFBP4 protein and CCL14 protein.

[0038] Further, the antibody against the 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 in SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.14. The amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.16.

[0039] The antibody against the 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 in SEQ ID NO.29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.30. The amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.32.

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

[0041] In some embodiments, the reagent for predicting the risk of colorectal cancer recurrence 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] This invention relates to biomarkers (IGFBP4 and CCL14 proteins) for predicting the risk of colorectal cancer recurrence, based on blood screening. These two biomarkers show significant differences in the blood of colorectal cancer patients and healthy individuals. By collecting blood samples, the levels of IGFBP4 and CCL14 proteins in an individual's blood can be detected to predict or assist in the diagnosis of that individual's likelihood of developing colorectal cancer. Alternatively, the levels of IGFBP4 and CCL14 proteins in the blood of a group can be detected, thereby classifying that group 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, the kit comprising 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, the antibody combination comprising an antibody against the IGFBP4 protein as described above, and an antibody against the CCL14 protein as described above.

[0046] Furthermore, this includes antibodies against IGFBP4 protein and CCL14 protein.

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

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

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

[0050] Furthermore, the data analysis module uses the detection values ​​of known samples as the training set, and divides colorectal cancer patients into non-recurrence group, in situ recurrence group and distant metastasis group according to their postoperative conditions. It analyzes the relationship between the detection values ​​of the non-recurrence group, in situ recurrence group and distant metastasis group to construct a model.

[0051] Furthermore, the system also 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 this invention are as follows:

[0053] 1. This invention provides an efficient method for recombinant expression of IGFBP4 and CCL14 proteins, which can be used as immunogens in experimental animals and as positive control materials in reagent kits. The IGFBP4 and CCL14 proteins provided by this invention have the characteristics of high yield, good stability, and strong immunogenicity.

[0054] 2. A novel antibody sequence specifically recognizing IGFBP4 is provided, which can be used to construct an IGFBP4 detection kit. Combined with a double antibody sandwich design, the cross-reactivity rate is extremely low (<2.0%), and the sensitivity reaches the ng / mL level, enabling high-precision detection of IGFBP4 protein.

[0055] 3. A novel antibody sequence specifically recognizing CCL14 is provided, which can be used to construct a CCL14 detection kit. Combined with a double antibody sandwich design, the cross-reactivity rate is extremely low (<3.0%), and the sensitivity reaches the ng / mL level, enabling high-precision detection of CCL14 protein.

[0056] 4. Diagnostic value of the kit alone or in combination in gastrointestinal tumors, especially in colorectal cancer; the combined detection model (IGFBP4+CCL14 logistic regression) significantly improves the discrimination ability compared with single markers (AUC≥0.92). Attached Figure Description

[0057] Figure 1 Flowchart of the process for recombinant expression of IGFBP4 and CCL14 proteins.

[0058] Figure 2 The results of SDS-PAGE analysis are for purified recombinant IGFBP4 and CCL14 antigens.

[0059] Figure 3 Flowchart of the preparation process for IGFBP4 monoclonal antibody and CCL14 monoclonal antibody.

[0060] Figure 4 This is a flowchart of animal immunization procedures.

[0061] Figure 5 Results of 4SDS-PAGE assay for IGFBP4 and CCL14 monoclonal antibodies.

[0062] Figure 6 Flowchart of purification process for IGFBP4 monoclonal antibody and CCL14 monoclonal antibody

[0063] Figure 7 The regression curve is for the IGFBP4 detection kit.

[0064] Figure 8 The regression curve is for the CCL14 detection kit.

[0065] Figure 9 ROC curves for different biomarkers in the diagnosis and detection of colorectal cancer. Detailed Implementation

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

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

[0068] I. Recombinant antigen expresses IGFBP4 and CCL14 proteins

[0069] For details of the implementation plan, please refer to... Figure 1 The flowchart of the recombinant protein expression and preparation process is shown below, with the following steps:

[0070] (1) Construction of recombinant antigen expression vectors: The nucleotide sequences of IGFBP4 (UniProt number: P22692) (SEQ ID NO.41) and CCL14 (UniProt number: Q16627) (SEQ ID NO.42) were obtained through bioinformatics analysis. The expression sequences of IGFBP4 and CCL14 (SEQ ID NO.43 and SEQ ID NO.44) were obtained through gene synthesis. The nucleotide sequences of IGFBP4 and CCL14 were cloned into the multiple cloning site of the pTT5 expression vector using molecular cloning methods. After screening for positive clones and verifying their accuracy by Sanger sequencing and double enzyme digestion, the vectors were transformed into host cells Escherichia coli TOP10 and finally cryopreserved. This part of the work was entrusted to Anhui General Biotechnology Co., Ltd.

[0071] (2) Recombinant antigen expression

[0072] 1. Required materials: LB medium (solid / liquid), ampicillin sodium 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 Procedure

[0074] 1) Carrier preparation

[0075] a. The outsourced synthesized expression vector (5ug of vector dry powder) was dissolved in 100ul of purified water and mixed well to obtain a 50ng / uL vector solution.

[0076] b. Transform TOP10 competent cells with 2 μL of vector solution; spread 100 μL of the transformation suspension onto LB agar plates and incubate overnight at 37°C.

[0077] c. After culturing for 16-18 h, pick single clones and transfer them to 5 mL of liquid LB medium. Incubate at 37 °C and 250 rpm for 8 h on a shaker.

[0078] d. After culturing for 8 h, the seed culture was inoculated 1:50 into 200 ml of liquid LB medium and cultured overnight (16-18 h) at 37°C and 250 rpm on a shaker.

[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 were placed in a 5% CO2 constant temperature shaker and cultured at 37°C and 120 rpm.

[0082] b. Use cells with a live cell percentage of ≥97% and a live cell density of 2*106 cells / mL for transfection. Prepare 200mL of cells one day in advance.

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

[0084] d. Gently mix the diluted vector with the transfection reagent and let 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 mix gently.

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

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

[0088] (3) Purification of recombinant antigens: The fermentation broth was purified by nickel affinity chromatography and desalted using G25. After passing quality inspection, the purified samples were aliquoted and frozen. The SDS-PAGE results of the purified IGFBP4 and CCL14 recombinant antigens are shown below. Figure 2 As shown, the amino acid sequences of the purified IGFBP4 and CCL14 recombinant antigens were verified by sequencing as SEQ ID NO.43 and SEQ ID NO.44, respectively.

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

[0090] For detailed implementation plans of monoclonal antibody screening, please refer to [link / reference]. Figure 3 The flowchart for monoclonal antibody preparation shown below illustrates the specific implementation steps:

[0091] (1) Animal immunization

[0092] The recombinant IGFBP4 and CCL14 antigens obtained above were used to immunize five BALB / c white blood cell mice. The mice were divided into primary, secondary, tertiary, and quaternary immunizations, and a booster immunization. Details regarding the injection time, adjuvant, and method of administration for each immunization are provided below. Figure 4 The immune flowchart shown.

[0093] (2) Serum titer detection

[0094] After immunization, blood was collected from the submandibular vein, and antibody titers were detected by indirect ELISA. Mice with an OD450 greater than 0.2 were selected for spleen fusion. The procedure for detecting antibody titers by indirect ELISA is as follows:

[0095] 1. Required materials: coating buffer (pH 9.6 carbonate buffer), blocking buffer (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, stop solution (2M H2SO4), and microplate reader.

[0096] 2. Experimental Procedure:

[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 wells of the ELISA plate, 100 μL / well, and incubate at 37°C for 2 h;

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

[0101] ②Blocking nonspecific 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 three times with PBST;

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

[0105] a) The sample to be tested (orbital blood) was serially diluted starting from a low concentration (1:1000, 1:4000, 1:16000, 1:64000, 1:256000).

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

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

[0108] ④ Add enzyme-labeled secondary antibody:

[0109] a) Dilute the secondary antibody with the recommended dilution 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 (to avoid secondary antibody residue causing increased background).

[0111] ⑤ Color development and termination 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 deepens, and avoid over-development).

[0113] b) Add 50 μL of stop solution (H2SO4) to each well. The solution will turn from blue to yellow. Immediately measure the absorbance at 450 nm (OD450) using a microplate reader.

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

[0115] Table 1. Serum titers of IGFBP4 mice detected by indirect ELISA.

[0116]

[0117] Table 2. Serum titers of CCL14 mice detected by indirect ELISA.

[0118]

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

[0120] (3) Cell fusion

[0121] Blood samples were collected from the eyeballs as a positive control. The mouse spleen was isolated, ground, counted, and fused according to the ratio of spleen cell number to sp2 / 0 cell number = 8:1. 1 mL of PEG1450 was added. The whole process was carried out under the condition of 37℃ water bath. Preheated blank medium was added to terminate the process. After centrifugation, HAT conditioned medium was added for pressure screening.

[0122] (4) Monoclonal antibody screening

[0123] Most of the fused cells were multiclonal cells, which needed to be plated into monoclonal cells. After three rounds of screening (ELISA, IgG), 10 hybridoma cells that efficiently secreted IGFBP4 and CCL14 specific antibodies were obtained.

[0124] After initial screening by indirect ELISA and verification by SPR, 10 hybridoma cell lines secreting IGFBP4 antibody (code: IGFBP4-mAb-10-01~IGFBP4-mAb-10-10) were selected from 55 clones. Their sequences and properties are shown in Table 3. 10 hybridoma cell lines secreting CCL14 antibody (code: CCL14-mAb-15-01~CCL14-mAb-15-10) were selected from 42 clones. Their sequences and properties are shown in Table 4. They are sorted from largest to smallest binding titer (OD450 value).

[0125] Table 3. Hybridoma cells secreting IGFBP4 antibody

[0126]

[0127] Table 4. Hybridoma cells secreting CCL14 antibodies

[0128]

[0129] As shown in Tables 3 and 4, the 10 antibodies obtained through screening for IGFBP4 and CCL14 exhibit excellent affinity and specificity. Among them, the two optimal antibodies for IGFBP4 are IGFBP4-mAb-10-01 and IGFBP4-mAb-10-02, with cross-reactivity to CCL14 both <2.0%; the two optimal antibodies for CCL14 are CCL14-mAb-15-01 and CCL14-mAb-15-02, with cross-reactivity to IGFBP4 both <3.0%.

[0130] (5) Monoclonal antibody purification

[0131] Hybridoma cells were expanded and cultured to ferment and secrete antibodies. The supernatant of the fermentation broth was collected, purified by Protein A affinity chromatography and G25 desalting and medium replacement, and after passing quality inspection, the samples were aliquoted and frozen. For detailed implementation procedures, please refer to [link to implementation plan]. Figure 6 The flowchart shown is a process flow diagram for the purification of monoclonal antibodies.

[0132] III. Ligand Antibody Screening

[0133] The 10 high-affinity IGFBP4 and CCL14 antibodies obtained from the above screening were then tested for antibody pairing to identify superior antibody pairs. The paired antibody screening employed a double-antibody sandwich ELISA assay to verify whether the two antibodies could simultaneously bind to the antigen, forming a "capture antibody-antigen-detection antibody" complex. The specific steps are as follows:

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

[0135] (2) Experimental steps:

[0136] ①Cover-capture antibody:

[0137] a) Dilute the capture antibody (unlabeled antibody) to 1 μg / mL and add it to the wells of the ELISA plate (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℃ for 1 h, and then wash the plate.

[0140] ③ Antigen binding: Add serially diluted antigen (e.g., 0.05 μg / mL, 100 μL / well), incubate at 37℃ for 1 h, and wash the plate.

[0141] ④ Detect antibody binding:

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

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

[0144] ⑤ Color development and reading:

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

[0146] b) Add stop solution (50 μL / well) and immediately measure absorbance (OD value) at 450 nm using a microplate reader.

[0147] The screening results for IGFBP4 paired antibodies are shown in Table 5, and the screening results for 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 Table 5, the optimal paired antibodies obtained from immunizing IGFBP4 were IGFBP4-mAb-10-05 (capture antibody) and IGFBP4-mAb-10-07 (labeled antibody), with a titer of 0.81. As shown in Table 6, the optimal paired antibodies obtained from immunizing CCL14 were CCL14-mAb-15-01 (capture antibody) and CCL14-mAb-15-08 (labeled antibody), with a titer of 0.96. It can be observed that when IGFBP4-mAb-10-07 was used as the capture antibody and IGFBP4-mAb-10-05 as the labeling antibody, the titer of the paired antibodies decreased significantly to 0.34; when CCL14-mAb-15-08 was used as the capture antibody and CCL14-mAb-15-01 as the labeling antibody, the binding titer of the paired antibodies also decreased significantly to 0.37. The reason may be that there is steric hindrance when antibodies recognize different epitopes. When IGFBP4 and CCL14 antigens are fixed by IGFBP4-mAb-10-05 and CCL14-mAb-15-01 capture antibodies, respectively, the other epitope may be exposed more fully, making it easier to detect antibody binding. Conversely, if the positions are exchanged, the epitope may not be exposed enough, resulting in low binding efficiency. Furthermore, different antibodies exhibit varying affinities. Capture antibodies require rapid and robust binding to antigens (high affinity), while detection antibodies may rely more on sustained antigen binding capacity (high binding capacity). As shown in Tables 3 and 4, the binding titer of IGFBP4-mAb-10-05 antibody is higher than that of IGFBP4-mAb-10-07, and the binding titer of CCL14-mAb-15-01 antibody is higher than that of CCL14-mAb-15-08. IGFBP4-mAb-10-05 and CCL14-mAb-15-01 have better affinity and are more suitable as capture antibodies.

[0153] IGFBP4-mAb-10-05 (capture antibody) is named IGFBP4 primary antibody, and IGFBP4-mAb-10-07 (labeled antibody) is named IGFBP4 secondary antibody. CCL14-mAb-15-01 (capture antibody) is named CCL14 primary antibody, and CCL14-mAb-15-08 (labeled antibody) is named CCL14 secondary antibody.

[0154] DNA sequencing and amino acid sequence analysis confirmed the following: The CDR1 sequence of the heavy chain of the IGFBP4 primary antibody is SEQ ID NO.1, the CDR2 sequence is SEQ ID NO.2, and 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 IGFBP4 secondary antibody is SEQ ID NO.7, the CDR2 sequence is SEQ ID NO.8, and 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 CCL14 primary antibody is SEQ ID NO.17, the CDR2 sequence is SEQ ID NO.18, and 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 CCL14 secondary antibody is SEQ ID NO.23, the CDR2 sequence is SEQ ID NO.24, and 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 Construction of recombinant antibody vector: Total RNA was extracted from hybridoma cells and reverse transcribed into cDNA using RT-PCR. The antibody light and heavy chain sequences were cloned and constructed into a T vector. DNA sequencing analysis was then performed to obtain the antibody gene sequence.

[0157] ⑦ Antibody Production and Purification: The antibody gene sequence obtained in step ⑥ was transfected into CHO cells and cultured on a large scale. The antibody was purified using protein A / G affinity chromatography. The purified antibody was then analyzed by SDS-PAGE. Figure 5 As shown, purified antibodies were preserved in phosphate-buffered saline (PBS) using a dialysis method.

[0158] In this embodiment, circular dichroism (CD) chromatography was used to determine the thermostability (Tm) value of the screened antibodies to evaluate their stability and compare them 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 detection results are shown in Tables 7 and 8. Tm represents the midpoint temperature of protein thermal denaturation, i.e., the temperature at which the protein unfolds to 50%, reflecting the trend of protein conformational changes during temperature variations. A higher Tm indicates better stability.

[0159] Table 7 Changes in IGFBP4 antibody stability

[0160]

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

[0162]

[0163] As can be seen from Tables 7 and 8, the IGFBP4 and CCL14 monoclonal antibodies obtained through screening have significantly better stability than commercially available IGFBP4 and CCL14 antibodies.

[0164] Example 2: Construction of an IGFBP4 and CCL14 antibody detection kit

[0165] This embodiment uses the IGFBP4 first antibody and second antibody screened in Example 1, as well as the CCL14 first antibody and second antibody to construct an IGFBP4 and CCL14 joint detection kit.

[0166] The kit includes:

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

[0168] Coating antibodies: Biotin-labeled IGFBP4 primary antibody; Biotin-labeled CCL14 primary antibody; Magnetic beads coated with streptavidin; 100 mM phosphate buffer, pH 7.4; Preservatives.

[0169] Labeled antibodies: acrylamide-labeled IGFBP4 secondary antibody; acrylamide-labeled CCL14 secondary antibody; preservatives; 100 mM phosphate buffer, pH 7.4; preservatives.

[0170] Standards: IGFBP4 standards (5ng / ml, 25ng / ml, 125ng / ml, 250ng / ml, 500ng / ml).

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

[0172] The IGFBP4 and CCL14 combined detection kit constructed in this embodiment includes a detection kit for detecting IGFBP4 and a detection kit for detecting CCL14. It can detect the concentrations of IGFBP4 and CCL14 in the sample to be tested, thereby obtaining the detection values ​​of IGFBP4 and CCL14 respectively. In other words, it can be used to detect IGFBP4, CCL14, or both simultaneously.

[0173] The method for detecting the concentration of the combined IGFBP4 and / or CCL14 markers in the sample using the combined detection kit provided in this embodiment is as follows:

[0174] The concentration of IGFBP4 in the sample to be tested was detected.

[0175] 1) Coating: Dilute the IGFBP4 primary antibody to 0.1-10ug / ml with carbonate CBS coating buffer (according to the corresponding test specifications), add 50ul to each well of a 96-well plate, and incubate overnight at 4°C.

[0176] 2) Sealing: The next day, spin dry the 96-well plate, add 200ul of sealing solution to each well, seal at room temperature (22-25℃, the same below) for 1 hour, then wash the plate 3 times with a plate washer, spin dry and use immediately, or store at -20℃ for later use.

[0177] 3) Add test samples: Set up control wells, 8 standard wells (for creating a standard curve), and test sample wells. Add blank diluent to the control wells. Add gradient concentrations of IGFBP4 working solution (diluted with diluent 8, with concentrations of 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, and 1000 ng / L) to the 8 standard wells. Add the test sample to the test sample wells, with a volume of 50 μL for each well. Seal the microplate with a blocking membrane and incubate at 37°C for 30 minutes. Set up replicates. Incubate at room temperature with shaking at 220 rpm for 1 hour. Wash the plate 3 times with a plate washer and pat dry.

[0178] 4) Add enzyme-labeled secondary antibody: Dilute the biotinylated IGFBP4 secondary antibody to the working concentration using dilution buffer. Add 50 μL to each well, and react at room temperature with shaking at 220 rpm for 1 hour. Wash the plate 3 times with a plate washer and pat dry.

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

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

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

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

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

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

[0185] III. Performance Evaluation of Chemiluminescence Reagent Kit

[0186] This embodiment also evaluates the performance of the IGFBP4 and CCL14 combined detection kit, mainly in the following aspects:

[0187] (1) Recovery rate

[0188] IGFBP4 protein at a final concentration of 500 ng / ml and CCL14 protein at a final concentration of 100 ng / ml were added to serum (mass spectrometry-based IGFBP4 concentration of 543 ng / ml and CCL14 concentration of 92 ng / ml) and plasma samples, respectively. The measurements were repeated and the mean values ​​were calculated. The recovery rate was examined (see Table 9). The recovery rate was the ratio of the measured value to the theoretical value.

[0189] Table 9 Recovery rate determination

[0190]

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

[0192] (2) Precision

[0193] Intra-assay and inter-assay precision were assessed using IGFBP4 protein control solutions and CCL14 protein control solutions (prepared from negative human serum) containing 5, 10, 50, and 250 ng / mL, respectively. Intra-assay precision: Quantitative analysis was performed on samples from the same batch of kits, with each sample measured 20 times consecutively. The SD and coefficient of variation (CV) for different concentrations were calculated. Inter-assay precision: Quantitative analysis was performed on samples from three different batches of kits, with each sample measured 8 times using the same kit. The SD and CV for different concentrations were calculated. The results are shown in Table 10.

[0194] Table 10 Intra-batch / Inter-batch Precision

[0195]

[0196] As can be seen, the chemiluminescent immunoassay kit constructed in this embodiment has a sensitivity of 5 ng / mL, and the intra-batch / inter-batch precision meets the detection requirements.

[0197] (3) Linear

[0198] Appropriate amounts of IGFBP4 protein (1000 ng / ml) and CCL14 protein (1000 ng / ml) were added to the fixed-value serum and plasma samples. The samples to be tested were serially diluted 10 times, 100 times, and 1000 times. The linear range is the ratio of the measured value of IGFBP4 and CCL14 content in the diluted sample to the theoretical value. The results are shown in Table 11.

[0199] Table 11 Linear Analysis Results

[0200]

[0201] As shown in Table 11, the linear detection range of IGFBP4 is 95-104%, and the linear detection range of CCL14 is 97-105%.

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

[0203] 1. Diagnostic efficacy of IGFBP4 and CCL14 chemiluminescent immunoassay kits, used alone or in combination.

[0204] This embodiment uses the IGFBP4 and CCL14 chemiluminescent immunoassay kit constructed in Example 2 to investigate the use of one kit alone and the combination of the two kits. The kits were used to test the levels of IGFBP4 and CCL14 in the serum of healthy individuals (60 cases) and colorectal cancer patients (55 cases). The ability of the kits to distinguish between diseased individuals was evaluated using SPSS 2.0 software.

[0205] When using the IGFBP4 chemiluminescent immunoassay kit alone, the kit uses an antibody combination of IGFBP4 primary antibody (heavy chain amino acid sequence SEQ ID NO. 33, light chain amino acid sequence SEQ ID NO. 34) and IGFBP4 secondary antibody (heavy chain amino acid sequence SEQ ID NO. 35, light chain amino acid sequence SEQ ID NO. 36) to construct the chemiluminescent immunoassay kit for distinguishing between healthy individuals and colorectal cancer patients. The detection results are shown in Table 12. The cutoff value for IGFBP4 content used to distinguish between colorectal cancer patients is 757.4 ng / mL. A level greater than or equal to 757.4 ng / mL is considered indicative of colorectal cancer patients, while a level less than 757.4 ng / mL is considered indicative of healthy individuals (therefore, the IGFBP4 detection accuracy for colorectal cancer screening needs to reach 0.1 ng / mL).

[0206] When using the CCL14 chemiluminescent immunoassay kit alone, the kit uses a combination of CCL14 primary antibody (heavy chain amino acid sequence SEQ ID NO. 37, light chain amino acid sequence SEQ ID NO. 38) and CCL14 secondary antibody (heavy chain amino acid sequence SEQ ID NO. 39, light chain amino acid sequence SEQ ID NO. 40) to construct the chemiluminescent immunoassay kit. The results for distinguishing between healthy individuals and colorectal cancer patients are shown in Table 12. The CCL14 cutoff value for distinguishing between colorectal cancer patients is 125.6 ng / mL. A level greater than or equal to 125.6 ng / mL is considered indicative of colorectal cancer, while a level less than 125.6 ng / mL is considered indicative of healthy individuals (therefore, the accuracy of CCL14 detection for colorectal cancer screening needs to reach 0.1 ng / mL).

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

[0208] Table 12 Diagnostic performance of the combined model

[0209]

[0210] As shown in Table 12, compared with using IGFBP4 or CCL14 chemiluminescent immunoassay kits alone, the logistic regression model constructed by combining IGFBP4 and CCL14 has better diagnostic performance and significant synergistic effect, and can better distinguish between cancer patients and healthy people.

[0211] Simultaneously, the IGFBP4-CCL14 combined detection kit of this invention was used to detect 66 suspected colorectal cancer patients, and the P-value was calculated according to the above formula. The results showed that among the 66 samples tested, 52 had P-values ​​greater than 0.487, of which 45 were histologically confirmed as colorectal cancer patients; 14 had P-values ​​less than 0.487, of which 4 were histologically confirmed as colorectal cancer patients, and the remaining 10 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%, indicating a good overall diagnostic effect.

[0212] 2. Differences in diagnostic efficacy among different combinations of IGFBP4 and CCL14 antibodies

[0213] This embodiment employs two antibody combinations: First, a combination of IGFBP4 primary and secondary antibodies with CCL14 primary and secondary antibodies; second, a combination of commercially available antibodies. Chemiluminescent immunoassay kits were constructed, and a combined diagnostic model (IGFBP4+CCL14) was used for the diagnosis of colorectal cancer. Test samples included serum from 200 healthy individuals and 360 colorectal cancer patients. Participants were randomly divided into a test group and a validation group. The test group consisted of serum from 100 healthy individuals and 180 colorectal cancer patients, while the validation group also consisted of serum from 100 healthy individuals and 180 colorectal cancer patients.

[0214] The first antibody combination includes: IGFBP4 primary antibody (heavy chain amino acid sequence SEQ ID NO.33, light chain amino acid sequence SEQ ID NO.34) and IGFBP4 secondary antibody (heavy chain amino acid sequence SEQ ID NO.35, light chain amino acid sequence SEQ ID NO.36), CCL14 primary antibody (heavy chain amino acid sequence SEQ ID NO.37, light chain amino acid sequence SEQ ID NO.38) and CCL14 secondary antibody (heavy chain amino acid sequence SEQ ID NO.39, light chain amino acid sequence SEQ ID NO.40). The model was constructed using 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 detection value of IGFBP4 (ng / mL); X2 is the actual detection 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 primary antibody for IGFBP4 (purchased from Abcam, catalog number ab205581) and a commercially available secondary antibody for IGFBP4 (purchased from Sino Biological, catalog number 10967-MM02), a commercially available primary antibody for CCL14 (purchased from Abcam, catalog number ab272382) and a commercially available secondary antibody for CCL14 (purchased from Abcam, catalog number ab272383). The 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 detection value of IGFBP4 (ng / mL); X2 is the actual detection 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 results of the two antibody combinations used to distinguish between healthy individuals and colorectal cancer patients are shown in Table 13.

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

[0218]

[0219] As shown in Table 13, although both IGFBP4 and CCL14 proteins are detected using the same combined diagnostic model, the diagnostic results vary significantly depending on the antibodies used. This may be because existing antibodies not only have low detection sensitivity but also exhibit significant differences in cross-reactivity and stability. For example, existing IGFBP4 antibodies cross-react with other members of the IGFBP family or chemokine analogs. Therefore, the chemiluminescent immunoassay kit constructed using the preferred antibody combination after mutation provided in this invention, including IGFBP4 primary and secondary antibodies and CCL14 primary and secondary antibodies, can significantly improve the diagnostic efficacy for colorectal cancer.

[0220] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

Claims

1. A kit for detecting IGFBP4 protein and CCL14 protein, characterized by, An antibody to an IGFBP4 protein and an antibody to a CCL14 protein, the antibody to the IGFBP4 protein comprising a first antibody and a second antibody; the first antibody comprising: (1) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 1, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 2, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 3 in a heavy chain variable region, and (2) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 4, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 5, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 6 in a light chain variable region; the second antibody comprising: (3) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 7, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 8, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 9 in a heavy chain variable region, and (4) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 10, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 11, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 12 in a light chain variable region; the antibody to the CCL14 protein comprising a first antibody and a second antibody; the first antibody comprising: (1) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 17, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 18, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 19 in a heavy chain variable region, and (2) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 20, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 21, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 22 in a light chain variable region; the second antibody comprising: (3) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 23, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 24, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 25 in a heavy chain variable region, and (4) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 26, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 27, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 28 in a light chain variable region.

2. The kit of claim 1, wherein In the antibody of the IGFBP4 protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16; in the antibody of the CCL14 protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 30; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

32.

3. Use of an antibody for the manufacture of a reagent for predicting whether an individual has colorectal cancer, characterized in that, The antibody includes the antibody of the IGFBP4 protein and the antibody of the CCL14 protein, and the antibody of the IGFBP4 protein includes a first antibody and a second antibody; the first antibody includes: (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 includes: (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; The antibody of the CCL14 protein includes a first antibody and a second antibody; the first antibody includes: (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 includes: (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. Use according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16; the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 30; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

32.

5. A kit for predicting whether an individual has colorectal cancer, characterized in that, The antibody of the IGFBP4 protein includes a first antibody and a second antibody; the first antibody includes: (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 includes: (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; The antibody of the CCL14 protein includes a first antibody and a second antibody; the first antibody includes: (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 includes: (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.

6. The kit of claim 5, wherein In the antibody of the IGFBP4 protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16; in the antibody of the CCL14 protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 29, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 30; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

32.

7. An antibody combination for predicting whether an individual has colorectal cancer, characterized in that, The antibody comprising the IGFBP4 protein includes a first antibody and a second antibody; the first antibody includes: (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 includes: (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. 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) 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; The antibody of the CCL14 protein includes a first antibody and a second antibody; the first antibody includes: (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 includes: (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.

8. The antibody combination as claimed in claim 7, wherein, The amino acid sequence of the heavy chain variable region of the first antibody of the antibody of the IGFBP4 protein 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 amino acid sequence of the heavy chain variable region of the first antibody of the antibody of the CCL14 protein 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.

9. A system for predicting whether an individual has colorectal cancer, characterized in that, The system includes a data analysis module for analyzing the detection value of an antigen detected by an antibody, the antibody including an antibody of an IGFBP4 protein and an antibody of a CCL14 protein, the antibody of the IGFBP4 protein including a first antibody and a second antibody; the first antibody includes: (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; The antibody of the CCL14 protein comprises a first antibody and 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.

10. The system of claim 9, wherein, In the antibody of the IGFBP4 protein, 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; in the antibody of the CCL14 protein, 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.

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