Application of TFF1 and AAT monoclonal antibodies and combination thereof in colorectal cancer diagnosis

Abstract

The invention provides application of TFF1 and AAT monoclonal antibodies and a combination thereof in colorectal cancer diagnosis, by optimizing immunogen sequences of TFF1 and AAT, screening the monoclonal antibodies with high affinity and high specificity, screening out the optimal TFF1 and AAT antibodies and combining the optimal TFF1 and AAT antibodies, the problems of low affinity and specificity and poor stability of the existing antibodies can be effectively solved, and the colorectal cancer diagnosis efficiency is improved. The kit has a wide detection range, shows an extremely high collaborative 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 belongs to the fields of antibody screening and disease diagnosis. Specifically, it relates to the application of a monoclonal antibody against TFF1 and AAT and their combination in the diagnosis of colorectal cancer. Background Art

[0002] Rectal cancer is the third most common malignant tumor globally, accounting for approximately 30% of colorectal cancer cases. In recent years, with the westernization of the diet structure (such as high-fat and low-fiber diet), lack of exercise, and the increasing trend of aging, the incidence of rectal cancer has been rising year by year. The current diagnostic methods mainly include: colonoscopy, fecal occult blood test (FOBT), blood biomarker detection, imaging examination, etc. Among them, colonoscopy is the gold standard for diagnosing rectal cancer, but it has the disadvantages of strong invasiveness and high cost. The fecal occult blood test has low specificity and is easily interfered by diseases such as hemorrhoids or digestive tract ulcers, and it is extremely prone to false positive or false negative results. In addition, the main deficiency of imaging examination is its limited ability to detect early micro-lesions and high cost. This patent uses the blood biomarker detection method, which has the advantages of non-invasiveness, low cost, and easy repeated detection. In addition, aiming at the problem of limited sensitivity and specificity of this detection method, this patent uses a more efficient and reliable method of combined detection of two biomarkers, which greatly improves the accuracy of early screening or diagnosis of rectal cancer.

[0003] The breast cancer-associated peptide (PS2, i.e., TFF1) belongs to the trefoil family members. The characteristics of this family are that it has at least one copy of the trefoil motif, a 40-amino acid domain containing three conserved disulfide bonds. They are stable secreted proteins expressed in the gastrointestinal mucosa. This gene is expressed in the goblet cells of the intestine and colon. The trefoil factor family is a class of secreted small peptides with a stable trefoil structure, including three members: TFF1, TFF2, and TFF3. Among these three patterns, the concentration of TFF1 compounds is the highest. In addition, TFF1 is closely related to the occurrence and development of various tumors. In patients with colorectal cancer, the content of TFF1 increases significantly, and it can be used as an early diagnostic biomarker for colorectal cancer.

[0004] Human alpha-1 antitrypsin (AAT), also known as serpin A1, also called alpha1-proteinase inhibitor (alpha1-Pi) and serine protease inhibitor 1 (serine protease inhibitor, group A, member 1), is a circulating glycoprotein whose main function is to inhibit neutrophil elastase and other serine proteases in blood and tissues. AAT deficiency is a genetic disease that usually predisposes to premature onset of chronic obstructive pulmonary disease (COPD), cirrhosis, recurrent panniculitis, systemic vasculitis, and a possible range of inflammatory and neoplastic diseases. In addition, some clinical studies have shown that patients with AAT deficiency have an increased risk of developing malignancies, including hepatocellular carcinoma, lung cancer, bladder tumors and gallbladder tumors, malignant lymphoma, and colon cancer. Therefore, it can be used as an early diagnostic biomarker for colorectal cancer.

[0005] Although both TFF1 and AAT can be used as early diagnostic biomarkers for colorectal cancer, the following gaps exist in the prior art: 1) Lack of standardized detection tools: The detection results of different kits vary greatly, making it difficult to accurately detect; 2) No exploration of the combined diagnostic value: There is no research data indicating that the combination of TFF1 and AAT can improve the diagnostic efficacy (no literature reports on indicators such as AUC value).

[0006] Therefore, there is an urgent need to find TFF1 antibodies and AAT antibodies with high specificity and high sensitivity for the early and accurate diagnosis of colorectal cancer respectively. Summary of the Invention

[0007] The present invention provides the use of TFF1 and AAT monoclonal antibodies and their combinations in the diagnosis of colorectal cancer. By optimizing the immunogen sequences of TFF1 and AAT, screening monoclonal antibodies with high affinity and high specificity, screening out the optimal TFF1 and AAT antibodies and combining them, it can effectively solve the problems of low affinity, low specificity, and poor stability of existing antibodies, has a wider detection range, shows extremely high combined diagnostic value during combined detection, significantly improves the diagnostic efficacy of early colorectal cancer, and has important clinical application value.

[0008] In the prior art solutions, when using traditional hybridoma technology, phage display technology, or recombinant protein immunization of animals to prepare monoclonal or polyclonal antibodies against TFF1 protein or AAT protein, there are problems of insufficient antibody affinity and specificity due to unoptimized immunogen design. Moreover, when using existing ELISA or chemiluminescence methods to detect TFF1 and AAT protein tumor markers, the kit is not optimized for the combination of TFF1 and AAT antibodies, resulting in low detection sensitivity and specificity and unable to achieve efficient diagnosis of colorectal cancer. The present invention has developed brand-new highly specific antibodies against TFF1 and AAT and their combinations, which can be used for the preparation of diagnostic kits to detect TFF1 and AAT in different samples to achieve clinical value.

[0009] On the one hand, the present invention provides a TFF1 monoclonal antibody, 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 relates to the technical field of antibody preparation, in particular to a specific targeting antibody of TFF1, its preparation method and application. By bioinformatics retrieval of the TFF1 nucleotide sequence, two antigens, TFF1-H01 and TFF1-H02, are designed for antigen plasmid synthesis. According to the obtained plasmids, TOP10 is transformed to pick positive clones and preserved. After activation and large-scale culture, the plasmids are extracted for eukaryotic cell transfection and expression, and the corresponding antigens are obtained by affinity purification. The TFF1-H01 antigen is used for immunization injection and hybridoma cell fusion, and TFF1-H02 is used for screening to obtain multiple pairs of monoclonal antibody strains. Through antigen detection and antibody pairing, finally, high-quality antibody pairs are selected for the development of a rapid detection kit for colorectal cancer.

[0016] The amino acid sequence of the TFF1-H01 antigen is as shown in SEQ ID NO.50, and the nucleotide sequence is as shown in SEQ ID NO.54; the amino acid sequence of the TFF1-H02 antigen is as shown in SEQ ID NO.51, and the nucleotide sequence is as shown in SEQ ID NO.55.

[0017] Further, the amino acid sequence of the heavy chain variable region of the first antibody of the TFF1 monoclonal 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.

[0018] The amino acid sequence of the heavy chain of the first antibody of the TFF1 monoclonal antibody is as shown in SEQ ID NO. 40, and the amino acid sequence of the light chain is as shown in SEQ ID NO. 41; the amino acid sequence of the heavy chain of the second antibody of the TFF1 monoclonal antibody is as shown in SEQ ID NO. 42, and the amino acid sequence of the light chain is as shown in SEQ ID NO. 43.

[0019] Further, the present invention provides a detection kit for TFF1, and the kit includes the antibody of the TFF1 protein described above.

[0020] It can be understood that the kit can be an ELISA detection kit, a chemiluminescent immunoassay detection kit, etc.

[0021] In some embodiments, the kit is an ELISA detection kit. By screening the optimal and suitable paired first and second antibodies of TFF1, a double-antibody combination ELISA detection kit is constructed.

[0022] On the other hand, the present invention provides an AAT monoclonal antibody, including a first antibody and / or a second antibody; the first antibody includes:

[0023] (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

[0024] (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:

[0025] (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 FDY in the heavy chain variable region, and

[0026] (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.25, CDR2 consisting of the amino acid sequence of SEQ ID NO.26, and CDR3 consisting of the amino acid sequence of SEQ ID NO.27 in the light chain variable region.

[0027] The present invention also relates to a specific targeting antibody against AAT, its preparation method and application. By bioinformatics retrieval of the nucleotide sequence of the AAT protein and designing 2 antigens: AAT-H01 and AAT-H02, antigen plasmids are synthesized. According to the obtained plasmids, TOP10 is transformed to pick positive clones, preserved, activated and amplified for culturing to extract plasmids for eukaryotic cell transfection and expression, and the corresponding antigens are obtained by affinity purification. The AAT-H01 antigen is used for immunization injection and hybridoma cell fusion, and AAT-H02 is used for screening to obtain multiple pairs of monoclonal antibody strains. Through antigen detection and antibody pairing, finally, high-quality antibody pairs are selected for the development of a rapid detection kit for colorectal cancer.

[0028] The amino acid sequence of the AAT-H01 antigen is as shown in SEQ ID NO.52, and the nucleotide sequence is as shown in SEQ ID NO.56; the amino acid sequence of the AAT-H02 antigen is as shown in SEQ ID NO.53, and the nucleotide sequence is as shown in SEQ ID NO.57.

[0029] Further, the amino acid sequence of the heavy chain variable region of the first antibody of the AAT monoclonal antibody is as shown in SEQ ID NO.28, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region of the second antibody of the AAT monoclonal antibody is as shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.31.

[0030] The heavy chain amino acid sequence of the first antibody of the AAT monoclonal antibody is as shown in SEQ ID NO.44, and the light chain amino acid sequence is as shown in SEQ ID NO.45; the heavy chain amino acid sequence of the second antibody of the AAT monoclonal antibody is as shown in SEQ ID NO.46, and the light chain amino acid sequence is as shown in SEQ ID NO.47.

[0031] On the other hand, the present invention provides a kit for detecting AAT protein, and the kit includes the antibody against AAT protein as described above.

[0032] It can be understood that the kit can be an ELISA detection kit, a chemiluminescence immunoassay detection kit, etc.

[0033] In some ways, the kit is an ELISA detection kit. By screening the optimal and suitable paired first and second antibodies against AAT, a double-antibody combination ELISA detection kit is constructed.

[0034] In another aspect, the present invention provides a kit for detecting TFF1 protein and / or AAT protein, comprising the above-mentioned TFF1 monoclonal antibody, and / or the above-mentioned AAT monoclonal antibody.

[0035] In some ways, combining the kit for detecting TFF1 protein and the kit for detecting AAT protein can be used to simultaneously detect TFF1 protein and AAT protein, and thus can be applied to the field of simultaneous detection of multiple markers, improving the efficiency of early diagnosis of colorectal cancer through combined detection.

[0036] In another aspect, the present invention provides the use of an antibody for preparing a reagent for predicting whether an individual has colorectal cancer, wherein the antibody comprises the above-mentioned TFF1 monoclonal antibody, and / or the above-mentioned AAT monoclonal antibody.

[0037] Traditional single-target antibody drugs have many limitations themselves. In existing cancer diagnosis technologies, the detection of a single biomarker (such as TFF1) has problems of low sensitivity and high false positive rate. Although TFF1 is related to the tumor microenvironment, its clinical application in single detection has not yet matured. The present invention uses TFF1 and AAT as combined markers, and solves the problem of insufficient diagnostic efficiency of a single marker through the combined detection of antibodies targeting TFF1 and AAT proteins, improving the accuracy and specificity of cancer diagnosis.

[0038] Although TFF1 and AAT are known colorectal cancer diagnostic markers, there are significant differences in their diagnostic efficiency when using different TFF1 antibodies and / or AAT antibodies to diagnose colorectal cancer. Because during the colorectal cancer diagnosis process, it is necessary to detect the blood sample through TFF1 antibody and / or AAT antibody. There are complex interfering substances in the blood sample, the matrix has a great influence, and cross-reactions or interference from some non-linear fragments are likely to occur. It is difficult to obtain ideal detection results by directly using existing antibodies, seriously affecting the accuracy of the diagnostic results.

[0039] Through the design, expression of antigens, and screening of monoclonal antibodies, the antibodies against TFF1 and AAT proteins finally obtained by the present invention have better detection sensitivity and specificity, and the signals are more stable and the cross-reactions are smaller. When used for the diagnosis of colorectal cancer, the diagnostic efficiency can be significantly improved.

[0040] Further, it includes a TFF1 monoclonal antibody and an AAT monoclonal antibody; the TFF1 monoclonal antibody 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; the AAT monoclonal antibody 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.28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.31.

[0041] Further, the reagent is used to detect the content of an antigen that can bind to an antibody in a body fluid sample; the body fluid sample includes any one or more of saliva, blood, urine, plasma, serum, and cerebrospinal fluid.

[0042] In some embodiments, the reagent for predicting colorectal cancer is a detection reagent prepared by using TFF1 and AAT proteins as detection targets, and the present invention uses antibodies to detect TFF1 and AAT proteins.

[0043] Further, the reagent is used to detect the presence, relative abundance or concentration of a biomarker in a body fluid sample.

[0044] The present invention has screened biomarkers (TFF1 and AAT proteins) for predicting the risk of colorectal cancer from blood. There are significant differences in the blood of colorectal cancer patients and healthy people for these two biomarkers. By collecting a blood sample, the content of TFF1 and AAT proteins in the individual's blood can be detected, so as to predict or assist in diagnosing the possibility of the individual suffering from colorectal cancer, or the content of TFF1 and AAT proteins in the blood of a certain group can be detected, and then this group can be divided into colorectal cancer patients and healthy people.

[0045] On the other hand, the present invention also provides a kit for predicting whether an individual has colorectal cancer, including the above-mentioned TFF1 monoclonal antibody and the above-mentioned AAT monoclonal antibody.

[0046] On the other hand, the present invention also provides an antibody combination for predicting whether an individual has colorectal cancer, including the above-mentioned TFF1 monoclonal antibody and the above-mentioned AAT monoclonal antibody.

[0047] In another aspect, the present invention also provides a system for predicting whether an individual has colorectal cancer. The system includes a data analysis module for analyzing the detection values of antigens, which are detected by antibodies, and the antibodies include the above-mentioned TFF1 monoclonal antibody and / or the above-mentioned AAT monoclonal antibody.

[0048] Further, the data analysis module uses the detection values of markers of known samples as a training set, and divides them into healthy people and colorectal cancer patients according to the situation after surgery of colorectal cancer patients, analyzes the relationship between the detection values of healthy people and colorectal cancer patients, and constructs a model.

[0049] Further, 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.

[0050] The present invention has the following beneficial effects:

[0051] 1. Design and prepare self-made TFF1 and AAT expression antigens, and immunize animals with the antigens to obtain specific targeted monoclonal antibodies, which have extremely high sensitivity;

[0052] 2. Recombine the prepared antibodies to improve the expression level and ensure the stable expression of the antibodies; introduce the detection of clinical blood samples in the antibody testing process, greatly increasing the reliability of experimental data; double-antibody detection, reducing the reagent dosage by 50%;

[0053] 3. Screen multiple pairs of monoclonal antibodies against the epitopes of the TFF1 antigen, construct a TFF1 detection kit, design it in combination with the double-antibody sandwich method, with an extremely low cross-reaction rate (<1%), a sensitivity reaching the ng / mL level, and capable of detecting the TFF1 protein with high precision;

[0054] 4. Screen multiple pairs of monoclonal antibodies against the epitopes of the AAT antigen, construct an AAT detection kit, design it in combination with the double-antibody sandwich method, with an extremely low cross-reaction rate (<1%), a sensitivity reaching the ng / mL level, and capable of detecting the AAT protein with high precision;

[0055] 5. Optimize the antibody combination and detection process to achieve efficient and economical clinical applications;

[0056] 6. The diagnostic value of the kit used alone or in combination in digestive tract tumors, especially in intestinal cancer; the combined detection model (TFF1 + AAT) is used for the first time, which significantly improves the discrimination ability compared with single markers (AUC≥0.91). Description of the Drawings

[0057] Figure 1SDS-PAGE test results after purification of TFF1-H01 antigen in Example 1;

[0058] Figure 2 SDS-PAGE test results after purification of TFF1-2 and TFF1-3 monoclonal antibodies in Example 1;

[0059] Figure 3 Fitted 4-parameter Logistic curve of the kit composed of TFF1-2 as the coated antibody and TFF1-3 as the enzyme-labeled antibody in Example 1;

[0060] Figure 4 SDS-PAGE test results after purification of AAT-H01 antigen in Example 2;

[0061] Figure 5 SDS-PAGE test results after purification of AAT-2 and AAT-6 monoclonal antibodies in Example 2;

[0062] Figure 6 Fitted 4-parameter Logistic curve of the kit composed of AAT-2 as the coated antibody and AAT-6 as the enzyme-labeled antibody in Example 2;

[0063] Figure 7 Receiver operating characteristic curve (ROC) and AUC values of ELISA kit 1 for TFF1 and ELISA kit 1 for AAT in Example 3 for combined detection and separate individual detections. Detailed implementation mode

[0064] The present invention will be further described in detail below in conjunction with the drawings and examples. It should be noted that the following examples are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0065] Example 1. Preparation, screening of TFF1 monoclonal antibody and preparation of its kit

[0066] 1. Preparation and screening of TFF1 monoclonal antibody

[0067] 1) Preparation of antigen

[0068] Retrieve the TFF1 sequence through bioinformatics and design two antigens: TFF1-H01 (amino acid sequence shown in SEQ ID NO.50, nucleotide sequence shown in SEQ ID NO.54) and TFF1-H02 (amino acid sequence shown in SEQ ID NO.51, nucleotide sequence shown in SEQ ID NO.55). To increase the immunogenicity of the antigens, during antigen design, purification, and immunization: 1. Add Kozak sequence, which can mainly enhance translation efficiency, ensure accuracy, optimize foreign gene expression, and regulate expression levels; 2. Immunize with dimers to make the immunogenicity stronger; 3. Monomer screening to ensure that the obtained antibodies are specific to a single epitope; 4. Add 6×His tag for easy purification to obtain a higher amount of antigen and significantly improve the purity of the antigen; 5. Boost immunization during the immunization process and directly inject into the spleen to obtain a higher antibody titer. Entrust GenScript to synthesize the protein plasmid, and transfect eukaryotic cells according to the obtained plasmid for expression and purification to obtain the corresponding antigen. The SDS-PAGE detection result of the purified TFF1-H01 antigen is as shown in Figure 1 shown. The TFF1-H01 antigen is used for immunization injection and hybridoma cell fusion, and the TFF1-H02 antigen is used for screening.

[0069] 2) Animal immunization

[0070] Immunize 5 BALB / c mice with the TFF1-H01 antigen, and the immunization process is as shown in Table 1 below.

[0071] Table 1. Immunization process of TFF1-H01 antigen

[0072]

[0073] 3) Preparation of hybridoma cells

[0074] After the immunization is completed, collect blood from the submandibular vein of the mice, and detect the antibody titer by indirect ELISA. The antigen coated is TFF1-H02, and the method for detecting the antibody titer by indirect ELISA is as follows:

[0075] a. Dilute the antigen TFF1-H02 to 2 μg / mL with carbonate buffer (pH 9.6), add 100 μL to each well of a 96-well ELISA plate; coat overnight at 4°C (12 - 16 hours);

[0076] b. Discard the liquid, pat dry, add 300 μL of washing buffer (PBS + 0.1% Tween20) to each well, and wash 3 times;

[0077] c. Add 200 μL of blocking solution (PBS containing 5% skim milk powder) to each well, and block at 37°C for 1 hour;

[0078] d. Discard the blocking solution, pat dry, and wash 3 times.

[0079] e. Add 100 μL serum to each well (i.e. primary antibody, serum diluted 1000, 4000, 16000, 64000, 256000 times respectively); incubate at 37°C for 1 hour, and wash 3 times;

[0080] f. Dilute the enzyme-labeled antibody (i.e., the secondary antibody, which is HRP-labeled goat anti-mouse IgG) to 1 μg / mL with PBS, and add 100 μL to each well; incubate at 37°C in the dark for 45 minutes, and wash 5 times;

[0081] g. Color development and termination reaction: Add 100 μL TMB substrate to each well and develop color at room temperature in the dark for 10 minutes; immediately add 50 μL stop solution (2M H2SO4) to each well;

[0082] h. Absorbance detection and data analysis: Use an ELISA reader to read the absorbance (OD value) of each well at 450 nm. Each well is detected three times in parallel and the average value is calculated.

[0083] The results of serum titer testing are shown in Table 2 below.

[0084] Table 2. Results of indirect ELISA test of antibody titers in serum of mice immunized with TFF1-H01 antigen

[0085]

[0086] According to the test results in Table 2, blood samples were collected from the eyeballs of mice with serum diluted 64,000 times and OD450 greater than 0.2 as positive controls, and the spleens of mice were separated. The spleens were ground and counted, and cell fusion was performed according to the ratio of spleen cell number: sp2 / 0 cell number = 8:1. 1 mL of PEG1450 was added, and the whole process was carried out in a 37°C water bath. Preheated blank culture medium was added for termination, and HAT conditioned culture medium was added after centrifugation for pressure screening.

[0087] 4) Monoclonal antibody screening

[0088] Most of the fused cells are cells of multiple clones and need to be further plated for monoclonalization. The positive hybridoma cells are diluted to a density of 0.5 - 1 cell per well using the limiting dilution method and inoculated into a 96-well plate for culture to ensure that the cells in each well are derived from a single ancestral cell. The titer is detected by indirect ELISA, with the coated antigen being TFF1-H02. The titer detection method is the same as that for detecting the mouse serum titer above (the primary antibody is the supernatant of positive hybridoma cells diluted at different multiples). Wells with a monoclonal cell mass and an OD450 value still greater than 2.0 when the positive hybridoma cell supernatant is diluted 1000-fold are selected and expanded to a 24-well plate for culture. The supernatant of the amplified monoclonal cells is repeatedly tested for antibodies to exclude false positive results caused by cell mixing. After ELISA OD450 titer detection and IgG antibody expression screening, ten monoclonal hybridoma cell lines with stable antibody secretion and strong proliferation ability are selected. The ELISA OD450 titers (positive hybridoma cell supernatant diluted 1000-fold) and IgG antibody expression levels of the ten monoclonal antibodies are shown in Table 3 below.

[0089] Table 3. ELISA titer detection results and IgG antibody expression levels of ten monoclonal antibodies

[0090]

[0091] According to the data in Table 3, the ELISA OD450 detection results show that these ten antibodies exhibit good binding ability to the TFF1-H02 antigen, especially TFF1-1, TFF1-2, TFF1-3, TFF1-4, TFF1-7, and TFF1-9.

[0092] Furthermore, the affinity of the ten monoclonal antibodies in Table 3 for TFF1-H02 and their cross-reactivity with AAT are detected. The affinity detection results are shown in Table 4 below. The smaller the KD value, the higher the affinity.

[0093] Table 4. Affinity and cross-reactivity results of ten monoclonal antibodies for TFF1-H02

[0094]

[0095] According to the data in Table 4, it was found that the antibody with the highest titer detected by OD450 in Table 3 did not necessarily have the best affinity. According to the affinity results, the smaller the KD value, the higher the affinity. Three preferred antibodies were screened out, namely TFF1-2 (the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are SEQ ID NO.1-3 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are SEQ ID NO.4-6 respectively; the amino acid sequence of the heavy chain variable region is SEQ ID NO.13, the amino acid sequence of the light chain variable region is SEQ ID NO.14, the amino acid sequence of the heavy chain is SEQ ID NO.40, and the amino acid sequence of the light chain is SEQ ID NO.41), TFF1-3 (the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are SEQ ID NO.7-9 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are SEQ ID NO.10-12 respectively; the amino acid sequence of the heavy chain variable region is SEQ ID NO.15, the amino acid sequence of the light chain variable region is SEQ ID NO.16, the amino acid sequence of the heavy chain is SEQ ID NO.42, and the amino acid sequence of the light chain is SEQ ID NO.43), and TFF1-9 (the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are SEQ ID NO.32-34 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are SEQ ID NO.35-37 respectively; the amino acid sequence of the heavy chain variable region is SEQ ID NO.38, the amino acid sequence of the light chain variable region is SEQ ID NO.39, the amino acid sequence of the heavy chain is SEQ ID NO.48, and the amino acid sequence of the light chain is SEQ ID NO.49). In addition, the cross-reactivity of the three preferred antibodies was <1%, indicating their high specificity.

[0096] TFF1-2, TFF1-3, and TFF1-9 antibodies were produced and purified through the corresponding hybridoma cells to obtain three highly pure TFF1 monoclonal antibodies. Taking the TFF1-2 and TFF1-3 monoclonal antibodies as examples, the detection results of the antibodies produced by hybridoma cells after purification by SDS-PAGE (4-12% 12-well protein gel purchased from GenScript, electrophoresis parameters: 180V, 25min) are as Figure 2 shown.

[0097] Meanwhile, in this embodiment, the thermal stability Tm value of the screened antibody was determined by circular dichroism (CD) to evaluate the stability of the antibody, and it was compared with the existing antibody. The existing TFF1 antibody was purchased from ABM Technology Co., Ltd., model MAB11350. The test results are shown in Table 5. Here, Tm represents the midpoint temperature of protein thermal denaturation, that is, the temperature when the protein unfolds by 50%, reflecting the trend of protein conformational change during the temperature change process. The higher the Tm, the better the stability.

[0098] Table 5. Tm values of different TFF1

[0099]

[0100] According to the results in Table 5, it was found that the Tm values of the TFF1 antibodies screened in this embodiment were all higher than those of the commercially available TFF1 antibodies, indicating that the screened TFF1 antibodies had better stability.

[0101] 2. Kit preparation

[0102] The TFF1 enzyme-linked immunosorbent assay (ELISA) kit includes: coated antibody, carbonate buffer (pH 9.6), washing buffer (PBS + 0.1% Tween20), sample diluent, blocking solution (PBS containing 5% skim milk powder), enzyme-labeled antibody, TMB substrate, termination solution (2M H2SO4).

[0103] The monoclonal antibodies TFF1-2, TFF1-3, and FF1-9 were paired pairwise and were respectively made into coated antibodies or enzyme-labeled antibodies and combined into kits as shown in Table 6 below.

[0104] Table 6. TFF1 kits composed of different coated antibodies and enzyme-labeled antibodies

[0105]

[0106] As shown in Table 6, six kits can be made by pairing the three monoclonal antibodies pairwise.

[0107] The detection steps of the kit are as follows:

[0108] (1) Fix the coated antibody:

[0109] 1) Dilute the coated antibody with carbonate buffer (pH 9.6) to 2 μg / mL, and add 100 μL to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate;

[0110] 2) Incubate overnight at 4°C (12 - 16 hours);

[0111] 3) Discard the liquid, pat dry, add 300 μL of washing buffer (PBS + 0.1% Tween20) to each well, and wash 3 times.

[0112] (2) Block non-specific sites:

[0113] 1) Add 200 μL of blocking solution (PBS containing 5% skim milk powder) to each well and block at 37°C for 1 hour;

[0114] 2) Discard the blocking solution, pat dry, and wash 3 times.

[0115] (3) Antigen capture and detection:

[0116] 1) Sample incubation: Add 100 μL to each well; incubate at 37°C for 1 hour and wash 3 times;

[0117] 2) Dilute the enzyme-labeled antibody to 1 μg / mL with sample diluent and add 100 μL to each well; incubate at 37°C in the dark for 45 minutes and wash 5 times (to improve specificity);

[0118] 3) Color development and termination reaction: Add 100 μL of TMB substrate to each well and develop color in the dark at room temperature for 10 - 15 minutes (the time needs to be optimized by preliminary experiment); immediately add 50 μL of termination solution (2M H2SO4) to each well;

[0119] 4) Absorbance detection and data analysis: Use an enzyme-linked immunosorbent assay reader to read the absorbance (OD value) of each well at 450 nm (main wavelength) and 630 nm (reference wavelength); calculate the concentration according to the fitted four-parameter Logistic curve.

[0120] The preparation steps of the described fitted four-parameter Logistic curve are as follows:

[0121] Prepare six concentrations of TFF1 standards at 0, 5, 25, 50, 100, and 200 ng / mL, detect the absorbance (OD value) at 450 nm (main wavelength) according to the above method, detect each concentration in parallel three times, calculate the average value, and simulate and fit the four-parameter Logistic curve through software to obtain 6 fitted four-parameter Logistic curves. Taking the kit composed of the coating antibody TFF1-2 and the enzyme-labeled antibody TFF1-3 in combination 1 as an example, its fitted four-parameter Logistic curve is as Figure 3 shown, with the four-parameter A estimate value of 0.052, standard deviation of 0.018, and confidence interval of [0.002, 0.102]; B estimate value of 1.111, standard deviation of 0.040, and confidence interval of [0.999, 1.224]; C estimate value of 1.310, standard deviation of 0.055, and confidence interval of [1.157, 1.462]; D estimate value of 2.475, standard deviation of 0.041, and confidence interval of [2.361, 2.589]. Calculated according to the four parameters, , R 2= 1.000, EC50 = 1.310 nM.

[0122] Furthermore, a TFF1 protein control solution (prepared with negative human serum) containing 5 ng / mL was used to investigate its within-batch / between-batch precision, and the test results are shown in Table 7 below.

[0123] Table 7. Within-batch / between-batch precision of the TFF1 detection kit

[0124]

[0125] According to the data in Table 7, the within-batch / between-batch CV values of Kits 1 and 2 are smaller, and the recovery rates are closer to 100%, indicating that Kits 1 and 2 are more stable and accurate in detection. The coating antibodies of Kits 1 and 2 are both TFF1-2, which is used to capture TFF1 in the test sample, with stronger specific capture ability, so the probability of producing false positives is low and the accuracy is higher. Therefore, the TFF1 kits are preferably Kits 1 and 2.

[0126] Example 2. Preparation, screening of monoclonal antibodies and preparation of their kits

[0127] 1. Preparation and screening of AAT monoclonal antibodies

[0128] 1) Preparation of antigen

[0129] The AAT sequence was retrieved through biological information and two antigens were designed: AAT-H01 (amino acid sequence shown in SEQ ID NO.52, nucleotide sequence shown in SEQ ID NO.56) and AAT-H02 (amino acid sequence shown in SEQ ID NO.53, nucleotide sequence shown in SEQ ID NO.57). To increase the immunogenicity of the antigen, during antigen design, purification and immunization: 1. Add Kozak sequence, introducing this sequence can mainly enhance translation efficiency, ensure accuracy, optimize foreign gene expression and regulate expression level; 2. Immunize with dimers to make the immunogenicity stronger; 3. Monomer screening to ensure that the obtained antibodies are against a single epitope; 4. Add 6×His tag to facilitate purification to obtain a higher amount of antigen and significantly improve the purity of the antigen; 5. Boost immunization during the immunization process and directly inject into the spleen to obtain a higher antibody titer. Entrust GenScript to synthesize protein plasmids, and eukaryotic cell transfection expression and purification are carried out according to the obtained plasmids to obtain the corresponding antigens. The SDS-PAGE test results of the purified AAT-H01 antigen are as Figure 4 shown. The AAT-H01 antigen is used for immunization injection and hybridoma cell fusion, and the AAT-H02 antigen is used for screening.

[0130] 2) Animal immunization

[0131] Five BALB / c mice were immunized with AAT-H01 antigen, and the immunization process was the same as that in Table 1 of Example 1.

[0132] 3) Preparation of hybridoma cells

[0133] After the immunization was completed, blood was collected from the submandibular vein of the mice, and the antibody titer was detected by indirect ELISA. The antigen for coating was AAT-H02. The method for detecting the antibody titer by indirect ELISA was as follows:

[0134] a. Dilute the antigen AAT-H02 to 2 μg / mL with carbonate buffer (pH 9.6), and add 100 μL to each well of a 96-well ELISA plate; coat overnight at 4°C (12 - 16 hours);

[0135] b. Discard the liquid, pat dry, add 300 μL of washing buffer (PBS + 0.1% Tween20) to each well, and wash 3 times;

[0136] c. Add 200 μL of blocking solution (PBS containing 5% skim milk powder) to each well, and block at 37°C for 1 hour;

[0137] d. Discard the blocking solution, pat dry, and wash 3 times.

[0138] e. Add 100 μL of serum (i.e., the primary antibody, and the serum was diluted 1000, 4000, 16000, 64000, and 256000 times respectively) to each well; incubate at 37°C for 1 hour, and wash 3 times;

[0139] f. Dilute the enzyme-labeled antibody (i.e., the secondary antibody, which is HRP-labeled goat anti-mouse IgG) to 1 μg / mL with PBS, and add 100 μL to each well; incubate at 37°C in the dark for 45 minutes, and wash 5 times;

[0140] g. Color development and termination reaction: Add 100 μL of TMB substrate to each well, and develop color at room temperature in the dark for 10 minutes; immediately add 50 μL of termination solution (2M H2SO4) to each well;

[0141] h. Absorbance detection and data analysis: Use an ELISA reader to read the absorbance (OD value) of each well at 450 nm. Each well was detected in parallel three times, and the average value was calculated.

[0142] The results of serum titer detection are shown in Table 8 below.

[0143] Table 8. Results of indirect ELISA for detecting antibody titer in the serum of mice immunized with AAT-H01 antigen

[0144]

[0145] According to the detection results in Table 8, select the mouse blood samples taken from the eyeballs with a serum dilution of 64,000 times and an OD450 greater than 0.2 as positive controls. Isolate the mouse spleens, grind the spleens, count the cells, and perform cell fusion according to the ratio of spleen cells: sp2 / 0 cells = 8:1. Add 1 mL of PEG1450. The whole process is carried out under the condition of a 37°C water bath. Add pre-warmed blank medium to terminate the reaction. After centrifugation, add HAT conditioned medium for pressure screening.

[0146] 4) Screening of monoclonal antibodies

[0147] Most of the fused cells are cells of multiple clones and need to continue with monoclonal plating. The positive hybridoma cells are diluted to a density of 0.5 - 1 cell per well using the limiting dilution method and inoculated into a 96-well plate for culture to ensure that the cells in each well are derived from a single ancestral cell. The titer is detected by indirect ELISA. The coated antigen is AAT-H02, and the titer detection method is the same as that for detecting the mouse serum titer above (the primary antibody is the supernatant of positive hybridoma cells diluted at different multiples). Select the wells where the OD450 value of the supernatant of positive hybridoma cells is still greater than 1.5 when diluted 1000 times and belongs to monoclonal cell clusters and expand them to a 24-well plate for culture. Repeat the antibody detection for the supernatant after amplification of the monoclonal cells to exclude false positive results caused by cell mixing. After ELISA OD450 titer detection and IgG antibody expression level screening, ten monoclonal hybridoma cell lines with stable antibody secretion and strong proliferation ability are selected. The ELISA OD450 titer (the supernatant of positive hybridoma cells diluted 1000 times) detection results and IgG antibody expression levels of the ten monoclonal antibodies are shown in Table 9 below.

[0148] Table 9. ELISA detection binding titer results and IgG antibody expression levels of ten monoclonal antibodies

[0149]

[0150] According to the data in Table 9, the ELISA OD450 detection results show that these ten monoclonal antibodies exhibit good binding ability to the AAT-H02 antigen, especially AAT-1, AAT-2, AAT-6, AAT-9, and AAT-10.

[0151] Furthermore, detect the affinity of the ten monoclonal antibodies in Table 9 for AAT-H02 and their cross-reactivity with TFF1. The affinity detection results are shown in Table 10 below. The smaller the KD value, the higher the affinity.

[0152] Table 10. Affinity results of ten monoclonal antibodies for AAT-H02

[0153]

[0154] According to the data in Table 10, it is found that the antibody with the highest titer detected by OD450 in Table 9 does not necessarily have the best affinity. According to the affinity results, the smaller the KD value, the higher the affinity. Two preferred antibodies were screened out, namely AAT-2 (the amino acid sequences of CDR1, CDR2, and CDR3 of the heavy chain are SEQ ID NO.17-19 respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are SEQ ID NO.20-22 respectively; the amino acid sequence of the heavy chain variable region is SEQ ID NO.28, the amino acid sequence of the light chain variable region is SEQ ID NO.29, the amino acid sequence of the heavy chain is SEQ ID NO.44, and the amino acid sequence of the light chain is SEQ ID NO.45), AAT-6 (the amino acid sequences of CDR1 and CDR2 of the heavy chain are SEQ ID NO.23-24 respectively, the amino acid sequence of CDR3 of the heavy chain is FDY, and the amino acid sequences of CDR1, CDR2, and CDR3 of the light chain are SEQ ID NO.25-27 respectively; the amino acid sequence of the heavy chain variable region is SEQ ID NO.30, the amino acid sequence of the light chain variable region is SEQ ID NO.31, the amino acid sequence of the heavy chain is SEQ ID NO.46, and the amino acid sequence of the light chain is SEQ ID NO.47). In addition, the cross-reactivity of the two preferred antibodies is <1%, indicating their high specificity.

[0155] AAT-2 and AAT-6 were produced and purified through the corresponding hybridoma cells to obtain two highly pure AAT monoclonal antibodies. Taking the AAT-2 and AAT-6 monoclonal antibodies as examples, the detection results of the antibodies produced by hybridoma cells after purification by SDS-PAGE (4-12% 12-well protein gel purchased from GenScript) are as Figure 5 shown.

[0156] Meanwhile, in this example, circular dichroism (CD) was also used to measure the thermal stability Tm value of the screened antibodies to evaluate the stability of the antibodies and compare them with the existing antibodies. The existing AAT antibody was purchased from ABM40300 of Abcam. The detection results are shown in Table 11. Among them, Tm represents the midpoint temperature of protein thermal denaturation, that is, the temperature when the protein unfolds by 50%, reflecting the trend of protein conformational change during the temperature change process. The higher the Tm, the better the stability.

[0157] Table 11. Tm values of different AAT antibodies

[0158]

[0159] According to the data in Table 11, it is found that the Tm values of the AAT antibodies screened in this example are all higher than those of the commercially available AAT antibodies, indicating that the screened AAT antibodies have better stability.

[0160] 2. Preparation of the kit

[0161] The AAT enzyme-linked immunosorbent assay kit includes: coated antibody, carbonate buffer (pH 9.6), washing buffer (PBS + 0.1% Tween20), sample diluent, blocking solution (PBS containing 5% skim milk powder), enzyme-labeled antibody, TMB substrate, termination solution (2M H2SO4).

[0162] AAT-2 and AAT-6 were respectively prepared into coated antibodies or enzyme-labeled antibodies and combined into a kit as shown in Table 12 below.

[0163] Table 12. AAT kits composed of different coated antibodies and enzyme-labeled antibodies

[0164]

[0165] As shown in Table 12, one pair of antibodies can be made into 2 kinds of kits.

[0166] The detection steps of the kit are as follows:

[0167] (1) Fix the coated antibody:

[0168] 1) Dilute the coated antibody with carbonate buffer (pH 9.6) to 2 μg / mL, and add 100 μL to each well of a 96-well enzyme-linked immunosorbent assay plate;

[0169] 2) Incubate at 4°C overnight (12 - 16 hours);

[0170] 3) Discard the liquid, pat dry, add 300 μL of washing buffer (PBS + 0.1% Tween20) to each well, and wash 3 times.

[0171] (2) Block non-specific sites:

[0172] 1) Add 200 μL of blocking solution (PBS containing 5% skim milk powder) to each well, and block at 37°C for 1 hour;

[0173] 2) Discard the blocking solution, pat dry, and wash 3 times.

[0174] (3) Antigen capture and detection:

[0175] 1) Sample incubation: Add 100 μL to each well; incubate at 37°C for 1 hour, and wash 3 times;

[0176] 2) Dilute the enzyme-labeled antibody with sample diluent to 1 μg / mL, add 100 μL to each well; incubate at 37°C in the dark for 45 minutes, and wash 5 times (to improve specificity);

[0177] 3) Color development and termination reaction: Add 100 μL of TMB substrate to each well and incubate at room temperature in the dark for 10 - 15 minutes (the time needs to be optimized by preliminary experiment); immediately add 50 μL of termination solution (2M H2SO4) to each well;

[0178] 4) Absorbance detection and data analysis: Use a microplate reader to read the absorbance (OD value) of each well at 450 nm (main wavelength) and 630 nm (reference wavelength); calculate the concentration according to the fitted 4-parameter Logistic curve.

[0179] The preparation steps of the fitted 4-parameter Logistic curve are as follows:

[0180] Prepare six concentrations of AAT standard products at 0, 10, 50, 100, 250, and 500 ng / mL, and detect the absorbance (OD value) at 450 nm (main wavelength) according to the above method. Each concentration is detected in parallel three times, and the average value is calculated. Use software to simulate and fit the 4-parameter Logistic curve to obtain 2 fitted 4-parameter Logistic curves. Taking the kit composed of the coating antibody of combination 1 as AAT-2 and the enzyme-labeled antibody as AAT-6 as an example, its fitted 4-parameter Logistic curve is as Figure 6 shown. The estimated value of its 4-parameter A is 0.083, the standard deviation is 0.043, and the confidence interval is [-0.054, 0.220]; the estimated value of B is 1.251, the standard deviation is 0.112, and the confidence interval is [0.896, 1.607]; the estimated value of C is 5.964, the standard deviation is 0.941, and the confidence interval is [2.970, 8.958]; the estimated value of D is 5.064, the standard deviation is 0.470, and the confidence interval is [3.567, 6.561]. According to the calculation of the four parameters, , R 2 = 0.999, EC50 = 5.964 nM.

[0181] Furthermore, use the AAT protein control product solution containing 10 ng / mL (prepared with negative human serum) to investigate its within-batch / between-batch precision. The test results are shown in Table 13 below.

[0182] Table 13. Within-batch / between-batch precision of the AAT detection kit

[0183]

[0184] According to the data in Table 13, the within-batch / between-batch CV values of Kit 1 are smaller, and the recovery rate is closer to 100%, indicating that Kit 1 has more stable detection and higher accuracy. Therefore, the AAT kit is preferably Kit 1.

[0185] Application of the TFF1 and AAT ELISA Kits Alone and in Combination in the Diagnosis of Colorectal Cancer

[0186] 1. Diagnostic Efficacy of Using the TFF1 and AAT ELISA Kits Alone and in Combination

[0187] In this experiment, the TFF1 and AAT ELISA kits constructed in Example 1 and Example 2 were used to test TFF1 and AAT in the sera of 100 healthy subjects and 100 colorectal cancer patients respectively, and their abilities to distinguish diseased populations were evaluated. Among them, the ages and genders of the 100 healthy subjects were matched with those of the colorectal cancer patient group, and they had no history of colorectal cancer or other digestive system diseases; the colorectal cancer patient group included 100 pathologically diagnosed colorectal cancer patients, covering different clinical stages (stages I-IV).

[0188] Venous blood was collected from healthy subjects and colorectal cancer patients at 5 mL per case, and the serum was separated by centrifugation (3000 rpm, 15 min), aliquoted and stored at -80 °C for later use. The preferred TFF1 ELISA kits 1 and 2 in Example 1 and the preferred AAT ELISA kit 1 in Example 2 were used to independently detect 200 samples respectively. The absorbance values were recorded and the concentrations were calculated to detect the target protein concentrations in the serum. At the same time, the commercially available TFF1 antibody (from Abcam Technology Co., Ltd., model MAB11350) in Example 1 and the commercially available AAT antibody (from Abcam Technology Co., Ltd., model ABM40300) in Example 2 were used to prepare corresponding ELISA kits for detection and comparison. Their abilities to distinguish diseased populations were evaluated by SPSS or R language respectively.

[0189] When using TFF1 alone as a biomarker, the kits were the preferred TFF1 ELISA kits 1 and 2 in Example 1 and the corresponding ELISA kits prepared with the commercially available TFF1 antibody in Example 1. The cut-off value of TFF1 content for distinguishing colorectal cancer patients was 0.5 ng / mL. Those with a TFF1 content greater than or equal to 0.5 ng / mL were judged as colorectal cancer patients, and those with a TFF1 content less than 0.5 ng / mL were judged as healthy individuals.

[0190] When using AAT alone as a biomarker, the kits were the preferred AAT ELISA kit 1 in Example 2 and the corresponding ELISA kits prepared with the commercially available AAT antibody in Example 2. The cut-off value of AAT content for distinguishing colorectal cancer patients was 5.0 ng / mL. Those with an AAT content greater than or equal to 5.0 ng / mL were judged as colorectal cancer patients, and those with an AAT content less than 5.0 ng / mL were judged as healthy individuals.

[0191] When using TFF1 and AAT as markers in combination, the kit is TFF1 enzyme-linked immunosorbent assay kit 1 + AAT enzyme-linked immunosorbent assay kit 1, and the combined diagnostic model (TFF1 + AAT) is used for the diagnosis of colorectal cancer. The Logistic regression formula for constructing TFF1 enzyme-linked immunosorbent assay kit 1 + AAT enzyme-linked immunosorbent assay kit 1 is: Risk score = 1.32×ln(TFF1) + 0.87×ln(AAT) (threshold = 0.5. When the risk score is greater than or equal to 0.5, it is judged as a colorectal cancer patient; when it is less than 0.5, it is judged as a healthy population). The receiver operating characteristic curve (ROC) and AUC values of the combined detection and individual detection of the said kit combination are as Figure 7 shown.

[0192] Table 14. Sample detection results of using TFF1 and AAT enzyme-linked immunosorbent assay kits alone

[0193]

[0194] According to the data in Table 14, it is found that the combined use of TFF1 enzyme-linked immunosorbent assay kit and AAT enzyme-linked immunosorbent assay kit for detection has a higher AUC value than the use of TFF1 enzyme-linked immunosorbent assay kit or AAT enzyme-linked immunosorbent assay kit alone for detection, indicating that the diagnostic efficacy of the combined use of the two kits for the diagnosis of colorectal cancer is higher. Among them, the diagnostic efficacy of the kit prepared with the TFF1 monoclonal antibody and AAT monoclonal antibody screened in the present invention for combined detection is significantly higher.

[0195] 2. Difference in diagnostic efficacy of combined use of TFF1 and AAT enzyme-linked immunosorbent assay kits

[0196] In this experiment, the kits for individual detection in Table 14 above were combined, which were respectively the following three kit combinations: The first: TFF1 enzyme-linked immunosorbent assay kit 1 + AAT enzyme-linked immunosorbent assay kit 1; The second: TFF1 enzyme-linked immunosorbent assay kit 2 + AAT enzyme-linked immunosorbent assay kit 1; The third: TFF1 enzyme-linked immunosorbent assay kit prepared with a commercially available TFF1 antibody + TFF1 enzyme-linked immunosorbent assay kit prepared with a commercially available AAT antibody. The combined diagnostic model (TFF1 + AAT) was used for the diagnosis of colorectal cancer. The test samples included the sera of 200 healthy people and 200 rectal cancer patients, which were randomly divided into a test group and a verification group. Among them, the test group included the sera of 100 healthy people and 100 colorectal cancer patients, and the verification group included the sera of 100 healthy people and 100 colorectal cancer patients.

[0197] Among the first kit combination, the Logistic regression formula for constructing the TFF1 enzyme-linked immunosorbent assay kit 1 + AAT enzyme-linked immunosorbent assay kit 1 is: Risk score = 1.35×ln(TFF1) + 0.89×ln(AAT) (threshold = 0.5. When the risk score is greater than or equal to 0.5, it is judged as a colorectal cancer patient; when it is less than 0.5, it is judged as a healthy person).

[0198] Among the second kit combination, the Logistic regression formula for constructing the TFF1 enzyme-linked immunosorbent assay kit 1 + AAT enzyme-linked immunosorbent assay kit 2 is: Risk score = 1.15×ln(TFF1) + 0.72×ln(AAT) (threshold = 0.5. When the risk score is greater than or equal to 0.5, it is judged as a colorectal cancer patient; when it is less than 0.5, it is judged as a healthy person).

[0199] Among the third kit combination, the Logistic regression formula for the TFF1 enzyme-linked immunosorbent assay kit prepared with a commercially available TFF1 antibody + the TFF1 enzyme-linked immunosorbent assay kit prepared with a commercially available AAT antibody is: Risk score = 0.83×ln(TFF1) + 0.65×ln(AAT) (threshold = 0.5. When the risk score is greater than or equal to 0.5, it is judged as a colorectal cancer patient; when it is less than 0.5, it is judged as a healthy person).

[0200] Table 15. Sample test results of jointly using TFF1 and AAT enzyme-linked immunosorbent assay kits

[0201]

[0202] According to the data in Table 15, although the detected markers are both TFF1 and AAT proteins and are based on the same combined diagnosis model, when using different antibodies for detection, the diagnostic results will be significantly different. The reason may be that the existing antibodies not only have low detection sensitivity, but also whether different antibodies are used as detection antibodies or enzyme-labeled antibodies respectively, and the detection results will also be significantly different. In addition, there are obvious differences in different antibodies in terms of stability (Tm), cross-reaction with the matrix, high-dose hook effect, etc. All these reasons will lead to a decline in the diagnostic efficacy for diagnosing colorectal cancer.

[0203] At the same time, during the diagnosis of colorectal cancer, it is necessary to detect its blood samples through TFF1 antibody and / or AAT antibody. There are complex interfering substances in the blood samples, and the matrix has a great influence, and there are easy cross-reactions or interferences from some non-linear fragments. Using different antibodies for the detection and diagnosis of colorectal cancer will inevitably produce completely different diagnostic effects. Therefore, it is necessary to select a better antibody combination to improve the diagnostic efficacy.

[0204] Therefore, the preferred antibody combination provided by the present invention, including the TFF1-2 antibody (i.e., the first antibody of the TFF1 monoclonal antibody) and the TFF1-3 antibody (i.e., the second antibody of the TFF1 monoclonal antibody), the AAT-2 antibody (i.e., the first antibody of the AAT monoclonal antibody) and the AAT-6 antibody (i.e., the second antibody of the AAT monoclonal antibody), is used to construct detection kits for two markers (TFF1 enzyme-linked immunosorbent assay kit 1 and AAT enzyme-linked immunosorbent assay kit 1), which can significantly improve the diagnostic efficacy of colorectal cancer.

[0205] Based on the above analysis, the strategy of jointly using the TFF1 and AAT enzyme-linked immunosorbent assay kits significantly improves the early diagnostic efficacy of rectal cancer, providing a reliable basis for clinical promotion.

[0206] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

Claims

1. A monoclonal antibody against TFF1, 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 TFF1 monoclonal antibody 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 AAT monoclonal antibody, 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 FDY in the heavy chain variable region, and (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.25, CDR2 consisting of the amino acid sequence of SEQ ID NO.26, and CDR3 consisting of the amino acid sequence of SEQ ID NO.27 in the light chain variable region.

4. The AAT monoclonal antibody according to claim 3, wherein The amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO.28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

31.

5. A kit for detecting TFF1 protein and / or AAT protein, characterized in that, Comprising the TFF1 monoclonal antibody as claimed in claim 1 or 2, and / or the AAT monoclonal antibody 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 TFF1 monoclonal antibody as claimed in claim 1 or 2, and / or the AAT monoclonal antibody as claimed in claim 3 or 4.

7. The use according to claim 6, characterized in that, Comprising a TFF1 monoclonal antibody and an AAT monoclonal antibody; the TFF1 monoclonal antibody comprises 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; The AAT monoclonal antibody comprises 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.28, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.29; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.30, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

31.

8. A kit for predicting whether an individual has colorectal cancer, characterized in that, Comprising the TFF1 monoclonal antibody as claimed in claim 1 or 2, and the AAT monoclonal antibody as claimed in claim 3 or 4.

9. An antibody combination for predicting whether an individual has colorectal cancer, characterized in that, Comprising the TFF1 monoclonal antibody as claimed in claim 1 or 2, and the AAT monoclonal antibody 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 TFF1 monoclonal antibody as claimed in claim 1 or 2, and / or the AAT monoclonal antibody as claimed in claim 3 or 4.

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