Use of tff1, aat monoclonal antibodies and combinations thereof in the diagnosis of colorectal cancer

Abstract

The application provides application of TFF1, AAT monoclonal antibodies and combinations thereof in colorectal cancer diagnosis, and solves the problems of low affinity and specificity and poor stability of existing antibodies by optimizing the immunogen sequence of TFF1 and AAT, screening high-affinity and high-specificity monoclonal antibodies, screening the optimal TFF1 and AAT antibodies and combining them, the detection range is wider, and the combination detection shows extremely high synergistic diagnostic value, the early colorectal cancer diagnosis efficiency is significantly improved, and the application has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of antibody screening and disease diagnosis. Specifically, it relates to the application of a TFF1, AAT monoclonal antibody and its combination in the diagnosis of colorectal cancer. Background Technology

[0002] Rectal cancer is the third most common malignant tumor worldwide, accounting for approximately 30% of colorectal cancer cases. In recent years, with the Westernization of dietary structures (such as high-fat, low-fiber diets), lack of exercise, and the aging trend, the incidence of rectal cancer has been rising year by year. Current diagnostic methods mainly include colonoscopy, fecal occult blood test (FOBT), blood biomarker testing, and imaging examinations. Among these, colonoscopy is the gold standard for diagnosing rectal cancer, but it has disadvantages such as high invasiveness and cost. The fecal occult blood test has low specificity and is easily interfered with by diseases such as hemorrhoids or peptic ulcers, resulting in false positives or false negatives. Furthermore, imaging examinations are limited in their ability to detect early, small lesions and are also expensive. This patent utilizes a blood biomarker testing method, which has advantages such as being non-invasive, low-cost, and easy to repeat. Moreover, addressing the limited sensitivity and specificity of this testing method, this patent employs a more efficient and reliable combined detection of two biomarkers, significantly improving the accuracy of early screening or diagnosis of rectal cancer.

[0003] Breast cancer-related peptide (PS2, or TFF1) belongs to the trefoil family, characterized by at least one copy of a trefoil motif, a 40-amino acid domain containing three conserved disulfide bonds. These are stable secretory proteins expressed in the gastrointestinal mucosa. The gene is expressed in goblet cells of the intestine and colon. The trefoil factor family is a class of secretory small peptides with a stable trefoil structure, including three members: TFF1, TFF2, and TFF3. Among these three, TFF1 has the highest concentration. Furthermore, TFF1 is closely related to the development and progression of various tumors; its levels are significantly increased in colorectal cancer patients, making it a potential biomarker for early diagnosis of colorectal cancer.

[0004] Human α-1 antitrypsin (AAT), also known as serpin A1, α1-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 the blood and tissues. AAT deficiency is a genetic disorder that often predisposes to the premature onset of chronic obstructive pulmonary disease (COPD), cirrhosis, recurrent panniculitis, systemic vasculitis, and a range of possible inflammatory and neoplastic diseases. Furthermore, some clinical studies have shown that patients with AAT deficiency have an increased risk of malignant tumors, including hepatocellular carcinoma, lung cancer, bladder and gallbladder tumors, malignant lymphoma, and colon cancer, thus serving as a potential biomarker for the early diagnosis of colorectal cancer.

[0005] Although both TFF1 and AAT can be used as biomarkers for the early diagnosis of colorectal cancer, the current technology has the following gaps: 1) Lack of standardized detection tools: different kits have large differences in detection results, making it difficult to detect accurately; 2) Lack of exploration of synergistic diagnostic value: there is no research data showing that the combination of TFF1 and AAT can improve diagnostic efficacy (indicators such as AUC value have not been reported in the literature).

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

[0007] This invention provides an application of TFF1 and AAT monoclonal antibodies and their combinations in the diagnosis of colorectal cancer. By optimizing the immunogenic sequences of TFF1 and AAT, high-affinity and high-specificity monoclonal antibodies are screened, and the optimal TFF1 and AAT antibodies are selected and combined. This effectively solves the problems of low affinity and specificity and poor stability of existing antibodies, resulting in a wider detection range. When combined with other antibodies, the combination demonstrates extremely high synergistic diagnostic value, significantly improving the diagnostic efficacy of early colorectal cancer and possessing important clinical application value.

[0008] Existing technologies that use traditional hybridoma techniques, phage display techniques, or recombinant protein immunization of animals to prepare monoclonal or polyclonal antibodies against TFF1 or AAT proteins suffer from insufficient antibody affinity and specificity due to unoptimized immunogen design. Furthermore, current ELISA or chemiluminescence methods for detecting TFF1 and AAT protein tumor markers do not optimize kits for combinations of TFF1 and AAT antibodies, resulting in low detection sensitivity and specificity, hindering efficient colorectal cancer diagnosis. This invention develops novel, highly specific antibodies against TFF1 and AAT, and their combinations, which can be used to prepare diagnostic kits for detecting TFF1 and AAT in different samples, thereby achieving clinical value.

[0009] On one hand, the present invention provides a TFF1 monoclonal antibody, 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 relates to the field of antibody preparation technology, and in particular to a specific targeting antibody for TFF1, its preparation method, and its application. The TFF1 nucleotide sequence was retrieved through bioinformatics, and two antigens, TFF1-H01 and TFF1-H02, were designed. Antigen plasmids were synthesized, and positive clones were selected from the top 10 transformants based on the obtained plasmids. These clones were then preserved, activated, and cultured to amplify the culture. The plasmids were then extracted and transfected into eukaryotic cells for expression, followed by affinity purification to obtain the corresponding antigens. The TFF1-H01 antigen was used for immunization and hybridoma cell fusion. Screening was performed using TFF1-H02 to obtain multiple pairs of monoclonal antibody strains. Through antigen detection and antibody pairing, the best antibody pairs were ultimately selected for the development of a rapid colorectal cancer detection kit.

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

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

[0019] Furthermore, the present invention provides a TFF1 detection kit, the kit comprising an antibody against the TFF1 protein described above.

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

[0021] In some embodiments, the kit is an ELISA assay kit. A dual-antibody combination ELISA assay kit is constructed by screening for optimal and appropriately paired TFF1 primary and secondary antibodies.

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

[0023] (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

[0024] (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; the second antibody comprises:

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

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

[0027] This invention also relates to a specific targeting antibody against AAT, its preparation method, and its application. The nucleotide sequence of the AAT protein is retrieved through bioinformatics, and two antigens, AAT-H01 and AAT-H02, are designed. Antigen plasmids are synthesized, and positive clones are selected from the top 10 transformants based on the obtained plasmids, preserved, activated, and amplified in culture. The plasmids are then extracted and transfected into eukaryotic cells for expression, followed by affinity purification to obtain the corresponding antigens. The AAT-H01 antigen is used for immunization and hybridoma cell fusion. Multiple monoclonal antibody pairs are obtained through screening using AAT-H02. Finally, through antigen detection and antibody pairing, the superior antibody pair is selected for the development of a rapid colorectal cancer detection kit.

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

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

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

[0032] It is understood that the kit may be an ELISA detection kit, a chemiluminescent immunoassay kit, etc.

[0033] In some embodiments, the kit is an ELISA assay kit. A dual-antibody combination ELISA assay kit is constructed by screening for optimal and appropriately paired AAT primary and secondary antibodies.

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

[0035] In some approaches, kits for detecting TFF1 protein and kits for detecting AAT protein are combined to simultaneously detect both TFF1 and AAT proteins. This allows for applications in fields requiring the simultaneous detection of multiple biomarkers, thereby improving the efficacy 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 to predict whether an individual has colorectal cancer, the antibody comprising the TFF1 monoclonal antibody described above, and / or the AAT monoclonal antibody described above.

[0037] Traditional single-target antibody drugs have many limitations. In existing cancer diagnostic techniques, the detection of a single biomarker (such as TFF1) suffers from low sensitivity and high false-positive rates. Although TFF1 is associated with the tumor microenvironment, its clinical application as a standalone marker is not yet mature. This invention uses TFF1 and AAT as combined biomarkers, employing combined detection of antibodies targeting TFF1 and AAT proteins to address the insufficient diagnostic efficacy of single biomarkers and improve the accuracy and specificity of cancer diagnosis.

[0038] While TFF1 and AAT are known diagnostic markers for colorectal cancer, the diagnostic efficacy varies significantly when using different TFF1 and / or AAT antibodies. This is because colorectal cancer diagnosis requires testing blood samples with TFF1 and / or AAT 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.

[0039] Through the design, expression, and screening of antigens and monoclonal antibodies, the antibodies for TFF1 and AAT 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.

[0040] Further, the antibody includes a TFF1 monoclonal antibody and an AAT monoclonal antibody; the TFF1 monoclonal antibody comprises a first antibody and a second antibody, wherein 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, 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.

[0041] 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 saliva, blood, urine, plasma, serum, and cerebrospinal fluid.

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

[0043] Furthermore, the reagent is used to detect the presence, relative abundance, or concentration of biomarkers in body fluid samples.

[0044] This invention relates to biomarkers (TFF1 and AAT proteins) for predicting colorectal cancer risk, 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 TFF1 and AAT 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 TFF1 and AAT proteins in the blood of a group can be detected, thereby dividing that group into colorectal cancer patients and healthy individuals.

[0045] In another aspect, the present invention also provides a kit for predicting whether an individual has colorectal cancer, comprising the TFF1 monoclonal antibody and the AAT monoclonal antibody described above.

[0046] In another aspect, the present invention also provides an antibody combination for predicting whether an individual has colorectal cancer, including the TFF1 monoclonal antibody and the AAT monoclonal antibody described above.

[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 value of an antigen, wherein the antigen is detected by an antibody, and the antibody includes the TFF1 monoclonal antibody described above, and / or the AAT monoclonal antibody described above.

[0048] Furthermore, the data analysis module uses the detection values ​​of markers from known samples as the training set. Based on the postoperative condition of colorectal cancer patients, they are divided into healthy individuals and colorectal cancer patients. The relationship between the detection values ​​of healthy individuals and colorectal cancer patients is analyzed to construct a model.

[0049] 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.

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

[0051] 1. Design and manufacture TFF1 and AAT expression antigens, and use these antigens to immunize animals to obtain specific targeting monoclonal antibodies with extremely high sensitivity;

[0052] 2. The prepared antibodies are recombined to increase expression levels and ensure stable antibody expression; the antibody testing process incorporates clinical blood sample testing, greatly increasing the reliability of experimental data; dual antibody detection reduces reagent consumption by 50%.

[0053] 3. Multiple pairs of monoclonal antibodies targeting the TFF1 antigenic epitope were screened and a TFF1 detection kit was constructed. Combined with the double antibody sandwich design, the cross-reactivity rate was extremely low (<1%), and the sensitivity reached the ng / mL level, enabling high-precision detection of TFF1 protein.

[0054] 4. Multiple pairs of monoclonal antibodies targeting AAT antigenic epitopes were screened and used to construct an AAT detection kit. Combined with a double-antibody sandwich design, the cross-reactivity rate was extremely low (<1%), and the sensitivity reached the ng / mL level, enabling high-precision detection of AAT protein.

[0055] 5. Optimize antibody combinations and detection processes to achieve efficient and economical clinical applications;

[0056] 6. Diagnostic value of the kit alone or in combination in gastrointestinal tumors, especially in colorectal cancer; the first use of the combined detection model (TFF1+AAT) significantly improved the discrimination ability compared with single markers (AUC≥0.91). Attached Figure Description

[0057] Figure 1The results of SDS-PAGE analysis of the purified TFF1-H01 antigen in Example 1 are shown.

[0058] Figure 2 The results of SDS-PAGE analysis of the purified TFF1-2 and TFF1-3 monoclonal antibodies from Example 1 are shown.

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

[0060] Figure 4 The results of SDS-PAGE analysis of the purified AAT-H01 antigen in Example 2 are shown.

[0061] Figure 5 The results of SDS-PAGE analysis of the purified AAT-2 and AAT-6 monoclonal antibodies from Example 2 are shown.

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

[0063] Figure 7 The receiver operating characteristic (ROC) curves and AUC values ​​of the TFF1 enzyme-linked immunosorbent assay kit 1 and the AAT enzyme-linked immunosorbent assay kit 1 used in Example 3 for combined detection and separate detection are shown. Detailed Implementation

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

[0065] Example 1: Preparation, screening, and kit preparation of TFF1 monoclonal antibodies.

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

[0067] 1) Preparation of antigen

[0068] TFF1 sequences were retrieved using bioinformatics, and two antigens were designed: TFF1-H01 (amino acid sequence as shown in SEQ ID NO. 50, nucleotide sequence as shown in SEQ ID NO. 54) and TFF1-H02 (amino acid sequence as shown in SEQ ID NO. 51, nucleotide sequence as shown in SEQ ID NO. 55). To enhance the immunogenicity of the antigens, the following measures were taken during antigen design, purification, and immunization: 1. A Kozak sequence was added, which mainly enhances translation efficiency, ensures accuracy, optimizes exogenous gene expression, and regulates expression levels; 2. Immunization was performed using dimers, resulting in stronger immunogenicity; 3. Monomer screening was conducted to ensure that the obtained antibodies target a single epitope; 4. A 6×His tag was added to facilitate purification, yielding higher antigen quantities and significantly improving antigen purity; 5. Boosting immunization was performed during the immunization process, with direct spleen injection to obtain higher antibody titers. Protein plasmid synthesis was commissioned to GenScript, and the obtained plasmids were transfected into eukaryotic cells for expression and purification to obtain the corresponding antigens. The SDS-PAGE results of the purified TFF1-H01 antigen are shown below. Figure 1 As shown. The TFF1-H01 antigen is used for immunization and hybridoma cell fusion, while the TFF1-H02 antigen is used for screening.

[0069] 2) Animal immunization

[0070] Five BALB / c mice were immunized with the TFF1-H01 antigen. The immunization procedure is shown in Table 1 below.

[0071] Table 1. TFF1-H01 Antigen Immunization Procedure

[0072]

[0073] 3) Preparation of hybridoma cells

[0074] After immunization, blood was collected from the submandibular vein of mice, and antibody titers were detected by indirect ELISA. The coating antigen was TFF1-H02. The method for detecting antibody titers by indirect ELISA is as follows:

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

[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 buffer (PBS containing 5% skim milk powder) to each well and block at 37°C for 1 hour;

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

[0079] e. Add 100 μL of serum (i.e., primary antibody, diluted 1000, 4000, 16000, 64000, and 256000 times respectively) to each well; 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) with PBS to 1 μg / mL, 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 of 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 stop solution (2M H2SO4) to each well.

[0082] h. Absorbance detection and data analysis: The absorbance (OD value) of each well was read at 450 nm using a microplate reader. Each well was measured in parallel three times, and the average value was calculated.

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

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

[0085]

[0086] Based on 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. The spleens of the mice were 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 entire process was carried out under a 37℃ water bath. The process was terminated by adding preheated blank culture medium. After centrifugation, HAT conditioned medium was added for pressure screening.

[0087] 4) Monoclonal antibody screening

[0088] Most of the fused cells were multiple clones and needed to be plated for monoclonalization. The positive hybridoma cells were diluted to a density of 0.5-1 cells per well using a limiting dilution method and seeded into 96-well plates to ensure that each well contained cells derived from a single ancestral cell. The titer was detected by indirect ELISA using the TFF1-H02 coating antigen, and the titer detection method was the same as that used for detecting mouse serum titers (the primary antibody was the supernatant of positive hybridoma cells diluted at different folds). Wells containing positive hybridoma cell supernatants with an OD450 value greater than 2.0 at a 1000-fold dilution and belonging to monoclonal cell clusters were expanded to 24-well plates for culture. The supernatant after monoclonal cell expansion was repeatedly tested for antibodies to eliminate false positive results caused by cell mixing. Ten monoclonal hybridoma cell lines with stable antibody secretion and strong proliferative capacity were selected through ELISA OD450 titer detection and IgG antibody expression level screening. The ELISA OD450 titers (positive hybridoma cell supernatant diluted 1000-fold) of ten monoclonal antibodies and the expression levels of IgG antibodies are shown in Table 3 below.

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

[0090]

[0091] According to the data in Table 3, the ELISA OD450 results showed that these ten antibodies exhibited 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 were tested. The affinity test 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 to TFF1-H02

[0094]

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

[0096] Three high-purity TFF1 monoclonal antibodies were obtained by producing and purifying TFF1-2, TFF1-3, and TFF1-9 antibodies using corresponding hybridoma cells. Taking TFF1-2 and TFF1-3 monoclonal antibodies as examples, the purified antibodies produced by hybridoma cells were analyzed by SDS-PAGE (4-12% 12-well protein gel purchased from GenScript, electrophoresis parameters: 180V, 25min). Figure 2 As shown.

[0097] In this embodiment, circular dichroism (CD) chromatography was used to determine the thermal stability (Tm) value of the screened antibodies to evaluate their stability and compare it with existing antibodies. The existing TFF1 antibody was purchased from AmyJet Scientific, model MAB11350. The test results are shown in Table 5. 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 variation. A higher Tm indicates better stability.

[0098] Table 5. Tm values ​​for different TFF1 values

[0099]

[0100] According to the results in Table 5, the Tm values ​​of the TFF1 antibodies screened in this embodiment are all higher than those of commercially available TFF1 antibodies, indicating that the screened TFF1 antibodies have better stability.

[0101] 2. Reagent kit preparation

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

[0103] The TFF1-2, TFF1-3, and FF1-9 monoclonal antibodies were paired up to prepare coating antibodies or enzyme-labeled antibodies, and then combined into a kit, as shown in Table 6 below.

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

[0105]

[0106] As shown in Table 6, the three monoclonal antibodies can be paired to create six different kits.

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

[0108] (1) Fixation of coated antibodies:

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

[0110] 2) Wrap the baby in a blanket at 4℃ overnight (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) Blocking nonspecific sites:

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

[0114] 2) Discard the sealing 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, then wash 3 times;

[0117] 2) Dilute the enzyme-labeled antibody to 1 μg / mL with sample diluent, 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 of reaction: Add 100 μL of TMB substrate to each well and develop color at room temperature in the dark for 10-15 minutes (preliminary experiments are needed to optimize the time); immediately add 50 μL of stop solution (2M H2SO4) to each well.

[0119] 4) Absorbance detection and data analysis: The absorbance (OD value) of each well was read using an ELISA reader at 450 nm (main wavelength) and 630 nm (reference wavelength); the concentration was calculated based on the fitted 4-parameter Logistic curve.

[0120] The steps for preparing the fitted 4-parameter Logistic curve are as follows:

[0121] TFF1 standard was prepared at six concentrations: 0, 5, 25, 50, 100, and 200 ng / mL. The absorbance (OD value) at 450 nm (main wavelength) was measured using the method described above. Each concentration was measured in triplicate, and the average value was calculated. A 4-parameter logistic curve was fitted using software simulation, resulting in six fitted 4-parameter logistic curves. Taking a kit consisting of combination 1 with TFF1-2 coated antibody and TFF1-3 enzyme-labeled antibody as an example, its fitted 4-parameter logistic curve is shown below. Figure 3 As shown, the estimated values ​​for the four parameters are: A = 0.052, standard deviation = 0.018, confidence interval = [0.002, 0.102]; B = 1.111, standard deviation = 0.040, confidence interval = [0.999, 1.224]; C = 1.310, standard deviation = 0.055, confidence interval = [1.157, 1.462]; D = 2.475, standard deviation = 0.041, confidence interval = [2.361, 2.589]. Based on the calculations of the four parameters... R 2=1.000, EC50=1.310 nM.

[0122] Furthermore, intra-batch / inter-batch precision was investigated using a TFF1 protein control solution (prepared from negative human serum) containing 5 ng / mL. The results are shown in Table 7 below.

[0123] Table 7. Intra-assay / Inter-assay Precision of TFF1 Detection Kit

[0124]

[0125] According to the data in Table 7, kits 1 and 2 have smaller intra-batch / inter-batch CV values ​​and closer to 100% recovery rates, indicating that kits 1 and 2 are more stable and accurate. Both kits 1 and 2 use TFF1-2 as the coating antibody, which is used to capture TFF1 in the test samples. This stronger specificity reduces the probability of false positives and increases accuracy. Therefore, kits 1 and 2 are preferred for detecting TFF1.

[0126] Example 2: Preparation, screening, and kit preparation of monoclonal antibodies

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

[0128] 1) Preparation of antigen

[0129] AAT sequences were retrieved using bioinformatics, and two antigens were designed: AAT-H01 (amino acid sequence as shown in SEQ ID NO. 52, nucleotide sequence as shown in SEQ ID NO. 56) and AAT-H02 (amino acid sequence as shown in SEQ ID NO. 53, nucleotide sequence as shown in SEQ ID NO. 57). To enhance the immunogenicity of the antigens, the following measures were taken during antigen design, purification, and immunization: 1. A Kozak sequence was added, which mainly enhances translation efficiency, ensures accuracy, optimizes exogenous gene expression, and regulates expression levels; 2. Immunization was performed using dimers, resulting in stronger immunogenicity; 3. Monomer screening was conducted to ensure that the obtained antibodies target a single epitope; 4. A 6×His tag was added to facilitate purification, yielding higher antigen quantities and significantly improving antigen purity; 5. Boosting immunization was performed during the immunization process, with direct spleen injection to obtain higher antibody titers. Protein plasmid synthesis was commissioned to GenScript, and the obtained plasmids were transfected into eukaryotic cells for expression and purification to obtain the corresponding antigens. The SDS-PAGE results of the purified AAT-H01 antigen are shown below. Figure 4 As shown. The AAT-H01 antigen is used for immunization and hybridoma cell fusion, while the AAT-H02 antigen is used for screening.

[0130] 2) Animal immunization

[0131] Five BALB / c mice were immunized with the AAT-H01 antigen, following the same immunization procedure as Table 1 in Example 1.

[0132] 3) Preparation of hybridoma cells

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

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

[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 buffer (PBS containing 5% skim milk powder) to each well and block at 37°C for 1 hour;

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

[0138] e. Add 100 μL of serum (i.e., primary antibody, 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) with PBS to 1 μg / mL, 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 stop solution (2M H2SO4) to each well.

[0141] h. Absorbance detection and data analysis: The absorbance (OD value) of each well was read at 450 nm using a microplate reader. Each well was measured in parallel three times, and the average value was calculated.

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

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

[0144]

[0145] Based on the test results in Table 8, blood samples were collected from the eyeballs of mice with serum diluted 64,000 times and OD450 greater than 0.2 as positive controls. The mouse spleens were isolated, ground, counted, and fused according to a ratio of spleen cell number:sp2 / 0 cell number = 8:1. 1 mL of PEG1450 was added. The entire process was carried out under 37℃ water bath conditions. Preheated blank culture medium was added to terminate the process. After centrifugation, HAT conditioned medium was added for pressure screening.

[0146] 4) Monoclonal antibody screening

[0147] Most of the fused cells were multiple clones and needed to be plated for monoclonalization. The positive hybridoma cells were diluted to a density of 0.5-1 cells per well using a limiting dilution method and seeded into 96-well plates to ensure that each well contained cells derived from a single ancestral cell. The titer was detected by indirect ELISA using AAT-H02 as the coating antigen. The titer detection method was the same as that used for detecting mouse serum titers (the primary antibody was the supernatant of positive hybridoma cells diluted at different folds). Wells containing positive hybridoma cell supernatants with an OD450 value greater than 1.5 at a 1000-fold dilution and belonging to monoclonal cell clusters were expanded to 24-well plates. The supernatant after monoclonal cell expansion was repeatedly tested for antibodies to eliminate false positives caused by cell mixing. Ten monoclonal hybridoma cell lines with stable antibody secretion and strong proliferative capacity were selected through ELISA OD450 titer detection and IgG antibody expression level screening. The ELISA OD450 titers (positive hybridoma cell supernatant diluted 1000-fold) of ten monoclonal antibodies and the expression levels of IgG antibodies are shown in Table 9 below.

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

[0149]

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

[0151] Furthermore, the affinity of the ten monoclonal antibodies in Table 9 for AAT-H02 and their cross-reactivity with TFF1 were tested. The affinity test 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 to AAT-H02

[0153]

[0154] Based on the data in Table 10, it was found that the antibody with the highest OD450 detection titer 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 selected: AAT-2 (the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 are SEQ ID NO. 17-19, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are SEQ ID NO. 20-22; 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) and AAT-6 (the amino acid sequences of the heavy chain CDR1 and CDR2 are SEQ ID NO. 23-24, the amino acid sequence of the heavy chain CDR3 is FDY, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are SEQ ID NO. 25-27; the amino acid sequence of the heavy chain variable region is SEQ ID NO. 29). 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 that they have high specificity.

[0155] Two high-purity AAT monoclonal antibodies, AAT-2 and AAT-6, were obtained through the production and purification of corresponding hybridoma cells. Taking AAT-2 and AAT-6 monoclonal antibodies as examples, the results of SDS-PAGE (4-12% 12-well protein gel purchased from GenScript) analysis after purification of the antibodies produced by hybridoma cells are shown below. Figure 5 As shown.

[0156] 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 AAT antibody was purchased from AmyJet Scientific, model ABM40300. The test results are shown in Table 11. 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 variation. A higher Tm indicates better stability.

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

[0158]

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

[0160] 2. Reagent kit preparation

[0161] The AAT enzyme-linked immunosorbent assay kit includes: coating antibody, carbonate buffer (pH 9.6), washing buffer (PBS + 0.1% Tween 20), sample diluent, blocking buffer (PBS containing 5% skim milk powder), enzyme-labeled antibody, TMB substrate, and stop solution (2M H2SO4).

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

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

[0164]

[0165] As shown in Table 12, a pair of antibodies can be used to make two kits.

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

[0167] (1) Fixation of coated antibodies:

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

[0169] 2) Wrap the baby in a blanket at 4℃ 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) Blocking nonspecific sites:

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

[0173] 2) Discard the sealing 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, then wash 3 times;

[0176] 2) Dilute the enzyme-labeled antibody to 1 μg / mL with sample diluent, 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 of reaction: Add 100 μL of TMB substrate to each well and develop color at room temperature in the dark for 10-15 minutes (preliminary experiments are needed to optimize the time); immediately add 50 μL of stop solution (2M H2SO4) to each well.

[0178] 4) Absorbance detection and data analysis: The absorbance (OD value) of each well was read using an ELISA reader at 450 nm (main wavelength) and 630 nm (reference wavelength); the concentration was calculated based on the fitted 4-parameter Logistic curve.

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

[0180] AAT standards were prepared at six concentrations: 0, 10, 50, 100, 250, and 500 ng / mL. The absorbance (OD value) at 450 nm (main wavelength) was measured using the method described above. Each concentration was measured in triplicate, and the average value was calculated. A 4-parameter logistic curve was fitted using software simulation, resulting in two fitted 4-parameter logistic curves. Taking a kit consisting of AAT-2 coated antibody and AAT-6 enzyme-labeled antibody as an example, its fitted 4-parameter logistic curve is shown below. Figure 6 As shown, the estimated values ​​for the four parameters are: A = 0.083, standard deviation = 0.043, confidence interval = [-0.054, 0.220]; B = 1.251, standard deviation = 0.112, confidence interval = [0.896, 1.607]; C = 5.964, standard deviation = 0.941, confidence interval = [2.970, 8.958]; D = 5.064, standard deviation = 0.470, confidence interval = [3.567, 6.561]. Based on the calculations of the four parameters... R 2 =0.999, EC50=5.964 nM.

[0181] Furthermore, intra-batch / inter-batch precision was investigated using AAT protein control solutions (prepared from negative human serum) containing 10 ng / mL. The results are shown in Table 13 below.

[0182] Table 13. Intra-assay / Inter-assay Precision of AAT Detection Kits

[0183]

[0184] Based on the data in Table 13, Kit 1 has a smaller intra-batch / inter-batch CV value and a recovery rate closer to 100%, indicating that Kit 1 is more stable and accurate. Therefore, Kit 1 is the preferred AAT kit.

[0185] Example 3: Application of TFF1 and AAT enzyme-linked immunosorbent assay kits, alone and in combination, in the diagnosis of colorectal cancer.

[0186] 1. Diagnostic efficacy of TFF1 and AAT enzyme-linked immunosorbent assay kits, used alone and in combination.

[0187] In this experiment, the TFF1 and AAT enzyme-linked immunosorbent assay (ELISA) kits constructed in Examples 1 and 2 were used to test TFF1 and AAT levels in the serum of healthy subjects (100 cases) and rectal cancer patients (100 cases), respectively, to evaluate their ability to distinguish between diseased populations. The 100 healthy subjects were matched for age and sex with the rectal cancer patient group and had no history of rectal cancer or other digestive system diseases; the rectal cancer patient group included 100 pathologically confirmed rectal cancer patients, covering different clinical stages (stages I-IV).

[0188] Venous blood was collected from healthy subjects and patients with rectal cancer, 5 mL / sample. Serum was separated by centrifugation (3000 rpm, 15 min), aliquoted, and stored at -80°C for later use. Two hundred samples were independently tested using the preferred TFF1 ELISA kits 1 and 2 from Example 1 and the preferred AAT ELISA kit 1 from Example 2. Absorbance values ​​were recorded, and the concentration of the target protein in the serum was calculated. Simultaneously, commercially available TFF1 antibody (AmyJet Pharmaceutical Industries Co., Ltd., model MAB11350) from Example 1 and commercially available AAT antibody (AmyJet Pharmaceutical Industries Co., Ltd., model ABM40300) from Example 2 were prepared into corresponding ELISA kits for comparison. The ability to differentiate between diseased populations was evaluated using SPSS or R.

[0189] When TFF1 is used alone as a biomarker, the kits are the preferred TFF1 enzyme-linked immunosorbent assay kits 1 and 2 in Example 1 and the corresponding enzyme-linked immunosorbent assay kits made from the commercially available TFF1 antibody in Example 1. The cutoff value for TFF1 content used to distinguish whether a patient is a colorectal cancer patient is 0.5 ng / mL. Patients with a TFF1 content greater than or equal to 0.5 ng / mL are judged to be colorectal cancer patients, and those with a TFF1 content less than 0.5 ng / mL are judged to be healthy individuals.

[0190] When AAT is used alone as a biomarker, the kit is the preferred AAT ELISA kit 1 in Example 2 and the corresponding ELISA kit made from the commercially available AAT antibody in Example 2. The cutoff value for AAT content used to distinguish whether a patient has colorectal cancer is 5.0 ng / mL. A level greater than or equal to 5.0 ng / mL is considered a colorectal cancer patient, and a level less than 5.0 ng / mL is considered a healthy person.

[0191] When TFF1 and AAT are used together as biomarkers, the kit consists of TFF1 ELISA kit 1 + AAT ELISA kit 1, and a combined diagnostic model (TFF1 + AAT) is used for the diagnosis of colorectal cancer. The logistic regression formula for constructing the TFF1 ELISA kit 1 + AAT ELISA kit 1 is: Risk score = 1.32 × ln(TFF1) + 0.87 × ln(AAT) (threshold = 0.5; a risk score greater than or equal to 0.5 indicates a colorectal cancer patient, and less than 0.5 indicates a healthy individual). The receiver operating characteristic (ROC) curves and AUC values ​​for the combined detection and individual detection of the kits are shown below. Figure 7 As shown.

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

[0193]

[0194] The data in Table 14 show that the AUC value of the TFF1 ELISA kit combined with the AAT ELISA kit is higher than that of either kit alone, indicating that the combined use of the two kits has higher diagnostic efficacy for colorectal cancer. In particular, the kit prepared from the TFF1 monoclonal antibody and AAT monoclonal antibody screened in this invention exhibits significantly higher diagnostic efficacy when used in combination.

[0195] 2. Differences in diagnostic efficacy when using TFF1 and AAT enzyme-linked immunosorbent assay kits in combination.

[0196] This experiment combined the individual detection kits listed in Table 14 above into three combinations: First: TFF1 ELISA kit 1 + AAT ELISA kit 1; Second: TFF1 ELISA kit 2 + AAT ELISA kit 1; Third: TFF1 ELISA kit prepared with commercially available TFF1 antibody + TFF1 ELISA kit prepared with commercially available AAT antibody. A combined diagnostic model (TFF1+AAT) was used to diagnose colorectal cancer. Test samples included serum from 200 healthy individuals and 200 colorectal cancer patients, randomly divided into a test group and a validation group. The test group included serum from 100 healthy individuals and 100 colorectal cancer patients, and the validation group included serum from 100 healthy individuals and 100 colorectal cancer patients.

[0197] In the first kit combination, the logistic regression formula for constructing the TFF1 ELISA kit 1 + AAT ELISA 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, the patient is judged to be a colorectal cancer patient; when it is less than 0.5, the patient is judged to be a healthy person).

[0198] In the second kit combination, the logistic regression formula for constructing the TFF1 ELISA kit 1 + AAT ELISA 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, the patient is judged to be a colorectal cancer patient, and when it is less than 0.5, the patient is judged to be a healthy person).

[0199] In the third kit combination, the logistic regression formula for the TFF1 enzyme-linked immunosorbent assay kit prepared with commercially available TFF1 antibody + the TFF1 enzyme-linked immunosorbent assay kit prepared with 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, the patient is judged to be a colorectal cancer patient, and when it is less than 0.5, the patient is judged to be a healthy person).

[0200] Table 15. Detection results of samples using the combined TFF1 and AAT enzyme-linked immunosorbent assay kit.

[0201]

[0202] According to the data in Table 15, although the biomarkers detected were both TFF1 and AAT proteins, and based on the same combined diagnostic model, the diagnostic results varied significantly depending on the antibodies used. This may be because existing antibodies not only have low detection sensitivity, but the results also differ significantly depending on whether the antibody is used as the detection antibody or the enzyme-labeled antibody. Furthermore, different antibodies exhibit significant differences in stability (Tm), cross-reactivity with the matrix, and high-dose hook effects, all of which contribute to decreased diagnostic efficacy for colorectal cancer.

[0203] Meanwhile, in the diagnosis of colorectal cancer, blood samples need to be tested using TFF1 antibody and / or AAT antibody. Blood samples contain complex interfering substances, have a significant matrix effect, are prone to cross-reaction, or are interfered by some nonlinear fragments. Using different antibodies for colorectal cancer detection and diagnosis will inevitably produce completely different diagnostic results. Therefore, it is necessary to select a better combination of antibodies to improve diagnostic efficacy.

[0204] Therefore, the preferred antibody combination provided by this invention, including TFF1-2 antibody (i.e., the first antibody of TFF1 monoclonal antibody) and TFF1-3 antibody (i.e., the second antibody of TFF1 monoclonal antibody), AAT-2 antibody (i.e., the first antibody of AAT monoclonal antibody) and AAT-6 antibody (i.e., the second antibody of AAT monoclonal antibody), constructs detection kits for two biomarkers (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] In summary, the combined use of TFF1 and AAT enzyme-linked immunosorbent assay kits significantly improves the early diagnostic efficacy of rectal cancer, providing a reliable basis for clinical application.

[0206] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A kit for detecting TFF1 protein and AAT protein, characterized in that, This includes a TFF1 monoclonal antibody and an AAT monoclonal antibody; the TFF1 monoclonal antibody comprises a first antibody and a second antibody; the first antibody comprises: (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 (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; The second antibody includes: (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 (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; The AAT monoclonal antibody comprises a first antibody and / or a second antibody; the first antibody comprises: (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 (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; The second antibody includes: (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 FDY in the variable region of the heavy chain, and (4) CDR1 composed of the amino acid sequence of SEQ ID NO.25, CDR2 composed of the amino acid sequence of SEQ ID NO.26 and CDR3 composed of the amino acid sequence of SEQ ID NO.27 in the variable region of the light chain.

2. The kit according to claim 1, characterized in that, In the TFF1 monoclonal 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. In the AAT monoclonal 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.

3. The use of an antibody in the preparation of a reagent for predicting whether an individual has colorectal cancer, characterized in that, The antibody includes a TFF1 monoclonal antibody and an AAT monoclonal antibody; the TFF1 monoclonal antibody includes a first antibody and a second antibody; the first antibody includes: (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 (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; The second antibody includes: (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 (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; The AAT monoclonal antibody comprises a first antibody and / or a second antibody; the first antibody comprises: (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 (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; The second antibody includes: (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 FDY in the variable region of the heavy chain, and (4) CDR1 composed of the amino acid sequence of SEQ ID NO.25, CDR2 composed of the amino acid sequence of SEQ ID NO.26 and CDR3 composed of the amino acid sequence of SEQ ID NO.27 in the variable region of the light chain.

4. The use as described in claim 3, characterized in that, In the TFF1 monoclonal 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. In the AAT monoclonal 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.

5. A kit for predicting whether an individual has colorectal cancer, characterized in that, Includes TFF1 monoclonal antibody and AAT monoclonal antibody; the TFF1 monoclonal antibody includes a first antibody and a second antibody; the first antibody includes: (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 (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; The second antibody includes: (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 (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; The AAT monoclonal antibody comprises a first antibody and / or a second antibody; the first antibody comprises: (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 (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; The second antibody includes: (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 FDY in the variable region of the heavy chain, and (4) CDR1 composed of the amino acid sequence of SEQ ID NO.25, CDR2 composed of the amino acid sequence of SEQ ID NO.26 and CDR3 composed of the amino acid sequence of SEQ ID NO.27 in the variable region of the light chain.

6. The reagent kit as described in claim 5, characterized in that, In the TFF1 monoclonal 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. In the AAT monoclonal 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.

7. An antibody combination for predicting whether an individual has colorectal cancer, characterized in that, Includes TFF1 monoclonal antibody and AAT monoclonal antibody; the TFF1 monoclonal antibody includes a first antibody and a second antibody; the first antibody includes: (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 (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; The second antibody includes: (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 (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; The AAT monoclonal antibody comprises a first antibody and / or a second antibody; the first antibody comprises: (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 (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; The second antibody includes: (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 FDY in the variable region of the heavy chain, and (4) CDR1 composed of the amino acid sequence of SEQ ID NO.25, CDR2 composed of the amino acid sequence of SEQ ID NO.26 and CDR3 composed of the amino acid sequence of SEQ ID NO.27 in the variable region of the light chain.

8. The antibody combination as described in claim 7, characterized in that, In the TFF1 monoclonal 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. In the AAT monoclonal 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.

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. The antigen is detected using antibodies, including TFF1 monoclonal antibody and AAT monoclonal antibody. The TFF1 monoclonal antibody includes a first antibody and a second antibody. The first antibody includes: (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 (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; The second antibody includes: (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 (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; The AAT monoclonal antibody comprises a first antibody and / or a second antibody; the first antibody comprises: (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 (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; The second antibody includes: (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 FDY in the variable region of the heavy chain, and (4) CDR1 composed of the amino acid sequence of SEQ ID NO.25, CDR2 composed of the amino acid sequence of SEQ ID NO.26 and CDR3 composed of the amino acid sequence of SEQ ID NO.27 in the variable region of the light chain.

10. The system as described in claim 9, characterized in that, In the TFF1 monoclonal 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. In the AAT monoclonal 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.

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