Antibodies to TFF3 and OSM proteins and their combination and use in the diagnosis of colorectal cancer

Abstract

The present invention provides antibodies to TFF3 and OSM proteins, their combination, and their application in the diagnosis of colorectal cancer. By optimizing the immunogenic sequences of TFF3 and OSM, phage display technology is used to screen monoclonal antibodies with high affinity and high specificity. Directed mutagenesis and combinatorial library screening are then used to introduce mutations in the CDR regions of the antibodies to screen out the optimal TFF3 and OSM antibodies. This can effectively solve the problems of low affinity and specificity of existing antibodies, extend the detection range, and demonstrate extremely high collaborative diagnostic value in combined detection, significantly improving the diagnostic efficacy of colorectal cancer and having important clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody screening and disease diagnosis, and in particular to antibodies against TFF3 and OSM proteins and combinations thereof and their applications in the diagnosis of colorectal cancer. Background Art

[0002] Digestive system tumors are a common cancer worldwide, with colorectal cancer (CRC) contributing particularly high morbidity and mortality. CRC is the third most common cancer worldwide, with over 1.9 million new cases expected in 2025 (accounting for 10% of all new cancers). While the five-year survival rate for patients with early-stage (Stage I) cancer can reach 90%, for patients with advanced (Stage IV) cancer, it is only 10-15%, highlighting the urgency of early diagnosis.

[0003] Recent proteomic studies have revealed that TFF3 (trefoil factor 3) is abnormally overexpressed in the serum of CRC patients (5-8 times higher than in healthy controls). OSM (oncostatin M), a cytokine of the IL-6 family, promotes CRC metastasis by activating the STAT3 pathway, and its serum level is positively correlated with tumor stage (3.5 times higher in advanced patients compared to early-stage patients). This suggests the diagnostic value of TFF3 and / or OSM in gastrointestinal tumors, particularly colorectal cancer.

[0004] Although both TFF3 and OSM have been reported to be associated with CRC, the existing technology has the following gaps: 1) Lack of standardized detection tools: Currently, only research institutions use academic methods such as ELISA for detection, and there are no commercial kits, making accurate detection difficult; 2) Unexplored collaborative diagnostic value: There is no evidence that the combination of TFF3 and OSM can improve diagnostic efficacy (indicators such as AUC values ​​have not been reported in the literature).

[0005] CN101706497A discloses an ELISA detection kit for human TFF3 for general laboratory or clinical research on gastrointestinal pathology. However, it lacks antigen optimization, resulting in an unstable immunogen sequence and a tendency for antibody performance to decline due to interference from glycosylation sites. Furthermore, the antibody sequence has not been optimized for mutations, making it difficult to accurately detect low-concentration targets. The kit also suffers from insufficient sensitivity and is prone to cross-reactions, making it prone to errors in colorectal cancer diagnosis.

[0006] Patent EP2236625A1 (active) discloses a method for detecting TFF3 gene or protein for establishing prognosis regarding progression of patients diagnosed with colorectal cancer after administration of therapy, but not for prediction of early colorectal cancer and not for use in combination with OSM.

[0007] Patent CN118670830B discloses that TFF3 can be used to predict early stage colorectal cancer, but it is not used in combination with OSM. Furthermore, there are currently no reports on the combined detection of TFF3 and OSM for the diagnosis of colorectal cancer.

[0008] In addition, current commercial anti-human TFF3 antibodies (such as ab202967 and ab108599) are only validated for use in tissue sections (IHC-P) or flow cytometry (Flow Cyt), and cannot meet the needs of TFF3 detection in serum samples.

[0009] Currently, the development of OSM (oncostatin M, a cytokine in the IL-6 family) antibodies is biased towards therapeutic applications. For example, CN103328508B discloses an antigen-binding protein (humanized antibody) targeting OSM for use in pharmaceutical compositions for the treatment and prevention of inflammatory diseases and conditions. It is not intended for the prediction of colorectal cancer and is not used in combination with TFF3. Furthermore, the antigen and antibody sequences have not been mutated or optimized, resulting in unstable immunogen sequences and the potential for antibody performance degradation due to interference from glycosylation sites. This makes it difficult to accurately detect low-concentration targets, and the assay lacks sensitivity and is prone to cross-reactivity, making it prone to errors in colorectal cancer diagnosis.

[0010] Therefore, there is an urgent need to find antibodies that can detect TFF3 protein and OSM protein with high sensitivity and specificity, and have stable performance, so as to more accurately diagnose colorectal cancer. Summary of the Invention

[0011] In response to the problems existing in the prior art, the present invention provides antibodies to TFF3 and OSM proteins, their combination, and their application in the diagnosis of colorectal cancer. By optimizing the immunogenic sequences of TFF3 and OSM, phage display technology is used to screen monoclonal antibodies with high affinity and high specificity. Directed mutagenesis and combinatorial library screening are then used to introduce mutations in the CDR regions of the antibodies to screen out the optimal TFF3 and OSM antibodies. These antibodies can effectively solve the problems of low affinity and specificity of existing antibodies, have a wider detection range, and demonstrate extremely high collaborative diagnostic value in combined detection, significantly improving the diagnostic efficacy of early colorectal cancer and having important clinical application value.

[0012] Existing approaches to producing monoclonal or polyclonal antibodies against TFF3 or OSM proteins using traditional hybridoma technology, phage display technology, or recombinant protein immunization in animals suffer from poor immunogen design, resulting in insufficient antibody affinity and specificity. Furthermore, existing ELISA or chemiluminescence assays for detecting TFF3, OSM proteins, or gastrointestinal tumor markers lack kit optimization for combinations of TFF3 and OSM antibodies, resulting in low sensitivity and specificity and the inability to efficiently diagnose gastrointestinal tumors (particularly colorectal cancer). The present invention has developed novel, highly specific antibodies against TFF3 and OSM, and their combinations, which can be used in the preparation of diagnostic kits to detect TFF3 and OSM in various samples, thereby realizing their clinical value.

[0013] In one aspect, the present invention provides an antibody against a TFF3 protein, comprising a first antibody and / or a second antibody; the first antibody comprising:

[0014] (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.9, a CDR2 consisting of the amino acid sequence of SEQ ID NO.10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.17, and

[0015] (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 19, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 2;

[0016] The second antibody comprises:

[0017] (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 13, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 14, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 20, and

[0018] (4) In the light chain variable region, CDR1 consists of the amino acid sequence of SEQ ID NO. 21, CDR2 consists of the amino acid sequence of SEQ ID NO. 16, and CDR3 consists of the amino acid sequence of SEQ ID NO. 8.

[0019] This study used bioinformatics analysis to perform a multiple sequence alignment of TFF3 amino acid sequences (UniProt No.: Q07654) from human, mouse, rat, and other species to identify human-specific and highly conserved linear epitopes, while avoiding the known disulfide bond core regions (C32-C58, C42-C57, and C52-C69) to ensure immunogen stability. The designed immunogen was prepared by immunizing BALB / c mice with complete and incomplete adjuvants 4-6 times, with 14-day intervals between immunizations. Immune lymphocytes were isolated from the spleens of mice immunized with TFF3 protein, and a phage display library was constructed.

[0020] Phage display technology was used to screen for high-affinity, high-specificity monoclonal antibodies targeting the EP1 and EP2 epitopes. The present invention employed four rounds of progressive screening. The resulting TFF3 primary and secondary antibodies both recognized native TFF3 clones, exhibited no EP2 dimerization obscuration, and exhibited no cross-reactivity with other members of the TFF family.

[0021] The antibodies obtained after four rounds of screening were then mutated in the CDR region to screen out mutants with higher affinity. After verification by ELISA and SPR, the sensitivity of the optimized antibodies was increased to the 100pg / mL level, and the specificity was further enhanced. It is suitable for the precise detection of low-concentration targets and can detect TFF3 protein with higher accuracy. It has higher diagnostic value when used alone or in combination with other antibodies to diagnose gastrointestinal tumors, such as colorectal cancer.

[0022] Furthermore, the amino acid sequence of the heavy chain variable region of the first antibody (TFF3-Ab1-1) is shown as SEQ ID NO.23, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody (TFF3-Ab4-1) is shown as SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.26.

[0023] Furthermore, the amino acid sequence of the heavy chain of the first antibody (TFF3-Ab1-1) is shown in SEQ ID NO.53, and the amino acid sequence of the light chain is shown in SEQ ID NO.54; the amino acid sequence of the heavy chain of the second antibody (TFF3-Ab4-1) is shown in SEQ ID NO.55, and the amino acid sequence of the light chain is shown in SEQ ID NO.56.

[0024] In another aspect, the present invention provides a kit for detecting TFF3 protein, wherein the kit comprises the antibody against TFF3 protein as described above.

[0025] It is understandable that the kit may be an ELISA detection kit, a chemiluminescence immunoassay kit, or the like.

[0026] In some embodiments, the kit is a chemiluminescent immunoassay kit, and a chemiluminescent immunoassay kit is constructed by screening the optimal and appropriately paired TFF3 primary antibody and secondary antibody.

[0027] In another aspect, the present invention provides an antibody against OSM protein, comprising a first antibody and / or a second antibody; the first antibody comprises:

[0028] (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 35, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 43, and

[0029] (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 45, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 28;

[0030] The second antibody comprises:

[0031] (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.39, a CDR2 consisting of the amino acid sequence of SEQ ID NO.40, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.46, and

[0032] (4) In the light chain variable region, CDR1 consists of the amino acid sequence of SEQ ID NO. 48, CDR2 consists of the amino acid sequence of SEQ ID NO. 42, and CDR3 consists of the amino acid sequence of SEQ ID NO. 32.

[0033] This study uses bioinformatics methods to analyze the OSM protein amino acid sequence, identifying stable structured regions and linear regions with high exposure. Furthermore, known glycosylation sites (glycosylated amino acid residues in proteins) are avoided. This optimized design resulted in the human-specific linear epitopes EP1 and EP2, significantly improving the affinity and specificity of the resulting antibodies and avoiding the performance degradation associated with interference from glycosylation sites.

[0034] In our immunogen design strategy targeting OSM protein, we specifically engineered two functional epitopes and optimized their KLH conjugation. The capture antibody targets an epitope located in the 69-88aa region of the OSM protein (KLREHARERPGAFPSEETLR). This epitope was mutated to alanine (C74A) at C74 to prevent disulfide bond interference. A CGG linker was added at the C-terminus, allowing conjugation to the KLH carrier protein via its free sulfhydryl (-SH) group (OSM immunogen 1). The detection antibody targets an epitope located in the 110-123aa region (LADLEQRLPKAQDL). A CGG sequence was pre-added to the N-terminus and conjugated to KLH via the sulfhydryl group (OSM immunogen 2). Mice were immunized with OSM immunogens 1 and 2, and splenocytes were harvested to construct two independent phage antibody libraries.

[0035] Four parallel rounds of screening were established for OSM immunogens 1 and 2, respectively, to obtain high-affinity and high-specificity monoclonal antibodies against OSM. The antibodies obtained through screening were then mutated in the CDR region to screen out mutants with higher affinity. After verification by ELISA and SPR, the sensitivity of the optimized antibodies was increased to the 0.1pg / mL level, and the specificity was further enhanced, making them suitable for the precise detection of low-concentration targets and capable of detecting OSM protein with higher precision. They have higher diagnostic value when used alone or in combination with other antibodies for the diagnosis of gastrointestinal tumors, such as colorectal cancer.

[0036] Furthermore, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO.49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.52.

[0037] Furthermore, the amino acid sequence of the heavy chain of the first antibody (OSM-Ab1-1) is shown in SEQ ID NO. 57, and the amino acid sequence of the light chain is shown in SEQ ID NO. 58; the amino acid sequence of the heavy chain of the second antibody (OSM-Ab1-3) is shown in SEQ ID NO. 59, and the amino acid sequence of the light chain is shown in SEQ ID NO. 60.

[0038] In another aspect, the present invention provides a kit for detecting OSM protein, wherein the kit comprises the antibody against OSM protein as described above.

[0039] It is understandable that the kit may be an ELISA detection kit, a chemiluminescence immunoassay kit, or the like.

[0040] In some embodiments, the kit is a chemiluminescent immunoassay kit, and a double-antibody combination chemiluminescent immunoassay kit is constructed by screening the optimal and appropriately paired OSM primary antibody and secondary antibody.

[0041] In yet another aspect, the present invention provides a kit for detecting TFF3 protein and / or OSM protein, wherein the kit comprises the antibody against TFF3 protein as described above, and / or the antibody against OSM protein as described above.

[0042] In some methods, a kit for detecting TFF3 protein and a kit for detecting OSM protein are combined to simultaneously detect TFF3 protein and OSM protein, thereby being applicable to fields requiring simultaneous detection of multiple markers, such as gastrointestinal tumor diagnosis, to improve diagnostic efficacy through combined detection.

[0043] In another aspect, the present invention provides a use of an antibody for preparing a reagent for predicting whether an individual has colorectal cancer, wherein the antibody comprises the antibody against the TFF3 protein as described above, and / or the antibody against the OSM protein as described above.

[0044] Although TFF3 and OSM are known diagnostic markers for colorectal cancer, the diagnostic efficacy of different TFF3 and / or OSM antibodies varies significantly. This is because the diagnosis of colorectal cancer requires testing blood samples with TFF3 and / or OSM antibodies, which contain complex interfering substances, a significant matrix effect, and are prone to cross-reactions and interference from nonlinear fragments. Direct testing with existing antibodies is difficult to achieve ideal results, severely impacting diagnostic accuracy.

[0045] After multiple rounds of screening, including screening of antigen epitopes, screening of monoclonal antibodies, and mutation of antibody CDR sequences, the antibodies against TFF3 and OSM proteins obtained in the present invention have better detection sensitivity and specificity, more stable signals, and smaller cross-reactions. When used in the diagnosis of colorectal cancer, they can significantly improve the diagnostic efficacy.

[0046] Furthermore, it includes antibodies against TFF3 protein and OSM protein.

[0047] Furthermore, the antibody to the TFF3 protein includes 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.23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.26;

[0048] The OSM protein 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.49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.52.

[0049] Furthermore, the reagent is used to detect the content of antigens capable of binding 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.

[0050] In some embodiments, the reagent for predicting the risk of colorectal cancer recurrence is a detection reagent prepared with TFF3 and OSM proteins as detection targets. The present invention uses antibodies to detect TFF3 and OSM proteins.

[0051] Furthermore, the reagent is used to detect the presence or relative abundance or concentration of biomarkers in a body fluid sample.

[0052] The present invention uses blood screening to identify biomarkers (TFF3 and OSM proteins) that predict the risk of colorectal cancer recurrence. These two markers show significant differences in the blood of colorectal cancer patients and healthy people. By collecting blood samples, the content of TFF3 and OSM proteins in the individual's blood can be detected. Based on the constructed prediction model, the possibility of the individual suffering from colorectal cancer can be predicted or assisted in diagnosis. Alternatively, the content of TFF3 and OSM proteins in the blood of a certain group of people can be detected, and the group can be divided into colorectal cancer patients and healthy people.

[0053] Furthermore, the Logistic regression formula of the model is:

[0054] Risk score = 0.23 × ln(TFF3) + 0.10 × ln(OSM);

[0055] Among them, TFF3 is the detection value of TFF3 protein, and OSM is the detection value of OSM protein. When the risk score is greater than or equal to 0.99, the patient is judged to be a colorectal cancer patient, and when it is less than 0.99, the patient is judged to be a healthy person.

[0056] In yet another aspect, the present invention provides a kit for predicting whether an individual has colorectal cancer, the kit comprising the antibody against the TFF3 protein as described above, and the antibody against the OSM protein as described above.

[0057] In another aspect, the present invention provides an antibody combination for predicting whether an individual has colorectal cancer, wherein the antibody combination comprises an antibody against the TFF3 protein as described above, and an antibody against the OSM protein as described above.

[0058] Furthermore, it includes antibodies against TFF3 protein and OSM protein.

[0059] Furthermore, the antibody to the TFF3 protein includes 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.23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.26;

[0060] The OSM protein 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.49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.52.

[0061] On the other hand, the present invention provides a system for predicting whether an individual has colorectal cancer, the system comprising a data analysis module for analyzing the detection value of an antigen, wherein the antigen is detected by an antibody, and the antibody comprises an antibody to the TFF3 protein as described above, and / or an antibody to the OSM protein as described above.

[0062] 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.

[0063] The beneficial effects of the present invention are:

[0064] 1. Innovative immunogen design: Optimize immunogen design through bioinformatics and avoid glycosylation sites (TFF3 / OSM) to enhance antibody stability and binding specificity (avoid nonspecific binding);

[0065] 2. Use phage display technology + affinity maturation (directed mutagenesis in the CDR region) to screen antibodies and increase the antibody affinity (KD value) to the nanomolar level;

[0066] 3. A new antibody sequence specifically recognizing TFF3 is provided, which can be used to construct a TFF3 detection kit. Combined with the double-antibody sandwich design, the cross-reactivity rate is extremely low (<2%), and the sensitivity reaches 100pg / mL, enabling high-precision detection of TFF3 protein.

[0067] 4. Provides a new antibody sequence that specifically recognizes OSM, which can be used to construct an OSM detection kit. Combined with the double-antibody sandwich design, it has an extremely low cross-reaction rate (<2%) and a sensitivity of 0.1 pg / mL, enabling high-precision detection of OSM protein.

[0068] 5. The diagnostic value of the test kit, alone or in combination, in gastrointestinal tumors, especially colorectal cancer; the combined detection model (TFF3+OSM logistic regression) significantly improved the discrimination ability compared with a single marker (AUC ≥ 0.96). BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The TFF3 detection standard curve of the TFF3 chemiluminescence kit constructed in Example 1;

[0070] Figure 2 The OSM detection standard curve of the OSM chemiluminescence kit constructed in Example 2;

[0071] Figure 3 The diagnostic efficacy of the colorectal cancer using the TFF3 and OSM chemiluminescent immunoassay kits in Example 3 alone or in combination;

[0072] Figure 4 This is the diagnostic efficacy of the optimal chemiluminescent immunoassay kit for colorectal cancer constructed in Example 3. DETAILED DESCRIPTION

[0073] The present invention will be described in further detail below in conjunction with the accompanying drawings and Examples. It should be noted that the following examples are intended to facilitate understanding of the present invention and do not serve to limit the present invention in any way. The reagents used in this example are all known products and were obtained by purchasing commercially available products.

[0074] Example 1. Screening of TFF3 antibodies and construction of a detection kit

[0075] 1. Immunogen Design and Animal Immunization

[0076] In this example, bioinformatics analysis was first used to perform a multiple sequence alignment of TFF3 amino acid sequences (UniProt number: Q07654) from species such as humans, mice, and rats to screen for human-specific and highly conserved linear epitopes, while avoiding the known disulfide bond core regions (C32-C58, C42-C57, and C52-C69) to ensure the stability of the immunogen. The designed immunogen was prepared and BALB / c mice were immunized 4 to 6 times with complete and incomplete adjuvants, with an interval of 14 days between each immunization. Immune lymphocytes were isolated from the spleens of mice immunized with TFF3 protein. The specific process is as follows:

[0077] 1. Immunogen Design

[0078] Based on the structural characteristics of TFF3 (59 aa, 3 disulfide bonds), two non-overlapping linear epitopes were selected for mouse immunization to ensure that sandwich ELISA pairings could be formed subsequently, as shown in Table 1.

[0079] Table 1. TFF3 immunogen epitope design

[0080]

[0081] 2. Mouse immunization protocol

[0082] (1) Immunization process

[0083] Animals: Balb / c mice (3 mice per group, immunized in two groups, EP1 and EP2); Adjuvant: Freund's complete adjuvant (first immunization) → Freund's incomplete adjuvant (boost); Immunization cycle: Day 0: 100 μg KLH-EP1 or GST-EP2 (subcutaneous multi-point injection); Day 14 / 28 / 42: Booster immunization (50 μg, intraperitoneal injection); Day 49: Orbital blood titer measurement (ELISA titer ≥1:64000); Day 56: Spleen collection (B cell isolation).

[0084] (2) Immunogen validation

[0085] ELISA: Validation was performed using native TFF3 (1 μg / mL) coated on the immunogen. Results from this validation experiment demonstrated that the immunogen could recognize intact TFF3 protein. Competition assay: Free EP1 / EP2 blocked serum binding, confirming the epitope specificity of the immunogen.

[0086] 2. Phage display technology for antibody screening

[0087] Phage display technology was used to screen for high-affinity, high-specificity monoclonal antibodies targeting the EP1 and EP2 epitopes. The specific steps involved constructing a phage display library and conducting multiple rounds of screening to obtain high-affinity monoclonal antibodies. Surface plasmon resonance (SPR) was used to measure the affinity constant (KD value) of the antibodies. Screening results showed that the antibodies obtained had significantly higher affinity and better specificity than those obtained using traditional methods.

[0088] 1. Screening Process

[0089] 1) Phage library construction

[0090] Source: Total RNA was extracted from the spleens of BALB / c mice immunized with N-terminal (EEYVGLSAN-KLH) and C-terminal (PLQDAECTF-GST) tags. cDNA was synthesized by reverse transcription, and the variable regions of the antibodies were amplified using universal mouse IgG VH / VK primers. Library Construction: scFv fragments (VH-linker-VL) were constructed using overlap extension PCR (OE-PCR) and cloned into a phage display vector (pComb3X). Library capacity: 2.5×10 9 CFU ensures coverage of diversity needs. Diversity verification: High-throughput sequencing analysis of the CDR3 regions of 200 clones confirmed that more than 90% of the sequences were unique and that the VH / VL families were evenly distributed (avoiding immune bias).

[0091] 2) Subtractive screening

[0092] Four rounds of progressive screening were performed for each of the two peptide epitopes, EP1 and EP2, using wild-type and mutant TFF3, along with competing antigens, to maximize the enrichment of highly specific clones. The screening targets were: 1. EP1 (EEYVGLSAN, 22-30aa): a linear epitope lacking Cys, necessitating the exclusion of clones with cross-reactivity with the TFF family; 2. EP2 (PLQEAECTF, 72-80aa): containing Cys78 (possibly involved in disulfide bonds), necessitating the determination of whether the antibody relies on a reduction-sensitive conformation. The four rounds of progressive screening for EP1 are shown in Table 2, and for EP2 in Table 3.

[0093] Table 2. Four-round progressive screening process for EP1

[0094]

[0095] Table 3. Four-round progressive screening process for EP2

[0096]

[0097] 2. Screening results (200 clones → 4 selected antibodies)

[0098] After initial ELISA screening (OD450 nm > 2.0) and SPR verification, four high-affinity antibodies (codenamed: TFF3-Ab1~4) were selected from 200 clones. Their sequences and performance comparisons are shown in Table 4.

[0099] Table 4. Four high-affinity antibodies obtained by screening

[0100]

[0101] As can be seen in Table 4, the four antibodies screened had superior affinity and specificity. Among them, the two best antibodies: TFF3-Ab1 (KD=3.8 nM) (heavy chain CDR1 sequence is SEQ ID NO.9, CDR2 sequence is SEQ ID NO.10, light chain CDR1 sequence is SEQ ID NO.11, CDR2 sequence is SEQ ID NO.12; heavy chain amino acid sequence is SEQ ID NO.61, light chain amino acid sequence is SEQ ID NO.62); and TFF3-Ab4 (KD=4.7 nM) (heavy chain CDR1 sequence is SEQ ID NO.13, CDR2 sequence is SEQ ID NO.14, light chain CDR1 sequence is SEQ ID NO.15, CDR2 sequence is SEQ ID NO.16; heavy chain amino acid sequence is SEQ ID NO.63, light chain amino acid sequence is SEQ ID NO.64), had affinity >20-fold higher than that of traditional hybridoma antibodies (KD>10 nM).

[0102] Specificity: The cross-reactivity of Ab1 and Ab4 to OSM was <1.5%, which was significantly lower than that of Ab2 (3.8%) and Ab3 (7.5%).

[0103] 3. Performance Verification Experiment

[0104] (1) Epitope competition ELISA

[0105] Ab1 and Ab4 can bind to TFF3 simultaneously (signal superposition), indicating that they recognize different epitopes and are suitable for double antibody sandwich kits.

[0106] (2) Thermal stability test

[0107] After storage at 4°C for 28 days, TFF3-Ab1 and TFF3-Ab4 retained >95% of their binding activity, which was superior to Ab2 (85%) and Ab3 (72%).

[0108] 3. Optimizing Antibodies Using Affinity Maturation Technology

[0109] Since the monoclonal antibodies obtained by direct screening have low affinity, low stability, and low expression levels, mutations are required to obtain better antibodies. In this example, affinity maturation optimization was performed on the monoclonal antibodies Ab1 and Ab4 obtained by screening. Using directed mutagenesis and combinatorial library screening strategies, mutations were introduced in the CDR regions of the antibodies to screen out variants with higher affinity. Validated by ELISA and SPR, the optimized antibodies showed an improved sensitivity of pg / mL and further enhanced specificity, making them suitable for accurate detection of low-concentration targets.

[0110] 1. Mutation design strategy

[0111] For the preliminarily screened TFF3-Ab1 and TFF3-Ab4, the following two methods were used to mutate the CDR regions: (1) Directed point mutagenesis: Based on the prediction of key residues based on structural simulation (Rosetta software), the heavy chain CDR3 (H3) and light chain CDR1 (L1) were targeted, and thus the heavy chain CDR3 (H3) and light chain CDR1 (L1) were mutated; (2) Random combination mutagenesis: The NNK degenerate codon library (covering 5 sites) was introduced into the H3 region or L1 region, with a library capacity of ≥10 8 .

[0112] 2. Sequence comparison before and after mutation

[0113] High-affinity variants were selected by phage display, and the optimal mutation patterns of TFF3-Ab1 and TFF3-Ab4 were finally obtained as shown in Table 5.

[0114] Table 5. Optimal mutation patterns of TFF3-Ab1 and TFF3-Ab4

[0115]

[0116] Note: The mutated amino acids are marked in bold.

[0117] Thus, the co-mutation obtained two preferred groups of TFF3 first antibodies: TFF3-Ab1-1 (H3 (CDR3)-1 and L1 (CDR1)), TFF3-Ab1-2 (H3 (CDR3)-2 and L1 (CDR1)); and two preferred groups of TFF3 second antibodies: TFF3-Ab4-1 (H3 (CDR3) and L1 (CDR1)-1), TFF3-Ab4-2 (H3 (CDR3) and L1 (CDR1)-2).

[0118] 3. Improved affinity and specificity after mutation

[0119] The performance of the two sets of TFF3 primary and secondary antibodies was tested using SPR, circular dichroism (CD), and ELISA, respectively. For SPR affinity testing, the antigen was immobilized on the chip surface, and varying concentrations of TFF3 antibody were injected into the mobile phase. The binding / dissociation signals (response units (RU)) were monitored in real time. The equilibrium dissociation constant (KD) was calculated from the association and dissociation rates, and a 1:1 binding model was fitted. Key parameters included flow rate (≤30 μL / min), temperature (25°C), and data subtraction of the reference channel. The results are shown in Table 6.

[0120] Table 6. Changes in SPR affinity

[0121]

[0122] As shown in Table 6, the dissociation constant of the mutant was reduced to <13.8 nM, and the affinity was increased by >3.0×. The affinity of the antibody after mutation was significantly improved.

[0123] The stability of the mutants was evaluated by measuring their thermal stability Tm values ​​using circular dichroism (CD) spectroscopy and compared with that of the original antibody. The results are shown in Table 7. Tm represents the midpoint temperature of thermal denaturation of the protein, that is, the temperature at which the protein unfolds by 50%, reflecting the tendency of protein conformational changes during temperature changes. A higher Tm indicates better stability.

[0124] Table 7. Stability changes

[0125]

[0126] As can be seen from Table 7, the stability of the antibody after mutation is significantly improved compared to before mutation.

[0127] ELISAs were used to detect the binding of mutants to TFF family members (TFF1 / TFF2) and OSM. The ELISA assays used TFF3-Ab1 and its mutants, TFF3-Ab1-1 or TFF3-Ab1-2, as primary antibodies, in a double-antibody sandwich assay with a secondary antibody (purchased from Thermofisher, model H00007033-M03) for detection of TFF1, TFF2, and OSM. A double-antibody sandwich assay was also established with TFF3-Ab4 and its mutants, TFF3-Ab4-1 or TFF3-Ab4-2, as primary antibodies (purchased from Abcam, model ab244735) for detection of TFF1, TFF2, and OSM. The results are shown in Table 8.

[0128] Table 8. Cross-reactivity verification results

[0129]

[0130] As can be seen from Table 8, after mutation, the cross-reactivity of both groups of TFF3 primary antibodies and TFF3 secondary antibodies was significantly reduced. For example, the cross-reactivity of Ab1 to OSM was 1.2% before mutation, which was reduced by 60% after mutation. It can be seen that the specificity of the antibodies obtained after mutation was significantly enhanced.

[0131] 4. Optimization of dual antibody combination kit

[0132] The kit was constructed by pairing two sets of TFF3 primary antibodies (TFF3-Ab1-1 and TFF3-Ab1-2) with two sets of TFF3 secondary antibodies (TFF3-Ab4-1 and TFF3-Ab4-2). The chemiluminescence kit reaction system was optimized, using an enhanced chemiluminescent substrate and a stable reaction buffer to enhance the stability and intensity of the detection signal.

[0133] 1. Optimization of chemiluminescent immunoassay reaction system

[0134] The chemiluminescent immunoassay kit consists of a TFF3 primary antibody coupled to carboxyl magnetic beads and a secondary antibody labeled with an isoluminol-NHS chemiluminescent group. Detection was performed using a Cosma 6500S fully automated chemiluminescent assay reader. An orthogonal experimental design (L16 array) was used to optimize four key parameters: ① Reaction buffer: Tris-HCl (pH 7.4-8.6) vs. PBS (containing 0.05% Tween-20); ② Enhancers: two commercial enhancers (Pierce® / Roche®) and two in-house formulations A or B (A: 0.2 mM p-iodophenol, B: 0.2 mM 4-imidazolephenol); ③ Substrate ratio: Luminol:H₂O₂ (1:1 to 1:5); and ④ Blocking agent: Casein (1-5%) / BSA (0.5-2%).

[0135] The four core parameters after optimization are: ① Reaction buffer: Tris-HCl (pH 8.2) + 0.1% CHAPS (signal-to-noise ratio increased by 3.5 times); ② Enhancer: Self-developed formula A (0.2 mM p-iodophenol) increases sensitivity by 40% compared with commercial reagents; ③ Substrate ratio: Luminol:H2O2 (1:3); ④ Blocking agent: Casein (3%).

[0136] 2. Screening of dual antibody combinations

[0137] Under the above optimized reaction system, antibody combinations were performed according to the methods shown in Table 9 to investigate the effects of different antibody combinations on the test results. Negative human serum was used to prepare quality control products for testing.

[0138] Table 9. Effects of different antibody combinations

[0139]

[0140] As shown in Table 9, different dual-antibody combinations significantly affect the performance of chemiluminescent immunoassays, with differences in background noise and minimum detection limits. Therefore, the combination of TFF3-Ab1-1 and TFF3-Ab4-1 is preferred for constructing a chemiluminescent immunoassay kit, as it offers lower background noise and higher detection sensitivity.

[0141] The optimal antibody combination TFF3-Ab1-1 (heavy chain variable region sequence is SEQ ID NO.23, light chain variable region sequence is SEQ ID NO.24; heavy chain amino acid sequence is SEQ ID NO.53, light chain amino acid sequence is SEQ ID NO.54) and TFF3-Ab4-1 (heavy chain variable region sequence is SEQ ID NO.25, light chain variable region sequence is SEQ ID NO.26; heavy chain amino acid sequence is SEQ ID NO.55, light chain amino acid sequence is SEQ ID NO.56) was used to construct a chemiluminescent immunoassay kit, and the standard curve was prepared. The linearity was very good, R²=0.998 ( Figure 1 The intra-batch and inter-batch precision of the TFF3 protein control solutions (prepared with negative human serum) containing 5 and 10 pg / mL were investigated, respectively. The test results are shown in Table 10.

[0142] Table 10. Intra-batch / inter-batch precision

[0143]

[0144] As can be seen, the chemiluminescent immunoassay kit constructed in this example, through optimization of the enhancer and buffer, achieved a sensitivity of 10 pg / mL, signal stability of over 2 hours, and intra- and inter-batch precision that met the assay requirements. Furthermore, this example also employed a high-dose quality control (200 ng / mL) for testing, yielding accurate results without a high-dose hook effect.

[0145] Example 2: Screening of OSM Antibodies and Construction of a Detection Kit

[0146] 1. Optimization of OSM protein immunogen sequence

[0147] In this example, the OSM protein amino acid sequence was first analyzed using bioinformatics methods to screen for stable structured regions and linear, highly exposed regions. Furthermore, known glycosylation sites (i.e., amino acid residues modified with sugar groups in proteins) were avoided. This optimized design significantly improved the affinity and specificity of the resulting antibody, avoiding the potential for decreased antibody performance due to interference from glycosylation sites.

[0148] 1. OSM structure analysis

[0149] The full-length precursor sequence of OSM (UniProt ID: P13725) is 252aa. The mature form (196aa) is removed, with a 25aa signal peptide and a 31aa C-terminal peptide (34-234aa). Key modifications include: disulfide bonds at C31-C152 (structural core) and C74-C192 (functionally critical); free sulfhydryl group at C105 (potential binding site); glycosylation sites at N100 and N217 (to be avoided); and restriction enzyme cleavage sites at positions 234-235 (which may affect C-terminal antibody design).

[0150] 2. Candidate epitopes

[0151] Epitope selection criteria: Epitope 1 (capture antibody): select stable structured regions (such as α-helices / β-folds containing disulfide bonds); Epitope 2 (detection antibody): select linear high-exposed regions (such as loops without glycosylation and away from disulfide bonds); Physical spacing: the distance between the two epitopes is >15 Å to avoid steric hindrance; Species specificity: give priority to human-specific sequences.

[0152] (1) Epitope 1 (capture antibody targeting): 69-88aa (KLREHCRERPGAFPSEETLR)

[0153] Reasons for selection: 1) Structural stability: contains α-helix (predicted by AlphaFold2) and disulfide bond anchor region (C74-C192); 2) Modification plan: To reduce disulfide bond interference, mutate to KLREHARERPGAFPSEETLR (C74→A).

[0154] (2) Epitope 2 (targeted by the detection antibody): 110-123aa (LADLEQRLPKAQDL)

[0155] Reasons for selection: 1) Surface accessibility: solvent-exposed loop region (surface accessibility >80%); 2) No modification interference: far away from glycosylation sites (N100 / N217) and free C105; 3) Modification scheme: adding an N-terminal CGG linker (CGGLADLEQRLPKAQDL).

[0156] 2. Phage Library Construction and Panning

[0157] 1. Immune library construction

[0158] Immunogens: In our immunogen design strategy for OSM protein, we specifically engineered two functional epitopes and optimized their KLH conjugation. The capture antibody targets an epitope located in the 69-88aa region of the OSM protein (KLREHARERPGAFPSEETLR). This was achieved by mutating C74 to alanine (C74A) to prevent disulfide bond interference. A CGG linker peptide was added to the C-terminus, allowing conjugation to the KLH carrier protein via its free thiol (-SH) group (OSM immunogen 1). The detection antibody targets an epitope located in the 110-123aa region (LADLEQRLPKAQDL). A CGG sequence was pre-added to the N-terminus and conjugated to KLH via the thiol group (OSM immunogen 2). This design preserves the native conformation of the epitope while significantly enhancing immunogenicity through the KLH carrier, paving the way for the subsequent generation of high-affinity and high-specificity antibodies.

[0159] Animal immunization: Mice were immunized with OSM immunogen 1 and immunogen 2, respectively. The immunization process was the same as in Example 1. Splenocytes were collected to construct two independent phage antibody libraries (Fab or scFv format).

[0160] Library diversity control: ensure library capacity > 10 8 , avoiding bias caused by epitope competition.

[0161] 2. Parallel four-round screening

[0162] Table 11. Four-round progressive screening process for EP1

[0163]

[0164] Table 12. Four-round progressive screening process for EP1

[0165]

[0166] 3. Post-screening verification and antibody pairing

[0167] 1. Monoclonal Verification

[0168] ELISA / SPR:

[0169] Epitope 1 antibodies: Binding to the C74A peptide and wild-type native OSM was verified, and clones that only bound the mutant peptide were excluded.

[0170] Epitope 2 Antibody: Validate binding to the CGG-linker peptide and native OSM; affinity should be similar (difference <20%).

[0171] 2. Double antibody sandwich pairing test

[0172] Sensitivity and specificity:

[0173] The limit of detection (LoD) must be ≤0.1 pg / mL and must not cross-react with LIF, a member of the IL-6 family.

[0174] 3. Positive clone identification and affinity maturation

[0175] (1) High-throughput screening

[0176] After initial ELISA screening (OD450 nm > 2.0) and SPR verification, four high-affinity antibodies (codenamed: OSM-Ab1~4) were selected from 300 clones. Their sequences and performance comparisons are shown in Table 13.

[0177] Table 13. Four high-affinity antibodies obtained by screening

[0178]

[0179] As can be seen in Table 13, the four antibodies screened had superior affinity and specificity. The two best antibodies, OSM-Ab1 (KD = 0.52 nM) (heavy chain CDR1 sequence: SEQ ID NO. 35, CDR2 sequence: SEQ ID NO. 36, light chain CDR1 sequence: SEQ ID NO. 37, CDR2 sequence: SEQ ID NO. 38; heavy chain amino acid sequence: SEQ ID NO. 65, light chain amino acid sequence: SEQ ID NO. 66) and OSM-Ab3 (KD = 0.64 nM) (heavy chain CDR1 sequence: SEQ ID NO. 39, CDR2 sequence: SEQ ID NO. 40, light chain CDR1 sequence: SEQ ID NO. 41, CDR2 sequence: SEQ ID NO. 42; heavy chain amino acid sequence: SEQ ID NO. 67, light chain amino acid sequence: SEQ ID NO. 68), exhibited affinities >20-fold higher than those of traditional hybridoma antibodies (KD > 10 nM).

[0180] (2) Directed evolution of CDR regions

[0181] Based on the prediction of key residues based on structural simulation (Rosetta software), the heavy chain CDR3 (H3) and light chain CDR1 (L1) were targeted and mutated. The optimal mutation patterns of OSM-Ab1 and OSM-Ab3 are shown in Table 14.

[0182] Table 14. Optimal mutation patterns for OSM-Ab1 and OSM-Ab3

[0183]

[0184] Note: The mutated amino acids are marked in bold.

[0185] Thus, the co-mutation obtained two preferred groups of OSM first antibodies: OSM-Ab1-1 (H3 (CDR3)-1 and L1 (CDR1)), OSM-Ab1-2 (H3 (CDR3)-2 and L1 (CDR1)); and two preferred groups of OSM second antibodies: OSM-Ab3-1 (H3 (CDR3)-1 and L1 (CDR1)), OSM-Ab3-2 (H3 (CDR3)-2 and L1 (CDR1)).

[0186] 5. Improved affinity and specificity after mutation

[0187] The performance of the two sets of OSM primary and secondary antibodies was tested using SPR and ELISA, respectively. For SPR affinity testing, OSM antigen was immobilized on a chip surface, and varying antibody concentrations were injected into the mobile phase. The binding / dissociation signals (response units (RU)) were monitored in real time. The equilibrium dissociation constant (KD) was calculated from the association and dissociation rates, and a 1:1 binding model was fitted. Key parameters included flow rate (≤30 μL / min), temperature (25°C), and data subtraction of the reference channel. The results are shown in Table 15.

[0188] Table 15. Changes in SPR affinity

[0189]

[0190] As can be seen from Table 15, the affinity of the mutated antibody was significantly improved. At the same time, the stability of the mutated antibody was significantly improved compared to the pre-mutation antibody, and the Tm was significantly increased.

[0191] ELISAs were used to detect the binding of mutants to the OSM family members LIF and TFF3. The ELISA assays used OSM-Ab1 and its mutants OSM-Ab1-1 or OSM-Ab1-2 as primary antibodies, respectively, in a double-antibody sandwich assay with a secondary antibody (purchased from Thermofisher, model MA5-31090) for TFF3 and LIF. A double-antibody sandwich assay was also established with OSM-Ab3 and its mutants OSM-Ab3-1 or OMS-Ab3-2 as primary antibodies for TFF3 and LIF. The results are shown in Table 16.

[0192] Table 16. Cross-reactivity verification results

[0193]

[0194] As can be seen from Table 16, after mutation, the cross-reactivity of both groups of OSM primary antibodies and OSM secondary antibodies was significantly reduced. For example, the cross-reactivity of Ab1 to TFF3 was 1.3% before mutation, but it was reduced by more than 50% after mutation. It can be seen that the specificity of the antibodies obtained after mutation was significantly enhanced.

[0195] 4. Optimization of dual antibody combination kit

[0196] The kit was constructed by pairing two sets of OSM primary antibodies (OSM-Ab1-1 and OSM-Ab1-2) with two sets of OSM secondary antibodies (OSM-Ab3-1 and OSM-Ab3-2). The chemiluminescence kit's reaction system was optimized, using an enhanced chemiluminescent substrate and a stable reaction buffer to enhance the stability and intensity of the detection signal.

[0197] 1. Optimization of chemiluminescent immunoassay reaction system

[0198] The chemiluminescent immunoassay kit consists of an OSM secondary antibody coupled to carboxyl magnetic beads and a primary antibody labeled with an isoluminol-NHS chemiluminescent group. Detection was performed using a Cosma 6500S fully automated chemiluminescent assay analyzer. An orthogonal experimental design (L9 array) was employed to optimize three key parameters: ① Reaction buffer: Tris-HCl (pH 7.8-9.0) vs. HEPES (0.05% Triton X-100); ② Enhancers: two commercial enhancers (Pierce® / Roche®) and two in-house formulations A or B (A: 0.2 mM p-iodophenol, B: 0.2 mM 4-imidazolephenol); and ③ Reaction time: 10-30 minutes.

[0199] The three core parameters after optimization are: ① Reaction buffer: HEPES (pH 8.6) + 0.05% BSA + 0.01% ProClin 950 (effectively suppressing background); ② Enhancer: self-developed formula B (0.2 mM 4-imidazolephenol), which increases the signal by more than 3 times that of the commercial substrate; ③ Reaction time: 25 minutes.

[0200] 2. Screening of dual antibody combinations

[0201] Under the above optimized reaction system, antibody combinations were performed according to the methods shown in Table 17 to investigate the effects of different antibody combinations on the test results. Negative human serum was used to prepare quality control products for testing.

[0202] Table 17. Effects of different antibody combinations

[0203]

[0204] Table 17 shows that different dual antibody combinations significantly affect the performance of chemiluminescent immunoassays, with differences in signal intensity, signal-to-noise ratio, and minimum detection limit. Therefore, the combination of OSM-Ab1-1 and OSM-Ab3-1 is preferred for constructing a chemiluminescent immunoassay kit, as it provides a stronger and more stable signal, lower background noise, and higher detection sensitivity.

[0205] The optimal antibody combination OSM-Ab1-1 (heavy chain variable region sequence is SEQ ID NO.49, light chain variable region sequence is SEQ ID NO.50; heavy chain amino acid sequence is SEQ ID NO.57, light chain amino acid sequence is SEQ ID NO.58) and OSM-Ab3-1 (heavy chain variable region sequence is SEQ ID NO.51, light chain variable region sequence is SEQ ID NO.52; heavy chain amino acid sequence is SEQ ID NO.59, light chain amino acid sequence is SEQ ID NO.60) was used to construct a chemiluminescent immunoassay kit, and a standard curve was prepared. The fitting curve R²=0.997( Figure 2 The intra-batch and inter-batch precision of OSM protein control solutions (prepared with negative human serum) containing 5 and 10 pg / mL were investigated, respectively. The test results are shown in Table 18.

[0206] Table 18. Intra-batch / inter-batch precision

[0207]

[0208] As can be seen, the chemiluminescent immunoassay kit constructed in this example, through optimization of the enhancer and buffer, achieved a sensitivity of 0.2 pg / mL, with intra- and inter-batch precision meeting the assay requirements and a recovery rate of 99.6-100.2% (compared to existing antibodies, which only achieve 75-88%). Furthermore, this example also employed a high-dose quality control (1 μg / mL) for testing, resulting in accurate results with no prozone or high-dose hook effect.

[0209] Example 3: Application of TFF3 and OSM Antibody Detection Kit in Diagnosis of Colorectal Cancer

[0210] 1. Diagnostic efficacy of TFF3 and OSM chemiluminescent immunoassay kits used alone or in combination

[0211] This example uses the TFF3 and OSM chemiluminescent immunoassay kits constructed in Example 1 and Example 2. The use of one kit alone and the combination of the two kits were investigated to test TFF3 and OSM in the sera of healthy subjects (124 cases) and colorectal cancer patients (64 cases). SPSS software (Version 22.0) was used to evaluate their ability to distinguish between diseased subjects.

[0212] When the TFF3 chemiluminescent immunoassay kit was used alone, the antibody combination used in the kit was TFF3-Ab1-1 (heavy chain amino acid sequence of SEQ ID NO. 53, light chain amino acid sequence of SEQ ID NO. 54) and TFF3-Ab4-1 (heavy chain amino acid sequence of SEQ ID NO. 55, light chain amino acid sequence of SEQ ID NO. 56) to construct a chemiluminescent immunoassay kit for distinguishing between healthy people and colorectal cancer patients. The test results are shown in Table 19. The TFF3 content cutoff value for distinguishing between patients with colorectal cancer was 15.7 ng / mL. Patients with a content greater than or equal to 15.7 ng / mL were classified as colorectal cancer patients, and those with a content less than 15.7 ng / mL were classified as healthy people.

[0213] Table 19. Analysis of detection results of TFF3 kit (chemiluminescence method)

[0214]

[0215] When the OSM chemiluminescent immunoassay kit was used alone, the antibody combination OSM-Ab1-1 (heavy chain amino acid sequence: SEQ ID NO. 57, light chain amino acid sequence: SEQ ID NO. 58) and OSM-Ab3-1 (heavy chain amino acid sequence: SEQ ID NO. 59, light chain amino acid sequence: SEQ ID NO. 60) were used to construct the chemiluminescent immunoassay kit for distinguishing between healthy individuals and colorectal cancer patients. The test results are shown in Table 20. The OSM content cutoff value for distinguishing between colorectal cancer patients was 34.8 pg / mL; patients with a content greater than or equal to 34.8 pg / mL were classified as colorectal cancer patients, while those with a content less than 34.8 pg / mL were classified as healthy individuals.

[0216] Table 20. Analysis of detection results of OSM kit (chemiluminescence method)

[0217]

[0218] When TFF3 and OSM chemiluminescent immunoassay kits were used together, a model was constructed using MedCalc software (Version 22.006). The constructed logistic regression formula was: risk score = 0.23×ln(TFF3) + 0.10×ln(OSM) (threshold = 0.99, when the risk score was greater than or equal to 0.99, it was judged as a colorectal cancer patient, and less than 0.99, it was judged as a healthy person). The diagnostic performance was compared with that of using TFF3 or OSM chemiluminescent immunoassay kits alone. The results are shown in Tables 21 and 22. Figure 3 shown.

[0219] Table 21. Joint model diagnostic performance

[0220]

[0221] As can be seen from Table 21, compared with using TFF3 or OSM chemiluminescence immunoassay kit alone, the logistic regression model constructed by combining TFF3 and OSM indicators (TFF3+OSM) has better diagnostic performance and obvious synergistic effect, which can better distinguish cancer patients from healthy people.

[0222] 2. Differences in diagnostic efficacy of different TFF3 and OSM antibody combinations

[0223] This example utilizes the following three antibody combinations: the first: an existing antibody combination; the second: the antibody combination screened in Examples 1 and 2 before mutation; and the third: the optimal antibody combination after mutation. Chemiluminescent immunoassay kits were constructed for each assay, and a combined diagnostic model (TFF3 + OSM) was employed for the diagnosis of colorectal cancer. The same test samples were used as before, testing TFF3 and OSM in sera from healthy individuals (184 subjects) and colorectal cancer patients (96 subjects). The samples were randomly divided into a test group (92 healthy individuals and 48 colorectal cancer patients) and a validation group (92 healthy individuals and 48 colorectal cancer patients).

[0224] The existing antibody combination includes the primary antibody for TFF3 (purchased from Abcam, model ab244735) and the secondary antibody for OSM (purchased from Thermofisher, model H00007033-M03), and the primary antibody for OSM (purchased from Abcam, model ab242851) and the secondary antibody for OSM (purchased from Thermofisher, model MA5-31090). A model was constructed using MedCalc software (Version 22.006) using the following logistic regression formula: risk score = 0.52 × ln(TFF3) + 0.33 × ln(OSM) (threshold = 0.72; a risk score greater than or equal to 0.72 indicates colorectal cancer, and less than 0.72 indicates healthy individuals).

[0225] The pre-mutation antibody combinations screened in Examples 1 and 2 included: TFF3-Ab1 (heavy chain amino acid sequence of SEQ ID NO. 61, light chain amino acid sequence of SEQ ID NO. 62) and TFF3-Ab4 (heavy chain amino acid sequence of SEQ ID NO. 63, light chain amino acid sequence of SEQ ID NO. 64); OSM-Ab1 (heavy chain amino acid sequence of SEQ ID NO. 65, light chain amino acid sequence of SEQ ID NO. 66) and OSM-Ab3 (heavy chain amino acid sequence of SEQ ID NO. 67, light chain amino acid sequence of SEQ ID NO. 68). A model was constructed using MedCalc software (Version 22.006), and the constructed logistic regression formula was: risk score = 0.35 × ln(TFF3) + 0.24 × ln(OSM) (threshold = 0.84; when the risk score is greater than or equal to 0.84, the patient is diagnosed as a colorectal cancer patient, and when it is less than 0.84, the patient is diagnosed as a healthy individual).

[0226] Preferred antibody combinations after mutation include TFF3-Ab1-1 (heavy chain amino acid sequence: SEQ ID NO. 53, light chain amino acid sequence: SEQ ID NO. 54) and TFF3-Ab4-1 (heavy chain amino acid sequence: SEQ ID NO. 55, light chain amino acid sequence: SEQ ID NO. 56); OSM-Ab1-1 (heavy chain amino acid sequence: SEQ ID NO. 57, light chain amino acid sequence: SEQ ID NO. 58) and OSM-Ab3-1 (heavy chain amino acid sequence: SEQ ID NO. 59, light chain amino acid sequence: SEQ ID NO. 60). A model was constructed using MedCalc software (Version 22.006), using the following logistic regression formula: risk score = 0.21 × ln(TFF3) + 0.11 × ln(OSM) (threshold = 0.97; a risk score greater than or equal to 0.97 indicates colorectal cancer, and less than 0.97 indicates healthy individuals).

[0227] The test results for distinguishing healthy people from colorectal cancer patients are shown in Table 22.

[0228] Table 22. Diagnostic performance of different TFF3 and OSM antibody combinations

[0229]

[0230] As can be seen from Table 22, although the markers detected are both TFF3 and OSM proteins, and based on the same joint diagnostic model, the diagnostic results will be significantly different when different antibodies are used for detection. The reason may be that the existing antibodies not only have low detection sensitivity, but also have significant differences in cross-reactivity and stability. For example, the existing TFF3 antibody easily cross-reacts with other members of the TFF family, and there are also problems such as EP1 being blocked by dimerization and dependence on the Cys78 disulfide bond, which seriously affect the diagnostic results; for example, the existing OSM antibody does not contain α-helix and disulfide anchor region (C74-C192), has poor structural stability, and is prone to cross-reactivity and high-dose hook effect; these reasons will lead to a decrease in diagnostic efficacy when used for diagnosing colorectal cancer. Therefore, the preferred antibody combination after mutation provided by the present invention includes TFF3-Ab1-1, TFF3-Ab4-1 and OSM-Ab1-1, OSM-Ab3-1, and the performance of the constructed chemiluminescent immunoassay kit is the best (the diagnostic efficacy of the test group is shown in Figure 2). Figure 4 ), which can significantly improve the diagnostic efficiency of colorectal cancer.

[0231] It can be understood that the embodiments described in the present invention are some preferred embodiments and features. Any person skilled in the art can make some changes and variations based on the essence of the description of the present invention. These changes and variations are also considered to fall within the scope of the present invention and the scope limited by the independent claims and the appended claims.

Claims

1. A kit for detecting TFF3 protein and OSM protein, characterized in that: The invention comprises an antibody against TFF3 protein and an antibody against OSM protein; the antibody against TFF3 protein comprises a first antibody and a second antibody; the first antibody comprises: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.9, a CDR2 consisting of the amino acid sequence of SEQ ID NO.10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.17, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 19, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 2; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.13, a CDR2 consisting of the amino acid sequence of SEQ ID NO.14, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.20, and (4) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 21, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 16, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 8; The antibodies against OSM protein include a first antibody and a second antibody; the first antibody includes: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.35, a CDR2 consisting of the amino acid sequence of SEQ ID NO.36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.43, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.45, a CDR2 consisting of the amino acid sequence of SEQ ID NO.38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.28; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.39, a CDR2 consisting of the amino acid sequence of SEQ ID NO.40, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.46, and (4) In the light chain variable region, CDR1 consists of the amino acid sequence of SEQ ID NO. 48, CDR2 consists of the amino acid sequence of SEQ ID NO. 42, and CDR3 consists of the amino acid sequence of SEQ ID NO.

32.

2. The kit according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the first antibody of the TFF3 protein is shown in SEQ ID NO.23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.26; The amino acid sequence of the heavy chain variable region of the first antibody against OSM protein is shown in SEQ ID NO. 49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

52.

3. Use of an antibody for preparing a reagent for predicting whether an individual has colorectal cancer, characterized in that: The antibodies include antibodies against TFF3 protein and antibodies against OSM protein; the antibodies against TFF3 protein include a first antibody and a second antibody; the first antibody includes: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.9, a CDR2 consisting of the amino acid sequence of SEQ ID NO.10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.17, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 19, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 2; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.13, a CDR2 consisting of the amino acid sequence of SEQ ID NO.14, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.20, and (4) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 21, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 16, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 8; The antibodies against OSM protein include a first antibody and a second antibody; the first antibody includes: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.35, a CDR2 consisting of the amino acid sequence of SEQ ID NO.36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.43, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.45, a CDR2 consisting of the amino acid sequence of SEQ ID NO.38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.28; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.39, a CDR2 consisting of the amino acid sequence of SEQ ID NO.40, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.46, and (4) In the light chain variable region, CDR1 consists of the amino acid sequence of SEQ ID NO. 48, CDR2 consists of the amino acid sequence of SEQ ID NO. 42, and CDR3 consists of the amino acid sequence of SEQ ID NO.

32.

4. The use according to claim 3, characterized in that The antibody to the TFF3 protein includes 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.23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.26; The OSM protein 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.49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

52.

5. A kit for predicting whether an individual has colorectal cancer, characterized in that: The invention relates to an antibody against TFF3 protein and an antibody against OSM protein; the antibody against TFF3 protein comprises a first antibody and a second antibody; the first antibody comprises: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.9, a CDR2 consisting of the amino acid sequence of SEQ ID NO.10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.17, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 19, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 2; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.13, a CDR2 consisting of the amino acid sequence of SEQ ID NO.14, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.20, and (4) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 21, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 16, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 8; The antibodies against OSM protein include a first antibody and a second antibody; the first antibody includes: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.35, a CDR2 consisting of the amino acid sequence of SEQ ID NO.36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.43, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.45, a CDR2 consisting of the amino acid sequence of SEQ ID NO.38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.28; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.39, a CDR2 consisting of the amino acid sequence of SEQ ID NO.40, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.46, and (4) In the light chain variable region, CDR1 consists of the amino acid sequence of SEQ ID NO. 48, CDR2 consists of the amino acid sequence of SEQ ID NO. 42, and CDR3 consists of the amino acid sequence of SEQ ID NO.

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6. The kit according to claim 5, wherein The antibody to the TFF3 protein includes 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.23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.26; The OSM protein 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.49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

52.

7. An antibody combination for predicting whether an individual has colorectal cancer, characterized in that: The invention comprises an antibody against TFF3 protein and an antibody against OSM protein; the antibody against TFF3 protein comprises a first antibody and a second antibody; the first antibody comprises: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.9, a CDR2 consisting of the amino acid sequence of SEQ ID NO.10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.17, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 19, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 2; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.13, a CDR2 consisting of the amino acid sequence of SEQ ID NO.14, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.20, and (4) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 21, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 16, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 8; The antibodies against OSM protein include a first antibody and a second antibody; the first antibody includes: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.35, a CDR2 consisting of the amino acid sequence of SEQ ID NO.36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.43, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.45, a CDR2 consisting of the amino acid sequence of SEQ ID NO.38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.28; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.39, a CDR2 consisting of the amino acid sequence of SEQ ID NO.40, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.46, and (4) In the light chain variable region, CDR1 consists of the amino acid sequence of SEQ ID NO. 48, CDR2 consists of the amino acid sequence of SEQ ID NO. 42, and CDR3 consists of the amino acid sequence of SEQ ID NO.

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8. The antibody combination according to claim 7, characterized in that The antibody to the TFF3 protein includes 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.23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.26; The OSM protein 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.49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

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9. A system for predicting whether an individual has colorectal cancer, characterized in that: The system includes a data analysis module, which is used to analyze the detection value of the antigen. The antigen is detected by antibodies, and the antibodies include antibodies to TFF3 protein and antibodies to OSM protein. The antibodies to TFF3 protein include a first antibody and a second antibody. The first antibody includes: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.9, a CDR2 consisting of the amino acid sequence of SEQ ID NO.10, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.17, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 19, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 2; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.13, a CDR2 consisting of the amino acid sequence of SEQ ID NO.14, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.20, and (4) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO. 21, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 16, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 8; The antibodies against OSM protein include a first antibody and a second antibody; the first antibody includes: (1) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.35, a CDR2 consisting of the amino acid sequence of SEQ ID NO.36, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.43, and (2) in the light chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.45, a CDR2 consisting of the amino acid sequence of SEQ ID NO.38, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.28; The second antibody comprises: (3) in the heavy chain variable region, a CDR1 consisting of the amino acid sequence of SEQ ID NO.39, a CDR2 consisting of the amino acid sequence of SEQ ID NO.40, and a CDR3 consisting of the amino acid sequence of SEQ ID NO.46, and (4) In the light chain variable region, CDR1 consists of the amino acid sequence of SEQ ID NO. 48, CDR2 consists of the amino acid sequence of SEQ ID NO. 42, and CDR3 consists of the amino acid sequence of SEQ ID NO.

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10. The system according to claim 9, wherein: The antibody to the TFF3 protein includes 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.23, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.26; The OSM protein 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.49, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.52.

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