Antibodies of TFF3 and OSM proteins as well as combination and application thereof in colorectal cancer diagnosis

Abstract

The invention provides antibodies of TFF3 and OSM proteins, a combination thereof and an application of the antibodies in colorectal cancer diagnosis, high-affinity and high-specificity monoclonal antibodies are screened through optimization of immunogen sequences of TFF3 and OSM and a phage display technology, then directed mutation and combinatorial library screening are adopted, mutation is introduced into CDR regions of the antibodies, the optimal TFF3 and OSM antibodies are screened out, and the colorectal cancer diagnosis is realized. The antibody can effectively solve the problems of low affinity and specificity of the existing antibody, is wider in detection range, shows extremely high collaborative diagnosis value during combined detection, remarkably improves the diagnosis efficiency of colorectal cancer, and has important clinical application value.

Description

Technical Field

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

[0002] Digestive system tumors are a high-incidence type of cancer globally, and among them, the incidence and mortality of colorectal cancer (CRC) are particularly serious. Colorectal cancer is the third most common cancer globally, and it is estimated that there will be more than 1.9 million new cases in 2025 (accounting for 10% of all new cancers). The 5-year survival rate of patients in the early stage (stage I) can reach 90%, but only 10 - 15% in the advanced stage (stage IV), highlighting the urgency of early diagnosis.

[0003] In recent years, proteomics research has found that TFF3 (trefoil factor 3) is abnormally highly expressed in the serum of CRC patients (5 - 8 times higher than that in healthy people); OSM (oncostatin M), as 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 than in early stage patients). This indicates the diagnostic value of TFF3 and / or OSM in digestive tract tumors, especially colorectal cancer.

[0004] Although both TFF3 and OSM have been reported to be related to CRC, the existing technologies have the following gaps: 1) Lack of standardized detection tools: Currently, only research institutions detect through academic methods such as ELISA, and there is no commercial kit, making it difficult to accurately detect; 2) The co-diagnostic value has not been explored: There is no evidence indicating that the combination of TFF3 and OSM can improve the diagnostic efficacy (no literature reports on indicators such as AUC value).

[0005] CN101706497A discloses an ELISA detection kit for human TFF3, which is for general laboratory or clinical research on gastrointestinal pathology. However, it does not optimize the antigen, the immunogen sequence is unstable, and it is easy to cause a decline in antibody performance due to interference by glycosylation sites. At the same time, it does not mutate and optimize the antibody sequence, making it difficult to ensure the accurate detection of low-concentration targets, with insufficient detection sensitivity and easy cross-reaction, and prone to errors when used for the diagnosis of colorectal cancer.

[0006] Patent EP2236625A1 (valid) discloses a method for detecting the TFF3 gene or protein, which is used to establish a prognosis for the progression of diagnosing patients with colorectal cancer after therapy, but it is not for the prediction of early colorectal cancer and is not used in combination with OSM.

[0007] Patent CN118670830B discloses that TFF3 can be used for the prediction of early carcinogenesis of colorectal cancer, but it is not used in combination with OSM. Moreover, there are currently no reports on the combined detection and diagnosis of colorectal cancer using the two markers TFF3 and OSM.

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

[0009] Currently, the development of OSM (oncostatin M, a cytokine belonging to the IL-6 family) antibodies is biased towards therapeutic uses. For example, CN103328508B discloses an antigen-binding protein (humanized antibody) against OSM for use in pharmaceutical compositions for the treatment and prevention of inflammatory diseases and disorders, not for the prediction of colorectal cancer, and it is not used in combination with TFF3. Moreover, the antigen and antibody sequences have not been mutated or optimized, and the immunogenic sequence is unstable, easily leading to a decline in antibody performance due to interference from glycosylation sites, making it difficult to ensure accurate detection of low-concentration targets, with insufficient detection sensitivity, prone to cross-reactions, and prone to errors when used for the diagnosis of colorectal cancer.

[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 diagnose colorectal cancer more accurately. Summary of the Invention

[0011] Aiming at the problems existing in the prior art, the present invention provides an antibody and its combination of TFF3 and OSM proteins and their application in the diagnosis of colorectal cancer. By optimizing the immunogenic sequences of TFF3 and OSM, high-affinity and high-specificity monoclonal antibodies are screened by phage display technology, and then directed mutagenesis and combinatorial library screening are used to introduce mutations into the CDR regions of the antibodies to screen out the optimal TFF3 and OSM antibodies, which can effectively solve the problems of low affinity and specificity of existing antibodies, have a wider detection range, show extremely high synergistic diagnostic value in combined detection, significantly improve the diagnostic efficiency of early colorectal cancer, and have important clinical application value.

[0012] In the existing technical solutions, when using traditional hybridoma technology, phage display technology or recombinant protein to immunize animals to prepare monoclonal or polyclonal antibodies against TFF3 protein or OSM protein, there are problems such as insufficient antibody affinity and specificity caused by unoptimized immunogen design. Moreover, when detecting TFF3, OSM protein or gastrointestinal tumor markers by existing ELISA or chemiluminescence methods, the kit is not optimized for the combination of TFF3 and OSM antibodies, resulting in low detection sensitivity and specificity, and it is impossible to achieve efficient diagnosis of gastrointestinal tumors (especially colorectal cancer). The present invention has developed a brand-new highly specific antibody of TFF3 and OSM and its combination, which can be used for the preparation of diagnostic kits to detect TFF3 and OSM in different samples to realize clinical value.

[0013] On the one hand, the present invention provides an antibody against TFF3 protein, and the antibody includes a first antibody and / or a second antibody; the first antibody includes:

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

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

[0016] The second antibody includes:

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

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

[0019] Through bioinformatics analysis, the present invention performs multiple sequence alignments on the TFF3 amino acid sequences (UniProt number: Q07654) of species such as humans, mice, and rats, screens out human-specific and highly conserved linear epitopes, and at the same time avoids known disulfide bond core regions (C32-C58, C42-C57, C52-C69) to ensure the stability of the immunogen. The designed immunogen is prepared and used to immunize BALB / c mice with complete adjuvant and incomplete adjuvant. The number of immunizations is 4-6 times, and the interval between each immunization is 14 days. Immunolymphocytes are isolated from the spleens of mice immunized with the TFF3 protein to construct a phage display library.

[0020] For the EP1 and EP2 epitopes, phage display technology is used to screen monoclonal antibodies with high affinity and high specificity. The present invention uses four rounds of progressive screening. The obtained anti-TFF3 primary antibody and anti-TFF3 secondary antibody can both recognize clones of native TFF3, there is no situation where EP2 is blocked by dimerization, and they do not cross-react with other members of the TFF family.

[0021] The antibodies obtained after four rounds of screening are further subjected to CDR region mutagenesis to screen out mutants with higher affinity. Verified by ELISA and SPR, the sensitivity of the optimized antibody is increased to the level of 100 pg / mL, the specificity is further enhanced, it is suitable for the accurate detection of low-concentration targets, can detect the TFF3 protein with higher precision, and has higher diagnostic value when diagnosing digestive tract tumors, such as colorectal cancer, alone or in combination with other antibodies.

[0022] Furthermore, the amino acid sequence of the heavy chain variable region of the primary antibody (TFF3-Ab1-1) 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 secondary antibody (TFF3-Ab4-1) 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.

[0023] Furthermore, the amino acid sequence of the heavy chain of the primary 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 secondary 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] On the other hand, the present invention provides a kit for detecting the TFF3 protein, and the kit includes the antibody of the TFF3 protein as described above.

[0025] It is understandable that the kit can be an ELISA detection kit, a chemiluminescent immunoassay detection kit, etc.

[0026] In some embodiments, the kit is a chemiluminescent immunoassay detection kit. By screening the optimal and suitable paired first and second antibodies against TFF3, a chemiluminescent immunoassay detection kit is constructed.

[0027] On the other hand, the present invention provides an antibody against OSM protein, the antibody comprising a first antibody and / or a second antibody; the first antibody comprising:

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

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

[0030] The second antibody comprises:

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

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

[0033] The present invention analyzes the amino acid sequence of OSM protein by bioinformatics methods, screens stable structured regions and linearly highly exposed regions. At the same time, known glycosylation sites (i.e., amino acid residues modified by sugars in proteins) are avoided. This optimized design results in human-specific linear epitopes EP1 and EP2, significantly improving the affinity and specificity of the prepared antibody and avoiding the decline in antibody performance caused by glycosylation site interference.

[0034] In the immunogen design for OSM protein, we specifically modified two functional epitopes and optimized the KLH conjugation. The epitope targeted by the capture antibody is located in the 69 - 88aa region of OSM protein (KLREHARERPGAFPSEETLR). By mutating C74 to alanine (C74A) to avoid disulfide bond interference and adding a CGG linker peptide at the C-terminus, its free sulfhydryl group (-SH) is used to conjugate with the KLH carrier protein (OSM immunogen 1). The epitope targeted by the detection antibody is located in the 110 - 123aa region (LADLEQRLPKAQDL), so a CGG sequence is pre-added directly at the N-terminus, and it is also conjugated with KLH through the sulfhydryl group (OSM immunogen 2). Mice were immunized with OSM immunogen 1 and immunogen 2 respectively, and splenocytes were taken to construct two independent phage antibody libraries.

[0035] For OSM immunogens 1 and 2, parallel four-round screening was respectively carried out to obtain high-affinity and high-specific monoclonal antibodies against OSM; for the antibodies obtained through screening, CDR region mutations were then carried out to screen out mutants with higher affinity. Verified by ELISA and SPR, the sensitivity of the optimized antibody was improved to the level of 0.1 pg / mL, the specificity was further enhanced, which was suitable for the precise detection of low-concentration targets, could detect OSM protein with higher precision, and had higher diagnostic value when diagnosing digestive tract tumors, such as colorectal cancer, alone or in combination with other antibodies.

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

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

[0038] On the other hand, the present invention provides a kit for detecting OSM protein, and the kit includes the antibody of OSM protein as described above.

[0039] It can be understood that the kit can be an ELISA detection kit, a chemiluminescent immunoassay detection kit, etc.

[0040] In some ways, the kit is a chemiluminescent immunoassay kit. By screening the optimal and suitable paired first antibody and second antibody of OSM, a dual-antibody combination chemiluminescent immunoassay kit is constructed.

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

[0042] In some ways, combining the kit for detecting TFF3 protein and the kit for detecting OSM protein can be used to simultaneously detect TFF3 protein and OSM protein, and thus can be applied to the field of simultaneous detection of multiple markers, such as the field of gastrointestinal tumor diagnosis, to improve the diagnostic efficiency through combined detection.

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

[0044] Although TFF3 and OSM are known diagnostic markers for colorectal cancer, there are significant differences in the diagnostic efficiency when using different TFF3 antibodies and / or OSM antibodies to diagnose colorectal cancer. Because during the diagnosis of colorectal cancer, it is necessary to detect the blood sample through TFF3 antibody and / or OSM antibody. There are complex interfering substances in the blood sample, the matrix has a great influence, cross-reaction is likely to occur, or there are interferences from some non-linear fragments. Directly using the existing antibodies for detection is difficult to obtain ideal detection results, seriously affecting the accuracy of the diagnostic results.

[0045] Through multiple rounds of screening such as screening of antigen epitopes, screening of monoclonal antibodies, and mutation of antibody CDR sequences, the antibodies of TFF3 and OSM proteins finally obtained by the present invention have better detection sensitivity and specificity, and the signal is more stable, with less cross-reaction. When used for the diagnosis of colorectal cancer, the diagnostic efficiency can be significantly improved.

[0046] Further, it includes the antibody of TFF3 protein and the antibody of OSM protein.

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

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

[0049] Furthermore, the reagent is used to detect the content of an antigen that can bind to the antibody 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 recurrence risk of colorectal cancer is a detection reagent prepared with TFF3 and OSM protein as detection targets. In the present invention, antibodies are used to detect TFF3 and OSM protein.

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

[0052] In the present invention, biomarkers (TFF3 and OSM protein) for predicting the recurrence risk of colorectal cancer are screened from blood. There are significant differences in the blood of colorectal cancer patients and healthy people for these two biomarkers. By collecting a blood sample, the content of TFF3 and OSM protein in the individual's blood can be detected, and according to the constructed prediction model, the possibility of the individual suffering from colorectal cancer can be predicted or used for auxiliary diagnosis. Or the content of TFF3 and OSM protein in the blood of a certain group can be detected, and then this 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] wherein, TFF3 is the detected value of the TFF3 protein, and OSM is the detected value of the OSM protein. When the risk score is greater than or equal to 0.99, it is judged as a colorectal cancer patient; when it is less than 0.99, it is judged as a healthy person.

[0056] On the other hand, the present invention provides a kit for predicting whether an individual has colorectal cancer. The kit includes 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, the antibody combination comprising an antibody against the TFF3 protein as described above and an antibody against the OSM protein as described above.

[0058] Further, it includes an antibody against the TFF3 protein and an antibody against the OSM protein.

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

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

[0061] In another aspect, 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, the antigen being detected by an antibody, the antibody including the antibody against the TFF3 protein as described above and / or the antibody against the OSM protein as described above.

[0062] Further, the system further includes a data storage module, a data input interface, and a data output interface; the data storage module is used for storing the detection values of biomarkers; the data input interface is used for inputting the detection values of biomarkers, and the data output interface is used for outputting the prediction result.

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

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

[0065] 2. Use phage display technology + affinity maturation (directed mutagenesis of CDR regions) to screen antibodies, so that the antibody affinity (KD value) is improved to the nanomolar level;

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

[0067] 4. A brand-new specific OSM antibody sequence is provided, which can be used to construct an OSM detection kit. Combining with the double-antibody sandwich method design, the cross-reactivity rate is extremely low (<2%), the sensitivity reaches the level of 01 pg / mL, and it can detect OSM protein with high precision;

[0068] 5. The diagnostic value of the kit used alone or in combination in digestive tract tumors, especially in colorectal cancer; The combined detection model (TFF3 + OSM logistic regression) significantly improves the discrimination ability (AUC≥0.96) compared with single markers. 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 colorectal cancer for the TFF3 and OSM chemiluminescence immunoassay kits used alone or in combination in Example 3;

[0072] Figure 4 The diagnostic efficacy of colorectal cancer for the optimal chemiluminescence immunoassay kit constructed in Example 3. Detailed Description of the Invention

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

[0074] Example 1. Screening of TFF3 Antibody and Construction of Detection Kit

[0075] I. Immunogen Design and Animal Immunization

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

[0077] 1. Immunogen design scheme

[0078] Based on the structural characteristics of TFF3 (59aa, 3 disulfide bonds), two non-overlapping linear epitopes were selected for mouse immunization to ensure the subsequent formation of a sandwich ELISA pair, as shown in Table 1 specifically.

[0079] Table 1. Design of TFF3 immunogen epitopes

[0080]

[0081] 2. Mouse immunization protocol

[0082] (1) Immunization process

[0083] Animals: Balb / c mice (3 mice per group, divided into two groups, EP1 and EP2, for immunization); Adjuvant: Freund's complete adjuvant (primary 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: Boost immunization (50 μg, intraperitoneal injection); Day 49: Orbital blood was collected to measure the titer (ELISA titer ≥ 1:64000); Day 56: Spleens were collected (B cells were isolated).

[0084] (2) Immunogen verification

[0085] ELISA detection: Verification was carried out using coated native TFF3 (1 μg / mL). The verification experimental results proved that this immunogen could recognize the complete TFF3 protein in the serum. Competition experiment: Free EP1 / EP2 blocked the serum binding to confirm the epitope specificity of this immunogen.

[0086] II. Screening antibodies by phage display technology

[0087] For the EP1 and EP2 epitopes, phage display technology was used to screen for high-affinity and high-specificity monoclonal antibodies. The specific steps included constructing a phage display library, performing multiple rounds of screening, and obtaining high-affinity monoclonal antibodies. The affinity constant (KD value) of the antibodies was determined by surface plasmon resonance (SPR) technology. The screening results showed that the affinity of the obtained antibodies was significantly improved compared with the traditional method, and the specificity was better.

[0088] 1. Screening process

[0089] 1) Phage library construction

[0090] Source: Total RNA was extracted from the spleens of BALB / c mice immunized with the N-terminal (EEYVGLSAN-KLH) and C-terminal (PLQDAECTF-GST), reverse transcribed into cDNA, and the antibody variable regions were amplified using murine IgG VH / VK universal primers. Library construction: The scFv fragment (VH-linker-VL) was constructed by overlap extension PCR (OE-PCR) and cloned into the phage display vector (pComb3X). Library capacity: 2.5×10 9 CFU to ensure coverage of the diversity requirements. Diversity verification: High-throughput sequencing analysis of the CDR3 regions of 200 clones confirmed that more than 90% were unique sequences, and the VH / VL family distribution was uniform (avoiding immune preference).

[0091] 2) Subtractive screening

[0092] For the two peptide epitopes of EP1 and EP2, four rounds of progressive screening were carried out respectively, combined with wild-type, mutant TFF3 and competitive antigens to maximize the enrichment of high-specificity clones. Screening targets: 1. EP1 (EEYVGLSAN, 22-30aa): Without Cys, linear epitope, clones that cross-react with the TFF family need to be excluded; 2. EP2 (PLQEAECTF, 72-80aa): Containing Cys78 (possibly involved in disulfide bonds), it is necessary to distinguish whether the antibody depends on the reduction-sensitive conformation. The four rounds of progressive screening for EP1 are shown in Table 2, and the four rounds of progressive screening for EP2 are shown in Table 3.

[0093] Table 2. Four rounds of progressive screening process for EP1

[0094]

[0095] Table 3. Four rounds of progressive screening process for EP2

[0096]

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

[0098] After primary screening by ELISA (OD450 nm > 2.0) and verification by SPR, 4 high-affinity antibodies (codes: TFF3-Ab1 to 4) were selected from 200 clones, and their sequences and performances are compared as shown in Table 4.

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

[0100]

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

[0102] Specificity: The cross-reactivity of Ab1 and Ab4 to OSM is both <1.5%, 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 storing at 4°C for 28 days, the binding activities of TFF3-Ab1 and TFF3-Ab4 are retained >95%, which is better than those of Ab2 (85%) and Ab3 (72%).

[0108] III. Optimize antibodies by affinity maturation technology

[0109] Since the affinity of the monoclonal antibodies obtained by direct screening is not high, the stability is relatively low, and the expression level is not high, mutations are still needed to obtain better antibodies. In this example, affinity maturation optimization was performed on the screened monoclonal antibodies Ab1 and Ab4. Using the strategies of directed mutagenesis and combinatorial library screening, mutations were introduced into the CDR regions of the antibodies to screen for variants with higher affinity. Verified by ELISA and SPR, the sensitivity of the optimized antibodies was increased to the pg / mL level, and the specificity was further enhanced, making them suitable for the precise 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 mutation: Based on structural simulation (Rosetta software), key residues were predicted, targeting the heavy-chain CDR3 (H3) and the light-chain CDR1 (L1), so mutations were made to the heavy-chain CDR3 (H3) and the light-chain CDR1 (L1); (2) Random combinatorial mutation: NNK degenerate codon libraries (covering 5 sites) were introduced into the H3 region or the L1 region respectively, and the library capacity was ≥10 8 .

[0112] 2. Sequence comparison before and after mutation

[0113] By phage display to sort out high-affinity variants, the optimal mutation methods of the finally obtained TFF3-Ab1 and TFF3-Ab4 are shown in Table 5.

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

[0115]

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

[0117] Thus, two preferred groups of TFF3 primary antibodies were co-mutated: TFF3-Ab1-1 (H3 (CDR3)-1 and L1(CDR1)), TFF3-Ab1-2 (H3 (CDR3)-2 and L1 (CDR1)); and two preferred groups of TFF3 secondary antibodies: TFF3-Ab4-1 (H3 (CDR3) and L1 (CDR1)-1), TFF3-Ab4-2 (H3 (CDR3) and L1 (CDR1)-2).

[0118] 3. Improvement of affinity and specificity after mutation

[0119] The performance of two groups of TFF3 primary antibodies and TFF3 secondary antibodies was detected by SPR, circular dichroism (CD), and ELISA respectively. Among them, the detection method for SPR affinity was to immobilize the antigen on the chip surface, inject mobile phases with different concentrations of TFF3 antibodies, and monitor the binding / dissociation signals (response unit RU) in real time. The equilibrium dissociation constant (KD) was calculated through the association rate and dissociation rate, and a 1:1 binding model was fitted. Key parameters: flow rate (≤30 μL / min), temperature (25 °C), and data deduction of the reference channel. The detection results are shown in Table 6.

[0120] Table 6. SPR Affinity Changes

[0121]

[0122] It can be seen from Table 6 that the dissociation constant of the mutant decreased to <13.8 nM, and the affinity increased by >3.0-fold. The affinity of the mutated antibody was significantly improved.

[0123] The thermal stability Tm value of the mutant was measured by circular dichroism (CD) to evaluate the stability of the above mutant, and it was compared with the original antibody. The detection results are shown in Table 7. Among them, Tm represents the midpoint temperature of protein thermal denaturation, that is, the temperature when 50% of the protein unfolds, reflecting the trend of protein conformational change during the temperature change process. The higher the Tm, the better the stability.

[0124] Table 7. Stability Changes

[0125]

[0126] It can be seen from Table 7 that the stability of the antibody after mutation was significantly improved compared with that before mutation.

[0127] ELISA was used to detect the binding of the mutant to TFF family members (TFF1 / TFF2) and OSM. The ELISA detection method was as follows: Using TFF3-Ab1 and its mutants TFF3-Ab1-1 or TFF3-Ab1-2 as the primary antibody, a sandwich ELISA was established with the secondary antibody (purchased from Thermofisher, model H00007033-M03) to detect TFF1, TFF2, and OSM. Using the primary antibody (purchased from Abcam, model ab244735) and the secondary antibody TFF3-Ab4 and its mutants TFF3-Ab4-1 or TFF3-Ab4-2 to establish a sandwich ELISA to detect TFF1, TFF2, and OSM. The detection results are shown in Table 8.

[0128] Table 8. Cross-Reactivity Verification Results

[0129]

[0130] As can be seen from Table 8, after the mutation, the cross-reactivity of the two groups of the first antibody against TFF3 and the second antibody against TFF3 both decreased significantly. For example, before the mutation, the cross-reactivity of Ab1 against OSM was 1.2%, and after the mutation, it decreased by 60%. It can be seen that the specificity of the antibody obtained after the mutation was significantly enhanced.

[0131] IV. Optimization of the double-antibody combination kit

[0132] The two groups of the first antibody against TFF3: TFF3-Ab1-1, TFF3-Ab1-2, and the two groups of the second antibody against TFF3: TFF3-Ab4-1, TFF3-Ab4-2 were respectively combined in pairs to construct the kit. And the reaction system of the chemiluminescence kit was optimized. An enhanced chemiluminescence substrate and a stable reaction buffer were used to improve the stability and intensity of the detection signal.

[0133] 1. Optimization of the chemiluminescent immunoassay reaction system

[0134] The chemiluminescent immunoassay kit includes: conjugating the first antibody against TFF3 to carboxyl magnetic beads and labeling the second antibody with isoluminol-NHS chemiluminescent group; the detection method is: using the Kosmai 6500S fully automatic chemical method detector for detection. Orthogonal experimental design (L16 array) was used to optimize the combination of 4 core parameters: ① Reaction buffer: Tris-HCl (pH 7.4 - 8.6) vs. PBS (containing 0.05% Tween-20); ② Enhancer: 2 commercial enhancers (Pierce® / Roche®), 2 self-developed formulations A or B (A: 0.2 mM p-iodophenol, B: 0.2 mM 4-imidazolylphenol); ③ Substrate ratio: Luminol:H2O2 (1:1 to 1:5); ④ Blocking agent: Casein (1 - 5%) / BSA (0.5 - 2%).

[0135] The 4 optimized core parameters were respectively: ① Reaction buffer: Tris-HCl (pH 8.2) + 0.1% CHAPS (the signal-to-noise ratio increased by 3.5 times); ② Enhancer: The self-developed formulation A (0.2 mM p-iodophenol) had 40% higher sensitivity than the commercial reagent; ③ Substrate ratio: Luminol:H2O2 (1:3); ④ Blocking agent: Casein (3%).

[0136] 2. Screening of the double-antibody combination

[0137] Under the above optimized reaction system, the antibody combinations were carried out respectively according to the method shown in Table 9, and the influence of different antibody combinations on the detection results was investigated. Quality control products were prepared with negative human serum for detection.

[0138] Table 9. Influence of different antibody combinations

[0139]

[0140] As can be seen from Table 9, using different double-antibody combinations has a significant impact on the effect of chemiluminescent immunoassay, and there are differences in both background noise and the lowest detection limit. Therefore, it is preferred to use the combination of TFF3-Ab1-1 and TFF3-Ab4-1 to construct a chemiluminescent immunoassay kit, which has lower background noise and higher detection sensitivity.

[0141] Using the optimal antibody combinations TFF3-Ab1-1 (the heavy-chain variable region sequence is SEQ ID NO.23, and the light-chain variable region sequence is SEQ ID NO.24; the heavy-chain amino acid sequence is SEQ ID NO.53, and the light-chain amino acid sequence is SEQ ID NO.54) and TFF3-Ab4-1 (the heavy-chain variable region sequence is SEQ ID NO.25, and the light-chain variable region sequence is SEQ ID NO.26; the heavy-chain amino acid sequence is SEQ ID NO.55, and the light-chain amino acid sequence is SEQ ID NO.56) to construct a chemiluminescent immunoassay kit and prepare a standard curve, the linearity is very good, R² = 0.998 ( Figure 1 ). Respectively using TFF3 protein quality control product solutions containing 5 and 10 pg / mL (prepared with negative human serum) to investigate the within-batch / between-batch precision, and the detection results are shown in Table 10.

[0142] Table 10, within-batch / between-batch precision

[0143]

[0144] It can be seen that for the chemiluminescent immunoassay kit constructed in this example, through the optimization of the enhancer and buffer, the sensitivity reaches 10 pg / mL, the signal stability reaches more than 2 h, and the within-batch / between-batch precision meets the detection requirements. At the same time, this example also uses a high-dose quality control product (200 ng / mL) for detection, and the detection results are accurate without the high-dose hook effect.

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

[0146] I. Optimization of the Immunogen Sequence of OSM Protein

[0147] In this example, the amino acid sequence of OSM protein was first analyzed by bioinformatics methods to screen for stable structured regions and linearly highly exposed regions. At the same time, known glycosylation sites (i.e., amino acid residues modified by sugars in proteins) were avoided. This optimized design significantly improved the affinity and specificity of the prepared antibodies and avoided the decline in antibody performance caused by glycosylation site interference.

[0148] 1. OSM Structure Analysis

[0149] The full-length precursor of OSM (UniProt ID: P13725) sequence is 252 amino acids (aa). The mature form is 196 aa, removing the 25 aa signal peptide and the 31 aa C-terminal peptide segment (34 - 234 aa). Key modifications: Disulfide bonds: C31 - C152 (structural core), C74 - C192 (functionally critical); Free thiol group: C105 (potential binding site); Glycosylation sites: N100, N217 (to be avoided); Cleavage site: positions 234 - 235 (may affect C-terminal antibody design).

[0150] 2. Candidate Epitopes

[0151] Epitope selection criteria: Epitope 1 (capture antibody): Select a stable structured region (such as an α-helix / β-sheet containing a disulfide bond); Epitope 2 (detection antibody): Select a linearly highly exposed region (such as a loop without glycosylation and far from disulfide bonds); Physical distance: The distance between the two epitopes > 15 Å to avoid steric hindrance; Species specificity: Prioritize human-specific sequences.

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

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

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

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

[0156] II. Phage Library Construction and Panning

[0157] 1. Immunization Library Construction

[0158] Immunogen: In the immunogen design for OSM protein, we specifically modified two functional epitopes and optimized the KLH conjugation. The epitope targeted by the capture antibody is located in the 69 - 88aa region of OSM protein (KLREHARERPGAFPSEETLR). By mutating C74 to alanine (C74A) to avoid disulfide bond interference and adding a CGG linker peptide at the C-terminus, it is conjugated to the KLH carrier protein using its free sulfhydryl group (-SH) (OSM immunogen 1). The epitope targeted by the detection antibody is located in the 110 - 123aa region (LADLEQRLPKAQDL), so a CGG sequence is directly added in advance at the N-terminus and also conjugated to KLH through the sulfhydryl group (OSM immunogen 2). This design not only retains the natural conformational characteristics of the epitope but also significantly enhances the immunogenicity through the KLH carrier, laying the foundation for the subsequent preparation of antibodies with high affinity and high specificity.

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

[0160] Library diversity control: Ensure that the library capacity > 10 8 , and avoid 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] III. Verification after screening and antibody pairing

[0167] 1. Monoclonal verification

[0168] ELISA / SPR:

[0169] Antibody against epitope 1: Verify the binding to C74A peptide and wild-type native OSM, and exclude clones that only bind to the mutant peptide.

[0170] Antibody against epitope 2: Verify the binding to CGG-linker peptide and native OSM, and the affinity should be close (difference < 20%).

[0171] 2. Bispecific sandwich pairing test

[0172] Sensitivity and specificity:

[0173] The limit of detection (LoD) should be ≤ 0.1 pg / mL and there should be no cross-reactivity with the IL-6 family member LIF.

[0174] 3. Identification of positive clones and affinity maturation

[0175] (1) High-throughput screening

[0176] After primary screening by ELISA (OD450 nm > 2.0) and verification by SPR, 4 high-affinity antibodies (codes: OSM-Ab1 - 4) were selected from 300 clones. Their sequences and performance are compared as shown in Table 13.

[0177] Table 13. 4 high-affinity antibodies obtained by screening

[0178]

[0179] As can be seen from Table 13, the 4 antibodies obtained by screening have better affinity and specificity. Among them, the two best antibodies are: OSM-Ab1 (KD = 0.52 nM) (the CDR1 sequence of the heavy chain is SEQ ID NO.35, the CDR2 sequence is SEQ ID NO.36, the CDR1 sequence of the light chain is SEQ ID NO.37, the CDR2 sequence is SEQ ID NO.38; the heavy chain amino acid sequence is SEQ ID NO.65, the light chain amino acid sequence is SEQ ID NO.66); and OSM-Ab3 (KD = 0.64 nM) (the CDR1 sequence of the heavy chain is SEQ ID NO.39, the CDR2 sequence is SEQ ID NO.40, the CDR1 sequence of the light chain is SEQ ID NO.41, the CDR2 sequence is SEQ ID NO.42; the heavy chain amino acid sequence is SEQ ID NO.67, the light chain amino acid sequence is SEQ ID NO.68). The affinity is improved by more than 20 times compared with traditional hybridoma antibodies (KD > 10 nM).

[0180] (2) Directed evolution of CDR regions

[0181] Based on structural simulation (Rosetta software), key residues were predicted, targeting the heavy chain CDR3 (H3) and the light chain CDR1 (L1). Therefore, mutations were made to the heavy chain CDR3 (H3) and the light chain CDR1 (L1). The optimal mutation methods for the finally obtained OSM-Ab1 and OSM-Ab3 are shown in Table 14.

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

[0183]

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

[0185] Thus, two preferred sets of OSM primary antibodies were co-mutated: OSM-Ab1-1 (H3 (CDR3)-1 and L1(CDR1)), OSM-Ab1-2 (H3 (CDR3)-2 and L1 (CDR1)); and two preferred sets of OSM secondary antibodies: OSM-Ab3-1 (H3 (CDR3)-1 and L1 (CDR1)), OSM-Ab3-2 (H3 (CDR3)-2 and L1 (CDR1)).

[0186] 5. Improvement of Affinity and Specificity after Mutation

[0187] The performance of the two sets of OSM primary antibodies and OSM secondary antibodies obtained was detected by SPR and ELISA respectively. The detection method for SPR affinity was to immobilize the OSM antigen on the chip surface, inject antibodies with different concentrations into the mobile phase, and monitor the binding / dissociation signals (response unit RU) in real time. The equilibrium dissociation constant (KD) was calculated through the association rate and dissociation rate, and a 1:1 binding model was fitted. Key parameters: flow rate (≤30 μL / min), temperature (25 °C), data subtraction of the reference channel. The detection results are shown in Table 15.

[0188] Table 15. Changes in SPR Affinity

[0189]

[0190] It can be seen from Table 15 that the affinity of the mutated antibody has been greatly improved. At the same time, compared with the antibody before mutation, the stability of the mutated antibody has also been significantly improved, and the Tm has increased significantly.

[0191] ELISA was used to detect the binding of the mutants to LIF and TFF3, members of the OSM family. The ELISA detection method was as follows: Using OSM-Ab1 and its mutants OSM-Ab1-1 or OSM-Ab1-2 as the primary antibody, a sandwich ELISA was established with the secondary antibody (purchased from Thermofisher, model MA5-31090) to detect TFF3 and LIF. Using the primary antibody (purchased from Abcam, model ab242851) and the secondary antibody OSM-Ab3 and its mutants OSM-Ab3-1 or OMS-Ab3-2 to establish a sandwich ELISA to detect TTFF3 and LIF. The detection results are shown in Table 16.

[0192] Table 16. Results of Cross-Reactivity Verification

[0193]

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

[0195] IV. Optimization of the dual-antibody combination kit

[0196] Two groups of OSM primary antibodies: OSM-Ab1-1, OSM-Ab1-2, and two groups of OSM secondary antibodies: OSM-Ab3-1, OSM-Ab3-2 were respectively combined in pairs to construct the kit. And the reaction system of the chemiluminescent kit was optimized. An enhanced chemiluminescent substrate and a stable reaction buffer were used to improve the stability and intensity of the detection signal.

[0197] 1. Optimization of the chemiluminescent immunoassay reaction system

[0198] The chemiluminescent immunoassay kit includes: conjugating the OSM secondary antibody to carboxyl magnetic beads and labeling the primary antibody with isoluminol-NHS chemiluminescent groups; the detection method is: using the Kosmai 6500S fully automatic chemical method detector for detection. Orthogonal experimental design (L9 array) was used to optimize the combination of 3 core parameters: ① Reaction buffer: Tris-HCl (pH 7.8 - 9.0) vs. HEPES (0.05% Triton X-100); ② Enhancer: 2 commercial enhancers (Pierce® / Roche®), 2 self-developed formulations A or B (A: 0.2 mM p-iodophenol, B: 0.2 mM 4-imidazolylphenol); ③ Reaction time: 10 - 30 minutes.

[0199] The optimized 3 core parameters are respectively: ① Reaction buffer: HEPES (pH 8.6) + 0.05% BSA + 0.01% ProClin 950 (effectively inhibiting background); ② Enhancer: self-developed formulation B (0.2 mM 4-imidazolylphenol), with the signal increased by more than 3 times compared to the commercial substrate; ③ Reaction time: 25 minutes.

[0200] 2. Screening of the dual-antibody combination

[0201] Under the above optimized reaction system, antibody combinations were carried out according to the methods shown in Table 17 respectively, and the effects of different antibody combinations on the detection results were investigated. Quality control products were prepared with negative human serum for detection.

[0202] Table 17. Effects of different antibody combinations

[0203]

[0204] As can be seen from Table 17, different combinations of double antibodies have obvious effects on chemiluminescence immunoassay, and there are differences in signal intensity, signal-to-noise ratio and lowest detection limit. Therefore, it is preferred to use the combination of OSM-Ab1-1 and OSM-Ab3-1 to construct a chemiluminescence immunoassay kit, which has stronger and more stable signals, lower background noise and higher detection sensitivity.

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

[0206] Table 18, within-run / between-run precision

[0207]

[0208] It can be seen that the chemiluminescence immunoassay kit constructed in this example, through the optimization of the enhancer and buffer, has a sensitivity of 0.2 pg / mL, the within-run / between-run precision meets the detection requirements, and the recovery rate is 99.6 - 100.2% (the existing antibodies can only reach 75 - 88%). At the same time, this example also uses a high-dose control product (1 μg / mL) for detection, and the test results are accurate, without the prozone phenomenon and without the high-dose hook effect.

[0209] Example 3, Application of TFF3 and OSM antibody detection kits in the diagnosis of colorectal cancer

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

[0211] In this example, the TFF3 and OSM chemiluminescence immunoassay kits constructed in Example 1 and Example 2 were used to investigate the use of one kit alone and the combination of two kits respectively. TFF3 and OSM in the sera of healthy people (124 cases) and colorectal cancer patients (64 cases) were tested, and their abilities to distinguish diseased populations were evaluated respectively through SPSS software (Version 22.0).

[0212] When the TFF3 chemiluminescence immunoassay kit is used alone, the antibody combination used in the kit is TFF3-Ab1-1 (the heavy chain amino acid sequence is SEQ ID NO.53, and the light chain amino acid sequence is SEQ ID NO.54) and TFF3-Ab4-1 (the heavy chain amino acid sequence is SEQ ID NO.55, and the light chain amino acid sequence is SEQ ID NO.56) to construct a chemiluminescence immunoassay kit. The detection results for distinguishing healthy people from colorectal cancer patients are shown in Table 19. The cut-off value of TFF3 content for distinguishing whether a person has colorectal cancer is 15.7 ng / mL. If it is greater than or equal to 15.7 ng / mL, the person is judged to be a colorectal cancer patient; if it is less than 15.7 ng / mL, the person is judged to be a healthy person.

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

[0214]

[0215] When the OSM chemiluminescence immunoassay kit is used alone, the antibody combination used in the kit is OSM-Ab1-1 (the heavy chain amino acid sequence is SEQ ID NO.57, and the light chain amino acid sequence is SEQ ID NO.58) and OSM-Ab3-1 (the heavy chain amino acid sequence is SEQ ID NO.59, and the light chain amino acid sequence is SEQ ID NO.60) to construct a chemiluminescence immunoassay kit. The detection results for distinguishing healthy people from colorectal cancer patients are shown in Table 20. The cut-off value of OSM content for distinguishing whether a person has colorectal cancer is 34.8 pg / mL. If it is greater than or equal to 34.8 pg / mL, the person is judged to be a colorectal cancer patient; if it is less than 34.8 pg / mL, the person is judged to be a healthy person.

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

[0217]

[0218] When the TFF3 and OSM chemiluminescence immunoassay kits are used in combination, a model (Version 22.006) is constructed using MedCalc software. The constructed Logistic regression formula is: Risk score = 0.23×ln(TFF3) + 0.10×ln(OSM) (threshold = 0.99. If the risk score is greater than or equal to 0.99, the person is judged to be a colorectal cancer patient; if it is less than 0.99, the person is judged to be a healthy person). Comparing its diagnostic performance with that of using the TFF3 or OSM chemiluminescence immunoassay kit alone, the results are shown in Table 21 and Figure 3 as follows.

[0219] Table 21, Diagnostic performance of the combined model

[0220]

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

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

[0223] In this example, the following three antibody combinations were used respectively: the first: the existing antibody combination; the second: the antibody combination before mutation screened in Examples 1 and 2; the third: the preferred antibody combination after mutation. Chemiluminescence immunoassay kits were constructed respectively, and the combined diagnostic model (TFF3 + OSM) was used for the diagnosis of colorectal cancer. The test samples were the same as before, and the TFF3 and OSM in the sera of healthy people (184 cases) and colorectal cancer patients (96 cases) were tested. The samples were randomly divided into a test group and a verification group. The test group included 92 healthy people and 48 colorectal cancer patients, and the verification group included 92 healthy people and 48 colorectal cancer patients.

[0224] The existing antibody combination includes: the first antibody of TFF3 (purchased from Abcam, model ab244735) and the second antibody (purchased from Thermofisher, model H00007033-M03), the first antibody of OSM (purchased from Abcam, model ab242851) and the second antibody (purchased from Thermofisher, model MA5-31090). A model (Version 22.006) was constructed through MedCalc software, and the constructed Logistic regression formula is: risk score = 0.52×ln(TFF3) + 0.33×ln(OSM) (threshold = 0.72, when the risk score is greater than or equal to 0.72, it is judged as a colorectal cancer patient, and when it is less than 0.72, it is judged as a healthy person).

[0225] The antibody combinations before screening in Examples 1 and 2 include: TFF3-Ab1 (heavy chain amino acid sequence is SEQ ID NO.61, light chain amino acid sequence is SEQ ID NO.62) and TFF3-Ab4 (heavy chain amino acid sequence is SEQ ID NO.63, light chain amino acid sequence is SEQ ID NO.64); OSM-Ab1 (heavy chain amino acid sequence is SEQ ID NO.65, light chain amino acid sequence is SEQ ID NO.66) and OSM-Ab3 (heavy chain amino acid sequence is SEQ ID NO.67, light chain amino acid sequence is SEQ ID NO.68). A model (Version 22.006) was constructed using MedCalc software, and the constructed Logistic regression formula is: 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, it is judged as a colorectal cancer patient, and when it is less than 0.84, it is judged as a healthy person).

[0226] The preferred antibody combinations after mutation include TFF3-Ab1-1 (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 amino acid sequence is SEQ ID NO.55, light chain amino acid sequence is SEQ ID NO.56); OSM-Ab1-1 (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 amino acid sequence is SEQ ID NO.59, light chain amino acid sequence is SEQ ID NO.60). A model (Version 22.006) was constructed using MedCalc software, and the constructed Logistic regression formula is: Risk score = 0.21×ln(TFF3) + 0.11×ln(OSM) (threshold = 0.97, when the risk score is greater than or equal to 0.97, it is judged as a colorectal cancer patient, and when it is less than 0.97, it is judged as a healthy person).

[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 detected markers are both the two proteins of TFF3 and OSM, and based on the same combined diagnostic model, when different antibodies are used for detection, there will be obvious differences in the diagnostic results. The reason may be that the existing antibodies not only have low detection sensitivity, but also have obvious differences in cross-reactivity and stability. For example, the existing TFF3 antibody is prone to cross-react with other members of the TFF family, and there are also problems such as EP1 being blocked by dimerization and relying on the Cys78 disulfide bond, which seriously affect the diagnostic results. Another example is that the existing OSM antibody has poor structural stability due to the lack of an α-helix and a disulfide bond anchoring region (C74-C192), and is prone to cross-reaction and high-dose hook effect. These reasons will all lead to a decrease in the diagnostic efficacy for diagnosing colorectal cancer. Therefore, the performance of the chemiluminescent immunoassay kit constructed with the preferred antibody combinations after mutation provided by the present invention, including TFF3-Ab1-1, TFF3-Ab4-1 and OSM-Ab1-1, OSM-Ab3-1, is the best (the diagnostic efficacy of the test group is shown in Figure 4 ), which can significantly improve the diagnostic efficacy of colorectal cancer.

[0231] It can be understood that the described embodiments of the present invention are all preferred embodiments and features. Any person of ordinary skill in the art can make some changes and variations according to the essence described in the present invention, and these changes and variations are also considered to be within the scope of the present invention and within the scope limited by the independent claims and the dependent claims.

Claims

1. An antibody against TFF3 protein, characterized in that, Comprising a first antibody and / or a second antibody; the first antibody comprises: (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.9, CDR2 consisting of the amino acid sequence of SEQ ID NO.10, and CDR3 consisting of the amino acid sequence of SEQ ID NO.17 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.19, CDR2 consisting of the amino acid sequence of SEQ ID NO.12, and CDR3 consisting of the amino acid sequence of SEQ ID NO.2 in the light chain variable region; The second antibody comprises: (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.13, CDR2 consisting of the amino acid sequence of SEQ ID NO.14, and CDR3 consisting of the amino acid sequence of SEQ ID NO.20 in the heavy chain variable region, and (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.21, CDR2 consisting of the amino acid sequence of SEQ ID NO.16, and CDR3 consisting of the amino acid sequence of SEQ ID NO.8 in the light chain variable region.

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

26.

3. An antibody against OSM protein, characterized in that, Comprising a first antibody and / or a second antibody; the first antibody comprises: (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.35, CDR2 consisting of the amino acid sequence of SEQ ID NO.36, and CDR3 consisting of the amino acid sequence of SEQ ID NO.43 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.45, CDR2 consisting of the amino acid sequence of SEQ ID NO.38, and CDR3 consisting of the amino acid sequence of SEQ ID NO.28 in the light chain variable region; The second antibody comprises: (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.39, CDR2 consisting of the amino acid sequence of SEQ ID NO.40, and CDR3 consisting of the amino acid sequence of SEQ ID NO.46 in the heavy chain variable region, and (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.48, CDR2 consisting of the amino acid sequence of SEQ ID NO.42, and CDR3 consisting of the amino acid sequence of SEQ ID NO.32 in the light chain variable region.

4. The antibody against OSM protein according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.49, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

52.

5. A kit for detecting TFF3 protein and / or OSM protein, characterized in that, An antibody comprising the TFF3 protein as claimed in claim 1 or 2, and / or an antibody comprising the OSM protein as claimed in claim 3 or 4.

6. Use of an antibody for the preparation of a reagent for predicting whether an individual has colorectal cancer, characterized in that, The antibody comprises an antibody against the TFF3 protein as claimed in claim 1 or 2, and / or an antibody against the OSM protein as claimed in claim 3 or 4.

7. The use according to claim 6, wherein An antibody against the TFF3 protein and an antibody against the OSM protein; the antibody against the TFF3 protein comprises a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.23, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.24; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.25, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.26; The antibody against the OSM protein comprises a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.49, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.50; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.51, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

52.

8. A kit for predicting whether an individual has colorectal cancer, characterized in that, An antibody comprising the TFF3 protein as claimed in claim 1 or 2, and an antibody comprising the OSM protein as claimed in claim 3 or 4.

9. An antibody combination for predicting whether an individual has colorectal cancer, characterized in that, An antibody comprising the TFF3 protein as claimed in claim 1 or 2, and an antibody comprising the OSM protein as claimed in claim 3 or 4.

10. A system for predicting whether an individual has colorectal cancer, characterized in that, The system comprises a data analysis module for analyzing the detection value of an antigen, the antigen being detected by an antibody, the antibody comprising an antibody against the TFF3 protein as claimed in claim 1 or 2, and / or an antibody against the OSM protein as claimed in claim 3 or 4.

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