Antibodies of ORM1 and CEA proteins as well as combination and application thereof in colorectal cancer diagnosis

Abstract

The invention provides an ORM1 and CEA protein antibody, a combination of the ORM1 and CEA protein antibody and an application of the ORM1 and CEA protein antibody in colorectal cancer diagnosis, and aims to effectively solve the problems of low affinity and specificity and poor stability of the existing antibody by optimizing the immunogen sequences of ORM1 and CEA, screening a monoclonal antibody with high affinity and high specificity, screening the optimal ORM1 and CEA antibody and combining the optimal ORM1 and CEA antibody. The kit has a wide detection range, shows an extremely high collaborative diagnosis value during combined detection, remarkably improves the diagnosis efficiency of early colorectal cancer, and has an important clinical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody screening and disease diagnosis. Specifically, it relates to antibodies against ORM1 and CEA proteins, their combinations, and their applications in the diagnosis of colorectal cancer. Background Art

[0002] Orosomucoid 1 (ORM1) is encoded by the region 32 on the long arm of chromosome 9 (9q32) and belongs to the Lipocalins family. Proteins in this family are usually involved in the transport and binding of lipids and hydrophilic small molecules. The protein product of the ORM1 gene is also known as ORM, AGP1, OMD 1, AGP-A, and α-1 acid glycoprotein 1. Recombinant human ORM1 produced in Escherichia coli is a single non-glycosylated polypeptide chain containing 184 amino acids (19 - 201) with a molecular weight of 21.7 kDa. ORM1 protein is mainly involved in the transport of lipids and hydrophilic small molecules in cells. It can bind to a variety of ligands, including fatty acids, steroids, and certain cytokines, thus playing a key role in the transport of substances inside and outside cells. It plays a key role in the occurrence and development of certain diseases, such as acute inflammatory reactions, cardiovascular diseases, diabetes, cancers (liver cancer, breast cancer, malignant mesothelioma, etc.). Through these complex regulatory mechanisms, the ORM1 gene ensures its timely expression under specific physiological conditions, maintaining the normal functions of cells and their adaptability to the external environment. The acute-phase plasma protein ORM1 synthesized by the liver can mediate the interaction between blood cells and endothelial cells. In addition, ORM1, together with hemopexin and C-reactive protein, regulates the extravasation of cells through infection and inflammation. The expression of ORM1 is induced by acute-phase stimulators such as bacterial lipopolysaccharide.

[0003] ORM1 promotes the progression and liver metastasis of CRC by regulating tumor cell growth, inducing macrophage M2 polarization, and mediating tumor immune tolerance. It is a potential predictive biomarker and therapeutic target for CRLM. Therefore, it is speculated that ORM1 can be used as a detection biomarker for intestinal cancer, and the early diagnosis of colorectal cancer can be achieved by detecting the expression level of ORM1.

[0004] CEA is the English abbreviation of carcinoembryonic antigen (carcinoembryonic antigen, CEA). Carcinoembryonic antigen is one of the tumor markers, belonging to an acidic glycoprotein. It exists on the surface of cancer cells differentiated from endodermal cells and can be detected in body fluids or excreta such as serum, cerebrospinal fluid, urine, and feces. Carcinoembryonic antigen is of great significance for the differential diagnosis of malignant tumors, disease monitoring, and evaluation of treatment effects. CEA was first extracted from colon cancer and embryonic tissues by Gold and Freedman in 1965. It is an acidic glycoprotein with the characteristics of human embryonic antigen, existing on the surface of cancer cells differentiated from endodermal cells and being a structural protein of the cell membrane. It is formed in the cytoplasm, secreted extracellularly through the cell membrane, and then enters the surrounding body fluids. Therefore, it can be detected in various body fluids and excreta such as serum, cerebrospinal fluid, milk, gastric juice, pleural effusion and ascites, urine, and feces. In the past, CEA was regarded as a specific marker for the early diagnosis of colorectal cancer. Through a large number of clinical practices, it was found that not only the CEA value can increase in gastrointestinal malignancies, but it also increases in the serum of breast cancer, lung cancer, and other malignancies. Therefore, carcinoembryonic antigen is a broad-spectrum tumor marker. Although it cannot be used as a specific indicator for diagnosing a certain malignant tumor, it still has important clinical value in the differential diagnosis of malignant tumors, disease monitoring, and efficacy evaluation.

[0005] The level of CEA is related to the following factors: ① It is related to the early, middle, and late stages of cancer. The higher the stage of cancer, the higher the carcinoembryonic antigen value, but the positive rate is not very high. ② It is related to tumor metastasis. When metastasis occurs, the concentration of carcinoembryonic antigen also increases. ③ It is related to the histological type of cancer. Adenocarcinoma is the most sensitive, followed by squamous cell carcinoma and poorly differentiated carcinoma. This shows that CEA is a differentiation antigen, and the higher the degree of differentiation, the higher the positive rate. ④ It is related to the improvement of the condition. When the condition improves, the serum carcinoembryonic antigen concentration decreases, and it increases when the condition deteriorates. Therefore, CEA can also be used as a detection marker for the early diagnosis of colorectal cancer, but the sensitivity for early cancer is insufficient when detected alone.

[0006] Although either ORM1 or CEA can be used for the early diagnosis of colorectal cancer, there is currently a lack of antibodies that can detect ORM1 or ECA with high sensitivity and high specificity. For the existing antibodies against ORM1 or ECA, due to the low expression level of the immunogen, the preparation cost is relatively high, and the sequence is not optimized during the antibody preparation process, the specificity of the antibody is not high, and it is easy to cross-react with other cell surface antigens. The sensitivity for the detection of early colorectal cancer is insufficient, making it difficult to meet the current scientific research and clinical needs.

[0007] Therefore, there is an urgent need to find antibodies that can detect ORM1 protein and CEA protein with high sensitivity and specificity, and have stable performance and a wider detection range, so as to more accurately diagnose early colorectal cancer. Summary of the Invention

[0008] In view of the problems existing in the prior art, the present invention provides an antibody against ORM1 and CEA proteins, their combination, and their application in the diagnosis of colorectal cancer. By optimizing the immunogen sequences of ORM1 and CEA, screening monoclonal antibodies with high affinity and high specificity, and screening out the optimal ORM1 and CEA antibodies and combining them, the problems of low affinity, low specificity, and poor stability existing in existing antibodies can be effectively solved. The detection range is wider, and extremely high synergistic diagnostic value is demonstrated during combined detection, significantly improving the diagnostic efficiency of early colorectal cancer and having important clinical application value.

[0009] ORM1 is an acute-phase response protein, the expression of which is increased in inflammation and cancer. CEA is a conventional serum marker for tumors such as colorectal cancer, and its expression also increases in inflammation. Due to the low expression level of the immunogens of ORM1 and CEA and the high preparation cost, the process of antibody preparation usually optimizes the eukaryotic expression and purification processes of recombinant antigens to improve the expression level, purity, and immunogenicity. However, their sequences are not optimized and screened, and the obtained antibodies have low specificity and are prone to cross-react with other cell surface antigens, making it impossible to achieve efficient diagnosis of gastrointestinal tumors (especially colorectal cancer). The present invention has developed novel high-affinity and specific antibodies against ORM1 and CEA and their combination, which can be used for the preparation of diagnostic kits to detect ORM1 and CEA in different samples, meeting the current scientific research and clinical needs.

[0010] On the one hand, the present invention provides an antibody against ORM1 protein, and the antibody includes a first antibody and / or a second antibody; the first antibody includes:

[0011] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.3, CDR2 consisting of the amino acid sequence of SEQ ID NO.4, and CDR3 consisting of the amino acid sequence of SEQ ID NO.5 in the heavy chain variable region, and

[0012] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.6, CDR2 consisting of the amino acid sequence of SEQ ID NO.7, and CDR3 consisting of the amino acid sequence of SEQ ID NO.8 in the light chain variable region;

[0013] The second antibody includes:

[0014] (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.11, CDR2 consisting of the amino acid sequence of SEQ ID NO.12, and CDR3 consisting of the amino acid sequence of SEQ ID NO.13 in the heavy chain variable region, and

[0015] (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.14, CDR2 consisting of the amino acid sequence of SEQ ID NO.15, and CDR3 consisting of the amino acid sequence of SEQ ID NO.16 in the light chain variable region.

[0016] The present invention relates to the technical field of antibody preparation, and particularly to a specific targeting antibody against ORM1, its preparation method and application. By bioinformatics retrieval of the ORM1 nucleotide sequence, 2 kinds of antigens, ORM1-H01 and ORM1-H02, are designed for antigen plasmid synthesis. According to the obtained plasmids, TOP10 is transformed to pick positive clones and preserved. After activation and large-scale culture, plasmids are extracted for eukaryotic cell transfection and expression, and the corresponding antigens are obtained by affinity purification. The ORM1-H01 antigen is used for immunization injection and hybridoma cell fusion, and ORM1-H02 is used for screening to obtain multiple pairs of monoclonal antibody strains. Through antigen detection and antibody pairing, finally, high-quality antibody pairs are selected for the development of a rapid detection kit for colorectal cancer.

[0017] Further, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.37; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.38, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.39.

[0018] Further, the heavy chain amino acid sequence of the first antibody is as shown in SEQ ID NO.9, and the light chain amino acid sequence is as shown in SEQ ID NO.10; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.18.

[0019] On the other hand, the present invention provides a kit for detecting ORM1 protein, and the kit includes the antibody against ORM1 protein as described above.

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

[0021] In some ways, the kit is an ELISA detection kit. By screening the optimal and suitable paired ORM1 first antibody and second antibody, a double-antibody combination ELISA detection kit is constructed.

[0022] On yet another aspect, the present invention provides an antibody against CEA protein, and the antibody includes a first antibody and / or a second antibody; the first antibody includes:

[0023] (1) CDR1 consisting of the amino acid sequence of SEQ ID NO.21, CDR2 consisting of the amino acid sequence of SEQ ID NO.22, and CDR3 consisting of the amino acid sequence VPD in the heavy chain variable region, and

[0024] (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.23, CDR2 consisting of the amino acid sequence of SEQ ID NO.24, and CDR3 consisting of the amino acid sequence of SEQ ID NO.25 in the light chain variable region;

[0025] The second antibody comprises:

[0026] (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.28, CDR2 consisting of the amino acid sequence of SEQ ID NO.29, and CDR3 consisting of the amino acid sequence of SEQ ID NO.30 in the heavy chain variable region, and

[0027] (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.31, CDR2 consisting of the amino acid sequence of SEQ ID NO.32, and CDR3 consisting of the amino acid sequence of SEQ ID NO.33 in the light chain variable region.

[0028] The present invention also relates to a specific targeting antibody against CEA, its preparation method and application. By bioinformatics retrieval of the CEA nucleotide sequence, 2 antigens, CEA-H01 and CEA-H02, are designed for antigen plasmid synthesis. According to the obtained plasmids, TOP10 is transformed to pick positive clones and preserved. The plasmids are activated, amplified and cultured to extract plasmids for eukaryotic cell transfection and expression, and the corresponding antigens are obtained by affinity purification. The CEA-H01 antigen is used for immunization injection and hybridoma cell fusion, and CEA-H02 is used for screening to obtain multiple pairs of monoclonal antibody strains. Through antigen detection and antibody pairing, finally, high-quality antibody pairs are selected for the development of a rapid detection kit for intestinal cancer.

[0029] Further, the amino acid sequence of the heavy chain variable region of the first antibody is as shown in SEQ ID NO.40, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.41; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.42, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.43.

[0030] Furthermore, the heavy chain amino acid sequence of the first antibody is as shown in SEQ ID NO.26, and the light chain amino acid sequence is as shown in SEQ ID NO.27; the heavy chain amino acid sequence of the second antibody is as shown in SEQ ID NO.34, and the light chain amino acid sequence is as shown in SEQ ID NO.35.

[0031] In another aspect, the present invention provides a kit for detecting CEA protein, which includes the antibody of CEA protein as described above.

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

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

[0034] In another aspect, the present invention provides a kit for detecting ORM1 protein and / or CEA protein, which includes the antibody of ORM1 protein as described above, and / or the antibody of CEA protein as described above.

[0035] In some embodiments, combining the kit for detecting ORM1 protein and the kit for detecting CEA protein can be used to simultaneously detect ORM1 protein and CEA protein, and thus can be applied to the field of simultaneous detection of multiple biomarkers, such as the diagnosis field of gastrointestinal tumors, to improve the diagnostic efficiency through combined detection.

[0036] In another aspect, the present invention provides the use of an antibody for preparing a reagent for predicting whether an individual has colorectal cancer, and the antibody includes the antibody of ORM1 protein as described above, and / or the antibody of CEA protein as described above.

[0037] Traditional single-target antibody drugs have many limitations themselves. In existing cancer diagnosis technologies, the detection of a single biomarker (such as CEA) has problems of low sensitivity and high false positive rate. Although ORM1 is related to the tumor microenvironment, its clinical application for single detection is not yet mature. The present invention uses ORM1 and CEA as combined biomarkers, and solves the problem of insufficient diagnostic efficiency of a single biomarker through the combined detection of antibodies targeting ORM1 and CEA proteins, thereby improving the accuracy and specificity of cancer diagnosis.

[0038] Although ORM1 and CEA are known diagnostic markers for colorectal cancer, there are significant differences in the diagnostic efficacy when different ORM1 antibodies and / or CEA antibodies are used to diagnose colorectal cancer. During the diagnosis of colorectal cancer, it is necessary to detect blood samples through ORM1 antibodies and / or CEA antibodies. There are complex interfering substances in the blood samples, and the matrix has a great influence. Cross-reactions are likely to occur, or there are interferences from some non-linear fragments. It is difficult to obtain ideal test results by directly using existing antibodies, which seriously affects the accuracy of the diagnostic results.

[0039] Through the design, expression of antigens and screening of monoclonal antibodies, the antibodies of ORM1 and CEA proteins finally obtained in the present invention have better detection sensitivity and specificity, and the signals are more stable and the cross-reactions are smaller. When used for the diagnosis of colorectal cancer, the diagnostic efficacy can be significantly improved.

[0040] Furthermore, the antibody includes an antibody of ORM1 protein and an antibody of CEA protein.

[0041] Furthermore, the antibody of ORM1 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.36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.37; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.38, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.39;

[0042] The antibody of CEA 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.40, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.41; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.42, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.43.

[0043] Furthermore, the reagent is used to detect the content of antigens that can bind to the antibody in body fluid samples; the body fluid samples include any one or more of saliva, blood, urine, plasma, serum, and spinal fluid.

[0044] In some embodiments, the reagent for predicting colorectal cancer is a detection reagent prepared with ORM1 and CEA proteins as detection targets, and the present invention uses antibodies to detect ORM1 and CEA proteins.

[0045] Furthermore, the reagent is used to detect the presence, relative abundance or concentration of biomarkers in body fluid samples.

[0046] The present invention screens for biomarkers (ORM1 and CEA proteins) for predicting the risk of colorectal cancer from blood. There are significant differences in the levels of these two biomarkers in the blood of colorectal cancer patients and healthy individuals. By collecting a blood sample, the likelihood of an individual having colorectal cancer can be predicted or diagnosed by detecting the levels of ORM1 and CEA proteins in the individual's blood. Alternatively, the levels of ORM1 and CEA proteins in the blood of a group can be detected, and then the group can be classified into colorectal cancer patients and healthy individuals.

[0047] In another aspect, the present invention provides a kit for predicting whether an individual has colorectal cancer, which comprises an antibody against the ORM1 protein as described above and an antibody against the CEA protein as described above.

[0048] In another aspect, the present invention provides an antibody combination for predicting whether an individual has colorectal cancer, which comprises an antibody against the ORM1 protein as described above and an antibody against the CEA protein as described above.

[0049] Further, it comprises an antibody against the ORM1 protein and an antibody against the CEA protein.

[0050] Further, the antibody against the ORM1 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.36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.37; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.38, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.39;

[0051] The antibody against the CEA 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.40, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.41; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.42, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.43.

[0052] In another aspect, the present invention provides a system for predicting whether an individual has colorectal cancer, which comprises a data analysis module for analyzing the detection value of an antigen detected by an antibody, and the antibody comprises an antibody against the ORM1 protein as described above and / or an antibody against the CEA protein as described above.

[0053] Further, the data analysis module uses the detection values of the biomarkers of known samples as the training set, divides them into healthy people and colorectal cancer patients according to the situation after surgery of colorectal cancer patients, analyzes the relationship between the detection values of healthy people and colorectal cancer patients, and constructs a model.

[0054] Further, the system further 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.

[0055] The beneficial effects of the present invention are as follows:

[0056] 1. Design self-made ORM1 and CEA expression antigens, immunize animals with this antigen to obtain specific targeted monoclonal antibodies, and have extremely high sensitivity;

[0057] 2. Recombine the prepared antibodies to increase the expression level and ensure the stable expression of the antibodies; the detection of clinical blood samples is introduced into the antibody inspection process, greatly increasing the reliability of experimental data; double-antibody detection reduces the reagent dosage by 50%;

[0058] 3. Screen multiple pairs of monoclonal antibodies against the ORM1 antigen epitope, construct an ORM1 detection kit, design in combination with the double-antibody sandwich method, with an extremely low cross-reaction rate (<2%), a sensitivity reaching the pg / mL level, and can detect ORM1 protein with high precision;

[0059] 4. Screen multiple pairs of monoclonal antibodies against the CEA antigen epitope, construct a CEA detection kit, design in combination with the double-antibody sandwich method, with an extremely low cross-reaction rate (<2%), a sensitivity reaching the pg / mL level, and can detect CEA protein with high precision;

[0060] 5. Optimize the antibody combination and detection process to achieve efficient and economical clinical applications;

[0061] 6. The diagnostic value of the kit used alone or in combination in digestive tract tumors, especially in colorectal cancer; the combined detection model (ORM1 + CEA logistic regression) significantly improves the discrimination ability compared with single biomarkers (AUC≥0.96). BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 is the flow chart for the screening and preparation of monoclonal antibodies;

[0063] Figure 2 is the flow chart for the preparation of mouse monoclonal antibodies;

[0064] Figure 3 is the SDS-PAGE detection result chart after antigen purification;

[0065] Figure 4 is the immune flow chart;

[0066] Figure 5 is the SDS-PAGE test result chart of the purified antibody;

[0067] Figure 6 is the detection regression curve of the ORM1 kit in Example 5;

[0068] Figure 7 is the detection regression curve of the CEA kit in Example 5;

[0069] Figure 8 is the performance result chart for diagnosing colorectal cancer patients with ORM1 and / or CEA in Example 6. Detailed implementation manners

[0070] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not impose any limitation on it. The reagents used in this embodiment are all known products and are obtained by purchasing commercially available products.

[0071] Example 1. Screening and preparation process of monoclonal antibody specifically binding to ORM1

[0072] The screening and preparation process of the monoclonal antibody specifically binding to ORM1 provided in this example is as Figure 1 and Figure 2 shown.

[0073] I. Preparation of ORM1 antigen

[0074] 1. Antigen expression

[0075] Through bioinformatics analysis, the nucleotide sequence of ORM1 (UniProt number: AAH26238) is obtained. By gene synthesis method, two antigens, ORM1-H01 (nucleotide sequence SEQ ID NO.44) and ORM1-H02 (nucleotide sequence SEQ ID NO.45), are designed. The antigen plasmids (pTT5) are synthesized by Shanghai Sangon. According to the obtained plasmids, TOP10 is transformed to pick positive clones, and the plasmids are amplified and cultured to extract plasmids for eukaryotic cell transfection and expression. The expression plasmid is transfected into HEK293 cells using Invitrogen Lipofectamine2000 transfection reagent. After 3 - 5 days (the expression time can be one of 3d, 4d, 5d, and 4d is preferred in this example), the culture supernatant is harvested.

[0076] Purified ORM1-H01 (SEQ ID NO.1) and ORM1-H02 (SEQ ID NO.2) proteins were obtained by affinity chromatography. The purification packing material can be one of Ni-NTA / Ni-IDA, and Ni-IDA is preferred in this example. The final Buffer can be one of PBS, TBS, and CBS, and PBS is preferred in this example. The protein was subjected to conventional physical and chemical analyses and biological activity analyses to select the best expression and purification conditions to ensure the reliability of the immunogen. After antigen purification, SDS-PAGE detection was performed, and the results are as Figure 3 shown.

[0077] 2. Animal immunization

[0078] Using the ORM1-H01 protein prepared in step 1 as an immunogen, 5 Balb / c mice were immunized respectively. The Freund's adjuvant immunization method was adopted to maintain the native conformation of the protein. The specific immunization process is shown in Figure 4 . After the immunization, the serum antibody titer of the immunized animals was detected by ELISA to determine the level of immune response. After the conventional immunization, if the immunized animals can reach the immune response level against the immunogen (OD value > 0.2, diluted 64,000 times), cell fusion can be carried out.

[0079] After animal immunization, serum titer detection was performed. The detection method was Elisa detection (OD450), and the results are shown in Table 1.

[0080] Table 1. Serum titer detection results

[0081]

[0082] According to Table 1, when diluted 64,000 times, the OD value > 0.2, and cell fusion and plating can be carried out. The preferred serum dilution method is stepwise dilution.

[0083] 3. Cell fusion and plating: The mice were sacrificed, and blood was taken from the eyeballs as a positive control. The spleens of the mice were isolated and cryopreserved. After grinding a part of the spleen and counting, cell fusion was carried out at a ratio of spleen cells: sp2 / 0 cells = 8:1, and 1 ml of PEG1450 was added to the preheated blank medium to terminate. After centrifugation, HAT conditioned medium was added for pressure screening. The whole process was carried out under the condition of a 37°C water bath. All the fused cells were plated into 96-well plates.

[0084] 4. Screening: The supernatant of the fused cells was screened by ELISA method. After three rounds of screening, hybridoma cells that were specifically positive for binding to the ORM1-H02 protein were selected respectively.

[0085] 5. Cloning, expansion culture and cryopreservation: Transfer the positive parental clones into a 24-well plate for expansion culture. Collect 2 mL of supernatant from each expanded clone for detection by indirect ELISA method. Cryopreserve these specific positive clone cells to avoid clone loss.

[0086] 6. Subcloning: Subclone the positive parental clones by the limiting dilution method to ensure that these positive parental clones are derived from single parental clone cells respectively, and screen the subclones by indirect ELISA method.

[0087] 7. Screening of paired antibodies: On the basis of antigen recognition confirmation, select 8 stable subclone cell lines from each parental clone for cryopreservation at low temperature and pair them up pairwise. According to the paired detection between antibodies, excellent antibody pairs are obtained. Collect 5 mL of supernatant from each subclone before cryopreservation at low temperature, and identify and preserve the subtypes of all subclones. The detection method for antibody pairing detection is ELISA detection (OD450), where the vertical column is the detection antibody (coating antibody), and the horizontal row is the enzyme-labeled antibody. The detection steps are as follows:

[0088] (1) Fixing the coating antibody:

[0089] 1) Dilute the coating antibody (detection antibody) to 2 μg / mL with carbonate buffer (pH 9.6), and add 100 μL to each well of a 96-well ELISA plate;

[0090] 2) Coat overnight at 4°C (12 - 16 hours);

[0091] 3) Discard the liquid, pat dry, and add 300 μL of washing buffer (PBS + 0.1% Tween20) to each well, and wash 3 times.

[0092] (2) Blocking non-specific sites:

[0093] 1) Add 200 μL of blocking solution (PBS containing 5% skim milk powder) to each well, and block at 37°C for 1 hour;

[0094] 2) Discard the blocking solution, pat dry, and wash 3 times.

[0095] (3) Antigen capture and detection:

[0096] 1) Sample incubation: Add 100 μL to each well; incubate at 37°C for 1 hour, and wash 3 times;

[0097] 2) Dilute the enzyme-labeled antibody to 1 μg / mL with sample diluent, and add 100 μL to each well; incubate at 37°C in the dark for 45 minutes, and wash 5 times (to improve specificity);

[0098] 3) Color development and termination reaction: Add 100 μL of TMB substrate to each well, and develop color in the dark at room temperature for 10 - 15 minutes (the time needs to be optimized by preliminary experiments); immediately add 50 μL of termination solution (2M H2SO4) to each well; measure the OD value, and the results are shown in Table 2.

[0099] Among the samples, the ORM1 antigen was diluted to 45 pg / ml, and the detection results are shown in Table 2.

[0100] Table 2. Detection results of antibody pairs

[0101]

[0102] It can be seen from Table 2 that the three optimal pairs of paired antibodies are antibody 4 and antibody 3 (the titer reaches 0.80), antibody 2 and antibody 7 (the titer reaches 1.13), and antibody 6 and antibody 8 (0.75).

[0103] 8. Construction of recombinant antibody plasmid: Extract the total RNA of hybridoma cells, and reverse transcribe the RNA into cDNA through RT-PCR reaction. Clone the antibody light chain and heavy chain sequences, construct the antibody light chain and heavy chain sequences onto the T vector, and then perform DNA sequencing analysis to obtain the antibody gene sequence.

[0104] 9. Antibody production and purification preparation: Transfect the antibody gene sequence obtained in step 8 into CHO cells, and perform expansion culture. Purify the antibody by protein A / G affinity chromatography. The SDS-PAGE detection of the purified antibody is as Figure 5 shown, and the purified antibody is stored in phosphate buffer (PBS) by dialysis method.

[0105] Example 2. Selection of ORM1 antibody pairs

[0106] Based on the three pairs of paired antibodies screened in Example 1, which are antibody 4 and antibody 3, antibody 2 and antibody 7, and antibody 6 and antibody 8, orthogonal pairing and QC detection are performed between the antibodies to obtain antibody pairs with excellent performance. The specific detection method is ELISA sandwich method, and the detection steps are as follows:

[0107] (1) Fix the coating antibody:

[0108] 1) Dilute the coating antibody (detection antibody) to 2 μg / mL with carbonate buffer (pH 9.6), and add 100 μL to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate;

[0109] 2) Coat overnight at 4°C (12 - 16 hours);

[0110] 3) Discard the liquid, pat dry, and add 300 μL of washing buffer (PBS + 0.1% Tween20) to each well, and wash 3 times.

[0111] (2)Blocking non-specific sites:

[0112] 1) Add 200 μL of blocking solution (PBS containing 5% skim milk powder) to each well and block at 37 °C for 1 hour;

[0113] 2) Discard the blocking solution, pat dry, and wash 3 times.

[0114] (3)Antigen capture and detection:

[0115] 1) Sample incubation: Add 100 μL to each well; incubate at 37 °C for 1 hour and wash 3 times;

[0116] 2) Dilute the enzyme-labeled antibody to 1 μg / mL with sample diluent and add 100 μL to each well; incubate at 37 °C in the dark for 45 minutes and wash 5 times (to improve specificity);

[0117] 3) Color development and termination reaction: Add 100 μL of TMB substrate to each well and develop color in the dark at room temperature for 10 - 15 minutes (the time needs to be optimized by preliminary experiments); immediately add 50 μL of termination solution (2M H2SO4) to each well; measure the OD value, and the results are shown in Table 3.

[0118] Table 3. Results of antibody pairs in detecting ORM1 antigen

[0119]

[0120] It can be seen from Table 3 that the most preferred antibody pair is Antibody 2 and Antibody 7. Among them, Antibody 2 is named ORM1 primary antibody (detection antibody), and Antibody 7 is named ORM1 secondary antibody (enzyme-labeled antibody). Thus, a detection kit for detecting the content of ORM1 can be constructed using ORM1 primary antibody and secondary antibody.

[0121] By DNA sequencing analysis, the CDR1 sequence of the heavy chain of the ORM1 first antibody is SEQ ID NO.3, the CDR2 sequence is SEQ ID NO.4, the CDR3 sequence is SEQ ID NO.5, the CDR1 sequence of the light chain is SEQ ID NO.6, the CDR2 sequence is SEQ ID NO.7, and the CDR3 sequence is SEQ ID NO.8; the heavy chain amino acid sequence is SEQ ID NO.9, and the light chain amino acid sequence is SEQ ID NO.10; the CDR1 sequence of the heavy chain of the ORM1 second antibody is SEQ ID NO.11, the CDR2 sequence is SEQ ID NO.12, the CDR3 sequence is SEQ ID NO.13, the CDR1 sequence of the light chain is SEQ ID NO.14, the CDR2 sequence is SEQ ID NO.15, and the CDR3 sequence is SEQ ID NO.16; the heavy chain amino acid sequence is SEQ ID NO.17, and the light chain amino acid sequence is SEQ ID NO.18.

[0122] Meanwhile, in this example, circular dichroism (CD) was also used to measure the Tm value of the thermal stability of the screened antibody to evaluate the stability of the antibody and compare it with the existing antibodies. The existing ORM1 antibody was purchased from Abnova, model H00005004-M01. The test results are shown in Table 4. Here, Tm represents the midpoint temperature of protein thermal denaturation, that is, the temperature when the protein unfolds by 50%, reflecting the trend of protein conformational change during the temperature change process. The higher the Tm, the better the stability.

[0123] Table 4. Stability change

[0124]

[0125] It can be seen from Table 4 that the stability of the screened ORM1 monoclonal antibody is significantly better than that of the commercially available ORM1 antibody.

[0126] Example 3. Screening and preparation process of monoclonal antibody specifically binding to CEA

[0127] The screening and preparation process of the monoclonal antibody specifically binding to CEA provided in this example is as Figure 1 and Figure 2 shown.

[0128] I. Preparation of CEA antigen

[0129] 1. Antigen expression

[0130] Through bioinformatics analysis, the nucleotide sequence of CEA (UniProt number: P06731) was obtained. By gene synthesis method, two antigens, CEA-H01 (nucleotide sequence SEQ ID NO.46) and CEA-H02 (nucleotide sequence SEQ ID NO.47), were designed. Shanghai Sangon was commissioned to synthesize antigen plasmids (pTT5). According to the obtained plasmids, TOP10 was transformed to pick positive clones, and the plasmids were amplified and cultured to extract plasmids for eukaryotic cell transfection and expression. The expression plasmid was transfected into HEK293 cells using Invitrogen Lipofectamine2000 transfection reagent, and the culture supernatant was harvested after 3 - 5 days (the expression time can be one of 3d, 4d, 5d, and 4d is preferred in this example).

[0131] Purified CEA-H01 (SEQ ID NO.19) and CEA-H02 (SEQ ID NO.20) proteins were obtained by affinity chromatography. The purification filler can be one of Ni-NTA / Ni-IDA, and Ni-IDA is preferred in this example. The final Buffer can be one of PBS, TBS, CBS, and PBS is preferred in this example. The protein was subjected to routine physical and chemical analysis and biological activity analysis to select the best expression and purification conditions to ensure the reliability of the immunogen. After antigen purification, SDS-PAGE detection was carried out, and the results are as Figure 3 shown.

[0132] 2. Animal Immunization

[0133] Using the CEA-H01 protein prepared in step 1 as the immunogen, 5 Balb / c mice were immunized respectively by means of Freund's adjuvant to maintain the native conformation of the protein. The specific immunization process is shown in Figure 4 . After the immunization, the serum antibody titer of the immunized animals was detected by ELISA method to determine the level of immune response. After the conventional immunization, if the immunized animals can reach the immune response level against the immunogen (OD value > 0.2, diluted 64000 times), cell fusion can be carried out.

[0134] After animal immunization, the serum titer was detected, and the results are shown in Table 5.

[0135] Table 5. Serum Titer Detection Results

[0136]

[0137] According to Table 5, when diluted 64000 times, the OD value > 0.2, cell fusion and plating can be carried out. The preferred serum dilution method is stepwise dilution.

[0138] 3. Cell fusion and plating: The mouse was sacrificed, and blood was collected from the eyeball as a positive control. The spleen of the mouse was isolated and cryopreserved. A part of the spleen was ground, counted, and cell fusion was carried out at a ratio of spleen cells: sp2 / 0 cells = 8:1. 1 ml of PEG1450 was added and terminated by adding preheated blank medium. After centrifugation, HAT conditioned medium was added for pressure screening. The whole process was carried out under the condition of a 37°C water bath. All the fused cells were plated into a 96-well plate.

[0139] 4. Screening: The supernatant of the fused cells was screened by ELISA method. After three rounds of screening, hybridoma cells that were positive for specific binding to CEA-H02 protein were selected respectively.

[0140] 5. Cloning, expansion culture and cryopreservation: The positive mother clone cells were transferred to a 24-well plate for expansion culture. 2 mL of supernatant was collected from each expanded clone for detection by indirect ELISA method. These specific positive clone cells were cryopreserved to avoid clone loss.

[0141] 6. Subcloning: The positive mother clones were subcloned by the limited dilution method to ensure that these positive mother clones were derived from single mother clone cells respectively, and indirect ELISA method was used for subcloning screening.

[0142] 7. Screening of paired antibodies: On the basis of antigen recognition confirmation, 8 stable subclone cell lines were selected from each mother clone for cryopreservation at low temperature and paired in pairs, a total of three pairs. According to the paired detection between antibodies, excellent antibody pairs were obtained. 5 mL of supernatant was collected from each subclone before cryopreservation at low temperature, and subtype identification and preservation were carried out for all subclones. The detection method for antibody paired detection was Elisa detection (OD450). In the sample, the CEA antigen was diluted to 45 pg / ml, and the detection results are shown in Table 6.

[0143] Table 6. Results of antibody paired detection

[0144]

[0145] It can be seen from Table 6 that the optimal three pairs of paired antibodies are antibody 1 and antibody 3 (titer reaches 0.83), antibody 7 and antibody 6 (titer reaches 1.21), and antibody 6 and antibody 8 (0.72).

[0146] 8. Construction of recombinant antibody plasmid: Total RNA of hybridoma cells was extracted, and RNA was reverse transcribed into cDNA by RT-PCR reaction. The light chain and heavy chain sequences of the antibody were cloned, and the light chain and heavy chain sequences of the antibody were constructed onto the T vector, and then DNA sequencing analysis was carried out to obtain the antibody gene sequence.

[0147] 9. Antibody production and purification preparation: Transfect the antibody gene sequence obtained in step 8 into CHO cells, perform scale-up culture, and purify the antibody by protein A / G affinity chromatography. The SDS-PAGE detection of the purified antibody is as shown in Figure 5 and store the purified antibody in phosphate buffer (PBS) by dialysis method.

[0148] Example 4. Selection of CEA antibody pairs

[0149] Based on the three pairs of paired antibodies screened in Example 1, namely antibody 1 and antibody 3, antibody 7 and antibody 6, antibody 6 and antibody 8, orthogonal pairing and QC detection are performed between the antibodies to obtain antibody pairs with excellent performance. The specific detection method is ELISA sandwich method, and the detection steps are as follows:

[0150] (1) Fix and coat the antibody:

[0151] 1) Dilute the coated antibody to 2 μg / mL with carbonate buffer (pH 9.6), and add 100 μL to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate.

[0152] 2) Incubate overnight at 4°C (12 - 16 hours).

[0153] 3) Discard the liquid, pat dry, add 300 μL of washing buffer (PBS + 0.1% Tween 20) to each well, and wash 3 times.

[0154] (2) Block non-specific sites:

[0155] 1) Add 200 μL of blocking solution (PBS containing 5% skim milk powder) to each well and block at 37°C for 1 hour.

[0156] 2) Discard the blocking solution, pat dry, and wash 3 times.

[0157] (3) Antigen capture and detection:

[0158] 1) Sample incubation: Add 100 μL to each well; incubate at 37°C for 1 hour and wash 3 times.

[0159] 2) Dilute the enzyme-labeled antibody to 1 μg / mL with sample diluent, add 100 μL to each well; incubate at 37°C in the dark for 45 minutes and wash 5 times (to improve specificity).

[0160] 3) Color development and termination reaction: Add 100 μL of TMB substrate to each well and develop color at room temperature in the dark for 10 - 15 minutes (the time needs to be optimized by preliminary experiment); immediately add 50 μL of termination solution (2M H2SO4) to each well; detect the OD value, and the results are shown in Table 7.

[0161] Table 7. Results of antibody pairs detecting CEA antigen

[0162]

[0163] As can be seen from Table 7, the most preferred antibody pair is antibody 7 and antibody 6. Among them, antibody 7 is named the first CEA antibody (detection antibody), and antibody 6 is named the second CEA antibody (enzyme-labeled antibody). Thus, the first CEA antibody and the second antibody can be used to construct a detection kit for detecting the CEA content respectively.

[0164] Through DNA sequencing analysis, the CDR1 sequence of the heavy chain of the first CEA antibody is SEQ ID NO.21, the CDR2 sequence is SEQ ID NO.22, the CDR3 sequence is: VPD, the CDR1 sequence of the light chain is SEQ ID NO.23, the CDR2 sequence is SEQ ID NO.24, and the CDR3 sequence is SEQ ID NO.25; the heavy chain amino acid sequence is SEQ ID NO.26, and the light chain amino acid sequence is SEQ ID NO.27; the CDR1 sequence of the heavy chain of the second CEA antibody is SEQ ID NO.28, the CDR2 sequence is SEQ ID NO.29, the CDR3 sequence is SEQ ID NO.30, the CDR1 sequence of the light chain is SEQ ID NO.31, the CDR2 sequence is SEQ ID NO.32, and the CDR3 sequence is SEQ ID NO.33; the heavy chain amino acid sequence is SEQ ID NO.34, and the light chain amino acid sequence is SEQ ID NO.35.

[0165] Meanwhile, in this example, circular dichroism (CD) was also used to measure the thermal stability Tm value of the screened antibodies to evaluate the stability of the antibodies and compare them with existing antibodies. The existing CEA antibody was purchased from Sigma, model HPA019758. The detection results are shown in Table 8. The Tm therein represents the midpoint temperature of protein thermal denaturation, that is, the temperature when the protein unfolds by 50%, reflecting the trend of protein conformational change during the temperature change process. The higher the Tm, the better the stability.

[0166] Table 8. Stability change

[0167]

[0168] As can be seen from Table 8, the stability of the screened CEA monoclonal antibody is significantly better than that of the commercially available CEA antibody.

[0169] Example 5. Construction of an ORM1 and CEA combined detection kit

[0170] In this example, the first antibodies and second antibodies of ORM1-1 and ORM1-2 screened in Example 2, and the first antibodies and second antibodies of CEA screened in Example 4 were used to construct the ORM1 and CEA combined detection kits.

[0171] Therefore, there are two kits in total. Kit 1 includes:

[0172] 1. Enzyme-labeled plate: A 96-well enzyme-labeled plate coated with the first antibody of ORM1-1 and the first antibody of CEA.

[0173] 2. Enzyme-labeled second antibody: The second antibody of ORM1-1 and the second antibody of CEA are biotinylated and then labeled with 0.5 μg / mL horseradish peroxidase.

[0174] 3. Diluent: By mass fraction, it includes 1% BSA, 0.05% Tween-20, and 0.02% NaN3, and the solvent is 1×PBS.

[0175] 4. Washing concentrate: 30×PBS buffer containing 4.5% Tween-20 by mass fraction.

[0176] 5. Chromogenic substrate: Chromogenic solution A, hydrogen peroxide with a mass fraction of 0.02%; chromogenic solution B, tetramethylbenzidine with a concentration of 4 g / L.

[0177] 6. Stop solution: Phosphoric acid with a concentration of 1 M.

[0178] 7. Sealing film.

[0179] Kit 2 includes:

[0180] 1. Enzyme-labeled plate: A 96-well enzyme-labeled plate coated with the first antibody of ORM1-2 and the first antibody of CEA.

[0181] 2. Enzyme-labeled second antibody: The second antibody of ORM1-2 and the second antibody of CEA are biotinylated and then labeled with 0.5 μg / mL horseradish peroxidase.

[0182] 3. Diluent: By mass fraction, it includes 1% BSA, 0.05% Tween-20, and 0.02% NaN3, and the solvent is 1×PBS.

[0183] 4. Washing concentrate: 30×PBS buffer containing 4.5% Tween-20 by mass fraction.

[0184] 5. Chromogenic substrate: Chromogenic solution A, hydrogen peroxide with a mass fraction of 0.02%; chromogenic solution B, tetramethylbenzidine with a concentration of 4 g / L.

[0185] 6. Stop solution: 1 M phosphoric acid.

[0186] 7. Blocking membrane.

[0187] The ORM1 and CEA combined detection kit constructed in this embodiment includes a detection kit for detecting ORM1 and a detection kit for detecting CEA, which can respectively detect the concentrations of ORM1 and CEA in the sample to be tested, so as to obtain the detection values of ORM1 and CEA respectively. That is, it can be used to detect ORM1, or to detect CEA, or to detect ORM1 and CEA simultaneously. Using the detected ORM1 and CEA as combined markers can effectively avoid the false positive and false negative results of using CEA alone as a marker for diagnosing colorectal cancer.

[0188] The method for detecting the concentrations of ORM1 and / or CEA combined markers in the sample to be tested using the combined detection kit provided in this embodiment is as follows:

[0189] Among them, to detect the concentration of ORM1 in the sample to be tested:

[0190] 1) Coating: Dilute the first antibody of ORM1 with carbonate CBS coating solution to 0.1 - 10 μg / ml (according to the corresponding inspection specifications), add 50 μl to each well of a 96-well plate, and incubate overnight at 4°C.

[0191] 2) Blocking: The next day, drain the 96-well plate, add 200 μl of blocking solution to each well, block at room temperature (22 - 25°C, the same below) for 1 h, then wash the plate 3 times with a plate washer, drain and use immediately, or store at -20°C for later use.

[0192] 3) Adding test samples: Set control wells, 8 standard wells (for making a standard curve), and wells for samples to be tested. Add blank diluent to the control wells, add working solutions of ORM1 with gradient concentrations (serially diluted from diluent 8 with gradient concentrations of 0 ng / L, 7.8125 ng / L, 15.625 ng / L, 31.25 ng / L, 62.5 ng / L, 125 ng / L, 250 ng / L, 500 ng / L, 1000 ng / L) to the 8 standard wells respectively, and add the sample to be tested to the wells for samples to be tested. The added volume is 50 μl for each. Seal the enzyme-labeled plate with a blocking membrane, incubate at 37°C for 30 minutes, and set duplicate wells. React with shaking at 220 rpm at room temperature for 1 h, wash the plate 3 times with a plate washer, and pat dry.

[0193] 4) Adding the enzyme-labeled second antibody: Dilute the biotinylated second antibody of ORM1 to the working concentration with diluent. Add 50 μl to each well, react with shaking at 220 rpm at room temperature for 1 h, wash the plate 3 times with a plate washer, and pat dry.

[0194] 5) Adding enzyme solution: Dilute the SA-HRP stock solution 10,000 times with the diluent to obtain the enzyme working solution. Add 50 μL to each well, react with shaking at 220 rpm at room temperature for 45 minutes to 1 hour, wash the plate 3 times with a plate washer, and pat dry.

[0195] 6) Adding chromogenic substrate: Add 50 μL of tetramethylbenzidine (TMB) with a concentration of 4 g / L and 5 μL of hydrogen peroxide with a mass fraction of 0.02% to each well, and incubate at 37 °C for 15 minutes.

[0196] 7) Adding stop solution: Add 50 μL of 1 M phosphoric acid to each well to terminate the reaction.

[0197] 8) Reading and calculation: Detect the absorbance (OD value) of each well at 450 nm.

[0198] 9) Fitting the standard curve by the four-parameter method to calculate the corresponding concentration: Use the concentration of the ORM1 working solution as the ordinate and its OD value as the abscissa to plot the standard curve, and obtain the regression curve as shown in Figure 6 shown, (R 2 = 0.999), and the regression equation is as follows: , the estimated value of its four-parameter A is 0.344, the standard deviation is 0.051, and the confidence interval is [0.181, 0.507]; the estimated value of B is 1.031, the standard deviation is 0.176, and the confidence interval is [0.471, 1.591]; the estimated value of C is 1.23e+4, the standard deviation is 8805, and the confidence interval is [-1.58e+4, 4.03e+4]; the estimated value of D is 6.203, the standard deviation is 2.470, and the confidence interval is [-1.656, 14.06]. Substitute the OD value of the test sample obtained by detection into the regression equation to calculate the concentration of ORM1 in the test sample.

[0199] The specific method for detecting the CEA concentration in the test sample is the same as above. The constructed regression curve is as shown in Figure 7 shown (R 2 = 0.998), and the regression equation is as follows: , the estimated value of its four-parameter A is 0.036, the standard deviation is 0.053, and the confidence interval is [-0.132, 0.205]; the estimated value of B is 1.241, the standard deviation is 0.168, and the confidence interval is [0.705, 1.776]; the estimated value of C is 2727, the standard deviation is 699.7, and the confidence interval is [500.2, 4954]; the estimated value of D is 4.116, the standard deviation is 0.603, and the confidence interval is [2.197, 6.035]. Substitute the OD value of the test sample obtained by detection into the regression equation to calculate the concentration of CEA in the test sample.

[0200] ORM1 protein quality control solutions and CEA protein quality control solutions (prepared with negative human serum) containing 5, 10, 50, and 250 pg / mL were used respectively to investigate their within-run / between-run precision. For within-run precision: The same batch of kits was used to quantitatively detect the certified samples. Each sample was continuously measured 20 times, and the SD values and coefficient of variation (CV) of samples with different concentrations were calculated respectively. For between-run precision: Three different batches of kits were selected to quantitatively detect the certified samples. Each sample was repeatedly measured 8 times using the same kit, and the SD values and coefficient of variation (CV) of samples with different concentrations were calculated respectively. The test results are shown in Table 9.

[0201] Table 9. Within-run / between-run precision

[0202]

[0203] It can be seen that for the chemiluminescent immunoassay kit constructed in this example, the detection sensitivity for ORM1 and CEA proteins can both reach 5 pg / mL, and the within-run / between-run precision meets the detection requirements.

[0204] Example 6. Application of the ORM1 and CEA combined detection kit in the diagnosis of colorectal cancer

[0205] 1. Diagnostic efficacy of using the ORM1 and CEA detection kits alone or in combination

[0206] In this example, the ORM1 and CEA detection kits constructed in Example 5 were used to investigate the use of ORM1 and CEA alone as markers or the combined detection of ORM1 and CEA as markers to test ORM1 and / or CEA in the sera of healthy individuals (158 cases) and colorectal cancer patients (89 cases). The ability to distinguish diseased populations was evaluated respectively using SPSS 22.0 software (Binary Logistic).

[0207] When using ORM1 alone as a marker, the antibody combination used in the kit was the ORM1 first antibody (heavy chain amino acid sequence is SEQ ID NO.9, light chain amino acid sequence is SEQ ID NO.10) and the ORM1 second antibody (heavy chain amino acid sequence is SEQ ID NO.17, light chain amino acid sequence is SEQ ID NO.18) to construct the detection kit. The detection results for distinguishing healthy individuals and colorectal cancer patients are shown in Table 10. The cut-off value of ORM1 content for distinguishing colorectal cancer patients was 320.5 pg / mL. Individuals with ORM1 content greater than or equal to 320.5 pg / mL were judged as colorectal cancer patients, and those with ORM1 content less than 320.5 pg / mL were judged as healthy individuals.

[0208] When using CEA alone as a biomarker, the antibody combination used in the kit is the first CEA antibody (the heavy chain amino acid sequence is SEQ ID NO.26, and the light chain amino acid sequence is SEQ ID NO.27) and the second CEA antibody (the heavy chain amino acid sequence is SEQ ID NO.34, and the light chain amino acid sequence is SEQ ID NO.35) to construct a detection kit. The detection results for distinguishing healthy people and colorectal cancer patients are shown in Table 10. The cut-off value of CEA content for distinguishing whether a person has colorectal cancer is 5.5 ng / mL. If it is greater than or equal to 5.5 ng / mL, the person is judged to be a colorectal cancer patient; if it is less than 5.5 ng / mL, the person is judged to be a healthy person.

[0209] When using two biomarkers, ORM1 and CEA, in combination for detection, the Logistic regression formula constructed by the SPSS 22.0 algorithm software is: Risk score = 1.82×ln(ORM1) + 0.95×ln(CEA) - 11.67 (threshold = 0.45. When the risk score is greater than or equal to 0.45, the person is judged to be a colorectal cancer patient; when it is less than 0.45, the person is judged to be a healthy person). Comparing its diagnostic performance with using ORM1 or CEA alone as a biomarker, the results are as shown in Table 10 and Figure 8 as follows.

[0210] Table 10. Comparison of diagnostic efficacy of using ORM1 and CEA alone or in combination

[0211]

[0212] According to Table 10 and Figure 8 it can be seen that compared with using ORM1 or CEA alone as a biomarker, combining the two biomarkers, ORM1 and CEA, to construct a logistic regression model (ORM1 + CEA) has better diagnostic performance and obvious synergistic effects, and can better distinguish colorectal cancer patients from healthy people.

[0213] 2. Differences in diagnostic efficacy of different ORM1 and CEA antibody combinations

[0214] In this embodiment, the following three antibody combinations are used respectively: The first one: the existing antibody combination; the second one: the first antibody (detection antibody) of ORM1, the second antibody (enzyme-labeled antibody), combined with the first antibody (detection antibody) of CEA, the second antibody (enzyme-labeled antibody); the third one: the first antibody (enzyme-labeled antibody) of ORM1, the second antibody (detection antibody), combined with the first antibody (enzyme-labeled antibody) of CEA, the second antibody (detection antibody), which is equivalent to that the third one exchanges the types of the detection antibody and the enzyme-labeled antibody. Detection kits for ORM1 and CEA are constructed respectively, and a combined diagnosis model (ORM1 + CEA) is used for the diagnosis of colorectal cancer. The test samples include the sera of 350 healthy people and 210 colorectal cancer patients, which are randomly divided into a test group and a verification group. Among them, the test group includes the sera of 175 healthy people and 105 colorectal cancer patients, and the verification group includes the sera of 175 healthy people and 105 colorectal cancer patients.

[0215] In the first one, the existing antibody combination includes: the first antibody of ORM1 (purchased from Abnova, model H00005004-M01) and the second antibody (purchased from Yuanmu, model YM-H0573), the first antibody of CEA (purchased from Sigma, model HPA019758) and the second antibody (purchased from Okai, model R181c1). A model is constructed through the SPSS 22.0 algorithm software, and the constructed Logistic regression formula is: Risk score = 1.65×ln(ORM1) + 0.75×ln(CEA) - 10.98 (threshold = 0.63. When the risk score is greater than or equal to 0.63, it is judged as a colorectal cancer patient; when it is less than 0.63, it is judged as a healthy person).

[0216] In the second one, the heavy chain amino acid sequence of the first antibody (detection antibody) of ORM1 is SEQ ID NO.9, the light chain amino acid sequence is SEQ ID NO.10; the heavy chain amino acid sequence of the second antibody (enzyme-labeled antibody) is SEQ ID NO.17, the light chain amino acid sequence is SEQ ID NO.18; the heavy chain amino acid sequence of the first antibody (detection antibody) of CEA is SEQ ID NO.26, the light chain amino acid sequence is SEQ ID NO.27, and the heavy chain amino acid sequence of the second antibody (enzyme-labeled antibody) of CEA is SEQ ID NO.34, the light chain amino acid sequence is SEQ ID NO.35. A model is constructed through the SPSS 22.0 algorithm software, and the constructed Logistic regression formula is: Risk score = 1.79×ln(ORM1) + 0.93×ln(CEA) - 11.54 (threshold = 0.44. When the risk score is greater than or equal to 0.44, it is judged as a colorectal cancer patient; when it is less than 0.44, it is judged as a healthy person).

[0217] In the third case, the heavy chain amino acid sequence of the ORM1 first antibody (enzyme-labeled antibody) is SEQ ID NO.9, and the light chain amino acid sequence is SEQ ID NO.10; the heavy chain amino acid sequence of the second antibody (detection antibody) is SEQ ID NO.17, and the light chain amino acid sequence is SEQ ID NO.18; the heavy chain amino acid sequence of the CEA first antibody (enzyme-labeled antibody) is SEQ ID NO.26, and the light chain amino acid sequence is SEQ ID NO.27; the heavy chain amino acid sequence of the CEA second antibody (detection antibody) is SEQ ID NO.34, and the light chain amino acid sequence is SEQ ID NO.35. The Logistic regression formula constructed by the SPSS 22.0 algorithm software is: Risk score = 0.65×ln(ORM1) + 0.25 ×ln(CEA) - 3.64 (threshold = 0.47. When the risk score is greater than or equal to 0.47, it is judged as a colorectal cancer patient; when it is less than 0.47, it is judged as a healthy person).

[0218] The test results of the three antibody combinations for distinguishing healthy people and colorectal cancer patients are shown in Table 11.

[0219] Table 11. Diagnostic performance of different ORM1 and CEA antibody combinations

[0220]

[0221] It can be seen from Table 11 that although the detected markers are both ORM1 and CEA proteins and are based on the same combined diagnostic model, when different antibodies are used for detection, the diagnostic results will also be significantly different. The reason may be that the existing antibodies not only have low detection sensitivity, but also different antibodies are used as detection antibodies or enzyme-labeled antibodies respectively, and the detection results will also be significantly different. In addition, there are obvious differences in stability (Tm), cross-reaction with the matrix, high-dose hook effect, etc. among different antibodies, and these reasons will all lead to a decrease in the diagnostic efficiency for diagnosing colorectal cancer.

[0222] At the same time, during the diagnosis of colorectal cancer, it is necessary to detect the blood samples through ORM1 antibodies and / or CEA antibodies. There are complex interfering substances in the blood samples, and the matrix has a great influence, and cross-reactions or interference from some non-linear fragments are likely to occur. Using different antibodies for the detection and diagnosis of colorectal cancer will inevitably produce completely different diagnostic effects. Therefore, a better antibody combination must be selected to improve the diagnostic efficiency.

[0223] Therefore, the preferred antibody combination provided by the present invention, including the ORM1 first antibody, the second antibody, the CEA first antibody, and the second antibody, is used to construct a detection kit (the second type) for two markers, which can significantly improve the diagnostic efficiency of colorectal cancer.

[0224] It can be understood that the embodiments described in the present invention are all preferred embodiments and features. Any person of ordinary skill in the art can make some changes and variations based on the essence described in the present invention, and these changes and variations are also considered to fall 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 ORM1 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.3, CDR2 consisting of the amino acid sequence of SEQ ID NO.4, and CDR3 consisting of the amino acid sequence of SEQ ID NO.5 in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.6, CDR2 consisting of the amino acid sequence of SEQ ID NO.7, and CDR3 consisting of the amino acid sequence of SEQ ID NO.8 in the light chain variable region; The second antibody comprises: (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.11, CDR2 consisting of the amino acid sequence of SEQ ID NO.12, and CDR3 consisting of the amino acid sequence of SEQ ID NO.13 in the heavy chain variable region, and (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.14, CDR2 consisting of the amino acid sequence of SEQ ID NO.15, and CDR3 consisting of the amino acid sequence of SEQ ID NO.16 in the light chain variable region.

2. The antibody against the ORM1 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.36, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.37; the amino acid sequence of the heavy chain variable region of the second antibody is as shown in SEQ ID NO.38, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.

39.

3. An antibody against CEA 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.21, CDR2 consisting of the amino acid sequence of SEQ ID NO.22, and CDR3 consisting of the amino acid sequence VPD in the heavy chain variable region, and (2) CDR1 consisting of the amino acid sequence of SEQ ID NO.23, CDR2 consisting of the amino acid sequence of SEQ ID NO.24, and CDR3 consisting of the amino acid sequence of SEQ ID NO.25 in the light chain variable region; The second antibody comprises: (3) CDR1 consisting of the amino acid sequence of SEQ ID NO.28, CDR2 consisting of the amino acid sequence of SEQ ID NO.29, and CDR3 consisting of the amino acid sequence of SEQ ID NO.30 in the heavy chain variable region, and (4) CDR1 consisting of the amino acid sequence of SEQ ID NO.31, CDR2 consisting of the amino acid sequence of SEQ ID NO.32, and CDR3 consisting of the amino acid sequence of SEQ ID NO.33 in the light chain variable region.

4. The antibody against CEA protein according to claim 3, characterized in that, The amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID NO.40, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.41; the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO.42, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

43.

5. A kit for detecting ORM1 protein and / or CEA protein, characterized in that, An antibody comprising the ORM1 protein as claimed in claim 1 or 2, and / or an antibody comprising the CEA 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 comprising the ORM1 protein as claimed in claim 1 or 2, and / or an antibody comprising the CEA protein as claimed in claim 3 or 4.

7. The use according to claim 6, wherein An antibody comprising the ORM1 protein and an antibody comprising the CEA protein; the antibody comprising the ORM1 protein comprises a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID NO.36, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.37; the amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO.38, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.39; The antibody comprising the CEA protein comprises a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody is shown as SEQ ID NO.40, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.41; The amino acid sequence of the heavy chain variable region of the second antibody is shown as SEQ ID NO.42, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.

43.

8. A kit for predicting whether an individual has colorectal cancer, characterized in that, An antibody comprising the ORM1 protein as claimed in claim 1 or 2, and an antibody comprising the CEA 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 ORM1 protein as claimed in claim 1 or 2, and an antibody comprising the CEA 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, which is used for analyzing the detection value of an antigen, the antigen is detected by an antibody, and the antibody comprises an antibody comprising the ORM1 protein as claimed in claim 1 or 2, and / or an antibody comprising the CEA protein as claimed in claim 3 or 4.

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