Antibodies to orm1, cea proteins and combinations thereof and use in diagnosis of colorectal cancer

Abstract

The application provides an antibody of ORM1 and CEA protein, a combination thereof and application in diagnosis of colorectal cancer, and solves the problems of low affinity and specificity and poor stability of existing antibodies by optimizing an immunogen sequence of ORM1 and CEA, screening a high-affinity and high-specificity monoclonal antibody, screening optimal ORM1 and CEA antibodies and combining the same. The combination has a wider detection range, exhibits extremely high synergistic diagnostic value in joint detection, significantly improves the diagnostic efficiency of early colorectal cancer, and has important clinical application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of antibody screening and disease diagnosis, in particular, to antibodies of ORM1, CEA protein and combinations thereof and application in diagnosis of colorectal cancer. BACKGROUND

[0002] Orosomucoid 1 (ORM1) is encoded by the long arm of chromosome 9 32 (9q32) and belongs to the Lipocalins family of proteins, which 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, alpha-1 acid glycoprotein 1. Recombinant human ORM1 produced in E. 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 some cytokines, thus playing a key role in the transport of substances inside and outside the cell. It plays a key role in the occurrence and development of certain diseases, such as acute inflammatory response, cardiovascular disease, diabetes, cancer (liver cancer, breast cancer, malignant mesothelioma, etc.). Through these complex regulatory mechanisms, ORM1 gene ensures its timely expression under certain physiological conditions, maintaining the normal function 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, mediates the extravasation of cells through infection and inflammation. The expression of ORM1 is induced by acute phase stimulatory mediators such as bacterial lipopolysaccharide.

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

[0004] CEA is the English abbreviation of carcinoembryonic antigen (CEA), which is one of the tumor markers, belongs to an acid glycoprotein, exists on the surface of cancer cells differentiated from endoderm cells, and can be detected in body fluids such as serum, cerebrospinal fluid, urine, feces and the like. Carcinoembryonic antigen has important significance for the differential diagnosis of malignant tumors, disease monitoring, and treatment effect evaluation. CEA is a tumor-related antigen first extracted from colon cancer and embryonic tissue by Gold and Freedman in 1965, which is an acid glycoprotein with human embryonic antigen characteristics, exists on the surface of cancer cells differentiated from endoderm cells, and is a structural protein of the cell membrane. It is formed in the cytoplasm, secreted outside the cell through the cell membrane, and then enters the surrounding body fluid. Therefore, it can be detected in various body fluids and excretions such as serum, cerebrospinal fluid, milk, gastric juice, pleural and peritoneal fluid, urine, feces and the like. In the past, CEA was used as a specific marker for early diagnosis of colorectal cancer. Through a large number of clinical practices, it is found that not only the CEA value of gastrointestinal malignant tumors can be elevated, but also the serum of breast cancer, lung cancer and other malignant tumors is elevated. Therefore, carcinoembryonic antigen is a broad-spectrum tumor marker, although it cannot be used as a specific indicator for the diagnosis of a certain malignant tumor, but it still has important clinical value in the differential diagnosis of malignant tumors, disease monitoring, and treatment effect evaluation.

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

[0006] Although ORM1 or CEA can be used for early diagnosis of colorectal cancer, there is still a lack of antibodies that can detect ORM1 or ECA with high sensitivity and specificity. The existing ORM1 or ECA antibodies have low immunogen expression, high preparation cost, and the antibody preparation process is not optimized for sequence, the specificity of the antibody is not high, and cross-reaction with other cell surface antigens is easy to occur, which has insufficient sensitivity for early colorectal cancer detection, and is difficult to meet the current scientific research and clinical needs.

[0007] Therefore, it is urgent to find antibodies that can detect ORM1 protein and CEA protein with high sensitivity and specificity, and have stable performance and wider detection range, so as to more accurately diagnose early colorectal cancer. SUMMARY

[0008] In view of the problems in the prior art, the present application provides an antibody of ORM1 and CEA protein, a combination thereof and application in diagnosis of colorectal cancer, by optimizing the immunogen sequence of ORM1 and CEA, screening a monoclonal antibody with high affinity and high specificity, screening the optimal ORM1 and CEA antibody and combining them, the problems of low affinity and specificity and poor stability of the existing antibody can be effectively solved, the detection range is wider, and the combination detection shows extremely high synergistic diagnostic value, which significantly improves the diagnostic efficiency of early colorectal cancer and has important clinical application value.

[0009] ORM1 is an acute phase response protein, which is increased in inflammation and cancer, and CEA is a conventional serum marker of tumors such as colorectal cancer, which is also increased in inflammation. Due to the low expression amount of the immunogen of ORM1 and CEA, the preparation cost is high, and the antibody preparation process is usually optimized to improve the expression amount, purity and immunogenicity of the eukaryotic expression and purification process of the recombinant antigen, but the sequence is not optimized and screened, the obtained antibody has low specificity and is prone to cross reaction with other cell surface antigens, and high-efficiency gastrointestinal tumor (especially colorectal cancer) diagnosis cannot be achieved. The present application develops a new high-affinity and specific antibody of ORM1 and CEA and a combination thereof, which can be used for preparation of a diagnostic kit, detection of ORM1 and CEA in different samples, and meets the current scientific research and clinical needs.

[0010] In one aspect, the present application provides an antibody of ORM1 protein, which comprises a first antibody and / or a second antibody; the first antibody comprises:

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

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

[0013] The second antibody comprises:

[0014] (3) CDR1 composed of the amino acid sequence of SEQ ID NO. 11, CDR2 composed of the amino acid sequence of SEQ ID NO. 12 and CDR3 composed 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 application relates to the technical field of antibody preparation, and particularly relates to a specific targeting antibody against ORM1 and a preparation method and application thereof. ORM1 nucleotide sequences are searched by bioinformatics, and two antigens ORM1-H01 and ORM1-H02 are designed. The antigen plasmids are synthesized, positive clones are selected from TOP10 transformed by the obtained plasmids, and the plasmids are preserved. The plasmids are extracted from activated amplification culture, and eukaryotic cells are transfected and expressed. The corresponding antigens are obtained by affinity purification. The ORM1-H01 antigen is subjected to immunization injection and hybridoma cell fusion, and ORM1-H02 is used for screening, so that a plurality of pairs of monoclonal antibody strains are obtained. Through antigen detection and antibody pairing, finally, high-quality antibody pairs are selected for the development of a colorectal cancer rapid detection kit.

[0017] Further, 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.

[0018] Further, 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.

[0019] In another aspect, the present application provides a kit for detecting ORM1 protein, which comprises the antibody of the ORM1 protein as described above.

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

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

[0022] In another aspect, the present application provides an antibody of CEA protein, which comprises a first antibody and / or a second antibody; the first antibody comprises:

[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 application also relates to a specific targeting antibody against CEA and a preparation method and application thereof, wherein CEA nucleotide sequences are searched by bioinformatics, two antigens, CEA-H01 and CEA-H02, are designed, antigen plasmids are synthesized, positive clones are selected from TOP10 transformed by the obtained plasmids, and are preserved, and the plasmids are extracted from activated amplification culture to perform eukaryotic cell transfection expression and affinity purification to obtain corresponding antigens. The CEA-H01 antigen is subjected to immunization injection and hybridoma cell fusion, and the 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 shown in SEQ ID NO. 40, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 41; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 42, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 43.

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

[0031] In another aspect, the present application provides a kit for detecting CEA protein, which comprises 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 modes, the kit is an ELISA detection kit. By screening the optimal and suitable pairing of CEA first antibody and second antibody, a double antibody combination ELISA detection kit is constructed.

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

[0035] In some modes, the kit for detecting ORM1 protein and the kit for detecting CEA protein are combined, which can be used for simultaneously detecting ORM1 protein and CEA protein, so as to be applied to the field of simultaneously detecting multiple markers, such as the field of gastrointestinal tumor diagnosis, so as to improve the diagnostic efficiency by combined detection.

[0036] In another aspect, the present application provides the use of an antibody for preparing a reagent for predicting whether an individual has colorectal cancer, which comprises 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. In the existing cancer diagnosis technology, the detection of a single biomarker (such as CEA) has the problems of low sensitivity and high false positive rate. ORM1 is related to tumor microenvironment, but its single detection has not yet matured in clinical application. The present application takes ORM1 and CEA as combined markers, and solves the problem of insufficient diagnostic efficiency of single marker by combined detection of antibodies targeting ORM1 and CEA protein, so as to improve the accuracy and specificity of cancer diagnosis.

[0038] Although ORM1 and CEA are known diagnostic markers for colorectal cancer, different ORM1 antibodies and / or CEA antibodies are used to diagnose colorectal cancer, and the diagnostic efficiency is significantly different. During the diagnosis of colorectal cancer, ORM1 antibodies and / or CEA antibodies are used to detect blood samples, and there are complex interfering substances in the blood samples, the matrix has a great influence, and there are cross reactions or the interference of some nonlinear fragments. Directly using existing antibodies for detection, it is difficult to obtain ideal detection results, which seriously affects the accuracy of the diagnosis results.

[0039] The ORM1 and CEA proteins of the present application are obtained through the design, expression of antigens, and screening of monoclonal antibodies, and the antibodies have better detection sensitivity and specificity, and the signal is more stable, the cross reaction is smaller, and the diagnostic efficiency can be significantly improved when used for the diagnosis of colorectal cancer.

[0040] Further, the antibodies include antibodies of the ORM1 protein and antibodies of the CEA protein.

[0041] Further, the antibodies of the ORM1 protein include a first antibody and a second antibody, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 37; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 38, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 39.

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

[0043] Further, the reagent is used to detect the content of the antigen capable of binding to the antibody in the body fluid sample; the body fluid sample includes 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 by taking ORM1 and CEA proteins as detection targets, and the present application uses antibodies to detect ORM1 and CEA proteins.

[0045] Further, the reagent is used to detect the presence or absence, relative abundance, or concentration of a biomarker in a body fluid sample.

[0046] The present application provides a biomarker (ORM1 and CEA protein) from blood screening to predict the risk of colorectal cancer, which is significantly different in the blood of colorectal cancer patients and healthy people. By collecting blood samples, the content of ORM1 and CEA protein in the blood of an individual can be detected to predict or assist in diagnosing the possibility of the individual suffering from colorectal cancer, or the content of ORM1 and CEA protein in the blood of a group can be detected to divide the group into colorectal cancer patients and healthy people.

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

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

[0049] Further, the antibody of the ORM1 protein and the antibody of the CEA protein are included.

[0050] Further, the antibody of the ORM1 protein comprises a first antibody and a second antibody, the first antibody has an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 36, and an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 37; the second antibody has an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 38, and an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 39.

[0051] The antibody of the CEA protein comprises a first antibody and a second antibody, the first antibody has an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 40, and an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 41; the second antibody has an amino acid sequence of a heavy chain variable region as shown in SEQ ID NO. 42, and an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 43.

[0052] In another aspect, the present application 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 comprising an antibody of the ORM1 protein as described above, and / or an antibody of the CEA protein as described above.

[0053] Further, the data analysis module uses the detection values of the markers of the known samples as a training set, and according to the conditions of the colorectal cancer patients after surgery, the healthy population and the colorectal cancer patients are divided, the relationship between the detection values of the healthy population and the colorectal cancer patients is analyzed, and a model is constructed.

[0054] Further, the system further comprises 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 the biomarkers; the data input interface is used for inputting the detection values of the biomarkers, and the data output interface is used for outputting the prediction results.

[0055] The beneficial effects of the present application are:

[0056] 1. Design self-made ORM1 and CEA expression antigens, and use the antigens to immunize animals to obtain specific target monoclonal antibodies, and the monoclonal antibodies have very high sensitivity;

[0057] 2. The prepared antibodies are recombined to improve the expression amount and ensure the stable expression of the antibodies; the detection of clinical blood samples is introduced into the antibody test process, which greatly increases the reliability of the experimental data; double antibody detection, and the reagent consumption is reduced by 50%;

[0058] 3. A plurality of pairs of monoclonal antibodies against ORM1 antigen epitopes are screened, an ORM1 detection kit is constructed, and a double antibody sandwich method is combined to design, the cross reaction rate is extremely low (<2%), the sensitivity reaches pg / mL level, and ORM1 protein can be detected with high precision;

[0059] 4. A plurality of pairs of monoclonal antibodies against CEA antigen epitopes are screened, a CEA detection kit is constructed, and a double antibody sandwich method is combined to design, the cross reaction rate is extremely low (<2%), the sensitivity reaches pg / mL level, and CEA protein can be detected with high precision;

[0060] 5. Antibody combination and detection process, efficient and economical clinical application is realized;

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

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

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

[0064] Figure 3 It is a SDS-PAGE detection result graph after antigen purification;

[0065] Figure 4 Figure 1 is an immunization flow chart;

[0066] Figure 5 Figure 2 is a SDS-PAGE detection result of the purified antibody;

[0067] Figure 6 Figure 3 is a regression curve of the ORM1 kit detection in Example 5;

[0068] Figure 7 Figure 4 is a regression curve of the CEA kit detection in Example 5;

[0069] Figure 8 Figure 5 is a performance result of the ORM1 and / or CEA in diagnosing colorectal cancer patients in Example 6. DETAILED DESCRIPTION

[0070] The present application will be further described in conjunction with the accompanying drawings and examples. It is to be noted that the following examples are intended to facilitate the understanding of the present application, and do not limit the present application in any way. The reagents used in the present examples are known products, which 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 the present example is shown in Figure 1 and Figure 2 .

[0073] I. Preparation of ORM1 antigen

[0074] 1. Antigen expression

[0075] Through bioinformatics analysis, the nucleotide sequence of ORM1 (UniProt number: AAH26238) was obtained. Through gene synthesis method, two antigens ORM1-H01 (nucleotide sequence SEQ ID NO. 44) and ORM1-H02 (nucleotide sequence SEQ ID NO. 45) were designed and obtained. The antigen plasmid (pTT5) was synthesized by Shanghai Generay Biotech Co., Ltd. The positive clones were selected by transforming TOP10 according to the obtained plasmid, and the plasmid was extracted by amplification culture and used for eukaryotic cell transfection expression. The expression plasmid was transfected into HEK293 cells by using Invitrogen Lipofectamine2000 transfection reagent. The culture supernatant was harvested after 3-5 days (the expression time can be one of 3d, 4d, 5d, and 4d is preferred in the present example).

[0076] Purified ORM1-H01 (SEQ ID NO. 1) and ORM1-H02 (SEQ ID NO. 2) proteins were obtained by affinity chromatography, wherein the purification filler can be one of Ni-NTA / Ni-IDA, and in this embodiment, Ni-IDA is preferred, and the final buffer can be one of PBS, TBS, CBS, and in this embodiment, PBS is preferred. The protein was subjected to conventional physicochemical analysis and biological activity analysis, and the optimal expression and purification conditions were selected to ensure the reliability of the immunogen. After the antigen was purified, SDS-PAGE detection was performed, and the results are shown in Figure 3 .

[0077] 2. Animal immunization

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

[0079] After animal immunization, the serum titer was detected by ELISA detection (OD450), and the results are shown in Table 1.

[0080] Table 1, serum titer detection results

[0081]

[0082] According to Table 1, if the OD value is > 0.2 after dilution of 64,000 times, cell fusion and plating can be performed, and the preferred serum dilution method is stepwise dilution.

[0083] 3. Cell fusion and plating: the mice were sacrificed, and the eyeball blood was taken as a positive control. The mouse spleen was isolated and frozen, and part of the spleen was ground and counted. According to the ratio of spleen cell number: sp2 / 0 cell number = 8:1, cell fusion was performed, and 1 ml PEG1450 was added to terminate the preheated blank medium. After centrifugation, HAT conditioned medium was added for pressure screening, and the whole process was carried out at 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, and three rounds of screening were performed to select hybridoma cells that specifically bind to ORM1-H02 protein.

[0085] 5. Clone expansion and cryopreservation: Positive master clone cells were transferred to 24-well plates for expansion. 2 mL supernatant was collected from each expanded clone for indirect ELISA detection. These specific positive clone cells were cryopreserved to avoid clone loss.

[0086] 6. Subcloning: The positive master clones were subcloned by limiting dilution to ensure that each positive master clone was derived from a single master clone cell. Subcloning screening was performed by indirect ELISA.

[0087] 7. Pairing antibody screening: Based on antigen recognition confirmation, 8 stable subclone cell lines were selected from each master clone for cryopreservation and pairing. Excellent antibody pairs were obtained based on antibody pairing detection. 5 mL supernatant was collected from each subclone before cryopreservation, and all subclones were identified and stored. The detection method for antibody pairing detection was Elisa detection (OD450), with vertical columns as detection antibodies (coating antibodies) and horizontal as enzyme-labeled antibodies. The detection steps were as follows:

[0088] (1) Fix 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 enzyme-labeled plate;

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

[0091] 3) Discard the liquid and pat dry. Add 300 μL of washing buffer (PBS + 0.1% Tween 20) to each well and wash 3 times.

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

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

[0094] 2) Discard the blocking solution and pat dry. 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 TMB substrate to each well, and develop color at room temperature for 10-15 minutes (the time needs to be optimized by pre-experiment); immediately add 50 μL termination solution (2M H2SO4) to each well; detect OD value, and the results are shown in Table 2.

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

[0100] Table 2, detection results of antibody pairing

[0101]

[0102] According to Table 2, the three pairs of optimal 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: total RNA of hybridoma cells is extracted, and RNA is reverse transcribed into cDNA by RT-PCR reaction. Antibody light chain and heavy chain sequences are cloned, and the antibody light chain and heavy chain sequences are constructed into T vector, and then DNA sequencing analysis is performed to obtain antibody gene sequence.

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

[0105] Example 2, ORM1 antibody pairing selection

[0106] Based on the three pairs of paired antibodies obtained by screening in Example 1, antibody 4 and antibody 3, antibody 2 and antibody 7, and antibody 6 and antibody 8, the orthogonal pairing between antibodies is detected, and the antibody pair with excellent performance is obtained, and the specific detection method is ELISA sandwich method detection, and the detection steps are as follows:

[0107] (1) Fixed 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-labeled plate;

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

[0110] 3) Discard the liquid, pat dry, 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 blocking solution (5% skim milk in PBS) per well, 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 per 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, add 100 μL per 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 TMB substrate per well, develop color at room temperature in the dark for 10-15 minutes (the time needs to be optimized by pre-experiment); immediately add 50 μL termination solution (2M H2SO4) per well; detect the OD value, and the results are shown in Table 3.

[0118] Table 3, results of antibody pairs for detecting ORM1 antigen

[0119]

[0120] According to Table 3, the most preferred antibody pair is antibody 2 and antibody 7, wherein antibody 2 is named as ORM1 first antibody (detection antibody), and antibody 7 is named as ORM1 second antibody (enzyme-labeled antibody). Thus, the ORM1 first antibody and the second antibody can be used to construct a detection kit for detecting the content of ORM1.

[0121] The CDR1 sequence of the heavy chain of the first ORM1 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 amino acid sequence of the heavy chain is SEQ ID NO. 9, and the amino acid sequence of the light chain is SEQ ID NO. 10; the CDR1 sequence of the heavy chain of the second ORM1 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 amino acid sequence of the heavy chain is SEQ ID NO. 17, and the amino acid sequence of the light chain is SEQ ID NO. 18.

[0122] Meanwhile, the thermal stability Tm value of the obtained antibody was determined by circular dichroism (CD) to evaluate the stability of the antibody, and the existing antibody was compared, wherein the existing ORM1 antibody was purchased from Abnova, model number H00005004-M01. The detection results are shown in Table 4. Tm represents the midpoint temperature of thermal denaturation of the protein, i.e. the temperature at which the protein is unfolded by 50%, reflecting the trend of protein conformation change during temperature change, and the higher the Tm, the better the stability.

[0123] Table 4, stability change

[0124]

[0125] According to Table 4, it can be seen that the stability of the screened ORM1 monoclonal antibody is obviously better than that of the ORM1 commercially available antibody.

[0126] Example 3, screening and preparation process of CEA-specific monoclonal antibody

[0127] The screening and preparation process of the CEA-specific monoclonal antibody provided in this embodiment is shown in Figure 1 and Figure 2 .

[0128] I. Preparation of CEA antigen

[0129] 1. Antigen expression

[0130] Through bioinformatics analysis, the nucleotide sequence of CEA (UniProt number: P06731) was obtained, and two antigens CEA-H01 (nucleotide sequence SEQ ID NO. 46) and CEA-H02 (nucleotide sequence SEQ ID NO. 47) were designed and obtained by gene synthesis method. The antigen plasmid (pTT5) was synthesized by Shanghai Generay Biotech Co., Ltd. According to the obtained plasmid, positive clones were transformed into TOP10, and the plasmid was extracted by amplification culture for eukaryotic cell transfection expression. Invitrogen Lipofectamine 2000 transfection reagent was used to transfect the expression plasmid into HEK293 cells, and the culture supernatant was harvested after 3-5 days (the expression time can be one of 3d, 4d, 5d, and the preferred one in this embodiment is 4d).

[0131] The purified CEA-H01 (SEQ ID NO. 19) and CEA-H02 (SEQ ID NO. 20) proteins were obtained by affinity chromatography method, wherein the purification filler can be one of Ni-NTA / Ni-IDA, and the preferred one in this embodiment is Ni-IDA. The final buffer can be one of PBS, TBS, CBS, and the preferred one in this embodiment is PBS. The protein was subjected to conventional physicochemical analysis and biological activity analysis, and the optimal expression and purification conditions were selected to ensure the reliability of the immunogen. After the antigen was purified, SDS-PAGE detection was performed, and the results are shown in Figure 3 .

[0132] 2、Animal immunization

[0133] The CEA-H01 protein prepared in step 1 was used as an immunogen to immunize 5 Balb / c mice, respectively, and Freund's adjuvant was used for immunization to maintain the natural conformation of the protein. The specific immunization process is shown in Figure 4 . After immunization, the antibody titer of the serum of the immunized animals was detected by ELISA method to determine the level of immune response. After conventional immunization, if the immunized animals can reach the immune response level (OD value > 0.2, diluted 64,000 times) against the immunogen, cell fusion can be performed.

[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, the OD value is greater than 0.2 when diluted 64,000 times, and cell fusion and plating can be performed. The preferred serum dilution method is stepwise dilution.

[0138] 3. Cell fusion and plating: The mice were sacrificed, and the eyeball blood was taken as a positive control. The mouse spleen was isolated and stored frozen. A portion of the spleen was ground and counted. The cells were fused at a ratio of 8:1 sp2 / 0 cells to spleen cells, and 1 ml of PEG1450 was added to terminate the reaction in preheated blank medium. After centrifugation, HAT conditioned medium was added for pressure screening. The entire process was carried out in a 37°C water bath. All the fused cells were plated in 96-well plates.

[0139] 4. Screening: The supernatant of the fused cells was screened by ELISA. After three rounds of screening, hybridoma cells that specifically bound to CEA-H02 protein were selected.

[0140] 5. Clone expansion and storage: The positive parent clone cells were transferred to a 24-well plate for expansion. 2 mL of supernatant was collected from each expanded clone for indirect ELISA detection. The specific positive clone cells were stored to avoid clone loss.

[0141] 6. Subcloning: The positive parent clones were subcloned using the limited dilution method to ensure that each positive parent clone came from a single parent clone cell. Subcloning was screened by indirect ELISA.

[0142] 7. Pairing antibody screening: Based on antigen recognition confirmation, eight stable subclone cell lines were selected from each parent clone for low-temperature storage and pairing. A total of three pairs were obtained based on antibody pairing detection. The optimal antibody pair was obtained. 5 mL of supernatant was collected from each subclone before low-temperature storage, and the subtypes of all subclones were identified and stored. The detection method for antibody pairing detection was Elisa detection (OD450). In the sample, CEA antigen was diluted to 45 pg / ml. The results are shown in Table 6.

[0143] Table 6. Antibody pairing detection results

[0144]

[0145] According to Table 6, 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. Recombinant antibody plasmid construction: Total RNA was extracted from hybridoma cells, and RNA was reverse transcribed into cDNA by RT-PCR reaction. The antibody light chain and heavy chain sequences were cloned and constructed into T vector. DNA sequencing analysis was performed to obtain the antibody gene sequence.

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

[0148] Example 4, CEA antibody pair selection

[0149] The three pairs of paired antibodies obtained based on the screening of example 1 are antibody 1 and antibody 3, antibody 7 and antibody 6, antibody 6 and antibody 8. The orthogonal pairing between antibodies is detected by QC, and the antibody pair with excellent performance is obtained. The specific detection method is ELISA sandwich method detection, and the detection steps are as follows:

[0150] (1) Fixed coating antibody:

[0151] 1) Dilute the coating antibody to 2 μg / mL with carbonate buffer (pH 9.6), and add 100 μL to each well of a 96-well enzyme-labeled plate;

[0152] 2) Coat overnight at 4°C (12-16 hours);

[0153] 3) Discard the liquid and pat dry. Add 300 μL of washing buffer (PBS + 0.1% Tween20) to each well and wash 3 times.

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

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

[0156] 2) Discard the blocking solution and pat dry. 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, and add 100 μL to each well; incubate at 37°C for 45 minutes in the dark, 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 for 10-15 minutes (the time needs to be optimized by pre-experiment); immediately add 50 μL of termination solution (2M H2SO4) to each well; detect OD value, and the results are shown in Table 7.

[0161] Table 7, results of antibody pair detection of CEA antigen

[0162]

[0163] According to Table 7, the most preferred antibody pair is antibody 7 and antibody 6, wherein antibody 7 is named as CEA first antibody (detection antibody), and antibody 6 is named as CEA second antibody (enzyme-labeled antibody). Thus, the CEA content detection kit can be constructed by using the CEA first antibody and the second antibody, respectively.

[0164] According to DNA sequencing analysis, the CDR1 sequence of the heavy chain of the CEA first antibody is SEQ ID NO. 21, the CDR2 sequence is SEQ ID NO. 22, and 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 amino acid sequence of the heavy chain is SEQ ID NO. 26, and the amino acid sequence of the light chain is SEQ ID NO. 27; the CDR1 sequence of the heavy chain of the CEA second antibody is SEQ ID NO. 28, the CDR2 sequence is SEQ ID NO. 29, and 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 amino acid sequence of the heavy chain is SEQ ID NO. 34, and the amino acid sequence of the light chain is SEQ ID NO. 35.

[0165] Meanwhile, the thermal stability Tm value of the obtained antibody is determined by circular dichroism (CD) to evaluate the stability of the antibody, and the existing antibody is compared, wherein the existing CEA antibody is purchased from Sigma, model number HPA019758. The detection results are shown in Table 8. The Tm in the table represents the thermal denaturation midpoint temperature of the protein, i.e. the temperature at which the protein is unfolded by 50%, reflecting the trend of protein conformation change in the process of temperature change, and the higher the Tm, the better the stability.

[0166] Table 8, stability change

[0167]

[0168] According to Table 8, the stability of the screened CEA monoclonal antibody is obviously better than that of the CEA commercially available antibody.

[0169] Example 5, construction of an ORM1 and CEA combined detection kit

[0170] The present embodiment respectively adopts the first antibody and the second antibody of ORM1-1 screened in Example 2, and the first antibody and the second antibody of ORM1-2; and the first antibody and the second antibody of CEA screened in Example 4 to construct the ORM1 and CEA combined detection kit.

[0171] Therefore, there are two kinds of kits, wherein the kit 1 comprises:

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

[0173] 2. Enzyme-labeled second antibody: ORM1-1 second antibody and CEA second antibody biotinylated and labeled with 0.5 ug / mL horseradish peroxidase.

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

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

[0176] 5. Color developing substrate: color developing solution A, 0.02% hydrogen peroxide by mass fraction; color developing solution B, 4g / L tetramethyl benzidine.

[0177] 6. Termination solution: 1M phosphoric acid.

[0178] 7. Sealing film.

[0179] The kit 2 comprises:

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

[0181] 2. Enzyme-labeled second antibody: ORM1-2 second antibody and CEA second antibody biotinylated and labeled with 0.5 ug / mL horseradish peroxidase.

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

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

[0184] 5. Color developing substrate: color developing solution A, 0.02% hydrogen peroxide by mass fraction; color developing solution B, 4g / L tetramethyl benzidine.

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

[0186] 7. Sealing film.

[0187] The constructed ORM1 and CEA combined detection kit 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 detected, so as to obtain the detection values of ORM1 and CEA respectively. That is, it can be used for detecting ORM1, CEA, or both ORM1 and CEA. Using the detected ORM1 and CEA as combined markers can effectively avoid the occurrence of false positive and false negative results in the diagnosis of intestinal cancer using CEA as a marker alone.

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

[0189] The concentration of ORM1 in the sample to be detected is detected as follows:

[0190] 1) Coating: dilute the ORM1 first antibody to 0.1-10 ug / ml (according to the corresponding test specification) with carbonate CBS coating solution, add 50ul per well to a 96-well plate, and incubate at 4°C overnight.

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

[0192] 3) Add test sample: set up control wells, 8 standard wells (for making a standard curve), and sample wells. Add blank diluent to the control wells, add gradient concentrations of ORM1 working solution (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, and add the sample to be detected to the sample wells. The volume added is 50uL. Seal the enzyme-labeled plate with a sealing film, incubate at 37°C for 30 minutes, and set up duplicate wells. Shake at 220 rpm for 1 hour at room temperature, wash the plate 3 times with a plate washer, and dry.

[0193] 4) Add enzyme-labeled secondary antibody: dilute the biotinylated ORM1 secondary antibody to the working concentration with diluent. Add 50ul per well, shake at 220 rpm for 1 hour at room temperature, wash the plate 3 times with a plate washer, and dry.

[0194] 5) Add enzyme solution: dilute SA-HRP stock solution with diluent to 10000 times as enzyme working solution. Add 50ul to each well, shake at 220rpm for 45min~1h at room temperature, wash 3 times with plate washer, and dry.

[0195] 6) Add color developing substrate: add 50uL of 4g / L tetramethyl benzidine (TMB) and 5uL of 0.02% hydrogen peroxide to each well, and incubate at 37℃ for 15min.

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

[0197] 8) Read and calculate: detect the absorbance (OD value) of each well at 450nm.

[0198] 9) Calculate the corresponding concentration by fitting the standard curve with four parameters: draw the standard curve with the concentration of ORM1 working solution as the vertical coordinate and the OD value as the horizontal coordinate, and obtain the regression curve as shown in Figure 6 , (R 2 =0.999), and the regression equation is as follows: , the 4-parameter A estimate is 0.344, the standard deviation is 0.051, and the confidence interval is [0.181, 0.507]; the B estimate is 1.031, the standard deviation is 0.176, and the confidence interval is [0.471, 1.591]; the C estimate is 1.23e+4, the standard deviation is 8805, and the confidence interval is [-1.58e+4, 4.03e+4]; and the D estimate is 6.203, the standard deviation is 2.470, and the confidence interval is [-1.656, 14.06]. Substitute the detected OD value of the sample to be tested into the regression equation to calculate the concentration of ORM1 in the sample to be tested.

[0199] The specific method for detecting the CEA concentration in the sample to be tested is the same as above, and the regression curve is as shown in Figure 7 , (R 2 =0.998), and the regression equation is as follows: , the 4-parameter A estimate is 0.036, the standard deviation is 0.053, and the confidence interval is [-0.132, 0.205]; the B estimate is 1.241, the standard deviation is 0.168, and the confidence interval is [0.705, 1.776]; the C estimate is 2727, the standard deviation is 699.7, and the confidence interval is [500.2, 4954]; and the D estimate is 4.116, the standard deviation is 0.603, and the confidence interval is [2.197, 6.035]. Substitute the detected OD value of the sample to be tested into the regression equation to calculate the concentration of CEA in the sample to be tested.

[0200] The ORM1 protein control solution and the CEA protein control solution (negative human serum preparation) containing 5, 10, 50, 250 pg / mL were used to investigate the intra-batch / inter-batch precision. The intra-batch precision: the same batch of kit was used to quantitatively detect the value sample, each sample was continuously measured for 20 times, and the SD value and coefficient of variation (CV) of different concentrations of samples were calculated respectively; the inter-batch precision: three different batches of kits were selected to quantitatively detect the value sample, each sample was repeatedly measured for 8 times using the same kit, and the SD value and coefficient of variation (CV) of different concentrations of samples were calculated respectively. The detection results are shown in Table 9.

[0201] Table 9, intra-batch / inter-batch precision

[0202]

[0203] It can be seen that the chemiluminescence immunoassay kit constructed in this embodiment can achieve a detection sensitivity of 5 pg / mL for ORM1 and CEA protein, and the intra-batch / inter-batch precision meets the detection requirements.

[0204] Example 6, application of ORM1 and CEA combined detection kit in diagnosis of colorectal cancer

[0205] 1. Diagnostic efficiency of ORM1 and CEA detection kit used alone or in combination

[0206] In this example, the ORM1 and CEA detection kit constructed in Example 5 was used to investigate the detection of ORM1 and CEA as markers alone or in combination, and the ORM1 and / or CEA in the serum of healthy people (158 cases) and colorectal cancer patients (89 cases) was tested. The ability of the kit for distinguishing between diseased and healthy populations was evaluated by SPSS 22.0 software (Binary Logistic).

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

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

[0209] When the two markers ORM1 and CEA are used for detection, the Logistic regression formula constructed by SPSS 22.0 algorithm software is: risk score = 1.82 x ln(ORM1) + 0.95 x ln(CEA) - 11.67 (threshold = 0.45, when the risk score is greater than or equal to 0.45, it is judged as a colorectal cancer patient, and less than 0.45, it is judged as a healthy person). Compared with the use of ORM1 or CEA alone as a marker, the diagnostic performance is shown in Table 10 and Figure 8 .

[0210] Table 10, comparison of diagnostic performance of ORM1 and CEA alone or in combination

[0211]

[0212] According to Table 10 and Figure 8 It can be seen that compared with the use of ORM1 or CEA alone as a marker, the combination of ORM1 and CEA as two markers, the constructed logistic regression model (ORM1+CEA), has better diagnostic performance, and has obvious synergistic effect, and can better distinguish colorectal cancer patients and healthy people.

[0213] 2. Difference in diagnostic performance of different ORM1 and CEA antibody combinations

[0214] The following three antibody combinations were used in this example: the first: the existing antibody combination; the second: ORM1 first antibody (detection antibody), second antibody (enzyme-labeled antibody), combined with CEA first antibody (detection antibody), second antibody (enzyme-labeled antibody); the third: ORM1 first antibody (enzyme-labeled antibody), second antibody (detection antibody), combined with CEA first antibody (enzyme-labeled antibody), second antibody (detection antibody), which is equivalent to the third, the detection antibody and the enzyme-labeled antibody are exchanged. The ORM1 and CEA detection reagent kits were constructed, and the combined diagnostic model (ORM1 + CEA) was used for the diagnosis of colorectal cancer. The test samples included 350 healthy people and 210 colorectal cancer patients, which were randomly divided into a test group and a validation group, wherein the test group included 175 healthy people and 105 colorectal cancer patients, and the validation group included 175 healthy people and 105 colorectal cancer patients.

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

[0216] In the second, the heavy chain amino acid sequence of the ORM1 first antibody (detection antibody) 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, and the light chain amino acid sequence is SEQ ID NO. 18; the heavy chain amino acid sequence of the CEA first antibody (detection antibody) is SEQ ID NO. 26, the light chain amino acid sequence is SEQ ID NO. 27, the heavy chain amino acid sequence of the CEA second antibody (enzyme-labeled 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 model is: risk score = 1.79 x ln(ORM1) + 0.93 x ln(CEA) -11.54 (threshold value = 0.44, when the risk score is greater than or equal to 0.44, it is judged as a colorectal cancer patient, and less than 0.44, it is judged as a healthy person).

[0217] In a third aspect, the heavy chain amino acid sequence of the ORM1 first antibody (enzyme-labeled antibody) 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 (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, 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 x ln(ORM1) + 0.25 x ln(CEA) - 3.64 (threshold value = 0.47, when the risk score is greater than or equal to 0.47, it is judged as a colorectal cancer patient, and when the risk score is less than 0.47, it is judged as a healthy person).

[0218] The detection results of the three antibody combinations for distinguishing between 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] As can be seen from Table 11, although the markers detected are both ORM1 and CEA proteins, based on the same joint diagnostic model, the diagnostic results will also be significantly different when different antibodies are used for detection. The reason may be that the existing antibodies not only have low detection sensitivity, but even different antibodies are used as detection antibodies or enzyme-labeled antibodies, and the detection results are also significantly different. In addition, different antibodies have significant differences in stability (Tm), cross-reaction with the matrix, high-dose hook effect, etc., which can all lead to a decrease in diagnostic efficiency for diagnosing colorectal cancer.

[0222] At the same time, in the process of diagnosing colorectal cancer, ORM1 antibodies and / or CEA antibodies are needed to detect blood samples, and there are complex interfering substances in the blood samples, the matrix has a large influence, and cross-reactions or interference of some non-linear fragments are prone to occur. Therefore, different antibodies are used for colorectal cancer detection and diagnosis, which will inevitably produce completely different diagnostic effects, and therefore a more optimal antibody combination must be selected to improve the diagnostic efficiency.

[0223] Therefore, the preferred antibody combination provided by the present application includes ORM1 first antibody, second antibody and CEA first antibody, second antibody, and the detection kit (second kind) of two markers constructed can significantly improve the diagnostic efficiency of colorectal cancer.

[0224] It is to be understood that the embodiments described herein are merely exemplary and that a person of ordinary skill in the art can make modifications and variations thereto without departing from the scope and spirit of the application.

Claims

1. A kit for detecting ORM1 protein and CEA protein, characterized by, An antibody to an ORM1 protein, and an antibody to a CEA protein; the antibody to the ORM1 protein includes a first antibody and a second antibody; the first antibody includes: (1) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 3, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 4, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 5 in a heavy chain variable region, and (2) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 6, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 7, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 8 in a light chain variable region; the second antibody includes: (1) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 11, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 12, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 13 in a heavy chain variable region, and (2) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 14, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 15, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 16 in a light chain variable region; the antibody to the CEA protein includes a first antibody and / or a second antibody; the first antibody includes: (1) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 21, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 22, and a CDR3 consisting of the amino acid sequence VPD in a heavy chain variable region, and (2) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 23, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 24, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 25 in a light chain variable region; the second antibody includes: (1) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 28, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 29, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 30 in a heavy chain variable region, and (2) a CDR1 consisting of the amino acid sequence of SEQ ID NO. 31, a CDR2 consisting of the amino acid sequence of SEQ ID NO. 32, and a CDR3 consisting of the amino acid sequence of SEQ ID NO. 33 in a light chain variable region.

2. The kit of claim 1, wherein In the antibody to the ORM1 protein, 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; The antibody of the CEA protein includes a first antibody and / or a second antibody; the first antibody includes:

3. Use of an antibody for the manufacture of a reagent for predicting whether an individual has colorectal cancer, characterized in that, (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 includes: (1) 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 (2) 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; The antibody of the CEA protein includes a first antibody and / or a second antibody; the first antibody includes: (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 includes: (1) 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 (2) 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. Use according to claim 3, characterized in that, In the antibody of the ORM1 protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 37; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 38, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 39; In the antibody of the CEA protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 40, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 41; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 42, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

43.

5. A kit for predicting whether an individual has colorectal cancer, characterized in that, The antibody of the ORM1 protein includes a first antibody and a second antibody; the first antibody includes: (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 includes: (1) 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 (2) 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; The antibody of the CEA protein includes a first antibody and / or a second antibody; the first antibody includes: (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 includes: (1) 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 (2) 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.

6. The kit of claim 5, wherein Among the antibodies of the ORM1 protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 36, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 37; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 38, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 39; Among the antibodies of the CEA protein, the amino acid sequence of the heavy chain variable region of the first antibody is shown in SEQ ID NO. 40, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 41; the amino acid sequence of the heavy chain variable region of the second antibody is shown in SEQ ID NO. 42, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

43.

7. An antibody panel for predicting whether an individual has colorectal cancer, characterized in that, The antibodies of the ORM1 protein and the antibodies of the CEA protein; the antibodies of the ORM1 protein include a first antibody and a second antibody; the first antibody includes: (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 includes: (1) 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 (2) 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; The antibodies of the CEA protein include a first antibody and / or a second antibody; the first antibody includes: (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: (1) 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 (2) 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.

8. The antibody combination of claim 7, wherein In the antibody of the ORM1 protein, 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; In the antibody of the CEA protein, 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.

9. A system for predicting whether an individual has colorectal cancer, the system comprising: The system comprises a data analysis module for analyzing the detection value of the antigen, wherein the antigen is detected by the antibody, and the antibody comprises the antibody of the ORM1 protein and the antibody of the CEA protein; The antibody of the ORM1 protein comprises a first antibody and 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: (1) 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 (2) 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) 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; The antibody of the CEA protein comprises 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: (1) 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 (2) 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.

10. The system of claim 9, wherein, In the antibody of the ORM1 protein, 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; In the antibody of the CEA protein, 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.

Images