Anti-idiotype antibody, preparation method thereof and application of anti-idiotype antibody in preparation of digestive tract tumor early screening product

Abstract

The invention relates to the field of biotechnology and medical diagnosis, and particularly discloses an anti-idiotype antibody, a preparation method thereof and application of the anti-idiotype antibody in preparation of a digestive tract tumor early screening product. The anti-idiotypic antibody used in the digestive tract tumor early screening product provided by the invention is prepared by the following steps: immunizing a rabbit with a vegetable protein (Zhengsi vegetable protein, abbreviated as ZPP) with tumor antigen activity as an antigen to obtain an antibody RZPP; the fragment F (ab ') 2 obtained by enzyme digestion after purification is used for immunizing a mouse to prepare a mouse anti-rabbit antibody, namely the anti-idiotype antibody. Furthermore, the anti-idiotype antibody is developed into a digestive tract tumor early screening product. The digestive tract tumor early screening product provided by the invention realizes early screening of digestive tract tumors on the basis of tumor antibody detection, and the screening means has the advantages of simplicity in operation, short detection period, good patient compliance, suitability for popularization and the like.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and medical diagnosis, and specifically discloses an anti-idiotypic antibody, a preparation method thereof and an application thereof in preparing an early screening product for digestive tract tumors. Background Art

[0002] Digestive tract tumors have the characteristics of high incidence, insidious onset, and poor prognosis. According to statistics, the five-year survival rate of early digestive tract cancer after surgical treatment can reach more than 90%, while the five-year survival rate of late digestive tract cancer is significantly lower, only about 30%.

[0003] Early screening of digestive tract tumors, especially screening at the stage of high-grade intraepithelial neoplasia, can avoid adverse consequences caused by delayed diagnosis. Because tumor tissue in the subclinical stage is fragile and blood vessels have not yet formed around the tumor, it is easy to "eliminate" it in its infancy by improving the body's function. The ideal early screening method for tumors should be minimally invasive, low-cost, and highly involved, and provide minimal false negatives or false positives. Existing tumor screening methods include serum tumor markers, imaging, endoscopic examinations, and histopathological examinations. Although these screening methods have reduced cancer-related mortality, they have low diagnostic efficacy (such as low sensitivity of directly detecting circulating tumor cells in the blood and low specificity of detecting circulating tumor cell DNA), high prices, strong invasiveness, and poor compliance (such as complications such as bleeding and perforation may occur in patients after biopsy), and the detection rate of early canceration of digestive tract-related tumors needs to be improved. For example, the existing serum tumor marker screening usually does not detect tumor antibodies but antigenic substances, and the antigenic substances in most cancer cells are high in the blood of patients with advanced cancer, and cancer cell antigens are not easy to be detected in the early stages of cancer. Therefore, there is an urgent need for a convenient and well-compliant method and related products for early screening of gastrointestinal tumors. Summary of the invention

[0004] In the subclinical stage of a tumor, the body's immune system will automatically recognize and produce cancer cell antibodies (antibodies produced by the body against cancer cells, defined as tumor antibodies in this patent). This type of antibody is secreted most in the early stages of cancer. As the number of cancer cells in patients with advanced cancer gradually increases, the antibodies in the body that resist cancer cells will decrease accordingly. In response to the deficiencies in existing technologies for early screening of digestive tract tumors, the present invention is based on the detection of tumor antibodies, and uses anti-idiotypic antibodies made from Zheng's plant protein (ZPP) to make a kit, test strips or test cards for early screening of digestive tract tumors, thereby achieving screening of early digestive tract tumors, especially high-grade intraepithelial neoplasia.

[0005] In order to achieve the above-mentioned object of the invention, the embodiment of the present invention adopts the following technical solution: In a first aspect, the present invention provides an anti-idiotypic antibody, wherein the anti-idiotypic antibody comprises a light chain variable region and a heavy chain variable region; wherein The amino acid sequence of the light chain variable region comprises the light chain CDR1 shown in SEQ ID NO: 1, the light chain CDR2 shown in SEQ ID NO: 2, and the light chain CDR3 shown in SEQ ID NO: 3; The amino acid sequence of the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO:4, a heavy chain CDR2 as shown in SEQ ID NO:5, and a heavy chain CDR3 as shown in SEQ ID NO:6.

[0006] In the amino acid sequence of the light chain variable region, the amino acid sequence shown in SEQ ID NO: 1 is specifically: KASQNVGTAVA; The amino acid sequence shown in SEQ ID NO:2 is specifically: LASNRYT; The amino acid sequence shown in SEQ ID NO:3 is specifically: QQYSSHPLT; In the amino acid sequence of the heavy chain variable region, the amino acid sequence shown in SEQ ID NO:4 is specifically: SGYYWN; The amino acid sequence shown in SEQ ID NO:5 is specifically: YISYDGINNYNPSLKN; The amino acid sequence shown in SEQ ID NO:6 is specifically: YFGDY.

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned anti-idiotypic antibody, the preparation method comprising: using Zheng's plant protein to immunize rabbits to obtain RZPP antibodies, and using the F(ab')2 fragment of the RZPP antibody to immunize mice to obtain the anti-idiotypic antibody.

[0008] Preferably, the RZPP antibody is subjected to enzymatic digestion, purification, and molecular sieve to obtain the F(ab')2 fragment of the RZPP antibody.

[0009] The preparation method of the F(ab')2 fragment of the RZPP antibody specifically includes: using Zheng's plant protein to immunize rabbits to obtain rabbit serum; purifying the rabbit serum through an A+G antibody purification column to obtain a pure IgG antibody (referred to as the RZPP antibody); dialyzing the above-mentioned pure IgG antibody in a sodium acetate (pH4.0) solution, cutting with pepsin, dialyzing in a PBS (pH8.0) solution, passing through a molecular sieve, collecting, and verifying by electrophoresis to obtain the F(ab')2 fragment of the RZPP antibody.

[0010] In a third aspect, the present invention provides the use of the above-mentioned anti-idiotypic antibody in the preparation of an early screening product for digestive tract tumors, wherein the test sample of the product is serum.

[0011] The technical principle of the anti-idiotypic antibody in the present invention for detecting tumor antibodies is as follows: the antibody RZPP produced by immunizing rabbits with ZPP (Zheng's plant protein, which has tumor antigen activity) is the image of tumor antibodies in tumor patients, and the specific fragment of the antibody RZPP, the F(ab')2 fragment, is used to immunize mice to produce two paired monoclonal antibodies, namely MRZPP (one of which is named anti-idiotypic antibody, and the anti-idiotypic antibody is the imaged ZPP antigen). In view of the fact that ZPP has tumor antigen activity, the anti-idiotypic antibody has the characteristic of being able to combine with tumor antibodies in the serum of early tumor patients, and is used for early screening of digestive tract tumors.

[0012] Preferably, the digestive tract tumor includes gastric cancer, esophageal cancer or cardiac cancer.

[0013] Preferably, the product comprises a test kit, a test strip or a test card.

[0014] Preferably, the product is prepared based on enzyme-linked immunosorbent assay, immunochromatography or immunochemiluminescence.

[0015] Further preferably, the product comprises at least one of an ELISA kit, a colloidal gold kit or a chemiluminescent immunoassay kit. The present invention is described by only taking an ELISA kit, a colloidal gold kit and a chemiluminescent immunoassay kit as examples. It should be understood that those skilled in the art can also use the anti-idiotypic antibody to prepare other types of products according to the prior art.

[0016] In a fourth aspect, the present invention provides a colloidal gold kit for early screening of digestive tract tumors, wherein the colloidal gold kit uses the above-mentioned anti-idiotypic antibody as a detection antibody.

[0017] In a fifth aspect, the present invention provides an ELISA kit for early screening of digestive tract tumors, wherein the ELISA kit uses the anti-idiotypic antibody as described in claim 1 as a detection antibody.

[0018] The present invention uses Zheng's plant protein (ZPP) to immunize rabbits to obtain RZPP antibodies, and immunize mice with F(ab')2 fragments obtained by purifying the RZPP antibodies and enzymatically cleaving them to obtain anti-idiotypic antibodies. The present invention creatively discovered that the anti-idiotypic antibodies obtained can specifically bind to tumor antibodies in patients with early digestive tract tumors, and can be further developed into corresponding kits, test strips or test cards for screening of early stages of digestive tract tumors (especially the stage of high-grade intraepithelial neoplasia diagnosed by pathology). This screening method has the advantages of simple operation, short detection cycle, good patient compliance, large market demand and suitable promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a picture of the F(ab')2 fragment of the RZPP antibody being passed through a molecular sieve during preparation in Example 1 of the present invention; Figure 2 The SDS-PAGE electrophoresis diagram of the anti-idiotypic antibody and antibody 26#8C2 in Example 1 of the present invention; wherein M is a marker, 1 is an anti-idiotypic antibody, and 2 is antibody 26#8C2; Figure 3 This is a graph showing the test results of the control group using a colloidal gold test kit in the effect example of the present invention; Figure 4 This is a diagram showing the test results of using a colloidal gold kit to test a digestive tract tumor in an advanced stage group in an effect example of the present invention; Figure 5 The results of the colloidal gold kit for detecting early cardiac cancer in the effect example of the present invention are as follows; Figure 6 This is a diagram showing the test results of using a colloidal gold kit to detect an early esophageal cancer group in an effect example of the present invention; Figure 7 This is a diagram showing the detection results of the early gastric cancer group using the colloidal gold kit in the effect example of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] In order to better illustrate the embodiments of the present invention, further examples are given below.

[0023] Example 1 Preparation of anti-idiotypic antibodies This example provides a method for preparing an anti-idiotypic antibody, which specifically includes two parts: preparation of the F(ab')2 fragment of the RZPP antibody and preparation of the anti-idiotypic antibody. The specific contents are as follows: 1. Preparation of F(ab')2 fragment of RZPP antibody Rabbits were immunized via ear vein using the tumor antigen active plant protein (also known as Zheng's plant protein, abbreviated as ZPP) described in Chinese patent CN 104650200B according to conventional techniques of those skilled in the art, with an immunization dose of 100 μL (100 μg / mL). After the first immunization, booster immunizations were performed on the 14th and 30th days to obtain rabbit serum.

[0024] 1. Preparation of RZPP Antibody The present invention uses the protein G method to separate and purify rabbit serum IgG, including using the GE AKTA pure system to separate and purify IgG, and the specific contents are as follows: The materials used include: Protein G chromatography column filler, binding buffer: PBS pH7.4, elution buffer: 100mM glycine, pH2-3; neutralization buffer: 1M Tris-HCl, pH 8.8; storage solution: 20% ethanol; 50 mL of rabbit serum was taken and purified by Protein G chromatography column to obtain 390 mg of pure IgG antibody (referred to as RZPP antibody), which was stored for future use.

[0025] 2. Preparation of RZPP Antibody F(ab')2 Fragment The specific preparation steps of the RZPP antibody F(ab')2 fragment are as follows: (1) Adjust the RZPP antibody concentration to about 20 mg / mL, place it in a dialysis bag with 0.2 M sodium acetate buffer and a cutoff volume of 9000 and dialyze it overnight (change the dialysis fluid every two hours from the beginning of the dialysis, and then dialyze overnight). After the dialysis, the protein concentration was measured to be 6.85 mg / mL; (2) Pepsin cleavage, pepsin: IgG (dialysis in the previous step) mass ratio = 1:20, 37 ° C water bath for 6 h, then adjust the pH to 8.0 with 1.8 M Tris base solution to terminate the reaction, and obtain the enzymatic solution, in which the pepsin concentration is 1 mg / mL; (3) Pour the above enzymatic hydrolysate into a dialysis bag with a cut-off volume of 9000 and dialyze it overnight at 4°C using PBS (pH 8.0) to remove small molecule impurities; (4) Pass the Protein A+G column, loading 1 mL each time and collecting the effluent; (5) Pass through molecular sieve, load 2 mL each time, and collect proteins with a molecular weight of about 110 KD. The molecular sieve picture is as follows Figure 1 As shown; (6) Electrophoresis verification was performed. The results showed that there was no protein residue in the Fc segment (15KD), proving that the molecular sieve could completely screen out Fc and obtain the pure F(ab')2 fragment in the RZPP antibody. The concentration was adjusted to 1 mg / mL and stored for later use.

[0026] 2. Preparation of anti-idiotypic antibodies The screening, preparation and sequencing of the anti-idiotypic antibodies in the present invention were entrusted to Wuhan Dian Biotechnology Co., Ltd. The specific process is as follows.

[0027] 1. Animal immunization Monoclonal antibodies can be obtained by using conventional immunological techniques, using the RZPP antibody F(ab')2 fragment as an antigen, immunizing animals, and then using cells from the immunized animals to prepare hybridoma cells. The type of immunized animals used in the preparation of hybridoma cells is not particularly limited, such as mice, rats, hamsters, rabbits, etc. In this example, BALB / c mice are used as immunized animals.

[0028] When an antigen is used for animal immunization, it is more effective to use an adjuvant in combination, and the adjuvant can improve the immune response of the immunized animal to the antigen. The type of adjuvant is not particularly limited, for example, Freund's complete adjuvant, Freund's incomplete adjuvant, pertussis vaccine, aluminum adjuvant, etc. can be used. In this embodiment, Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the second and subsequent immunizations (all for multiple injections at the back). Take an equal volume of RZPP antibody F(ab')2 fragment and Freund's complete adjuvant, mix them and fully emulsify them as the primary immunization reagent.

[0029] Six BALB / c mice aged 5 to 8 weeks (recorded as mouse L, mouse R, mouse O, mouse LR, mouse R2 and mouse L2) were injected with the primary immunization reagent at multiple points on the back. The primary immunization injection dose was 100 μg antigen / experimental mouse, and the booster immunization injection dose was 50 μg antigen / experimental mouse. The second immunization was performed 2 weeks after the first immunization. The third and fourth immunizations were performed 2 weeks after the last immunization. The second immunization began with an equal volume of mixed and fully emulsified RZPP antibody F(ab')2 fragment and Freund's incomplete adjuvant; 10 days after the fourth immunization, the mouse tail vein blood was taken to prepare serum, and the antiserum titer was determined by indirect ELISA. The results are shown in Table 1. The immunogen was 50 ng / well.

[0030] Table 1

[0031] As can be seen from Table 1, the antibody titer of mouse O reached above 1:32K, which was the best, and this mouse was selected for subsequent experiments.

[0032] 2. Cell fusion and subcloning 2.1 Myeloma cell preparation One week before fusion, SP2 / 0 cells were revived and cultured normally to the logarithmic phase.

[0033] 2.2 Preparation of splenocytes The mice to be fused were selected and killed by cervical dislocation on the day of fusion. The spleens were removed and spleen cells were collected and counted according to standard procedures.

[0034] 2.3 Cell fusion Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10, and cell fusion was performed according to the standard procedure. Then, they were cultured with HATDMEM complete medium. Hybridoma cells were visible 3 days after fusion. On the 7th day, 1 / 2HAT complete medium was replaced, and on the 8th day, 1 / 2HT medium was replaced. Screening tests were started about 10 days after fusion.

[0035] Cell fusion results: After fusion, the cells were cultured in HAT selective medium and observed under a microscope, where multiple growing hybridoma cells were seen, proving that the fusion operation was successful.

[0036] 2.4 Fusion screening Pipette 100 μL / well of cell supernatant for indirect ELISA detection. Determine the positive wells based on the ELISA results. Use a single-channel pipette to pick out the positive wells detected on the entire plate and conduct a second re-test to further confirm the positive wells.

[0037] 2.5 Subcloning Perform two rounds of subcloning on the positive well cells from the rescreening. (Because the positive well cell lines obtained from the first subcloning are not stable yet and may contain multiple hybridoma cells, it is generally believed that the hybridoma cells after the second subcloning are a single cell line and are confirmed to be positive).

[0038] For the first subclone, the cells in the limited release positive wells were transferred to multiple wells and cultured with HTDMEM medium. The wells were observed under a microscope for about 7 days and the wells with clone growth were detected by indirect ELISA. The wells with high OD values ​​were taken as positive wells. The cells in the positive wells were picked for the second subcloning, and the stable positive hybridoma cell lines were detected as the cells for the final preparation of monoclonal antibodies, and the culture was expanded.

[0039] 2.6 Monoclonal Antibody Subtype Identification Use the monoclonal antibody subtype identification kit of SouthernBiotech in the United States to measure the subtype of each supernatant. Prepare the strips coated with immunogenic protein, 50ng / well, collect 600μL supernatant from each clone, and add them to 6 enzyme-labeled wells of the corresponding protein, 100μL / well. Incubate at 37℃ for 1h, wash three times with PBST, add the diluted typing secondary antibodies against IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 to 6 wells, incubate at 37℃ for 1h, wash three times with PBST, and develop with TMB. The subtype of the secondary antibody corresponding to the well with signal reaction is the subtype of the antibody.

[0040] 2.7 After two rounds of subcloning and retesting, 16 positive cell lines were identified, corresponding to the antibody subtypes. After measurement, their titers are shown in Table 2.

[0041] Table 2

[0042] 3. Ascites Preparation and Antibody Purification 3.1 Ascites preparation The above 16 positive cells were expanded and cultured and injected into the peritoneal cavity of BALB / c mice (sensitized with Freund's incomplete adjuvant). Generally, bulging of the abdomen of the mice was seen in 7-10 days, indicating the formation of ascites. When the mice had obvious ascites, the ascites was extracted in time.

[0043] 3.2 Ascites purification The ascites of the above cells was purified, and the purity of the antibody after purification was greater than 90%; Purification method: Protein A / G column affinity purification (IgG1, IgG2a, IgG2b, IgG3 subtype antibodies) 3.2.1. Centrifuge the ascites, aspirate the light yellow liquid and calculate the volume; 3.2.2. Load the Protein A / G filler into a gravity purification column and wash three times with PBS, each time using 10x column volume of PBS.

[0044] 3.2.3. Load the ascites into the column and mix gently at 4℃ for 2-4h.

[0045] 3.2.4. Release the flow-through liquid and set aside.

[0046] 3.2.5. Wash the ProteinA / G filler three times with PBS, elute the antibody with pre-cooled pH 1.0 HCL-Glycine eluent, and immediately neutralize the collected antibody with 10xPBS neutralizing solution.

[0047] 3.2.6. Detect antibody concentration and combine high concentration collection tubes.

[0048] 3.2.7. Load the eluted antibody with a higher concentration.

[0049] 3.2.8. Dialyze against PBS at 4°C overnight.

[0050] 3.3 After testing the concentration and purity, adjust the concentration to 2 mg / mL.

[0051] 4. Antibody Pairing 4.1 Biotinylated Antibodies 16 purified antibodies were taken out, each with a weight of 0.5 mg, and biotin-labeled according to the requirements of the biotin labeling kit, and the titer of the labeled antibodies was tested. The specific results are shown in Table 3.

[0052] Table 3

[0053] The results in Table 3 show that 16 self-produced antibodies were successfully labeled.

[0054] 4.2 Antibody pairing for double antibody sandwich ELISA chessboard method 16 purified antibodies and 16 biotin-labeled antibodies were taken out and paired in sandwich ELISA according to the chessboard method. The specific results are shown in Table 4.

[0055] Table 4

[0056] The chessboard pairing results preliminarily showed that the OD value was higher when coating antibody 3#1E10 was paired with detection antibody 4#2A2, coating antibody 9#2B11 was paired with detection antibody 3#1E10, coating antibody 6#2B4 was paired with detection antibody 3#1E10, coating antibody 16#4G1 was paired with detection antibody 3#1E10, coating antibody 26#6C6 was paired with detection antibody 3#1E10, and coating antibody 3#1E10 was paired with detection antibody 9#2B11.

[0057] 4.3 Double antibody sandwich ELISA detection of natural samples The natural samples were tested using double antibody sandwich ELISA, and the specific results are shown in Table 5 below.

[0058] Table 5

[0059] As can be seen from Table 5, the double antibody sandwich method was used to detect rabbit negative serum. The results showed that the coated antibody 3#1E10 was paired with the detection antibody 4#2A2, the coated antibody 3#1E10 was paired with the detection antibody 9#2B11, the coated antibody 6#2B4 was paired with the detection antibody 3#1E10, the coated antibody 9#2B11 was paired with the detection antibody 3#1E10, and the coated antibody 16#4G11 was paired with the detection antibody 3#1E10. Although the above paired antibodies can recognize the immunogen well, they can also recognize the IgG in the negative serum. The coated antibody 26#8C2 is paired with the detection antibody 3#1E10, which can not only better recognize the immunogen, but also basically does not recognize the negative serum, and can also resist interference, so it is practical. Therefore, this application names the 3#1E10 antibody as an anti-idiotypic antibody for further study.

[0060] 5. Determination of variable region sequences of anti-idiotypic antibodies The marker, the purified anti-idiotypic antibody and antibody 26#8C2 were determined by SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis is shown in the figure below. Figure 2 shown. Figure 2 In the figure, M is a marker, 1 is an anti-idiotypic antibody, and 2 is antibody 26#8C2. Combining the electrophoresis results and the protein molecular weight regression equation, the molecular weights of the two staining bands of the anti-idiotypic antibody were calculated to be approximately 55 kD and 25 kD, which are consistent with the molecular weights of the IgG heavy chain and light chain reported in the literature. The amino acid sequence of the variable region of the anti-idiotypic antibody was determined.

[0061] The amino acid sequence of the light chain is shown in SEQ ID NO: 7, specifically: DIVMTQSQKFMSASVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYLASNRYTGVPDRFTGSASGTDFTLTISNVQSEDLADYFCQQYSSHPLTFGGGTKVELK.

[0062] Comparing with the mouse proteome, determining the sequence of the light chain variable region of the anti-idiotypic antibody; the amino acid sequence of the light chain variable region comprises the light chain CDR1 shown in SEQ ID NO: 1, the light chain CDR2 shown in SEQ ID NO: 2, and the light chain CDR3 shown in SEQ ID NO: 3; In the amino acid sequence of the light chain variable region, the amino acid sequence shown in SEQ ID NO: 1 is specifically: KASQNVGTAVA; The amino acid sequence shown in SEQ ID NO:2 is specifically: LASNRYT; The amino acid sequence shown in SEQ ID NO:3 is specifically: QQYSSHPLT.

[0063] After determination, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 8, specifically: QMQPKESGPGLVKPSQSLSLTCSVTGYSVTSGYYWNWIRQFPGNKLEWMGYISYDGINNYNPSLKNRISITRDTSKNQFFLKLNSVTTADTATYYCARYFGDYWGQGTTLKVSS.

[0064] By comparing with the mouse proteome, the sequence of the heavy chain variable region of the anti-idiotypic antibody was determined; the amino acid sequence of the heavy chain variable region included the heavy chain CDR1 shown in SEQ ID NO:4, the heavy chain CDR2 shown in SEQ ID NO:5 and the heavy chain CDR3 shown in SEQ ID NO:6.

[0065] In the amino acid sequence of the heavy chain variable region, the amino acid sequence shown in SEQ ID NO:4 is specifically: SGYYWN; The amino acid sequence shown in SEQ ID NO:5 is specifically: YISYDGINNYNPSLKN; The amino acid sequence shown in SEQ ID NO:6 is specifically: YFGDY.

[0066] Example 2 A colloidal gold kit for early screening of digestive tract tumors The present invention prepares a colloidal gold kit for early screening of digestive tract tumors based on anti-idiotypic antibodies. The consumables used in preparing the colloidal gold kit include a card shell, a sample pad (GS12), a gold label pad (8964), an NC membrane (Sartorius 140) and absorbent paper. The materials used include goat anti-mouse secondary antibody, anti-idiotypic antibody and antibody 26#8C2, wherein antibody 26#8C2 is used as a coating antibody and anti-idiotypic antibody is used as a labeling antibody. The specific preparation process includes the following contents.

[0067] 1. Preparation of conjugate pad 1. Preparation of Colloidal Gold The colloidal gold solution was prepared by the trisodium citrate reduction method. The specific method includes: a round-bottom flask is filled with 100 mL of 1% chloroauric acid aqueous solution, and a digital heating magnetic stirrer is refluxed and heated to boiling for 20 minutes. Then, a 1% trisodium citrate solution (prepared immediately after use) with a volume percentage of 1.5% (i.e., 1.5 ml) of chloroauric acid aqueous solution is added, and heating and boiling are continued for 10 minutes. The color of the solution turns into wine red. After cooling, the water volume is supplemented with deionized water to obtain a colloidal gold solution. 2. Preparation of Colloidal Gold Labeled Anti-idiotypic Antibody Solution Take 100mL of colloidal gold solution in a centrifuge tube, add 400μL of 0.2M K2CO3, shake well while adding, and mix thoroughly. Let stand for 15 minutes. Add 500μL of anti-idiotypic antibody working solution with a concentration of 1mg / mL, shake well while adding, and cover tightly, and let stand for 15 minutes. Add 1mL of 10% BSA blocking solution, shake well while adding, and cover tightly, and let stand for 15 minutes; centrifuge at 11000rpm and 4℃ for 20 minutes, remove the supernatant, take 10mL of reconstitution solution (containing 0.076% borax, 1% trehalose, 1% BSA, and 0.03% preservative) and add it to the above centrifuge tube, and shake gently to fully dissolve it.

[0068] 3. Preparation of the Conjugate Pad Soak the gold-labeled pad (8964) in the treatment solution (0.09% sodium dihydrogen phosphate dihydrate, 0.5% sodium dihydrogen phosphate dodecahydrate, 2% trehalose, 2% BSA, 0.5% Tween 20, 0.1% PEG20000 and appropriate amount of preservative) for 5 seconds, dry at room temperature for 4 hours, and then transfer to a 37℃ constant temperature drying oven for baking for 14 hours to obtain the pretreated gold-labeled pad; finally, spray gold according to the "Film Scraper Operation Procedure", and the environmental requirements for gold spraying are: temperature 25℃, humidity <30%. Gold spraying concentration: 2.0μL / cm (gold spraying parameters fluctuate within a certain range, and the concentration is determined by actual testing); after gold spraying, place it in a constant temperature oven for drying for 2 hours, and the humidity is <30%. Stack it in order and seal it in an aluminum foil bag with desiccant for storage.

[0069] 2. Preparation of Chromatography Pad NC membrane (Sartorius 140) was selected as the chromatography pad material, and the positions of a test line T and a quality control line C were marked on the NC membrane. Antibody 26#8C2 was diluted to 0.4 mg / mL, and goat anti-mouse secondary antibody was diluted to 0.2 mg / mL. The test line T and quality control line C of the NC membrane were marked with a film marker at a dosage of 1 μL / cm, and then baked in a 37°C constant temperature drying oven for 20 hours.

[0070] 3. Preparation of sample pad The sample pad (GS12) was completely immersed in the treatment solution (0.086% sodium dihydrogen phosphate dihydrate, 0.514% disodium hydrogen phosphate dodecahydrate, and 0.5% Tween 20) for 5 seconds, dried at room temperature for 4 hours, and then transferred to a 37°C drying oven and baked for 14 hours.

[0071] 4. Assembly of Colloidal Gold Kit The colloidal gold test kit consists of a sample pad, a binding pad, a chromatography pad, a sample suction pad and a cartridge. The sample suction pad is made of absorbent filter paper. The sticking standards for absorbent paper, gold label pad and sample pad, as well as the cutting standards are as follows: Pasting standards for absorbent paper: ① Specifications of absorbent paper: 30cm long, 1.7cm wide; ② Tear off the backing on the upper end of the NC film; ③ When pasting, first check the uniformity of the cut of the absorbent paper, and paste the smooth surface on the backboard; ④ Press the NC film with 2mm±0.05mm of the absorbent paper, align the other end with the edge of the backboard, and press it tightly after passing the test.

[0072] The sticking standard of gold label pad: ① Specification of gold label pad: 30cm long and 1.0cm wide; ② Tear off the backing under the NC film, check that the width of the gold label pad is uniform, without damage, and the gold is evenly sprayed without marks; ③ Press the upper end of the gold label pad against the NC film 1.0mm±0.05mm, and press it tightly after passing the test.

[0073] The sticking standard of the sample pad: ①Specimen pad specifications: 30cm long, 1.5cm wide; ②Tear off the backing under the NC film and check that the width of the sample pad is uniform and there is no damage. ③The upper end of the sample pad is pressed with a gold pad of 1.0mm±0.05mm, and the lower end is aligned with the edge of the back plate, and the surface is flat; press it tightly after passing the test.

[0074] Strip cutting standards: ① Plate trimming: cut off the marked part at 5mm±0.05mm within the prepared marking point, and the cut should be neat; ② Strip cutting: Strip cutting width: 3.96mm, and strip cutting shall be carried out in accordance with the "Strip Cutting Machine Operating Procedures".

[0075] The specific assembly process of the colloidal gold test kit is as follows: Lay the card: Place the bottom of the card shell in the direction of absorbent paper on the top and sample pad on the bottom vertical strip side by side; Loading the strip: pinch the absorbent paper end of the test strip, check whether there are any marks or scratches on the chromatography pad, binding pad (gold label pad), and sample pad, and make sure they are firmly pasted. First put the sample pad end into the card slot and close to the bottom of the slot, then put the absorbent paper end. The test strip must be completely placed in the card slot and parallel to the slot.

[0076] Capping: Check the appearance and cleanliness of the card surface, align the sample hole with the sample pad and press the two card surfaces together. The test strip is fixed in the card slot in the center of the card observation window, and the absorbent paper cannot be seen at the upper end and the binding pad cannot be seen at the lower end. This is the colloidal gold test kit for early screening of digestive tract tumors.

[0077] 5. How to use the colloidal gold test strips Add the serum sample to be tested into the sample well of the colloidal gold test kit, place the test kit flat, observe the color changes of the test line and the quality control line within 5 to 15 minutes and record the results.

[0078] 6. Determination of the test results of colloidal gold test paper box Positive result: Color appears on the chromatography pad detection line T of the colloidal gold kit and the quality control line C, and the test result is determined to be positive.

[0079] Negative result: The test line T on the chromatography pad of the colloidal gold test kit did not show color, and only the quality control line C showed color, and the test result was determined to be negative.

[0080] Invalid result: The quality control line C did not show color, and the test result was judged to be invalid and needs to be retested.

[0081] Example 3 An ELISA kit for early screening of digestive tract tumors This example is based on anti-idiotypic antibodies to prepare an ELISA kit for early screening of digestive tract tumors, in which antibody 26#8C2 is used as a coating antibody and anti-idiotypic antibodies are used as detection antibodies. The specific contents are as follows: 1. Experimental materials and reagents: Coating solution: 0.16% anhydrous sodium carbonate, 0.29% sodium bicarbonate, the solvent is deionized water; Blocking solution: sodium chloride 0.8%, sodium dihydrogen phosphate dihydrate 0.059%, disodium hydrogen phosphate dodecahydrate 0.58%, sucrose 5%, sodium casein 0.25%, bovine serum 5% and preservative 0.04%; Washing solution: disodium hydrogen phosphate dodecahydrate 2.32%, sodium chloride 6.4%, potassium dihydrogen phosphate 0.2%, potassium chloride 0.0036%, Tween-20 2%, preservative 0.03%; Sample diluent: sodium chloride 0.8%, potassium dihydrogen phosphate 0.02%, potassium chloride 0.02%, disodium hydrogen phosphate dodecahydrate 0.54%; Enzyme label diluent: sodium chloride 0.8%, sodium dihydrogen phosphate dihydrate 0.05%, disodium hydrogen phosphate dodecahydrate 0.58%, sucrose 5%, BSA 0.25%, glycerol 5% and preservative 0.03%; Enzyme-labeled antibody: The anti-idiotypic antibody was labeled with horseradish peroxidase to prepare a horseradish peroxidase-labeled anti-idiotypic antibody with a concentration of 2 mg / mL.

[0082] Color developing solution A: Take 9.2 g of disodium hydrogen phosphate dodecahydrate, 2.55 g of citric acid, and 0.5 g of urea peroxide and dissolve them in 500 mL of ultrapure water; Color developing solution B: 1 g of citric acid, 0.15 g of disodium ethylenediaminetetraacetate, 10 mL of glycerol, 20 mL of anhydrous ethanol, 1.5 mL of acetone, 0.5 g of methylbenzidine (TMB) and 468.5 mL of ultrapure water.

[0083] Stop solution: Take 14 mL of concentrated sulfuric acid and slowly add it into 486 mL of deionized water and stir evenly.

[0084] 2. Preparation of coated ELISA plate Dissolve the antibody 26#8C2 in the coating solution and mix thoroughly to prepare a 26#8C2 coating solution with a concentration of 1 μg / mL.

[0085] Coating ELISA plate: Add the prepared 26#8C2 coating solution to the sample wells of the ELISA plate, with the sample volume of 100μL / well; incubate in a constant temperature incubator at 37℃ for 2h or at 4℃ overnight, then remove the coating solution, wash once with washing solution, leave for 1min, shake out the washing solution in the plate wells and pat dry.

[0086] Blocking: Add blocking solution to the sample wells of the coated ELISA plate at a volume of 150 μL / well, incubate at 37°C for 2 hours, and then remove the blocking solution.

[0087] Drying and packaging: Place the sealed ELISA plate in a 37°C oven for drying, package, and obtain the coated ELISA plate, which is then stored at 4°C for future use.

[0088] 3. How to use the kit Serum sample incubation: dilute the serum sample to be tested with sample diluent at a ratio of 1:5, then add the diluted serum sample to the sample wells coated with the ELISA plate, with a sample volume of 100 μL / well, incubate in a 37°C constant temperature incubator for 1 hour, then discard the liquid in the sample wells, wash five times with washing solution, 3 minutes each time.

[0089] Enzyme-labeled antibody incubation: dilute the horseradish peroxidase-labeled anti-idiotypic antibody with enzyme-labeled diluent at a ratio of 1:8000, then add the diluted horseradish peroxidase-labeled anti-idiotypic antibody to the sample wells of the ELISA plate at a volume of 100 μL / well, incubate in a 37°C constant temperature incubator for 30 minutes, then discard the liquid in the sample wells and wash five times with washing solution, each time for 3 minutes.

[0090] Color development and termination reaction: Mix color development solution A and color development solution B in a 1:1 ratio of equal volumes, then quickly add the mixed color development solution to the sample wells of the ELISA plate at a volume of 100 μL / well, place at 37°C in the dark for 15 minutes, then add 50 μL of stop solution to each sample well to terminate the color development reaction, set the ELISA instrument at dual wavelengths of 450nm and 630nm, measure the OD value, and use the blank control well to adjust to zero.

[0091] Example 4 A chemiluminescent immunoassay kit for early screening of digestive tract tumors Based on the anti-idiotypic antibody and the preparation method of the ELISA kit described in Example 3, those skilled in the art can prepare a chemiluminescent immunoassay kit for early screening of gastrointestinal tumors according to common knowledge and existing technologies, and use a chemiluminescence instrument to read the results.

[0092] Effect example The colloidal gold kit prepared in Example 2 of the present invention or the ELISA kit prepared in Example 3 was used to detect serum samples of patients with early gastrointestinal tumors confirmed by pathology, patients with late gastrointestinal tumors and normal people, and the value of the colloidal gold kit or ELISA kit provided by the present invention for early screening or diagnosis of gastrointestinal tumors was evaluated and analyzed.

[0093] 1. Sample source A total of 96 serum samples were collected from a hospital in Hebei Province, including 39 normal human sera (control group), 4 sera from patients with early cardiac cancer (early cardiac cancer group), 21 sera from patients with early esophageal cancer (early esophageal cancer group), 12 sera from patients with early gastric cancer (early gastric cancer group), and 20 sera from patients with advanced digestive tract tumors (recorded as the advanced digestive tract tumor group, including 10 gastric cancer samples, 6 esophageal cancer samples, and 4 cardiac cancer samples).

[0094] 2. Application of colloidal gold test kit for early screening of digestive tract tumors The colloidal gold test kit for early screening of digestive tract tumors prepared in Example 2 of the present invention was used to test serum samples of a control group, an early cardiac cancer group, an early esophageal cancer group, an early gastric cancer group, and a late digestive tract tumor group. The specific detection method is: drop the serum sample to be tested on the sample addition hole of the test kit, place the test kit flat, observe the color changes of the test line and the quality control line within 5 to 15 minutes, and record the results. Figure 3~Figure 7 All of the pictures show that only the test strip portion is retained after the casing of the colloidal gold test kit is removed.

[0095] The test results of the control group are as follows: Figure 3 As shown in Figure 2, the test results of the advanced digestive tract tumor group are as follows: Figure 4 The test results of the early cardiac cancer group are shown in Figure 5 The results of the early esophageal cancer group are shown in Figure 6 As shown in Figure 2, the test results of the early gastric cancer group are as follows: Figure 7 shown.

[0096] Depend on Figures 3 to 7 The results show that the overall positive rate of early gastrointestinal tumors (such as early cardia cancer, early esophageal cancer and early gastric cancer) detection results is more than 75%; the test results of normal people are all negative; the test results of late gastrointestinal tumors are either positive or negative, but the vast majority are negative.

[0097] 3. Application of ELISA kit for early screening of digestive tract tumors The ELISA kit for early screening of digestive tract tumors prepared in Example 3 of the present invention was used to detect serum samples of the control group, early cardiac cancer group, early esophageal cancer group, early gastric cancer group and advanced digestive tract tumor group, respectively. The specific detection method is to react the serum sample to be tested with the coated antibody and the enzyme-labeled antibody, add a color developing solution for color development, and put it on the microplate reader to record the results.

[0098] By testing the serum of the control group, the reference range of normal human serum OD values ​​was established through the industry standard W / S402-2012 "Establishment of Reference Ranges for Clinical Laboratory Test Items", with a reference range of 0.26~0.36, a mean of 0.3155, and 95% of normal people fall within this range. People with early or late digestive tract tumors can be screened out if they exceed this reference range. According to statistics, the serum OD values ​​of patients with late digestive tract tumors fall within the range of 0.52~0.81, with a mean of 0.678; the serum OD values ​​of patients with early digestive tract tumors fall within the range of 0.9~2.1, and the OD values ​​of digestive tract tumors at different stages vary greatly. The OD value test results and clinical diagnosis results of some patients with early digestive tract tumors are shown in Table 6.

[0099] Table 6

[0100] By combining the detection data of the ELISA kit for early screening of digestive tract tumors provided by the present invention with the normal reference area, early gastric cancer, cardia cancer or esophageal cancer can be screened.

[0101] The present invention uses anti-idiotypic antibodies to prepare screening products (such as ELISA kits or colloidal gold kits) suitable for early screening of digestive tract tumors, especially screening of high-grade intraepithelial neoplasia of digestive tract tumors. And the ELISA kit or colloidal gold kit provided by the present invention is used for screening, which has the advantages of simple operation, short detection cycle, good patient compliance, and suitable for promotion.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-idiotypic antibody, characterized in that The anti-idiotypic antibody comprises a light chain variable region and a heavy chain variable region; wherein, The amino acid sequence of the light chain variable region comprises the light chain CDR1 shown in SEQ ID NO: 1, the light chain CDR2 shown in SEQ ID NO: 2, and the light chain CDR3 shown in SEQ ID NO: 3; The amino acid sequence of the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO:4, a heavy chain CDR2 as shown in SEQ ID NO:5, and a heavy chain CDR3 as shown in SEQ ID NO:

6.

2. A method for preparing an anti-idiotypic antibody according to claim 1, characterized in that: The preparation method comprises: using Zheng's plant protein to immunize rabbits to obtain RZPP antibodies, and using the F(ab')2 fragment of the RZPP antibody to immunize mice to obtain the anti-idiotypic antibody.

3. The method for preparing an anti-idiotypic antibody according to claim 2, characterized in that: The RZPP antibody is subjected to enzyme digestion, purification, and molecular sieve to obtain the F(ab')2 fragment of the RZPP antibody.

4. Use of the anti-idiotypic antibody according to claim 1 in the preparation of a product for early screening of digestive tract tumors, wherein the test sample of the product is serum.

5. The use according to claim 4, characterized in that The digestive tract tumors include gastric cancer, esophageal cancer or cardiac cancer.

6. The use according to claim 4, characterized in that The products include test kits, test strips or test cards.

7. The use according to claim 6, characterized in that The product is prepared based on immunochemiluminescence, enzyme-linked immunosorbent assay or immunochromatography.

8. The use according to claim 7, characterized in that The product comprises at least one of an ELISA kit, a colloidal gold kit or a chemiluminescent immunoassay kit.

9. A colloidal gold kit for early screening of digestive tract tumors, characterized in that: The colloidal gold kit uses the anti-idiotypic antibody as claimed in claim 1 as the detection antibody.

10. An ELISA kit for early screening of digestive tract tumors, characterized in that: The ELISA kit uses the anti-idiotypic antibody as claimed in claim 1 as the detection antibody.

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