Novel anti-acridinium ester monoclonal antibody, bispecific antibody thereof and application of novel anti-acridinium ester monoclonal antibody in ELISA (Enzyme-Linked Immunosorbent Assay) kit

Abstract

The invention provides a novel anti-acridinium ester monoclonal antibody which is a nano antibody, and the amino acid sequence of the novel anti-acridinium ester monoclonal antibody is shown as any one of SEQ ID NO.1-8. The invention also provides a bispecific antibody which is formed by carrying out in-vitro fusion expression on the anti-acridinium ester monoclonal antibody and a CHI3L1 antibody. Wherein the anti-acridinium ester monoclonal antibody is located at the C end of the CHI3L1 antibody, and a (G4S) 3 protein linker is connected between the anti-acridinium ester monoclonal antibody and the CHI3L1 antibody. In addition, the invention also provides an application of the antibody in preparation of an ELISA kit. The method has the advantages that the anti-acridinium ester monoclonal antibody is designed, and the anti-acridinium ester monoclonal antibody and a target antigen-antibody are subjected to fusion expression to form a bispecific antibody; the bispecific antibody not only can be combined with a specific target antigen, but also can be combined with acridinium ester coupled with HRP (horse radish peroxidase) to obtain a signal, so that the link of marking a traditional second antibody is omitted, the ELISA detection process is simplified, intermediate steps are reduced, and the cost is saved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and in particular to a novel anti-acridinium ester monoclonal antibody, a bispecific antibody thereof and application in an ELISA kit. Background Art

[0002] ELISA, the full name of which is Enzyme-Linked Immunosorbent Assay, is a commonly used detection technique in immunological experiments. The basic principle of ELISA is to connect the test substance with the enzyme through the specific immune reaction between the antigen and the antibody, and then produce a color reaction through the enzyme and the substrate for quantitative determination. Its basis is the solid phase of the antigen or antibody and the enzyme labeling of the antigen or antibody. The antigen or antibody bound to the surface of the solid phase carrier still retains its immunological activity, and the enzyme-labeled antigen or antibody retains both its immunological activity and the activity of the enzyme. During the determination, the specimen to be tested (the antibody or antigen to be measured) reacts with the antigen or antibody on the surface of the solid phase carrier. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. Then the enzyme-labeled antigen or antibody is added and also bound to the solid phase carrier through reaction. At this time, the amount of enzyme on the solid phase is in a certain ratio to the amount of the substance to be tested in the specimen. After adding the substrate of the enzyme reaction, the substrate is hydrolyzed or oxidized and reduced by the enzyme to become a colored product. The amount of the product is directly related to the amount of the substance being tested in the specimen, so qualitative or quantitative analysis can be performed based on the depth of color.

[0003] ELISA can be used to determine antigens or antibodies. Different types of detection methods can be designed based on the source of the reagents, the condition of the specimens, and the specific conditions of the test. The main types of ELISA used for clinical testing are as follows:

[0004] 1. Double Antibody Sandwich Method

[0005] Detection of antigens: The operation includes combining specific antibodies with solid phase carriers, adding the sample to be tested and keeping it warm to react, so that the antigen in the sample combines with the solid phase antibody to form a solid phase antigen-antibody complex; then adding enzyme-labeled antibodies and keeping them warm to react, so that the antigen on the solid phase immune complex combines with the enzyme-labeled antibodies; after thoroughly washing the unbound enzyme-labeled antibodies, adding substrates for color development, and the enzyme on the solid phase catalyzes the substrate to become a colored product. The amount of antigen in the sample can be measured by colorimetry. This method is suitable for testing various macromolecular antigens such as proteins, such as HBeAg, HBsAg, hCG, AFP, etc.

[0006] Detection antibody: The reaction mode is similar to the double antibody sandwich method, using specific antigens for coating and preparation of enzyme conjugates to detect the corresponding antibodies.

[0007] 2. Indirect method

[0008] It is a commonly used method for detecting antibodies. Its principle is to use enzyme-labeled anti-antibodies (anti-human immunoglobulin antibodies) to detect the test antibody bound to the solid phase antigen. The operation includes linking the specific antigen with the solid phase carrier to form a solid phase antigen; adding diluted test serum and keeping it warm to react, so that the specific antibody in the serum binds to the solid phase antigen to form a solid phase antigen-antibody complex; adding enzyme-labeled anti-antibody after washing; and then adding substrate for color development. This method is mainly used for the detection of pathogen antibodies for the diagnosis of infectious diseases.

[0009] 3. Competition Law

[0010] Antibody detection: When interfering substances in the antigen material are difficult to remove, or it is difficult to obtain sufficient purified antigen, this method can be used to detect specific antibodies. The principle is that the antibodies in the specimen compete with a certain amount of enzyme-labeled antibodies for binding to the solid phase antigen. The more antibodies in the specimen, the less enzyme-labeled antibodies are bound to the solid phase, so the positive reaction is lighter in color than the negative reaction.

[0011] Antigen detection: Small molecule antigens or half-antibodies lack more than two sites for sandwich method, so they cannot be determined by double antibody sandwich method. The competitive method can be used. The principle is that the antigen in the sample and a certain amount of enzyme-labeled antigen compete with the solid phase antibody for binding. The more antigen content in the sample, the less enzyme-labeled antigen is bound to the solid phase, and the lighter the final color is.

[0012] 4. Capture method

[0013] Take the measurement of IgM antibodies as an example: the detection of IgM antibodies is used in the early diagnosis of infectious diseases. Indirect ELISA is generally only suitable for the detection of total antibodies or IgG antibodies. Therefore, if anti-human IgM is used as a secondary antibody to indirectly measure IgM antibodies, the specimen must first be treated with protein A or anti-IgG antibodies to remove the interference of IgG. The capture coating method is often used to measure antibody IgM. First, the solid phase is coated with anti-human IgM antibodies to capture IgM in the serum specimen (including specific IgM antibodies and non-specific IgM against the antigen). Then the antigen is added, and this antigen only binds to the specific IgM. Then the specific antibody against the antigen is labeled with an enzyme, and then reacts with the substrate, and the color is positively correlated with the IgM in the specimen.

[0014] The application trend of the above-mentioned double antibody sandwich ELISA detection method has increased significantly in recent years, but there are still some problems. For example, in the traditional double antibody sandwich method, the second antibody is usually labeled with HRP and the first antibody detects the antigen. The corresponding test method is affected by two factors: "second antibody production batch difference" and "HRP labeled antibody process", and has the disadvantages of complex operation, high labeling cost, and difficult to control batch difference.

[0015] Based on this, there is still room for improvement in the double antibody sandwich type ELISA detection method, and there is still a strong demand for better ELISA reagents. Summary of the invention

[0016] The technical problem to be solved by the present invention is to provide a novel anti-acridinium ester monoclonal antibody, a bispecific antibody thereof and an application thereof in an ELISA kit; the present invention forms a bispecific antibody by fusion expression of a self-developed anti-acridinium ester monoclonal antibody and a specific target antigen antibody-CHI3L1 antibody; the production process of the bispecific antibody is highly controllable and has a small batch difference; at the same time, the antibody (used as a second antibody for ELISA detection) can not only bind to a specific target antigen, but also directly bind to "HRP-coupled acridinium ester" to obtain a signal, thereby eliminating the traditional second antibody labeling link, greatly simplifying the ELISA detection process, reducing intermediate steps, and thus saving costs.

[0017] The present invention adopts the following technical solutions to solve the above technical problems:

[0018] A novel anti-acridinium ester monoclonal antibody is a nanobody, and the amino acid sequence is shown in any one of SEQ ID NOs. 1 to 8.

[0019] As one of the preferred embodiments of the present invention, the anti-acridinium ester monoclonal antibody is expressed by in vitro fusion with the CHI3L1 antibody; wherein the anti-acridinium ester monoclonal antibody is located at the C-terminus of the CHI3L1 antibody, and a (G4S)3 protein linker is connected between the CHI3L1 antibody and the anti-acridinium ester monoclonal antibody.

[0020] A use of the anti-acridinium ester monoclonal antibody or bispecific antibody in the preparation of an ELISA kit.

[0021] An ELISA kit comprises a bispecific antibody composed of an anti-acridinium ester monoclonal antibody and a CHI3L1 antibody fused and expressed in vitro, and an HRP-coupled acridinium ester; wherein the amino acid sequence of the anti-acridinium ester monoclonal antibody is shown in any one of SEQ ID NOs. 1 to 8.

[0022] As one of the preferred embodiments of the present invention, the bispecific antibody can simultaneously bind to the “target antigen CHI3L1” and the “HRP-conjugated acridinium ester”.

[0023] As one of the preferred embodiments of the present invention, the preparation method of the HRP-coupled acridinium ester is as follows:

[0024] (1) Coupling

[0025] After HRP was dissolved in MES buffer, acridinium ester NSP-SA-NHS was added and incubated at room temperature for 2 to 4 h;

[0026] (2) Fluid exchange

[0027] Use an ultrafiltration concentration tube or dialysis bag to change the HRP solution after coupling with acridinium ester and adjust the protein concentration to 0.4-0.6 mg / ml; finally, add the preservation solution and store it at low temperature and away from light for later use.

[0028] As one of the preferred embodiments of the present invention, in step (1), the MES buffer used has a pH of 6.5 and a concentration of 20 mM.

[0029] As one of the preferred embodiments of the present invention, in step (1), the mixing molar ratio of HRP to acridinium ester is 1:20.

[0030] As one of the preferred embodiments of the present invention, in step (1), the incubation is performed at room temperature for 3 hours.

[0031] As one of the preferred embodiments of the present invention, in step (2), a 3 kDa ultrafiltration concentration tube or a 3 kDa dialysis bag is used to replace the HRP solution after coupling with acridinium ester into a mixed solution containing 20 mM MES 6.5 and 150 mM NaCl.

[0032] As one of the preferred embodiments of the present invention, in step (2), the protein concentration is adjusted to 0.5 mg / ml.

[0033] As one of the preferred embodiments of the present invention, in step (2), the storage solution has a final concentration of 0.1% BSA and 40% glycerol.

[0034] The advantages of the present invention compared to the prior art are:

[0035] (1) The present invention independently designs an anti-acridinium ester monoclonal antibody, and fuses it with a specific target antigen antibody - CHI3L1 antibody to form a bispecific antibody; when used as the second antibody in the ELISA platform, the bispecific antibody can not only bind to the specific target antigen, but also directly bind to the "HRP-coupled aridinium ester" to obtain a signal, eliminating the traditional second antibody labeling link (the corresponding antibody of the present invention does not need to be additionally coupled to Biotin or HRP by chemical methods), greatly simplifying the ELISA detection process, reducing intermediate steps, and thus saving costs;

[0036] (2) The "HRP-coupled acridinium ester" composed of the acridinium ester of the present invention and horseradish peroxidase is very easy to obtain, and this feature makes the labeling process more controllable for reagent manufacturers during the reagent development process;

[0037] (3) The anti-acridinium ester monoclonal antibody of the present invention is a nanobody, which has a strong affinity for aridinium ester and good specificity. The ELISA reagent prepared by the bispecific antibody expressed by fusion with a specific target antibody also has a stronger signal value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a structural diagram of a bispecific antibody formed by fusion expression of an anti-acridinium ester monoclonal antibody and a CHI3L1 antibody of the present invention;

[0039] Figure 2 It is an ELISA affinity test diagram of the anti-acridinium ester monoclonal antibody of the present invention and aridinium ester (in the figure, SEQ ID NO.1 refers to the bispecific antibody test corresponding to the SEQ ID NO.1 sequence, SEQ ID NO.2 refers to the bispecific antibody test corresponding to the SEQ ID NO.2 sequence, and SEQ ID NO.3 to SEQ ID NO.8 and so on). DETAILED DESCRIPTION

[0040] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagent products and experimental methods used in the following embodiments are conventional reagents or methods in the art unless otherwise specified, and will not be repeated.

[0041] Example 1

[0042] A novel anti-acridinium ester monoclonal antibody is a nanobody, and the amino acid sequence is shown in any one of SEQ ID NOs. 1 to 8.

[0043] Example 2

[0044] A symmetrical bispecific antibody CHI3L1-AEab is formed by in vitro fusion expression of the anti-acridinium ester monoclonal antibody of Example 1 and the CHI3L1 antibody (Anhui Qiancheng Biotechnology Co., Ltd., product number: MC01902). The anti-acridinium ester monoclonal antibody is located at the C-terminus of the CHI3L1 antibody, and a (G4S)3 protein linker is connected between the CHI3L1 antibody and the anti-acridinium ester monoclonal antibody, such as Figure 1 shown.

[0045] Example 3

[0046] Method for obtaining the above-mentioned bispecific antibody CHI3L1-AEab:

[0047] (1) Construction of CHO-K1-CHI3L1-AEab protein antibody stable cell line

[0048] a. Cell recovery

[0049] Take a liquid nitrogen frozen cell (about 1×10 7 ), and quickly thaw in a 37°C water bath. After thawing, wipe the surface of the cryotube with alcohol cotton and place it in a clean bench for operation. Centrifuge at 300g for 5 minutes, discard the supernatant, add 20mL of CHO complete medium preheated to 37°C, resuspend the cells and transfer to a 125mL shake flask for culture. The culture conditions were set at 37°C and 5% CO 2 , rotation speed 120~130rpm.

[0050] b. Plasmid extraction

[0051] The "DH5α strain stably expressing the CHI3L1-AEab bispecific antibody" (provided by Gene Synthesis Company) was inoculated in advance. The strain was inoculated with 30 mL of 2×YT medium, and after shaking culture at 37°C overnight, the stable transfection plasmid pXC-CHI3L1-AEab was extracted using a commercial plasmid extraction kit.

[0052] c. Cell transfection

[0053] The viability was greater than 95% and the cell density reached 2-3×10 6 The CHO cells without obvious aggregation were centrifuged at 300g for 5 min and the supernatant was discarded. 7 Cells were resuspended in 200 μL Celetrix commercial electroporation solution. 25 μg of pXC-CHI3L1-AEab plasmid was transfected each time. The transfection operation was performed on a Celetrix electroporator with a voltage set at 1250V.

[0054] d. Cell recovery and screening

[0055] The transfected cells were resuspended in CHO complete medium and placed at 37°C and 5% CO 2 After recovery, the cells were centrifuged at 300 g for 5 min, the culture medium was discarded (1 mL was left for measuring the expression level), and CHO complete culture medium and 25 μM MSX were added to adjust the cell density to 1×10 6 The cells were placed at 37°C and 5% CO 2 Culture in an incubator for 8 to 10 days until the viability exceeds 30%.

[0056] e. Monoclonal screening

[0057] Using Kangsheng commercial monoclonal culture medium, cells with a viability of more than 30% were diluted to 2.5 cells / mL, 200 μL per well, and spread over 96-well plates, a total of 30 plates. The culture conditions were 37°C and 5% CO 2, until the monoclonal growth completely covers the bottom of the well. The monoclonal cells with higher expression levels are screened and transferred to shake flasks for large-scale culture. Finally, a CHO-K1 monoclonal working cell bank is constructed and frozen in a liquid nitrogen tank for subsequent antibody expression.

[0058] (2) Expression of “CHI3L1-AEab bispecific antibody” in CHO-K1 suspension expression system

[0059] a. Cell recovery

[0060] The CHO-K1 working cell line constructed in step (1) was taken out from the liquid nitrogen tank and quickly placed in a 37°C water bath for thawing. After thawing, the cells were resuspended in CHO cell culture medium and placed in a cell culture shaker for 48 hours.

[0061] b. Cell transfer and fed-batch culture

[0062] The revived cells were transferred to the fermentation medium and the cell density was adjusted to 0.5×10 6 / mL. On the 5th day (Day 5), the feeding operation was started, and on the 7th day (Day 7), the culture temperature was lowered to extend the cell production period.

[0063] c. Collection of supernatant

[0064] The culture supernatant was collected on day 15 or when the cell viability dropped below 60%, the cell precipitate was removed by centrifugation, and the culture supernatant was retained for subsequent purification steps.

[0065] (3) Affinity purification of CHI3L1-AEab bispecific antibody

[0066] a. Affinity column filling

[0067] Calculate the required amount of Protein A filler according to experimental requirements, load the commercial Protein A filler into the column, and wash the filler with PBS equilibration buffer to ensure that the column is in equilibrium.

[0068] b. Loading and cleaning

[0069] The cell culture supernatant collected by centrifugation was loaded onto the affinity column at a low flow rate. After loading, the column was washed with equilibration buffer for 10 column volumes. Then, the column was washed again with pre-elution buffer A at pH 5.0 for 10 column volumes to remove non-specific binding substances.

[0070] c. Elution and Neutralization

[0071] The target antibody protein was eluted with a citric acid eluent at pH 3.2. The eluted protein solution was immediately neutralized with 2M Tris buffer to maintain protein activity. After the neutralized antibody solution was measured for concentration, it was dialyzed to PBS buffer and finally stored in aliquots.

[0072] Example 4

[0073] Acquisition of AE-BSA protein:

[0074] (1) Coupling

[0075] 10 mg of BSA was dissolved in 20 mM MES 6.5, and NSP-SA-NHS (Suzhou Yake, catalog number: Y0080) was added at a BSA: acridinium ester molar ratio of "1:20", and incubated at room temperature for 3 h.

[0076] (2) Fluid exchange

[0077] Use a 3kDa ultrafiltration concentrator or 3KDa dialysis bag to replace the BSA solution after coupling with acridinium ester into a mixture containing 20mM MES6.5 and 150mM NaCl, and adjust the protein concentration to 0.5mg / ml; finally, add a final concentration of 0.1% BSA and 40% glycerol, and store at low temperature and away from light for later use.

[0078] Example 5

[0079] ELISA affinity test of the above bispecific antibody CHI3L1-AEab nanobody end:

[0080] (1) Coating

[0081] AE-BSA protein (prepared in Example 4) was diluted to 0.5 μg / mL using carbonate buffer, 100 μL was added to each well, and added to the Elisa plate, and allowed to stand overnight at 4°C to complete coating.

[0082] (2) Closed

[0083] The next day, the Elisa plate was washed three times with PBST (PBS + 0.1% Tween 20) washing solution, and then a blocking solution prepared with PBST and 3% BSA was added, 200 μL per well, and blocked at 37° C. for 1 hour.

[0084] (3) Addition of bispecific antibody CHI3L1-AEab

[0085] After blocking, wash the Elisa plate three times with PBST. Dilute the bispecific antibody CHI3L1-AEab with blocking solution, with a starting concentration of 1 μg / mL, and dilute it in a 3-fold gradient to form 8 different gradients. Add the diluted antibody to the Elisa plate, add 100 μL to each well, and incubate at 37°C for 2 hours.

[0086] (4) Add "HRP-labeled goat anti-mouse secondary antibody"

[0087] Wash the Elisa plate three times with PBST again. Then dilute the "HRP-labeled goat anti-mouse secondary antibody" with blocking solution. Add the diluted secondary antibody to the Elisa plate, 100 μL per well, and incubate at 37°C for 1 hour.

[0088] (5) Color rendering

[0089] After washing the Elisa plate three times with PBST, add 100 μL OPD colorimetric solution for colorimetric reaction. The colorimetric reaction time is 5 to 15 minutes, and the reaction is stopped according to the colorimetric effect. When the reaction is terminated, add 100 μL 2M H 2 SO 4 Finally, use a microplate reader at wavelength OD 450 Detect absorbance value.

[0090] (6) Data processing

[0091] Import the Elisa test results into GraphPad software for data processing, such as Figure 2 (In the figure, SEQ ID NO.1 refers to the bispecific antibody detection corresponding to the SEQ ID NO.1 sequence, SEQ ID NO.2 refers to the bispecific antibody detection corresponding to the SEQ ID NO.2 sequence, and SEQ ID NO.3 to SEQ ID NO.8 and so on). Figure 2 The analysis results show that CHI3L1-AEab has good binding activity to acridinium esters.

[0092] Example 6

[0093] Acquisition of AE-HRP (Acridinium ester coupled to HRP):

[0094] (1) Coupling

[0095] 10 mg HRP was dissolved in 20 mM MES 6.5, and NSP-SA-NHS (Suzhou Yake, catalog number: Y0080) was added according to the HRP: acridinium ester molar ratio of "1:20", and incubated at room temperature for 3 h.

[0096] (2) Fluid exchange

[0097] Use a 3kDa ultrafiltration concentrator or 3kDa dialysis bag to replace the HRP solution after coupling with acridinium ester into a mixture containing 20mM MES6.5 and 150mM NaCl, adjust the protein concentration to 0.5mg / ml, add 0.1% BSA and 40% glycerol to a final concentration, and store at low temperature and away from light for later use.

[0098] Example 7

[0099] CHI3L1-AEab bispecific antibody is used for paired ELISA to detect CHI3L1 antigen calibrator:

[0100] (1) Coating

[0101] CHI3L1 paired antibody (Anhui Qiancheng Biotechnology Co., Ltd., product number: MC01901) was diluted to 0.5 μg / mL using carbonate buffer, 100 μL was added to each well, and added to the Elisa plate. The coating was completed by standing overnight at 4°C.

[0102] (2) Closed

[0103] The next day, the Elisa plate was washed three times with PBST (PBS + 0.1% Tween 20) washing solution, and then a blocking solution prepared with PBST and 3% BSA was added, 200 μL per well, and blocked at 37° C. for 1 hour.

[0104] (3) Adding antigen

[0105] After blocking, wash the Elisa plate three times with PBST. Dilute the CHI3L1-AEab bispecific antibody to a concentration of 1ug / mL with blocking solution, and then dilute the CHI3L1 antigen (Anhui Qiancheng Biotechnology Co., Ltd., product number: AG01901) with this mixture. The starting concentration of the antigen is 1μg / mL, and it is continuously diluted in a 2-fold gradient to form 7 different gradients. The last gradient is 0, added to the Elisa plate, 100μL is added to each well, and incubated at 37°C for 2 hours.

[0106] (4) Add AE-HRP

[0107] The Elisa plate was washed again with PBST for three times. AE-HRP (Acridinium ester coupled to HRP, prepared in Example 6) was then diluted with blocking solution. The diluted AE-HRP was added to the Elisa plate, 100 μL per well, and incubated at 37° C. for 1 hour.

[0108] (5) Color rendering

[0109] After washing the Elisa plate three times with PBST, add 100 μL OPD colorimetric solution for colorimetric reaction. The colorimetric reaction time is 5 to 15 minutes, and the reaction is stopped according to the colorimetric effect. When the reaction is terminated, add 100 μL 2M H 2 SO 4 Finally, use a microplate reader at wavelength OD 450 Detect absorbance value.

[0110] (6) Data processing

[0111] The ratio of the CHI3L1-AEab group standard to the blank control was calculated. When the ratio was greater than 2, it indicated that the ELISA assay method could determine a certain concentration range of CHI3L1 standards. The lowest concentration was the sensitivity of the reagent. The average value was taken after three parallel tests. The data are shown in Tables 1 to 8 (wherein, CHI3L1-AEab1 antibody refers to a bispecific antibody based on the anti-acridinium ester monoclonal antibody of SEQ ID NO.1 sequence, CHI3L1-AEab2, CHI3L1-AEab3... and so on).

[0112] Table 1 Results of using CHI3L1-AEab1 bispecific antibody for paired ELISA to detect CHI3L1 antigen calibrator

[0113]

[0114] Table 2 Results of using CHI3L1-AEab2 bispecific antibody for paired ELISA to detect CHI3L1 antigen calibrator

[0115]

[0116]

[0117] Table 3 Results of using CHI3L1-AEab3 bispecific antibody for paired ELISA detection of CHI3L1 antigen calibrator

[0118]

[0119] Table 4 Results of using CHI3L1-AEab4 bispecific antibody for paired ELISA to detect CHI3L1 antigen calibrator

[0120]

[0121] Table 5 Results of using CHI3L1-AEab5 bispecific antibody for paired ELISA to detect CHI3L1 antigen calibrator

[0122]

[0123] Table 6 Results of using CHI3L1-AEab6 bispecific antibody for paired ELISA detection of CHI3L1 antigen calibrator

[0124]

[0125] Table 7 Results of using CHI3L1-AEab7 bispecific antibody for paired ELISA to detect CHI3L1 antigen calibrator

[0126]

[0127]

[0128] Table 8 Results of using CHI3L1-AEab8 bispecific antibody for paired ELISA to detect CHI3L1 antigen calibrator

[0129]

[0130] Example 8

[0131] Preparation of HRP-labeled CHI3L1 monoclonal antibody (control antibody):

[0132] (1) Weigh 2 mg of HRP and dissolve it in 0.5 mL of H 2 O.

[0133] (2) Add freshly prepared 0.06 M NaIO 4 The solution was 0.5 mL and stored at 4°C in the dark for 30 min.

[0134] (3) Add 0.5 mL of 160 mM ethylene glycol and incubate at room temperature for 30 min.

[0135] (4) Add 2 mg of CHI3L1 monoclonal antibody (Anhui Qiancheng Biotechnology Co., Ltd., product number: MC01902) to the supernatant and mix well.

[0136] (5) The above solution was placed in a dialysis bag and dialyzed against 0.05 mM CB buffer at 4°C overnight.

[0137] (6) Transfer to a centrifuge tube and add freshly prepared 5 mg / mL NaBH 4 0.2mL, shipped, placed at 4℃ for 2h.

[0138] (7) Add an equal volume of saturated ammonium sulfate solution and incubate at 4°C for 30 min. Centrifuge at 4°C, 4000 rpm, for 20 min. Discard the supernatant, filter and dissolve the precipitate in PBS.

[0139] (8) The above liquid is placed in a dialysis bag and dialyzed against 0.02 M PBS at 4°C overnight; (the liquid can be changed multiple times).

[0140] (9) Transfer to a centrifuge tube, add an equal volume of glycerol and 0.1% PC300, and store at -20°C.

[0141] Example 9

[0142] CHI3L1 monoclonal antibody is used for paired ELISA detection of CHI3L1 antigen calibrator:

[0143] (1) Coating

[0144] CHI3L1 antibody (Anhui Qiancheng Biotechnology Co., Ltd., product number: MC01901) was diluted to 0.5 μg / mL using carbonate buffer, 100 μL was added to each well, and added to the Elisa plate. The coating was completed by standing overnight at 4°C.

[0145] (2) Closed

[0146] The next day, the Elisa plate was washed three times with PBST (PBS + 0.1% Tween 20) washing solution, and then a blocking solution prepared with PBST and 3% BSA was added, 200 μL per well, and blocked at 37° C. for 1 hour.

[0147] (3) Adding antigen

[0148] After blocking, wash the Elisa plate three times with PBST. Dilute the "HRP-labeled CHI3L1 monoclonal antibody" with blocking solution to a concentration of 1ug / mL, and then use this mixture to dilute the CHI3L1 antigen protein (Anhui Qiancheng Biotechnology Co., Ltd., product number: AG01901). The starting concentration of the antigen is 1μg / mL, and it is continuously diluted in a 2-fold gradient to form 7 different gradients. The last gradient is 0, and added to the Elisa plate, 100μL is added to each well, and incubated at 37°C for 2 hours.

[0149] (4) Color rendering

[0150] After washing the Elisa plate three times with PBST, add 100 μL OPD colorimetric solution for colorimetric reaction. The colorimetric reaction time is 5 to 15 minutes, and the reaction is stopped according to the colorimetric effect. When the reaction is terminated, add 100 μL 2M H 2 SO 4 Finally, use a microplate reader at wavelength OD 450 Detect absorbance value.

[0151] (5) Data processing

[0152] The ratio of the CHI3L1 antibody group standard to the blank control was calculated. When the ratio was greater than 2, it indicated that the ELISA assay method could determine CHI3L1 standards within a certain concentration range. The lowest concentration was the sensitivity of the reagent. The average value was obtained by performing three parallel tests. The data are shown in Table 9.

[0153] Table 9 Results of using CHI3L1 monoclonal antibody for paired ELISA to detect CHI3L1 antigen calibrator

[0154]

[0155] Combining the data results in Examples 8 and 9, it can be seen that when used as the second antibody in the ELISA platform, the bispecific antibody of the present invention can not only bind to a specific target antigen, but also directly bind to the "HRP-coupled acridinium ester" to obtain a signal, eliminating the traditional second antibody labeling step, simplifying the ELISA detection process, and having a strong signal value, and can replace the reagents in the traditional sandwich ELISA.

[0156] Example 10

[0157] An ELISA kit for detecting CHI3L1 antigen, comprising the bispecific antibody CHI3L1-AEab, AE-HRP, CHI3L1 paired antibody and other components required for ELISA detection, such as buffer, blocking solution, color developing solution, etc. (see Example 7).

[0158] In summary, the present invention has independently developed and designed an anti-acridinium ester monoclonal antibody, and fused it with a specific target antigen antibody-CHI3L1 antibody to form a bispecific antibody; when used as the second antibody in the ELISA platform, the bispecific antibody can not only bind to the specific target antigen, but also directly bind to the "HRP-coupled aridinium ester" to obtain a signal, eliminating the traditional second antibody labeling link (the corresponding antibody of the present invention does not need to be additionally coupled to Biotin or HRP by chemical methods), greatly simplifying the ELISA detection process, reducing intermediate steps, and thus saving costs. At the same time, the anti-acridinium ester monoclonal antibody of the present invention is a nano antibody, which has a strong affinity with aridinium ester and good specificity, and the ELISA reagent prepared by the fused and expressed bispecific antibody also has a stronger signal value.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A novel anti-acridinium ester monoclonal antibody, characterized in that: It is a nanobody, and its amino acid sequence is shown in any one of SEQ ID NO.1-8.

2. A bispecific antibody, characterized in that: The method is composed of in vitro fusion expression of the anti-acridinium ester monoclonal antibody of claim 1 and the CHI3L1 antibody; wherein the anti-acridinium ester monoclonal antibody is located at the C-terminus of the CHI3L1 antibody, and a (G4S)3 protein linker is connected between the CHI3L1 antibody and the anti-acridinium ester monoclonal antibody.

3. Use of the anti-acridinium ester monoclonal antibody according to claim 1 or the bispecific antibody according to claim 2 in the preparation of an ELISA kit.

4. An ELISA kit, characterized in that: The invention comprises using an anti-acridinium ester monoclonal antibody and a CHI3L1 antibody to express in vitro a bispecific antibody and an HRP-coupled aridinium ester; wherein the amino acid sequence of the anti-acridinium ester monoclonal antibody is shown in any one of SEQ ID NOs. 1 to 8.

5. The ELISA kit according to claim 4, characterized in that The bispecific antibody is capable of simultaneously binding to the "target antigen CHI3L1" and the "HRP-conjugated acridinium ester".

6. The ELISA kit according to claim 4, characterized in that The preparation method of the HRP-coupled acridinium ester is as follows: (1) Coupling After HRP was dissolved in MES buffer, acridinium ester NSP-SA-NHS was added and incubated at room temperature for 2 to 4 h; (2) Fluid exchange Use an ultrafiltration concentration tube or dialysis bag to change the HRP solution after coupling with acridinium ester and adjust the protein concentration to 0.4-0.6 mg / ml; finally, add the preservation solution and store it at low temperature and away from light for later use.

7. The ELISA kit according to claim 6, characterized in that In the step (1), the MES buffer used has a pH of 6.5 and a concentration of 20 mM.

8. The ELISA kit according to claim 6, characterized in that In the step (1), the mixing molar ratio of HRP to acridinium ester is 1:

20.

9. The ELISA kit according to claim 6, characterized in that In the step (2), a 3 kDa ultrafiltration concentration tube or a 3 kDa dialysis bag is used to replace the HRP solution after coupling with acridinium ester into a mixed solution containing 20 mM MES 6.5 and 150 mM NaCl.

10. The ELISA kit according to claim 6, characterized in that In the step (2), the storage solution has a final concentration of 0.1% BSA and 40% glycerol.

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