An acridinium ester or alkaline phosphatase antibody and its application and chemiluminescence kit

Abstract

The present invention provides an acridinium ester antibody and an alkaline phosphatase antibody. The acridinium ester antibody is a monoclonal antibody against acridinium ester, and its amino acid sequence is shown as any one of SEQ ID NO.1 to SEQ ID NO.8; the alkaline phosphatase antibody is a monoclonal antibody against alkaline phosphatase, and its amino acid sequence is shown as any one of SEQ ID NO.9 to SEQ ID NO.14. The present invention also provides the applications of the above acridinium ester antibody and alkaline phosphatase antibody in the preparation of a chemiluminescence detection kit and a chemiluminescence detection kit. The antibody of the present invention can directly form a complex with acridinium ester / alkaline phosphatase without additional chemical coupling, greatly reducing the operation steps and R & D investment; at the same time, the antibody of the present invention has strong affinity and good specificity with acridinium ester / alkaline phosphatase, and the chemiluminescence reagent prepared from the bispecific antibody obtained by its fusion expression also has better sensitivity, specificity, stronger signal value and stability.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and particularly to an acridinium ester or alkaline phosphatase antibody and its application and a chemiluminescence kit. Background Art

[0002] Chemiluminescent immunoassay (CLIA) is a modern analytical method with extremely high sensitivity and specificity in biomedical detection. Compared with traditional immunoassay techniques (such as enzyme-linked immunosorbent assay, ELISA, and radioimmunoassay, RIA), CLIA overcomes its limitations in sensitivity, specificity, and safety. Therefore, CLIA has become a highly efficient tool widely used in clinical diagnosis and biomedical research.

[0003] The research on the principle of chemiluminescence began in the 1950s and was applied in the field of immunoassay in the late 1970s. The basic principle of chemiluminescence is that a specific substrate reacts with a catalyst to generate a light signal (usually visible light or fluorescence), and this process does not rely on an external excitation light source. This discovery has led to the rapid development of CLIA technology.

[0004] With the continuous progress of immunology, antibody technology, and detection instruments, the specificity and sensitivity of CLIA technology have been significantly improved. Especially the innovation of antibody preparation technology has enabled CLIA to succeed in a wider range of application fields, including the detection of biomarkers and the early diagnosis of diseases.

[0005] In recent years, with the increasing demand for clinical testing, the upgrading of automation technology and instrumentation has enabled the CLIA method to maintain high accuracy and efficiency while processing a large sample volume. These advancements have significantly improved laboratory work efficiency and have enabled CLIA technology to occupy an important position in clinical diagnosis.

[0006] Currently, chemiluminescent immunoassay methods mainly include three types: direct chemiluminescent immunoassay, chemiluminescent enzyme immunoassay, and electrochemiluminescent immunoassay.

[0007] Among them, in direct chemiluminescent immunoassay, an antibody or antigen (such as acridinium ester) is directly labeled to react immunologically with the corresponding target substance in the sample; after the reaction, an oxidant (such as hydrogen peroxide) and an alkaline environment are added to stimulate the decomposition reaction of acridinium ester to generate a light signal without the need for an external catalyst.

[0008] Chemiluminescent enzyme immunoassay uses enzymes (such as horseradish peroxidase or alkaline phosphatase) to label antibodies or antigens; after forming a solid-phase complex through an immune reaction, a chemiluminescent substrate is added, and the enzyme catalyzes the decomposition of the substrate to generate a light signal; this signal is processed by a photomultiplier tube and then converted into an electrical signal for concentration determination by a computer system.

[0009] Electrochemiluminescence immunoassay uses electrochemiluminescent agents (such as ruthenium tris(bipyridyl)) to label antibodies or antigens; after an immune reaction, a chemiluminescent reaction triggered by an electron transfer process generates a light signal; this signal is received and amplified by a photon detection system, and finally the concentration of the target substance in the sample is calculated.

[0010] The above three chemiluminescent immunoassay methods have wide applications in the biomedical field, not only being suitable for the early diagnosis of diseases, but also playing important roles in drug research and development, immune monitoring, and biological research.

[0011] However, despite the numerous advantages of the above chemiluminescent immunoassay techniques, there are still some challenges. For example, direct chemiluminescent immunoassay has the disadvantages of low labeling stability and high background interference. Chemiluminescent enzyme immunoassay has the disadvantages of more operation steps and a complicated enzyme labeling process.

[0012] Therefore, there is still much room for improvement in chemiluminescent immunoassay technology at present, and there is still a strong demand for better chemiluminescent reagents in this field. Summary of the Invention

[0013] The technical problem to be solved by the present invention is to provide an acridinium ester or alkaline phosphatase antibody and its application and a chemiluminescent kit; the antibody of the present invention is used for chemiluminescent detection, and can directly form a complex with acridinium ester / alkaline phosphatase without additional chemical coupling, greatly reducing operation steps and R & D investment; at the same time, the antibody of the present invention has strong affinity and good specificity for acridinium ester / alkaline phosphatase, and the chemiluminescent reagent prepared from the bispecific antibody expressed by fusion also has better sensitivity, specificity, stronger signal value and stability.

[0014] The present invention adopts the following technical solutions to solve the above technical problems:

[0015] An acridinium ester antibody, which is an anti-acridinium ester monoclonal antibody, and the antibody form is a nanobody, and the amino acid sequence is shown as any one of SEQ ID NO.1 to 8.

[0016] As one of the preferred embodiments of the present invention, the anti-acridinium ester monoclonal antibody is in vitro fusion expression with a target antigen antibody to form a bispecific antibody, and the bispecific antibody is used for simultaneously binding a target antigen and acridinium ester.

[0017] As one of the preferred embodiments of the present invention, the acridinium ester is a small molecule acridinium ester or a modified acridinium ester; more preferably, it is NSP-SA-NHS.

[0018] An alkaline phosphatase antibody, which is a monoclonal antibody against alkaline phosphatase, and the antibody form is a nanobody, and the amino acid sequence is shown in any one of SEQ ID NO.9-14.

[0019] As one of the preferred embodiments of the present invention, the monoclonal antibody against alkaline phosphatase and the target antigen antibody are fused and expressed in vitro to form a bispecific antibody, and the bispecific antibody is used to simultaneously bind the target antigen and alkaline phosphatase.

[0020] As one of the preferred embodiments of the present invention, the alkaline phosphatase is bovine intestinal alkaline phosphatase.

[0021] Use of an above-mentioned acridinium ester antibody or alkaline phosphatase antibody in the preparation of a chemiluminescence detection kit.

[0022] As one of the preferred embodiments of the present invention, the bispecific antibody composed of the monoclonal antibody against acridinium ester / monoclonal antibody against alkaline phosphatase and the target antigen antibody is used as a detection antibody and applied to a chemiluminescence detection kit.

[0023] As one of the preferred embodiments of the present invention, the composed bispecific antibody can be symmetric or asymmetric; more preferably, it is symmetric.

[0024] As one of the preferred embodiments of the present invention, in the composed bispecific antibody, the monoclonal antibody against acridinium ester / monoclonal antibody against alkaline phosphatase is located at the N-terminus of the target antigen antibody or can also be at the C-terminus of the target antigen antibody, and more preferably, it is at the C-terminus.

[0025] As one of the preferred embodiments of the present invention, in the composed bispecific antibody, a protein linker can be added or not added between the target antigen antibody and the corresponding monoclonal antibody against acridinium ester / monoclonal antibody against alkaline phosphatase, and this linker can be any form of protein liner, such as: (G4S)n, (G3S)n, etc.; more preferably, a (G4S)3 protein linker is added.

[0026] A chemiluminescence detection kit, which includes a bispecific antibody formed by fusing and expressing a monoclonal antibody against acridinium ester / monoclonal antibody against alkaline phosphatase and a target antigen antibody in vitro; wherein, the amino acid sequence of the monoclonal antibody against acridinium ester is shown in any one of SEQ ID NO.1-8, and the amino acid sequence of the monoclonal antibody against alkaline phosphatase is shown in any one of SEQ ID NO.9-14.

[0027] As one of the preferred embodiments of the present invention, the target antigen antibodies are specifically cTnI, PCT, myo, CKMB, GP73, ST2, 25-OH-VD, TSH, anti-TSHR, anti-TPO, anti-TG, TT3, and TT4 antibodies.

[0028] The advantages of the present invention compared with the prior art are as follows:

[0029] (1) The antibodies of the present invention can be fused and expressed with target antigen antibodies to form bispecific antibodies, which are applied to chemiluminescent kits; the formed bispecific antibodies can simultaneously bind to the target antigen and acridinium ester / alkaline phosphatase. This characteristic enables the antibodies to bind to the chemiluminescent substance acridinium ester / alkaline phosphatase and form a stable complex without additional chemical labeling and modification processes. This makes the operation process of reagent manufacturers in the development and production of kits simpler and more convenient. At the same time, it can more conveniently and quickly replace the detection antibodies used in existing chemiluminescent kits, shortening the manufacturer's development cycle and replacement cost and reducing R & D investment.

[0030] (2) The anti-acridinium ester monoclonal antibody or anti-alkaline phosphatase monoclonal antibody in the present invention is a nanobody, which has strong affinity and good specificity for acridinium ester / alkaline phosphatase. The chemiluminescent reagent prepared from the bispecific antibody formed by its fusion expression also has better sensitivity, specificity, stronger signal value, and stability.

[0031] (3) Most of the reagents in the kit of the present invention can be mutually adapted for the two chemiluminescent platforms of the alkaline phosphatase platform and the acridinium ester platform (reagents other than antibodies), which further reduces the management cost of reagents and platforms at the downstream hospital end. Description of the Drawings

[0032] Figure 1 It is a structural diagram of the bispecific antibody formed by the anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody and the target antigen antibody of the present invention;

[0033] Figure 2 It is an ELISA affinity detection chart of the anti-acridinium ester monoclonal antibody and acridinium ester of the present invention;

[0034] Figure 3 It is an ELISA affinity detection chart of the anti-alkaline phosphatase monoclonal antibody and alkaline phosphatase of the present invention;

[0035] Figure 4 It is a clinical correlation detection chart of the kit of the present invention. Detailed Embodiments

[0036] The following is a detailed description of the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, 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, unless otherwise specified, are conventional reagents or methods in the art and will not be elaborated.

[0037] Example 1

[0038] An anti-acridinium ester monoclonal antibody, which is a nanobody, and its amino acid sequence is shown as any one of SEQ ID NO.1-8.

[0039] Example 2

[0040] A symmetric bispecific antibody TSH-AE is composed of the anti-acridinium ester monoclonal antibody of Example 1 and a target antigen antibody (thyroid-stimulating hormone TSH monoclonal antibody, Anhui Qiancheng Biotechnology Co., Ltd., product number: MC04101) by in vitro recombination.

[0041] Moreover, the anti-acridinium ester monoclonal antibody is located at the C-terminus of the target antigen antibody. At the same time, a (G4S)3 protein linker is also added between the target antigen antibody and the corresponding anti-acridinium ester monoclonal antibody, as Figure 1 shown.

[0042] Example 3

[0043] An anti-alkaline phosphatase monoclonal antibody, which is a nanobody, and its amino acid sequence is shown as any one of SEQ ID NO.9-14.

[0044] Example 4

[0045] A symmetric bispecific antibody TSH-AP is composed of the anti-alkaline phosphatase monoclonal antibody of Example 3 and a target antigen antibody (thyroid-stimulating hormone TSH monoclonal antibody, Anhui Qiancheng Biotechnology Co., Ltd., product number: MC04101) by in vitro recombination.

[0046] Moreover, the anti-alkaline phosphatase monoclonal antibody is located at the C-terminus of the target antigen antibody. At the same time, a (G4S)3 protein linker is also added between the target antigen antibody and the corresponding anti-alkaline phosphatase monoclonal antibody, as Figure 1 shown.

[0047] Example 5

[0048] Methods for obtaining the above two bispecific antibodies TSH-AE and TSH-AP:

[0049] (1) Construction of CHO-K1-TSH-AE / AP protein antibody stable cell lines

[0050] a. Cell resuscitation

[0051] Take a vial of cells cryopreserved in liquid nitrogen (about 1×10 7 ), and quickly thaw it in a 37°C water bath. After thawing, wipe the surface of the cryovial with an alcohol swab and perform the operation in a laminar flow hood. Centrifuge at 300g for 5 min, discard the supernatant, add 20 mL of pre-warmed (to 37°C) CHO complete medium, resuspend the cells, and transfer them to a 125 mL shake flask for culture. The culture conditions are set as 37°C, 5% CO2, and a rotational speed of 120 - 130 rpm.

[0052] b. Plasmid extraction

[0053] Inoculate the "DH5α strain stably expressing TSH-AE / AP bispecific antibody" (provided by a gene synthesis company) in advance. After culturing this strain overnight at 37°C with shaking, extract the plasmid using a commercial plasmid extraction kit. The plasmids are pXC17.4-TSH-AE and pXC17.4-TSH-AP stable transfection plasmids respectively.

[0054] c. Cell transfection

[0055] Centrifuge the cells with a viability greater than 95%, a cell density of 2 - 3×10 6 and no obvious aggregation at 300g for 5 min, and discard the supernatant. Take 4×10 7 cells and resuspend them in 400 uL of Celetrix commercial electroporation buffer. Transfect 25 ug of pXC17.4-TSH-AE or pXC17.4-TSH-AP plasmid each time, consuming 2×10 7 cells respectively. The transfection operation is carried out on a Celetrix electroporator, and the set voltage is 1250V.

[0056] d. Cell recovery and screening pressure

[0057] Resuspend the transfected cells in CHO complete medium and place them in a 37°C, 5% CO2 incubator to recover statically for 24 h. After recovery, centrifuge the cells at 300g for 5 min, discard the medium, re-add CHO medium and add 25 μM MSX, and adjust the cell density to 1×10 6 . Culture the cells in a 37°C, 5% CO2 incubator for 8 - 10 days until the viability exceeds 30%.

[0058] e. Monoclonal screening

[0059] Dilute the cells with a viability rate of over 30% to 2.5 cells / mL. Use the commercial monoclonal medium from Consen, 200 μL per well, to cover a 96-well plate completely, with a total of 30 plates. The culture conditions are 37°C and 5% CO2 until the monoclonal growth completely covers the bottom of the wells. Screen the monoclonal cells with higher expression levels and transfer them to a shake flask for large-scale culture. Finally, construct a CHO-K1 monoclonal working cell bank and store it in a liquid nitrogen tank for subsequent antibody expression.

[0060] (2) Expression of "TSH-AE and TSH-AP bispecific antibody" in the CHO-K1 suspension expression system

[0061] a. Cell resuscitation

[0062] Take out the CHO-K1 working cell line constructed in step (1) from the liquid nitrogen tank and quickly thaw it in a 37°C water bath. After thawing, resuspend the cells with CHO cell medium and place them in a cell culture shaker for culture. The resuscitation time is 48 h.

[0063] b. Cell transfer and fed-batch culture

[0064] Transfer the resuscitated cells to the fermentation medium and adjust the cell density to 0.5×10 6 / mL. Start the fed-batch feeding operation on the 5th day (Day 5) of culture and lower the culture temperature on the 7th day (Day 7) to extend the cell production period.

[0065] c. Supernatant collection

[0066] When culturing to the 15th day (Day 15) or when the cell viability drops below 60%, collect the culture supernatant. Remove the cell precipitate by centrifugation and retain the culture supernatant for subsequent purification steps.

[0067] (3) Affinity purification of TSH-AE and TSH-AP bispecific antibody

[0068] a. Affinity column packing

[0069] Calculate the required amount of Protein A packing according to the experimental requirements, load the commercial Protein A packing into the column, and wash the packing with PBS balance buffer to ensure that the column is in a balanced state.

[0070] b. Sample loading and washing

[0071] Load the centrifuged cell culture supernatant onto the affinity column at a low flow rate. After sample loading, wash the column with the balance buffer, with a washing volume of 10 column volumes. Then, wash it again with the pre-elution buffer A with a pH of 5.0 for 10 column volumes to remove non-specific binding substances.

[0072] c. Elution and Neutralization

[0073] 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 concentration of the neutralized antibody solution was determined, it was dialyzed and changed to PBS buffer, and finally aliquoted and stored.

[0074] Example 6

[0075] ELISA Affinity Detection of the Nanobody End of "TSH-AE or TSH-AP Bispecific Antibody":

[0076] (1) Coating

[0077] The BSA-AE protein or bovine intestinal alkaline phosphatase was diluted to 0.5 μg / mL with carbonate buffer, and 100 μL was added to each well and added to the ELISA plate. Coating was completed by standing overnight at 4°C.

[0078] (2) Blocking

[0079] The next day, the ELISA plate was washed three times with a washing solution containing 0.1% Tween-20 in PBS. Then, a blocking solution prepared with PBS (PBS + 0.1% Tween-20) and 2.5% skim milk powder was added, 200 μL per well, and blocked at 37°C for 1 h.

[0080] (3) Adding the Primary Antibody

[0081] After blocking, the ELISA plate was washed three times with PBS + 0.1% Tween-20. The antibody was diluted with PBST and 2.5% skim milk powder, starting at a concentration of 1 μg / mL, and serially diluted in a 3-fold gradient to form 8 different gradients. The diluted antibody was added to the ELISA plate, 100 μL per well, and incubated at 37°C for 2 h.

[0082] (4) Adding the Secondary Antibody

[0083] The ELISA plate was washed three times again with PBS + 0.1% Tween-20. Then, the "goat anti-mouse HRP-labeled secondary antibody" was diluted with PBST and 2.5% skim milk powder. The diluted secondary antibody was added to the ELISA plate, 100 μL per well, and incubated at 37°C for 1 h.

[0084] (5) Color Development

[0085] After the ELISA plate was washed three times with PBS + 0.1% Tween-20, 100 μL of OPD color development solution was added for color development reaction. The color development reaction time was 5 - 15 min, and the reaction was stopped according to the color development effect. When the reaction was terminated, 100 μL of 2M H2SO4 was added to each well. Finally, the absorbance was detected with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of OD 450 value.

[0086] (6) Data processing

[0087] Import the Elisa test results into GraphPad software for data processing (see Figure 2 、 Figure 3 ). From the analysis results of Figure 2 、 Figure 3 , it can be seen that the TSH-AE antibody and the TSH-AP antibody have good binding activities to acridinium ester and alkaline phosphatase respectively.

[0088] Example 7

[0089] Preparation of streptavidin magnetic beads:

[0090] (1) Magnetic bead cleaning and activation

[0091] Weigh 100 mg of 1 μm magnetic beads (Suzhou Weidu Biotechnology Co., Ltd., product number CMP1001CA), place them in a magnetic field for separation, and wash them twice with 10 mL of 0.1 M MES (pH 6.5) buffer. Subsequently, add 10 mL of 0.1 M MES buffer (pH 6.5) and 20 mg of EDC, and react at room temperature for 20 min to complete the magnetic bead activation.

[0092] (2) Antibody conjugation

[0093] Add 2 mg of streptavidin protein (ROCHE, product number 28903727103) to the activated magnetic beads, stir and incubate at room temperature for 3 h to complete the conjugation.

[0094] (3) Blocking treatment

[0095] After the conjugation is completed, place the magnetic beads in a magnetic field for separation, add 5 mL of 5% BSA solution to resuspend the magnetic beads, and incubate at room temperature for 3 h to complete the blocking.

[0096] (4) Magnetic bead cleaning and storage

[0097] After blocking, place the magnetic beads in a magnetic field for separation, and wash them three times with a buffer containing 50 mM MOPS (pH 8.0), 5% betaine, 0.1% Tween-20, 0.1% BSA, and 0.1% PC300. Finally, resuspend the magnetic beads to a final concentration of 1% and store for later use.

[0098] Example 8

[0099] Biotinylation of the capture antibody:

[0100] (1) Ultrafiltration concentration and buffer exchange

[0101] 1 mg of anti-TSH antibody (Anhui Qiancheng Biotechnology Co., Ltd., product number MC04102) was concentrated and exchanged into PBS using a 10 kDa ultrafiltration concentrator tube.

[0102] (2) Coupling

[0103] According to the molar ratio of antibody:biotin of 1:10, NHS-PEG12-biotin (Thermo Fisher, product number 21312) was added to the antibody solution, and the coupling was carried out at room temperature for 1 h.

[0104] (3) Ultrafiltration concentration and buffer exchange

[0105] Using a 10 kDa ultrafiltration concentrator tube, the biotinylated antibody was concentrated and exchanged to remove free biotin, and then exchanged into PBS. The antibody concentration was adjusted to 0.5 mg / mL, and glycerol was added and stored for later use.

[0106] Example 9

[0107] Obtaining of BSA-acridinium ester component:

[0108] (1) Coupling

[0109] 10 mg of BSA was dissolved in 20 mM MES 6.5, and NSP-SA-NHS (Suzhou Yak, product number Y0080) was added according to the molar ratio of BSA:acridinium ester of 1:20, and incubated at room temperature for 3 h.

[0110] (1) Buffer exchange

[0111] Using a 3 kDa ultrafiltration concentrator tube or 3 kDa dialysis bag, the BSA conjugated with acridinium ester was exchanged into "20 mM MES (pH 6.5) + 150 mM NaCl solution". The protein concentration was adjusted to 0.5 mg / mL, and 0.1% BSA and 40% glycerol were added at the final concentration, and then stored under low temperature and light protection conditions for later use.

[0112] Example 10

[0113] A chemiluminescent reagent in this example, used for the detection of thyroid stimulating hormone antigen, comprises the following components:

[0114] Component M: Composed of streptavidin magnetic beads (prepared in Example 7) and buffer. Among them, the working concentration of the magnetic beads is 0.75 mg / mL, and the buffer is composed of 50 mM Tris-HCl PH7.4, 150 mM NaCl, 0.1% Tween-20, 0.5% BSA, 0.1% proclin 300. During preparation, according to the component content of reagent M, each component was mixed in the same container and mixed evenly to obtain it.

[0115] Component R1: Composed of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% BSA, 1% trehalose, 0.1% proclin 300, and 2.3 μg / mL biotinylated antibody (prepared in Example 8). When preparing, according to the component contents of reagent R1, mix each component in the same container and mix evenly to obtain it.

[0116] Component R2: Composed of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% BSA, 1% trehalose, 0.1% proclin 300, 2 μg / mL TSH-AE bispecific antibody / TSH-AP bispecific antibody, 0.2 μg / mL BSA-AE (prepared in Example 9) / 0.1 μg / mL bovine intestinal alkaline phosphatase. When preparing, according to the component contents of reagent R2, mix each component in the same container and mix evenly to obtain it.

[0117] Test Example 1

[0118] In this test example, a chemiluminescence detection kit for detecting thyroid stimulating hormone (TSH) was composed of components M, R1, and R2 in the reagent of Example 10, and its overall performance was tested. Among them, the nanobody in the TSH-AE bispecific antibody was taken as SEQ ID NO.3, and the nanobody in the TSH-AP was taken as SEQ ID NO.13.

[0119] I. Detection method of chemiluminescence analyzer:

[0120] Taking the FEIPENG Shine i1000 fully automatic chemiluminescence immunoassay analyzer as an example, first add 50 μL of component R1, add 50 μL of sample, add 50 μL of component M, add 50 μL of component R2, incubate at 37°C for 5 min; the magnetic separator separates and removes the supernatant, washes 2 times with the washing solution, adds the substrate to react and detect the luminescence value.

[0121] The system will analyze the set concentration of the calibrator and the measured luminescence value, and automatically generate a calibration curve. When measuring the sample, the instrument will automatically calculate the concentration value corresponding to the sample through the calibration curve according to the luminescence signal of the sample.

[0122] II. Sensitivity detection:

[0123] Using a 5% bovine serum albumin solution as a blank sample, repeat the measurement more than 20 times according to the above detection method of the fully automatic chemiluminescence immunoassay analyzer, and use the blank mean plus twice the standard deviation as the lowest detection limit. The results show that the lowest detection limit of the kit of the present invention is 0.008 uIU / L.

[0124] III. Linear range detection:

[0125] A high-concentration sample close to 150 uIU / mL was serially diluted with normal saline, and the dilution factors were 1 / 1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, and 1 / 256, respectively, to prepare 9 sample solutions with different concentrations. The samples were detected using an automated chemiluminescence immunoassay analyzer, and the concentrations of the samples at each dilution were measured. Each concentration was measured 3 times in duplicate, and the average value of each measurement was calculated.

[0126] Using the dilution concentration as the independent variable and the average value of the measurement results as the dependent variable, linear regression analysis was performed, and the regression equation and the correlation coefficient r were calculated. The test data are shown in Tables 1 and 2, indicating that the linear performance of the chemiluminescent reagent prepared with TSH-AE bispecific antibody and the chemiluminescent reagent prepared with TSH-AP bispecific antibody was maintained at 150 uIU / mL. The regression equations were y = 1.0292x - 0.0954 and y = 1.0292x - 0.0954, respectively, and the correlation coefficients were R 2 = 1, R 2 = 0.9996.

[0127] Table 1. Detection of the linear range of the kit - acridinium ester platform

[0128] Dilution ratio Theoretical concentration (uIU / L) Detected concentration (uIU / L) Recovery rate (%) 1 / 256 0.6 0.73 124.6 1 / 128 1.2 1.25 106.7 1 / 64 2.3 2.56 109.2 1 / 32 4.7 5.1 108.8 1 / 16 9.4 9.73 103.8 1 / 8 18.8 19.2 102.4 1 / 4 37.5 38.97 103.9 1 / 2 75.0 76.82 102.4 1 / 1 150.0 154.66 103.1

[0129] Table 2. Detection of the linear range of the kit - alkaline phosphatase platform

[0130]

[0131]

[0132] IV. Repeatability and precision:

[0133] A quality control product with a TSH concentration of 1.5 uIU / mL was used as a sample and measured according to the detection method of the automated chemiluminescent immunoassay analyzer described above. Each concentration was measured 10 times in duplicate, and the mean value and standard deviation of the measurements were calculated, respectively. The results are shown in Table 3.

[0134] Table 3. Detection of the repeatability and precision of the kit

[0135]

[0136]

[0137] As can be seen from Table 3, the repeatability and accuracy of the detection kits prepared with TSH-AE bispecific antibody or TSH-AP bispecific antibody can meet the requirements of clinical applications.

[0138] V. Clinical correlation detection:

[0139] Using 20 Roche electrochemiluminescence calibrated clinical samples, the reagents in Example 10 were used, and the correlation detection of the clinical samples was carried out according to the detection method of the chemiluminescence analyzer. The detection results are shown in Figure 4 .

[0140] In addition, it should be noted that in addition to the above-mentioned thyroid stimulating hormone TSH monoclonal antibody, the anti-acridinium ester / anti-alkaline phosphatase monoclonal antibody of the present invention can also bind to other target antigen antibodies, such as cTnI, PCT, myo, CKMB, GP73, ST2, 25-OH-VD, anti-TSHR, anti-TPO, anti-TG, TT3, TT44 antibodies, etc., and is suitable for the detection of corresponding target antigens.

[0141] In summary, the bispecific antibody composed of the anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody of the present invention and the target antigen can be directly applied to the chemiluminescence platform, and can bind to the luminescent substance to form a complex without additional chemical modification and labeling, greatly reducing the R & D investment of reagent manufacturers in the labeling process and raw material screening, and the reagent production process is simpler and more controllable. At the same time, the antibody of the present invention has strong affinity and good specificity for acridinium ester / alkaline phosphatase, and the chemiluminescence reagent prepared by the bispecific antibody prepared by its fusion expression also has better sensitivity, specificity, stronger signal value and stability.

[0142] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An acridinium ester antibody, characterized in that, It is an anti-acridinium ester nanobody, and its amino acid sequence is shown as SEQ ID NO.

3.

2. The acridinium ester antibody according to claim 1, characterized in that, The anti-acridinium ester nanobody and the target antigen antibody are fused and expressed in vitro to form a bispecific antibody, and the bispecific antibody is used to simultaneously bind the target antigen and acridinium ester.

3. Application of the acridinium ester antibody according to claim 1 or 2 in the preparation of a chemiluminescence detection kit.

4. The application according to claim 3, characterized in that The bispecific antibody composed of the anti-acridinium ester nanobody and the target antigen antibody is used as a detection antibody and applied to a chemiluminescence detection kit; the anti-acridinium ester nanobody is the acridinium ester antibody according to claim 1 or 2.

5. The application according to claim 4, wherein In the bispecific antibody thus formed, the anti-acridinium ester nanobody is located at the C-terminus of the target antigen antibody.

6. The application according to claim 4, wherein In the bispecific antibody thus formed, there is also a (G4S)3 protein linker connected between the target antigen antibody and the anti-acridinium ester nanobody.

7. A chemiluminescence detection kit, characterized in that, It includes fusing and expressing the anti-acridinium ester nanobody and the target antigen antibody in vitro to form a bispecific antibody; among them, the amino acid sequence of the anti-acridinium ester nanobody is shown as SEQ ID NO.

3.

8. The chemiluminescence detection kit according to claim 7, characterized in that, The target antigen antibody is specifically cTnI, PCT, myo, CKMB, GP73, ST2, 25-OH-VD, TSH, anti-TSHR, anti-TPO, anti-TG, TT3, TT4 antibody.

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