Acridinium ester or alkaline phosphatase antibody, application thereof and chemiluminescence kit

Abstract

The invention provides an acridinium ester antibody and an alkaline phosphatase antibody. The acridinium ester antibody is an anti-acridinium ester monoclonal antibody, and the amino acid sequence of the acridinium ester antibody is shown as any one of SEQ ID NO.1-8; the alkaline phosphatase antibody is an anti-alkaline phosphatase monoclonal antibody, and the amino acid sequence of the alkaline phosphatase antibody is shown as any one of SEQ ID NO.9-14. The invention also provides application of the acridinium ester antibody and the alkaline phosphatase antibody in preparation of a chemiluminescence detection kit and the chemiluminescence detection kit. The antibody disclosed by the invention can directly form a compound with acridinium ester / alkaline phosphatase without coupling by an additional chemical method, so that the operation steps and research and development investment are greatly reduced; meanwhile, the antibody disclosed by the invention has strong affinity and good specificity with acridinium ester / alkaline phosphatase, and a chemiluminescent reagent prepared from the bispecific antibody subjected to fusion expression also has better sensitivity and specificity and stronger signal value and stability.

Description

Technical Field

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

[0002] Chemiluminescent Immunoassay (CLIA) is a modern analytical method with extremely high sensitivity and specificity in biomedical testing. 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 an efficient tool widely used in clinical diagnosis and biomedical research.

[0003] The study of chemiluminescence began in the 1950s and was applied in the field of immunoassay in the late 1970s. The basic principle of chemiluminescence is to generate light signals (usually visible light or fluorescence) through the reaction of specific substrates and catalysts, and this process does not rely on external excitation light sources. 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. In particular, the innovation of antibody preparation technology has enabled CLIA to succeed in a wider range of applications, including biomarker detection and early diagnosis of diseases.

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

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

[0007] Among them, direct chemiluminescent immunoassay directly labels antibodies or antigens (such as acridinium esters) to undergo an immune reaction 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 the acridinium ester and 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 a solid-phase complex is formed through an immune reaction, a chemiluminescent substrate is added, and the enzyme catalyzes the decomposition of the substrate to produce a light signal; this signal is processed by a photomultiplier tube and converted into an electrical signal, and the concentration is measured by a computer system.

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

[0010] The above three chemiluminescent immunoassay methods are widely used in the biomedical field. They are not only suitable for early diagnosis of diseases, but also play an important role in drug development, immune monitoring and biological research.

[0011] However, despite the numerous advantages of the above-mentioned chemiluminescent immunoassay technology, 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 operating steps and complicated enzyme labeling process.

[0012] Based on this, there is still much room for improvement in chemiluminescent immunoassay technology, and there is still a strong demand in this field for better chemiluminescent reagents. 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 chemiluminescence kit; the antibody of the present invention is used for chemiluminescence detection, and can directly form a complex with acridinium ester / alkaline phosphatase without the need for additional chemical coupling, thereby greatly reducing the operation steps and research and development investment; at the same time, the antibody of the present invention has a strong affinity with acridinium ester / alkaline phosphatase and good specificity, and the chemiluminescence reagent prepared by the fusion-expressed bispecific antibody also has better sensitivity, specificity, and 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 is an anti-acridinium ester monoclonal antibody in the form of a nanobody, and the amino acid sequence is shown in any one of SEQ ID NOs. 1 to 8.

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

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

[0018] An alkaline phosphatase antibody is an anti-alkaline phosphatase monoclonal antibody, the antibody form is a nanobody, and the amino acid sequence is shown in any one of SEQ ID NOs. 9 to 14.

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

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

[0021] A use of the 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 anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody 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 bispecific antibody of the composition can be symmetrical or asymmetrical, and more preferably symmetrical.

[0024] As one of the preferred embodiments of the present invention, in the bispecific antibody of the composition, the anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody is located at the N-terminus of the target antigen antibody, or at the C-terminus of the target antigen antibody, more preferably at the C-terminus.

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

[0026] A chemiluminescent detection kit comprises a bispecific antibody composed of an anti-acridinium ester monoclonal antibody / an anti-alkaline phosphatase monoclonal antibody and a target antigen antibody for in vitro fusion expression; wherein the amino acid sequence of the anti-acridinium ester monoclonal antibody is shown in any one of SEQ ID NOs. 1 to 8, and the amino acid sequence of the anti-alkaline phosphatase monoclonal antibody is shown in any one of SEQ ID NOs. 9 to 14.

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

[0028] The advantages of the present invention compared to the prior art are:

[0029] (1) The antibody of the present invention can be fused with the target antigen antibody to form a bispecific antibody, which can be used in a chemiluminescent kit; the bispecific antibody can bind to the target antigen and acridinium ester / alkaline phosphatase at the same time. This feature allows the antibody to bind to the chemiluminescent substance acridinium ester / alkaline phosphatase and form a stable complex without additional chemical labeling modification, making the operation process of the reagent manufacturer in the development and production of the kit simpler and more convenient, and can more conveniently and quickly replace the detection antibody used in the existing chemiluminescent kit, 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 a strong affinity with aridinium ester / alkaline phosphatase and good specificity. The chemiluminescent reagent prepared by the fusion-expressed bispecific antibody also has better sensitivity, specificity, and stronger signal value and stability;

[0031] (3) Most of the reagents in the kit of the present invention are compatible with both the alkaline phosphatase platform and the acridinium ester platform (reagents other than antibodies), which further reduces the management costs of reagents and platforms at the downstream hospital end. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0035] Figure 4 It is a clinical correlation detection diagram of the kit of the present invention. DETAILED DESCRIPTION

[0036] 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, unless otherwise specified, are conventional reagents or methods in the art and will not be repeated.

[0037] Example 1

[0038] An 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.

[0039] Example 2

[0040] A symmetrical bispecific antibody TSH-AE is formed by in vitro recombination 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).

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

[0042] Example 3

[0043] An anti-alkaline phosphatase monoclonal antibody is a nanobody, and the amino acid sequence is shown in any one of SEQ ID NOs. 9 to 14.

[0044] Example 4

[0045] A symmetrical bispecific antibody TSH-AP is formed by in vitro recombination 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).

[0046] The anti-alkaline phosphatase monoclonal antibody is located at the C-terminus of the target antigen antibody. Meanwhile, a (G4S)3 protein linker is added between the target antigen antibody and the corresponding anti-alkaline phosphatase monoclonal antibody. 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 line

[0050] a. Cell recovery

[0051] Take a liquid nitrogen frozen cell (about 1×10 7 ), and quickly thaw in a 37℃ 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℃, resuspend the cells and transfer to a 125mL shake flask for culture. The culture conditions are set at 37℃, 5% CO2, and a speed of 120-130rpm.

[0052] b. Plasmid extraction

[0053] The "DH5α strain stably expressing TSH-AE / AP bispecific antibody" (provided by Gene Synthesis Company) was inoculated in advance. The strain was cultured at 37°C overnight with shaking, and the plasmids were extracted using a commercial plasmid extraction kit. The plasmids were pXC17.4-TSH-AE and pXC17.4-TSH-AP stable transfection plasmids.

[0054] c. Cell transfection

[0055] The viability was greater than 95% and the cell density reached 2-3×10 6 The cells without obvious aggregation were centrifuged at 300g for 5 min and the supernatant was discarded. 7 Cells were resuspended in 400uL Celetrix commercial electroporation solution. Each transfection of pXC17.4-TSH-AE or pXC17.4-TSH-AP plasmid 25ug, consuming 2×10 cells respectively. 7 The transfection operation was performed on a Celetrix electroporator with a voltage set at 1250V.

[0056] d. Cell recovery and screening

[0057] The transfected cells were resuspended in CHO complete medium and placed in a 37°C, 5% CO2 incubator to recover for 24 h. After recovery, the cells were centrifuged at 300 g for 5 min, the medium was discarded, CHO medium was added and 25 μm MSX was added to adjust the cell density to 1×10 6 The cells were cultured in a 37°C, 5% CO2 incubator for 8 to 10 days until the viability exceeded 30%.

[0058] e. Monoclonal screening

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

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

[0061] a. Cell recovery

[0062] 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.

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

[0064] 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.

[0065] c. Collection of supernatant

[0066] 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.

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

[0068] a. Affinity column filling

[0069] 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.

[0070] b. Loading and cleaning

[0071] 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.

[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 2MTris buffer to maintain protein activity. After the neutralized antibody solution was measured for concentration, it was dialyzed and replaced with PBS buffer and finally stored in aliquots.

[0074] Example 6

[0075] ELISA affinity test of the nanobody end of "TSH-AE or TSH-AP bispecific antibody":

[0076] (1) Coating

[0077] Dilute BSA-AE protein or calf intestinal alkaline phosphatase to 0.5ug / mL using carbonate buffer, add 100uL to each well, and add to the Elisa plate. Incubate overnight at 4°C to complete the coating.

[0078] (2) Closed

[0079] The next day, the Elisa plate was washed three times with PBS containing 0.1% Tween-20, and then a blocking solution prepared with PBS (PBS + 0.1% Tween-20) and 2.5% skim milk powder was added, 200uL per time, and blocked at 37°C for 1 hour.

[0080] (3) Add primary antibody

[0081] After blocking, wash the Elisa plate three times with PBS + 0.1% Tween-20. Dilute the antibody with PBST and 2.5% skim milk powder, with a starting concentration of 1ug / mL, and dilute it in 3-fold gradients to form 8 different gradients. Add the diluted antibody to the Elisa plate, add 100uL to each well, and incubate at 37°C for 2h.

[0082] (4) Add secondary antibody

[0083] Wash the Elisa plate three times with PBS + 0.1% Tween-20 again. Then dilute the "goat anti-mouse HRP labeled secondary antibody" with PBST and 2.5% skim milk powder. Add the diluted secondary antibody to the Elisa plate, 100uL per well, and incubate at 37℃ for 1h.

[0084] (5) Color rendering

[0085] After washing the Elisa plate three times with PBS + 0.1% Tween-20, add 100uL 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 100uL 2MH2SO4 to each well. Finally, use an enzyme reader at a wavelength of OD 450 Detect absorbance value.

[0086] (6) Data processing

[0087] Import the Elisa test results into GraphPad software for data processing (see Figure 2 , Figure 3 ).from Figure 2 , Figure 3 The analysis results showed that TSH-AE antibody and TSH-AP antibody had good binding activity 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 1um magnetic beads (Suzhou Weidu Biotechnology Co., Ltd., catalog number CMP1001CA), separate them in a magnetic field, and wash them twice with 10 mL of 0.1 M MES (pH 6.5) buffer. Then, add 10 mL of 0.1 M MES buffer (pH 6.5) and 20 mg of EDC, and react at room temperature for 20 minutes to complete the activation of the magnetic beads.

[0092] (2) Antibody conjugation

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

[0094] (3) Closed treatment

[0095] After the coupling is completed, the magnetic beads are placed in a magnetic field for separation, 5 mL of 5% BSA solution is added to resuspend the magnetic beads, and incubated at room temperature for 3 h to complete the blocking.

[0096] (4) Magnetic bead cleaning and storage

[0097] After blocking, the magnetic beads were separated in a magnetic field and washed 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, the magnetic beads were resuspended to a final concentration of 1% and stored for later use.

[0098] Example 8

[0099] Biotinylation of capture antibody:

[0100] (1) Ultrafiltration concentration and liquid replacement

[0101] 1 mg of anti-TSH antibody (Anhui Qiancheng Biotechnology Co., Ltd., catalog number MC04102) was concentrated using a 10 kDa ultrafiltration concentration tube and replaced with PBS.

[0102] (2) Coupling

[0103] NHS-PEG12-biotin (Thermo Fisher, Catalog No. 21312) was added to the antibody solution at a molar ratio of antibody to biotin of 1:10 and coupled at room temperature for 1 h.

[0104] (3) Ultrafiltration concentration and liquid replacement

[0105] Use a 10kDa ultrafiltration concentrator to concentrate the biotinylated antibody and replace the solution with PBS to remove free biotin. Adjust the antibody concentration to 0.5 mg / mL and add glycerol to store for later use.

[0106] Example 9

[0107] Obtaining BSA-acridinium ester fraction:

[0108] (1) Coupling

[0109] 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.

[0110] (1) Fluid exchange

[0111] Use a 3kDa ultrafiltration concentration tube or a 3KDa dialysis bag to replace the BSA solution after coupling with acridinium ester into "20mM MES (pH6.5) + 150mM NaCl solution", 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.

[0112] Example 10

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

[0114] Component M: It is composed of streptavidin magnetic beads (prepared in Example 7) and buffer. 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, and 0.1% proclin 300. During preparation, the components of reagent M are mixed in the same container according to the component content, and the mixture is uniformly mixed.

[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 ug / mL biotinylated antibody (prepared in Example 8). During preparation, the components of reagent R1 are mixed in the same container according to their contents, and the mixture is uniformly mixed.

[0116] Component R2: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% BSA, 1% trehalose, 0.1% proclin 300, 2 ug / mL TSH-AE bispecific antibody / TSH-AP bispecific antibody, 0.2 ug / mL BSA-AE (prepared in Example 9) / 0.1 ug / mL calf intestinal alkaline phosphatase. During preparation, the components of reagent R2 are mixed in the same container according to the component contents, and the mixture is uniformly mixed to obtain the reagent.

[0117] Test Example 1

[0118] This test example uses components M, R1, and R2 in the reagent of Example 10 to form a chemiluminescent detection kit for detecting thyroid stimulating hormone (TSH), and tests its overall performance. Among them, the nanobody in the TSH-AE bispecific antibody is SEQ ID NO.3, and the nanobody in TSH-AP is SEQ ID NO.13.

[0119] 1. Chemiluminescence analyzer detection method:

[0120] Taking the Feipeng Shine i1000 fully automatic chemiluminescence immunoassay as an example, first add 50uL component R1, add 50uL sample, add 50uL component M, add 50uL component R2, and incubate the reaction at 37°C for 5 minutes; use a magnetic separator to separate and remove the supernatant, wash twice with cleaning solution, add substrate to react and detect the luminescence value.

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

[0122] 2. Sensitivity detection:

[0123] 5% bovine serum albumin solution was used as blank sample, and the detection was repeated more than 20 times according to the above automatic chemiluminescence immunoassay detection method, and the blank mean plus two times the standard deviation was used as the minimum detection limit. The results showed that the minimum detection limit of the kit of the present invention was 0.008uIU / L.

[0124] 3. Linear range detection:

[0125] The high-concentration sample of nearly 150uIU / mL was diluted with physiological saline in a gradient manner, and the dilution multiples 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 of different concentrations. The concentration of each diluted concentration sample was measured using a fully automatic chemiluminescence immunoassay analyzer, and each concentration was measured three times, and the average value of each measurement was calculated.

[0126] The dilution concentration was used as the independent variable and the average value of the measurement results was used as the dependent variable. Linear regression analysis was performed and the regression equation and correlation coefficient r were calculated. The test data are shown in Tables 1 and 2, indicating that the linear performance of the luminescent reagent prepared by TSH-AE dual antibody and the luminescent reagent prepared by TSH-AP dual antibody was maintained at 150uIU / 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. Linear range of the kit - acridinium ester platform

[0128] Dilution ratio Theoretical concentration (uIU / L) Detection 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. Linear range of the kit - alkaline phosphatase platform

[0130]

[0131]

[0132] 4. Repeatability and precision:

[0133] The quality control product with a TSH concentration of 1.5 uIU / mL was used as a sample and measured according to the fully automatic chemiluminescence immunoassay detection method. Each concentration was repeated 10 times, and the mean and standard deviation of the measurement were calculated respectively. The results are shown in Table 3.

[0134] Table 3. Repeatability and precision test of the kit

[0135]

[0136]

[0137] It can be seen from Table 3 that the repeatability and accuracy of the detection kit prepared by TSH-AE bispecific antibody or TSH-AP bispecific antibody can meet the requirements of clinical application.

[0138] 5. Clinical relevance testing:

[0139] Twenty Roche electrochemiluminescence value clinical samples were used, and the reagents in Example 10 were used to perform correlation tests on the clinical samples according to the chemiluminescence analyzer detection method. The test 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 be combined with 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 are suitable for corresponding target antigen detection.

[0141] In summary, the bispecific antibody composed of the anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody and the target antigen of the present invention can be directly applied to the chemiluminescent platform, and can be combined with the luminescent substance to form a complex without additional chemical modification and labeling, which greatly reduces the research and development investment of reagent manufacturers in labeling technology and raw material screening, and the reagent production process is simpler and controllable. At the same time, the antibody of the present invention has a strong affinity with acridinium ester / alkaline phosphatase and good specificity, and the chemiluminescent reagent prepared by the fusion-expressed bispecific antibody also has better sensitivity, specificity, and stronger signal value and stability.

[0142] 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. An acridinium ester antibody, characterized in that: It is an anti-acridinium ester monoclonal antibody, and its amino acid sequence is shown in any one of SEQ ID NOs. 1 to 8.

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

3. An alkaline phosphatase antibody, characterized in that It is an anti-alkaline phosphatase monoclonal antibody, and its amino acid sequence is shown in any one of SEQ ID NOs. 9 to 14.

4. The alkaline phosphatase antibody according to claim 3, characterized in that The anti-alkaline phosphatase monoclonal antibody is fused with the target antigen antibody in vitro to form a bispecific antibody, and the bispecific antibody is used to simultaneously bind to the target antigen and alkaline phosphatase.

5. Use of the acridinium ester antibody according to any one of claims 1 to 2, or the alkaline phosphatase antibody according to any one of claims 3 to 4 in the preparation of a chemiluminescent detection kit.

6. The use according to claim 5, characterized in that: The bispecific antibody composed of the anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody and the target antigen antibody is used as a detection antibody and applied to a chemiluminescence detection kit.

7. The use according to claim 6, characterized in that: In the bispecific antibody of the composition, the anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody is located at the C-terminus of the target antigen antibody.

8. The use according to claim 6, characterized in that: In the bispecific antibody of the composition, a (G4S)3 protein linker is further connected between the target antigen antibody and the corresponding anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody.

9. A chemiluminescent detection kit, characterized in that: The invention comprises using an anti-acridinium ester monoclonal antibody / anti-alkaline phosphatase monoclonal antibody to express in vitro with a target antigen antibody to form a bispecific antibody; wherein the amino acid sequence of the anti-acridinium ester monoclonal antibody is shown in any one of SEQ ID NOs. 1 to 8, and the amino acid sequence of the anti-alkaline phosphatase monoclonal antibody is shown in any one of SEQ ID NOs. 9 to 14.

10. The chemiluminescent detection kit according to claim 9, characterized in that: 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.

Images