25-OH-VD paired antibody and application thereof in chemiluminescence platform

Abstract

The invention provides a 25-OH-VD paired antibody, which comprises an anti-25-OH-VD monoclonal antibody, wherein the anti-25-OH-VD monoclonal antibody is a monoclonal antibody; the anti-25-OH-VD monoclonal antibody is a nano antibody, and the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The invention also provides a nucleic acid molecule or an expression vector, and a code of the nucleic acid molecule or the expression vector contains the 25-OH-VD paired antibody molecule. Meanwhile, the invention also provides an application of the 25-OH-VD paired antibody as a detection antibody in preparation of a chemiluminescence detection kit, and correspondingly provides the chemiluminescence detection kit and a chemiluminescence detection method of 25-OH-VD. The kit breaks through the limitation of a traditional competition method, realizes the detection of 25-OH-VD by a double-antibody sandwich method, can rapidly measure 25-OH-VD, and has the advantages of high sensitivity and high clinical coincidence rate.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and in particular to a 25-hydroxyvitamin D (25-OH-VD) paired antibody and an application thereof in a chemiluminescence platform. Background Art

[0002] Vitamin D is a fat-soluble steroid hormone precursor that is primarily produced by the skin in response to exposure to sunlight. The two most important forms of vitamin D are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). In contrast to vitamin D3, the body cannot produce vitamin D2, which can be ingested through fortified food or supplements. In the blood, vitamins D3 and D2 are bound to vitamin D binding protein (VDBP) and transported to the liver, where both are hydroxylated to form 25-hydroxyvitamin D (25-OH-VD).

[0003] 25-hydroxyvitamin D (25-OH-VD) is the main storage form of vitamin D. It has a high content, a long half-life, and is stable in nature. It is not affected by blood calcium and parathyroid hormone (PTH) levels, and can fully reflect the total amount of food intake and self-synthesized vitamin D in the body, as well as the conversion capacity of vitamin D. Therefore, 25-OH-VD is often considered to be the best indicator for objectively evaluating vitamin D nutritional status.

[0004] Currently, the latest detection method for 25-OH-VD is immunoassay. However, due to the very small molecular weight of 25-OH-VD, it is difficult to develop antibodies against its different epitopes for immunoassay using the double antibody sandwich method. Therefore, most of the current diagnostic detection methods for this small molecule use the principle of competition. However, the sensitivity of competitive immunoassay is low and the clinical compliance rate is poor. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a 25-hydroxyvitamin D (25-OH-VD) paired antibody and its application in a chemiluminescence platform, wherein the antibody can specifically bind to the 25-OH-VD / first antibody complex to realize the double antibody sandwich method for detecting 25-OH-VD, and has the advantages of rapid detection, high sensitivity and high clinical compliance rate.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A 25-OH-VD paired antibody comprises an anti-25-OH-VD monoclonal antibody; the anti-25-OH-VD monoclonal antibody is a nano antibody, and the amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] As one of the preferred embodiments of the present invention, the anti-25-OH-VD monoclonal antibody specifically selects the amino acid sequence shown in SEQ ID NO.1.

[0009] A nucleic acid molecule or expression vector, which encodes the above-mentioned 25-OH-VD paired antibody molecule.

[0010] As one of the preferred methods of the present invention, the expression of the coding gene in the recipient cell can be achieved by transient expression and stable expression; the expression strategy includes transfecting mammalian cells with one or more expression vectors carrying DNA fragments encoding antibodies, so that the antibodies are expressed and assembled in the recipient cells; but a more preferred expression method is secretion into the culture medium, and the antibodies are recovered therefrom by methods such as chromatography well known to those skilled in the art.

[0011] An application of the above 25-OH-VD paired antibody as a detection antibody in the preparation of a chemiluminescence detection kit.

[0012] A chemiluminescent detection kit comprises the above-mentioned 25-OH-VD paired antibody.

[0013] As one of the preferred embodiments of the present invention, it also includes a 25-OH-VD / first antibody complex; the 25-OH-VD paired antibody can specifically bind to the 25-OH-VD / first antibody complex.

[0014] As one of the preferred embodiments of the present invention, the first antibody of the 25-OH-VD / first antibody complex and the 25-OH-VD paired antibody respectively bind to different epitopes of 25-OH-VD.

[0015] A chemiluminescent detection method for 25-OH-VD comprises the following steps:

[0016] (1) contacting the sample with a first reagent comprising an acidic pH buffer to separate 25-OH-VD in the sample from its binding protein;

[0017] (2) contacting the 25-OH-VD in the sample obtained in step (1) with a second reagent; the second reagent comprises magnetic particles coated with a first antibody, the first antibody having a specific binding affinity to the 25-OH-VD, thereby forming a 25-OH-VD / first antibody complex;

[0018] (3) contacting the 25-OH-VD / first antibody complex with a third reagent; the third reagent comprises a second antibody labeled with a chemiluminescent detection signal generating molecule, i.e., the 25-OH-VD paired antibody described above; the second antibody has binding affinity with the 25-OH-VD / first antibody complex to form a complex between the 25-OH-VD, the first antibody, and the 25-OH-VD paired antibody labeled with a signal generating molecule;

[0019] (4) After incubation and washing, the concentration of 25-OH-VD in the sample is determined based on the chemiluminescence signal intensity (RLU) and the calibration curve.

[0020] As one of the preferred embodiments of the present invention, in step (3), the chemiluminescent detection signal generating molecule is selected from at least one of acridinium ester, isoluminol, alkaline phosphatase, horseradish peroxidase, and fluorescein, and more preferably acridinium ester.

[0021] As one of the preferred embodiments of the present invention, in step (4), the 25-OH-VD calibrator is tested using a fully automatic chemiluminescence immunoassay analyzer, a standard curve is drawn and built into computer software; then the clinical samples are tested and the sample concentration is calculated based on the sample luminescence value.

[0022] As one of the preferred embodiments of the present invention, the calibrator can use standard buffer to prepare 25-OH-VD into 25-OH-VD solutions with concentrations of 2 ng / mL, 18.75 ng / mL, 37.5 ng / mL, 75 ng / mL, 150 ng / mL and 300 ng / mL, respectively.

[0023] The advantages of the present invention compared to the prior art are:

[0024] The present invention screened a 25-OH-VD paired antibody that can specifically bind to the 25-OH-VD / first antibody complex and is used to detect the level of 25-OH-VD in the blood, develop a chemiluminescent detection kit suitable for the double antibody sandwich method, and apply it to the chemiluminescent detection platform in the field of medical diagnosis;

[0025] The present invention breaks through the limitations of the traditional competition method and realizes the double antibody sandwich method for detecting 25-OH-VD. It can quickly measure 25-OH-VD (with results in 10 minutes or less) and has the advantages of high sensitivity and high clinical compliance rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a paired binding activity curve diagram of the 25-OH-VD antibody of the present invention;

[0027] Figure 2is a graph showing the binding activity of the 25-OH-VD antibody of the present invention and 25-OH-VD BSA;

[0028] Figure 3 Graph showing the clinical relevance of the 25-OH-VD antibody pair of the present invention. DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1: Preliminary screening of 25-OH-VD paired antibodies:

[0031] (1) Alpacas were immunized with a complex of 25-OH-VD monoclonal antibody (Bioventix, represented by Shenzhen Yawei Century Technology Co., Ltd., catalog number: BVX.vitD3.2F4, referred to as VD mAb) and 25-OH-VD small molecule at a molar ratio of 1:4, and peripheral blood was taken to isolate PBMC cells, extract RNA and reverse transcribe (RNA extraction kit is B518651-0050 from Sangon Biotechnology, and reverse transcription kit is AE311-02 from Quanshijin Biotechnology). According to the information of the alpaca antibody database on the IMGT website, corresponding library construction primers were designed, light and heavy chain fragments were amplified, constructed onto phagemid vectors, and electroporated into TG1 competent cells to obtain an antibody library with a library capacity of about 1*10E8. Recombinant phages were prepared for the above antibody library strains, and a recombinant phage library with a titer of about 1*10E13 was obtained for subsequent alpaca antibody screening.

[0032] (2) Using the prepared VD mAb biotin protein (prepared in Example 7), the Dynabeads from Thermo TM M-280 streptavidin was combined, and then incubated with 25-OH-VD small molecules, and multiple rounds of liquid phase screening were performed on the recombinant phages of the alpaca immune library. The screening process was referred to books (Therapeutic antibody engineering, William R. Strohl and Lilam M. Strohl, 2012; Antibody Engineering Methods and Protocols, Patrick Chames, 2012.). A large number of monoclonal bacterial colonies were picked from the second and subsequent bacterial culture plates in 96 deep-well plates to prepare the supernatant expressing soluble antibodies, that is, when the density of monoclonal bacteria reached OD600 At about 0.3, IPTG was added for overnight induction at 30°C, thereby obtaining a supernatant containing monomeric soluble antibody with a Flag tag.

[0033] Example 2: ELISA to detect soluble antibody binding activity and further screen antibodies:

[0034] (1) VD mAb protein (Bioventix, Shenzhen Yawei Century Technology Co., Ltd., catalog number: BVX.vitD3.2F4) was plated and NaHCO was used. 3 Dilute it to 1 μg / mL with buffer and spread on double 96-well ELISA plates, i.e., each 96-deep-well plate corresponds to two 96-well ELISA plates, 100 μL per well, and store in a 4°C refrigerator overnight; TPBS (PBS + 0.1% Tween 20) Wash three times, 3% skim milk powder dissolved in TPBS as a blocking agent, fill each well (about 250 μL), and block at 37°C for 1 hour; wash three times with TPBS, add 25-OH-VD small molecules with a final concentration of 1 μg / mL, 100 μL / well, and incubate at room temperature for 1 hour with shaking. Skip this step for the control ELISA; wash three times with TPBS, add soluble expression supernatant, 100 μL / well, and incubate at room temperature for 1 hour with shaking; wash three times with TPBS, add 1:5000 diluted Anti-Flag / HRP secondary antibody (purchased from Sigma, product number A8592), 100 μL per well, and incubate at room temperature for 30 minutes with shaking; wash three times with TPBS, add 100 μL of OPD o-phenylenediamine (purchased from Sigma, product number 78412) substrate working solution containing 0.1% hydrogen peroxide to each well for color development, and add 100 μL after about 1 to 20 minutes. 1M sulfuric acid was used to terminate the reaction and OD was measured by microplate reader 450 Absorbance value.

[0035] (2) According to the ELISA results, the monoclonal bacteria corresponding to the positive wells were selected, and the absorbance values ​​of the corresponding wells of the control ELISA were compared. The smaller the absorbance value of the latter, the greater the ratio of the experimental group to the control group, and the more suitable it was to be selected as a positive clone. The plasmid contained in the positive clone bacteria was purified and sequenced. After sequence analysis, two nanobodies with strong binding activity were obtained, and the amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0036] Example 3: Construction of two eukaryotic expression plasmids for screening nanobodies:

[0037] (1) Using the two nanoantibody DNA sequences (SEQ ID NO.3, SEQ ID NO.4) in Example 2 as templates, suitable PCR primers (SEQ ID NO.5, SEQ ID NO.6) were designed, and restriction sites BamHI and XbaI were set at both ends. The PCR system was based on the full-form gold FastPfu fast high-fidelity DNA polymerase (Cat. No. AP221-01), including 25 μL 2*FastPfuMix, 1 μL forward primer (SEQ ID NO.5), 1 μL reaction primer (SEQ ID NO.6), an appropriate amount of gene template, and ddH 2 The PCR reaction program was as follows: 95°C, 2 min; 95°C 20 s, 56°C 20 s, 72°C 15 s, 30 cycles; 72°C, 5 min.

[0038] (2) After recovering the PCR product, pre-mix 50uL of the enzyme digestion system, use BamHI and XbaI enzymes to digest the PCR product, and construct the variable region of the nanobody into the pcDNA3.4 vector that was also digested by T4 DNA Ligase. The ligation system is 10μL.

[0039] (3) Screening positive clones for restriction digestion verification and sequencing to obtain eukaryotic expression plasmids of two nanobodies. The plasmids of the two clones were transiently transfected into Expi293 suspension cells respectively, and the expression supernatant after transfection was purified using Protein A affinity chromatography gel from Tiandi Renhe Company to obtain the pure monoclonal antibody proteins of the corresponding clones, namely VD mAb1 and VD mAb2, and the amino acid sequences corresponded to SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0040] Example 4: ELISA to detect the paired binding activity of 25-OH-VD antibody:

[0041] (1) Coating

[0042] Use of NaHCO 3 Dilute VD mAb (Bioventix, Shenzhen Yawei Century Technology Co., Ltd., catalog number: BVX.vitD3.2F4) to 1 μg / mL in buffer, add 100 μL to each well, and add to the ELISA plate. Let stand overnight at 4°C to complete coating.

[0043] (2) Closed

[0044] 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 (PBS + 0.1% Tween 20) and 2.5% skim milk powder was added, 250 μL per well, and blocked at 37° C. for 1 h.

[0045] (3) Add small molecules (skip this step for control ELISA)

[0046] After blocking, wash the ELISA plate three times with PBST washing solution. Dilute the 25-OH-VD small molecule with blocking solution to a final concentration of 1ug / mL, add 100μL to each well of the Elisa plate, and incubate at 37℃ for 1h.

[0047] (4) Add primary antibody

[0048] Wash the ELISA plate three times with PBST cleaning solution. Dilute the antibodies VD mAb1 and VD mAb2 with blocking solution, with a starting concentration of 1 μg / mL, and dilute them in a 3-fold gradient to form 8 different gradients. Add the diluted antibodies VD mAb1 and VD mAb2 to the ELISA plate, add 100 μL to each well, and incubate at 37°C for 2 hours.

[0049] (5) Add secondary antibody

[0050] Wash the ELISA plate three times with PBST cleaning solution. Then dilute the HRP-labeled goat anti-human 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.

[0051] (6) Color rendering

[0052] After washing the ELISA plate three times with PBST cleaning solution, add 100 μL OPD color development solution for color development reaction. The color development reaction time is 5 to 15 minutes, and the reaction is stopped according to the color development effect. When the reaction is terminated, add 100 μL 2M H 2 SO 4 Finally, use a microplate reader at wavelength OD 450 Detect the absorbance value. Figure 1 .

[0053] The analysis results show that both VD mAb1 and VD mAb2 antibodies can be paired with VD mAb to detect the small molecule 25-OH-VD, among which the non-specific binding signal of VD mAb2 and VD mAb is relatively strong.

[0054] Example 5: ELISA to detect the binding activity of 25-OH-VD antibody and 25-OH-VD BSA:

[0055] 25-OH-VD BSA (purchased from Nanjing Ruibaiyuan Biotechnology Co., Ltd.) protein plate, NaHCO 3 Buffer was diluted to 1 μg / mL, and 96-well ELISA plates were plated, 100 μL per well, and placed in a 4°C refrigerator overnight; TPBS (PBS + 0.1% Tween 20) was washed three times, 3% BSA was dissolved in TPBS as blocking solution, each well was filled (about 250 μL), and blocked at 37°C for 1 hour; TPBS was washed three times, and antibodies VD mAb, VD mAb1 and VD mAb2 were diluted with blocking solution, with a starting concentration of 1 μg / mL, and diluted continuously in a 3-fold gradient to form 8 different gradients, 100 μL / well, and incubated at room temperature for 1 hour; TPBS was washed three times, and rabbit anti-sheep HRP secondary antibody diluted 1:5000 with blocking solution was added to the wells corresponding to VD mAb, and VD mAb1 and VD The wells corresponding to mAb2 were added with 1:5000 diluted goat anti-human HRP secondary antibody, 100 μL per well, and incubated at room temperature with shaking for 30 min; washed three times with TPBS, 100 μL of OPD o-phenylenediamine (purchased from Sigma, catalog number 78412) substrate working solution containing 0.1% hydrogen peroxide was added to each well for color development, and 100 μL of 1 M sulfuric acid was added after about 1-20 min to terminate the reaction, and the OD450 absorbance was measured with an enzyme reader.

[0056] ELISA results are shown in Figure 2 From the results, it can be seen that VD mAb binds to 25-OH-VD BSA, while VD mAb1 and VDmAb2 do not bind to 25-OH-VD BSA, proving that the two paired antibodies bind to the composite epitope of "VD mAb and 25-OH-VD small molecule".

[0057] Example 6, Preparation of Streptavidin Magnetic Beads:

[0058] (1) Magnetic bead cleaning and activation

[0059] Weigh 100 mg of 1 μm magnetic beads (provided by Suzhou Weidu Biotechnology Co., Ltd., product 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 min to complete the activation of the magnetic beads.

[0060] (2) Coupling

[0061] 2 mg of streptavidin protein (supplier: ROCHE, catalog number 28903727103) was added to the activated magnetic beads, and incubated with stirring at room temperature for 3 h to complete the coupling.

[0062] (3) Closed treatment

[0063] After coupling, the magnetic beads were separated in a magnetic field, 5 mL of 5% BSA solution was added to resuspend the magnetic beads, and incubated at room temperature for 3 h to complete blocking.

[0064] (4) Magnetic bead cleaning and storage

[0065] 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.

[0066] Example 7, biotinylation of capture antibody (VD mAb biotin):

[0067] (1) Ultrafiltration concentration and liquid replacement

[0068] 1 mg of 25-OH-VD monoclonal antibody (Bioventix, Shenzhen Yawei Century Technology Co., Ltd., catalog number: BVX.vitD3.2F4), i.e., VD mAb, was concentrated using a 10 kDa ultrafiltration concentrator and replaced with PBS.

[0069] (2) Coupling

[0070] NHS-PEG12-biotin (Thermo Fisher, catalog number: 21312) was added to the antibody solution at a molar ratio of "antibody:biotin" of "1:10" and coupled at room temperature for 1 hour.

[0071] (3) Ultrafiltration concentration and liquid replacement

[0072] Use a 10kDa ultrafiltration concentrator to concentrate the biotinylated antibody, remove free biotin, and replace the solution with PBS. Adjust the antibody concentration to 0.5 mg / mL, add glycerol and store for later use, which is VD mAb biotin.

[0073] Example 8, Obtaining the Antibody-Acridinium Ester Component:

[0074] (1) Coupling

[0075] 10 mg of VD mAb1 was dissolved in 20 mM MES 6.5, and NSP-SA-NHS (Suzhou Yake, catalog number: Y0080) was added at a molar ratio of antibody: acridinium ester of 1:20, and incubated at room temperature for 3 h.

[0076] (2) Fluid exchange

[0077] Use a 3kDa ultrafiltration concentrator or a 3KDa dialysis bag to replace the antibody solution after coupling with acridinium ester to 20mM MES 6.5150mM 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, which is the chemiluminescent marker VD mAb1-acridinium ester.

[0078] Example 9, chemiluminescent detection reagent:

[0079] When the detection antigen is 25-OH-VD, the chemiluminescent detection reagent of this embodiment includes component M, component R1 and component R2.

[0080] Among them, the M component is the streptavidin magnetic beads prepared in Example 5, the working concentration of the magnetic beads is 0.75 mg / mL, the buffer solution is composed of 50 mM Tris-HCl PH7.4, 150 mM NaCl, 0.1% Tween 20, 0.5% BSA, and 0.1% proclin 300. According to the component content of the M component, the components are mixed in the same container and mixed evenly to obtain the product.

[0081] The R1 component is composed of 50mM Tris-HCl PH7.4, 150mM NaCl, 0.5% BSA, 1% trehalose, 0.1% proclin 300, and 2.3ug / mL of biotinylated antibody VD mAb biotin (Example 7). According to the component content of the R1 component, the components are mixed in the same container and mixed evenly to obtain the product.

[0082] The R2 component consists of 50 mM Tris-HCl PH7.4, 150 mM NaCl, 0.5% BSA, 1% trehalose, 0.1% proclin 300, and 0.2 ug / mL VD mAb1-acridinium ester (Example 8). According to the component content of the R2 component, the components are mixed in the same container and mixed evenly to obtain the product.

[0083] Test Example 1

[0084] This test example uses the components M, R1, and R2 in Example 9 to form a chemiluminescent detection kit for detecting 25-OH-VD, and tests its related performance.

[0085] 1. Chemiluminescence analyzer detection method:

[0086] Take the Feipeng Shine i1000 fully automatic chemiluminescence immunoassay as an example: first add 50uL R1 component, add 50uL sample, add 50ul M component, add 50ul R2 component, and incubate at 37℃ for 5min; remove the supernatant by magnetic separator, wash twice with cleaning solution, add substrate to react and detect the luminescence value.

[0087] 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.

[0088] 2. Linear range detection:

[0089] The 25-OH-VD small molecule was prepared into samples with concentrations of 2ng / mL, 18.75ng / mL, 37.5ng / mL, 75ng / mL, 150ng / mL and 300ng / mL using standard buffer (40mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0), and detected using a fully automatic chemiluminescence immunoassay analyzer. Each dilution concentration was measured separately, and each concentration was repeated 3 times to calculate the average value of each measurement. With 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 correlation coefficient r were calculated. The detection data are shown in Table 1. The results show that the linear range of the luminescence kit prepared using VD mAb1 for the detection of 25-OH-VD samples is 2ng / mL to 150ng / mL, and the regression equation corresponding to the first five concentrations is y=152965x-727363, and the correlation coefficient is R 2 =0.994.

[0090] Table 1. Test Example 1 Kit Linear Range Detection

[0091] Theoretical concentration (ng / mL) Detection value (RLU) 2 9026 18.75 1260346 37.5 4851881 75 11671928 150 21897570 300 26361472

[0092] 3. Clinical relevance testing:

[0093] Using 20 Roche electrochemiluminescence valued clinical samples, 5 μL of serum sample was mixed with 180 μL of dissociation agent (i.e., acidic pH buffer) and incubated for 5 minutes to separate vitamin D in the sample from its binding protein. The reagent in Example 9 was used to perform correlation detection on the clinical samples using a fully automatic chemiluminescence immunoassay. The content of 25-OH-VD in the sample was determined based on the RLU and the calibration curve established under the same analytical conditions, and the sample concentration was calculated based on the sample luminescence value. The test results are shown in Figure 3 .

[0094] In summary, the antibody pair consisting of VD mAb1 and VD mAb of the present invention can be directly applied to the chemiluminescence platform.

[0095] 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 25-OH-VD paired antibody, characterized in that: It comprises an anti-25-OH-VD monoclonal antibody; the anti-25-OH-VD monoclonal antibody is a nano antibody, and the amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The 25-OH-VD paired antibody according to claim 1, characterized in that: The anti-25-OH-VD monoclonal antibody specifically selects the amino acid sequence shown in SEQ ID NO.

1.

3. A nucleic acid molecule or expression vector, characterized in that: The encoding comprises the 25-OH-VD paired antibody molecule according to claim 1 or 2.

4. Use of the 25-OH-VD paired antibody as claimed in claim 1 or 2 as a detection antibody in the preparation of a chemiluminescence detection kit.

5. A chemiluminescent detection kit, characterized in that: It comprises the 25-OH-VD paired antibody according to claim 1 or 2.

6. The chemiluminescent detection kit according to claim 5, characterized in that: Also included is a 25-OH-VD / first antibody complex; the 25-OH-VD paired antibody can specifically bind to the 25-OH-VD / first antibody complex.

7. The chemiluminescent detection kit according to claim 6, characterized in that: The first antibody of the 25-OH-VD / first antibody complex and the 25-OH-VD paired antibody respectively bind to different epitopes of 25-OH-VD.

8. A chemiluminescent detection method for 25-OH-VD, characterized in that: The steps include: (1) contacting the sample with a first reagent comprising an acidic pH buffer to separate 25-OH-VD in the sample from its binding protein; (2) contacting the 25-OH-VD in the sample obtained in step (1) with a second reagent; the second reagent comprises magnetic particles coated with a first antibody, the first antibody having a specific binding affinity to the 25-OH-VD, thereby forming a 25-OH-VD / first antibody complex; (3) contacting the 25-OH-VD / first antibody complex with a third reagent; the third reagent comprises a second antibody labeled with a chemiluminescent detection signal generating molecule, i.e., the 25-OH-VD paired antibody according to claim 1 or 2; the second antibody has binding affinity with the 25-OH-VD / first antibody complex to form a complex between the 25-OH-VD, the first antibody, and the 25-OH-VD paired antibody labeled with a signal generating molecule; (4) After incubation and washing, the concentration of 25-OH-VD in the sample is determined based on the chemiluminescence signal intensity and the calibration curve.

9. The chemiluminescent detection method of 25-OH-VD according to claim 8, characterized in that: In the step (3), the chemiluminescent detection signal generating molecule is selected from at least one of acridinium ester, isoluminol, alkaline phosphatase, horseradish peroxidase, and fluorescein.

10. The chemiluminescent detection method of 25-OH-VD according to claim 9, characterized in that: The chemiluminescent detection signal generating molecule is specifically acridinium ester.

Images