An immunoassay kit and its preparation method and application

Abstract

The present invention discloses a kit for detecting the expression level of RBP4 protein, the kit is composed of an effective amount of RBP4 protein and two effective amounts of anti-RBP4 protein monoclonal antibodies and matching detection reagents, the two anti-RBP4 protein monoclonal antibodies are monoclonal antibody 1 and monoclonal antibody 2, the heavy chain variable region and light chain variable region sequences encoding monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2, and the heavy chain variable region and light chain variable region sequences encoding monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.4. The kit of the present invention has the advantages of high sensitivity, wide linear range and fast detection speed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an immunoassay kit and a preparation method and application thereof. Background Art

[0002] Retinol binding protein (RBP) is a small molecule protein secreted by the liver in plasma with a short half-life. It is a specific transporter of vitamin A in the blood and can sensitively reflect the disease status of the body. In the past, RBP was considered to be a sensitive indicator for early diagnosis and efficacy observation of kidney disease, but in recent years, more and more studies have found that serum RBP or urine RBP levels are closely related to human renal function, liver function, clinical nutritional status, epidemic hemorrhagic fever, tumor diagnosis, etc.

[0003] RBP is mainly synthesized in the liver, supplemented by serum, cerebrospinal fluid, urine and other body fluids. Seven different RBP subtypes have been discovered, mainly distributed in the blood circulation system (RBP4, referred to as RBP), cells (RBP1,

[0004] RBP2, RBP5, RBP6, RBP7, referred to as RBPs) and retinal interphotoreceptor (RBP3, referred to as IRBP).

[0005] At present, the detection methods of retinol binding protein include enzyme-linked immunosorbent assay (ELISE), immunoturbidimetry, radioimmunoassay (IRMA) and immunoelectrophoresis. Among them, immunoturbidimetry uses a fully automatic biochemical analyzer for detection, has no radioactive source, is more environmentally friendly, and has good stability, precision and accuracy. It is widely used in routine clinical testing. However, the concentration of RBP in body fluids is low, resulting in poor specificity and sensitivity, which is not convenient for widespread clinical application.

[0006] Monoclonal antibodies are produced by artificially prepared hybridoma cells, which are formed by the fusion of an antigen-activated B cell and a myeloma cell. Advantages of monoclonal antibodies: high purity, high sensitivity, strong specificity, few cross-reactions, and low preparation cost. Disadvantages: certain technical requirements, and the epitope is easily lost through chemical treatment of the antigen.

[0007] In 1975, molecular biologists GJF Koehler and C. Milstein created hybridoma technology based on natural hybridization technology. They fused mouse myeloma cells that can be cultured and proliferated in large quantities in vitro with pure mouse B cells after antigen immunization to form a hybrid cell line, which has the characteristics of tumor cells that are easy to proliferate indefinitely in vitro, and the characteristics of antibody-forming cells that synthesize and secrete specific antibodies. Cultivating this hybridoma as a single cell can form a single cell line, that is, a monoclonal antibody. By using the method of culture or intraperitoneal inoculation of mice, a large amount of high-concentration, very uniform antibodies can be obtained, and their structure, amino acid sequence, specificity, etc. are consistent. Moreover, during the culture process, as long as there is no mutation, the antibodies secreted at different times can maintain the same structure and function. This monoclonal antibody cannot be obtained by other methods. Summary of the invention

[0008] In order to make up for the deficiencies of the prior art, the object of the present invention is to provide a detection kit for human RBP and a preparation method thereof.

[0009] Therefore, on the one hand, the present invention discloses a kit for detecting the expression level of RBP4, wherein the kit is composed of an effective amount of RBP4 protein, two effective amounts of anti-RBP4 protein monoclonal antibodies and a matching detection reagent, wherein the two anti-RBP4 protein monoclonal antibodies are monoclonal antibody 1 and monoclonal antibody 2, the heavy chain variable region and light chain variable region sequences encoding monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2, and the heavy chain variable region and light chain variable region sequences encoding monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] Preferably, the antigen epitope sequence bound by the monoclonal antibody 1 described in the kit of the present invention is SFRVKENFDKARFSGTWYAMAKKDP.

[0011] Preferably, the antigen epitope sequence bound by the monoclonal antibody 2 described in the kit of the present invention is TDTEDPAKFKMKYWGVASFLQKGND.

[0012] Preferably, the monoclonal antibody 1 described in the kit of the present invention is coupled to magnetic particles, and the concentration of the magnetic particles coupled to the monoclonal antibody 1 is 0.2 mg / ml.

[0013] Preferably, the monoclonal antibody 2 described in the kit of the present invention is labeled with acridinium ester, and the concentration of the acridinium ester-labeled monoclonal antibody 2 is 20 ng / ml.

[0014] Preferably, the RBP4 protein described in the kit of the present invention is used to prepare a calibrator, and the calibrator consists of a serum calibrator and a urine calibrator, wherein:

[0015] The serum calibrator concentrations are: 140.0, 70.0, 35.0, 15.0, 5.0, 0.0 mg / L;

[0016] The concentrations of the urine calibrators are: 20.0, 10.0, 5.0, 2.5, 1.0, and 0.0 mg / L.

[0017] In another aspect, the present invention also discloses a use of the anti-RBP4 protein monoclonal antibody in the preparation of an RBP4 detection reagent.

[0018] The kit provided by the present invention has the advantages of simultaneous detection of blood and urine, simple operation, high sensitivity, wide linear range, low cost, and prospect of large-scale and automated application (automated detection can be realized by integration into existing chemiluminescence detection instruments).

[0019] In addition, the monoclonal antibody prepared by the present invention has good specificity and sensitivity, and is suitable for the detection of RBP4 protein, providing good support for the subsequent development of detection reagents for RBP4 protein.

[0020] Furthermore, the monoclonal antibody prepared by the present invention adopts the method of designing antigenic polypeptides and immunizing mice, which can directly determine the antigenic epitope recognized by the monoclonal antibody, and has the advantages of strong targeting and good specificity; the antigenic epitope polypeptide disclosed by the present invention also has an important reference role in the subsequent research on RBP4 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Analysis results of RBP4 protein signal peptide.

[0022] Figure 2 Linear range measurement diagram of serum samples of the kit of the present invention.

[0023] Figure 3 Linear range measurement diagram of urine samples in the kit of the present invention. DETAILED DESCRIPTION

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0025] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0026] Example 1: Preparation of retinol binding protein 4 (RBP4)

[0027] The human RBP4 protein (P02753 · RET4_HUMAN) was selected and its amino acid sequence was analyzed. First, the signal peptide was analyzed, and the results showed that ( Figure 1 ), the probability that the sequence has a signal peptide is 99.919%, signal peptide type: SP (Sec / SPI); cleavage site: 18-19, signal peptide sequence: MKWVWALLLLAALGSGRA.

[0028] Therefore, the amino acid sequence of the RBP4 recombinant protein was determined as follows (20 amino acids were removed from the N-terminus). The amino acid sequence of the RBP4 recombinant protein (180aa) is:

[0029] DCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRSERNL.

[0030] The amino acid sequence of the above-mentioned RBP4 recombinant protein was further analyzed to design an antigen epitope polypeptide for use in the preparation of RBP4 protein monoclonal antibodies. The sequence of the designed antigen epitope polypeptide is shown in Table 1:

[0031] Table 1 Epitope peptide sequences

[0032]

[0033] Example 2: Preparation and screening of monoclonal antibodies

[0034] The 4 polypeptides in Table 1 were synthesized by artificial synthesis, and the 4 polypeptides were emulsified with adjuvants to prepare vaccines (100 ng / ml), and mice were immunized. Monoclonal antibodies were prepared by conventional hybridoma cell technology. The specific methods are as follows:

[0035] 2.1 Immunization of animals Immunization of animals is the process of immunizing mice with target antigens to produce sensitized B lymphocytes. Generally, female BALB / c mice aged 6 to 8 weeks are selected and immunized according to the established immunization scheme (the four polypeptides are fully emulsified with equal weight of Freund's complete adjuvant and injected into the dorsal abdomen of mice at multiple points, 0.2 ml / mouse; after an interval of 2 weeks, take the same amount of antigen as the first immunization and the same weight of Freund's incomplete adjuvant, fully emulsify, and inject 0.2 ml / mouse into the abdomen for the second time. After 2 weeks, double the dose of antigen is used for intraperitoneal injection immunization). Antigens enter the peripheral immune organs through the blood circulation or lymphatic circulation, stimulate the corresponding B lymphocyte clones, activate and proliferate them, and differentiate them into sensitized B lymphocytes.

[0036] 2.2 Cell fusion The mice were killed by carbon dioxide gas, and the spleen was removed aseptically, squeezed and ground in a plate to prepare a spleen cell suspension. The prepared syngeneic myeloma cells were mixed with mouse spleen cells in a certain proportion, and the fusion promoter polyethylene glycol was added. Under the action of polyethylene glycol, various lymphocytes can fuse with myeloma cells to form hybridoma cells.

[0037] 2.3 Selective culture The purpose of selective culture is to screen fused hybridoma cells, and HAT selective culture medium is generally used. In HAT culture medium, the de novo DNA synthesis pathway in unfused mouse myeloma cells will be blocked; unfused myeloma cells cannot synthesize DNA using the salvage pathway due to the lack of hypoxanthine-guanine-phosphoribosyltransferase (HGPRT); thus, both DNA synthesis pathways of unfused mouse myeloma cells are blocked, and myeloma cell DNA cannot replicate and die. Although unfused B lymphocytes have hypoxanthine-guanine-phosphoribosyltransferase, they themselves cannot survive in vitro for a long time and gradually die. Only fused hybridoma cells can synthesize DNA through the salvage pathway because they have obtained hypoxanthine-guanine-phosphoribosyltransferase from B lymphocytes, and have the characteristics of myeloma cells that can proliferate indefinitely. Therefore, hybridoma cells can survive and proliferate in HAT culture medium.

[0038] 2.4 Screening and cloning of hybridoma positive clones Among the hybridoma cells grown in HAT medium, only a few are cells that secrete the predetermined specific monoclonal antibodies. Therefore, screening and cloning are necessary. The limiting dilution method is usually used for cloning and culturing of hybridoma cells. A sensitive, rapid and specific immunological method is used to screen out positive hybridoma cells that can produce the required monoclonal antibodies, and clone and expand them. After comprehensive identification of the immunoglobulin type, subclass, specificity, affinity, epitope of the antigen recognized and its molecular weight of the secreted monoclonal antibodies, they are frozen in time.

[0039] 2.5 Large-scale preparation of monoclonal antibodies Hybridoma cells are placed in culture bottles for culture. During the culture process, hybridoma cells produce and secrete monoclonal antibodies. The culture supernatant is collected and centrifuged to remove cells and their debris to obtain the required monoclonal antibodies. However, the amount of antibodies produced by this method is limited. Various new culture techniques and devices continue to emerge, greatly increasing the production of antibodies.

[0040] 2.6 After preparation and testing, one good hybridoma cell and monoclonal antibody were screened for each polypeptide, as shown in Table 2.

[0041] Table 2 Correspondence between hybridoma cells, monoclonal antibodies and antigen epitopes

[0042]

[0043] Example 3: Detection and pairing of monoclonal antibodies

[0044] The prepared monoclonal antibodies were screened and detected using ELISA. The specific method was briefly described as follows: RBP4 protein (purchased from Nanjing Okey Biotechnology Co., Ltd., K5916) was used to coat the ELISA plate (1 μg / ml), 0.1 ml per well, and coated overnight at 4 degrees; after washing 3 times, it was blocked with 5% skim milk, 0.2 ml per well, and coated at 37 degrees for 2 hours; after washing 3 times, 10-fold serial dilutions of monoclonal antibodies were added (all adjusted to a concentration of 1 mg / ml before dilution), 0.1 per well, and then the monoclonal antibodies were added to the plate. ml, 37 degrees for 1h; after washing 3 times, add HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution) to each well; incubate at 37 degrees for 0.5h; after washing 3 times, add color developing solution, 0.1ml per well, 37 degrees for 10min; add stop solution (2M sulfuric acid), 0.05ml per well, and detect OD450nm value; when P / N value (sample OD450nm value / blank control OD value) > 2.1, and OD450nm value > 0.1, it is judged as positive, otherwise it is negative.

[0045] The test results showed (as shown in Table 2) that monoclonal antibodies 1, 2 and 3 all had good titers (all ≥10 5 ), monoclonal antibody 4 was relatively low (titer was 10 4 ); therefore, monoclonal antibodies 1, 2 and 3 were selected as monoclonal antibodies for subsequent kit development, but the antigen epitope polypeptides recognized by monoclonal antibodies 2 and 3 were relatively close, and the potency of monoclonal antibody 3 was lower than that of monoclonal antibody 2, so monoclonal antibody 1 and monoclonal antibody 2 were selected as target monoclonal antibodies for kit development.

[0046] We analyzed the variable region sequences of monoclonal antibody 1 and monoclonal antibody 2, wherein the sequences encoding the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2; the sequences encoding the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0047] Example 4: Preparation and characterization of RBP4 protein detection kit

[0048] 4.1 Preparation of Calibrators

[0049] RBP4 protein (purchased from Nanjing Oukai Biotechnology Co., Ltd., K5916) was used as the stock solution, and diluted with diluent (TBST buffer containing 2% BSA) for preparation.

[0050] The concentrations of serum calibrators are: 140.0, 70.0, 35.0, 15.0, 5.0, and 0.0 mg / L.

[0051] The concentrations of urine calibrators are: 20.0, 10.0, 5.0, 2.5, 1.0, and 0.0 mg / L.

[0052] 4.2 The magnetic particles were coupled to the monoclonal antibody 1 in a conventional EDC-activated carboxyl manner. The specific process is briefly described as follows:

[0053] 1) Take 1 mg of carboxyl magnetic particles (purchased from ThermoFisher), wash them three times with 0.1 M MES buffer and resuspend them in 0.5 ml MES (pH 6.0) buffer.

[0054] 2) Add 10 μg of monoclonal antibody 1, mix well, and incubate at room temperature for 20 min.

[0055] 3) Add 10 μl EDC (10 mg / ml), mix well, and incubate at room temperature for 2 h.

[0056] 4) After centrifugation, discard the supernatant, add 500 μl of washing buffer (TBST), and wash three times.

[0057] 5) Block the cells with TBST buffer containing 1% BSA at room temperature for 2 h.

[0058] 6) After washing three times with TBST, resuspend in 0.5 ml of TBST buffer containing 1% BSA and store at 2-8°C until use.

[0059] 4.3 Acridinium ester labeling of monoclonal antibody 2 is a conventional labeling method, and the specific process is briefly described as follows:

[0060] 1) In the dark, mix 10 μg of monoclonal antibody 2 with 190 μl of acridinium ester solution (6.5 mM), react at room temperature for 1 h, add 0.1 ml of lysine (10 g / L), and continue the reaction for 10 min.

[0061] 2) Inject the labeled solution into a Sephadex G-25 desalting column and wash with PBS.

[0062] 3) Detect protein concentration at 280 nm. When a clear protein peak appears on the chromatograph, collect the effluent.

[0063] 4) The effluent (about 0.5 ml) was stored at -20℃ for later use.

[0064] 4.4 Kit Testing

[0065] 4.4.1 Preparation of test kit

[0066] Dilute the magnetic particles coupled to monoclonal antibody 1 in 4.2 to 0.2 mg / ml and label it as reagent R1;

[0067] Dilute the acridinium ester-labeled monoclonal antibody 2 in 4.3 by 1000 times (the concentration of monoclonal antibody 2 is about 20 ng / ml) and label it as reagent R2;

[0068] The diluent was 0.1% BSA TBS pH 7.4.

[0069] 4.4.2 Testing steps

[0070] 1) 10μl sample + 50μl reagent R1 + 50μl reagent R2 + 90μl wash buffer;

[0071] 2) Incubate at 37°C for 15 min (37°C 1000 rpm);

[0072] 3) Wash with wash buffer 3 times, 500 μl each time;

[0073] 4) Add 200 μl of substrate (acridinium ester colorimetric substrate purchased from Abbott or other manufacturers) and immediately detect the RLU value using a chemiluminescence instrument.

[0074] The wash buffer is TBST buffer, and the sample can be serum, urine, etc. When the sample is serum, the calibrator used is the serum calibrator; when the sample is urine, the calibrator used is the urine calibrator.

[0075] Example 5: Performance evaluation of the kit

[0076] 5.1 Evaluation of kit sensitivity

[0077] 5.1.1 Evaluation of sensitivity of serum samples

[0078] First, calibrate with serum calibrators, then use 0.9% NaCl as blank sample, repeat the test 20 times, take the average value of blank sample plus 3 times the standard deviation, which is the detection limit of this kit. After testing and calculation, the detection limit of this kit is 0.5mg / L.

[0079] 5.1.2 Urine sample sensitivity evaluation

[0080] First, calibrate with urine calibrant, then use 0.9% NaCl as blank sample, repeat the test 20 times, take the average value of blank sample plus 3 times the standard deviation, which is the detection limit of this kit. After testing and calculation, the detection limit of this kit is 0.05mg / L.

[0081] 5.2 Evaluation of the linear performance of the kit

[0082] 5.2.1 Serum linearity evaluation

[0083] The high-value RBP serum samples were diluted with 0.9% NaCl at a dilution ratio of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and 0. Each concentration was measured 3 times, and the mean was calculated. The measured values ​​of the samples were correlated with the dilution ratio, and the regression equation was obtained. The theoretical value of the sample was calculated by the regression equation. The results are shown in Figure 2 As can be seen from the figure, good linearity can be maintained within the range of sample concentration less than 150 mg / L, that is, R 2 Greater than 0.99.

[0084] 5.2.2 Urine linearity evaluation

[0085] The high-value URBP urine samples were diluted with 0.9% NaCl at dilution ratios of 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, and 0. Each concentration was measured three times, and the mean was calculated. The measured values ​​of the samples were correlated with the dilution ratios, and the regression equation was obtained. The theoretical value of the sample was calculated using the regression equation. The results are shown in Figure 3 As can be seen from the figure, good linearity can be maintained within the range of sample concentration less than 25 mg / L, that is, R 2 Greater than 0.99.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A kit for detecting the expression level of RBP4, characterized in that: The kit is composed of an effective amount of RBP4 protein, an effective amount of two anti-RBP4 protein monoclonal antibodies and a matching detection reagent. The two anti-RBP4 protein monoclonal antibodies are monoclonal antibody 1 and monoclonal antibody 2. The sequences encoding the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The sequences encoding the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

2. The kit according to claim 1, characterized in that The antigen epitope sequence recognized by the monoclonal antibody 1 is SFRVKENFDKARFSGTWYAMAKKDP.

3. The kit according to claim 1, characterized in that The antigen epitope sequence recognized by the monoclonal antibody 2 is TDTEDPAKFKMKYWGVASFLQKGND.

4. The kit according to claim 1, characterized in that The monoclonal antibody 1 is coupled to magnetic particles, and the concentration of the magnetic particles coupled to the monoclonal antibody 1 is 0.2 mg / ml.

5. The kit according to claim 1, characterized in that The monoclonal antibody 2 is labeled with acridinium ester, and the concentration of the monoclonal antibody 2 labeled with acridinium ester is 20 ng / ml.

6. The kit according to claim 1, characterized in that The RBP4 protein is used to prepare a calibrator, and the calibrator consists of a serum calibrator and a urine calibrator, wherein: The serum calibrator concentrations are: 140.0, 70.0, 35.0, 15.0, 5.0, 0.0 mg / L; The concentrations of the urine calibrators are: 20.0, 10.0, 5.0, 2.5, 1.0, and 0.0 mg / L.

7. Use of two anti-RBP4 protein monoclonal antibodies in the preparation of RBP4 detection reagents, characterized in that: The two anti-RBP4 protein monoclonal antibodies are monoclonal antibody 1 and monoclonal antibody 2. The sequences encoding the heavy chain variable region and the light chain variable region of monoclonal antibody 1 are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The sequences encoding the heavy chain variable region and the light chain variable region of monoclonal antibody 2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

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