Hybrid creatine kinase isoenzyme double-antibody sandwich ELISA kit and preparation method thereof

Abstract

The invention provides a hybrid creatine kinase isoenzyme double-antibody sandwich ELISA kit and a preparation method thereof, and belongs to the technical field of CK-MB antibody and creatine kinase isoenzyme quantitative detection. The process comprises the following steps: respectively immunizing Japanese big-ear rabbits and mice by using CK-MB heterodimer protein as an immunogen, screening positive monoclonal cells by adopting a Beacon single-cell screening platform, and preparing a rabbit (mouse) anti-human CK-MB gene engineering antibody; the double-antibody sandwich ELISA detection kit is established by further screening optimal reaction conditions. The detection method disclosed by the invention has excellent performance in the aspects of intra-batch and inter-batch repeatability, sensitivity and specificity, and is suitable for CK-MB detection in clinical and scientific research.

Description

Technical Field

[0001] The invention relates to the technical field of CK-MB antibody and creatine kinase isoenzyme quantitative detection, and in particular to a hybrid creatine kinase isoenzyme double antibody sandwich ELISA kit and a preparation method thereof. Background Art

[0002] Hybrid creatine kinase isoenzyme (CK-MB) is mainly present in normal myocardial tissue. When myocardial cells are damaged, the CK-MB contained in them will be released into the blood, causing the blood content to rise sharply in a short period of time. Therefore, CK-MB can be used for early auxiliary diagnosis of acute myocardial infarction (AMI), and is also of great significance in judging the effect of thrombolysis and estimating the area of ​​myocardial infarction.

[0003] AMI develops rapidly, has a low cure rate, many complications, poor prognosis and high costs. Therefore, establishing an efficient and rapid detection method is a key measure for timely detection and early treatment of AMI. At present, electrocardiogram (ECG), coronary angiography (CAG), computed tomography (CT), magnetic resonance imaging (MRI) and other methods are commonly used in clinical diagnosis of AMI. However, due to the low accuracy of ECG, the low sensitivity of CAG for plate stability diagnosis, and the long time required for MRI, misdiagnosis, missed diagnosis and delayed diagnosis of AMI often occur in clinical practice. Therefore, myocardial injury markers such as CK-MB, lactate dehydrogenase, and troponin can be used to assist in the diagnosis of patients with no obvious clinical symptoms and severe conditions, which can achieve rapid diagnosis of AMI and effectively improve the detection rate.

[0004] In recent years, clinical detection of CK-MB in patient serum is mainly in the form of activity detection and quality detection. Chemiluminescence method can detect the serum content of CK-MB. This method is based on the principle of antigen-antibody specific binding, effectively avoiding the influence of substances such as CK-BB on the test results, and has the advantages of high specificity and high accuracy.

[0005] ELISA technology uses the property of organic materials (such as polystyrene) to effectively adsorb proteins and other substances. Microplates made of organic materials are used as solid phase carriers, proteins (antigens or antibodies) are coated in them, and each reaction substance is added in turn. The unbound components must be washed off each time, and finally the color development and result determination are performed through the interaction between the color developing solution and peroxidase (Engvall et al., 1971). Traditional ELISA test kits usually rely on single-species antibodies and are easily interfered by cross-reactions, resulting in reduced sensitivity and specificity of the test. In addition, the variability between batches is large and cannot meet strict clinical testing standards. The double antibody sandwich ELISA method uses two different species of antibodies to pair, so the detection accuracy is higher.

[0006] At present, most CK-MB quality detection kits are imported from abroad, with high detection costs, and the diagnostic raw materials in the kits are usually traditional monoclonal antibodies derived from hybridomas. Traditional monoclonal antibodies are usually cultured in vitro or in vivo on hybridoma cells to secrete antibodies, but during the culture of hybridoma clones, antibody genes may be lost or mutated, resulting in a weakened ability of hybridoma cell lines to produce antibodies and a decrease in antibody titer. The emergence of genetically engineered antibodies can significantly shorten the antibody preparation cycle and improve the specificity and affinity of antibodies. Therefore, the preparation of high-specificity diagnostic antibodies for CK-MB and their application in the development of quality detection kits has important clinical significance and can also generate social and economic benefits. Summary of the invention

[0007] In view of the content of the background technology, the purpose of the present invention is to provide a hybrid creatine kinase isozyme double antibody sandwich ELISA kit and its preparation method. The present invention provides a specific mouse and rabbit anti-human CK-MB genetic engineering antibody, which increases the specificity and affinity, and the prepared double antibody sandwich ELISA detection kit of different species has higher sensitivity, specificity and stability, which can more effectively solve the shortcomings of the prior art, especially in terms of antibody selection and detection condition optimization, and achieve higher detection accuracy.

[0008] To achieve the above purpose, the present invention specifically adopts the following technical solutions: The present invention provides a hybrid creatine kinase isozyme double antibody sandwich ELISA kit, comprising: a capture antibody, a biotin-labeled detection antibody, a CKMB protein, an antibody diluent, a washing solution, a blocking solution, a color developing solution and a stop solution; Among them, the capture antibody is the mouse anti-human CK-MB genetically engineered monoclonal antibody CKMB-mouse-F1R2, and its heterodimer CK-MB amino acid sequences CKM and CKB are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the biotin-labeled detection antibody is the biotin-labeled rabbit anti-human CK-MB genetically engineered monoclonal antibody CKMB-rabbit-G1R3.

[0009] SEQ ID NO.1: PFGNTHNKFKLNYKPEEEYPDLSKHNNHMAKVLTLELYKKLRDKETPSGFTVDDVIQTGVDNPGHPFIMTVGCVAGDEESYEVFKELFDPIISDRHGGYKPTDKHKTDLNHENLKGGDDLDPNYVLSSRVRTGRSIKGYTLPPHCSRGERRAVEKLSVEALNSLTGEFKGKYYPLKSMTEKEQQQLIDDH FLFDKPVSPLLLASGMARDWPDARGIWHNDNKSFLVWVNEEDHLRVISMEKGGNMKEVFRRFCVGLQKIEEIFKKAGHPFMWNQHLGYVLTCPSNLGTGLRGGVHVKLAHLSKHPKFEEILTRLRLQKRGTGGVDTAAVGSVFDVSNADRLGSSEVEQVQLVVDGVKLMVEMEKKLEKGQSIDDMIPAQK SEQ ID NO.2: PFSNSHNALKLRFPAEDEFPDLSAHNNHMAKVLTPELYAELRAKSTPSGFTLDDVIQTGVDNPGHPYIMTVGCVAGDEESYEVFKDLFDPIIEDRHGGYKPSDEHKTDLNPDNLQGGDDLDPNYVLSSRVRTGRSIRGFCLPPHCSRGERRAIEKLAVEALSSLDGDLAGRYYALKSMTEAEQQQLIDDH FLFDKPVSPLLLASGMARDWPDARGIWHNDNKTFLVWVNEEDHLRVISMQKGGNMKEVFTRFCTGLTQIETLFKSKDYEFMWNPHLGYILTCPSNLGTGLRAGVHIKLPNLGKHEKFSEVLKRLRLQKRGTGGVDTAAVGGVFDVSNADRLGFSEVELVQMVVDGVKLLIEMEQRLEQGQAIDDLMPAQK Furthermore, the antibody diluent is PBS buffer; the washing solution is PBST; the blocking solution is BSA; the color developing solution is TMB; and the stop solution is HCl.

[0010] The present invention also provides a method for preparing the hybrid creatine kinase isozyme double antibody sandwich ELISA kit, comprising the following steps: S1, dilute the capture antibody CKMB-mouse-F1R2 with CB and coat it; S2: Wash the plate with washing solution, add blocking solution to block the plate, and pat dry; S3, add sample diluent, incubate, wash the plate with washing solution, and spin dry; S4, adding diluted biotin-labeled detection antibody for incubation, washing the plate with washing solution, and drying; S5, add streptavidin-HRP diluent, incubate, wash the plate with washing solution, and spin dry; S6, add color developing solution, and perform color development reaction in the dark; S7, add stop solution to terminate the reaction, OD 450 reading; S8. Use an ELISA reader to measure the known concentration of hybrid creatine kinase isoenzyme and obtain the corresponding OD 450 The linear standard curve was obtained by fitting the logarithmic value of CK-MB concentration.

[0011] Furthermore, the coating parameters of S1 are 4° C., 12-20 h; and the final concentration of the capture antibody after dilution is 1 μg / mL.

[0012] Furthermore, the final concentration of the sample diluent in S3 is 1 μg / mL, and the incubation is performed at 37° C. for 1 h.

[0013] Furthermore, the biotin-labeled detection antibody in S4 is diluted 1:4000, and the incubation is carried out at 37° C. for 1 hour.

[0014] Further, streptavidin-HRP was diluted 2000 times as the working concentration, the streptavidin-HRP diluent in S5 was 1% BSA in 0.01 M PBS (pH 7.4), and the incubation was carried out at 37° C. for 30 min.

[0015] The beneficial effects of the present invention are: 1. The genetically engineered antibody prepared by the present invention has specific recognition ability for the natural protein CK-MB, laying a foundation for further developing a high-specificity and high-sensitivity hybrid creatine kinase isozyme CK-MB quality detection kit.

[0016] 2. The kit prepared by the present invention has the advantages of high sensitivity, wide detection range, good repeatability, high specificity, good stability, etc., which can reduce the detection cost and generally help clinical practice to carry out efficient and accurate quantitative detection.

[0017] 3. The present invention lays a theoretical foundation for the development of colloidal gold immunochromatographic test strips and chemiluminescence technology.

[0018] 4. The CK-MB double antibody sandwich ELISA rapid detection kit and detection system of the present invention are simple to operate, directly add serum samples, and have a short detection time, which is suitable for popularization and use. It overcomes the shortcomings of existing detection methods that are expensive, time-consuming, and require professional operators, and can independently process data for quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the effect of eukaryotic expression in Example 1 of the present invention.

[0020] Figure 2 This is a graph showing the titer of the genetically engineered antibody in Example 2 of the present invention.

[0021] Figure 3 This is a graph showing the affinity constant determination of the genetically engineered antibody in Example 2 of the present invention.

[0022] Figure 4 It is a schematic diagram of the sandwich ELISA pairing principle in Example 3 of the present invention.

[0023] Figure 5 It is a standard curve diagram of the kit in Example 4 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used 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.

[0026] Example 1: Preparation of paired monoclonal antibodies The CKMB mouse monoclonal antibody included in the kit was obtained by our laboratory through Beacon platform screening and recombinant expression. The preparation method of CKMB rabbit monoclonal antibody is as follows: 1. Preparation of CK-MB immunogen: Take the No. 9 clone of CK-MB provided by our laboratory and culture it in 500 mL. Induce it to express a large amount of target protein CK-MB at 37℃ with 0.5 mmol / L IPTG for 4 h. Release the protein from the bacteria by ultrasonic disruption and add it to the nickel affinity chromatography column. The nickel ions can bind to the His tag on the protein, thereby adsorbing the target protein CK-MB. The supernatant, flow-through, washing liquid and nickel column were sampled for polyacrylamide gel electrophoresis. The SDS-Page results showed that there were double bands of 40 kD~55 kD of different thicknesses in the lane of the nickel column electrophoresis. The protein provided by our laboratory uses the co-expression vector pETDuet-1 to achieve the expression of heterodimeric proteins. The hybrid creatine kinase isozyme CK-MB prepared in this way is similar to the natural protein in structure. As an immunogen, it helps to obtain highly specific antibodies that recognize natural proteins.

[0027] 2. Animal immunization: Japanese big-eared rabbits were used as immunized animals. In order to produce plasma cells that can secrete specific antibodies against CK-MB in the rabbits, the rabbits were immunized by subcutaneous injection. The prokaryotic expression protein CK-MB obtained in step 1 was used as the antigen, and Freund's adjuvant was used as the immune adjuvant. The power of the tissue grinder was set to 60 Hz, and the working time was 15 minutes. The solution in the tube was emulsified to a milky white opaque state. The rabbits were immunized with the emulsified protein. 300 μg, 1 mL per rabbit for the first immunization; 200 μg, 1 mL per rabbit for the second to fourth immunizations; the interval between each immunization was two weeks. Blood was collected from the ear vein after the fourth immunization. The titer test could reach 1:243K. After the titer test was qualified, blood was collected from the carotid artery for subsequent antibody preparation.

[0028] 3. Isolation of rabbit memory B cells: PBMCs were isolated from the whole blood of immunized rabbits, and rabbit memory B cells were sorted out by magnetic bead sorting technology. After in vitro stimulation and culture, single effector B cells that secreted specific antibodies against CK-MB were screened out using the Beacon platform.

[0029] 4. Antibody preparation: To obtain the gene sequence of a single plasma cell, the RNA in a single plasma cell was extracted by magnetic bead method and reverse transcribed into cDNA. To obtain the gene sequence of the variable region of the antibody light and heavy chains, the gene sequence of the variable region of the antibody light and heavy chains was amplified by polymerase chain reaction using degenerate primers and recovered. After agarose gel electrophoresis, the amplified DNA band was located between 300 bp and 500 bp, which was consistent with the size of the variable region of the antibody light and heavy chains. The variable regions of the antibody light and heavy chains were constructed with T vectors respectively. After overnight culture, several blue and white single colonies grew on the plate. The white single colony was picked for expansion culture and the heavy chain clone was named 718S-VH-X1Tn (X represents the well number, n represents the clone number), and the light chain clone was named 718S-VK-X1Tn (X represents the well number, n represents the clone number). Subsequently, the variable region of the antibody was constructed on the PCDNA3.4 vector to realize the vector construction of the rabbit anti-human CK-MB full-length antibody. Then, eukaryotic cells were transfected and expressed to obtain large amounts of stable genetically engineered antibodies. The eukaryotic expression effect diagram is shown in the figure below. Figure 1 As shown, the prepared recombinant genetic engineering antibody has good expression effect and high antibody purity and can be used for subsequent applications.

[0030] Example 2: Characterization of genetically engineered antibodies The antibody characterization is introduced using the two genetically engineered antibodies CKMB-rabbit-G1R3 and CKMB-rabbit-G1R5, which are the preferred detection antibodies for subsequent kit pairing.

[0031] 1. Determination of the titer of genetically engineered antibodies: The ELISA method was used to determine the antibody titer. CKMB-rabbit-G1R3 and CKMB-rabbit-G1R5 were diluted to 1 µg / mL with 1×PBS, and then the antibodies were diluted in a 3-fold gradient. The diluted antibodies were added to the ELISA plate coated with 2µg / mL immunogen CK-MB, and two parallel control groups were set up. After the color development was completed, the reading was performed at a wavelength of 450 nm. The titer results are detailed in Figure 2 .

[0032] 2. Determination of affinity constant of genetically engineered antibodies: Use 0.5 μg / mL and 0.1 μg / mL of immunogen CK-MB to coat the ELISA plate, dilute the genetically engineered antibodies by 3 times and add them to the ELISA plate for incubation, set up 2 sets of parallel controls, and read the absorbance at 450 nm after color development. Draw an affinity curve and calculate the antibody concentration at ODmax / 2. Calculate the affinity constant of the genetically engineered antibody according to formula 1.

[0033] Formula 1 Ka = (n-1) / 2(n[Ab]1-[Ab]2) In formula 1, n is the ratio of the two groups of immunogen coating concentrations (n>1), [Ab]1 corresponds to the antibody concentration at the low coating concentration at ODmax / 2, and [Ab]2 corresponds to the antibody concentration at the high coating concentration at ODmax / 2. Figure 3 As shown; the binding constant of CKMB-rabbit-G1R3 can reach 3.57×10 9 The binding constant of CKMB-rabbit-G1R5 can reach 3.79×10 9 , so both are >10 7 High affinity antibodies with high affinity are used for subsequent testing.

[0034] Comparison of antibody titer and affinity results showed that both genetically engineered antibodies could efficiently recognize CKMB protein and could be used for the development of sandwich ELISA methods.

[0035] Example 3: Pairing of Sandwich ELISA The CK-MB antigen was coated onto the ELISA plate, and the prepared mouse / rabbit anti-human genetic engineering antibodies were paired for sandwich ELISA. The principle of sandwich ELISA experiment is shown in Figure 4 OD was measured by microplate reader 450 Read the samples, select the positive wells, and record the corresponding antibody pairs. The pairing results are shown in Table 1.

[0036] Table 1

[0037] It can be seen from the pairing results in Table 1 that in the forward pairing, the capture antibody CKMB-mouse-F1R2 was paired with the detection antibodies CKMB-rabbit-G1R3, CKMB-rabbit-G1R5, and CKMB-rabbit-B1R1. At the same time, the P / N value of CKMB-mouse-F1R2 paired with the detection antibody CKMB-rabbit-G1R3 was the highest, and the pairing effect was the best. Finally, CKMB-mouse-F1R2 and CKMB-rabbit-G1R3 were selected for the preparation of the kit.

[0038] Example 4: Preparation method of double antibody sandwich ELISA kit for hybrid creatine kinase isozyme CK-MB 1. Biotin-labeled detection antibody The CKMB-rabbit-G1R3 antibody was dialyzed against 0.1 M sodium bicarbonate, pH = 8, to remove glycerol to avoid affecting the labeling reaction. The antibody concentration after dialysis was determined by BCA reagent at 1.6 mg / mL. The antibody was coupled with Thermo Fisher NHS-LC-Biotin at a molar ratio of 20:1 and placed in an oscillator at 800 rpm and 25°C for 1 h. Biotin-Ab was dialyzed against 0.01 M, pH = 7.4 PBS (2 days) to remove excess biotin and prevent competition reaction of biotin in ELISA. The antibody was stored at 4°C for future use.

[0039] 2. Kit detection reaction procedure 1) Coating: Capture antibody mouse monoclonal antibody CKMB-mouse-F1R2 was coated (diluted with 1× CB buffer, final concentration 1.0 μg / mL, 100 μL / well) at 4°C overnight (12-20 h). To prevent liquid evaporation or contamination, cover with sealing film.

[0040] 2) Blocking: Wash the coated wells twice with PBST, and block with 2% BSA (prepared with 1× PBS, 200 μL / well) at 37°C for 2 h. Discard the liquid in the wells and pat dry.

[0041] 3) Standard sample incubation: Set up sample wells and blank control wells separately. Add 100 μL of 1× PBS buffer to the blank wells, and add sample diluent (diluted with 1× PBS buffer, final concentration 1 μg / mL, 100 μL / well) to the sample wells. Incubate at 37℃ for 1 h, then wash the plate 3 times with 1× PBST, 200 μL each time, shake gently for 2 min, then discard the liquid in the wells and spin dry.

[0042] 4) Detection antibody incubation: The detection antibody CKMB-rabbit-G1R3-Biotin was diluted 1:4000 (1×PBS dilution, 100 μL / well), incubated at 37°C for 1 h, and then washed three times with 1×PBST, 200 μL each time, gently shaken for 2 min, and then the liquid in the wells was discarded and dried.

[0043] 5) Streptavidin incubation: Add streptavidin-HRP, diluted 2000 times with 1% BSA, 100 μL / well, incubate at 37°C for 30 min, then wash the plate 5 times with 1× PBST, 200 μL each time, gently shake for 2 min, discard the liquid in the wells, and spin dry.

[0044] 6) Color development: Add 100 μL of TMB and incubate at 37°C in a dark incubator for 10 min.

[0045] 7) Stop reading: Add 50 μL / well 1 M HCl to stop the reaction, OD 450 nm readings.

[0046] 8) Result judgment: Establishment of standard equation: Use microplate reader to measure the known concentration of hybrid creatine kinase isoenzyme CK-MB to obtain the corresponding OD 450 -CK-MB concentration logarithm, fitted to obtain the standard curve, detailed results see Figure 5 .

[0047] According to the standard curve, the detection limit (sensitivity) of the CKMB sandwich ELISA kit was calculated to be 15.50 ng / mL, and the linear range was 31.25-1000 ng / mL. At the same time, the SA-HRP amplification system used was 2 times more sensitive than the secondary antibody system, further improving the analytical sensitivity of the kit.

[0048] in conclusion: In summary, the preparation of a hybrid creatine kinase isozyme rabbit genetic engineering antibody of the present invention provides a new idea for the development of raw materials for sandwich ELISA kits. The preparation method of CKMB monoclonal antibodies of different species from Beacon sources is efficient, and large-scale preparation in vitro is carried out through recombinant expression, which reduces endogenous interference from animals. Further development of double-antibody sandwich ELISA paired detection is carried out, which has higher sensitivity and simple operation. At the same time, the discovery of paired antibodies also lays a good pairing foundation for the method development of chemiluminescence, immunofluorescence and other platforms.

[0049] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A hybrid creatine kinase isozyme double antibody sandwich ELISA kit, characterized in that: include: Capture antibody, biotin-labeled detection antibody, CKMB protein, antibody diluent, washing solution, blocking solution, color development solution and stop solution; Among them, the capture antibody is the mouse anti-human CK-MB genetically engineered monoclonal antibody CKMB-mouse-F1R2, and its heterodimer CK-MB amino acid sequences CKM and CKB are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively; the biotin-labeled detection antibody is the biotin-labeled rabbit anti-human CK-MB genetically engineered monoclonal antibody CKMB-rabbit-G1R3.

2. The hybrid creatine kinase isozyme double antibody sandwich ELISA kit according to claim 1, characterized in that: The antibody diluent is PBS buffer; the washing solution is PBST; the blocking solution is BSA; the color developing solution is TMB; and the stop solution is HCl.

3. The method for preparing the hybrid creatine kinase isozyme double antibody sandwich ELISA kit according to claim 1 or 2, characterized in that: The steps include: S1, dilute the capture antibody CKMB-mouse-F1R2 with CB and coat it; S2: Wash the plate with washing solution, add blocking solution to block the plate, and pat dry; S3, add sample diluent, incubate, wash the plate with washing solution, and spin dry; S4, adding diluted biotin-labeled detection antibody for incubation, washing the plate with washing solution, and drying; S5, add streptavidin-HRP diluent, incubate, wash the plate with washing solution, and spin dry; S6, add color developing solution, and develop color reaction in the dark; S7, add stop solution to terminate the reaction, OD 450 reading; S8. Use an ELISA reader to measure the known concentration of hybrid creatine kinase isoenzyme and obtain the corresponding OD 450 The linear standard curve was obtained by fitting the logarithmic value of CK-MB concentration.

4. The method for preparing the hybrid creatine kinase isozyme double antibody sandwich ELISA kit according to claim 3, characterized in that: The coating parameters described in S1 are 4°C and 12-20 h; the final concentration of the capture antibody after dilution is 1 μg / mL.

5. The method for preparing the hybrid creatine kinase isozyme double antibody sandwich ELISA kit according to claim 3, characterized in that: The final concentration of the sample diluent in S3 is 1 μg / mL, and the incubation is carried out at 37° C. for 1 h.

6. The method for preparing the hybrid creatine kinase isozyme double antibody sandwich ELISA kit according to claim 3, characterized in that: The biotin-labeled detection antibody in S4 was diluted 1:4000, and the incubation was carried out at 37° C. for 1 h.

7. The method for preparing the hybrid creatine kinase isozyme double antibody sandwich ELISA kit according to claim 3, characterized in that: Streptavidin-HRP was diluted 2000 times as the working concentration. The streptavidin-HRP diluent in S5 was 0.01 M PBS with 1% BSA. The incubation was carried out at 37° C. for 30 min.

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