ELISA kit for detecting nuclease and use method thereof

Abstract

The invention provides an ELISA kit for detecting nuclease and a use method thereof, and relates to the technical field of biology. The kit comprises a detection plate coated with a rabbit nuclease polyclonal antibody (rabbit polyclonal antibody), a nuclease standard substance, a biotin-labeled mouse nuclease monoclonal antibody (mouse monoclonal antibody), streptavidin with an HRP (horse radish peroxidase) label, a sample diluent, a scrubbing solution, a confining solution, a developing solution and a stop solution. The use method of the kit comprises the following steps: respectively adding a sample to be detected and a nuclease standard substance into the detection plate, incubating, and washing the plate; adding the murine monoclonal antibody, incubating, and washing the plate; adding the streptavidin, incubating, and washing the plate; sequentially adding a developing solution and a stop solution, and reading an OD450 value; and calculating the nuclease concentration in the to-be-detected sample through the nuclease standard curve. The kit provided by the invention is strong in specificity, high in accuracy and sensitivity and good in stability; the application range is wide, and nuclease of different manufacturers can be detected.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an ELISA kit for detecting nuclease and a method for using the same. Technical Background

[0002] Nuclease, also known as Universal Nuclease (UN), is a genetically engineered enzyme derived from Serratia Marcescen bacteria. It can degrade nucleic acids (DNA and RNA) in all forms (single-stranded, double-stranded, linear, circular, natural or denatured), digesting them into 5-monophosphate oligonucleotides with a length of 3-8 bases, and the enzyme does not have base recognition specificity. In addition, the digestion function of nuclease is less affected by external conditions, that is, it can maintain high stability and digestion activity under the conditions of 6M urea, 0.1M Guanidine HCl, 0.4% Triton X-100, 0.1% SDS, 1mM EDTA, and 1mM PMSF. It is very suitable as an enzyme preparation for various scientific research and the pharmaceutical industries of vaccines, proteins, and polysaccharides to remove nucleic acid residues in samples or products and improve the purity of samples and the efficacy of biological products.

[0003] At present, nuclease is increasingly widely used in the field of biological products. After using Nuclease to remove nucleic acids during the production process of biological products, it is also necessary to remove Nuclease and detect the removal effect to ensure that the product meets the production standard requirements. Since the detection specificity and accuracy of the kit prepared with polyclonal antibodies against nuclease in China are not high enough, major domestic biological product manufacturers mainly use the Universal Nuclease ELISA detection kit of Merck. The detection range of this kit is 1-10 ng / mL, and the detection sensitivity is 24 pg / mL. At the same time, with the rise of domestic vaccines, cells, and gene therapies, the demand for Nuclease residue detection kits has increased rapidly, resulting in a shortage of supply and high prices. More importantly, the imported Universal Nuclease residue detection kit is subject to purchase restrictions in China, which affects the development of China's biological product industry. In summary, there is an urgent need for a new detection method with high accuracy and stability for detecting residual nuclease. Summary of the Invention

[0004] In view of the above problems, the present invention provides an ELISA kit for detecting nuclease and its use method. The kit includes: a detection plate coated with a rabbit-derived polyclonal antibody against nuclease (rabbit-derived polyclonal antibody), a standard product of nuclease, a mouse-derived monoclonal antibody against nuclease labeled with biotin (mouse-derived monoclonal antibody), streptavidin labeled with HRP, a sample diluent, a washing solution, a blocking solution, a chromogenic solution, and a termination solution. The steps of using the kit are as follows: add the sample to be detected and the nuclease standard product to the detection plate respectively, incubate, and wash the plate; add the mouse-derived monoclonal antibody, incubate, and wash the plate; add the streptavidin, incubate, and wash the plate; add the TMB chromogenic solution for color development, add the termination solution to terminate the reaction, and use an enzyme-linked immunosorbent assay reader to read the OD 450 value; calculate the concentration of nuclease in the sample to be detected through the nuclease standard curve. The kit provided by the present invention has strong specificity, high accuracy, and good stability; high sensitivity, with a detection lower limit of 12 pg / mL and a quantitative detection range of 0.047 - 3 ng / mL; wide application range, and can detect nucleases from different manufacturers.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] On the one hand, the present invention provides an ELISA kit for detecting nuclease. The kit includes a detection plate coated with a capture antibody and a detection antibody. The detection antibody is prepared by immunizing an animal with inactivated nuclease. The inactivated nuclease (SEQ ID NO: 2) refers to a nuclease that does not have nuclease activity and cannot degrade nucleic acids, and is obtained by point mutation on the basis of the sequence of active nuclease (SEQ ID NO: 1).

[0007] In order to maximize the comprehensive performance of the kit, the present invention has optimized the components of the kit, their related parameters, and the implementation methods of the kit in all aspects.

[0008] Further, the detection antibody is a mouse-derived monoclonal antibody against targeted nuclease labeled with biotin, and its subtype is any one of IgG1, IgG2a, and IgG3, preferably IgG3.

[0009] It should be understood that using the IgG3 antibody subtype to detect antigens in a double antibody sandwich ELISA has the following advantages:

[0010] 1. High specificity: The IgG3 antibody subtype has a unique antigen-binding site and can bind to the target antigen with high specificity. This specific binding ability enables the IgG3 antibody to effectively avoid cross-reactions in the double antibody sandwich ELISA experiment and ensure the accuracy of the detection results;

[0011] 2. IgG3 subtype antibodies have high sensitivity, that is, they can bind to antigens and be detected even at low concentrations. This high-sensitivity characteristic makes IgG3 perform excellently in detecting low-abundance antigens and be able to detect antigens that other subtypes may not be able to detect;

[0012] 3. Suitable for complex samples, the IgG3 antibody subtype does not require sample purification in the double-antibody sandwich ELISA. Due to its high specificity, it can accurately detect the target antigen even in complex biological samples (such as serum, cell culture supernatant, etc.);

[0013] 4. Good stability and repeatability. The IgG3 antibody subtype shows good stability and repeatability in the double-antibody sandwich ELISA, ensuring the consistency and reliability of experimental results.

[0014] In summary, the IgG3 antibody subtype has advantages such as high specificity, high sensitivity, suitability for complex samples, applicability to various application scenarios, and good stability and repeatability when detecting antigens in the double-antibody sandwich ELISA. These characteristics make it an ideal choice for detecting antigens.

[0015] Furthermore, the capture antibody is a rabbit polyclonal antibody targeting nuclease.

[0016] Furthermore, the concentration of the detection antibody is 0.2 - 1.0 μg / mL, preferably 0.3 μg / mL; the concentration of the capture antibody is 1.0 - 5.0 μg / mL, preferably 2 μg / mL.

[0017] Furthermore, the buffer for coating the capture antibody is CBS or PBS, preferably CBS; after coating the capture antibody, the detection plate needs to be blocked; the blocking solution contains any one or more of 1% BSA and 5% skim milk powder, preferably 1%. Specifically, the blocking solution is a PBS buffer containing 0.05% Tween-20, 0.05% Proclin300, and 1% BSA.

[0018] Furthermore, the temperature of coating / blocking is 4°C - 37°C, preferably, the coating / blocking temperature is 4°C; the duration of coating / blocking is 0.5 - 20 h, preferably, coating / blocking for 20 h, that is, overnight.

[0019] Furthermore, the kit also includes a standard product of nuclease, streptavidin labeled with HRP, sample diluent, washing solution, chromogenic solution, and termination solution.

[0020] On the other hand, the present invention provides a method for detecting the concentration of nuclease, using the kit described above to detect the concentration of nuclease.

[0021] Furthermore, the method comprises the following steps:

[0022] (1) Add the sample to be tested and the nuclease standard to the detection plate coated with the capture antibody, incubate, and wash the plate;

[0023] (2) Add the biotin-labeled murine monoclonal antibody, incubate, and wash the plate;

[0024] (3) Add the diluted streptavidin labeled with HRP, incubate, and wash the plate;

[0025] (4) Add the chromogenic solution for color development, add the stop solution to terminate the reaction, and read the OD 450 value;

[0026] (5) Prepare a nuclease concentration standard curve, substitute the absorbance value of the sample to be detected into the curve, and calculate the nuclease concentration in the sample.

[0027] Further, in step (1), the incubation temperature is 25°C to 37°C, preferably 37°C; the incubation time is 1 to 5 h, preferably 2 h;

[0028] Further, in step (3), the dilution factor of the streptavidin is 2,000 to 80,000 times, preferably 10,000 times;

[0029] Further, the incubation time is 20 min to 1 h, preferably 40 min; in step (4), the color development time is 5 to 30 min, preferably 10 min.

[0030] On the other hand, the present invention provides a use of an ELISA kit for improving the accuracy of detecting nuclease concentration.

[0031] The beneficial effects of the present invention include:

[0032] 1. The ELISA detection kit of the present invention uses a murine monoclonal antibody prepared by immunizing experimental animals with inactivated nuclease. In the method for preparing the antibody, when screening antibodies by immunizing with inactivated nuclease, the positive rate of the antibody is high and the subtypes are diverse; the advantage of using inactivated antigen is that, firstly, there are differences in animal behavior: when immunizing animals with active nuclease antigen, the animals show manic (circling) and hair loss, and in severe cases, they may die, which affects the preparation of antibodies; secondly, the antibody preparation cycle is different: the present invention proves that under the conditions of the same immunization dose and the same injection method, using active nuclease to immunize animals 8 times can obtain antibodies with the target titer (1:256,000), while inactivated nuclease immunization reaches the same titer after 4 times.

[0033] 2. Different mouse strains were used in the preparation of nuclease murine monoclonal antibodies: CD-1 mice were used as the animal strain for immunizing mice before fusion. This strain of mice increased the positive rate of hybridoma cell fusion and shortened the immunization cycle at the same time. Immunodeficient mice Balb / c were used for ascites preparation. Using them in combination made it easier to screen monoclonal antibodies with good specificity and high affinity, laying a foundation for improving the detection sensitivity of the kit.

[0034] 3. The combined use of rabbit polyclonal antibodies and murine monoclonal antibodies was to avoid non-specific reactions of antibodies between the same species and reduce the false positive rate during kit detection.

[0035] 4. Each of the two antibodies in the kit of the present invention has its own advantages. Among them, rabbit polyclonal antibodies can recognize multiple epitopes on the nuclease antigen, so they can bind as much nuclease as possible, and the production cost of rabbit polyclonal antibodies is relatively low. On the other hand, murine monoclonal antibodies only target one antigen epitope, so they have higher specificity and fewer cross-reactions. In addition, the screened murine monoclonal antibody is of IgG3 subtype, and this antibody has high purity and good affinity, and can bind the target antigen more effectively.

[0036] 5. Since rabbit polyclonal antibodies can recognize multiple epitopes on the antigen, they can more sensitively recognize the target antigen in the ELISA detection system, but this may also lead to false positive results. Murine monoclonal antibodies have higher specificity and can more accurately recognize the target antigen, reducing false positive results. However, the sensitivity of murine monoclonal antibodies is not as good as that of rabbit polyclonal antibodies. Therefore, pairing two antibodies from different genera complements the disadvantages of the two antibodies, that is, it maximally ensures the specific recognition between antigen and antibody and reduces the non-specific binding of antibodies of the same genus, further reducing false positive results. All in all, this pairing method improves sensitivity, reduces the false positive rate, and enhances the specificity of detection.

[0037] 6. The kit provided by the present invention has strong specificity, high accuracy and good stability; high sensitivity, with a detection lower limit of 12 pg / mL and a quantitative detection range of 0.047 - 3 ng / mL; wide application range, and can detect nucleases from different manufacturers. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0039] Figure 1 : Standard curve of nuclease concentration, where y = 0.4754x + 0.1897 (R 2= 0.9902);

[0040] Figure 2 : Sensitivity test of the ELISA kit provided by the present invention;

[0041] Figure 3 : SDS-PAGE result diagram of rabbit polyclonal antibody after Protein A purification; among them, the Marker is in lane 1, and the samples in lanes 2-4 are eluates;

[0042] Figure 4 : SDS-PAGE result diagram of rabbit polyclonal antibody after UN antigen affinity purification; the sample in lane 1 is the flow-through, lane 2 is the Marker, and the samples in lanes 3-8 are eluates;

[0043] Figure 5 : SDS-PAGE result diagram of concentrated rabbit polyclonal antibody; lane 1 is the Marker, and the sample in lane 2 is the concentrated rabbit polyclonal antibody;

[0044] Figure 6 : BCA protein concentration standard curve; linear regression equation y = 0.0009x + 0.0788 (R 2 = 0.9968);

[0045] Figure 7 : SDS-PAGE result diagram of purified mouse monoclonal antibody; lane 1 is the Marker, and the samples in lanes 2-7 are eluates. Detailed implementation manners

[0046] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention; based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0047] The OD values shown in the tables in the following embodiments are all average OD values, that is, 3 replicates are set for each experimental group, and the average OD values are calculated therefrom; the experimental methods used in the embodiments are all conventional methods unless otherwise specified; the materials and reagents used are shown in Table 1, and the active nuclease (i.e., Benzonase nuclease) selected in the present invention has been disclosed in the patent "A Preparation Method and Application of a Protein-Tag-Free Nuclease" (Application No.: 202411428187.5); unless otherwise specified, the reagents and materials are commercially available.

[0048] Table 1 Sources of materials and reagents

[0049]

[0050] Example 1: An ELISA kit for detecting nuclease

[0051] This example provides a double antibody sandwich detection kit for nuclease residues. The kit contains: a detection plate (preferably an enzyme-linked immunosorbent assay (ELISA) plate) coated with a rabbit-derived polyclonal antibody against nuclease (rabbit-derived polyclonal antibody), a Benzonase nuclease (SEQ ID NO:1) standard (standard concentration is 500 ng / mL), a biotin-labeled monoclonal antibody against nuclease (mouse-derived monoclonal antibody, 3B5) at a concentration of 0.3 μg / mL, streptavidin labeled with HRP (dilution ratio is 1:10,000), sample diluent, washing solution, blocking solution, chromogenic solution, and termination solution; the sample diluent is a PBS buffer solution (pH = 7.4) containing 1% BSA, 0.05% NP40, and 0.05% Proc1in; the washing solution is a PBS buffer solution (pH = 7.4) containing 0.05% Tween 20 (i.e., PBST); the blocking solution is a PBS buffer solution (pH = 7.4) containing 0.05% Tween-20, 0.05% Proclin300, and 1% BSA; the chromogenic solution is a tetramethylbenzidine (TMB) chromogenic solution; the termination solution is 2M sulfuric acid; the kit is stored at 4°C for long-term preservation.

[0052] The preparation method of the detection plate coated with the rabbit-derived polyclonal antibody is as follows: the concentration of the capture antibody (rabbit-derived polyclonal antibody against nuclease) is 2 μg / mL, and 100 μL is added to each well; 100 μL of the coating solution is added to each well. The coating solution is a 0.05M carbonate buffer solution (pH = 9.6), and the coating is carried out overnight at 4°C; the plate is washed 5 times with the washing solution (PBS buffer solution containing 0.05% Tween 20 (PBST)); the ELISA plate is blocked overnight (20 h) at 4°C with the blocking solution (1% BSA), and the volume of the blocking solution added to each well is 200 μL. The plate is washed 5 times with the washing solution and then reserved.

[0053] The preparation methods of the rabbit-derived polyclonal antibody and the mouse-derived monoclonal antibody are shown in Example 4.

[0054] Example 2: A method for detecting nuclease concentration

[0055] This example provides a method (sandwich ELISA) for detecting nuclease concentration using the kit described in Example 1. The operations are as follows: adding samples, washing the plate, adding the biotin-labeled mouse monoclonal antibody; washing the plate, adding streptavidin labeled with HRP, washing the plate; adding TMB for color development, adding the termination solution to terminate the reaction, and OD 450 reading.

[0056] Specifically, 100 μL of the sample to be detected and standards at various concentration gradients (4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, and 0.125 ng / mL) (to prepare a nuclease concentration standard curve) were added respectively. Meanwhile, a positive control (PBS solution containing 1 ng / mL Benzonase) and a negative control (PBS solution) were set up. Each sample was set with three replicates and incubated for 2 hours. Then, the plate was washed 3 - 5 times with a washing solution (PBS buffer (PBST) containing 0.05% Tween 20); 100 μL of a detection antibody (biotin-labeled murine nuclease monoclonal antibody) at 0.3 μg / mL was added to each well, incubated at 37°C for 1 hour, and the plate was washed 3 - 5 times with the washing solution; streptavidin labeled with HRP was diluted at a ratio of 1:10,000 with a sample diluent (the diluent components were the same as those described in Example 1), 100 μL of the diluted streptavidin was added to each well, incubated at room temperature for 40 minutes, and the plate was washed 3 - 5 times with the washing solution; 100 μL of TMB chromogenic solution was added to each well. After chromogenic reaction at 37°C for 10 minutes, 100 μL of 2M sulfuric acid termination solution was added to each well to terminate the chromogenic reaction, and the absorbance value (OD 450 value) at a wavelength of 450 nm was read with an enzyme-linked immunosorbent assay (ELISA) reader. According to the absorbance values of the nuclease standards, a nuclease concentration standard curve y = 0.4754x + 0.1897 (R 2 = 0.9902)( Figure 1 ) was obtained, and the absorbance value of the detected sample was substituted into this curve, and then the nuclease concentration in the detected sample was calculated.

[0057] It should be noted that the preparation process and usage steps of the kits described in Examples 1 - 2 have been optimized to a certain extent. The specific optimization process can be seen in Examples 5 - 12.

[0058] Example 3: Performance test of the sandwich ELISA method for detecting nuclease

[0059] In this example, the kit described in Example 1 was subjected to a performance test. Without special instructions, the specific experimental steps were the same as those described in Example 2.

[0060] 3.1 Specificity test

[0061] The positive samples used in this example were 0.25 ng / mL Benzonase solution and negative sample diluents. The negative sample diluents were 0.5 mg / mL E. coli HCP (Beijing Bomed Gene Technology Co., Ltd.), 1.8 mg / mL 293 HCP (Jiaxing Anyu Biotechnology Co., Ltd.), 1 mg / mL CHO HCP (Tian Guang Biotechnology Co., Ltd.), and 0.5 mg / mL Vero HCP (Jiaxing Anyu Biotechnology Co., Ltd.), as well as the corresponding negative sample solutions diluted with the sample diluent at dilution factors of 2, 4, and 8 times respectively. The remaining experimental steps were the same as those described in Examples 1-2. The specific experimental results are shown in Table 2, and the values in the table are OD 450 values.

[0062] Table 2 Specificity test results

[0063]

[0064] As can be seen from Table 2, the OD 450 values of the sample diluent (negative control) and the positive control were 0.1879 and 0.3825 respectively, while the detected OD 450 values of the contents of E. coli, 293 cells, Vero cells, and CHO cells were concentrated between 0.18 and 0.19, and the highest did not exceed 0.2. That is, the test results can be determined to be negative, indicating that the sandwich ELISA kit provided by the present invention has high detection specificity.

[0065] 3.2 Sensitivity and standard curve preparation

[0066] In this example, a nuclease (inactivated nuclease after genetic modification, Benzonase) standard was diluted with a sample diluent to obtain nuclease solutions with concentrations of 3 ng / mL, 1.5 ng / mL, 0.75 ng / mL, 0.375 ng / mL, 0.188 ng / mL, 0.093 ng / mL, 0.047 ng / mL, 0.024 ng / mL, 0.012 ng / mL, and 0.006 ng / mL respectively. And they were detected by the ELISA method described in Example 2 to determine the lowest detectable Benzonase concentration by the ELISA method, so as to determine the sensitivity of this method. The experimental results are shown in Table 3 and Figure 2 .

[0067] Table 3 Sensitivity test results

[0068] Nuclease detection concentration <![CDATA[OD 450 value]]> 3 ng / mL 2.328 1.5 ng / mL 1.5361 0.75 ng / mL 0.9298 0.375 ng / mL 0.5863 0.188 ng / mL 0.4148 0.093 ng / mL 0.2774 0.047 ng / mL 0.2248 0.024 ng / mL 0.189 0.012 ng / mL 0.1572 0.006 ng / mL 0.1597

[0069] As shown in Table 3 and Figure 2 , as the nuclease content decreased, its OD 450The absorbance value shows a downward trend. When the nuclease concentration is 12 pg / mL, the OD 450 value tends to be stable, indicating that the lowest limit of the nuclease concentration detection method provided by the present invention is 12 pg / mL.

[0070] 3.3 Experimental sample recovery rate test

[0071] The actual sample recovery rate reflects the accuracy of the detection method. Generally, the kit detection recovery rate should be between 80% and 110%. In this example, nucleases with concentrations of 1.5 ng / mL and 0.75 ng / mL (inactivated nucleases after genetic modification, Benzonase) were added to the actual sample adenovirus purification solution (Jiaxing Anyu Biotechnology Co., Ltd.), and the established ELISA method (Example 2) was used for detection. The detection results are shown in Table 4, where the recovery rate = detected concentration / theoretical concentration × 100%.

[0072] Table 4 Test results of actual sample recovery rate

[0073] Theoretical concentration 1.5 ng / mL 0.75 ng / mL Detection concentration 1.53 ng / mL 0.77 ng / mL Recovery rate 102％ 103％

[0074] As shown in Table 4, the recovery rates of the adenovirus samples added with 1.5 ng / mL and 0.75 ng / mL nucleases are between 100%, indicating that the kit provided by the present invention has a high recovery rate.

[0075] 3.4 Precision test

[0076] 3.4.1 Intra-plate difference test

[0077] In this example, the curve construction of the standard products was repeated 6 times on the same ELISA plate. The concentrations of the Benzonase standard products were 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, and 0.125 ng / mL. The specific experimental steps were the same as those described in Example 2, and the detection results are shown in Table 5.

[0078] Table 5 Intra-plate test results

[0079]

[0080] As can be seen from Table 5, in the same ELISA plate, the coefficient of variation (CV) of each concentration in the 6 repeated standard curves is less than 5%, indicating that the intra-plate difference of the kit provided by the present invention is very small and the repeatability is high.

[0081] 3.4.2 Inter-batch difference test

[0082] In this example, three batches of ELISA kits were produced using rabbit polyclonal antibodies prepared at different times, and the inter-batch differences of these three batches of kits were tested. That is, these three batches of kits were used to detect adenovirus samples containing His nuclease (Benzonase nuclease) at 3 ng / mL and 1.5 ng / mL respectively. The detection steps were the same as those described in Example 2. The detection results are shown in Table 6, where the recovery rate = (detected concentration / theoretical concentration) × 100%.

[0083] Table 6 Test results between batches

[0084] Batch 3 ng / mL 1.5 ng / mL 1 81.60％ 93.70％ 2 93.80％ 97.30％ 3 90.80％ 91.00％ Average value 88.70％ 94.00％ Standard deviation 0.063 0.031 CV 7.10％ 3.20％

[0085] As can be seen from Table 6, the recovery rates of the enzyme-labeled plates coated with rabbit polyclonal antibodies of three different batches for samples added with 3 ng / mL and 1.5 ng / mL nuclease respectively are all between 80% and 100%, and the CV < 10%. It can be seen that the inter-batch differences of the kits provided by the present invention are very small.

[0086] 3.5 Stability test

[0087] In this example, the coated enzyme-labeled plates were placed at 4°C (control) and 37°C for 7 days respectively, and then detected for Benzonase nuclease standards at 4 ng / mL, 2 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, and 0.125 ng / mL. The detection method was the same as that described in Example 2. The detection results are shown in Table 7, where OD 450 = (OD 450 detection value at 37°C - OD 450 detection value at 4°C) / OD 450 detection value at 4°C × 100%.

[0088] Table 7 Stability test

[0089]

[0090] As shown in Table 7, the decline rate of the enzyme-labeled plate coated with nuclease rabbit polyclonal antibody after being placed at 37°C for 7 days is about 10%, which basically meets the stability standard of the kit.

[0091] 3.6 Application range of the kit

[0092] In this example, nucleases from different manufacturers at 0.3 ng / mL and 0.6 ng / mL were respectively added to the actual sample of adenovirus purification solution (Jiaxing Anyu Biotechnology Co., Ltd.), and the ELISA kit described in Example 1 was used for detection. The detection steps were the same as those described in Example 2. The detection results are shown in Table 8, where the recovery rate = (detected concentration / theoretical concentration) × 100%.

[0093] Table 8 Recovery rates of nucleases from different manufacturers

[0094]

[0095] As can be seen from Table 8, the sample recovery rates of different manufacturers are between 80% and 110%, meeting the sample recovery requirements, indicating that the kit used has high detection accuracy and a wide application range. That is, the ELISA kit provided by the present invention can not only detect the Benzonase nuclease produced by its own company, but also detect the nucleases of other manufacturers sold on the market.

[0096] Example 4: Preparation of Antibodies

[0097] The preparation steps of the rabbit-derived nuclease polyclonal antibody (rabbit polyclonal antibody) and the mouse-derived nuclease monoclonal antibody labeled with avidin HRP (mouse monoclonal antibody) described in Example 1 are as follows:

[0098] 4.1 Preparation of Rabbit-Derived Nuclease Polyclonal Antibody

[0099] 4.1.1 Preparation of Rabbit Serum

[0100] 4.1.1.1 Animal Immunization: The active nuclease antigen obtained by prokaryotic expression and two Ni column purifications (SEQ ID NO: 1, the detailed preparation method of the nuclease is the same as that described in the patent "A Method for Preparing a Protein-Tag-Free Nuclease and Its Application" (Application No.: 202411428187.5)) is mixed with Freund's adjuvant in equal volume and completely emulsified with an emulsifier; the emulsified antigen is injected subcutaneously into the back of New Zealand rabbits at 6 weeks of age and weighing 2.3 - 2.5 kg at multiple points, immunized once every 2 weeks for 12 consecutive weeks. For the primary immunization, the His-tagged nuclease antigen (Benzonase nuclease) is emulsified with Freund's complete adjuvant, and for the booster immunization, the antigen is emulsified with Freund's incomplete adjuvant; the antigen dose for each immunization is 0.5 mg / rabbit.

[0101] 4.1.1.2 Detection of Serum Titer by ELISA: After 4 immunizations, rabbit venous blood is taken to detect the serum titer by indirect ELISA. The specific operation is as follows:

[0102] (1) Coat the antigen with carbonate coating buffer (pH = 9.6), add 100 μL / well to a 96-well enzyme-linked immunosorbent assay (ELISA) plate, incubate overnight at 4°C, set two replicates for each dilution, and set PBS control and negative serum control;

[0103] (2) Wash 3 times with PBST, 5 minutes each time;

[0104] (3) Block with 1% fetal bovine serum (diluted with PBS), add 100 μL to each well, incubate overnight at 4°C;

[0105] (4) Wash 4 times with PBST, 5 minutes each time;

[0106] (5) Add the sample, 100 μL per well, incubate at 37 °C for 45 min;

[0107] (6) Wash 5 times with PBST, 5 min each time;

[0108] (7) Dilute the HRP-labeled goat anti-rabbit secondary antibody with PBS at a dilution ratio of 1:20,000, then add 100 μL per well to the above wells, incubate at 37 °C for 45 min;

[0109] (8) Wash 3 times with PBST, 5 min each time;

[0110] (9) Add 100 μL of TMB chromogenic solution to each well, place in the dark for 15 min of chromogenesis, add 100 μL per well of the stop solution (2 M sulfuric acid), then read the value of each well at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMax M5). Each group of experiments has 3 replicates, and the corresponding average value is calculated for subsequent analysis. Stop immunization after the serum titer reaches 1:256,000 or above. Take the whole blood of the rabbit through the carotid artery. After standing at room temperature for 1 hour, centrifuge at 10,000 rpm for 5 minutes to initially separate the serum, and place the serum at -20 °C for later use.

[0111] The detection results of antibody titers in rabbit sera are shown in Table 9. The positive well determination criterion: P (average OD value of the test sample) / N (average OD value of the negative control) ≥ 2.1; The results show that the serum titers of both rabbits reached 512,000. Further, the serum titer of Rabbit 1# is relatively higher, so the serum was used for subsequent experiments.

[0112] Table 9 Detection Results of Rabbit Serum Titers

[0113]

[0114] 4.1.2 The first purification of rabbit serum was carried out by Protein A affinity method

[0115] (1) Column packing: Pack the GE Protein A packing material into a 5 mL purification column;

[0116] (2) Sample treatment: Take 2 mL of rabbit serum in a 1.5 mL centrifuge tube, centrifuge at 12,000 rpm at 4 °C for 15 min to remove cell debris and some protein aggregates; Mix the serum supernatant with the equilibration buffer (dilution ratio of 1:10), then filter through a 0.22 μm membrane, and adjust the pH value to 7.4 with 6 M hydrochloric acid, which is similar to the pH of the equilibration buffer;

[0117] (3) Washing: Set the pumps of 50% A and 50% B to pump water of about 3 - 5 column volumes, which has been filtered through a 0.22 μm filter membrane, into the purification column to wash the purification column until the OD 280 is stable at 0;

[0118] (4) Equilibrating the column: Flush the column with 3 - 5 column volumes of equilibration buffer (1×PBS + 2% trehalose + 5% glycerol) until the conductivity remains stable at 13 - 15 mS / cm;

[0119] (5) Loading: Load the filtered sample, loading 2 - 3 times;

[0120] (6) Equilibration: Flush the column with 3 - 5 column volumes of equilibration buffer (1×PBS + 2% trehalose + 5% glycerol) until the OD 280 value remains stable;

[0121] (7) Elution: Use 0.1 M glycine (pH = 2.7 - 3.0) to flush the column to elute the rabbit polyclonal antibody; Pre - add 1 M Tris - HCl (pH = 9.0) to the receiving sample tube to neutralize the pH of the eluted antibody solution to about 7.0;

[0122] (8) Dialysis: As soon as possible, dialyze the eluted antibody with equilibration buffer (1×PBS + 2% trehalose + 5% glycerol), dialyzing 4 times;

[0123] (9) Use SDS - PAGE to preliminarily detect its purity and concentration. The specific operation steps of SDS - PAGE are as follows:

[0124] A. Preparation of gel solution: Take 10 mL of 30% acrylamide solution, 25 mL of Tris - HCl buffer (pH 8.8), 1 mL of 10% SDS, 0.5 mL of 10% APS, 0.05 mL of TEMED (tetramethylethylenediamine), and add deionized water to 50 mL to prepare 10% separating gel; Take 1.67 mL of 30% acrylamide solution, 3 mL of Tris - HCl buffer (pH 6.8), 0.1 mL of 10% SDS, 0.05 mL of 10% APS, 0.005 mL of TEMED, and add deionized water to 5 mL to prepare 5% stacking gel;

[0125] B. Gel casting: Wash and dry the glass plates, seal them with rubber strips, pour the prepared separating gel solution into the sandwich of the glass plates, pay attention to avoid generating bubbles, and then let it stand for a period of time until the gel polymerizes. After the gel polymerizes, carefully pour the prepared stacking gel solution on the separating gel, insert the comb, and let it stand until the gel polymerizes;

[0126] C. Preparation of sample buffer: Take 2.5 mL of glycerol, 0.01 g of bromophenol blue, 2.5 mL of Tris-HCl buffer (pH 6.8), 2.5 mL of 10% SDS, and 0.5 mL of β-mercaptoethanol, and add deionized water to make up to 10 mL to prepare the sample buffer;

[0127] D. Treating the sample: Mix the protein sample to be tested with the sample buffer at a ratio of 1:1 (v / v), then boil in a water bath at 100 °C for 5 minutes to denature the protein, and then quickly cool it;

[0128] E. Electrophoresis: Place the gel in the electrophoresis tank, add an appropriate amount of electrophoresis buffer (Tris-glycine buffer) to ensure that the gel is completely immersed; use a micropipette to add the treated sample to the gel wells, generally adding 10 - 20 μL of sample to each well, and at the same time load the protein Marker (Beijing Bomed Biotechnology). First, perform electrophoresis at a lower voltage (about 80 V). After the sample enters the separation gel, increase the voltage to 120 - 150 V and continue electrophoresis until the bromophenol blue front approaches the bottom of the gel; the electrophoresis time is generally 1 - 3 hours, and the specific time depends on the gel concentration and electrophoresis conditions;

[0129] F. Staining and decolorization: After electrophoresis, take out the gel, put it into the staining solution, gently shake it, and stain for 1 - 2 hours. The staining solution formula is: 0.25 g of Coomassie Brilliant Blue R-250 (Solarbio), 50 mL of methanol, and 10 mL of glacial acetic acid, and add deionized water to make up to 100 mL); after staining, put the gel into the decolorizing solution, gently shake it, and decolorize for several hours to several days until the background is clear. The preparation method of the decolorizing solution: Take 50 mL of methanol and 10 mL of glacial acetic acid, mix them evenly, and then add deionized water to make up to 100 mL.

[0130] The results are as Figure 3 shown. After the rabbit serum is purified by Protein A and stained with Coomassie Brilliant Blue, a protein band of about 55 kDa can be clearly seen on the protein gel, which is the rabbit polyclonal antibody. The purity of the rabbit polyclonal antibody in lanes 2 - 3 is about 90%. Although the purity of the rabbit polyclonal antibody meets the requirements, its background value is relatively high and it cannot reach the coating ELISA test plate. Therefore, affinity chromatography purification is needed to perform secondary purification on the rabbit polyclonal antibody purified by Protein A. After the second purification, SDS-PAGE and ELISA tests are performed on the purified product to determine whether it meets the requirements of the later double-antibody sandwich ELISA.

[0131] 4.1.3 Cross-linking of nuclease (UN) antigen with filler

[0132] 4.1.3.1 Preparation of coupling buffer: The coupling buffer contains 0.2M NaHCO 3and 0.5 M NaCl, and filter with a 0.22 μm filter membrane;

[0133] 4.1.3.2 Dialysis of UN antigen: Load the UN antigen (prepared as described above) into a dialysis bag, and dialyze it with the prepared coupling buffer three times under the condition of 4°C;

[0134] 4.1.3.3 Concentration of UN antigen: Concentrate the dialyzed UN antigen with an ultrafiltration tube, and replace it with the coupling buffer twice to concentrate the antigen 14 - 15 times.

[0135] 4.1.3.4 Activation of NHS-Sepharose 4 Fast Flow packing: Load the NHS-Sepharose 4 Fast Flow packing into a purification gravity column, wash the packing with 10 - 15 column volumes of filtered water to remove 20% ethanol, and then quickly wash the packing with 10 - 15 column volumes of precooled 1 mM HCl to activate the packing. After activation, quickly wash the packing with 3 - 5 column volumes of coupling buffer.

[0136] 4.1.3.5 Crosslinking of antigen and packing: Add the replaced UN antigen to the gravity column, and place the gravity column in a shaker at 4°C and crosslink overnight.

[0137] 4.1.3.6 Blocking: Discard the liquid in the gravity column and wash it with filtered water, and collect the washing solution for later measurement of the packing coupling rate. Add 0.5 M ethanolamine, 0.5 M NaCl, and a buffer solution with pH = 8.3 for blocking, and place the gravity column in a shaker at room temperature for reaction for 2 - 3 hours.

[0138] 4.1.3.7 Medium cleaning: Wash the packing alternately with two buffer solutions with different pH values (0.5 M ethanolamine, 0.5 M NaCl, pH = 8.3; 0.1 M acetic acid, 0.5 M NaCl, pH = 4.0). The specific operation is as follows: After blocking, wash the packing with 3 - 5 column volumes of filtered water, add 3 - 5 column volumes of buffer solution containing 0.1 M acetic acid and 0.5 M NaCl (pH = 4.0), wash the packing with 3 - 5 column volumes of filtered water, and then add 3 - 5 column volumes of buffer solution containing 0.5 M ethanolamine and 0.5 M NaCl (pH = 8.3). The two buffer solutions are alternately washed and circulated 5 - 6 times.

[0139] 4.1.4 Purification of rabbit polyclonal antibody by antigen affinity chromatography

[0140] 4.1.4.1 Column packing: Load the NHS-Sepharose 4 Fast Flow packing crosslinked with UN antigen into a 5 mL purification column.

[0141] 4.1.4.2 Sample treatment: Mix the rabbit polyclonal antibody purified by Protein A (step 1.2) after dialysis 4 times above with the equilibration buffer (1×PBS + 2% trehalose + 5% glycerol) (dilution ratio is 1:1), filter it using a 0.22 μm membrane, and adjust the pH value to 7.4, which should be similar to the pH of the equilibration buffer.

[0142] 4.1.4.3 Washing: Use 100% B pump to apply high pressure to wash the column with water for about 3 - 5 column volumes after filtering through a 0.22 μm filter membrane until the OD 280 value stabilizes at 0.

[0143] 4.1.4.4 Equilibrating the column: Wash the column with 3 - 5 column volumes of the equilibration buffer (1×PBS + 2% trehalose + 5% glycerol) until the conductivity remains stable.

[0144] 4.1.4.5 Loading: Load the treated sample, and load it 2 - 3 times.

[0145] 4.1.4.6 Re - equilibration: Wash the column with 3 - 5 column volumes of the equilibration buffer (1×PBS + 2% trehalose + 5% glycerol) until the OD 280 value remains stable.

[0146] 4.1.4.7 Elution: Wash the column with 0.1M glycine (pH = 2.7 - 3.0) to elute the rabbit polyclonal antibody.

[0147] 4.1.4.8 Dialysis: Dialyze the eluted antibody as soon as possible with the equilibration buffer (1×PBS + 2% trehalose + 5% glycerol) for 4 times.

[0148] 4.1.4.9 Concentration: Pre - clean a 15 mL 50KDa ultrafiltration tube (Millipore). Since there is trace glycerol on the ultrafiltration membrane of the ultrafiltration tube, it needs to be rinsed with pure water or buffer before use. It should be noted that once the ultrafiltration centrifuge tube is wetted, it should be prevented from drying out; add 10 mL of the sample to the ultrafiltration centrifuge tube and cover the lid; place the covered ultrafiltration centrifuge tube in the rotor of the centrifuge, and place a balance tube on the opposite side, centrifuge at 3,000 r / min for 10 - 30 min. After centrifugation, take out the whole centrifuge tube from the centrifuge, take out the inner tube, discard the centrifugate in the outer tube, and repeatedly aspirate the antibody concentrate with a pipette (note: avoid touching the filter membrane with the pipette when aspirating to prevent damage to the filter membrane), to wash the antibody adsorbed on the membrane, and finally transfer the antibody concentrate to a sterile 1.5 mL centrifuge tube.

[0149] 4.1.4.10 Use polyacrylamide gel electrophoresis (SDS - PAGE) experiments to detect the purity of the rabbit polyclonal antibody after purification and concentration ( Figures 4 - 5);The titer of the concentrated rabbit polyclonal antibody was detected by ELISA (the detection steps are the same as those described in step "4.1.1.2", and the results are shown in Table 10) to determine the activity of the antibody. The criterion for judging positive wells was P / N≥2.1; the concentration of the concentrated rabbit polyclonal antibody was detected by the BCA method (Sangon Biotech, C503021-0500, and the specific operation steps can be found in the instruction manual on its official website) (the BCA protein concentration standard curve is shown in Figure 6 , and the detection results are shown in Table 11).

[0150] Table 10 Detection results of the titer of the concentrated rabbit polyclonal antibody

[0151] Potency <![CDATA[OD of rabbit polyclonal antibody 450 value]]> <![CDATA[OD of negative control 450 value]]> 1:4,000 2.3762 0.1969 1:8,000 2.1056 0.1946 1:16,000 1.605 0.1432 1:32,000 1.0253 0.1512 1:64,000 0.5233 1:128,000 0.3984 1:256,000 0.1593 1:512,000 0.164

[0152] Table 11 Results of detecting the concentration of the concentrated rabbit polyclonal antibody by the BCA method

[0153]

[0154] It can be seen from Figure 4 that only a single band was shown in SDS-PAGE, and the size of the band was in line with expectations, about 55 kDa. Moreover, the purity of the rabbit polyclonal antibody after successive purification by protein A and antigen affinity purification was >98%; the purity, titer and concentration of the concentrated rabbit polyclonal antibody were >98%, 128,000 and 1,750 μg / mL ( Figure 5 and Tables 10 - 11). In summary, the rabbit polyclonal antibody after two purifications and concentration can be used as the coating antibody / capture antibody in the nuclease detection kit.

[0155] 4.2 Preparation of murine nuclease monoclonal antibody

[0156] 4.2.1 Immunize mice with nuclease

[0157] The preparation method of the active His-tagged nuclease (SEQ ID NO: 1) is the same as described above. The inactive (inactivated) nuclease protein sequence (SEQ ID NO: 2) is obtained by point mutation based on the amino acid sequence of the active nuclease (SEQ ID NO: 1), and the point mutation can cause the nuclease to lose its function of cleaving nucleic acids. The preparation method of the inactive nuclease is the same as that of the active nuclease; the active / inactivated His-tagged nuclease antigens are respectively mixed with Freund's adjuvant in equal volume, and the mixture is completely emulsified with an adjuvant emulsifier (Suzhou Bioron Technology Co., Ltd.); the emulsified antigens are subcutaneously injected at multiple points on the back of 4-6-week-old Balb / c or CD-1 female mice, and immunized once every 2 weeks. It should be noted that Freund's complete adjuvant is used to emulsify the nuclease antigen for the primary immunization, and Freund's incomplete adjuvant is used to emulsify the nuclease antigen for the booster immunization; the antigen dose for each immunization is 100 μg / animal; after 4 immunizations, venous blood is taken, and the serum titer is detected by the indirect ELISA method. The specific steps are the same as those described in step "4.1.1.2"; immunization is stopped until the serum titer reaches 1:256,000 or above, and cell fusion is carried out. The detection results of the titer are shown in Tables 12 to 13, and the positive well determination criterion is P / N ≥ 2.1.

[0158] Table 12 Detection results of the titer of murine monoclonal antibodies prepared by immunization with active nuclease

[0159]

[0160] (Note: The bold OD value is determined as a positive well)

[0161] Table 13 Detection results of the titer of murine monoclonal antibodies prepared by immunization with inactivated nuclease

[0162]

[0163] (Note: The bold OD value is determined as a positive well)

[0164] As can be seen from Table 12 and Table 13, when immunizing mice of the same strain (Balb / c), the inactivated nuclease (reaching the target titer of 1:256,000 after six immunizations) has a 4-week shorter immunization cycle compared to the active nuclease (reaching the target titer of 1:256,000 after eight immunizations). In addition, the active nuclease has a greater impact on the behavior and habits of mice, causing hair loss and neurological effects (such as mania and circling), and even leading to the death of mice, thus affecting the immunization cycle. From the data in Table 12, it can also be concluded that compared with Balb / c strain mice, the immunization cycle of CD-1 strain mice can be further shortened by 4 weeks based on six immunizations with inactivated nuclease. This is because CD-1 mice belong to outbred mice, with strong adaptability and vitality, and their immune systems are relatively "general", capable of quickly reacting to foreign antigens and producing sufficient immune cells and antibodies. This characteristic enables CD-1 mice to achieve better immune effects during immunization without undergoing multiple long-term immune stimulations like inbred mice (BALB / c strain). In summary, immunizing mice with inactivated nuclease can shorten the antibody preparation cycle, so inactivated nuclease is used for subsequent immunization. The antibody titers in the sera of mice immunized with nuclease antigens in two forms (inactivated and active) have both reached 256,000, and subsequent cell fusion experiments can be carried out. Specifically, the B cells of Balb / c mice immunized with inactivated nuclease six times and CD-1 mice immunized four times are respectively fused with SP2 / 0 tumor cells, and then the positive rate of cell fusion is statistically analyzed and the subtypes of positive cell lines are screened out.

[0165] 4.2.2 Cell Fusion

[0166] 4.2.2.1 Preparation of dissection instruments: 2 pairs of 14-cm surgical scissors, 1 pair of 18-cm surgical scissors, 1 10-cm curved forceps, 4 14-cm forceps, 2 14-cm hemostats, 10 5-mL syringes. All the above instruments need to be autoclaved and dried.

[0167] 4.2.2.2 Experimental reagents: Serum-free DMEM medium, DMEM + 10 - 15% FBS + glutamine (Gln) + sodium pyruvate + HAT, 100× double antibody (penicillin-streptomycin), 70% or 75% alcohol; all the above reagents are sterile.

[0168] 4.2.2.3 Experimental instruments: Alcohol cotton, 1.5-mL centrifuge tubes, 1.5-mL centrifuge tube racks, 50-mL centrifuge tube racks, 500-mL beakers, foam box lids (for fixing dissected mice); all the above instruments need to be autoclaved and dried.

[0169] 4.2.2.4 Preparation of feeder cells: Take a normally growing 8-week-old Balb / c strain mouse, extract its eyeballs with curved forceps, take negative serum, and then decapitate the mouse. Immerse the treated mouse in 75% ethanol for 10 min, fix the mouse on a foam board with thumb tacks. First, make a small horizontal incision in the abdomen, then longitudinally cut upward along the midline of the abdomen to cut the cortex. After cutting, expose the peritoneum. After disinfecting with an alcohol cotton ball, use a 5 mL syringe to aspirate 5 mL of serum-free DMEM medium. Gently press with an alcohol cotton ball to suspend the peritoneal cells, then aspirate the cell suspension with a syringe and inject it into a 15 mL sterile centrifuge tube. After centrifuging at 1,000 r / min for 10 - 15 min, discard the supernatant, wash once with serum-free medium, and resuspend with DMEM + 10 - 15% FBS + Gln + sodium pyruvate + HAT. After cell counting, plate in a 96-well plate at 2×10 4 cells / well. Prepare one day before the feeder cells fuse.

[0170] 4.2.2.5 Preparation of SP2 / 0 tumor cells: Resuscitate SP2 / 0 cells (Institute of Basic Medicine, Chinese Academy of Medical Sciences). The culture time of SP2 / 0 cells before fusion should not exceed 2 weeks. Always ensure that the cells are in the logarithmic growth phase, and the density should not exceed 1×10 6 cells / mL. Culture them with DMEM complete medium containing 10% FBS, passage 3 times a week, and the cell density is 2.5×10 4 -1×10 5 cells / mL. At the same time, take a small amount of SP2 / 0 cells and culture them in HAT medium to determine whether all cells will die or whether the cells are contaminated, so as to judge whether the selection / screening reagent is effective. Since the protein in the serum will interfere with membrane fusion, SP2 / 0 cells need to be washed three times with serum-free DMEM medium before fusion, counted, and the cell concentration is adjusted to 1×10 7 / mL, and the cell viability is over 95%.

[0171] 4.2.2.6 Preparation of immune B cells: Decapitate and inactivate nuclease-immunized Balb / c mice that have been immunized six times or CD-1 mice that have been immunized four times, and immerse them in 75% alcohol for 5 min. Fix the mice on a foam board with thumb tacks, disinfect with an alcohol cotton ball after cutting the cortex, hold the muscle layer with forceps and gently lift it upward, cut the muscle layer, and take the mouse spleen. Rinse with serum-free DMEM, then place the spleen in a petri dish and add 5 - 10 mL of serum-free DMEM medium. Squeeze the spleen with the inner core of a syringe, and then filter the medium through a 200-mesh filter into a 50 mL centrifuge tube. Centrifuge at 1,000 r / min for 10 min, discard the supernatant, rinse the cell pellet three times with serum-free DMEM medium, count, and adjust the cell concentration to 1×10 8 / mL, and then resuspend with 20 mL of serum-free DMEM medium.

[0172] 4.2.2.7 Cell fusion: Mix B cells / tumor cells at a number ratio of 5:1 - 10:1 (preferably 10:1 in this example), centrifuge at 1,000 r / min for 10 min, and discard the supernatant as much as possible; gently flick the precipitate to loosen it, and all subsequent fusion steps are carried out under the condition of a 37°C water bath. Add 1 mL of 50% PEG 4000 within 60 s, shake well while adding, and then complete the aspiration of the cell suspension with a pipette tip within 30 s; add 15 mL of serum-free DMEM medium within 5 min, shake well while adding, centrifuge at 1,000 r / min for 10 min, and discard the supernatant; after resuspending with serum-free DMEM medium, centrifuge at 1,000 r / min for 10 min again, and discard the supernatant; add HAT medium containing 20% FBS, adjust the cell concentration to 1.3×10 6 / mL, and add 75 μL / well to the culture plate containing the prepared feeder layer cells; place it in a CO 2 incubator (37°C and 5% CO 2 ) for culture; for 96-well plates, a total of 9 plates are plated, 60 wells / plate, and the side wells are filled with 1×PBS as a protective solution, with the same volume as the culture wells; observe the cell changes every day, and change to HAT screening medium for culture after the 4th day.

[0173] 4.2.3 Detection of fused cells

[0174] 4.2.3.1 Sampling: Observe and mark under the microscope, take 100 μL of the supernatant in the well positions where cells are growing. It should be noted that the sampling pipette tip should not be inserted to the bottom of the well, and tube labels should be made.

[0175] 4.2.3.2 Plate detection: Coat Benzonase nuclease onto a 96-well enzyme-linked immunosorbent assay (ELISA) plate at a concentration of 1 μg / mL, add 100 μL of the cell supernatant sample, and incubate at 37°C for 30 min; wash the plate 3 times with PBST, add HRP-labeled goat anti-mouse secondary antibody diluted 1:10,000, incubate at 37°C for 30 min, wash 3 times with PBST, add 100 μL of TMB chromogenic solution, develop color at 37°C for 30 min, and then add the stop solution (50 mM dilute sulfuric acid) to terminate the reaction; use an ELISA reader to detect the absorbance value at a wavelength of 450 nm. The wells with sample OD values close to the OD values of the positive control (i.e., the mouse serum obtained before fusion) are considered positive cell wells and are used for the next subcloning.

[0176] As can be seen from Table 14, there are 128 strongly positive cell wells among about 600 cell wells; combining Table 14 and Table 15, it can be seen that compared with Balb / c mice (only 2 strongly positive wells among 94 cell wells), the positive rate of hybridoma cells obtained by fusing CD-1 mouse cells immunized with inactivated nuclease is higher. Therefore, the strongly positive cell wells prepared from CD-1 mice were expanded into a 48-well plate for re-examination. The re-examination steps are the same as those described in the above "plate inspection". The re-examination results are shown in Table 16. In this example, 32 cell lines were re-examined, and 21 strongly positive cells (such as cells 3C4 / 12E2 / 3B5, etc.) were selected for subsequent subtype identification and subcloning screening. The subtype identification refers to the subtype identification of the antibodies secreted by the selected hybridoma positive cell lines. The specific identification steps refer to the instruction manual of the monoclonal antibody subclass identification enzyme ready-to-use kit produced by Beijing Boaolong Immunotechnology Co., Ltd. The judgment criteria are as described in the instruction manual. The blue wells observed with the naked eye are positive. As can be seen from Table 17, the subtype of 3C4 / 9D11 / 4B2 is IgG1, the subtype of 3E3 / 12E2 is IgG2a, and the subtype result of 3B5 is IgG3.

[0177] Table 14 Preliminary Screening Results of CD-1 Mouse Fusion Cells

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188] (Note: The bold font in the table is the positive control well, and the OD value with a gray background and enlarged font is determined as the positive well)

[0189] Table 15 Screening Results of Balb / c Mouse Fusion Cells

[0190]

[0191] (Note: The wells with bold font in the table are positive control wells, and the OD values with gray background and enlarged font are determined as positive wells)

[0192] Table 16 Secondary screening results of fused cells

[0193]

[0194] (Note: The wells with bold font in the table are positive control wells, and the OD values with gray background and enlarged font are determined as positive wells) Table 17 Antibody subtype identification

[0195]

[0196] 4.2.3.3 Preservation: Randomly selected 6 cell strains with the highest OD 450 values (i.e., 3C4, 12E2, 3B5, 9D11, 4B2, 3E3 cell strains) for preservation, with 9 tubes preserved for each cell strain; The specific operation steps are as follows:

[0197] A. Open the biosafety cabinet and disinfect with ultraviolet light for 30 min;

[0198] B. After turning on the ventilation in the biosafety cabinet, prepare the cryopreservation solution (DMEM + 10% FBS + 10% DMSO), place it at 4°C for more than 4 h, and set aside for later use;

[0199] C. After the cryopreservation solution is prepared, place the cells in the biosafety cabinet;

[0200] D. Gently aspirate the culture supernatant and keep it for later use;

[0201] E. Aspirate 2 - 3 mL of complete medium containing 10% serum and gently pipette the cells;

[0202] F. After pipetting and mixing evenly, take a small amount of the sample for trypan blue staining and counting with a hemocytometer;

[0203] G. According to the final cell density of 3 - 5×10 6 cells / mL, mix the resuspended cell suspension with the previously reserved supernatant in a centrifuge tube, and centrifuge at 200 g at room temperature for 5 min;

[0204] H. After centrifugation, discard the supernatant, resuspend the cells with 1 mL of the prepared cryopreservation solution, mix evenly, then aliquot into 1.5 mL cryogenic tubes and tighten, 1 mL / tube, place in a programmable gradient cooling box, and store overnight in an -80°C refrigerator;

[0205] I. The next day, take out the cells from the programmable gradient cooling box, place them in a liquid nitrogen tank for long-term storage and make corresponding records.

[0206] 4.2.4 Clonal screening

[0207] 4.2.4.1 Cloning and Plating: Count the 3B5 cell line in the logarithmic growth phase, then dilute the cells to 10 cells / mL with complete DMEM + HT medium, and plate 100 μL / well (i.e., approximately 1 cell / well); add 100 μL of 1×PBS buffer to the four side wells of the 96-well plate; culture at 37 °C and 5% CO 2 for 7 - 10 days; observe under the microscope. When there is only one cell cluster growing aggregately in the screened cell well plate, it is determined that this cell cluster is formed by the division and proliferation of one cell, and it can be preliminarily determined that the cells growing in this well are monoclonal, and make relevant marks.

[0208] 4.2.4.2 Plate Inspection and Re-inspection: The coating concentration of the monoclonal antibody against nuclease is 2 μg / mL, and the remaining steps are the same as those described in "4.2.3.2". The positive detection rate of the first subclone of 3B5 is about 40% (Table 18), and cells such as 3B5-E2, 3B5-A8, 3B5-D10, and 3B5-C8 are selected and expanded into a 48-well plate for re-inspection. The re-inspection steps are the same as those described in "4.2.3.2"; the closer the OD 450 value of the sample is to the positive control, the more it means that the sample is a positive cell. As can be seen from Table 19, the cells expanded into the 48-well plate are all strongly positive cells in the re-inspection. Select the 3B5-D10 cell line for secondary subcloning.

[0209] Table 18 Initial Inspection Results of the First Subclone of the Fusion Cells

[0210]

[0211]

[0212] (Note: The wells corresponding to the bold fonts in the table are positive control wells, and the OD values with gray background and enlarged fonts are determined as positive wells)

[0213] Table 19 Re-inspection Results of the First Subclone of the Fusion Cells

[0214]

[0215]

[0216] 4.2.4.3 Second Cloning: Take the positive clone 3B5-D10 screened in the first cloning and continue with the second cloning. The plating density this time is 0.2 - 0.5 cells / well, and the volume is 100 μL / well; repeat the first cloning operation with other conditions unchanged.

[0217] 4.2.4.4 Inspection of plates: In addition to the positive control, a negative control (i.e., the complete DMEM+HT medium used for culturing subclones) was also set up, and the rest was the same as described in step "4.2.3.2"; the positive rate of the second subcloning of 3B5-D10 was about 90% (Table 20). 3B5-D10-B5, 3B5-D10-D5, 3B5-D10-D6, 3B5-D10-H6, 3B5-D10-A10, and 3B5-D10-E10 were expanded into a 48-well plate for re-inspection, and the re-inspection steps were the same as those described in step "4.2.3.2"; the cells expanded into the 48-well plate were all strongly positive cells in the re-inspection (Table 21). The 3B5-D10-D6 cell line was selected for the third subcloning. When detecting the third subcloning, both a positive control (mouse serum obtained before fusion) and a negative control (the complete DMEM+HT medium used for culturing subclones) were set up.

[0218] Table 20 Initial inspection results of the second subcloning of fused cells

[0219]

[0220] (Note: The wells corresponding to the bold font in the table are positive control wells, and the wells in the 1st and 2nd positions (from top to bottom) in the 12th column are negative control wells. The OD values with a gray background and enlarged font are determined as

[0221] positive wells)

[0222] Table 21 Re-inspection results of the second subcloning of fused cells

[0223]

[0224] (Note: The wells corresponding to the bold font in the table are positive control wells. The OD values with a gray background and enlarged font are determined as positive wells)

[0225] 4.2.4.5 Third cloning: The same as described in steps "4.2.4.1", "4.2.4.2", and "4.2.4.4". The positive rate of the third subcloning of 3B5-D10-D6 reached 100% (Table 22). Subsequently, the 3B5-D10-D6-C7 cells were selected for expansion culture to be injected into mice to prepare ascites.

[0226] Table 22 Inspection results of the third subcloning of fused cells

[0227]

[0228]

[0229] (Note: The holes corresponding to the bold fonts in the table are positive control holes. The 1st and 2nd holes (from top to bottom) in the 12th column are negative control holes. The OD values with gray background and enlarged fonts are determined as

[0230] positive holes)

[0231] 4.2.5 Ascites Preparation

[0232] 4.2.5.1 Observe the cell status of 3B5-D10-D6-C7 in the T25 flask. When the cell status is good (i.e., the cells are round and transparent), perform cell injection.

[0233] 4.2.5.2 Wash the cells with serum-free DMEM medium, then add serum-free DMEM medium to resuspend the cells, and then collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 1,000 rpm for 8 min.

[0234] 4.2.5.3 Resuspend the cells with normal saline, take out a part of the cell suspension for cell counting, and then centrifuge at 1,000 rpm for 8 min.

[0235] 4.2.5.4 According to the cell density of 2×10 6 / mL, calculate the amount of normal saline required for resuspension, and resuspend the cells with normal saline.

[0236] 4.2.5.5 Use a 1 mL syringe to aspirate 500 μL of the resuspended solution, hold the back of the neck of an 8-week-old female Balb / c mouse, inject the resuspended solution into the abdomen of the mouse, and then gently massage the abdomen of the mouse for 1 - 2 min to prevent the formation of solid tumors.

[0237] 4.2.5.6 Wait until the abdomen of the mouse is full of ascites, then aspirate it, and detect the titer of the antibody in the ascites by ELISA method. The specific steps are the same as those described in "4.1.1.2". As shown in Table 23, the ascites titers of the 3 mice obtained are all 256,000, which can be used for subsequent purification experiments.

[0238] Table 23 Detection Results of Monoclonal Antibody Titers in Mouse Ascites

[0239] Potency Mouse 1 Mouse 2 Mouse 3 Negative control 1:4,000 2.4263 2.4617 2.4554 0.1481 1:8,000 2.3752 2.3505 2.4148 0.1272 1:16,000 2.2422 2.2607 2.2706 0.1472 1:32,000 1.9575 2.1328 1.9018 0.1455 1:64,000 1.2643 1.4187 1.1234 1:128,000 0.3871 0.5181 0.551 1:256,000 0.3483 0.3213 0.3585

[0240] 4.2.6 Ascites Purification

[0241] 4.2.6.1 Column packing: Pack Protein A packing into a 5 mL purification column.

[0242] 4.2.6.2 Sample treatment: Take 2 mL of mouse ascites into a 15 mL centrifuge tube, centrifuge at 4°C and 12,000 rpm for 15 min to remove cell debris and large protein aggregates; after mixing the serum supernatant with the equilibration solution (1×PBS + 2% trehalose + 5% glycerol) (dilution ratio is 1:10), filter it using a 0.22 μm membrane, and adjust the pH value to 7.4, which is similar to the pH of the equilibration solution.

[0243] 4.2.6.3 Washing: Wash the column with water filtered through a 0.22 μm filter membrane and applied with high pressure by the 100% B pump for 3 - 5 column volumes until the OD 280 reaches 0 and remains stable.

[0244] 4.2.6.4 Equilibrating the column: Wash the column with 3 - 5 column volumes of the equilibration solution (1×PBS + 2% trehalose + 5% glycerol) until the conductivity remains stable.

[0245] 4.2.6.5 Loading: Load the filtered sample, load it 2 - 3 times, and the loading time and number of loading times can be adjusted according to the protein purity.

[0246] 4.2.6.6 Re - equilibration: Wash the column with 3 - 5 column volumes of the equilibration solution (1×PBS + 2% trehalose + 5% glycerol) until the OD 280 remains stable.

[0247] 4.2.6.7 Elution: Wash the column with 0.1 M glycine (pH = 2.7 - 3.0) to elute the rabbit polyclonal antibody. Before receiving the sample, 1 M Tris - HCl (pH = 9.0) should be added to the receiving tube to neutralize the pH of the eluted antibody to about 7.0.

[0248] 4.2.6.8 Dialysis: Dialyze the eluted antibody as soon as possible using the equilibration solution (1×PBS + 2% trehalose + 5% glycerol), and dialyze 4 times.

[0249] 4.2.6.9 Use SDS - PAGE experiment to verify the purity of the antibody in the flow - through and eluate; use ELISA method to detect the titer of the purified antibody. The specific steps are the same as those described in "4.1.1.2", and a positive well is determined when P / N ≥ 2.1.

[0250] According to the results of sub - clone subtype identification, the cell line produces IgG3 antibody. Mouse IgG antibody contains two chains: heavy chain (about 55 kDa) and light chain (about 25 kDa). Therefore, theoretically, two bands of different sizes will appear in the same lane of the protein gel. Figure 7As shown, two bands of the expected size appeared in the lanes of the eluate sample, especially in lanes 5 - 6. Based on this, it can be determined that the eluate sample is a murine IgG monoclonal antibody, and its purity is as high as about 98%. As can be seen from Table 24, the titer of the concentrated murine monoclonal antibody is about 256,000, and it can be used for subsequent biotin labeling experiments.

[0251] Table 24 Detection results of the titer of the concentrated murine monoclonal antibody

[0252] Potency 3B5 - D10 - D6 - C7 Negative control 1:4,000 2.5486 0.1432 1:8,000 2.5044 0.1461 1:16,000 2.436 0.1398 1:32,000 2.22 1:64,000 1.443 1:128,000 0.5445 1:256,000 0.3239

[0253] 4.2.7 Biotin labeling of murine monoclonal antibody:

[0254] 4.2.7.1 Preparation of 10 mM biotin reagent solution: Dissolve 2 mg of NHS - Biotin in 590 μL of DMSO.

[0255] 4.2.7.2 Mix biotin and antibody in a 1.5 mL centrifuge tube at a molar ratio of 20:1.

[0256] 4.2.7.3 Place the centrifuge tube containing the mixture of biotin and antibody in a shaker at 4°C and incubate for 2 hours.

[0257] 4.2.7.4 Use a desalting column to remove the excess biotin reagent.

[0258] 4.2.7.5 Detection of antibody labeling effect

[0259] After coating the ELISA plate with 1 μg / mL His - removing nuclease, block it with 1% BSA. Dilute the biotin - labeled antibody with a concentration of 0.7 mg / mL from 1,000 - fold in a two - fold dilution to form 7 gradients. After incubation and washing the plate, add HRP - labeled avidin, develop color with TMB. The remaining steps are the same as described in "4.1.1.2"; Determine the labeling effect by comparing the OD 450 value of the highest dilution - multiple well with the OD 450 value of the negative control. As can be seen from Table 25, when the HRP - labeled antibody is serially diluted 64,000 - fold, the detection result is still positive, indicating that the antibody labeling is successful.

[0260] Table 25 Detection of the effect of HRP labeling of the antibody

[0261] Dilution factor <![CDATA[OD 450 value]]> 1K 2.7382 2K 2.6473 4K 2.544 8K 2.2881 16K 1.8836 32K 0.8981 64K 0.449 Negative control 0.2076

[0262] Example 5: Screening of the nuclease form of the immunized animal

[0263] As can be seen from Example 4, in the present invention, two forms of nucleases were used to immunize mice respectively. When the antibodies reached the same target titer (1:256,000), the number of times the animals were immunized with active nucleases was more than that with inactivated nucleases; and the murine polyclonal antibodies prepared by immunizing CD-1 mice with inactivated nucleases had multiple subtypes, increasing the success rate of antibody pairing in the double-antibody sandwich ELISA method, thereby shortening the preparation cycle of the kit; at the same time, it was also found during the experiment that immunization with active nucleases would cause hair loss and some neurological reactions (circling) in animals, but the effects of the form of nucleases on the antibody activity of animals and on the sensitivity of the detection kit were still unclear. Therefore, in this example, sera with the same titer (1:256,000) prepared by immunizing CD-1 mice with inactivated nucleases and active nucleases respectively as described in Example 4 were used to explore this. Among them, the detection objects were the PBS solution containing 1 ng / mL Benzonase as the positive control and the PBS solution as the negative control, and the remaining experimental steps were the same as those described in Examples 1-2. Subsequently, the detection sensitivity was determined according to the P / N value, that is, the larger the P / N, the higher the detection efficiency of the serum under this immunization condition. The specific results are shown in Table 26.

[0264] Table 26 Influence of the form of nucleases used to immunize animals on the detection results

[0265]

[0266] As shown in Table 26, the P / N value of the serum obtained by immunizing mice with inactivated nucleases was greater than that obtained by immunizing with active nucleases, indicating that the serum prepared by immunizing with inactivated nucleases had a better detection effect. This may be because the inactivated nucleases would not degrade the nucleic acids in the mice or affect other protein functions, and thus had less impact on the normal physiological activities of cells and less harm to the animals; at the same time, animals in good growth condition had strong immunity, and the immune response generated would be stronger, and thus the antibodies produced had a higher affinity for antigens, resulting in higher sensitivity for detecting nucleases. In summary, in order to improve the sensitivity of the kit for detecting nucleases described in the present invention, it is preferred to use inactivated nucleases to immunize mice to prepare murine monoclonal antibodies.

[0267] Example 6: Screening of murine monoclonal antibodies

[0268] Since the method for detecting the concentration of nucleases provided by the present invention is the double-antibody sandwich ELISA method, which requires the use of two antibodies in combination, in this example, an antibody pairing experiment was carried out, that is, purified rabbit polyclonal antibodies were paired with preliminarily screened murine monoclonal antibodies to further screen out murine monoclonal antibodies with better pairing and capable of improving the detection efficiency of the method.

[0269] As can be seen from Example 4, three subtypes of antibodies (IgG1, IgG2a, and IgG3) were screened by subcloning of murine monoclonal antibodies. In this example, the antibodies of different subtypes were respectively used to perform antibody pairing experiments with rabbit polyclonal antibodies to explore the optimal combination of rabbit polyclonal antibodies - murine monoclonal antibodies; each murine monoclonal antibody was serially diluted to obtain murine monoclonal antibody solutions of 1,000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, and 0 ng / mL (negative control). The remaining experimental steps were the same as those described in Examples 1 - 2, and the sensitivity of its pairing was determined according to P / N≥2.1. As can be seen from the detection results (Table 27), the pairing effect of the 3B5 murine monoclonal antibody of the IgG3 subtype with the rabbit polyclonal antibody was the best, and its initial sensitivity was 100 ng / mL. This may be because the IgG3 antibody has a longer hinge region, making it more flexible and having higher affinity when binding to the antigen, thus improving the detection sensitivity. In addition, antibodies of this subtype also have advantages such as improving the specificity, stability, and repeatability of the kit, and being suitable for detecting complex samples (Example 3); followed by the 3E3 murine monoclonal antibody belonging to the IgG2a subtype, and the detection sensitivity of its combination with the rabbit polyclonal antibody was 1,000 ng / mL; therefore, in order to ensure the sensitivity in the above - mentioned detection, murine monoclonal antibodies of the IgG3 and IgG2a subtypes are preferably selected when used in combination with rabbit polyclonal antibodies, more preferably, the IgG3 subtype, specifically, the 3B5 murine monoclonal antibody.

[0270] Table 27 Influence of murine monoclonal antibody types on detection sensitivity

[0271]

[0272] Note: The wells corresponding to the bold font in the table are positive wells

[0273] Example 7: Optimization of antibody concentration

[0274] In this example, the checkerboard titration method was used to explore the optimal concentrations of the capture antibody (rabbit polyclonal antibody) and the detection antibody (mouse monoclonal antibody) in the sandwich ELISA detection system provided by the present invention. Among them, the concentrations of the capture antibody were set at 4 μg / mL, 2 μg / mL, and 1 μg / mL respectively, and the concentrations of the detection antibody were set at 0.3 μg / mL and 0.5 μg / mL respectively; the detection objects were the PBS solution containing 1 ng / mL Benzonase as the positive control and the PBS solution as the negative control, and the remaining experimental steps were the same as those described in Examples 1 to 2. The larger the P / N value, the greater the corresponding detection value and the lower the negative background value, indicating the best antibody coating amount. That is, when the P / N value is the largest, it indicates that the antibody coating amount and non-specific binding reach the best balance point; if the coating amount is too low, the detection value will be low, and if the coating amount is too high, there may be non-specific binding, resulting in a high negative background value. Therefore, the optimal concentrations of the capture antibody and the detection antibody can be determined according to the P / N value, that is, the larger the P / N value, the better the concentration combination of the corresponding capture antibody and detection antibody; as can be seen from the detection results (Table 28), when the concentrations of the capture antibody (rabbit polyclonal antibody) and the detection antibody (mouse monoclonal antibody) are 2 μg / mL and 0.3 μg / mL respectively, the P / N value (i.e., 5.28186) is the largest, indicating that the above concentrations are the optimal concentrations of the capture antibody and the detection antibody. In order to improve the detection effect of the kit provided by the present invention, the concentrations of the capture antibody and the detection antibody in the kit are preferably 2 μg / mL and 0.3 μg / mL respectively.

[0275] Table 28 Optimization of the Concentrations of the Capture Antibody and the Detection Antibody

[0276]

[0277]

[0278] (P is the positive control, N is the negative control; 3 replicates were set in this experiment, and the values in the table are the averages)

[0279] Example 8: Optimization of the Coating Buffer, Coating Temperature and Time

[0280] In this example, the capture antibody (rabbit polyclonal antibody) was diluted with 2 kinds of coating diluents, and the coating diluents were 0.01M PBS buffer (pH = 7.4) and 0.05M CBS buffer (pH = 9.6) respectively. The remaining experimental steps were the same as those described in Examples 1, 2 and 7. The results are shown in Table 29. The best coating buffer is the CBS buffer when the P / N value is the largest. This may be because the CBS buffer provides a suitable pH environment for the activity and stability of the rabbit polyclonal antibody.

[0281] Table 29 Optimization of the Coating Buffer

[0282]

[0283] (P is the positive control and N is the negative control; this experiment was set up with 3 replicates, and the values in the table are the averages)

[0284] Next, based on the optimization results of the above coating buffer, this example also explored the effects of temperature and time on the ELISA test results when coating with rabbit polyclonal antibody. Three coating conditions were set, namely coating at 37°C for 1 hour, coating at 37°C for 2 hours, and coating at 4°C for 20 hours (overnight). The remaining experimental steps were the same as those described in Examples 1, 2, and 7. The results are shown in Table 30. When the coating condition was overnight at 4°C, the P / N was the largest, that is, this condition was the best coating condition. In summary, to improve the detection effect of the kit provided by the present invention, it is preferred to coat the rabbit polyclonal antibody overnight at 4°C with 0.05M CBS buffer (pH = 9.6).

[0285] Table 30 Optimization of Coating Temperature and Time

[0286]

[0287] (P is the positive control and N is the negative control; this experiment was set up with 3 replicates, and the values in the table are the averages)

[0288] Example 9: Optimization of Blocking Conditions

[0289] In this example, 1% BSA and 5% skim milk powder were respectively selected as the blocking solutions, and the other components of the blocking solution were the same as those in Example 1 (i.e., PBS buffer containing 0.05% Tween-20 and 0.05% Proclin300). Two blocking conditions were set, namely blocking at 37°C for 2 hours and blocking overnight at 4°C respectively. The remaining experimental steps were the same as those described in Examples 1, 2, and 7. The detection results are shown in Table 31. From the detection results, it can be seen that when using 1% BSA solution to block the enzyme-labeled plate overnight at 4°C, the P / N is the largest, indicating that this condition is the best blocking condition. Therefore, to improve the detection effect of the kit provided by the present invention, it is preferred to block with 1% BSA solution overnight at 4°C.

[0290] Table 31 Optimization of Blocking Conditions

[0291]

[0292] (P is the positive control and N is the negative control; this experiment was set up with 3 replicates, and the values in the table are the averages)

[0293] Example 10: Optimization of Sample Incubation Temperature and Time

[0294] The sample incubation temperature in this example was set to room temperature or 37°C, and incubated for 1 hour and 2 hours respectively. The remaining experimental steps were the same as those described in Examples 1, 2, and 7. The test results are shown in Table 32. The best detection sample incubation condition was when the P / N was the largest, that is, incubating at 37°C for 2 hours was the best incubation condition. Therefore, in order to improve the detection effect of the kit provided by the present invention, the sample was preferably incubated at 37°C for 2 hours.

[0295] Table 32 Optimization of Sample Incubation Temperature and Time

[0296]

[0297] (P is the positive control, N is the negative control; 3 replicates were set in this experiment, and the values in the table are the averages)

[0298] Example 11: Optimization of the Concentration and Incubation Time of HRP-Labeled Avidin

[0299] In this example, the dilution ratios of streptavidin labeled with HRP were set to 1:5,000 and 1:10,000 respectively, and incubated at 37°C for 30 minutes and 40 minutes respectively. The remaining experimental steps were the same as those described in Examples 1, 2, and 7. The test results are shown in Table 33. The best HRP-labeled avidin incubation condition was when the P / N was the largest, that is, the HRP-labeled avidin was diluted 10,000 times and incubated at room temperature for 40 min was the best working condition. Therefore, in order to improve the detection effect of the kit provided by the present invention, the above best working condition was preferably adopted.

[0300] Table 33 Optimization of the Concentration and Incubation Time of HRP-Labeled Avidin

[0301]

[0302] (P is the positive control, N is the negative control; 3 replicates were set in this experiment, and the values in the table are the averages)

[0303] Example 12: Optimization of the TMB Color Development Time

[0304] In this example, the color development times of the TMB color development solution were 10 min and 15 min respectively. The remaining experimental steps were the same as those described in Examples 1, 2, and 7. The test results are shown in Table 34. The best TMB color development time was when the P / N was the largest, that is, the reaction of the TMB color development solution for 10 min was the best working condition. Therefore, in order to improve the detection effect of the kit provided by the present invention, the above best working condition was preferably adopted.

[0305] Table 34 Optimization of the TMB Color Development Time

[0306] 10 min 15 min P 0.95945 0.9512 N 0.18165 0.1912 P / N 5.28186 4.9749

[0307] (P is the positive control, N is the negative control; this experiment was set up with 3 replicates, and the values in the table are the averages)

[0308] The foregoing has shown and described the basic principles, main features and advantages of the present invention. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, it is intended to embrace all changes that fall within the meaning and scope of the equivalent elements of the claims in the present invention, and any reference signs in the claims should not be regarded as limiting the claims involved.

Claims

1. An ELISA kit for detecting nucleases, characterized in that: The kit comprises a detection plate coated with a capture antibody and a detection antibody, wherein the detection antibody is prepared from an animal immunized with inactivated nuclease.

2. The kit according to claim 1, characterized in that The detection antibody is a biotin-labeled mouse monoclonal antibody targeting nuclease, and its subtype is any one of IgG1, IgG2a and IgG3; the capture antibody is a rabbit polyclonal antibody targeting nuclease.

3. The kit according to claim 1, characterized in that The concentration of the detection antibody is 0.2-1.0 μg / mL; the concentration of the capture antibody is 1.0-5.0 μg / mL.

4. The kit according to claim 1, characterized in that The buffer for coating the capture antibody is CBS or PBS; after coating the capture antibody, the detection plate needs to be blocked; the blocking solution contains any one or more of 1% BSA and 5% skim milk.

5. The kit according to claim 4, characterized in that The coating / sealing temperature is 4° C. to 37° C., and the coating / sealing time is 0.5 to 20 hours.

6. The kit according to claim 5, characterized in that The kit also includes a standard substance for nuclease, streptavidin labeled with HRP, a sample diluent, a washing solution, a color developing solution and a stop solution.

7. A method for detecting nuclease concentration, characterized in that: The nuclease concentration is detected using the kit according to any one of claims 1 to 6.

8. The method according to claim 7, characterized in that The method comprises the following steps: (1) adding the sample to be tested and the nuclease standard to the detection plate coated with the capture antibody, incubating, and washing the plate; (2) adding biotin-labeled mouse monoclonal antibody, incubating, and washing the plate; (3) Add diluted HRP-labeled streptavidin, incubate, and wash the plate; (4) Add colorimetric solution to develop color, add stop solution to terminate the reaction, and read the OD 450 value; (5) Prepare a nuclease concentration standard curve, substitute the absorbance value of the sample to be tested into the curve, and calculate the nuclease concentration in the sample.

9. The method according to claim 8, characterized in that In step (1), the incubation temperature is 25°C to 37°C, and the incubation time is 1 to 5 hours; in step (3), the dilution factor of the avidin is 2,000 to 80,000 times; the incubation time is 20 minutes to 1 hour; and in step (4), the color development time is 5 to 30 minutes.

10. Use of an ELISA kit for improving the accuracy of detecting nuclease concentration.

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