CN113777307A - All-round nuclease Benzonase ELISA detection kit - Google Patents

Abstract

The invention provides a Benzonase ELISA detection kit of a totipotent nuclease, which comprises: rabbit-derived Benzonase polyclonal antibodies; a biotin-labeled rabbit-derived Benzonase polyclonal antibody; avidin-labeled HRP; a standard Benzonase; developing solution, stopping solution, sealing solution and washing solution. The detection lower limit of the detection kit is 24 pg/ml, the quantitative detection range is 0.047-3 ng/ml, and the kit can be used for detecting Benzonase of different manufacturers and replacing imported Benzonase detection kits. Meanwhile, a methodology system evaluation is carried out on the constructed ELISA detection system, the kit has good specificity and high detection accuracy.

Description

All-round nuclease Benzonase ELISA detection kit

Technical Field

The invention relates to an ELISA detection kit for a full-functional nuclease Benzonase, belonging to the technical field of biology.

Background

With the increasing introduction of biological products (recombinant proteins, Vero cell vaccines, etc.) into the therapeutic field, quality control of biological products is becoming more and more strict, wherein nucleic acid residues are the most important of various quality control standards, because of better stability of DNA, residues are easily formed in the production process, and when the biological products are applied to the prevention and treatment of human diseases, uncontrollable risk factors can be brought about. The host nucleic acid residue of the final product must comply with WHO, FDA and EMEA and NMPA regulatory regulations in China.

Benzonase is a genetically engineered endonuclease derived from Serratia marcescens (Serratia marcescens). Benzonase can degrade double-stranded, single-stranded, linear and circular DNA and RNA, and completely degrade nucleic acid into 5' -monophosphate oligonucleotide with the length of 3-5 basic groups. It is also called "totipotent nuclease" because it can degrade DNA and RNA in any form with high efficiency. Benzonase is an effective tool enzyme for the removal of all forms of DNA and RNA from biological products, whether in laboratory studies or during industrial production. Through efficient nucleic acid removal, the effects and the yield of subsequent experiments and production can be obviously improved, and the performance is superior to that of other nucleic acid removal methods.

Benzonase belongs to recombinant protein expression products, and residues cannot be left in biological products, so after nucleic acid is removed by using Benzonase in the production process of the biological products, the Benzonase needs to be removed and detected, and the product is ensured to meet the requirements of production standards. At present, large domestic biological product manufacturers mainly use Merck's Benzonase ELISA detection kit (product number: 101681), the detection quantitative range is 1-10 ng/ml, the detection sensitivity is 0.2 ng/ml, no good kit exists at home, the detection sensitivity of the kit prepared by using polyclonal antibodies as raw materials at home is not high, monoclonal antibodies are used as raw materials, and the kit has no compatibility with Benzonase of different manufacturers. In recent years, with the rise of vaccine and cell therapy markets in China, the demand of the imported Benzonase ELISA detection kit is rapidly increased, the supply is short and the price is high, and the invention can provide a good method for producing the product.

Disclosure of Invention

The invention aims to provide a Benzonase ELISA detection kit for a totipotent nuclease, which has the advantages of good specificity, high sensitivity and high detection accuracy.

The technical scheme adopted by the invention is as follows:

a universal nuclease Benzonase ELISA detection kit, comprising:

rabbit-derived Benzonase polyclonal antibodies;

a biotin-labeled rabbit-derived Benzonase polyclonal antibody;

avidin-labeled HRP;

a standard Benzonase;

a sample diluent;

developing solution, stopping solution, sealing solution and washing solution;

wherein the biotin-labeled rabbit-derived polyclonal antibody is preserved in a biotin-labeled antibody stock solution;

the preparation method of the rabbit-derived Benzonase polyclonal antibody comprises the following steps:

the method comprises the following steps of carrying out subcutaneous multipoint immunization on the back of a New Zealand rabbit by using Benzonase Protein for 12 weeks, immunizing once every two weeks, separating serum after carotid blood sampling, and sequentially purifying by Protein G affinity chromatography and antigen affinity chromatography to obtain the rabbit-derived Benzonase polyclonal antibody, wherein the primary immunization dose is 0.5 mg/rabbit, and the booster immunization dose is 0.25 mg/rabbit.

Preferably, the blocking solution contains 0.05% Tween-20, 0.05% Proclin300, and 1% PEG6000 in PBS.

Preferably, the biotinylated antibody stock solution contains 1% BSA, 0.05% Proclin300, 0.01% Tween-20, 10% MPD in PBS.

Preferably, the sample dilutions contained 3% lactose, 1% BSA, 0.05% NP40, 0.5% gelatin, 0.05% Proclin, 0.5% PEG400 in PBS.

The Protein G affinity chromatography purification specifically comprises the following steps: diluting rabbit serum with PBS, balancing Protein G affinity purification medium with PBS, mixing the diluted rabbit serum with the Protein G affinity purification medium, incubating, loading the incubated Protein G purification medium into a gravity chromatography column, collecting flow-through liquid, washing with PBS, eluting an antibody with glycine elution buffer solution after washing, and obtaining Protein G purification eluent.

The antigen affinity chromatography purification specifically comprises the following steps: balancing an antigen affinity purification medium by PBS, mixing the antigen affinity purification medium with Protein G purification eluent, incubating for 2 hours, loading the incubated medium into a gravity chromatographic column, collecting flow-through liquid, washing by PBS, eluting an antibody by glycine elution buffer solution after washing, concentrating the eluent by an ultrafiltration concentration tube, dialyzing and replacing the concentrated liquid by PBS, adding glycerol, and storing at-20 ℃ for later use.

The preparation steps of the antigen affinity purification medium are as follows:

(1) vertically placing a column containing NHS activated agarose resin, balancing the column to room temperature, allowing the resin to settle, washing with a washing solution by suction filtration for three times, and washing with a coupling buffer solution for one time;

(2) adding the dissolved Benzonase to the washed NHS activated agarose;

(3) mixing and shaking reaction to couple Benzonase to NHS activated agarose;

(4) washing the coupled NHS activated agarose with deionized water, and adding a sealing solution with twice column volume for oscillation reaction;

(5) draining and removing confining liquid, adding deionized water to wash the resin, washing the resin by sequentially using a washing liquid 1, the deionized water, a washing liquid 2 and the deionized water, then adding a protection buffer solution with the same volume, and storing at 2-8 ℃;

wherein the washing solution: 1 mM hydrochloric acid;

coupling liquid: 0.2M NaHCO3, 0.5M NaCl, pH 8.0 or other amine free buffers;

washing solution 1: 0.1M acetic acid-sodium acetate, 0.5M NaCl, pH 8.0;

washing solution 2: 0.1M Tris-HCl, 0.5M NaCl, pH 8.0;

protection buffer: 1xPBS comprising 20% ethanol;

dissolving 20 mg of Benzonase in 2 ml of coupling solution, and dialyzing to replace the coupling solution;

sealing liquid: ethanolamine 0.5M, NaCl 0.5M, pH 8.3.

The preparation method of the biotin-labeled rabbit-derived Benzonase polyclonal antibody comprises the following steps:

1) diluting rabbit-derived Benzonase polyclonal antibody with PBS;

2) biotin is dissolved in DMSO;

3) mixing the biotin reagent solution with the antibody solution in a ratio such that biotin is in excess, and incubating for a period of time;

4) and (4) dialyzing to remove the excessive biotin reagent to obtain the biotin-labeled rabbit-derived Benzonase polyclonal antibody.

The detection kit has the lower detection limit of 24 pg/ml and the quantitative detection range of 0.047-3 ng/ml, can detect Benzonase of different manufacturers, and can replace imported Benzonase detection kits. Meanwhile, a methodology system evaluation is carried out on the constructed ELISA detection system, and the kit has good specificity, high sensitivity and high detection accuracy. The rabbit immune dose of the invention is half less than the conventional dose, the immune cycle is improved to 12 weeks, and the rabbit polyclonal antibody with high affinity can be prepared. The kit can be developed by preparing polyclonal antibody, and the development cycle is short. In the purification method, Protein G affinity purification and antigen affinity chromatography purification are combined, so that high-quality multi-antibody raw materials can be obtained, and the detection sensitivity is improved.

Drawings

FIG. 1 is a graph comparing the effects of different immunization doses.

FIG. 2 is a SDS-PAGE identification of Benzonase rabbit polyclonal antigen affinity chromatography purification.

FIG. 3 is a test chart showing the specificity of the sandwich ELISA assay.

FIG. 4 is a graph of a sensitivity test of a sandwich ELISA assay.

FIG. 5 is a graph showing the standard curve of a sandwich ELISA assay.

FIG. 6 is a graph of anti-interference test in a sandwich ELISA assay.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The main materials of the embodiment of the invention are as follows:

benzonase from Saint Biotechnology, Shanghai, Inc., of the next.

Freund's complete adjuvant, Freund's incomplete adjuvant, purchased from Sigma.

New Zealand white rabbits (2-2.5 kg), purchased from Shanghai Dry biology Ltd.

NHS activated agarose, purchased from Biotechnology engineering (Shanghai) GmbH.

Biotin-N-succinimidyl esters, available from Biotechnology engineering (Shanghai) Ltd.

HRP-labeled avidin, purchased from Biotechnology engineering (Shanghai) GmbH.

HRP-labeled goat anti-mouse IgG secondary antibody from shinkansen biotechnology (shanghai) ltd.

96-well microplate, purchased from Thermo Fisher Scientific.

TMB color developing solution from santa sequosa, next (shanghai) co.

PBS pH7.4, glycine eluent pH2.7, Tween 20, BSA, skimmed milk powder, DMSO, Tris Base, NaCO₃NaHCO₃Sulfuric acid, gelatin, Tween-20, NP40, Proclin300, PEG400, PEG6000, MPD, lactose, available from Biotechnology engineering (Shanghai) GmbH.

Purified or unpurified adenovirus fluid was obtained from pureggin biotechnology limited.

Benzonase ELISA kit purchased from Beijing Yinqiao Biotech Co., Ltd.

EXAMPLE 1 preparation and purification of anti-Benzonase polyclonal antibody

1.1 preparation of Rabbit immune serum

Equal volumes of Benzonase and freund's complete adjuvant were mixed and the emulsification was pushed to completion with a syringe. The emulsified antigen was used for subcutaneous multiple immunizations in the back of New Zealand rabbits for 12 weeks, once every two weeks. The primary immunization emulsified Benzonase with Freund's complete adjuvant and the booster immunization emulsified Benzonase with Freund's incomplete adjuvant. The primary immunization antigen dose is 0.5 mg/vaccine, the boosting immunization antigen dose is 0.25 mg/vaccine, the other group immunization dose is the conventional immunization dose, the primary immunization dose is 1 mg/vaccine, and the boosting immunization dose is 0.5 mg/vaccine. Taking venous blood after 3 times of immunization, performing ELISA to detect serum titer, and performing titer detection every 2 weeks later, wherein the serum titer reaches 1: stopping immunization for more than 20 ten thousand, taking whole blood of the rabbit from carotid artery, standing for 1 hour at room temperature, 10000 rpm, centrifuging for 5 minutes, taking serum, adding glycerol with one time volume, mixing uniformly, and standing at-20 ℃ for later use.

ELISA serum titer detection: benzonase was dissolved in PBS, 100. mu.l of enzyme well-coated microplate at a concentration of 1. mu.g/ml, left at 37 ℃ for 1 hour for coating or overnight at 4 ℃, and the plate was washed 3 times with PBST (PBS containing 0.05% Tween 20). 5% skimmed milk powder, 200. mu.l per well, left at 37 ℃ for 2 hours and sealed. Rabbit serum was diluted 2-fold with PBS and set up as negative control in 11 gradients of 1000-fold, 2000-fold, 4000-fold, etc. Each well was diluted with 100. mu.l of serum, and after washing the plate for 5 times at 37 ℃ for 1 hour in PBST, a secondary goat anti-mouse IgG antibody labeled with HRP (1: 5000 dilution) was added at 37 ℃ for 40 minutes, and after washing the plate for 5 times in PBST, a TMB developing solution was added in an amount of 100. mu.l per well.

As shown in table 1, 1# -7# rabbits showed 0.5 mg/rabbit for prime, 0.25 mg/rabbit for boost, and the titer exceeded 1: 20 ten thousand, the serum titer of some rabbits even exceeded 1: 100 ten thousand. 1 mg of rabbit 8# is initially immunized, 0.5 mg of rabbit is boosted, and the titer is 1 after 6 times of immunization: 25.6 ten thousand. As shown in FIG. 1, the rabbit was immunized with the high dose of antigen, and the titer was higher than that of the low dose after 3 weeks of immunization, but the low dose immunization resulted in serum with higher titer as the number of immunizations increased.

TABLE 18 serum titer test after 6 immunizations of rabbits

1.2 Protein G purified Rabbit serum

Protein G affinity purification rabbit serum step: rabbit serum was diluted 10-fold with PBS. Protein G affinity purification media were equilibrated with PBS. The diluted rabbit sera were incubated with Protein G affinity purification media for 2 hours at 4 ℃. The incubated Protein G purification medium was loaded onto a gravity chromatography column, the flow-through was collected, washed with 20 column volumes of PBS, and the antibody was eluted with 10 column volumes of glycine elution buffer after washing. Placing the mixture into an antigen affinity chromatographic column at 4 ℃ for further purification.

1.3 antigen affinity chromatography column preparation

The preparation method of the antigen affinity chromatographic column comprises the following steps:

preparation of Buffer

All water and buffer were filtered through 0.22 μm or 0.45 μm filters.

Required additional materials

(1) Empty columns and compatible centrifuge/collection tubes.

(2) Washing liquid: 1 mM hydrochloric acid.

(3) Coupling liquid: 0.2M NaHCO₃0.5M NaCl, pH 8.0 or other amine-free buffers.

(4) Washing solution 1: 0.1M acetic acid-sodium acetate, 0.5M NaCl, pH 8.0.

(5) Washing solution 2: 0.1M Tris-HCl, 0.5M NaCl, pH 8.0.

(6) Protection solution: (vii) KHz-1 PBS containing 20% ethanol.

(7) 20 mg of Benzonase was dissolved in 2 ml of the coupling solution, and the solution was replaced with the coupling solution by dialysis.

(8) Sealing liquid: ethanolamine 0.5M, NaCl 0.5M, pH 8.3.

Antigen coupling process

Mix agarose gel several times by inversion

(1) The column containing the NHS activated resin was placed vertically and equilibrated to room temperature to allow the resin to settle. Washed three times with washing solution by suction filtration and once with coupling buffer. The resin bed cannot be dried during use, the lumps are dissolved by blowing or shaking, and the resin bed can be quickly cleaned by cold solution to prevent hydrolysis.

(2) Dissolved Benzonase was added to washed NHS activated agarose (volume NHS activated agarose 4 FF: volume sample solution 1: 2.

(3) The mixing and shaking reaction was carried out at 28 ℃ for 4 hours, or at 4 ℃ overnight. NHS-Activated SefiniseTM 4 FF must be in a floating state, otherwise coupling efficiency is affected.

(4) A coupling sample was collected to examine the coupling efficiency. The filler is washed with deionized water. Two column volumes of blocking solution were added and the reaction was shaken at 28 ℃ for 1 hour.

(5) The blocking solution was drained and removed and three column volumes of deionized water were added to wash the resin. And (3) washing the resin twice by using a washing solution 1, deionized water, a washing solution 2 and deionized water in sequence, adding a protection buffer solution with the same volume, and storing at 2-8 ℃.

Lanes 1 and 2 of FIG. 2 show that there were fewer totipotent nucleases after coupling than before coupling, indicating that the totipotent nucleases were coupled to the agarose gel.

1.4 antigen affinity chromatography purification of Rabbit polyclonal antibody

Antigen affinity chromatography column purification antibody step:

antigen affinity purification media was equilibrated with PBS and the purification media was mixed with Protein G purification eluent and incubated at 4 ℃ for 2 hours. The incubated medium was loaded onto a gravity chromatography column, the flow-through was collected, washed with 20 column volumes of PBS, and after washing the antibody was eluted with 10 column volumes of glycine elution buffer. The eluate was concentrated in a 50 kD ultrafiltration tube and the displacement concentrate was dialyzed against PBS. Adding glycerol with one volume and storing at-20 deg.C for use. The results of purification of 3 rabbit sera are shown in table 2. The purity of antibody purification is shown in lane 4 of FIG. 1.

TABLE 23 purification results of rabbits

Rabbit number	Rabbit serum (ml)	ProteinG purification (mg)	Antigen affinity chromatography (mg)
				1#	47	356.1	4.9
2#	49	346.6	5.2
				3#	45	369.7	5.5

Example 2 Biotin-labeled antibody

(1) The biotin reagent was removed from the cold room and allowed to equilibrate to room temperature before opening in step 3.

(2) The antibody was diluted with PBS and 1-10 mg of antibody was dissolved in 0.5-2.0 ml PBS.

(3) A10 mM biotin reagent solution was prepared in DMSO. 2 mg of NHS-Biotin was dissolved in 590. mu.l of DMSO.

(4) And (2) mixing the biotin and the antibody in a molar ratio of 20: 1, the biotin reagent is mixed with the antibody solution.

(5) Incubate on ice for 2 hours or at room temperature for 30 minutes.

(6) At this point the antibody labeling was complete and dialysis was used to remove excess biotin reagent.

ELISA was used to detect the labeling effect of the antibody. The enzyme label plate is coated with 1 mug/ml Benzonase, 5% skimmed milk powder is used for blocking, the labeled antibody is diluted into 7 gradients from 1000 times (the final concentration of the antibody is 1 mug/ml) in a double dilution mode, and HRP labeled avidin is added after incubation and plate washing. TMB color development. The labeling effect was judged by observing the positive wells with the highest dilution factor, as shown in table 3, which were positive when diluted 16000 times, indicating successful antibody labeling.

TABLE 3 detection of the potency of the labeled antibodies

Dilution factor	1K	2K	4K	8K	16K	32K	64K	Negative control
									OD450Value of	2.132	1.654	0.978	0.456	0.384	0.269	0.162	0.154

Example 3 sandwich ELISA assay Benzonase method was performed.

The sandwich ELISA comprises the following steps: coating rabbit polyclonal antibody, washing plate, sealing enzyme label plate, adding sample, washing plate, adding biotin-labeled rabbit polyclonal antibody, washing plate, adding HRP-labeled avidin, washing plate, adding TMB for color development, adding stop solution for stopping, and reading OD 450. At the beginning of debugging, the OD value of the negative sample is found to be more than 0.2, which is related to the simultaneous use of multiple antibodies as the coated antibody and the detection antibody, and only the OD value of the negative sample is reduced to be less than 0.2 by searching the optimal concentration of the coated antibody and the optimal concentration of the detection antibody, and then other conditions are debugged and optimized is meaningful. The debugging sequence in the invention is as follows: optimizing the concentration of the coating antibody and the concentration of the detection antibody, optimizing the coating buffer solution, the coating temperature and time, optimizing the optimal sealing condition, optimizing the incubation temperature and time of the sample, optimizing the concentration and incubation time of the HRP-labeled avidin and optimizing the time of TMB color development.

3.1 the concentration of the coating antibody and the working concentration of the detection antibody are optimized.

Using a checkerboard titration method, the concentration of coating antibody was set at 1. mu.g/ml, 1.5. mu.g/ml, 2. mu.g/ml, 2.5. mu.g/ml, 3. mu.g/ml, 3.5. mu.g/ml, 4. mu.g/ml, 100. mu.l per well. The concentration of the detection antibody was set at 0.4. mu.g/ml, 0.6. mu.g/ml, 0.8. mu.g/ml, 1. mu.g/ml, 100. mu.l per well. The coating solution is PBS firstly, the coating is carried out overnight at 4 ℃, the plate washing solution is PBS containing 0.05 percent Tween 20 and is named as PBST, the plate is washed for 5 times, the blocking solution is 5 percent skim milk powder firstly, each hole is 200 mu l, the blocking solution is blocked for 2 hours at 37 ℃, the coating antibody is incubated for 1 hour at 37 ℃, and the HRP-labeled avidin 1: diluting with 5000 solution, incubating at 37 deg.C for 45 min with 100 μ l per well, allowing TMB color development solution to act at 37 deg.C for 15 min with 50 μ l 2M sulfuric acid per well, and reading OD₄₅₀The value is obtained. The positive sample was PBS containing 1 ng/ml Benzonase, and the negative sample was PBS containing 100. mu.l per well. Rabbit polyclonal antibodies from different rabbit sources, i.e., different batches of rabbit polyclonal antibodies, were tested at concentrations used, and the polyclonal results for # 1 and # 2 rabbits are shown in tables 4 and 5. As can be seen from Table 4, the antibody coating concentration of the No. 1 rabbit was 2. mu.g/ml, and the OD value of the negative sample (N) was low and the OD value was 0.2 or less since the P/N value was the largest at the detection antibody concentration of 0.6. mu.g/ml, the concentrations were determined as the optimum antibody coating concentration and the optimum working concentration of the detection antibody. Table 5 shows that the concentration of the coating antibody in the No. 2 rabbit was 2. mu.g/ml, and the concentration of the detection antibody was 0.8. mu.g/ml, which is the optimum working concentration. The invention also tests other batches of rabbit polyclonal antibodies, and finds that the working concentrations of the coating antibody and the detection antibody of each batch of rabbit polyclonal antibodies are different, so that the optimal working concentration of each batch of rabbit polyclonal antibodies needs to be adjusted. It is known that for each lot of polyclonal antibodies,the optimal detection effect can be achieved only by searching the optimal usage amount ratio of the coated antibody and the detection antibody, the incubation condition of the detection antibody needs to be fixed when the optimal working concentration of the rabbit polyclonal antibody of each batch is optimized, and the method is fixed at 37 ℃ for 1 hour.

TABLE 41 Rabbit polyclonal antibody concentration and test results of the test

TABLE 52 Rabbit polyclonal antibody concentration and test results for detection antibody concentration

3.2 optimize optimal coating buffer, coating temperature and time.

The coated antibody was diluted with 3 coating dilutions, Tris-HCl (0.02M), PBS (0.01M) and CBS (0.05M), using a working concentration of 2. mu.g/ml, 100. mu.l per well, and incubated at 4 ℃ for 16 hours. The plate washing liquid is PBST, and the plate washing is carried out for 5 times. 200. mu.l of 5% skim milk powder was added to each well and the plate was blocked after incubation for 2 hours at 37 ℃. Positive samples were treated with Benzonase solution containing 1 ng/ml, and negative samples were treated with PBS, 100. mu.l per well. The samples were incubated at 37 ℃ for 1 hour. The detection antibody was incubated at 37 ℃ for 1 hour using 0.6. mu.g/ml, 100. mu.l per well. HRP-labeled avidin 1: dilutions were used at 5000 dilutions, 100. mu.l per well, and incubated for 45 min at 37 ℃. The color developing solution of TMB is 100 mul per well, the reaction is carried out for 15 minutes at 37 ℃, the color developing is stopped by 50 mul 2M ammonium sulfate per well, and OD is read₄₅₀Values, as shown in table 6. The negative value is low, and the optimal coating buffer solution is PBS when the P/N is maximum.

Table 6 test results for optimal coating dilutions

Selecting PBS as the best coatingThe coating time was set at three times, 37 ℃ for 1 hour, 37 ℃ for 2 hours and 4 ℃ for 16 hours. The working concentration of coating antibody was 2. mu.g/ml, 100. mu.l per well, and incubated at 4 ℃ for 16 hours. The plate washing liquid is PBST. 200. mu.l of 5% skim milk powder was added to each well and the plate was blocked after incubation for 2 hours at 37 ℃. Positive samples were treated with Benzonase solution containing 1 ng/ml, and negative samples were treated with PBS, 100. mu.l per well. The samples were incubated at 37 ℃ for 1 hour. The detection antibody was incubated at 37 ℃ for 1 hour using 0.6. mu.g/ml, 100. mu.l per well. HRP-labeled avidin 1: dilutions were used at 5000 dilutions, 100. mu.l per well, and incubated for 45 min at 37 ℃. The TMB developing solution is applied to each well at 37 ℃ for 15 minutes in an amount of 100. mu.l, the development is stopped with 50. mu.l of 2M ammonium sulfate per well, and the OD is read₄₅₀Values, as shown in table 7. The maximum P/N is the optimal coating condition, namely the coating effect is optimal after 16 hours at 4 ℃.

TABLE 7 test results of optimal coating conditions

3.3 optimization of the optimal sealing conditions.

The concentration of the coating antibody was 2. mu.g/ml, and the enzyme-labeled plate was coated with 100. mu.l of PBS per well at 4 ℃ for 16 hours. The blocking solution adopts two types of BSA and skimmed milk powder, the concentration of the blocking solution is respectively 1%, 3% and 5%, each hole is 200 mu l, and the blocking conditions are set to be 1 hour at 37 ℃, 2 hours at 37 ℃ and overnight at 4 ℃. The plate washing liquid is PBST, and the plate washing is carried out for 5 times. Positive samples were treated with Benzonase solution containing 1 ng/ml, and negative samples were treated with PBS, 100. mu.l per well. The samples were incubated at 37 ℃ for 1 hour. The detection antibody was incubated at 37 ℃ for 1 hour using 0.6. mu.g/ml, 100. mu.l per well. HRP-labeled avidin 1: dilutions were used at 5000 dilutions, 100. mu.l per well, and incubated for 45 min at 37 ℃. The color developing solution of TMB is 100 mul per well, the reaction is carried out for 15 minutes at 37 ℃, the color developing is stopped by 50 mul 2M ammonium sulfate per well, and OD is read₄₅₀Values, as shown in table 8. The maximum P/N is the best sealing condition, namely 5 percent of skimmed milk powder is used, and the sealing effect is best at 37 ℃ for 2 hours.

TABLE 8 test results for optimum blocking conditions

3.4 optimization of incubation temperature and time of test samples

The concentration of the coating antibody was 2. mu.g/ml, and the enzyme-labeled plate was coated with 100. mu.l of PBS per well at 4 ℃ for 16 hours. The plate washing solution was washed 5 times with PBST. The sealing liquid is 5% skimmed milk powder, and sealing is carried out at 37 deg.C for 2 hr. The positive sample is 1 ng/ml Benzonase solution, the negative sample is PBS, and the stability of the sample at 37 ℃ is not determined, so 6 conditions of incubation for 1 hour and 2 hours at 16 ℃, 25 ℃ and 37 ℃ are set. The detection antibody was incubated at 37 ℃ for 1 hour using 0.6. mu.g/ml, 100. mu.l per well. HRP-labeled avidin 1: dilutions were used at 5000 dilutions, 100. mu.l per well, and incubated for 45 min at 37 ℃. The color developing solution of TMB is 100 mul per well, the reaction is carried out for 15 minutes at 37 ℃, the color developing is stopped by 50 mul 2M ammonium sulfate per well, and OD is read₄₅₀Values, as shown in table 9. The maximum P/N is the best incubation condition of the detected sample, namely the detection effect is the best when the sample is incubated at 37 ℃ for 2 hours, and the stability of the whole Benzonase at 37 ℃ in the detection system of the invention can be seen.

TABLE 9 incubation temperature and time test results for the best test samples

3.5 the concentration of HRP-labeled avidin and the incubation time were optimized.

The concentration of the coating antibody was 2. mu.g/ml, and the enzyme-labeled plate was coated with 100. mu.l of PBS per well at 4 ℃ for 16 hours. The plate washing solution was washed 5 times with PBST. The sealing liquid is 5% skimmed milk powder, and sealing is carried out at 37 deg.C for 2 hr. Positive samples were treated with Benzonase solution containing 1 ng/ml, and negative samples were treated with PBS, 100. mu.l per well. HRP-labeled avidin was set to 1: 3000,1: 5000,1: 7000 dilution, incubation conditions set at 37 ℃ for 30 minutes, 45 minutes, 1 hour three time period, 100. mu.l per well. The color developing solution of TMB is 100 mul per well, the reaction is carried out for 15 minutes at 37 ℃, the color developing is stopped by 50 mul 2M ammonium sulfate per well, and OD is read₄₅₀Values, as shown in table 10. The P/N maximum is the optimal incubation condition for the HRP-labeled avidin, i.e.HRP-labeled avidin was diluted 5000-fold and incubated at 37 ℃ for 45 minutes as the optimal working condition.

TABLE 10 working concentration and incubation time test results for HRP-labeled avidin

3.6 optimization of TMB color development time.

The concentration of the coating antibody was 2. mu.g/ml, and the enzyme-labeled plate was coated with 100. mu.l of PBS per well at 4 ℃ for 16 hours. The plate washing solution was washed 5 times with PBST. The sealing liquid is 5% skimmed milk powder, and sealing is carried out at 37 deg.C for 2 hr. Positive samples were treated with Benzonase solution containing 1 ng/ml, and negative samples were treated with PBS, 100. mu.l per well. The samples were incubated at 37 ℃ for 1 hour. The detection antibody was incubated at 37 ℃ for 1 hour using 0.6. mu.g/ml, 100. mu.l per well. HRP-labeled avidin 1: dilutions were used at 5000 dilutions, 100. mu.l per well, and incubated for 45 min at 37 ℃. TMB developing solution 100. mu.l per well, set at 37 ℃ for 15 minutes, 30 minutes, 45 minutes, 60 minutes, stop developing color with 50. mu.l per well of 2M ammonium sulfate, read OD₄₅₀Values, as shown in table 11. The maximum P/N is the optimal TMB color development time. TMB was allowed to act for 30 minutes as the optimum working condition.

TABLE 11 optimal TMB color development time

Example 4 kit stability optimization

The stability of the kit is related to the stability of each component of the kit, and comprises the following components: coating the closed ELISA plate, biotin-labeled antibody, avidin-labeled HRP, Benzonase standard, TMB color developing solution and sulfuric acid. The stability of each component was tested using a heat accelerated test at 37 ℃ for 6 days and then at 4 ℃ for one day. Each time with one heat-accelerated fraction, the other fractions were left at 4 ℃ (microplate coated for the day's block and Benzonase standard for the day) and sandwich ELISA was used with a Benzonase concentration of 1 ng/ml.

TABLE 12 results of thermally accelerated stability test for each component

Thermally accelerated component	Enzyme label plate after being coated and sealed	Biotin-labeled antibodies	Avidin-labeled HRP	Benzonase standard substance	TMB color development liquid	Sulfuric acid stop solution
							37℃（OD450）	0.854	0.754	1.211	0.879	1.214	1.213
4℃(OD450)	1.211	1.198	1.217	1.218	1.210	1.209

As shown in Table 12, the stability of the enzyme-labeled plate, the biotin-labeled antibody, and the Benzonase standard, which were coated and sealed, was not stabilized by thermal acceleration, and the stability of the avidin-labeled antibody, the TMB developing solution, and the sulfuric acid stop solution was not affected by thermal acceleration. According to the invention, a large number of tests are carried out on the formula of the confining liquid, and finally, the result shows that the PBS containing 5% of skimmed milk powder, 0.05% of tween-20, 0.05% of Proclin300 and 1% of PEG6000 can stabilize the enzyme label plate after confinement. The invention searches for the stock solution culture of the biotin-labeled antibody, and finally discovers that PBS containing 1% BSA, 0.05% Proclin300, 0.01% Tween-20 and 10% MPD can stably store the biotin-labeled antibody. The present inventors performed a number of tests on the stock solutions of Benzonase standards and finally found that PBS containing 3% lactose, 1% BSA, 0.05% NP40, 0.5% gelatin, 0.05% Proclin, 0.5% PEG400 can stabilize Benzonase, and Buffer of this formulation was used to formulate the sample dilutions of the kit.

Example 5 ELISA specificity test

The detection is carried out by an established ELISA method, specifically, the concentration of the coating antibody adopts 2 mu g/ml, the coating solution uses PBS, each hole is 100 mu l, and the ELISA plate is coated at 4 ℃ for 16 hours. The plate washing solution was washed 5 times with PBST. The sealing liquid is 5% skimmed milk powder, and sealing is carried out at 37 deg.C for 2 hr. The samples were incubated at 37 ℃ for 1 hour. The detection antibody was incubated at 37 ℃ for 1 hour using 0.6. mu.g/ml, 100. mu.l per well. HRP-labeled avidin 1: dilutions were used at 5000 dilutions, 100. mu.l per well, and incubated for 45 min at 37 ℃. The color of the TMB developing solution was developed for 30 minutes in an amount of 100. mu.l per well. Color development was stopped with 50. mu.l of 2M sulfuric acid per well, and OD was read₄₅₀The value is obtained. Positive samples containing Benzonase (0.1 ng/ml), negative sample dilutions, 5% rabbit serum samples, 5% mouse serum samples, 5% sheep serum samples, e.coli HCP (0.5 mg/ml), 293 HCP (1.8 mg/ml), CHO HCP (1 mg/ml), Vero HCP (0.5 mg/ml), 1% BSA, and 8 concentration gradients of these samples diluted 2-fold with sample dilutions. The detection results are shown in fig. 3 and table 13, the detected OD values are below 0.15 at both high protein concentration and low protein concentration of the sample, and the low OD value of the high-concentration protein sample is not caused by the inhibition of sandwich ELISA reaction by the high-concentration protein, which indicates that the sandwich ELISA method of the kit has strong specificity.

TABLE 13 ELISA specificity test results

	Rabbit serum	Mouse serum	Sheep serum	E.coli HCP	293 HCP	CHO HCP	Vero HCP	BSA	Sample diluent	Positive sample
											Concentration of original sample	0.142	0.137	0.133	0.129	0.132	0.140	0.132	0.138	0.139	0.301
2 times dilution	0.152	0.134	0.136	0.133	0.139	0.137	0.136	0.143	0.140	0.296
											4 times dilution	0.149	0.140	0.135	0.133	0.137	0.135	0.134	0.147	—	—
8 times dilution	0.145	0.136	0.141	0.131	0.141	0.134	0.140	0.142	—	—
											16 times dilution	0.138	0.139	0.133	0.139	0.133	0.131	0.142	0.137	—	—
32 times dilution	0.140	0.141	0.140	0.136	0.139	0.132	0.135	0.138	—	—
											64 times dilution	0.138	0.136	0.138	0.141	0.138	0.138	0.134	0.130	—	—

Example 6 ELISA detection sensitivity test and preparation of detection Standard Curve

Benzonase standards were diluted with PBS at concentrations of 3ng/ml, 1.5 ng/ml, 0.75 ng/ml, 0.375 ng/ml, 0.188 ng/ml, 0.093ng/ml, 0.047 ng/ml, 0.024 ng/ml, 0.012 ng/ml, 0.006 ng/ml, respectively. The sensitivity of the method was determined by performing the assay using the established ELISA method and determining the lowest detectable Benzonase concentration. As shown in FIG. 4, the value of OD450nm decreased with the decrease of the Benzonase content, and OD450nm leveled off when the Benzonase content was below 0.024 ng/ml, indicating that the detection limit of Benzonase by the double-antibody sandwich ELISA method was 0.024 ng/ml.

Several concentrations of 3ng/ml, 1.5 ng/ml, 0.75 ng/ml, 0.375 ng/ml, 0.188 ng/ml, 0.093ng/ml, 0.047 ng/ml and their corresponding OD values were taken and fitted to the curve by quadratic regression on ELISA Calc software, as shown in FIG. 5. The equation: y = a + bx + cx²，a=0.19034，b=1.11350，c=-0.13038，r²=0.9995。

Example 7 actual sample recovery test

The actual sample recovery reflects the readiness of the assay. For an actual sample adenovirus purified solution given by Puruijin Biotechnology Co., Ltd, the concentration of the purified solution is diluted by 2 times from 3ng/ml, Benzonase is added, and the detection recovery rate of the kit is between 80% and 110% between the Benzonase concentration of 3ng/ml and 0.093ng/ml, as shown in Table 14, the kit disclosed by the invention is high in detection accuracy.

TABLE 14 actual sample recovery test results

Example 8 ELISA assay precision test

8.1 in-Board Difference test

The curve construction of the standard substance is repeated 6 times on the same ELISA plate, and the result is shown in Table 15, wherein the CV value is less than 5%, and the difference in the plate of the kit of the invention is very small.

TABLE 15 in-board variation test results

8.2 run-to-run differential test

3 batches of ELISA kits were produced from rabbit polyclonal antibodies prepared at different times, and the batch-to-batch differences of the three batches of kits were tested. For the non-purified adenovirus harvest, Benzonase is added at a concentration of 2 times from 3ng/ml, and the detection recovery rate of the kit is between 70% and 110% and CV is less than 10% as shown in Table 16 when the concentration of the Benzonase is between 3 and 0.188 ng/ml, so that the kit has small batch-to-batch difference.

TABLE 16 run-to-run test results

8.3 intermediate precision measurement

The test was carried out by 3 panelists independently, and the samples of unpurified adenovirus diluted from 3ng/ml Benzonase standard to 1.5 ng/ml, 0.375 ng/ml, 0.094 ng/ml Benzonase were tested. As shown in table 17, CV < 15%.

TABLE 17 intermediate precision test results

Theoretical concentration of sample (ng/ml)	1.5	0.375	0.094
				Concentration detected by the laboratory technician (ng/ml)	1.388	0.382	0.079
Concentration detected by the second laboratory technician (ng/ml)	1.458	0.363	0.068
				Concentration of three tests (ng/ml) by the laboratory technician	1.318	0.370	0.063
Mean value (ng/ml)	1.388	0.372	0.070
				Standard deviation of	0.070	0.010	0.008
CV value	5.04%	2.59%	11.69%

Example 9 ELISA assay anti-interference test

BSA solutions containing 1 ng/ml Benzonase were assayed by ELISA method under test at 500. mu.g/ml, 200. mu.g/ml, 50. mu.g/ml, 5. mu.g/ml BSA concentrations, respectively. Compared with a competitive Benzonase kit (the raw material of the antibody of the kit is rabbit monoclonal antibody). The results of the experiment are shown in FIG. 6, and the recovery rate of ELISA detection is within the normal range (70% -120%) under different BSA concentrations. It can be seen that the anti-interference ability of the ELISA of the invention is stronger than that of the competitors, which may be an advantage of the polyclonal antibody as a raw material.

Example 10 kit stability test

The coated strips and the universal reagent were allowed to stand at 37 ℃ for 6 days, then transferred to 4 ℃ for 1 day and tested. As shown in Table 18, the results show that the performance of the kit is not obviously reduced, and the falling amplitude is within 20 percent, which is qualified.

TABLE 18 kit stability test results

Claims (8)

1. A universal nuclease Benzonase ELISA detection kit, comprising:

rabbit-derived Benzonase polyclonal antibodies;

a biotin-labeled rabbit-derived Benzonase polyclonal antibody;

avidin-labeled HRP;

a standard Benzonase;

a sample diluent;

developing solution, stopping solution, sealing solution and washing solution;

wherein the biotin-labeled rabbit-derived polyclonal antibody is preserved in a biotin-labeled antibody stock solution;

the preparation method of the rabbit-derived Benzonase polyclonal antibody comprises the following steps:

the method comprises the following steps of carrying out subcutaneous multipoint immunization on the back of a New Zealand rabbit by using Benzonase Protein for 12 weeks, immunizing once every two weeks, separating serum after carotid blood sampling, and sequentially purifying by Protein G affinity chromatography and antigen affinity chromatography to obtain the rabbit-derived Benzonase polyclonal antibody, wherein the primary immunization dose is 0.5 mg/rabbit, and the booster immunization dose is 0.25 mg/rabbit.

2. The kit for detecting the Benzonase ELISA using the all-round nuclease as claimed in claim 1, wherein the blocking solution contains PBS containing 0.05% Tween-20, 0.05% Proclin300 and 1% PEG 6000.

3. The kit according to claim 1, wherein the stock solution of biotinylated antibodies comprises 1% BSA, 0.05% Proclin300, 0.01% Tween-20, 10% MPD in PBS.

4. The kit according to claim 1, wherein the sample diluent contains 3% lactose, 1% BSA, 0.05% NP40, 0.5% gelatin, 0.05% Proclin, 0.5% PEG400 in PBS.

5. The kit for detecting Benzonase ELISA using a totipotent nuclease as claimed in claim 1, wherein the Protein G affinity chromatography purification specifically comprises: diluting rabbit serum with PBS, balancing Protein G affinity purification medium with PBS, mixing the diluted rabbit serum with the Protein G affinity purification medium, incubating, loading the incubated Protein G purification medium into a gravity chromatography column, collecting flow-through liquid, washing with PBS, eluting an antibody with glycine elution buffer solution after washing, and obtaining Protein G purification eluent.

6. The kit for detecting the Benzonase ELISA using the pluripotent nuclease according to claim 1, wherein the antigen affinity chromatography purification specifically comprises: balancing an antigen affinity purification medium by PBS, mixing the antigen affinity purification medium with Protein G purification eluent, incubating for 2 hours, loading the incubated medium into a gravity chromatographic column, collecting flow-through liquid, washing by PBS, eluting an antibody by glycine elution buffer solution after washing, concentrating the eluent by an ultrafiltration concentration tube, dialyzing and replacing the concentrated liquid by PBS, adding glycerol, and storing at-20 ℃ for later use.

7. The kit for detecting the Benzonase ELISA using the pluripotent nuclease as claimed in claim 6, wherein the antigen affinity purification medium is prepared by the steps of:

(1) vertically placing a column containing NHS activated agarose resin, balancing the column to room temperature, allowing the resin to settle, washing with a washing solution by suction filtration for three times, and washing with a coupling buffer solution for one time;

(2) adding the dissolved Benzonase to the washed NHS activated agarose;

(3) mixing and shaking reaction to couple Benzonase to NHS activated agarose;

(4) washing the coupled NHS activated agarose with deionized water, and adding a sealing solution with twice column volume for oscillation reaction;

(5) draining and removing confining liquid, adding deionized water to wash the resin, washing the resin by sequentially using a washing liquid 1, the deionized water, a washing liquid 2 and the deionized water, then adding a protection buffer solution with the same volume, and storing at 2-8 ℃;

wherein the washing solution: 1 mM hydrochloric acid;

coupling liquid: 0.2M NaHCO3, 0.5M NaCl, pH 8.0 or other amine free buffers;

washing solution 1: 0.1M acetic acid-sodium acetate, 0.5M NaCl, pH 8.0;

washing solution 2: 0.1M Tris-HCl, 0.5M NaCl, pH 8.0;

protection buffer: 1xPBS comprising 20% ethanol;

dissolving 20 mg of Benzonase in 2 ml of coupling solution, and dialyzing to replace the coupling solution;

sealing liquid: ethanolamine 0.5M, NaCl 0.5M, pH 8.3.

8. The kit for detecting the Benzonase ELISA using the totipotenic nuclease as claimed in claim 1, wherein the preparation steps of the rabbit-derived Benzonase polyclonal antibody labeled with biotin comprise:

1) diluting rabbit-derived Benzonase polyclonal antibody with PBS;

2) biotin is dissolved in DMSO;

3) mixing the biotin reagent solution with the antibody solution in a ratio such that biotin is in excess, and incubating for a period of time;

4) and (4) dialyzing to remove the excessive biotin reagent to obtain the biotin-labeled rabbit-derived Benzonase polyclonal antibody.

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