CN117665292A - anti-DNase B detection kit and preparation method thereof - Google Patents

Abstract

The invention relates to the technical field of anti-DNase B detection, and discloses an anti-DNase B detection kit and a preparation method thereof, wherein the anti-DNase B detection kit comprises a reagent R1 and a reagent R2, and the reagent R1 comprises: tris; sodium chloride; tween; bovine serum albumin; sodium azide; polyethylene glycol; the solvent is purified water; reagent R2 includes: 3-morpholinopropane sulfonic acid; tween; sodium azide; a protective agent; antigen latex particles; the solvent is purified water. The invention provides the anti-DNase B detection kit which is simple, convenient, quick and accurate to operate, is suitable for clinical application and can be applied to a biochemical instrument, and the stability of the kit is improved by screening different protective agents.

Description

anti-DNase B detection kit and preparation method thereof

Technical Field

The invention relates to the technical field of anti-DNase B detection, in particular to an anti-DNase B detection kit and a preparation method thereof.

Background

Streptolysin O, streptokinase, hyaluronidase, deoxyribonuclease AND other substances produced by the streptococcus A can be paired AND combined with corresponding antibodies of anti-streptolysin O (ASO), anti-streptolysin (ASK), anti-hyaluronidase (AHD), anti-deoxyribonuclease B (AND-B) AND the like. Among them, type A Streptococcus produces type 4 deoxyribonuclease (A, B, C AND type D) in the maximum amount of type B deoxyribonuclease, so that the determination of AND-B is of great importance for diagnosis of Streptococcus infection. In some specific cases, the AND-B assay has the following advantages over the ASO assay: (1) AND-B is significantly elevated at streptococcal skin infection, while ASO titers rarely change; (2) In the case of acute rheumatic fever, acute glomerulonephritis AND other streptococcal infections, the duration of the increase in AND-B is longer than in ASO. In addition, AND-B is also of great importance in diagnosing chronic valvular disease where chorea or onset is not apparent. The world health organization has formally listed the AND-B assay as the second method for detecting Streptococcus antibodies following ASO.

The current method for clinically detecting group A hemolytic streptococcus (GAS) infected serum only comprises the steps of determination of anti-hemolysin O (ASO) and bacterial culture, and the positive rate of bacterial culture is reduced due to early application of antibiotics. In addition, the kinetics of the antibodies against ASO and DNase-B are different, the ASO reaction occurs earlier, peaks 3-5 weeks after GAS infection, and falls to normal for most 2 months; the anti-DNase-B antibody reaction is slightly behind ASO, the peak time is 4-6 weeks, the high concentration state of the antibody is longer than ASO, and the reaction can last for 3-6 months. Because of how light or atypical the clinical manifestations of rheumatic fever are, when ASO is detected, its peak period is passed, the positive rate is low, and the anti-DNase-B antibody exists in the body for a long time, and because of the different antibody dynamics of the two, it not only has important complementary action between them, but also has parallel and independent diagnostic action. The Jones standard revised in 1992 again underscores the diagnosis of RF, which must have evidence of GAS infection, otherwise the diagnosis cannot be established, and recommends anti-DNase-B detection as the best option for ASO removal.

The current methods for measuring the anti-DNase B antibody mainly comprise an enzyme-linked immunosorbent assay, a micro-titration method, an immunoturbidimetry method and the like. The detection methods have more or less defects, firstly, the ELISA method has complicated operation and long detection time, and is very inconvenient for a laboratory of a hospital; the microtitration also has the problem of longer detection time and is troublesome to operate; the conventional immunoturbidimetry has a problem of low sensitivity, and may not be accurately measured for early-stage infected persons. There is an urgent need for a detection method that is simple to operate and has high sensitivity to detect anti-DNase B antibodies.

Disclosure of Invention

(one) solving the technical problems

The invention provides a detection reagent which is simple, convenient, quick and accurate to operate, is suitable for clinical application and can be applied to a biochemical analyzer, and the development of the reagent fills the gap. The invention also improves the stability of the kit by screening different protective agents.

In order to achieve the above purpose, the present invention provides the following technical solutions:

in a first aspect, the present invention provides an anti-deoxyribonuclease B assay kit comprising a reagent R1 and a reagent R2;

wherein the reagent R1 comprises the following components in percentage by mass: sodium chloride 0.5-10%, tween 0.1-10%, benzenesulfonamide (benzene sulfonamide, BSA) 0.1-2%, preservative 0.05-0.2% and polyethylene glycol 0.1-5%, wherein the buffer solution of the reagent R1 is GOOD' S buffer solution;

the reagent R2 comprises 1-10mg/mL antigen latex particles, and the reagent R2 further comprises, in mass fraction: tween 0.1-10%, preservative 0.05-0.2% and protective agent 0.5-10%, wherein the buffer solution of the reagent R2 is GOOD' S buffer solution.

In the invention, the detection principle of the anti-DNase B detection kit is as follows: the anti-DNase B in the sample is combined with the DNase B antigen coated by the latex particles in the reagent to form an antigen-antibody complex, the complex is aggregated to generate a certain turbidity, the turbidity is proportional to the content of the antibody in the presence of the certain antigen, the turbidity is measured at the wavelength of 600nm, and the quantitative measurement of the anti-DNase B can be performed through a multi-point calibration curve.

The mass fraction of sodium chloride in the reagent R1 is 0.5-10%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, etc.

The mass fraction of tween in the reagent R1 is 0.1-10%, for example, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% and so on.

The mass fraction of benzenesulfonamide in the reagent R1 is 0.1-2%, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%, 1.2%, 1.3%, 1.5%, 1.6%, 1.7%, 1.8% or 2%, etc.

The mass fraction of the preservative in the reagent R1 is 0.05-0.2%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.1%, 0.12%, 0.14%, 0.15%, 0.16%, 0.18% or 0.2%, etc.

The mass fraction of polyethylene glycol in the reagent R1 is 0.1-5%, for example, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 1.8%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, etc.

The mass fraction of tween in the reagent R2 is 0.1-10%, for example, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% and so on.

The mass fraction of the preservative in the reagent R2 is 0.05-0.2%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.1%, 0.12%, 0.14%, 0.15%, 0.16%, 0.18% or 0.2%, etc.

The mass fraction of the protective agent in the reagent R2 is 0.5-10%, for example, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, etc.

The content of the antigen latex particles in the reagent R2 is 1-10mg/mL, and may be, for example, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, or the like.

As a preferable technical scheme of the invention, the protective agent is a combination of trehalose and glycerol, and the mass ratio of the trehalose to the glycerol is 1 (0.8-1.2), for example, 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2 or the like can be adopted.

Preferably, the buffer solution of the reagent R1 is any one of Tris buffer solution, 3-morpholinopropane sulfonic acid buffer solution, MES buffer solution or HEPES buffer solution.

Preferably, the reagent R1 is Tris buffer solution, and the molar concentration of Tris in the reagent R1 is 10-200mmol/L, for example, 10mmol/L, 20mmol/L, 30mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 70mmol/L, 80mmol/L, 90mmol/L, 100mmol/L, 110mmol/L, 120mmol/L, 130mmol/L, 140mmol/L, 150mmol/L, 160mmol/L, 170mmol/L, 180mmol/L, 190mmol/L or 200mmol/L, etc.

The reagent R1 has a pH of 6 to 8 and may be, for example, 6, 6.5, 7, 7.4, 7.5 or 8, etc.

The buffer solution in the reagent R2 is 3-morpholinopropane sulfonic acid (MOPS) buffer solution.

The molar concentration of 3-morpholinopropane sulfonic acid (MOPS) in the reagent R2 is 10-200mmol/L, and can be 10mmol/L, 20mmol/L, 30mmol/L, 50mmol/L, 60mmol/L, 80mmol/L, 100mmol/L, 120mmol/L, 140mmol/L, 150mmol/L, 160mmol/L, 180mmol/L or 200mmol/L, for example.

The reagent R2 has a pH of 6.9-7.5, and may be, for example, 6.9, 7.0, 7.05, 7.1, 7.2, 7.3, 7.4, 7.5, etc.

Preferably, tween in the reagent R1 is tween 20.

Polyethylene glycol in the reagent R1 is polyethylene glycol PEG-20000.

Tween in the reagent R2 is tween 20.

The preservative in the reagent R1 and the reagent R2 is respectively and independently selected from sodium azide, thimerosal or Proclin300.

In the invention, the antigen in the antigen latex particles is purified deoxyribonuclease B or deoxyribonuclease B expressed by genetic engineering;

the antigen is wild, mutant or modified deoxyribonuclease B.

As a preferable technical scheme, the antigen in the antigen latex particles is deoxyribonuclease B expressed by genetic engineering and subjected to mutation;

the invention selects the antigen with higher antigenicity and specificity and expressed on the escherichia coli, and the specific preparation is as follows:

the protein sequence coded by DNase B gene is mutated by adopting PCR in vitro site-directed mutagenesis (PCR-SDM), and mutation sites are H264R, S238P and I167V, specifically the mutation: mutation of 264 His (CAT) to Arg (CGT), 238 Ser (TCA) to Pro (CCC), 167 Ile (ATT) to Val (GTT) was confirmed by determining the nucleotide sequence of the target gene fragment after ligation with the T vector. The restriction enzymes EcoRI and HindIII are used for cutting the gene fragment of the plasmid Pmd19-T-DNase B, and then the gene fragment is directionally cloned into the expression vector pET-28a (+) cut by the same way, thus constructing the recombinant expression plasmid pET-28a (+) -DNase B. Extracting the plasmid, and carrying out colony PCR and enzyme digestion identification to prove that the DNase B gene fragment is inserted into an expression vector in the correct direction;

NCBI accession number WP_011055059,streptodornase B[Streptococcus pyogenes of the protein sequence of DNase B), the specific sequence is:

MNLLGSRRVFSKKCRLVKFSMVALVSATMAVTTVTLENTALARQTQVSNDVVLNDGASKYLNEALAWTFNDSPNYYKTLGTSQITPALFPKAGDILYSKLDELGRTRTARGTLTYANVEGSYGVRQSFGKNQNPAGWTGNPNHVKYKIEWLNGLSYVGDFWNRSHLIADSLGGDALRVNAVTGTRTQNVGGRDQKGGMRYTEQRAQEWLEANRDGYLYYEVAPIYNADELIPRAVVVSMQSSDNTINEKVLVYNTANGYTINYHNGTPTQK(SEQ ID NO.1)

extracting recombinant plasmid containing mutant DNase B gene, converting the recombinant plasmid into escherichia coli BL21 (DE 3), inducing fusion protein expressed by IPTG, detecting the molecular weight of about 29KD by SDS-PAGE, and scanning by gel imager software to obtain DNase B protein and obtaining high-efficiency expression in escherichia coli; after fermentation of the target protein is completed, the target protein is obtained through centrifugation, cell breaking, centrifugation, washing and column elution, and the obtained target protein can meet the preparation requirement of the kit through SDS-PAGE electrophoresis detection.

In the present invention, the anti-deoxyribonuclease B assay kit comprises:

reagent R1 includes: tris, 10-200mmol/L; sodium chloride, 0.5-10%; tween, 0.1-10%; benzene sulfonamide, 0.1-2%; sodium azide, 0.05-0.2%; polyethylene glycol, 0.1-5%; the solvent is purified water;

reagent R2 includes: 3-morpholinopropane sulfonic acid (MOPS), 10-200mmol/L; tween, 0.1-10%; sodium azide, 0.05-0.2%; 0.5-10% of protective agent; antigen latex particles, 1-10mg/mL; the solvent is purified water.

On the other hand, the invention also provides a preparation method of the anti-DNase B detection kit, which comprises the following steps:

the preparation process of the reagent R1 comprises the following steps: adding sodium chloride after preparing a buffer solution, adding bovine serum albumin, polyethylene glycol, tween and a preservative after dissolving, adjusting pH after complete dissolution, and fixing volume to obtain a reagent R1;

the preparation process of the reagent R2 comprises the following steps: measuring purified water, adding antigen latex particles, adding a protective agent after dissolution, adding a buffer solution solvent, tween and a preservative after dissolution, and regulating pH and fixing volume after complete dissolution to obtain a reagent R2.

For example, the kit of the present invention can be prepared as follows:

the preparation process of the reagent R1 comprises the following steps: weighing purified water, adding Tris, dissolving, adding sodium chloride, dissolving, adding BSA, dissolving, adding PEG, dissolving, adding Tween, adding sodium azide, completely dissolving, regulating pH with hydrochloric acid, and fixing volume to obtain reagent R1;

the preparation process of the reagent R2 comprises the following steps: weighing purified water, adding antigen latex particles, dissolving, adding a protective agent, dissolving, adding MOPS, dissolving, adding tween, adding sodium azide, completely dissolving, regulating pH with hydrochloric acid, fixing volume to obtain a reagent R2, and standing the obtained R2 at 4 ℃ for preservation;

wherein, the main component of R2 is latex particles coated with anti-DNase B antigen, and the following steps are specific preparation steps of the latex particles coated with antigen:

(1) activation of microspheres: adding purified water into the microspheres, shaking and mixing uniformly, adding EDC, shaking and mixing uniformly, and shaking in a shaking table;

(2) antigen coupling: adding antigen after activation, shaking and mixing uniformly, and shaking in a shaking table;

(3) closing: centrifuging the coupled antigen microsphere mixed solution by using a centrifuge, removing supernatant to obtain precipitate, re-dissolving and re-suspending the precipitate by using a cleaning solution, performing ultrasound, adding a sealing solution, uniformly oscillating and mixing, and oscillating in a shaking table to prepare the antigen latex particles.

In a third aspect, the present invention provides a method for using the anti-dnase B detection kit of the first aspect in non-disease diagnosis, where the anti-dnase B detection kit is used in combination with a biochemical analyzer, and the specific steps include:

and after mixing and reacting a sample to be detected with the reagent R1 and the reagent R2, detecting the absorbance of the solution, and obtaining the concentration of the anti-DNase B in the sample to be detected according to the change value of the absorbance.

Compared with the prior art, the invention has the following beneficial technical effects:

firstly, the invention obtains the composition taking trehalose and glycerol as protective agents, the trehalose can activate DNaseI at 37 ℃, the trehalose stabilizes and activates proteins by influencing protein hydration, the hydration of proteins in solution can be reduced, the glycerol can reduce the activity of water, degradation is prevented and soluble proteins are protected, and the stability and activity of heat-sensitive enzymes at high temperature can be increased by the synergistic effect between the trehalose and the glycerol, so that the stability of the kit is improved.

Secondly, the invention selects the antigen with higher antigenicity and specificity and expressed on the escherichia coli, and effectively solves the technical problems that the natural crude extract contains known and unknown streptococcus products, can interfere the detection result and amplify the detection data, and leads the detection result to deviate, thereby failing to meet the clinical detection requirement.

Thirdly, the anti-DNase B detection kit provided by the invention can be used in a common biochemical analyzer, and compared with the detection kit in the prior art, which needs to be analyzed in a specific protein analyzer, the application range of the detection kit is wider, and for a common detection center such as a hospital and a physical examination center, the biochemical analyzer is a common device, and the detection kit does not need to purchase an expensive specific protein analyzer, so that the detection cost is reduced, and the popularization rate of the anti-DNase B detection is further effectively improved.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. Embodiments and features of embodiments in this application may be combined with each other without conflict. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The reagents used in the examples below are all commercially available from manufacturers routine in the art; the experimental methods used are all conventional experimental methods known to the person skilled in the art.

Example 1

The invention provides an anti-DNase B detection kit, which comprises:

the reagent R1 comprises the following components in percentage by weight: TRIS buffer, 30mmol/L; tween 20, 0.5%; benzene sulfonamide BSA, 0.5%; sodium chloride, 2%; sodium azide, 0.1%; polyethylene glycol PEG-20000, 1%; the pH was 7.40; the solvent is purified water;

the preparation process of the reagent R1 comprises the following steps: weighing 800mL of purified water, adding 3.6342g of Tris, dissolving, adding 20g of sodium chloride, dissolving, adding 5g of BSA, dissolving, adding 10g of PEG20000, dissolving, adding 5mL of Tween 20, adding 1g of sodium azide, regulating the pH to 7.40 by using hydrochloric acid after complete dissolution, and fixing the volume to 1L;

the reagent R2 comprises the following components in percentage by weight: 3-morpholinopropane sulfonic acid (MOPS), 100mmol/L; tween 20, 0.5%; sodium azide, 0.1%; a protectant (a combination of 5% trehalose and 5% glycerol); antigen latex particles, 1mg/mL; the solvent is purified water;

the preparation process of the reagent R2 comprises the following steps: weighing 800mL of purified water, adding 1mg of antigen latex particles, dissolving, adding 5g of trehalose and 5g of glycerol as a combined protective agent, dissolving, adding 2.09g of MOPS, dissolving, adding 0.5mL of Tween 20, adding 0.1g of sodium azide, regulating the pH to 7.05 by using hydrochloric acid after complete dissolution, and fixing the volume to 100mL;

wherein, the main component of R2 is latex particles coated with anti-DNase B antigen, the particle diameter of the latex microsphere of the reagent is 141nm, and the specific preparation steps of the latex particles coated with antigen are as follows:

(1) activation of microspheres: taking 400 mu L of microspheres (P0112 microspheres of JSR company), adding 2mL of purified water, shaking and mixing uniformly, adding 300 mu L of EDC (10 mg/mL), shaking and mixing uniformly, and shaking in a shaking table at 25 ℃ for 15 minutes;

(2) antigen coupling: after activation, 450 mu L (1 mg/mL) of antigen is added, mixed evenly by shaking, and the mixture is shaken for 90 minutes in a shaking table at 25 ℃;

(3) closing: centrifuging the coupled antigen microsphere mixed solution by using a centrifuge, removing supernatant, taking precipitate, re-dissolving and re-suspending by using 15mL of cleaning solution (formula of the cleaning solution: 100mmol/L of Tris buffer solution, pH=7.05), performing ultrasound, adding 400 mu L of sealing solution (finished product purchased to JSR: CE 210), oscillating and mixing uniformly, and oscillating in a shaking table at 25 ℃ for 120 minutes to prepare antigen latex particles;

example 2

The invention provides a use method of an anti-DNase B detection kit, which specifically comprises the following steps:

the anti-DNase B assay kit provided in example 1 was used to perform anti-DNase B assay on a biochemical analyzer, the parameters of which are shown in Table 1 below, and the steps of which are shown in Table 2 below;

TABLE 1

Dominant wavelength	600nm	Secondary wavelength	/
				Measurement method	Endpoint method	Reaction direction	Positive and negative reactions

TABLE 2

Calibration procedure: and (3) calibrating by using a calibration standard with a fixed value, diluting the calibration standard with a multiple ratio to 5 points of 1, 1/2, 1/4, 1/8 and 0, and calibrating to obtain a calibration curve.

Kit feasibility test protocol:

in order to prove that the anti-DNase B detection kit provided by the invention can be used for measuring the anti-DNase B antibody on a biochemical instrument, the invention implements the following test scheme, and the test scheme is as follows:

and (3) comparing the reagent: beckmann reagent, instrument IMMAGE;

the instrument used for self-made reagent is Hitachi 7180 biochemical instrument;

experiment one: 50 normal human samples were tested (negative for anti-DNase B concentration < 187U/mL), and specific test results are shown in Table 1-1 below;

TABLE 1-1

As can be seen from Table 1-1, both detection methods were negative in 50 normal human samples.

Experiment II: 25 positive samples were assayed (anti-DNase B concentration >187U/mL positive) and specific test results are shown in tables 1-2 below;

TABLE 1-2

As is clear from tables 1 to 2, all of the 25 positive samples were detected as positive.

As can be seen from the measurement results in tables 1-1 and 1-2, the kit of the present invention can completely measure the anti-DNase B antibody on a biochemical analyzer.

Kit thermal stability test protocol:

because the latex microspheres in the R2 reagent are coated with the enzyme type anti-deoxyribonuclease B, the anti-deoxyribonuclease B is sensitive to temperature in the system, and a protective agent such as bovine serum albumin, sucrose, trehalose, glycerol and the like is required to be added in a 37 ℃ stability test, in order to obtain a kit with better thermal stability, the following protective agent screening test is carried out.

The experimental scheme is designed as follows:

experiment one: bovine serum albumin is selected as a protective agent in the R2 reagent, and the mass fraction is 5%;

experiment II: sucrose is selected as a protective agent in the R2 reagent, and the mass fraction is 10%;

experiment III: trehalose is selected as a protective agent in the R2 reagent, and the mass fraction is 10%;

experiment IV: glycerol is selected as a protective agent in the R2 reagent, and the mass fraction is 10%;

experiment five: the combination of trehalose and glycerol is selected as a protective agent in the R2 reagent, the content of trehalose is 5%, and the content of glycerol is 5%.

Experiment one: bovine serum albumin was selected as a protective agent in the R2 reagent, and the effect of the protective agent on reagent stability is shown in table 2-1 below; wherein, the content of bovine serum albumin is 5%.

TABLE 2-1

Experiment II: sucrose was selected as a protective agent in the R2 reagent, and the effect of this protective agent on reagent stability is shown in table 2-2 below; wherein the content of sucrose is 10%.

TABLE 2-2

Serum	Low value	Median value	High value
				Accelerating for one day at 37 DEG C	-8.5％	-7.9％	-8.1％
Accelerating for two days at 37 DEG C	-11.2％	-10.8％	-10.4％
				Accelerating for three days at 37 DEG C	-14.5％	-13.8％	-14.3％
Accelerating for four days at 37 DEG C	-18.6％	-18.3％	-17.9％
				Accelerating for five days at 37 DEG C	-21.5％	-22.3％	-20.6％
Accelerating for six days at 37 DEG C	-23.4％	-25.1％	-23.1％
				Accelerating for seven days at 37 DEG C	-24.9％	-25.8％	-24.9％
Accelerating for ten days at 37 DEG C	-27.8％	-28.2％	-27.9％

Experiment III: trehalose was selected as a protective agent in the R2 reagent, and the effect of this protective agent on reagent stability is shown in tables 2 to 3 below; wherein the content of trehalose is 10%.

Tables 2 to 3

Serum	Low value	Median value	High value
				Accelerating for one day at 37 DEG C	-4.8％	-3.7％	-4.1％
Accelerating for two days at 37 DEG C	0.6％	0.2％	0.4％
				Acceleration at 37 DEG CThree days	1.5％	1.4％	1.7％
Accelerating for four days at 37 DEG C	1.9％	2.0％	2.1％
				Accelerating for five days at 37 DEG C	2.8％	2.6％	2.8％
Accelerating for six days at 37 DEG C	3.5％	3.1％	3.0％
				Accelerating for seven days at 37 DEG C	4.1％	4.0％	3.8％
Accelerating for ten days at 37 DEG C	4.9％	4.6％	4.2％

Experiment IV: glycerol was selected as a protective agent in the R2 reagent, and the effect of this protective agent on reagent stability is shown in tables 2-4 below; wherein the content of glycerol is 10%.

Tables 2 to 4

Serum	Low value	Median value	High value
				Accelerating for one day at 37 DEG C	-5.9％	-4.8％	-5.1％
Accelerating for two days at 37 DEG C	-6.2％	-5.8％	-6.0％
				Accelerating for three days at 37 DEG C	-6.8％	-6.3％	-6.5％
Accelerating for four days at 37 DEG C	-7.2％	-7.1％	-6.9％
				Accelerating for five days at 37 DEG C	-7.5％	-7.4％	-7.4％
Accelerating for six days at 37 DEG C	-7.5％	-7.5％	-7.3％
				Accelerating for seven days at 37 DEG C	-7.2％	-7.0％	-7.6％
Accelerating for ten days at 37 DEG C	-6.9％	-6.6％	-7.0％

Experiment five: the combination of trehalose and glycerol was selected as a protectant in the R2 reagent, the effect of which on reagent stability is shown in tables 2-5 below; wherein the content of trehalose is 5%, and the content of glycerol is 5%.

Tables 2 to 5

Serum	Low value	Median value	High value
				Accelerating for one day at 37 DEG C	-2.8％	-3.0％	-2.8％
Accelerating for two days at 37 DEG C	-2.5％	-2.8％	-2.6％
				Accelerating for three days at 37 DEG C	-2.0％	-2.2％	-2.3％
Accelerating for four days at 37 DEG C	-1.8％	-1.7％	-2.0％
				Accelerating for five days at 37 DEG C	-1.3％	-1.5％	-1.7％
Accelerating for six days at 37 DEG C	-0.9％	-0.9％	-1.0％
				Accelerating for seven days at 37 DEG C	0.2％	0.1％	-0.3％
Accelerating for ten days at 37 DEG C	0.8％	0.6％	0.4％

As can be seen from the effect data in tables 2-1, 2-2, 2-3, 2-4 and 2-5, the heat stability effect of the reagent was better with the combination of 5% trehalose and 5% glycerol as the protective agent.

The action principle of the combination of trehalose and glycerol as a protective agent is as follows: trehalose can activate DNaseI at 37 ℃, trehalose is added, enzyme activity is still obviously improved when the temperature is increased to 50 ℃, the trehalose stabilizes and activates proteins by influencing protein hydration, the hydration of proteins in a solution can be reduced, glycerol can reduce the activity of water, degradation is prevented and soluble proteins are protected, and trehalose and glycerol are simultaneously added into a reaction system, so that the stability and activity of heat-sensitive enzymes at high temperature can be improved through the synergistic effect between the trehalose and the glycerol.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (8)

1. An anti-deoxyribonuclease B detection kit, which is characterized by comprising a reagent R1 and a reagent R2;

wherein the reagent R1 comprises the following components in percentage by mass: 0.5-10% of sodium chloride, 0.1-10% of tween, 0.1-2% of bovine serum albumin, 0.05-0.2% of preservative and 0.1-5% of polyethylene glycol, wherein the buffer solution of the reagent R1 is GOOD' S buffer solution;

the reagent R2 comprises 1-10mg/mL antigen latex particles, and the reagent R2 further comprises, in mass fraction: tween 0.1-10%, preservative 0.05-0.2% and protective agent 0.5-10%, wherein the buffer solution of the reagent R2 is GOOD' S buffer solution.

2. The anti-deoxyribonuclease B assay kit of claim 1 wherein the protectant is a combination of trehalose and glycerol;

the mass ratio of the trehalose to the glycerol is 1 (0.8-1.2);

the buffer solution of the reagent R1 is any one of Tris buffer solution, 3-morpholinopropane sulfonic acid buffer solution, MES buffer solution or HEPES buffer solution;

the pH value of the reagent R1 is 6-8;

the buffer solution in the reagent R2 is 3-morpholinopropane sulfonic acid buffer solution;

the molar concentration of the 3-morpholinopropane sulfonic acid MOPS in the reagent R2 is 10-200mmol/L;

the pH of the reagent R2 is 6.9-7.5.

3. The kit for detecting the anti-DNase B according to claim 1, wherein tween in the reagent R1 is tween 20, tween 40, tween 60 or tween 80;

the polyethylene glycol in the reagent R1 is polyethylene glycol PEG-4000, polyethylene glycol PEG-8000 or polyethylene glycol PEG-20000;

tween in the reagent R2 is tween 20, tween 40, tween 60 or tween 80;

the preservative in the reagent R1 and the reagent R2 is respectively and independently selected from sodium azide, thimerosal or Proclin300.

4. The kit for detecting the anti-DNase B according to claim 1, wherein the antigen in the antigen latex particles is purified DNase B or DNase B expressed by genetic engineering;

the antigen is wild, mutant or modified deoxyribonuclease B.

5. The kit for detecting the anti-DNase B according to claim 4, wherein the antigen in the antigen latex particles is the DNase B expressed by genetic engineering and subjected to mutation;

the mutation sites are H264R, S238P and I167V.

6. A method for preparing an anti-dnase B assay kit according to any one of claims 1 to 5, comprising the steps of:

the preparation process of the reagent R1 comprises the following steps: adding sodium chloride after preparing a buffer solution, adding benzenesulfonamide, polyethylene glycol, tween and a preservative after dissolving, adjusting pH after complete dissolution, and fixing volume to obtain a reagent R1;

the preparation process of the reagent R2 comprises the following steps: measuring purified water, adding antigen latex particles, adding a protective agent after dissolution, adding a buffer solution solvent, tween and a preservative after dissolution, and regulating pH and fixing volume after complete dissolution to obtain a reagent R2.

7. The method of claim 6, wherein the step of preparing the antigen latex particles comprises:

adding purified water into the microspheres, oscillating and mixing, then adding EDC for activation, and adding antigen for coupling after the activation is finished;

centrifuging the antigen microsphere mixed solution obtained after coupling, removing supernatant to obtain precipitate, re-dissolving and re-suspending the precipitate with a cleaning solution, performing ultrasound, adding a sealing solution, and uniformly oscillating and mixing to obtain the antigen latex particles.

8. A method of using the anti-dnase B assay kit of any one of claims 1 to 5 in non-disease diagnosis, wherein the anti-dnase B assay kit is used in combination with a biochemical analyzer, comprising the steps of:

and after mixing and reacting a sample to be detected with the reagent R1 and the reagent R2, detecting the absorbance of the solution, and obtaining the concentration of the anti-DNase B in the sample to be detected according to the change value of the absorbance.