CN104614523A - A mycobacterium tuberculosis TB-SA antibody enzyme-linked immunosorbent assay detection kit and a preparing method thereof - Google Patents

Abstract

A mycobacterium tuberculosis TB-SA antibody enzyme-linked immunosorbent assay detection kit and a preparing method thereof are disclosed. The detection kit comprises a TB-SA antibody-coated plate, a sample diluting solution, an enzyme labeling solution, a concentration washing solution, a color developing agent A solution, a color developing agent B solution, a negative control, a positive control and a stop solution. The preparing method includes: preparing the TB-SA antibody-coated plate, preparing the sample diluting solution, preparing the enzyme labeling solution, preparing the concentration washing solution, preparing the controls and preparing the color developing solutions. The kit is high in sensitivity and high in specificity, so that interferences caused by false-positive or false-negative phenomena to clinical diagnosis are reduced, and the kit can rapidly and accurately detect a mycobacterium tuberculosis TB-SA antibody and is small in error and high in detection sensitivity. The kit is convenient to prepare and the preparing method is simple in operation, so that the kit and the preparing method are suitable for industrial large-scale production.

Description

Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit and preparation method thereof

Technical field

The present invention relates to Biological Detection technical field, be specifically related to Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit and preparation method thereof.

Background technology

Tuberculosis, acquired immune deficiency syndrome (AIDS) are together with malaria, and be regarded as affecting the whole world sanitarian three large diseases so far, tuberculosis is one of major disease of China's priority control, is also public health problem and the social concern of global concern.

According to the 5th the tuberculosis epidemiology investigation display of the World Health Organization (WHO) (WHO) and the whole nation: the whole world has the population of nearly 1/3 to infect Much's bacillus at present, and 8,000,000 new cases that have an appointment every year occur, and die from this disease close to 3,000,000 people.China is one of 22 tuberculosis high burden countries in the whole world, and tuberculosis patient quantity occupies second place of the world.Have pulmonary tuberculosis patient about 4,500,000 at present, tuberculosis year number of the infected about 1,300,000, the phthisical incidence of disease of China is 4,59/,100,000 people, accounts for 14.3% of whole world morbidity, every year because tuberculosis death toll reaches 130,000, substantially exceeds the summation of animal infectious diease death toll.

Tuberculosis is infectious diseases common to human beings and animals, can infect the crowd in each stage, and the 5th epidemiology survey shows, and with advancing age, the incidence of disease of tuberculosis patient increases.Have report display, infectious M patient may infect 10 ~ 15 people in 1 year.In the face of so severe situation, we should take measures early.Early diagnosis lungy and effectively treatment are the most important things of tuberculosis prophylaxis work, and propagating for control tulase has important clinical meaning.Current most domestic hospital diagnoses activity combination according to direct smear microscopy and Much's bacillus culture technique.Although tulase smear detects simple and accuracy is high, positive rate is low.Tulase cultivation detection confidence is high, but consuming time longer, can not meet clinical demand.In addition, it is the antigen mixture that Much's bacillus is slightly carried that tuberculin skin test (PPD) tests antigen used, all there is common antigen composition with many non-tuberculous mycobacterias and Bacille Calmette-Guerin (BCG), thus cause this method for diagnosis of tuberculosis poor specificity.China is tuberculosis high prevalence district, owing to extensively inoculating BCG, also causes PPD to test false positive rate very high, so the reaction power can only testing skin according to PPD clinically gives auxiliary diagnosis.

Summary of the invention

Namely object of the present invention is to overcome the deficiencies in the prior art, provides a kind of and can detect Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit in sample by fast qualitative.

Another object of the present invention is to the preparation method that Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit is provided.

The object of the invention is to be achieved through the following technical solutions: Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit, described detection kit is made up of TB-SA antigen coated microplate, sample diluting liquid, enzyme labeling liquid, concentrated washing lotion, developer A liquid, developer B liquid, negative controls, positive reference substance and stop buffer.

Further, described TB-SA antigen coated microplate is the microwell plate being coated with TB-SA antigen, and described TB-SA antigen phosphate buffer dilutes, and its bag is 0.01mg/ml by concentration.

Further, described sample diluting liquid is the damping fluid containing Tween-20 and albumen.

Further, described enzyme labeling liquid is the staphylococcal protein A solution containing horseradish peroxidase-labeled.

Further, described concentrated washing lotion is the damping fluid containing Tween-20.

Further, described developer A liquid is the solution containing urea peroxide, and developer B liquid is the solution containing TMB.

Further, described positive reference substance is the serum solution containing TB-SA antibody positive, and negative controls is the serum solution containing TB-SA negative antibody.

Further, described stop buffer is vitriolated solution.

The preparation method of Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit, it comprises the following steps:

The preparation of S1.TB-SA antigen coated microplate: extremely being wrapped by concentration by TB-SA antigen diluent with phosphate buffer is 0.01mg/ml, is coated in by coating buffer on microwell plate, preserves after 35 ~ 40 DEG C of dryings;

S2. the preparation of sample diluting liquid: with 10 times of concentrated phosphate buffer 500ml, add casein 5.00g, adds purified water and is stirred to casein and fully dissolves; Add the Tween-20 of 3ml again, add purified water after mixing to 1000ml, fully mix, preserve;

S3. the preparation of enzyme labeling liquid: be 10 times of concentrated phosphate buffer 300ml with pH, adds purified water to 3000ml, horseradish peroxidase target staphylococcal protein A is diluted to 1:4 ten thousand ~ 1:6 ten thousand, preserve;

S4. the preparation of concentrated washing lotion: take sodium chloride 300g, sodium hydrogen phosphate 85g adds after 1000ml purified water dissolves, then add Tween-20 15ml, add to 1300ml by purified water, stir and evenly mix rear preservation;

S5. the preparation of reference substance

S51. the preparation of positive reference substance: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA antibody positive after stirring and evenly mixing, preserve after mixing;

S52. the preparation of negative controls: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA negative antibody after stirring and evenly mixing, preserve after mixing;

S6. the preparation of nitrite ion:

S61. the preparation of developer A liquid: take citric acid 0.9g, urea peroxide 0.1g, after adding purified water 100ml dissolving, adds purified water to 170ml, fully preserves after mixing;

S62. the preparation of developer B liquid: add glycerine 17ml after being dissolved by 3g citric acid by 100ml purified water, add the 0.04g TMB dissolved with 3ml DMSO after mixing, add purified water to 170ml, preserve;

S7. the preparation of stop buffer: add purified water to 180ml after purified water 150ml and the sulfuric acid 18ml measured being mixed, preserve.

The Cleaning Principle of Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit of the present invention:

This kit is at microwell plate endoperidium TB-SA antigen, if have TB-SA antibody to combine with it in sample to be tested, then react with enzymic-labelled antibody, formed " Ag-Ab-multienzyme complex ", enzymatic chromogenic reagent, can judge the presence or absence of TB-SA antibody according to colour developing degree.

It is serum that this kit detects sample, and the sample of separation can store 7 days under 2-8 DEG C of preservation, and longer-term storage should put-20 DEG C, avoids multigelation, should balance to room temperature, and fully mix before the sample use of refrigeration or freezen protective.

The detection method of this kit is:

1. reagent prepares: by kit balance to room temperature, by purified water, concentrated washing lotion is done 1:20 dilution, is namely made into wash operating solution for subsequent use.

2. application of sample: antigen coated for the TB-SA of requirement lath is fixed on grillage, each hole adds 100 μ l Sample dilution, adds 5 μ l samples to be tested according to the order of sequence; Blank (can not establish blank with during double UV check), each 2 holes of negative control, positive control are established in each experiment, add Sample dilution, negative controls, each 100 μ l/ holes of positive reference substance respectively; Concussion mixing, shrouding, 37 DEG C of incubations 10 minutes, wash plate 5 times with wash operating solution, pat dry.

3. enzyme-added: every hole adds enzyme labeling liquid 100 μ l, concussion mixing, shrouding, 37 DEG C of incubations 10 minutes, wash plate 5 times with wash operating solution, pat dry.

4. value is read in colour developing: every hole adds each 50 μ l of developer A, B liquid, and mixing, shrouding, puts 37 DEG C of lucifuges and develop the color 10 minutes.Every hole adds stop buffer 50 μ l, mixing, completes result and measure in 10 minutes.Single wavelength measures: available blank contrast is withered, measures the OD value in each hole under microplate reader 450nm wavelength; Double UV check: measure each hole OD value under microplate reader 450nm/630nm wavelength.

The OD value of negative control answers <0.15.The OD value of positive control answers >0.80.

The explanation of assay:

1. critical value (Cutoff) calculates: Cutoff=negative controls OD value+0.2

2. the judgement of assay:

[the OD value of sample]≤[Cutoff] is: TB-SA negative antibody.

[the OD value of sample] > [Cutoff] is: TB-SA antibody positive.

The invention has the beneficial effects as follows: this kit is at microwell plate endoperidium TB-SA antigen, if there is TB-SA antibody to combine with it in sample to be tested, react with enzymic-labelled antibody again, formed " Ag-Ab-multienzyme complex ", enzymatic chromogenic reagent, can judge the presence or absence of TB-SA antibody according to colour developing degree.The present invention is highly sensitive, high specificity, therefore, the present invention not only decreases the interference that false positive or false negative phenomenon are brought clinical diagnosis, this kit is detected fast and accurately to Much's bacillus TB-SA antibody, and error is little, detection sensitivity is high; This kit is easy to prepare, and preparation method is simple to operate, is suitable for industrialization large-scale production.

Accompanying drawing explanation

Fig. 1 is the P/N curve map of sample differential responses time in embodiment 1;

Fig. 2 is the P/N curve map of enzyme labeling liquid differential responses time in embodiment 1.

Embodiment

Below in conjunction with embodiment, the present invention will be further described, and protection scope of the present invention is not limited to the following stated:

Embodiment 1:

1. the determination in sample reaction time

We have selected positive reference material and the negative reference product of different antibodies level, according to the dilution ratio determined, (dilution ratio ask clear and definite different antibodies level, positive reference material, negative reference product, determining) measures the result of 10min, 20min, 30min, 45min and 1 hour, the 10min of experimental result display for several times best results, add up as shown in table 1:

Table 1: the result difference of sample differential responses time

The sample reaction time	Positive reference material mean value (P)	Negative reference product mean value (N)	P/N
				10min	1.40	0.148	9.40
20min	1.41	0.156	9.09
				30min	1.44	0.162	8.89
45min	1.46	0.166	8.82
				60min	1.50	0.173	8.63

2. the determination in enzyme labeling liquid reaction time

In enzyme labeling liquid, the main material participating in reaction is horseradish peroxidase mark SPA, SPA can specificly be attached on IgG, forming IgG-SPA-horseradish peroxidase complex is linked on microwell plate, in final apertures, the content of horseradish peroxidase mark SPA and IgG is proportionate, so the content suitably improving enzyme can reduce the reaction time.We determine 10min, 20min, 30min result, experimental result display 10min best results, add up as shown in table 2:

Table 2: the result difference of enzyme labeling liquid differential responses time

The sample reaction time	Positive reference material mean value (P)	Negative reference product mean value (N)	P/N
				10min	1.234	0.137	9.03
20min	1.308	0.147	8.91

30min

1.450

0.163

8.88

3. the determination of chromogenic reaction time

The Color Appearance System that kit uses is current general TMB Color Appearance System, in like product, developing time is substantially at 10min and 15min, we design the developing time of 10min, guarantee to develop the color when 10min linear, through the adjustment to the adjustment of pH value, key reaction composition urea peroxide and TMB, make under the catalytic action of horseradish peroxidase, 10min developer presents good linear relationship, R ²reach 0.99, meet the request for utilization of qualitative product completely.The chromogenic reaction time is for being defined as 10min.

4. the determination of enzyme labeling liquid concentration

According to the requirement of " euzymelinked immunosorbent assay (ELISA) detects reagent registration technology examination governing principle ", measure the result under different enzyme concentration, guaranteeing that positive reference material and negative reference product all pass through, and under the prerequisite that negative background is lower, positive and negative ratio (P/N value) is large as far as possible, pulls open differentiation effect.Concrete deterministic process is as follows:

1. according to technological requirement, wrap by good TB-SA bag by plate and other component fluids.

2. horseradish peroxidase mark SPA is made gradient dilution, as 1:2 ten thousand, 1:3 ten thousand, 1:4 ten thousand, 1:5 ten thousand, 1:6 ten thousand etc.

3. detect the negative reference product and positive reference material that prepare, compare the difference that concentration is different, be decided to be best working concentration with what meet above-mentioned requirements most.

Test result display for several times, current process uses the concentration of enzyme labeling liquid between 1:4 ten thousand ~ 1:6 ten thousand, but requires often to criticize to debug by this scheme, determines best enzyme concentration.

Embodiment 2: Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit, described detection kit is made up of TB-SA antigen coated microplate, sample diluting liquid, enzyme labeling liquid, concentrated washing lotion, developer A liquid, developer B liquid, negative controls, positive reference substance and stop buffer.Specification and quantity are: 48T.

TB-SA antigen coated microplate is the microwell plate being coated with TB-SA antigen, and TB-SA antigen phosphate buffer dilutes, and its bag is 0.01mg/ml by concentration.48 hole × 1 piece.

Sample diluting liquid is the damping fluid containing Tween-20 and albumen.5ml × 1 bottle.

Enzyme labeling liquid is the staphylococcal protein A solution containing horseradish peroxidase-labeled.5ml × 1 bottle.

Concentrated washing lotion is the damping fluid containing Tween-20.25ml × 1 bottle.

Developer A liquid is the solution containing urea peroxide, 3ml × 1 bottle.Developer B liquid is the citric acid solution containing TMB, 3ml × 1 bottle.

Positive reference substance is the serum solution containing TB-SA antibody positive, 1ml × 1 bottle.Negative controls is the serum solution containing TB-SA negative antibody, 1ml × 1 bottle.

Stop buffer is vitriolated solution, 3ml × 1 bottle.

Storage condition and the term of validity: kit puts 2 ~ 8 DEG C

The preparation method of above-mentioned Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit, it comprises the following steps:

The preparation of S1.TB-SA antigen coated microplate: extremely being wrapped by concentration by TB-SA antigen diluent with phosphate buffer is 0.01mg/ml, is coated in by coating buffer on microwell plate, preserves after 37 DEG C of dryings;

S2. the preparation of sample diluting liquid: with 10 times of concentrated phosphate buffer 500ml, add casein 5.00g, adds purified water and is stirred to casein and fully dissolves; Add the Tween-20 of 3ml again, add purified water after mixing to 1000ml, fully mix, preserve;

S3. the preparation of enzyme labeling liquid: be 10 times of concentrated phosphate buffer 300ml with pH, adds purified water to 3000ml, horseradish peroxidase target staphylococcal protein A is diluted to 1:4 ten thousand ~ 1:6 ten thousand, preserve;

S4. the preparation of concentrated washing lotion: take sodium chloride 300g, sodium hydrogen phosphate 85g adds after 1000ml purified water dissolves, then add Tween-20 15ml, add to 1300ml by purified water, stir and evenly mix rear preservation;

S5. the preparation of reference substance

S51. the preparation of positive reference substance: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA antibody positive after stirring and evenly mixing, preserve after mixing;

S52. the preparation of negative controls: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA negative antibody after stirring and evenly mixing, preserve after mixing;

S6. the preparation of nitrite ion:

S61. the preparation of developer A liquid: take citric acid 0.9g, urea peroxide 0.1g, after adding purified water 100ml dissolving, adds purified water to 170ml, fully preserves after mixing;

S62. the preparation of developer B liquid: add glycerine 17ml after being dissolved by 3g citric acid by 100ml purified water, add the 0.04g TMB dissolved with 3ml DMSO after mixing, add purified water to 170ml, preserve;

S7. the preparation of stop buffer: add purified water to 180ml after purified water 150ml and the sulfuric acid 18ml measured being mixed, preserve.

Embodiment 3: Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit, described detection kit is made up of TB-SA antigen coated microplate, sample diluting liquid, enzyme labeling liquid, concentrated washing lotion, developer A liquid, developer B liquid, negative controls, positive reference substance and stop buffer.Specification and quantity are: 96T.

TB-SA antigen coated microplate is the microwell plate being coated with TB-SA antigen, and TB-SA antigen phosphate buffer dilutes, and its bag is 0.01mg/ml by concentration.96 hole × 1 piece.

Sample diluting liquid is the damping fluid containing Tween-20 and albumen.10ml × 1 bottle.

Enzyme labeling liquid is the staphylococcal protein A solution containing horseradish peroxidase-labeled.10ml × 1 bottle.

Concentrated washing lotion is the damping fluid containing Tween-20.50ml × 1 bottle.

Developer A liquid is the solution containing urea peroxide, 6ml × 1 bottle.Developer B liquid is the citric acid solution containing TMB, 6ml × 1 bottle.

Positive reference substance is the serum solution containing TB-SA antibody positive, 2ml × 1 bottle.Negative controls is the serum solution containing TB-SA negative antibody, 2ml × 1 bottle.

Stop buffer is vitriolated solution, 6ml × 1 bottle.

The preparation method of above-mentioned Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit, it comprises the following steps:

The preparation of S1.TB-SA antigen coated microplate: extremely being wrapped by concentration by TB-SA antigen diluent with phosphate buffer is 0.01mg/ml, is coated in by coating buffer on microwell plate, preserves after 37 DEG C of dryings;

S2. the preparation of sample diluting liquid: with 10 times of concentrated phosphate buffer 500ml, add casein 5.00g, adds purified water and is stirred to casein and fully dissolves; Add the Tween-20 of 3ml again, add purified water after mixing to 1000ml, fully mix, preserve;

S3. the preparation of enzyme labeling liquid: be 10 times of concentrated phosphate buffer 300ml with pH, adds purified water to 3000ml, horseradish peroxidase target staphylococcal protein A is diluted to 1:4 ten thousand ~ 1:6 ten thousand, preserve;

S4. the preparation of concentrated washing lotion: take sodium chloride 300g, sodium hydrogen phosphate 85g adds after 1000ml purified water dissolves, then add Tween-20 15ml, add to 1300ml by purified water, stir and evenly mix rear preservation;

S5. the preparation of reference substance

S51. the preparation of positive reference substance: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA antibody positive after stirring and evenly mixing, preserve after mixing;

S52. the preparation of negative controls: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA negative antibody after stirring and evenly mixing, preserve after mixing;

S6. the preparation of nitrite ion:

S61. the preparation of developer A liquid: take citric acid 0.9g, urea peroxide 0.1g, after adding purified water 100ml dissolving, adds purified water to 170ml, fully preserves after mixing;

S62. the preparation of developer B liquid: add glycerine 17ml after being dissolved by 3g citric acid by 100ml purified water, add the 0.04g TMB dissolved with 3ml DMSO after mixing, add purified water to 170ml, preserve;

S7. the preparation of stop buffer: add purified water to 180ml after purified water 150ml and the sulfuric acid 18ml measured being mixed, preserve.

Embodiment 4: the detection method of kit of the present invention

1. storage condition and the term of validity:

Kit puts 2-8 DEG C of preservation, is sure not freezing, the term of validity 12 months.Use that rear liquid reagent is airtight puts back to 2-8 DEG C of preservation, unspent micropore lath needs to reinstate with drying agent one valve bag and seals 2-8 DEG C and preserve.

2. be suitable for instrument:

Incubator, there is the microplate reader of Single wavelength 450nm or dual wavelength 450nm/630nm.

3. sample requirement: it is serum that this kit detects sample, the sample of separation can store 7 days under 2-8 DEG C of preservation, and longer-term storage should put-20 DEG C, avoids multigelation, should balance to room temperature, and fully mix before the sample use of refrigeration or freezen protective.

4. detection method is:

(1) reagent prepares: by kit balance to room temperature, by purified water, concentrated washing lotion is done 1:20 dilution, is namely made into wash operating solution for subsequent use.

(2) application of sample: antigen coated for the TB-SA of requirement lath is fixed on grillage, each hole adds 100 μ l Sample dilution, adds 5 μ l samples to be tested according to the order of sequence; Blank (can not establish blank with during double UV check), each 2 holes of negative control, positive control are established in each experiment, add Sample dilution, negative controls, each 100 μ l/ holes of positive reference substance respectively; Concussion mixing, shrouding, 37 DEG C of incubations 10 minutes, wash plate 5 times with wash operating solution, pat dry.

(3) enzyme-added: every hole adds enzyme labeling liquid 100 μ l, concussion mixing, shrouding, 37 DEG C of incubations 10 minutes, wash plate 5 times with wash operating solution, pat dry.

(4) value is read in colour developing: every hole adds each 50 μ l of developer A, B liquid, and mixing, shrouding, puts 37 DEG C of lucifuges and develop the color 10 minutes.Every hole adds stop buffer 50 μ l, mixing, completes result and measure in 10 minutes.Single wavelength measures: available blank contrast is withered, measures the OD value in each hole under microplate reader 450nm wavelength; Double UV check: measure each hole OD value under microplate reader 450nm/630nm wavelength.

The OD value of negative control answers <0.15.The OD value of positive control answers >0.80.

The explanation of assay:

1. critical value (Cutoff) calculates: Cutoff=negative controls OD value+0.2

2. the judgement of assay:

[the OD value of sample]≤[Cutoff] is: TB-SA negative antibody.

[the OD value of sample] > [Cutoff] is: TB-SA antibody positive.

5. performance index:

(1) coincidence rate: use enterprise's reference material to detect, its positives reference material and negative reference product coincidence rate are 100%.

(2) precision: imprecision <15% in test; Test bay imprecision <15%.

(3) Common drugs, pathological state and endogenous material interference:

A. agents does not affect testing result at 2.5 times for the treatment of levels: Rimactazid, pyrazinamide, streptomysin, amikacin, penicillin, ceftriaxone, erythromycin, digoxin, nifepine, ranitidine, frusemide, lidocaine, ketoconazole, methylprednisolone, prednisone.

B. following situations is noiseless for testing result: minor hemolysis, blood triglyceride raise (>980mg/dL), blood sugar raises (>11.1mmol/L), cholerythrin raises (>6.3mg/ml), injected current influenza vaccine, HBV is positive, HCV is positive, HIV is positive, rheumatoid factor positive, pneumonia, lung cancer etc.

Claims (9)

1. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit, it is characterized in that, described detection kit is made up of TB-SA antigen coated microplate, sample diluting liquid, enzyme labeling liquid, concentrated washing lotion, developer A liquid, developer B liquid, negative controls, positive reference substance and stop buffer.

2. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, it is characterized in that, described TB-SA antigen coated microplate is the microwell plate being coated with TB-SA antigen, and described TB-SA antigen phosphate buffer dilutes, and its bag is 0.01mg/ml by concentration.

3. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, it is characterized in that, described sample diluting liquid is the damping fluid containing Tween-20 and albumen.

4. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, is characterized in that, described enzyme labeling liquid is the staphylococcal protein A solution containing horseradish peroxidase-labeled.

5. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, is characterized in that, described concentrated washing lotion is the damping fluid containing Tween-20.

6. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, it is characterized in that, described developer A liquid is the solution containing urea peroxide, and developer B liquid is the solution containing TMB.

7. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, is characterized in that, described positive reference substance is the serum solution containing TB-SA antibody positive, and negative controls is the serum solution containing TB-SA negative antibody.

8. Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, it is characterized in that, described stop buffer is vitriolated solution.

9. the preparation method of Much's bacillus TB-SA abzyme linked immunosorbent assay (ELISA) kit as claimed in claim 1, it is characterized in that, it comprises the following steps:

S1. the preparation of TB-SA antigen coated microplate: extremely being wrapped by concentration by TB-SA antigen diluent with phosphate buffer is 0.01mg/ml, is coated in by coating buffer on microwell plate, preserves after 37 DEG C of dryings;

S2. the preparation of sample diluting liquid: with 10 times of concentrated phosphate buffer 500ml, add casein 5.00g, adds purified water and is stirred to casein and fully dissolves; Add the Tween-20 of 3ml again, add purified water after mixing to 1000ml, fully mix, preserve;

S3. the preparation of enzyme labeling liquid: be 10 times of concentrated phosphate buffer 300ml with pH, adds purified water to 3000ml, horseradish peroxidase target staphylococcal protein A is diluted to 1:4 ten thousand ~ 1:6 ten thousand preservation;

S4. the preparation of concentrated washing lotion: take sodium chloride 300g, sodium hydrogen phosphate 85g adds after 1000ml purified water dissolves, then add Tween-20 15ml, add to 1300ml by purified water, stir and evenly mix rear preservation;

S5. the preparation of reference substance

S51. the preparation of positive reference substance: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA antibody positive after stirring and evenly mixing, preserve after mixing;

S52. the preparation of negative controls: with 10 times of concentrated phosphate buffer 70ml, add trehalose 0.15g, Tween-20 0.35ml, glycerine 35ml, add purified water after dissolving to 650ml, add the serum 6.5ml containing TB-SA negative antibody after stirring and evenly mixing, preserve after mixing;

S6. the preparation of nitrite ion:

S61. the preparation of developer A liquid: take citric acid 0.9g, urea peroxide 0.1g, after adding purified water 100ml dissolving, adds purified water to 170ml, fully preserves after mixing;

S62. the preparation of developer B liquid: add glycerine 17ml after being dissolved by 3g citric acid by 100ml purified water, add the 0.04g TMB dissolved with 3ml DMSO after mixing, add purified water to 170ml, preserve;

S7. the preparation of stop buffer: add purified water to 180ml after purified water 150ml and the sulfuric acid 18ml measured being mixed, preserve.