CN115902217A - Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for bovine paratuberculosis and application of indirect ELISA detection kit - Google Patents

Abstract

The invention discloses an indirect ELISA detection kit for bovine paratuberculosis and application thereof. The invention belongs to the technical field of microorganism detection. The invention successfully screens out the envelope antigen with good antigenicity, and simultaneously establishes the bovine paratuberculosis indirect ELISA detection kit and the detection method thereof by applying the antigen. According to the invention, 100 parts of MAP positive serum are detected, and the sensitivity of the method is 93.0%. The detection of 200 parts of MAP negative serum shows that the specificity of the method is 95.5 percent and is obviously higher than that of other detection methods. In a word, the established indirect ELISA method for detecting bovine paratuberculosis has the characteristics of rapidness, accuracy, high sensitivity and high specificity. The invention provides a technical support for preventing and removing bovine paratuberculosis and protecting the healthy and rapid development of the livestock industry in China.

Description

Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for bovine paratuberculosis and application of indirect ELISA detection kit

Technical Field

The invention relates to an indirect ELISA detection kit for bovine paratuberculosis and application thereof. The invention belongs to the technical field of microorganism detection.

Background

Mycobacterium avium subspecies paratuberculosis (A)Mycobacterium aviumsubsp.paratuberculosisMAP) is a pathogenic bacterium of Paratuberculosis (PTB) or Johne's Disease (JD), a progressive, infectious, granulomatous chronic enteritis, mainly in the host range of ruminants, in particular cows and various domestic animals. In the early part of the last century, the development of vehicles has enabled animals to move around the world, which has led to the global spread of PTB, and in most important dairy production countries, the prevalence rate of MAP infection in cows is over 50%, sick cows infected with paratuberculosis are not suitable for breeding, and the sale of sick cows easily infects other cattle, causing irreparable loss, and having a serious impact on the global economy. This long-distance, progressive, indiscernible transmission of PTB is mainly due to the large number of cases of latent infection, and the high resistance of the pathogenic bacterium MAP to adverse environmental factors, which all constitute a risk of MAP transmission. Also, PTB is a disease susceptible to a wide range of hosts, including non-human primates.

In recent years, cattle raising industry in China is continuously expanded, but most cattle farms are not managed properly, the raising density is too high, the sanitary environment is poor, so that the transmission of PTB is more and more rapid, the incidence rate is obviously increased, after cattle are infected with MAP, the MAP can be vertically transmitted through a placenta, the sick cattle are not suitable for breeding, the milk yield is reduced, and meanwhile, the selling of the sick cattle is easy to infect other cattle herds, so that the inestimable loss is brought to the animal husbandry. According to the regulation of the Ministry of agriculture in China, PTB is a second class animal epidemic disease, and MAP belongs to a third class animal pathogenic microorganism.

At present, no effective method for preventing the disease exists, the sick cattle generally show clinical symptoms at the later stage of the disease, the medicine cannot be used for treating the disease, and the active immunization method is not suitable for being adopted because the spreading route is wide and the detection of tuberculosis is easy to interfere. Aiming at the disease, while the feeding management is enhanced and the breeding method is standardized, the only method is to detect the disease regularly, isolate suspected animals and eliminate the diseased animals.

In the existing diagnostic method, microscopic examination has the defects of low sensitivity and specificity, and meanwhile, the microscopic examination is influenced by other mycobacteria in the environment, so that the detection result is false positive. In the subclinical stage, the bacteria content discharged from sick animals is low, so that the bacterial culture sensitivity is low, and meanwhile, the bacteria culture is negative, and the detection needs to be carried out again, so that the time consumption is long, the cost is high, and the rapid identification and identification of MAP are limited. Immunohistochemistry has good sensitivity but cross-reactivity may be present.

Among them, many studies have been made on the detection of the immune aspect of MAP cells, but mainly limited to 3 reasons: (1) No specific antigen was found in all animals infected with MAP that could universally stimulate cytokines or other indicators of cells; (2) Whether an animal has a strong cellular immune response after being infected with MAP is not clear; (3) this detection is costly. At the same time, control of diagnostic costs is also an important factor in the detection and prevention of PTB, and the cost of detection cannot exceed the losses due to the disease itself. Therefore, ELISA is very suitable for detecting PTB in cattle farms due to the characteristics of simple operation, high sensitivity, good specificity and suitability for large-scale screening and qualification. However, in the detection process, tuberculosis can interfere the detection of PTB, so that no effective antigen exists in China at present, and PTB can be detected without being interfered by tuberculosis.

Therefore, at present, the urgent need exists for finding a coating antigen with good antigenicity and establishing an indirect ELISA detection method for bovine paratuberculosis by using the antigen.

Disclosure of Invention

The invention aims to provide an indirect ELISA detection kit for bovine paratuberculosis and application thereof.

In order to achieve the purpose, the invention adopts the following technical means:

according to the invention, the MAP culture medium components and culture conditions are optimized, the biological characteristics of various subcellular components of MAP are analyzed, the subcellular components with good reactionogenicity are successfully screened out, then the subcellular components are subjected to component separation, nonspecific protein adsorption treatment and other methods to obtain the coating antigen with the best effect, and the subcellular components are used as the coating antigen to optimize the conditions of the indirect ELISA antibody detection method for bovine paratuberculosis, determine the negative and positive critical values and evaluate the effect. The results show that: the sensitivity of the established indirect ELISA method is 92.0 percent, the specificity is 95.5 percent, the variation coefficient in batches and among batches is less than 10 percent, and the kit can be stably stored for 12 months after being assembled. 1900 clinical serum samples from different provinces were tested, and the positive rate of PTB was 5.05%.

On the basis of the research, the invention provides an indirect ELISA detection kit for bovine paratuberculosis, which comprises an ELISA plate coated by culture filtrate of mycobacterium avium paratuberculosis subspecies, wherein the culture filtrate of the mycobacterium avium paratuberculosis subspecies is prepared by the following method:

(1) Will comprise 10 ⁹ Inoculating CFU/mL mycobacterium avium subspecies paratuberculosis into a 7H9 culture medium containing 0.05% v/v Tween-80, 0.2% v/v glycerol, 5mM asparagine and 10% v/v self-made ADC, culturing for 10 weeks at constant temperature of 37 ℃ and 160rpm, and collecting a bacterial liquid; the homemade ADC is prepared by the following method: naCl 8.5g, dextrorotation-Dextrose 20.0g and catalase 0.03g, and filtering and sterilizing through a filter with the constant volume of 1L and 0.22 mu m;

(2) The collected cell suspension was centrifuged, and the supernatant was filtered through a 0.22 μm filter to obtain a culture filtrate.

Among them, the concentration of the culture filtrate coated on the microplate is preferably 50. Mu.g/mL.

Wherein, the centrifugation in the step (2) is preferably 10000 rpm centrifugation at 4 ℃ for 30 min.

Preferably, the kit further comprises a coating buffer solution, a confining solution, a concentrated washing solution, an HRP-labeled rabbit anti-bovine IgG antibody, a developing solution and a stop solution.

Preferably, the coating buffer solution is 20 mmol/L Tris-HCl buffer solution, the blocking solution is 1% v/v skim milk +5% v/v pig serum, the concentrated washing solution is PBST, the color development solution is TMB color development solution, and the stop solution is 0.05% v/v hydrofluoric acid.

Furthermore, the invention also provides application of the indirect ELISA kit for detecting bovine paratuberculosis in preparing a reagent for detecting or diagnosing bovine paratuberculosis.

Preferably, the reagent is an indirect ELISA detection reagent.

Compared with the prior art, the invention has the beneficial effects that:

the invention successfully screens out the envelope antigen with good antigenicity, and simultaneously establishes the bovine paratuberculosis indirect ELISA detection kit and the detection method thereof by applying the antigen. In the experimental process, 3 types of serum including paratuberculosis positive serum, tuberculosis positive serum and negative serum are adopted for detection in the whole process, so that an indirect ELISA method capable of eliminating the influence of tuberculosis on PTB detection is established. In the experiment, when the optimal conditions of each step are established, a method combining extensive screening and important repetition is adopted to screen the optimal conditions as much as possible, and when partial conditions are screened, an orthogonal test design method is selected, so that the operation steps are reduced, and the accuracy of the result is improved. The sensitivity of the method is 93.0 percent by detecting 100 parts of MAP positive serum. The detection of 200 MAP negative serums shows that the specificity of the method is 95.5 percent. Is obviously higher than other detection methods.

The indirect ELISA detection method for bovine paratuberculosis established in the experiment has the characteristics of rapidness, accuracy, high sensitivity and high specificity. Provides technical support for preventing and eliminating the disease and protecting the healthy and rapid development of the animal husbandry in China.

Drawings

FIG. 1 shows the results of primary screening of envelope antigens;

FIG. 2 shows the preliminary optimization results of the envelope antigen culturing process;

FIG. 3 shows the results of purification by CF gel filtration chromatography;

FIG. 4 is a schematic of the collection of components after sucrose density gradient centrifugation;

figure 5 is a rabbit serum titer test prepared from m.plei and Msg;

FIG. 6 is a preliminary optimization of antigen coating concentration;

FIG. 7 is a preliminary screening of the blocking solution;

FIG. 8 is a graph of the effect of various factors on the A/N value;

FIG. 9 shows the dilution factor A/N values of five secondary antibodies;

FIG. 10 shows the values of the dilution factor A/B for five secondary antibodies;

FIG. 11 shows the optimization of the time of action of the serum sample and the secondary antibody;

FIG. 12 is a screen of a developing solution and a stopping solution;

FIG. 13 is a histogram and normal distribution graph of the OD630nm values of negative serum;

FIG. 14 is a specific assay.

Description of the symbols:

(symbol)	english full scale	Chinese full scale
			BCG	Bacillus Calmette-Guerin	BCG vaccine
BSA	Bovine serum albumin	Bovine serum albumin
			CF	Culture filtrate	Culture filtrate
HRP	Horseradish peroxide	Horseradish peroxidase
			IgG	Immunoglobulin G	Immunoglobulin G
kDa	kilODalton	Kilodalton (kilodalton)
			M. Phlei	Mycobacterium phlei	Mycobacterium phlei
Msg	Mycobacterium smegmatis	Mycobacterium smegmatis
			MTB	Mycobacterium tuberculosis	Mycobacterium tuberculosis
PB	Phosphate buffer	Phosphoric acid buffer solution
			PBS	Phosphate buffered saline	Phosphate buffer
PBST	Phosphate buffered saline with Tween-20	Phosphate buffer solution added with 5 per mill tween-20
			PEG2000	Polyethylene glycol 2000	Polyethylene glycol 2000
PMSF	Phenylmethanesulfonyl fluoride	Phenylmethylsulfonyl fluoride
			TMB	3,3',5,5'-tetramethyl benzidine	Tetramethyl benzidine

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 screening and optimization of antigens

1. Materials and reagents

1.1 materials

Strains for experiments: the antigen strain for developing the kit is MAP 18 strain purchased from China institute of biological products, and kept and applied by Harbin veterinary institute. 5363 healthy calves of 4~8 months old are provided by the experimental animal center of the Harbin veterinary research institute of Chinese academy of agricultural sciences.

1.2 reagents

Enzyme-labeled plates were purchased from costar corporation; TMB color developing solutions were purchased from tiangen corporation; 7H9 Medium was purchased from BD; PROCLIN-300 preservative and Freund's incomplete adjuvant were purchased from sigma, and Protein A resin was purchased from GE; the BCA protein quantitative assay kit was purchased from Abcam.

1.3 instruments

Microplate reader-ELX 800 was purchased from BioTeK corporation, usa; AKTA protein purification system was purchased from GE.

2. Method for producing a composite material

2.1 serum sources for testing and preparation

(1) Negative serum source and preparation

4~8 months old healthy and nonimmunized 1 calf is selected, and both MAP and MTB antibodies are negative by detection (both antibodies detected by Mycobacterium paratuberculosis antibody detection kit and Mycobacterium bovis antibody detection kit are negative and are purchased from IDEXX, USA). Collecting blood from jugular vein, standing at 37 ℃ for 2 hours, separating serum (faint yellow clear liquid), inactivating the separated serum by 56 ℃ water bath for 30 minutes, adding PROCLIN-300 preservative in an amount of 0.04% of the amount of the serum, filtering and sterilizing by a filter with the size of 0.22 mu m, carrying out sterile quantitative subpackaging, 3 mL/bottle, and storing at 2-8 ℃.

(2) MAP positive serum source and preparation

From known infected livestock, clinical observation and caesarean examination results prove that MAP infected sick cattle have positive intestinal content culture, meanwhile, the result shows that MAP is strong positive by using a Mycobacterium paratuberculosis antibody detection kit of IDEXX company, and the cattle jugular vein is subjected to blood collection, and a serum separation method and negative serum are adopted.

(3) MTB positive serum source and preparation

Selecting 4~8 month-old healthy and nonimmunized calf 1, immunizing 7083E6 protein (specific protein of Mycobacterium tuberculosis provided by bacteriological laboratory of Harbin veterinary institute of Chinese academy of agricultural sciences), collecting blood from jugular vein after 2 weeks of secondary immunization, and separating serum.

2.2 Culture of MAP colonies

Preparation of 2.2.1W-R potato culture medium

(1) Preparation of liquid W-R's Medium (1L)

Asparagine	5.0 g
		KH 2 PO 4	2.0 g
MgSO 4	1.0 g (containing 7H) 2 O is 2.045 g)
		Ammonium citrate	2.0 g
NaCl	2.0 g
		Citric acid ferric ammonium salt	0.075 g
Dextrose (LD-)	10.0 g (if is C) 6 H 12 O 6 ·H 2 O is 11.0 g)
		Glycerol	48 mL
Mineral additive A and B liquid	Each 1.33 mL

Distilled water 950 mL (excluding the volume of glycerol) was divided into two portions, which were placed in 100 ℃ water baths, and asparagine was added to one portion of the water bath to dissolve it. Then, another part of distilled water after water bath was taken out from a 100mL beaker (bottle) and the above reagents were dissolved in the above order, and the dissolved reagents were added to asparagine solution in the above order, then, 48mL of glycerin was added, and 1.33 mL of solution A and solution B, respectively, was adjusted to pH 7.2 with 1M NaOH and was filtered.

Solution A:

ZnSO 4 （ZnSO 4 ×7H 2 O）	2.0 g（3.56 g）
		CuSO 4 （CuSO 4 ×5 H 2 O）	0.2 g（0.313 g）
Co(NO 3 ) 2 （Co(NO 3 ) 2 ×6 H 2 O）	0.1 g（0.16 g）
		distilled water	100 mL

And B, liquid B:

5% CaCl ₂ solutions of

(2) Preparation of fresh potato blocks

The middle round column part of the fresh potato is taken and divided into two parts along the diagonal line, and the two parts are soaked in clean water flowing water for 24 h.

(3) High pressure of culture medium

Soaking a part of W-R liquid culture medium in potato, heating in water bath at 70 deg.C for 2h, and cooling for half an hour. Pouring the rest W-R liquid culture medium into test tubes, wherein each tube is about 40 mL, placing the potato blocks subjected to water bath treatment on the upper parts of the test tubes to clamp, tightly sealing by using a cover, and carrying out autoclaving at 121 ℃ for 15-20 min.

2.2.2 culture of 2MAP W-R Potato culture Medium

Inoculating MAP P18 strain on a slant of a W-R potato culture medium, culturing at a constant temperature of 37 ℃ for 4-6 weeks, and growing an irregular colony which is fine, has the diameter of about 1mm, is slightly white, has a circular raised surface and a slightly thin edge, and is the MAP.

2.3 Screening of antigens

2.3.1MAP cultivation

100. Mu.L (hereinafter, unless otherwise mentioned, the inoculated strains were all the disrupted bacterial solutions of dry bacteria retaining the activity) was contained in 10. Mu.L ⁹ CFU/mL of the strain was inoculated into 7H9 medium, and since no resistance was added to the medium, contamination was prevented in each case in triplicate, and after culturing at 37 ℃ at constant temperature of 160rpm for 8 weeks, the bacterial suspension was collected.

2.3.2 bacterial subcellular fraction isolation

The collected bacterial liquid is subjected to subcellular component separation, 50 mL of MAP culture is taken, centrifugation is carried out at 10,000 rpm and 4 ℃ for 30min, the supernatant is filtered by a filter membrane with the aperture of 0.22 mu m to obtain Culture Filtrate (CF), and a part of CF is subjected to boiling inactivation treatment. After the cells were washed with 1 × PBS, they were resuspended in PBS containing PMSF (final concentration 1 mM) and PBS containing no PMSF, sonicated in ice bath for 15 min (sonication 5s, stop 5 s), allowed to stand on ice for 15 min and then sonicated for 15 min. The ultrasonic product is centrifuged at 3000 g at 4 ℃ for 5min, then centrifuged at 100,000 g at 4 ℃ for 4h, and the supernatant is transferred for standby. The supernatant is the cytoplasmic component and precipitates as a mixture of cell walls and cell membranes. Another 10mL of MAP culture was taken, subjected to micro-sonication, and then CF and thalli were separated, and then the thalli were resuspended in 1mL of 20mM Tris-HCl (pH = 8.0), and a final concentration of 0.05% Tween-80 was added, and centrifuged at 10000 rpm 4 ℃ for 2 min after 5% sonication for 30s (sonication for 3s, stop for 3 s), and supernatant was collected, which was a cell wall component, and mixed with CF to form a cell wall and CF mixed component, and the concentrations of CF (component 1), inactivated CF (component 2), cytoplasm (component 3), cytoplasm (component 4) which was not treated with PMSF, a mixed component of cell membrane and cell wall (component 5), a mixed component of cell membrane and cell wall which was not treated with PMSF (component 6), and a mixed component of cell wall and CF (component 7) were measured using a BCA protein quantitative detection kit.

2.3.3 Primary screening of coating antigens

Detection is carried out by adopting an indirect ELISA method, CBS is used as a coating buffer solution, a component 1, a component 2, a component 3, a component 4, a component 5, a component 6, a component 7 and whole bacteria (a component 8) are used as a coating antigen, the coating concentration of the coating antigen is initially set to 50 mug/mL, 5% skim milk is initially used as a sealing solution, negative serum (N), MAP positive serum (A) and MTB positive serum (B) are diluted by 100 times to be used as a primary antibody, rabbit anti-bovine secondary antibody (HRP mark) is diluted by 5,000 times to be used as a secondary antibody, TMB is used as a color developing solution (in a specific embodiment, all color developing solutions are TMB color developing solutions of Tiangen company), 2M sulfuric acid is used as a stopping solution, the diluent and the washing solution are PBST in the whole operation process, washing is carried out 3 times each time at an interval of 3min, each hole is provided with two parallel controls, and the OD450nm value is read after the color development is stopped. The strength of antigenicity is judged according to the magnitude of A/B and A/N values. This part of the experiment was performed in triplicate and the most dominant antigen was finally screened.

2.3.4 optimization of coating antigens

2.3.4.1 coated antigen culture process optimization

100 μ L of 10 ⁹ Respectively inoculating CFU/mL strains into 100mL of 7H9 culture medium (culture medium 1), 7H9+ ADC culture medium (culture medium 2), 7H9+ self-made ADC culture medium (culture medium 3) and beef extract culture medium (culture medium 4), respectively culturing at constant temperature of 37 ℃ and 160rpm for 4 weeks, 6 weeks, 8 weeks, 10 weeks and 12 weeks, collecting bacterial liquid, separating subcellular components to obtain advantageous antigen components, respectively coating enzyme label plates with the advantageous antigen components obtained by different treatment groups, carrying out indirect ELISA detection, judging the strength of the antigenicity of the advantageous antigen components of different culture groups according to the A/N and A/B values, and screening out the culture antigen, wherein the self-made ADC is prepared by the following method: 8.5g of NaCl, 20.0g of dextrorotation-Dextrose and 0.03g of catalase, and filtering and sterilizing the mixture by using a filter with the volume constant being up to 1L and 0.22 mu m. The antigen of the culture mode is further optimized, and the culture medium under the culture condition is processed into the following groups of original components (component 1), glycerol-free components (component 2), double-glycerol-content components (component 3), tween-80-free components (component 4) and double-Tween-80-content components(component 5) and an anaerobic culture group (component 6), culturing at constant temperature of 37 ℃ and 160rpm, collecting bacterial liquid, separating subcellular components to obtain dominant antigen components, respectively coating the enzyme-labeled plate with the dominant antigen components obtained by different treatment groups, carrying out indirect ELISA detection, judging the strength of the dominant antigen antigenicity of different components according to the values of A/B and A/N, and screening out a better method for culturing the antigen. The preferred cultivation method was again optimized to include 10 in 100. Mu.L ⁹ And (2) inoculating CFU/mL strains into the better culture medium, respectively adding serine, lysine, cysteine, glycine, valine, leucine, glutamine, phenylalanine, glutamic acid, methionine, asparagine, alanine, proline, threonine, arginine, tyrosine, histidine, isoleucine and ascorbic acid into the culture medium, wherein the final concentration of the additives is 5mM, simultaneously setting a control group without additives, repeating the steps for two groups, culturing according to the better culture mode, collecting bacterial liquid, separating subcellular components to obtain dominant antigen components, respectively coating the dominant antigen components obtained by adding different component treatment groups on the enzyme label plate, carrying out indirect ELISA detection, judging the strength of the dominant antigen components of different antigen additive culture groups according to the values of A/B and A/N, and screening the optimal additive components of the culture antigens.

Individual optimization of 2.3.4.2 coated antigen

In order to further optimize the envelope antigen, the envelope antigen is separated according to the above optimization conditions, and then the following treatments are respectively performed on the envelope antigen.

(1) Gel filtration chromatography

After all the working solution (20% ethanol, 20mM Tris-HCl (pH = 8.0), deionized water) and the coating antigen are filtered by a 0.45 mu m filter membrane, the instrument is opened, the cleaning and pipeline preparation of an AKTA machine are firstly carried out, a chromatographic column is installed after the pump cleaning is finished, a pre-packed column of Superdex 200 (separation range of 20 kDa-200 kDa) is installed on the AKTA, the detection wavelength is 280 nm, the pre-packed column is loaded with a sample ring after being balanced, the operation program is started, and the sample is collected after the program operation is finished. Collecting samples under each peak, respectively coating the ELISA plate at the concentration of 50 mu g/mL, setting untreated coating antigen as a control group, carrying out indirect ELISA detection, and judging the strength of each protein antigenicity under different peaks according to the A/B and A/N values.

(2) Retention of proteins below 20kDa in the envelope antigen

Intercepting proteins below 20kDa in the coated antigen by a hollow fiber ultrafiltration experimental device, leaving mixed components of proteins with small molecular weights, coating an ELISA plate according to concentration gradients of 5 mu g/mL,10 mu g/mL, 20 mu g/mL, 35 mu g/mL, 50 mu g/mL,100 mu g/mL and 150 mu g/mL, setting the non-intercepted coated antigen as a control group, carrying out indirect ELISA detection, and judging the strength of antigenicity under different concentrations according to the values of A/B and A/N.

(3) Preparation of envelope antigen pure protein derivatives

Taking 10mL as a coating antigen, adding 3.0 g of phenol per milliliter, uniformly mixing, standing for 4 hours at 4-8 ℃, reserving a supernatant (sample 1), discarding a precipitate, removing incompatible protein, adding 40% trichloroacetic acid with a final concentration of 2-4% into the supernatant, uniformly mixing, standing for 4-6 hours, discarding a supernatant (sample 2), obtaining a precipitate, washing the precipitate with 1% trichloroacetic acid for three times, and centrifuging at 3,000 rpm to collect the precipitate (sample 3). The precipitate was completely dissolved with PB, an equal amount of saturated ammonium sulfate solution was added thereto, overnight at 4 ℃ (sample 4), the liquid was centrifuged and the precipitate was collected, PB was added to dissolve to obtain a crude pure protein derivative (sample 5), and the crude extract was dialyzed twice against PBS (phenol 3.0 g per ml) to obtain a purified pure protein derivative (sample 6). Collecting the sample coated ELISA plate of each step in the preparation process, setting untreated coated antigen as a control group, carrying out indirect ELISA detection, and judging the antigenicity of the coated antigen after each step of treatment according to the A/B and A/N values.

(4) Sucrose density gradient centrifugation

Selecting three gradient (20%, 40% and 60%) sucrose solutions, sequentially adding the sucrose solutions into a centrifuge tube by using a long needle tube, finally slowly adding the coating antigen into the uppermost layer, centrifuging for 2h at 120,000g, then gently taking out the centrifuge tube, finding that a bright band exists between 20% and 40% and between 40% and 60%, respectively sucking 8 parts of the solutions shown in figure 4, respectively dialyzing the solutions overnight at 4 ℃ in a PBS buffer solution to remove sugar, respectively coating the 8 solutions on an enzyme label plate, setting the untreated coating antigen as a control group, carrying out indirect ELISA detection, and judging the strength of the antigenicity of the coating antigen after each step of treatment according to the A/B and A/N values.

(5) Adsorption of non-specific reactive proteins

First of all byM. PhleiPreparation of rabbit hyperimmune serum with Msg

Male New Zealand white rabbits, each of which is about 3kg, are bought back to be fed for a week and are immunized after adapting to the environment, and each bacterium is used for immunizing two rabbits. The rabbit ear marginal vein before immunization was sampled and used as a negative control after serum isolation. The rabbits were immunized with sterile PBS diluted to 2 mg/mL m.phlei and Msg mixed with freund's incomplete adjuvant 1:1 (calculated as 0.5mg/kg body weight) once every two weeks. After 7-10 days of three immunizations, 2mL of serum is collected from the marginal veins of the ears, and subjected to antibody titer detection, wherein the serum to be tested is subjected to double dilution (1. After the antibody titer meets the experimental requirements, all sera were collected.

Primary purification of rabbit serum antibody by caprylic acid-ammonium sulfate method

Centrifuging the collected rabbit serum at 12,000 rpm for 5min, collecting the supernatant 5mL, adding 20mL of acetic acid buffer solution, mixing well, placing into a stirrer, adding 1,875 μ L of n-octanoic acid while slowly stirring, stirring for 30min, immediately centrifuging at 4 deg.C at 12,000 rpm for 30min, and collecting the supernatant. 0.227g of ammonium sulfate was added to each ml of the supernatant, and the mixture was further stirred for 30min, left to stand for 5h, centrifuged at 12,000 rpm at 4 ℃ for 30min, and the supernatant was discarded, and the precipitate was resuspended in 2.5 mL of PBS. And (3) putting the suspension into a dialysis card, dialyzing with PBS (phosphate buffer solution), dialyzing at 4 ℃ overnight, changing the solution for 3 times, and finally collecting the solution in the dialysis card to obtain the primarily purified rabbit serum antibody.

Adsorption of non-specific proteins in coated antigens

A 3-tube ProteinA a resin was prepared, after equilibrating the resin with buffer a for 10 column volumes, the primarily purified m.phlei and Msg rabbit serum antibodies were split into m.phlei group (sample 1), msg group (sample 2) and m.phlei + Msg (1:1 isoconcentrated mix) mix group (sample 3), and passed through the resin separately until saturation. Respectively adding equivalent coating antigens into three resins, after 10 min of action, collecting penetrating liquid, washing impurities by using buffer B, after collecting impurity washing liquid, slowly adding the penetrating liquid into the resins, after 10 min of action, collecting penetrating liquid again, washing impurities by using buffer B, repeating the process for 5 times, collecting all the penetrating liquid (sample 4) and the impurity washing liquid (sample 5) of the sample 1 group, the penetrating liquid (sample 6) and the impurity washing liquid (sample 7) of the sample 2 group, the penetrating liquid (sample 8) and the impurity washing liquid (sample 9) of the sample 3 group, respectively coating the ELISA plates with the collected samples 4-9, simultaneously setting untreated coating antigens as a control group, carrying out indirect detection, and judging the antigenicity intensity of the 6 samples according to the values of A/B and A/N.

3. Results

3.1 screening of antigens

3.1.1 Primary screening of coating antigens

The detection is carried out by adopting an indirect ELISA method, CBS is used as a coating buffer solution, a component 1, a component 2, a component 3, a component 4, a component 5, a component 6, a component 7 and whole bacteria (a component 8) are used as a coating antigen, the coating concentration of the coating antigen is 50 mug/mL, 100-fold dilution of negative serum (N), MAP positive serum (A) and MTB positive serum (B) is used as a primary antibody, 5,000-fold dilution of rabbit anti-bovine secondary antibody (HRP mark) is used as a secondary antibody, and the OD450nm value is measured after the color development is terminated. The values of A/B and A/N are analyzed after the values are read by a microplate reader, and the result shows that (shown in figure 1), the value of A/B in the component 1 is relatively high, and the value of A/N is highest, so that the effect of the component 1 is best, namely CF is the coating antigen with the optimal potential.

3.1.2 optimization of the Process for preparation of coating antigen

100 μ L of 10 ⁹ Inoculating CFU/mL strain into 100mL culture medium 1-4, culturing at 37 deg.C and 160rpm for 4 weeks, 6 weeks, 8 weeks, 10 weeks, and 12 weeks, collectingAnd (2) separating the subcellular components to obtain CF, respectively coating the ELISA plates with the CF obtained from different treatment groups, carrying out indirect ELISA detection, and displaying analysis results of A/B and A/N values after reading the values by an ELISA reader (see figure 2), wherein the A/N value is optimal and the A/B value is excellent when the culture is carried out for 10 weeks under the condition of a culture medium 3, so that the culture condition is excellent when 7H9+ self-prepared ADC culture medium is cultured for 10 weeks. Considering that the effect of secreted CF may be affected by the added components (Tween-80, glycerol) in the culture medium, under the culture condition, the culture medium additives are optimized by the components 1-6, after 10 weeks of culture, CF coated ELISA plates with different components are separated, indirect ELISA detection is carried out, the values of A/B and A/N are analyzed after the values are read by an ELISA reader, the analysis result shows (see Table 1), the results of the components 1-5 are not very different, but in the culture process, the component 2 can affect the turbidity of bacteria, the bacteria are cultured according to the component 4, the bacteria are more easily polluted than other groups, and meanwhile, considering that materials are saved as much as possible in the experimental process, the components of the original culture medium are selected. Therefore, the primary optimized culture condition is 7H9 (0.05% v/v Tween-80, 0.2% v/v glycerol) + the self-made ADC culture medium is cultured for 10 weeks at the constant temperature of 160rpm at 37 ℃. Considering that different amino acids may affect the types and the contents of proteins in the bacterial growth process, the culture mode of the coating antigen in the previous step is optimized again, 18 amino acids and ascorbic acid are respectively added into the culture medium, the final concentration is 5mM, simultaneously, a control group without additives is arranged, each group is repeated for two times, and after the culture is carried out at the constant temperature of 37 ℃ and the constant speed of 160rpm for 10 weeks, the bacterial liquid is collected. After separating CF, respectively coating the ELISA plates, carrying out indirect ELISA detection, analyzing the A/B and A/N values after reading the values by an ELISA reader, and taking the A/N values and the A/B values into consideration, wherein the A/B and A/B values show that lysine, cysteine, phenylalanine, asparagine, alanine, histidine and isoleucine additive groups can improve the sensitivity and specificity of coated antigen detection; among the components that reduce the sensitivity and specificity of detection of the coating antigen are the glutamate and tyrosine additive package. When repeated experiments were carried out under the same conditions, the results showed (see Table 2) that the reproducibility of the lysine and asparagine groups was better, but considering the lower A/B values of the lysine group, the detection specificity was reduced, so the conclusion was that the addition of asparagine enabled the presence ofEffectively improve the detection sensitivity and specificity of the CF.

Therefore, the culture filtrate of the mycobacterium avium subspecies paratuberculosis which is the final optimized culture condition is prepared by the following method:

(1) Will comprise 10 ⁹ Inoculating CFU/mL mycobacterium avium paratuberculosis subspecies into a 7H9 culture medium containing 0.05% v/v Tween-80, 0.2% v/v glycerol, 5mM asparagine and 10% v/v homemade ADC, culturing at constant temperature of 37 ℃ and 160rpm for 10 weeks, and collecting bacterial liquid; the homemade ADC is prepared by the following method: naCl 8.5g, dextrorotation-Dextrose 20.0g and catalase 0.03g, and filtering and sterilizing through a filter with the constant volume of 1L and 0.22 mu m;

(2) The collected cell suspension was centrifuged, and the supernatant was filtered through a 0.22 μm filter to obtain Culture Filtrate (CF).

3.2 comparison of the detection Effect after separation of the envelope antigens

3.2.1 gel filtration chromatography

After the filtered CF passes through a gel filtration chromatography column Superdex 200, after the program operation is finished, it can be seen from FIG. 3 that the absorption peaks at 47 mL, 67 mL and 116 mL are more obvious and are distributed in D region, F region and J region, the tube samples (D8, D9, F4, F5, J5 and J6) under the highest peaks are respectively collected and coated on an ELISA plate at the concentration of 50 mug/mL, meanwhile, the CF which is not subjected to gel filtration chromatography is used as a contrast for indirect ELISA detection, and the values of A/B and A/N are analyzed after the value is read by an ELISA reader, and the result shows that the A/N and A/B results of the 6 tube samples (see Table 3) are not as the CF detection values before gel filtration chromatography. Indicating that no more advantageous coating antigen was isolated during the gel filtration chromatography purification of CF than before.

3.2.2 entrapment of proteins under 20kDa in the envelope antigen

Intercepting proteins below 20kDa in the envelope antigen by a hollow fiber ultrafiltration experimental device, leaving mixed components of proteins with small molecular weight, respectively coating an enzyme label plate by setting 7 gradients according to the concentration of 5-150 mu g/mL, simultaneously setting the non-intercepted envelope antigen (50 mu g/mL) as a control group, carrying out indirect ELISA detection, analyzing the values of A/B and A/N after reading the value by an enzyme label instrument, and analyzing the values of A/B and A/N after reading the value by the enzyme label instrument, wherein the results show that (see table 4) the values of A/N and A/B of the control group are obviously superior to the values of the concentration gradients of the mixed components of the small molecular weight proteins after interception. The result shows that the entrapped small molecular weight protein has no effective envelope antigen component obviously superior to the original CF.

3.2.3 detection of pure protein derivatives of envelope antigens

Respectively coating the ELIAS plate with samples (total 6 types) collected in each step in the preparation process of the pure protein derivatives coated with the antigens, simultaneously setting untreated coated antigens as a control group, and carrying out indirect ELISA detection, wherein the results show that (table 5) the comprehensive determination results of the A/N and A/B values of the 6 samples are not as same as the values of the control group, and the results show that the pure protein derivatives coated with the antigens and the pure protein derivatives do not produce more effective components which can be used as the coated antigens than the original CF in the preparation process.

3.2.4 sucrose Density gradient centrifugation of coated antigen

The sucrose solution with three gradients of 20%,40% and 60% is selected to carry out density gradient centrifugation on CF, 8 parts of solution shown in figure 4 are respectively absorbed, dialysis is respectively carried out in PBS buffer solution to remove sugar, the 8 solutions are respectively coated on an enzyme label plate, meanwhile, untreated coated antigen is arranged as a control group to carry out indirect ELISA detection, the values of A/B and A/N are analyzed after the value is read by an enzyme label instrument, and the result shows that (see table 6) the sucrose density gradient centrifugation does not separate out more effective antigen components than the original antigen.

3.3 nonspecific protein adsorption treatment of coating antigen

3.3.1 Rabbit hyperimmune serum titer detection

Performing antibody titer detection on collected serum prepared from M.Phlei and Msg, performing multiple dilution on the serum to be detected (the first hole is subjected to 1. The results show (see fig. 5), both the serum titers can reach 102,400, and the requirements of the experiment are met.

3.3.2 adsorption of non-specific proteins in coated antigens

After the prepared rabbit hyperimmune serum is subjected to primary antibody purification by an octanoic acid-ammonium sulfate method, protein A resin is added until saturation, CF is added into the resin, samples in the purification process are collected, samples 4-9 are respectively coated with an enzyme label plate, meanwhile, untreated coated antigen is set as a control group for indirect ELISA detection, and the values of A/B and A/N are analyzed after the value is read by an enzyme label instrument, and the result shows that (see table 7), the values of A/B and A/N of the control group are obviously superior to the values of the groups of the samples 4-9. Indicating that untreated CF is more antigenic.

Example 2 establishment of Indirect ELISA detection method for bovine paratuberculosis

1. Materials and reagents

1.1 materials

Specific quality control sera were preserved by the harbin veterinary institute. Male New Zealand white rabbits-3 kg, SPF grade, purchased from the Harbin veterinary institute engineering center.

1.2 reagents

Goat anti-Niu Duo cloned antibody (HRP labeled) was purchased from KPL corporation; four rabbit anti-bovine secondary antibodies were purchased from sigma; TMB color developing solutions of 5 different brands were purchased from sigma, shanghai Biotech, amresco, thermo and Tiangen.

1.3 Instrument

Microplate reader-ELX 800 was purchased from BioTeK corporation, usa; the hollow fiber ultrafiltration experimental apparatus was purchased from Beijing Asahi Membrane Equipment, LLC.

2. Method of producing a composite material

2.1 operating procedure of Indirect ELISA detection method

(1) Coating: and (3) taking optimized CF as a coating antigen, diluting to an optimal concentration, adding the optimized CF into an ELISA plate (100 mu L/hole), incubating for a period of time, discarding liquid in the ELISA plate, washing by PBST, and then patting the ELISA plate dry.

(2) And (3) sealing: adding a sealing solution into the flap-dried ELISA plate, incubating for a period of time at 200. Mu.L/well, discarding the liquid in the ELISA plate, washing by PBST, and flap-drying the ELISA plate.

(3) Primary antibody (or test sample): diluting the negative serum (N), the MAP positive serum (A) and the MTB positive serum (B) or the collected clinical serum sample (after optimization treatment) by PBST with the optimal dilution times, adding the diluted serum sample into an ELISA plate with 100 mu L/hole, repeating each sample for 2-3 times, after incubating for a period of time, discarding the liquid in the ELISA plate, washing by PBST, and then drying the ELISA plate.

(4) Enzyme-labeled secondary antibody: diluting the optimal secondary antibody with PBST, adding the diluted secondary antibody into an ELISA plate with 100 mu L/hole, incubating for a period of time, discarding the liquid in the ELISA plate, washing with PBST, and then patting the ELISA plate dry.

(5) Color development: add 100. Mu.L of optimal color developing solution to each well, develop for a period of time (protect from light).

(6) And (3) terminating the reaction: 100 μ L of stop solution was added to each well.

(7) And (4) reading the value.

2.2 optimization of conditions in Indirect ELISA detection Process

2.2.1 Screening of coating buffer

Diluting CF with PBS (0.01 mol/L), CBS (0.05 mol/L) and Tris-HCl (20 mmol/L) as coating buffer solutions, diluting the coating raw materials to 50 mu g/mL with the 3 coating solutions in a primary test, adding 100 mu L/well into an ELISA plate, incubating for 12h at 4 ℃, washing with PBST three times at intervals of 3min, drying the ELISA plate after the last washing (the step is called washing for short), diluting skim milk to 5% with PBST as a blocking solution, adding 200 mu L/well into the ELISA plate, blocking for 2h at 37 ℃, repeating the washing procedure, diluting negative serum (N), MAP positive serum (A) and MTB positive serum (B) with PBST as 100-fold dilution as a primary antibody, incubating for 100 mu L/well, repeating the washing procedure after incubating for 1h at 37 ℃, rabbit anti-bovine secondary antibody (HRP label) as 5,000-fold dilution as a secondary antibody, repeating the washing procedure after 1h at 37 ℃, incubating for 1h, using PBS (day) as a color developing solution, adding 10 mu L as a color developing solution after incubating for 1h, and reading the two parallel stop-developing values with sulfuric acid at each time and each of 100 nm. And screening out the optimal coating buffer according to the sizes of the A/B and A/N values.

2.2.2 preliminary optimization of coating antigen concentration

The selected optimal coating buffer was used to dilute the CF concentration to 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL, 40. Mu.g/mL, 50. Mu.g/mL, 75. Mu.g/mL, 100. Mu.g/mL, 150. Mu.g/mL, and 200. Mu.g/mL for 10 gradients, and the plates were coated with each other. After color development of TMB (Tiangen), reaction was stopped with 2M sulfuric acid, two parallel controls were placed in each well, and after completion of the test, OD450nm was read. And screening out the optimal coating concentration according to the sizes of the A/B and A/N values.

2.2.3 optimization of coating time

After dilution of CF to optimal concentration, 5 conditions were tested on the microplate, 1h at 37 ℃, 2h at 37 ℃, overnight at 4 ℃, 1h at 37 ℃ overnight at 4 ℃ and 2h at 37 ℃ overnight at 4 ℃, respectively. After color development of TMB (Tiangen), the reaction was stopped with 2M sulfuric acid, two parallel controls were placed in each well, and after completion of the assay, OD450nm was read. And screening out the optimal coating time according to the sizes of the A/B and A/N values.

2.2.4 preliminary screening of confining liquids

And diluting the CF at the optimal concentration by using the optimal coating buffer solution, coating the ELISA plate according to the optimal coating time, and optimizing the sealing solution. The primary selection was 5% v/v skim milk, 1% v/v PEG-2000, 5% v/v gelatin, 5% v/v sheep serum, 5% v/v horse serum, 5% v/v pig serum, 1% v/v skim milk +5% v/v pig serum, 5% v/v rabbit serum, 1% v/v skim milk +5% v/v rabbit serum, 5% v/v chicken serum, 5% v/v BCG immunized rabbit serum, 5% v/v Msg immunized rabbit serum, 1% v/v BSA, surMODics-Fish, thermo-PBS as blocking fluid, blocking for 2h at 37 ℃,100 fold dilution of serum, 5,000 fold dilution of rabbit anti-bovine secondary antibody (HRP marker) as secondary antibody, after TMB (Tiangen) color development, stop reaction with 2M sulfuric acid, two parallel controls were placed in each well, and after the experiment was completed, OD450nm was read. And screening out better confining liquid according to the A/B and A/N values.

2.2.5 adsorption of non-specific factors in serum antibodies

Separately culturing Msg,M. PhleiAnd BCG, after diluting the serum to be detected, adding three bacteria CF with equal volume (refer to the method of step 2.3.2 of example 1), respectively acting at 4 ℃ overnight (12 h), acting at 37 ℃ for 30min, acting at 37 ℃ for 2h, then adding into an enzyme label plate, diluting rabbit anti-bovine secondary antibody (marked by HRP) by 5,000 times to be used as a secondary antibody, after color development of TMB (Tiangen), terminating the reaction by using sulfuric acid of 2M, setting two parallel controls for each hole, and reading OD450nm value after the test is finished. And selecting the optimal CF antigen which can be used for absorbing and removing serum antibodies and can cross react with other mycobacteria according to the sizes of A/B and A/N values.

2.2.6 optimization of coating concentration, blocking solution, blocking time, and serum dilution factor

According to the previous experimental results, three levels of four factors of coating concentration, blocking solution, blocking time and serum dilution factor are selected as L ₉ （3 ⁴ ) And (4) orthogonal experimental design. Selecting the optimal three concentrations in the primary screening of the coating concentration; selecting three different concentration ratios of the optimal components in the primary screening by the confining liquid; the sealing time is respectively selected from 37 ℃ for 30min,37 ℃ for 1h and 37 ℃ for 2 h; the serum dilution was selected from three dilutions of 20-fold, 50-fold and 100-fold. Rabbit anti-bovine secondary antibody (HRP labeled) was diluted 5,000 times as a secondary antibody, and after color development of TMB (tiangen), the reaction was stopped with 2M sulfuric acid, two parallel controls were placed in each well, and after completion of the assay, OD450nm was read. The results of the experiment were statistically analyzed to determine the optimal coating concentration, blocking solution, blocking time and serum dilution.

2.2.7 screening of different Secondary antibodies and optimization of dilution factor of the Secondary antibodies

Five secondary antibodies, i.e., HRP-labeled goat anti-bovine polyclonal antibody (secondary antibody 1), rabbit anti-bovine (produced in whole serum) IgG antibody (secondary antibody 2), rabbit anti-bovine (Fc-specific) IgG antibody (secondary antibody 3), HRP-labeled rabbit anti-bovine (affinity separation antibody) IgG antibody (secondary antibody 4), and HRP-labeled rabbit anti-bovine (anti-serum IgG fraction) IgG antibody (secondary antibody 5), were selected, respectively, and since the secondary antibody 2 and the secondary antibody 3 were not HRP-labeled, the preparation of the enzyme-labeled antibody was first performed.

2.5 mg of HRP was dissolved in 0.25 mL deionized water, 0.25 mL of sodium periodate (0.06 mol/L) (freshly prepared) aqueous solution was added thereto, 0.25 mL of ethylene glycol (0.16 mol/L) aqueous solution 0.25 zxft 3252 was added thereto after mixing at 4 ℃ for 30min, the mixture was left at room temperature for 30min, 0.25 zxft 3425 of aqueous solution containing 2.5 mg purified rabbit anti-bovine IgG was added thereto, the mixture was mixed and added to a dialysis card, and the dialysis card was placed in 0.05 mol/L CBS (pH = 9.5) for overnight dialysis at 4 ℃. The liquid was aspirated from the dialysis card, and 0.1 mL was added sodium borohydride solution (5 mg/mL) and allowed to stand 2h at 4 ℃. An equal volume of saturated ammonium sulfate solution (pH = 7.0) was added thereto, the mixture was allowed to stand at 4 ℃ for 30min, and then centrifuged at 3,500 rpm/min for 30min, the supernatant was discarded, the precipitate was suspended in PBS (0.02 mol/L; pH = 7.4) and added to a dialysis card, the card was put into PBS and dialyzed overnight at 4 ℃ for three times, during which time the solution was exchanged and the liquid in the card was aspirated, and then the secondary HRP-labeled antibody was obtained by centrifugation to remove insoluble matter.

After dilution of CF at optimal concentration with optimal coating buffer, elisa plates were coated for optimal coating time, optimized blocking solution was added and after incubation for a period of time, optimal dilutions of serum samples were added, and 5 antibodies were raised according to 1:50,000 and 1,000, and after a certain period of action, the reaction was quenched with TMB (Tiangen), 2M sulfuric acid, two parallel controls per well, and after completion of the assay, the OD450nm was read. And screening out the optimal secondary antibody and the dilution multiple thereof according to the A/B and A/N values.

2.2.8 optimization of serum samples and duration of action of Secondary antibodies

Diluting CF at the optimal concentration by using an optimal coating buffer solution, coating an ELISA plate according to the optimal coating time, adding an optimized blocking solution, incubating for a period of time, adding an optimal dilution serum sample, incubating for 30min, 45min and 60 min at 37 ℃, adding an optimized secondary antibody, diluting according to the optimal dilution times, adding the ELISA plate, and incubating for 30min, 45min and 60 min at 37 ℃. After the color development of TMB (Tiangen), the reaction was stopped with 2M sulfuric acid, two parallel controls were placed in each well, the rest were run by indirect ELISA, and after the assay was complete, the OD450nm values were read. And screening the optimal action time of the serum sample and the secondary antibody according to the A/B and A/N values.

2.2.9 color development liquid and termination liquid screening method

Diluting CF with an optimal coating buffer solution, coating an enzyme label plate according to optimal coating time, adding an optimized blocking solution, incubating for a period of time, adding an optimal diluted serum sample and a secondary antibody, respectively selecting 5 TMB color developing solutions of different brands from sigma, shanghai worker, amresco, thermo and Tiangen company during color development, respectively selecting 2M sulfuric acid and 0.05% hydrofluoric acid as stop solutions during termination, setting two parallel controls for each well, operating the rest according to an indirect ELISA method, and reading OD450nm or OD630nm values after the test is completed. And screening out the optimal color developing solution and the optimal stopping solution according to the A/B and A/N values.

2.2.10 optimization of color development time and color development temperature

Diluting CF at the optimal concentration by using an optimal coating buffer solution, coating an enzyme label plate according to the optimal coating time, adding an optimized confining liquid, incubating for a period of time, adding a serum sample and a secondary antibody with the optimal dilution, adding an optimal developing liquid, setting the action time to be 5min, 10 min and 15 min respectively, and selecting the action temperature to be 22 ℃ (normal temperature) and 37 ℃ respectively to perform a two-factor cross experiment. After the addition of the stop solution, the OD450nm or OD630nm values were read. Two parallel controls are arranged in each well, the rest are operated according to an indirect ELISA method, and the optimal developing time and developing temperature are screened out according to the A/B and A/N values.

Optimization of 2.2.11 washing step

Diluting CF at the optimal concentration by using an optimal coating buffer solution, coating an enzyme label plate according to the optimal coating time, adding an optimized confining liquid, incubating for a period of time, adding an optimal diluted serum sample and a secondary antibody, developing according to the optimized condition, reading the OD450nm or OD630nm value after termination, and setting two parallel controls for each well. The washing steps in the whole operation process are set to be in the following 3 modes, continuous washing is carried out for 3 times, and the washing is carried out for the last time; washing for 3 times, each time at an interval of 1 min, and drying for the last time; washing 3 times (3 min/time), and drying in the last time. The rest of the procedures were performed according to the indirect ELISA method, and the optimal washing method was selected according to the magnitude of A/B and A/N values.

2.3 determination of Positive and negative cutoff values

According to the optimized result in 2.2, 200 bacteria cultured as MAP negative sera were detected, and the average value of OD of all sera was calculated after reading (

) And Standard Deviation (SD), calculating a cut-off value based on the sum SD, and determining the OD value (S or ^) based on the clinical sample when the clinical sample is detected>

) (the average value was calculated for the samples with duplicate wells), the OD value of the standard positive serum (P) and the OD value of the standard negative serum (N) were calculated according to the following formulas:

S/P（%）=100×

to be provided with

The value of +3 XSD is taken as the MAP positive cutoff value for ^ 4>

The value of +2 × SD is used as the negative critical value of MAP, if the value is between the two, the MAP antibody is judged to be suspicious, and the detection is required again.

2.4 Effect evaluation of the Indirect ELISA method established

2.4.1 specificity assays

(1) Cross-reaction detection

Selecting positive serum of 10 cattle commonly suffered from diseases in clinic, such as mycobacterium tuberculosis positive serum (MTB), bovine infectious rhinotracheitis positive serum (IBR), bovine parainfluenza virus positive serum (BPIV), brucella positive serum (brucellosis), foot-and-mouth disease positive serum (foot-and-mouth disease), bovine agalactia positive serum (bovine agalactia), bovine viral diarrhea positive serum (BVDV), bovine escherichia coli (28 a) positive serum (large intestine 28 a), bovine escherichia coli (empty vector) positive serum (large intestine empty), bovine mycoplasma positive serum (bovine mycoplasma), and the like, detecting by an optimized ELISA method, repeating the experiment for 4 holes for each serum, judging the result according to S/P (%) value, and if the S/P (%) value of the MAP positive serum is not less than or equal to that of the MAPs

The value of +3 XSD, the S/P (%) value of the other 10 disease-positive sera are not higher than ^ H>

The value of +2 XSD shows no cross reaction and good specificity.

(2) Detection of 200 negative sera

200 parts of serum which is cultured by bacteria and is MAP negative is selected, detection is carried out according to an optimized indirect ELISA operation program, each serum is repeated for 2 times, negative and positive results are judged according to S/P (%) values, and the positive detection rate is calculated.

2.4.2 sensitivity test

(1) Positive quality control serum detection

MAP-positive sera were diluted with PBST according to seven gradients of 1,200, 1,500, 1,000, 1,500, 1, 3,000, 1,5,000, and the other steps were performed according to the optimized indirect ELISA method, and sensitivity was calculated from S/P (%) values after reading with an microplate reader.

(2) Detection of 50 positive sera

Selecting 50 parts of serum with MAP positive bacteria, detecting according to an optimized indirect ELISA operation program, repeating each serum for 2 times, judging a negative and positive result according to an S/P (%) value, and calculating a positive detection rate.

2.4.3 repeatability experiments

(1) In-batch repeatability experiments

Randomly selecting 4 blocks from the same batch of prepared CF-coated ELISA plates, and detecting 20 parts of serum (including 6 parts of MAP strong positive serum, 8 parts of MAP weak positive serum and 6 parts of MAP negative serum) samples according to an optimized method. The mean of each serum (3 replicate wells) on each plate was compared to the mean of the sera on the other plates and the total of each serum in 4 plates was calculated

And SD according to the formula SD/>

The intra-batch coefficient of variation (C.times.V%) was calculated at 100% and the intra-batch reproducibility was determined.

(2) Batch to batch repeatability experiments

Randomly selecting 3 enzyme label plates coated with CF prepared from different batches, and detecting the 20 serum samples according to an optimized method. The mean of each serum (3 replicate wells) on each plate was compared to the mean of the sera on the other plates and the total of each serum in the 3 plates was calculated

And SD according to the formula SD/>

The coefficient of variation between batches (C.times.V%) was calculated at 100% and the repeatability between batches was determined.

2.4.4 compliance experiments

After the CF-coated ELISA plate is sealed, 13 parts of MAP positive serum and 19 parts of MAP negative serum, secondary antibody, TMB color development liquid, stop solution, washing solution and diluent are assembled into a kit, three representative areas of Shanghai, xinjiang and fertilizer combination are selected for coincidence experiments, each area is subjected to experiment operation by different people, and negative and positive results are judged according to S/P (%) values to calculate the positive coincidence rate.

2.4.5 comparative test

250 parts of MAP serum with known negative and positive after bacterial culture are selected, the assembled detection kit and the detection kit for the antibody of the mycobacterium paratuberculosis of IDEXX are respectively used for detection, and the experimental results are summarized and compared.

2.4.6 shelf life test

And (3) extracting 6 boxes from the assembled kit, storing the assembled kit at 2~8 ℃ for 12 months, extracting one box at 1,2, 3, 6, 9 and 12 months respectively to perform relevant experiments on each part, comparing the relevant experiments with the newly assembled kit, firstly detecting whether the volume and the appearance color of each solution are changed, whether a precipitate is generated, whether the ELISA plate is well sealed, selecting 20 parts of serum in 2.3.3, performing conformity rate, specificity and sensitivity experiments on the ELISA plate, and judging the stability of the kit according to the experimental results.

Detection of 2.4.7 clinical serum samples

Selecting 1900 parts of clinical serum samples (collected or locally inspected) including 585 parts of Ulluquinazi bovine serum, 1015 parts of Duyashan bovine serum, 123 parts of Changchun bovine serum, 58 parts of Shanghai bovine serum, 10 parts of Zhejiang bovine serum, 44 parts of Henan bovine serum and 65 parts of fertile bovine serum. The 1900 parts of serum come from different provinces and cities, spread throughout northeast, northwest, east of China and central regions, and can represent the popular condition of MAP in China to a certain extent.

3. Results

3.1 optimization of conditions in Indirect ELISA detection Process

3.1.1 screening of coating buffers

The CF was diluted to 50. Mu.g/mL with PBS (0.01 mol/L), CBS (0.05 mol/L) and Tris-HCl (20 mmol/L) as coating buffers, and the experiments were performed according to the procedure of indirect ELISA, and the values of A/B and A/N were analyzed after reading with a microplate reader, which revealed (see Table 8) that the values of A/B and A/N were the best when the coating buffer was Tris-HCl (20 mmol/L). Therefore, tris-HCl (20 mmol/L) was chosen as the coating buffer for subsequent experiments.

3.1.2 preliminary optimization of antigen coating concentration

The selected optimal coating buffer solution is used for diluting CF to 10 concentration gradients in the range of 5 mu g/mL-200 mu g/mL, the indirect ELISA detection is carried out after the ELISA plates are respectively coated, the values of A/B and A/N are analyzed after the value reading of an ELISA reader, and the result shows that the values of A/B and A/N are higher when the coating concentration is 30 mu g/mL, 40 mu g/mL and 50 mu g/mL (see figure 6). Therefore, these three concentrations were initially selected for subsequent experiments.

3.1.3 optimization of coating time

The CF coating time was divided into 1h at 37 deg.C, 2h at 37 deg.C, overnight at 4 deg.C (12 h), overnight at 4 deg.C after 1h at 37 deg.C, and overnight at 4 deg.C after 2h at 37 deg.C. The values of A/B and A/N were analyzed after the values read by the microplate reader according to the indirect ELISA procedure, and the results showed that (see Table 9) the values of A/B and A/N were both higher when the coating was incubated overnight at 4 ℃. Therefore, the coating antigen was selected for optimal coating time by incubation overnight at 4 ℃.

3.1.4 preliminary screening of the confining liquid

The method comprises the steps of initially selecting 15 materials such as 5% of skim milk, 1% of PEG-2000 and the like as confining liquid, carrying out 2h confinement at 37 ℃, detecting according to an indirect ELISA operation step, analyzing values of A/B and A/N after reading by an enzyme-linked immunosorbent assay (ELISA) instrument, and indicating that (shown in figure 7), when the confining liquid is 1% v/v of skim milk and 5% v/v of pig serum, the values of A/B and A/N are high, so that the components of the confining liquid, namely the skim milk and the pig serum, are initially selected and added to carry out subsequent optimization.

3.1.5 adsorption of non-specific factors in serum antibodies

Respectively selecting Msg,M. PhleiAnd CF of BCG three kinds of bacteria is used as an antigen which can generate cross reaction with other mycobacteria in the absorption and removal serum antibody, after being mixed with the diluted serum with the same volume, the mixture is respectively interacted under three conditions of 4 ℃ overnight (12 h), 37 ℃ 30min and 37 ℃ 2h, then detection is carried out according to the operation steps of indirect ELISA, the values of A/B and A/N are analyzed after the value is read by an ELISA reader, the result shows (see table 10) and the values of A/B and A/N are comprehensively considered,M. Phleithe CF has good absorption and removal effects on nonspecific factors in serum antibodies, but the interaction time and temperature have little influence on the result, and considering that the time is saved as much as possible in the practical experimental operation process, the CF of M.Phlei is selected to act on the diluted serum antibodies, and the action time is 37 ℃ for 30 min.

3.1.6 optimization of coating concentration, blocking solution, blocking time, and serum dilution factor

According to the previous experimental results, three levels of four factors of coating concentration, blocking solution, blocking time and serum dilution factor are selected as L ₉ （3 ⁴ ) Orthogonal experimental design (as shown in table 11). The antigen coating concentration is selected from 30 mug/mL, 40 mug/mL and 50 mug/mL; the confining liquid is selected from 1% v/v skimmed milk and 1% v/v pig serum (confining liquid 1), 1% skimmed milk and 3% pig serum (confining liquid 2) and 1% v/v skimmed milk and 5% v/v pig serum (confining liquid 3); the sealing time is respectively selected from 30min at 37 ℃, 1h at 37 ℃ and 2h at 37 ℃; serum dilutions were selected from three dilutions, 20-, 50-and 100-fold, with two parallel controls per well. Then pressDetecting according to the operation steps of indirect ELISA, and reading after the experiment is finishedOD450nm value. Statistical analysis was performed on the experimental results, as shown in table 12, analysis of variance showed that the effect of coating concentration and serum dilution factor on a/N value was very significant (α = 0.01) and the effect of coating concentration, blocking solution and serum dilution factor on a/N value was significant (α = 0.05). The effect of the blocking time on the results was not significant. As can be seen in FIG. 8, the differences mainly result from 30. Mu.g/mL and 40. Mu.g/mL in coating concentration, 1 and 3 in blocking solution, and also 20-fold and 100-fold dilutions in serum dilution. Thus, it was determined that the optimal coating concentration was 50. Mu.g/mL, 1% v/v skim milk +3% v/v pig serum for the blocking solution, 1h at 37 ℃ for the blocking time, and 50-fold dilution for the serum dilution.

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3.1.7 screening of different Secondary antibodies and optimization of dilution factor of the Secondary antibodies

The five antibodies were diluted 5,000, 10,000, 20,000, 50,000, 100,000 times, and the values of a/B and a/N were analyzed after the values were read by an ELISA reader, as a result, according to the detection method of indirect ELISA, it was shown (see fig. 9 and fig. 10) that when the secondary antibody was a secondary antibody 2, i.e., an HRP-labeled rabbit anti-bovine (Fc-specific) IgG antibody, the values of a/B and a/N were significantly superior to those of the other secondary antibodies, and when the HRP-labeled rabbit anti-bovine (Fc-specific) IgG antibody was used as the secondary antibody, the values of a/B and a/N were optimal when the dilution factor was 10,000. Thus, an HRP-labeled rabbit anti-bovine (Fc-specific) IgG antibody was selected as the secondary antibody, diluted by 10,000.

3.1.8 optimization of serum samples and duration of action of Secondary antibodies

The action time of a serum sample and the action time of a secondary antibody are respectively set as 37 ℃ for incubation for 30min, 45min and 60 min for a two-factor cross test, after the test is finished according to the operation method of indirect ELISA, the values of A/B and A/N are analyzed by an enzyme-labeling instrument OD450nm reading value, as shown in figure 11, when the serum sample acts for 45min and the secondary antibody acts for 45min, the value of A/N is obviously superior to the combination of other times, meanwhile, the value of A/B is higher, the values of A/B and A/N are comprehensively considered, and the time is saved as much as possible in the operation process of the test, so that the serum sample and the secondary antibody are finally selected to be incubated for 45min as the optimal incubation time.

3.1.9 color development liquid and termination liquid screening method

TMB from 5 different companies is respectively selected as developing solution, sulfuric acid of 2M and hydrofluoric acid of 0.05% are selected as stop solution, two parallel controls are arranged on each hole, the rest is operated according to an indirect ELISA method, the values of A/B and A/N are analyzed after the value is read by an enzyme-linked immunosorbent assay, and the result shows that (shown in figure 12), the value of OD630nm is read when the TMB developing by amresco is stopped by the hydrofluoric acid of 0.05%, and the values of A/B and A/N are obviously superior to those of other combinations.

Optimization of 3.1.10 color development time and color development temperature

Selecting development time of 5min, 10 min and 15 min, developing temperature of 22 ℃ (normal temperature) and 37 ℃ to perform a two-factor cross experiment, setting two parallel controls for each hole, operating the rest according to an indirect ELISA method, analyzing values of A/B and A/N after reading by an enzyme-linked immunosorbent assay (ELISA) instrument, and showing that (see table 13), when the development time is 10 min, the values of A/B and A/N are obviously higher than those of other times, the values of A/B and A/N at 37 ℃ are obviously better than 22 ℃, although the value of A is higher than that of other times when the development time is 15 min, the value of B and the value of N are also relatively higher, comprehensively considering, selecting development temperature of 37 ℃ for 10 min as an optimal development condition.

3.1.11 optimization of the washing step

The washing steps in the ELISA operation process are set as the following 3 modes, the washing is continuously carried out for 3 times, and the drying is carried out for the last time; washing for 3 times (1 min/time), and drying for the last time; washing 3 times (3 min/time), and drying in the last time. The rest of the washing steps are operated according to an indirect ELISA method, after the A/B and A/N values are read, no obvious difference exists between all the groups, and in consideration of time cost and operation convenience, continuous washing is selected for 3 times, and the last drying step is used as a washing method.

3.2 determination of Positive and negative cut-off values

According to the optimized result of 2.2, 200 bacteria cultured as MAP negative sera were detected, OD mean value and standard deviation of all sera were calculated after OD630nm reading, and plotted according to mean value and standard deviation (see FIG. 13), as shown in the figure, it can be analyzed that the data distribution tends to normal distribution, the mean value of the group of data: (A), (B)

) It was 0.286, and the Standard Deviation (SD) was 0.0695. To be provided with

The value of +3 XSD is the positive MAP cutoff value, i.e. 0.5, for->

The value of +2 × SD is the MAP negative cutoff, i.e., 0.425. Thus, when the detection of the clinical sample is complete, the reading at OD630nm (S or `) is taken as a function of the clinical sample>

) (the average was calculated for the samples with duplicate wells), OD630nm of the standard positive serum (P) and OD630nm of the standard negative serum (N) were calculated according to the following formula:

S/P（%）=100×

when the S/P value is more than or equal to 50%, the clinical sample is judged to be MAP positive, when the S/P value is less than or equal to 42.5%, the clinical sample is judged to be MAP negative, when the S/P value is more than 42.5% and less than 50%, the clinical sample is judged to be suspicious, and the clinical sample is required to be rechecked.

3.3 evaluation of Effect of Indirect ELISA method

3.3.1 specificity experiments

(1) Cross-reaction detection

The method is characterized in that 10 positive sera of the cattle frequently suffering from diseases in clinic are selected, the detection is carried out by an optimized ELISA method, and S/P (%) values are analyzed after values are read by an enzyme-linked immunosorbent assay (ELISA), and the results show that (shown in figure 14), S/P (%) values of MAP positive sera are more than 50%, S/P (%) values of other 10 disease positive sera are less than 42.5%, and the detection method is proved to have good specificity.

(2) Detection of 200 negative sera

200 parts of bacteria are selected to be cultured as MAP negative serum, detection is carried out according to an optimized indirect ELISA operation procedure, each serum is repeated for 2 times, the S/P value of the detection result is calculated, 9 parts of the 200 parts of the sera have S/P (%) values larger than 50 percent and are positive sera, and the S/P (%) values of the rest of the sera are all smaller than 42.5 percent. Thus, the method specificity was 95.5% (9/200).

3.3.2 sensitivity test

(1) Positive quality control serum detection

The MAP positive serum is diluted by PBST for seven gradients, other steps are detected according to an optimized indirect ELISA method, after a value is read by an enzyme-linked immunosorbent assay (ELISA) instrument, an S/P (%) value is calculated, and the result shows that (shown in table 14), when the positive serum is diluted to 1,000 times, the S/P (%) value is still more than 50%, and the positive serum is positive, so that the detection method of the experiment has high sensitivity.

(2) Detection of 50 positive sera

Selecting 50 parts of serum with positive MAP by bacterial culture, detecting according to an optimized indirect ELISA operation program, repeating each serum for 2 times, and calculating the S/P value of the detection result, wherein the S/P (%) value of 46 parts of serum in 100 parts of serum is more than 50%, the serum is positive serum, and the S/P (%) values of the rest of serum are less than 42.5%. Thus, the sensitivity of the process was 92.0% (46/50).

3.3.3 repeatability experiments

(1) In-batch repeatability experiments

Calculate the total serum content of each of the 4 plates

And SD according to the formula SD/>

The coefficient of variation (CxV%) in batches calculated by multiplying 100% is shown in Table 15, and the CxV% is between 1.04% and 9.97%, and is less than 10%, which indicates that the method has good repeatability in batches. />

(2) Batch to batch repeatability experiments

Calculate the total serum content of each of the 3 plates

And SD according to the formula SD/>

The coefficient of variation (CxV%) between batches is calculated by multiplying 100%, as shown in Table 16, the CxV% is between 3.16% and 9.44%, and is less than 10%, which shows that the method has good repeatability between batches. />

3.3.4 compliance experiments

The results of the coincidence experiment detection in Shanghai, xinjiang and Hefei are 13 parts of MAP positive serum and 19 parts of MAP negative serum. The experiment is proved to have good repeatability.

3.3.5 comparative test

250 parts of serum are detected by using the kit assembled by the method and the kit for detecting the mycobacterium paratuberculosis antibody of IDEXX respectively, the coincidence rate is calculated, and the results are shown in Table 17. Therefore, the coincidence rate of the kit assembled by the method and the Mycobacterium paratuberculosis antibody detection kit of IDEXX is (%) = (47 + 189)/250 × 100% =94.4%.

3.3.6 shelf life test

And (3) extracting 6 boxes from the assembled kit, storing the assembled kit at 2~8 ℃ for 12 months, extracting one box at 1,2, 3, 6, 9 and 12 months respectively to perform relevant experiments on each part, comparing the relevant experiments with the newly assembled kit, and firstly detecting the volume of each solution, wherein the dosage and the appearance color of all reagents have no obvious change, no precipitate is generated, and the enzyme label plate is well sealed. The compliance rate experiment is carried out on the ELISA plate, 20 sera completely conform to the negative and positive, no cross reaction is caused with the positive sera of the above 10 other diseases, the specificity sensitivity is completely consistent with that of the newly assembled kit, and the kit is good in stability and can be stably stored for 12 months.

5363 detection of clinical serum samples in the 3,4

1900 clinical serum samples from different provinces and cities are taken, and the serum samples spread in northeast, northwest, east of China and central regions and can represent the prevalence situation of MAP in China to a certain extent. The detection result shows that 96 parts of MAP positive serum exist in 1900 parts of serum, and the positive rate is 5.05%.

3.5 preliminary Assembly of the kit

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Claims (7)

1. A bovine Paratuberculosis (PTB) indirect ELISA detection kit is characterized in that the kit comprises Mycobacterium avium subspecies Paratuberculosis (PTB)Mycobacterium avium subsp. paratuberculosisMAP), wherein the culture filtrate of the mycobacterium avium subspecies paratuberculosis is prepared by the following method:

(1) Will comprise 10 ⁹ Inoculating CFU/mL mycobacterium avium paratuberculosis subspecies into a 7H9 culture medium containing 0.05% v/v Tween-80, 0.2% v/v glycerol, 5mM asparagine and 10% v/v self-made ADC, culturing for 10 weeks at constant temperature of 37 ℃ and 160rpm, and collecting a bacterial solution; the homemade ADC is prepared by the following method: naCl 8.5g, dextrorotation-Dextrose 20.0g and catalase 0.03g, and the volume is 1L and the volume is 0.22 mu mFiltering and sterilizing by using a m filter;

(2) The collected cell suspension was centrifuged, and the supernatant was filtered through a 0.22 μm filter to obtain a culture filtrate.

2. The kit for detecting bovine Paratuberculosis (PTB) indirect ELISA according to claim 1, wherein the concentration of the culture filtrate coated enzyme label plate is 50 μ g/mL.

3. The kit for indirect ELISA detection of bovine Paratuberculosis (PTB) according to claim 1, wherein the centrifugation in step (2) is centrifugation at 10000 rpm and 4 ℃ for 30 min.

4. The kit for indirect ELISA detection of bovine Paratuberculosis (PTB) according to claim 1, wherein the kit further comprises Mycobacterium phlei (Mycobacterium phlei) (for adsorption of nonspecific factors in serum antibodies), (PTB) for indirect ELISA detection of Paratuberculosis in bovine Paratuberculosis (PTB) according to claim 1, and the kit further comprises a reagent kit for detecting the presence of nonspecific factors in serum antibodiesMycobacterium phlei) The culture filtrate, coating buffer solution, sample diluent, confining liquid, concentrated washing liquid, rabbit anti-bovine IgG antibody marked by HRP, developing liquid and stop solution.

5. The kit for the indirect ELISA detection of bovine Paratuberculosis (PTB) according to claim 4, wherein the coating buffer is 20 mmol/L Tris-HCl buffer, the sample diluent is PBST, the blocking solution is 1% v/v skim milk +5% v/v pig serum, the concentrated washing solution is 25 XPBST, the color developing solution is TMB color developing solution, and the stop solution is 0.05% v/v hydrofluoric acid.

6. Use of the indirect ELISA kit for bovine paratuberculosis of any of claims 1-5 in the preparation of a reagent for detecting or diagnosing bovine paratuberculosis.

7. The use of claim 6, wherein the reagent is an indirect ELISA detection reagent.

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