CN115902217B - Indirect ELISA (enzyme-Linked immuno sorbent assay) detection kit for bovine paratuberculosis and application thereof - 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 coating antigen with good antigenicity, and simultaneously establishes the bovine paratuberculosis indirect ELISA detection kit and the detection method thereof by using the antigen. The sensitivity of the method of the invention is 93.0% by detecting 100 MAP positive serum. The specificity of the method of the invention is 95.5% by detecting 200 MAP negative serum, which is obviously higher than other detection methods. In a word, the method for indirectly ELISA detection of bovine paratuberculosis, which is established by the invention, has the characteristics of rapidness, accuracy, high sensitivity and high specificity. The invention provides technical support for preventing and eliminating bovine paratuberculosis and protecting the healthy and rapid development of animal husbandry in China.

Description

A bovine paratuberculosis indirect ELISA detection kit and its application

Technical Field

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

Background Art

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB) or Johne's disease (JD), a progressive, infectious, granulomatous, chronic enteritis that primarily affects ruminants, especially dairy cows and various domesticated animals. In the early 20th century, the development of transportation allowed animals to move around the world, leading to a global prevalence of PTB. In most major dairy producing countries, the herd prevalence of MAP infection in dairy cows exceeded 50%. Paratuberculosis-infected cows are unsuitable for breeding, and the sale of sick cows can easily infect other herds, causing irreparable losses and having a serious impact on the global economy. This long-distance, gradual, and unnoticeable spread of PTB is mainly due to the presence of a large number of latent infection cases and the high resistance of the pathogen to adverse environmental factors, all of which constitute the risk of MAP transmission. At the same time, PTB is a disease with a wide range of susceptible hosts, including non-human primates.

In recent years, my country's cattle industry has been expanding. However, due to improper management of most cattle farms, high stocking density and poor sanitary environment, PTB has spread more and more rapidly, and the incidence rate has increased significantly. After cattle are infected with MAP, MAP can be transmitted vertically through the placenta, so sick cows are no longer suitable for breeding, and milk production is reduced. At the same time, the sale of sick cows is very likely to infect other herds, causing immeasurable losses to the animal husbandry industry. According to the Ministry of Agriculture of my country, PTB is a Class II animal epidemic disease and MAP is a Class III animal pathogenic microorganism.

At present, there is no effective way to prevent the disease. Sick cattle generally show clinical symptoms in the late stage of the disease, and drugs cannot treat it. At the same time, due to its wide transmission routes, it is easy to interfere with the detection of tuberculosis, so active immunization is not suitable. In response to this disease, while strengthening feeding management and standardizing breeding methods, the only way is to conduct regular testing, isolate suspected animals, and eliminate sick animals.

Among the existing diagnostic methods, microscopic examination has the disadvantages of low sensitivity and specificity, and is also affected by other mycobacteria in the environment, resulting in false positive test results. Due to the subclinical stage, the amount of bacteria excreted from the body by sick animals is small, making bacterial culture sensitivity low. At the same time, negative bacterial cultures need to be retested, which is time-consuming and costly, limiting the rapid identification of MAP. Immunohistochemistry has good sensitivity but may have cross-reactions.

Among them, a lot of research has been done on the detection of MAP cellular immunity, but it is mainly limited by three reasons: (1) No specific antigens have been found in all animals infected with MAP that can universally stimulate cytokines or other indicators of cells; (2) It is not clear whether animals have a strong cellular immune response after being infected with MAP; (3) The cost of this test is very high. At the same time, the control of diagnostic costs is also an important factor in the detection and prevention of PTB. The cost of the test cannot exceed the loss caused by the disease itself. Therefore, ELISA is easy to operate, highly sensitive, and specific, and is suitable for large-scale screening and qualitative analysis. It is very suitable for the detection of PTB in cattle farms. However, since tuberculosis can interfere with the detection of PTB during the detection process, there is currently no very effective antigen in China that can detect PTB without being interfered with by tuberculosis.

Therefore, it is urgent to find a coating source with good antigenicity and establish an indirect ELISA detection method for bovine paratuberculosis using the antigen.

Summary of the invention

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

In order to achieve the above object, the present invention adopts the following technical means:

The present invention optimizes the MAP culture medium components and culture conditions, analyzes the biological characteristics of various subcellular components of MAP, successfully screens out subcellular components with good reactivity, and then uses component separation, non-specific protein adsorption treatment and other methods to obtain the best coating antigen. The subcellular component is used as the coating antigen to optimize the conditions of the indirect ELISA antibody detection method for bovine paratuberculosis, determine the positive and negative critical values, and evaluate the effect. The results show that the established indirect ELISA method has a sensitivity of 92.0%, a specificity of 95.5%, and a coefficient of variation within and between batches of less than 10%. After being assembled into a kit, it can be stably stored for 12 months. 1900 clinical serum samples from different provinces and cities were tested, and the positive rate of PTB was 5.05%.

Based on the above research, the present invention proposes an indirect ELISA detection kit for bovine paratuberculosis, wherein the kit comprises an ELISA plate coated with a culture filtrate of avian Mycobacterium paratuberculosis subspecies, wherein the culture filtrate of avian Mycobacterium paratuberculosis subspecies is prepared by the following method:

(1) Inoculate 10 ⁹ CFU/mL of Mycobacterium paratuberculosis subspecies into 7H9 medium containing 0.05% v/v Tween-80, 0.2% v/v glycerol, 5 mM asparagine and 10% v/v homemade ADC, culture at 37°C and 160 rpm for 10 weeks, and collect the bacterial liquid; wherein the homemade ADC is prepared by the following method: NaCl 8.5 g, Dextrose 20.0 g, catalase 0.03 g, dilute to 1 L, and filter and sterilize with a 0.22 µm filter;

(2) The collected bacterial liquid is centrifuged and the supernatant is filtered through a 0.22 μm pore size filter membrane to obtain the culture filtrate.

Preferably, the concentration of the culture filtrate coated on the ELISA plate is 50 μg/mL.

Preferably, the centrifugation in step (2) refers to centrifugation at 10,000 rpm and 4°C for 30 min.

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

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

Furthermore, the present invention also proposes the use of the bovine paratuberculosis indirect ELISA detection kit in preparing a reagent for detecting or diagnosing bovine paratuberculosis.

Among them, preferably, the reagent is an indirect ELISA detection reagent.

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

The present invention successfully screens out a coating source with good antigenicity, and at the same time uses the antigen to establish an indirect ELISA detection kit for bovine paratuberculosis and a detection method thereof. During the experiment, three types of sera, paratuberculosis positive serum, tuberculosis positive serum and negative serum, were used for detection throughout the process to establish an indirect ELISA method that can eliminate the influence of tuberculosis on PTB detection. In the experiment, when establishing the optimal conditions for each step, a method combining extensive screening and focused repetition was adopted to screen out the optimal conditions as much as possible. When screening some conditions, an orthogonal experimental design method was selected, which not only reduced the operating steps, but also improved the accuracy of the results. Through the detection of 100 MAP positive sera, it was concluded that the sensitivity of this method was 93.0%. Through the detection of 200 MAP negative sera, it was concluded that the specificity of this method was 95.5%. It is significantly higher than other detection methods.

The indirect ELISA method for bovine paratuberculosis established in this experiment is rapid, accurate, sensitive and specific, providing technical support for preventing and eliminating the disease and protecting the healthy and rapid development of my country's animal husbandry.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the preliminary screening results of coating antigens;

FIG2 is a preliminary optimization result of the coated antigen culture process;

Fig. 3 is the purification result of CF gel filtration chromatography;

Fig. 4 is a schematic diagram of fraction collection after sucrose density gradient centrifugation;

Figure 5 shows the titer test of rabbit serum prepared by M.Phlei and Msg;

Figure 6 shows the preliminary optimization of antigen coating concentration;

Figure 7 shows the preliminary screening of blocking solutions;

Figure 8 shows the influence of various factors on the A/N value;

Figure 9 shows the A/N values of five secondary antibodies at different dilution factors;

Figure 10 shows the A/B values of five secondary antibodies at different dilution factors;

Figure 11 shows the optimization of serum samples and secondary antibody action time;

FIG12 is a screen of a color developing solution and a stop solution;

Figure 13 is a histogram and normal distribution diagram of negative serum OD630nm values;

Figure 14 shows specificity detection.

Explanation of symbols:

symbol English full name Chinese full name BCG Bacillus Calmette-Guerin BCG BSA Bovine serum albumin BSA CF Culture filter Culture filtrate HRP Horseradish peroxide Horseradish peroxidase IgG Immunoglobulin G IgG 1 kDa kilODalton Kilodaltons M. Phlei Mycobacterium phlei Mycobacterium phlei Msg Mycobacterium smegmatis Mycobacterium smegmatis MTB Mycobacterium tuberculosis Mycobacterium tuberculosis PB Phosphate buffer Phosphate buffer solution PBS Phosphate buffered saline Phosphate buffered saline PBST Phosphate buffered saline with Tween-20 Phosphate buffered saline with 5‰ Tween-20 PEG2000 Polyethylene glycol 2000 Polyethylene glycol 2000 PMSF Phenylmethanesulfonyl fluoride Phenylmethylsulfonyl fluoride TMB 3,3',5,5'-tetramethyl benzidine Tetramethylbenzidine

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Screening and optimization of antigens

1 Materials and reagents

1.1 Materials

Experimental strains: The strain used for the antigen development of this kit is MAP P18, purchased from the China Institute for the Identification of Biological Products, and kept and used by the Harbin Veterinary Research Institute. Two healthy calves aged 4 to 8 months were provided by the Experimental Animal Center of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

1.2 Reagents

ELISA plates were purchased from Costar; TMB colorimetric solution was purchased from Tiangen; 7H9 culture medium was purchased from BD; PROCLIN-300 preservative and Freund's incomplete adjuvant were purchased from Sigma, Protein A resin was purchased from GE; BCA protein quantitative detection kit was purchased from Abcam.

1.3 Instruments

The ELX800 microplate reader was purchased from BioTeK, USA; the AKTA protein purification system was purchased from GE.

2 Methods

2.1 Source and preparation of serum for the experiment

(1) Source and preparation of negative serum

A healthy non-immunized calf aged 4 to 8 months was selected, and the MAP and MTB antibodies were negative (both the Mycobacterium paratuberculosis antibody detection kit and the Mycobacterium bovis antibody detection kit were negative, both purchased from IDEXX, USA). Blood was collected from the jugular vein, and the serum (light yellow clear liquid) was separated after standing at 37°C for 2 hours. The separated serum was inactivated in a 56°C water bath for 30 minutes, and PROCLIN-300 preservative was added at 0.04% of the serum volume, filtered and sterilized with a 0.22 µm filter, and aseptically quantitatively packaged in 3 mL/bottle and stored at 2-8°C.

(2) Source and preparation of MAP-positive serum

The sick cows came from known infected herds. Clinical observations and autopsy results proved that they were infected with MAP, and the intestinal contents were cultured positive. At the same time, the Mycobacterium paratuberculosis antibody detection kit from IDEXX was used for testing, and the results showed a strong positive for MAP. Blood was collected from the jugular vein of the cow, and the serum was separated using the same method as the negative serum.

(3) Source and preparation of MTB positive serum

A healthy non-immunized calf aged 4 to 8 months was selected and immunized with 7083E6 protein (a Mycobacterium tuberculosis-specific protein provided by the Bacterial Disease Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences). Blood was collected from the jugular vein 2 weeks after the second immunization, and the serum was separated.

2.2 Cultivation of MAP colonies

2.2.1 Preparation of W-R potato culture medium

(1) Preparation of liquid W-R medium (1 L)

Asparagine 5.0 g _{KH2PO4 ​} 2.0 g MgSO ₄ 1.0 g (2.045 g with 7H ₂ O) Ammonium citrate 2.0 g NaCl 2.0 g Ammonium Ferric Citrate 0.075 g Dextrose (LD-) 10.0 g (11.0 g if C ₆ H ₁₂ O ₆ ·H ₂ O) glycerin 48 mL Mineral Additives A and B Fluids 1.33 mL each

Take 950 mL of distilled water (excluding the volume of glycerol) and divide it into two parts. Put them into a 100℃ water bath respectively. Add asparagine to one part and dissolve it. Then use a 100 mL beaker (bottle) to take another part of the distilled water after the water bath to dissolve the above reagents in the above order, and add the dissolved reagents to the asparagine solution in the above order. Then add 48 mL of glycerol, 1.33 mL of solution A and solution B respectively, adjust the pH to 7.2 with 1 M NaOH, and filter.

Liquid 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

Liquid B:

5% CaCl ₂ solution

(2) Preparation of fresh potato cubes

Take the round columnar part in the middle of a fresh potato, cut it into two parts along the diagonal line, and soak them in clean running water for 24 hours.

(3) High pressure culture medium

Take a portion of W-R liquid medium, soak the potatoes in it, place in a 70℃ water bath for 2 hours, and cool for half an hour. Pour the remaining W-R liquid medium into test tubes, about 40 mL per tube, place the potato pieces treated in the water bath on the top of the test tube, seal the cap tightly, and sterilize at 121℃ for 15-20 minutes.

2.2.2 Cultivation of MAP W-R potato medium

The MAP P18 strain was inoculated on a slope of a W-R potato culture medium and cultured at 37°C for 4-6 weeks. Small, slightly white, irregular colonies with a diameter of about 1 mm and a rounded surface and slightly thin edges grew out. These colonies were MAP.

2.3 Antigen screening

2.3.1 Cultivation of MAP

100 μL (all inoculated bacteria are broken dry bacteria that retain activity, unless otherwise specified) containing 10 ⁹ CFU/mL of bacteria was inoculated into 7H9 medium. Since no resistance was added to the medium, three replicates were made for each type to prevent contamination. After culturing at 37°C and 160 rpm for 8 weeks, the bacterial solution was collected.

2.3.2 Separation of bacterial subcellular components

The collected bacterial liquid was separated into subcellular components. 50 mL of MAP culture was centrifuged at 10,000 rpm and 4℃ for 30 min. The supernatant was filtered through a 0.22 μm pore size filter membrane to obtain the culture filtrate (CF). A portion of the CF was inactivated by boiling. The bacteria were washed with 1×PBS, resuspended with PBS containing PMSF (final concentration 1 mM) and PBS without PMSF, respectively, and ultrasonicated for 15 min in an ice bath (ultrasonication for 5s, stop for 5s), and then placed on ice for 15 min and ultrasonicated for another 15 min. The ultrasonic product was centrifuged at 3000 g at 4℃ for 5 min and then at 100,000 g at 4℃ for 4h. The supernatant was transferred for use. At this time, the supernatant was the cytoplasmic component, and the precipitate was a mixed component of the cell wall and cell membrane. Another 10 mL of MAP culture was taken and subjected to microsonic treatment to separate CF and bacteria. The bacteria were resuspended in 1 mL of 20 mM Tris-HCl (pH = 8.0), and a final concentration of 0.05% Tween-80 was added. After ultrasonication at an amplitude of 5% for 30 s (ultrasonication for 3 s, stop for 3 s), the mixture was centrifuged at 10,000 rpm and 4°C for 2 min. The supernatant was collected, which was the cell wall component. After mixing with CF, a mixed component of cell wall and CF was formed. The concentrations of CF (component 1), inactivated CF (component 2), cytoplasm (component 3), cytoplasm without PMSF treatment (component 4), a mixed component of cell membrane and cell wall (component 5), a mixed component of cell membrane and cell wall without PMSF treatment (component 6), and a mixed component of cell wall and CF (component 7) were determined using a BCA protein quantitative detection kit.

2.3.3 Preliminary screening of coating antigens

The indirect ELISA method was used for detection, with CBS as the coating buffer, component 1, component 2, component 3, component 4, component 5, component 6, component 7, and whole bacteria (component 8) as the coating source, the coating source coating concentration was initially set to 50μg/mL, 5% skim milk was initially used as the blocking solution, negative serum (N), MAP positive serum (A), MTB positive serum (B) diluted 100 times as the primary antibody, rabbit anti-bovine secondary antibody (HRP labeled) diluted 5,000 times as the secondary antibody, TMB as the colorimetric solution (all colorimetric solutions in the specific implementation method are TMB colorimetric solutions of Tiangen Company), 2M sulfuric acid as the stop solution, the diluent and washing solution during the entire operation process were PBST, each washing was performed 3 times, with an interval of 3min, and two parallel controls were set for each well. The OD450nm value was read after the color development was terminated. The strength of the antigenicity was determined according to the size of the A/B and A/N values. This part of the experiment was repeated three times, and the most dominant antigen was finally screened out.

2.3.4 Optimization of coating antigen

2.3.4.1 Optimization of the coating antigen culture process

100 μL of the bacterial strain containing 10 ⁹ CFU/mL was inoculated into 100 mL of 7H9 medium (medium 1), 7H9+ADC medium (medium 2), 7H9+self-made ADC medium (medium 3), and beef extract medium (medium 4), respectively. After culturing at 37°C and 160 rpm for 4 weeks, 6 weeks, 8 weeks, 10 weeks, and 12 weeks, the bacterial liquid was collected, and the dominant antigen components were obtained by subcellular component separation. The dominant antigen components obtained from different treatment groups were coated on enzyme-labeled plates, respectively, and indirect ELISA detection was performed. The antigenicity of the dominant antigen components in different culture groups was determined according to the size of A/N and A/B values, and the method of culturing antigens was screened. The self-made ADC was prepared by the following method: NaCl 8.5 g, Dextrose 20.0 g, catalase 0.03 g, fixed volume to 1 L, and filtered and sterilized with a 0.22 μm filter. The antigen of this culture method was further optimized, and the culture medium under this culture condition was treated with the following groups: original component group (component 1), group without glycerol addition (component 2), group with double glycerol content (component 3), group without Tween-80 addition (component 4), group with double Tween-80 content (component 5), and anaerobic culture group (component 6). The culture was carried out at a constant temperature of 37°C and 160rpm, and the bacterial liquid was collected. The dominant antigen components were obtained by subcellular component separation. The dominant antigen components obtained from different treatment groups were coated on ELISA plates, and indirect ELISA detection was performed. The strength of the antigenicity of the dominant antigens of different components was determined according to the size of the A/B and A/N values, and the better way to cultivate antigens was screened out. The optimal culture method was optimized again, 100 μL of bacteria containing 10 ⁹ CFU / mL was inoculated into the above-mentioned optimal culture medium, and serine, lysine, cysteine, glycine, valine, leucine, glutamine, phenylalanine, glutamic acid, methionine, asparagine, alanine, proline, threonine, arginine, tyrosine, histidine, isoleucine and ascorbic acid were added to the culture medium respectively, with a final concentration of 5 mM. A control group without additives was set up at the same time, and two replicates were made for each group. After culturing according to the above-mentioned optimal culture method, the bacterial liquid was collected, and the dominant antigen components were obtained by subcellular component separation. The dominant antigen components obtained from the treatment groups with different components were coated on enzyme-labeled plates, and indirect ELISA detection was performed. The strength of the antigenicity of the dominant antigen components in the culture groups with different additives was determined according to the size of A/B and A/N values, and the optimal additive components for culturing antigens were screened out.

2.3.4.2 Individual Optimization of Coating Antigen

In order to further optimize the coated antigen, after separating the coated antigen according to the above-mentioned optimized conditions, the coated antigen was subjected to the following treatments.

(1) Gel filtration chromatography

After filtering all working solutions (20% ethanol, 20 mM Tris-HCl (pH=8.0), deionized water) and coated antigens through a 0.45 μm filter membrane, turn on the instrument, first clean the AKTA machine and prepare the pipeline. After the pump is cleaned, install the chromatography column, install the pre-loaded column of Superdex 200 (separation range 20 kDa-200 kDa) on the AKTA, the detection wavelength is 280 nm, the pre-loaded column is balanced and loaded with a sample loop, start running the program, and collect samples after the program runs. Collect samples under each peak and coat the ELISA plate at a concentration of 50 μg/mL. At the same time, set the untreated coated antigen as the control group for indirect ELISA detection, and determine the strength of the antigenicity of each protein under different peaks according to the size of the A/B and A/N values.

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

The proteins below 20 kDa in the coated antigen were retained by a hollow fiber ultrafiltration experimental device, leaving a mixed component of small molecular weight proteins, and coated on an ELISA plate according to a concentration gradient of 5 μg/mL, 10 μg/mL, 20 μg/mL, 35 μg/mL, 50 μg/mL, 100 μg/mL, and 150 μg/mL. At the same time, the unretained coated antigen was set as a control group for indirect ELISA detection, and the strength of the antigenicity at different concentrations was determined according to the A/B and A/N values.

(3) Preparation of pure protein derivatives for coating antigens

Take 10 mL of the coated antigen, add 3.0 g of phenol per mL, mix well and let stand at 4-8°C for 4 hours, retain the supernatant (sample 1) and discard the precipitate, remove the insoluble protein, add 40% trichloroacetic acid with a final concentration of 2%~4% to the supernatant, mix well and let stand for 4-6 hours, discard the supernatant (sample 2), obtain the precipitate, wash the precipitate three times with 1% trichloroacetic acid and collect the precipitate by centrifugation at 3,000rpm (sample 3). Dissolve all the precipitate with PB, add an equal amount of saturated ammonium sulfate solution to it, and let it stand at 4°C overnight (sample 4), collect the precipitate after centrifugation of the liquid, dissolve it with PB to obtain a crude extracted pure protein derivative (sample 5), and dialyze the crude extract twice in PBS (3.0 g of phenol added per mL) to obtain a refined pure protein derivative (sample 6). The samples from each step of the preparation process were collected and coated on ELISA plates. Untreated coated antigens were set as a control group for indirect ELISA testing. The strength of the antigenicity of the coated antigen after each step of treatment was determined based on the A/B and A/N values.

(4) Sucrose density gradient centrifugation

Select three gradients (20%, 40%, 60%) of sucrose solution, add them into the centrifuge tube in sequence with a long needle, and finally slowly add the coated antigen to the top layer. After centrifugation at 120,000g for 2 hours, gently take out the centrifuge tube. It can be found that there is a bright band between 20% and 40% and between 40% and 60%. Pipette the solutions of the eight parts shown in Figure 4 respectively, dialyze them in PBS buffer at 4°C overnight to remove sugar, and coat the eight solutions on ELISA plates respectively. At the same time, set the untreated coated antigen as the control group for indirect ELISA detection. The strength of the antigenicity of the coated antigen after each step of treatment is determined according to the A/B and A/N values.

(5) Adsorption of non-specific reactive proteins

First, rabbit high immune serum was prepared using M. Phlei and Msg

Male New Zealand white rabbits, weighing about 3 kg each, were purchased and raised for one week to adapt to the environment before immunization. Two rabbits were immunized with each bacterium. Blood was collected from the ear vein of the rabbit before immunization, and the serum was separated as a negative control. M.Phlei and Msg were diluted to 2 mg/mL with sterile PBS, mixed with Freund's incomplete adjuvant in a ratio of 1:1 (calculated based on body weight 0.5 mg/kg), and then immunized with rabbits every two weeks. 7-10 days after the third immunization, 2 mL of blood was collected from the ear vein to separate the serum for antibody titer detection. The serum to be tested was diluted in multiples (1:100; 1:200; 1:400; 1:800; 1:1,600; 1:3,200; 1:6,400; 1:12,800; 1:25,600; 1:51,200; 1:102,400; 1:204,800). The ratio of the OD value of the reaction well to the negative control was greater than or equal to 2 and was judged as positive. The dilution multiple of the highest dilution multiple was used as the titer of the immune serum. After the antibody titer reached the requirements of this experiment, all serum was collected.

Preliminary purification of rabbit serum antibodies by caprylic acid-ammonium sulfate method

After the collected rabbit serum was centrifuged at 12,000 rpm for 5 min, 5 mL of supernatant was taken, 20 mL of acetate buffer was added, and the mixture was placed in a stirrer. While slowly stirring, 1,875 μL of octanoic acid was added. After continuing to stir for 30 min, the mixture was immediately centrifuged at 4°C 12,000 rpm for 30 min, and the supernatant was collected. 0.227 g of ammonium sulfate was added to each milliliter of supernatant, and the mixture was continued to stir for 30 min. After standing for 5 h, the mixture was centrifuged at 4°C 12,000 rpm for 30 min, the supernatant was discarded, and the precipitate was resuspended in 2.5 mL of PBS. The suspension was placed in a dialysis card and dialyzed with PBS. The dialyzation was performed overnight at 4°C and the solution was changed 3 times. Finally, the solution in the dialysis card was collected as the preliminarily purified rabbit serum antibody.

Non-specific protein adsorption in coated antigen

Three tubes of Protein A resin were prepared. After equilibrating the resin with buffer A for 10 column volumes, the preliminarily purified M.Phlei and Msg rabbit serum antibodies were divided into M.Phlei group (sample 1), Msg group (sample 2) and M.Phlei+Msg (1:1 equal concentration mixture) mixed group (sample 3), and passed through the resin separately until saturation. An equal amount of coated antigen was added to the three resins respectively. After 10 min of action, the flow-through was collected and washed with buffer B. After collecting the wash solution, the flow-through was slowly added to the resin. After 10 min of action, the flow-through was collected again and washed with buffer B. After repeating this process 5 times, all the flow-throughs (sample 4) and wash solutions (sample 5) of sample 1, the flow-throughs (sample 6) and wash solutions (sample 7) of sample 2, and the flow-throughs (sample 8) and wash solutions (sample 9) of sample 3 were collected. The collected samples 4-9 were coated on ELISA plates respectively. At the same time, untreated coated antigen was set as the control group for indirect ELISA detection. The strength of the antigenicity of the six samples was determined according to the A/B and A/N values.

3 Results

3.1 Antigen screening

3.1.1 Preliminary screening of coating antigens

The indirect ELISA method was used for detection, with CBS as the coating buffer, and components 1, 2, 3, 4, 5, 6, 7, and whole bacteria (component 8) as coating antigens, with a coating concentration of 50 μg/mL, negative serum (N), MAP positive serum (A), and MTB positive serum (B) diluted 100 times as primary antibodies, and rabbit anti-bovine secondary antibody (HRP labeled) diluted 5,000 times as secondary antibodies. The OD450nm value was measured after color development was terminated. After the A/B and A/N values were read by the microplate reader, the results showed (see Figure 1) that the A/B value in component 1 was relatively high, and the A/N value was the highest, so component 1 had the best effect, that is, CF was the most dominant coating antigen.

3.1.2 Optimization of the coating antigen preparation process

100 μL of the bacterial strain containing 10 ⁹ CFU /mL was inoculated into 100 mL of culture medium 1-4, and cultured at 37°C and 160 rpm for 4, 6, 8, 10 and 12 weeks, respectively. The bacterial liquid was collected and CF was obtained by subcellular component separation. The CF obtained from different treatment groups were coated on ELISA plates and tested by indirect ELISA. The A/B and A/N values were analyzed after the ELISA instrument read the values. The results showed (see Figure 2) that under the conditions of culture medium 3, the A/N value was optimal and the A/B value was better after 10 weeks of culture. Therefore, 7H9+self-made ADC culture medium for 10 weeks was the optimal culture condition. Considering that the added components (Tween-80, glycerol) in the culture medium may affect the effect of secreted CF, under this culture condition, the additives of the culture medium were optimized to components 1-6. After 10 weeks of culture, the CF of different components were separated and coated on the ELISA plate for indirect ELISA detection. The A/B and A/N values were analyzed after the ELISA instrument read the values. The analysis results showed (see Table 1) that the results of components 1-5 were not much different, but during the culture process, component 2 would affect the turbidity of bacteria. When cultured according to component 4, bacteria were more easily contaminated than other groups. At the same time, considering the need to save materials as much as possible during the experiment, the original culture medium components were selected. Therefore, the initial optimized culture conditions were 7H9 (0.05% v/v Tween-80, 0.2% v/v glycerol) + homemade ADC culture medium at 37°C and 160 rpm for 10 weeks. Considering that different amino acids may affect the types and contents of proteins in the bacterial growth process, the culture method of the coated antigen in the previous step was optimized again. 18 kinds of amino acids and ascorbic acid were added to the culture medium, with a final concentration of 5 mM. A control group without additives was set up at the same time. Two replicates were made in each group. After culturing at 37℃ and 160 rpm for 10 weeks, the bacterial liquid was collected. After the CF was separated, they were coated with enzyme-labeled plates and indirect ELISA was performed. After the values were read by the enzyme-labeled instrument, the A/B and A/N values were analyzed. The results showed (see Table 2) that considering the A/N value and A/B value, the groups with lysine, cysteine, phenylalanine, asparagine, alanine, histidine and isoleucine additives could improve the sensitivity and specificity of the coated antigen detection; the groups with glutamic acid and tyrosine additives could reduce the sensitivity and specificity of the coated antigen detection. The experiment was repeated under the same conditions, and the results showed (see Table 2) that the repeatability of the lysine and asparagine groups was good, but considering that the A/B value of the lysine group was lower and the detection specificity was reduced, it was concluded that the addition of asparagine could effectively improve the detection sensitivity and specificity of CF.

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

(1) Inoculate 10 ⁹ CFU/mL of Mycobacterium paratuberculosis subspecies into 7H9 medium containing 0.05% v/v Tween-80, 0.2% v/v glycerol, 5 mM asparagine and 10% v/v homemade ADC, culture at 37°C and 160 rpm for 10 weeks, and collect the bacterial liquid; wherein the homemade ADC is prepared by the following method: NaCl 8.5 g, Dextrose 20.0 g, catalase 0.03 g, dilute to 1 L, and filter and sterilize with a 0.22 µm filter;

(2) The collected bacterial liquid is centrifuged and the supernatant is filtered through a 0.22 μm pore size filter membrane to obtain the culture filtrate (CF).

3.2 Comparison of detection effects after separation of coated antigens

3.2.1 Gel filtration chromatography

The filtered CF was passed through the gel filtration column Superdex 200. After the program was run, it can be seen from Figure 3 that there were obvious absorption peaks at 47 mL, 67 mL and 116 mL, distributed in the D, F and J regions. The samples of each tube under the highest peak (D8, D9, F4, F5, J5, J6) were collected and coated with the ELISA plate at a concentration of 50 μg/mL. At the same time, the CF without gel filtration chromatography was used as a control for indirect ELISA detection. After the ELISA instrument read the values, the A/B and A/N values were analyzed. The results showed (see Table 3) that the A/N and A/B results of the 6 tubes of samples were not as good as the CF detection values before gel filtration chromatography. This shows that in the process of CF gel filtration chromatography purification, no more advantageous coated antigens were separated than before purification.

3.2.2 Retention of proteins below 20 kDa in the coated antigen

The proteins below 20 kDa in the coated antigen were retained by the hollow fiber ultrafiltration experimental device, leaving the mixed components of small molecular weight proteins. Seven gradients were set according to the concentration of 5 μg/mL-150 μg/mL to coat the ELISA plate respectively. At the same time, the unretained coated antigen (50 μg/mL) was set as the control group for indirect ELISA detection. The values of A/B and A/N were analyzed after the ELISA instrument read the values. The results showed (see Table 4) that the values of A/N and A/B of the control group were significantly better than the values of each concentration gradient of the mixed components of the small molecular weight proteins after the retention. This shows that there is no effective coated antigen component in the small molecular weight protein after the retention that is significantly better than the original CF.

3.2.3 Detection of pure protein derivatives of coated antigens

The samples collected at each step in the preparation process of the pure protein derivatives of the coated antigen (a total of 6 types) were coated on ELISA plates respectively, and the untreated coated antigen was set as the control group for indirect ELISA detection. The results showed (Table 5) that the comprehensive judgment results of the A/N and A/B values of the 6 samples were not as good as those of the control group, indicating that the pure protein derivatives of the coated antigen and the preparation process of the pure protein derivatives did not produce components that were more effective than the original CF and could serve as coating antigens.

3.2.4 Sucrose density gradient centrifugation of coated antigen

Three gradients of sucrose solutions of 20%, 40%, and 60% were selected to perform density gradient centrifugation on CF. The solutions of the eight parts shown in Figure 4 were respectively aspirated and dialyzed in PBS buffer to remove sugar. The eight solutions were coated on ELISA plates respectively, and untreated coated antigens were set as the control group for indirect ELISA detection. After the ELISA instrument read the values, the A/B and A/N values were analyzed. The results showed (see Table 6) that sucrose density gradient centrifugation did not separate antigen components that were more effective than the original antigens.

3.3 Nonspecific protein adsorption treatment of coated antigens

3.3.1 Rabbit high immune serum titer test

The collected serum prepared from M.Phlei and Msg was tested for antibody titer, and the serum to be tested was diluted in multiples (1:100 dilution in the first well, and then diluted in multiples to 1:204,800), and the pre-immune serum was used as a negative control, and M.Phlei and Msg were used as coating antigens for indirect ELISA detection. The results were analyzed after the microplate reader read the values. The results showed (see Figure 5) that the titers of both sera could reach 102,400, which met the requirements of this experiment.

3.3.2 Adsorption of nonspecific proteins in coated antigens

After the prepared rabbit high immune serum was initially purified by the caprylic acid-ammonium sulfate method, Protein A resin was added to saturation, CF was added to the resin, and all samples in the purification process were collected. Samples 4-9 were coated with ELISA plates respectively, and untreated coated antigens were set as the control group for indirect ELISA detection. The A/B and A/N values were analyzed after the ELISA instrument read the values. The results showed (see Table 7) that the A/B and A/N values of the control group were significantly better than those of sample groups 4-9, indicating that the untreated CF had better antigenicity.

Example 2 Establishment of indirect ELISA detection method for bovine paratuberculosis

1 Materials and reagents

1.1 Materials

Specific quality control serum was stored by Harbin Veterinary Research Institute. Male New Zealand white rabbits-3 kg, SPF grade, were purchased from the Engineering Center of Harbin Veterinary Research Institute.

1.2 Reagents

Sheep anti-bovine polyclonal antibody (HRP labeled) was purchased from KPL Company; four rabbit anti-bovine secondary antibodies were all purchased from Sigma Company; five different brands of TMB colorimetric solutions were purchased from Sigma, Shanghai Bioengineering, Amresco, Thermo and Tiangen Company.

1.3 Instruments

The ELX800 microplate reader was purchased from BioTeK, USA; the hollow fiber ultrafiltration experimental device was purchased from Beijing Xubang Membrane Equipment Co., Ltd.

2 Methods

2.1 Operation steps of indirect ELISA detection method

(1) Coating: Use the optimized CF as the coating antigen, dilute it to the optimal concentration and add it to the ELISA plate (100 μL/well). After incubation for a period of time, discard the liquid in the ELISA plate, wash it with PBST and pat the plate dry.

(2) Blocking: Add blocking solution to the ELISA plate after tapping dry, 200 μL/well. After incubation for a period of time, discard the liquid in the ELISA plate, wash with PBST and tap the plate dry.

(3) Primary antibody (or test sample): Dilute the negative serum (N), MAP positive serum (A), MTB positive serum (B) or the collected clinical serum sample (after optimization treatment) with PBST at the optimal dilution multiple and add it to the ELISA plate, 100 μL/well, and perform 2-3 replicates for each sample. After incubation for a period of time, discard the liquid in the ELISA plate, wash with PBST and pat the ELISA plate dry.

(4) Enzyme-labeled secondary antibody: Dilute the optimal secondary antibody with PBST and add it to the ELISA plate at 100 μL/well. After incubation for a period of time, discard the liquid in the ELISA plate, wash with PBST, and pat the plate dry.

(5) Color development: Add 100 μL of the best color developing solution to each well and allow the color to develop for a period of time (avoid light).

(6) Stop reaction: Add 100 μL of stop solution to each well.

(7) Reading value.

2.2 Optimization of various conditions during indirect ELISA detection

2.2.1 Screening of coating buffer

PBS (0.01 mol/L), CBS (0.05 mol/L) and Tris-HCl (20 mmol/L) were used as coating buffer to dilute CF. In the preliminary experiment, the three coating solutions were used to dilute the coating source to 50 μg/mL, 100 μL/well was added to the ELISA plate, incubated at 4°C for 12 hours, washed three times with PBST, each time with an interval of 3 minutes, and the ELISA plate was patted dry after the last wash (this step is referred to as washing). Skim milk was diluted to 5% with PBST as blocking solution, 200 μL/well was added to the ELISA plate, blocked at 37°C for 2 hours, and the washing procedure was repeated. Negative serum (N), MAP positive serum (A), and MTB positive serum (B) were diluted 100 times with PBST as primary antibodies, 100 μL/well, incubated at 37°C for 1 hour, and the washing procedure was repeated. Rabbit anti-bovine secondary antibody (HRP labeled) was diluted 5,000 times as secondary antibody and incubated at 37°C for 1 hour. After incubation for 1 hour, the washing procedure was repeated, TMB (Tiangen) was used as the color developing solution, and 2M sulfuric acid was used as the stop solution after color development at 37°C for 10 minutes, and 100 μL was added to each well. Two parallel controls were set up for each well, and the OD450nm value was read after color development was terminated. The best coating buffer was screened according to the size of A/B and A/N values.

2.2.2 Preliminary optimization of coating antigen concentration

The optimal coating buffer was used to dilute the CF concentration to 5 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL, 150 μg/mL, and 200 μg/mL, a total of 10 gradients, and the ELISA plate was coated separately. 5% skim milk was used as the blocking solution, serum was diluted 100 times, rabbit anti-bovine secondary antibody (HRP labeled) was diluted 5,000 times as the secondary antibody, TMB (Tiangen) was used for color development, and 2M sulfuric acid was used to terminate the reaction. Two parallel controls were set up for each well. After the experiment was completed, the OD450nm value was read. The optimal coating concentration was screened according to the size of the A/B and A/N values.

2.2.3 Optimization of coating time

After CF was diluted to the optimal concentration, five conditions were tested for coating the ELISA plate, namely, incubation at 37℃ for 1 h, incubation at 37℃ for 2 h, incubation at 4℃ overnight, incubation at 37℃ for 1 h and then at 4℃ overnight, and incubation at 37℃ for 2 h and then at 4℃ overnight. 5% v/v skim milk was used as the blocking solution, serum was diluted 100 times, rabbit anti-bovine secondary antibody (HRP labeled) was diluted 5,000 times as the secondary antibody, TMB (Tiangen) was used for color development, and 2M sulfuric acid was used to terminate the reaction. Two parallel controls were set for each well. After the test was completed, the OD450nm value was read. The optimal coating time was screened according to the size of the A/B and A/N values.

2.2.4 Preliminary screening of blocking solution

After diluting CF at the optimal concentration with the optimal coating buffer, the ELISA plate was coated according to the optimal coating time and the blocking solution was optimized. 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, and Thermo-PBS were initially selected as blocking solutions. The cells were blocked at 37°C for 2 hours, the serum was diluted 100 times, and the rabbit anti-bovine secondary antibody (HRP labeled) was diluted 5,000 times as the secondary antibody. After color development with TMB (Tian Gen), the reaction was terminated with 2M sulfuric acid. Two parallel controls were set up in each well. After the experiment was completed, the OD450nm value was read. The better blocking solution was screened out according to the A/B and A/N values.

2.2.5 Adsorption of nonspecific factors in serum antibodies

Msg, M. Phlei and BCG were cultured separately, and the serum to be tested was diluted and then added with equal volumes of three kinds of bacterial CF (refer to the method of step 2.3.2 of Example 1), and then added to the ELISA plate after being exposed to 4°C overnight (12 h), 37°C for 30 min, and 37°C for 2 h, respectively. Rabbit anti-bovine secondary antibody (HRP labeled) was diluted 5,000 times as the secondary antibody, and TMB (Tiangen) was used for color development, and 2 M sulfuric acid was used to terminate the reaction. Two parallel controls were set up for each well. After the test was completed, the OD450nm value was read. According to the size of the A/B and A/N values, the better CF antigens that can be used to absorb and remove serum antibodies that can cross-react with other mycobacteria were screened.

2.2.6 Optimization of coating concentration, blocking solution, blocking time and serum dilution multiple

According to the previous experimental results, four factors, namely, coating concentration, blocking solution, blocking time and serum dilution multiple, were selected with three levels for L ₉ (3 ⁴ ) orthogonal experimental design. The coating concentration selected the three best concentrations in the initial screening; the blocking solution selected three different concentration ratios of the best components in the initial screening; the blocking time selected 37℃ 30min, 37℃ 1h and 37℃ 2h respectively; the serum dilution multiple selected 20 times, 50 times and 100 times. Rabbit anti-bovine secondary antibody (HRP labeled) was diluted 5,000 times as the secondary antibody. After TMB (Tiangen) color development, the reaction was terminated with 2M sulfuric acid. Two parallel controls were set for each well. After the experiment was completed, the OD450nm value was read. The experimental results were statistically analyzed to determine the optimal coating concentration, blocking solution, blocking time and serum dilution multiple.

2.2.7 Screening of different secondary antibodies and optimization of secondary antibody dilution multiples

Five secondary antibodies were selected, including 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 (antiserum IgG part) IgG antibody (secondary antibody 5). Since secondary antibody 2 and secondary antibody 3 were not HRP-labeled, enzyme-labeled antibodies were prepared first.

Dissolve 2.5 mg HRP in 0.25 mL deionized water, add 0.25 mL sodium periodate (0.06 mol/L) (freshly prepared) aqueous solution, mix at 4°C for 30 min, add 0.25 mL ethylene glycol (0.16 mol/L) aqueous solution, let stand at room temperature for 30 min, add 0.25 mL aqueous solution containing 2.5 mg purified rabbit anti-bovine IgG, mix well, add to dialysis card, put the dialysis card in 0.05 mol/L CBS (pH=9.5) at 4°C overnight for dialysis. Aspirate the liquid from the dialysis card, add 0.1 mL sodium borohydride solution (5 mg/mL), and let stand at 4°C for 2 h. An equal volume of saturated ammonium sulfate solution (pH=7.0) was added thereto, and the mixture was allowed to stand at 4°C for 30 min. The mixture was centrifuged at 3,500 rpm/min for 30 min, and the supernatant was discarded. The precipitate was suspended in PBS (0.02 mol/L; pH=7.4) and added to a dialysis card. The dialysis card was placed in PBS and dialyzed overnight at 4°C. During the period, the liquid was changed three times, and the liquid in the dialysis card was aspirated. After centrifugation to remove insoluble matter, the HRP-labeled secondary antibody was obtained.

After diluting CF at the optimal concentration with the optimal coating buffer, the ELISA plate was coated according to the optimal coating time. After adding the optimized blocking solution and incubating for a period of time, the serum sample with the optimal dilution was added, and the five antibodies were diluted at 1:5,000, 1:10,000, 1:20,000, 1:50,000 and 1:100,000 respectively. After a period of action, TMB (Tiangen) was used for color development, and 2M sulfuric acid was used to terminate the reaction. Two parallel controls were set up for each well. After the test was completed, the OD450nm value was read. The best secondary antibody and its dilution multiple were selected according to the size of the A/B and A/N values.

2.2.8 Optimization of serum samples and secondary antibody action time

After diluting CF at the optimal concentration with the optimal coating buffer, the ELISA plate was coated according to the optimal coating time. After adding the optimized blocking solution and incubating for a period of time, the serum sample with the optimal dilution was added, and incubated at 37°C for 30 min, 45 min and 60 min, respectively. After adding the optimized secondary antibody, it was diluted according to the optimal dilution multiple and added to the ELISA plate and incubated at 37°C for 30 min, 45 min and 60 min. After TMB (Tiangen) color development, the reaction was terminated with 2M sulfuric acid. Two parallel controls were set up in each well, and the rest were operated according to the indirect ELISA method. After the test was completed, the OD450nm value was read. The optimal serum sample and the action time of the secondary antibody were screened according to the size of the A/B and A/N values.

2.2.9 Screening of colorimetric and stop solutions

After diluting CF at the optimal concentration with the optimal coating buffer, the ELISA plate was coated according to the optimal coating time. After adding the optimized blocking solution and incubating for a period of time, the serum sample and secondary antibody at the optimal dilution were added. When developing the color, 5 different brands of TMB color developing solutions from Sigma, Shanghai Shenggong, Amresco, Thermo and Tiangen were selected. When stopping, 2M sulfuric acid and 0.05% hydrofluoric acid were selected as stop solutions. Two parallel controls were set for each well, and the rest were operated according to the indirect ELISA method. After the test was completed, the OD450nm or OD630nm value was read. The optimal color developing solution and stop solution were selected according to the size of the A/B and A/N values.

2.2.10 Optimization of color development time and color development temperature

After diluting CF at the optimal concentration with the optimal coating buffer, the ELISA plate was coated according to the optimal coating time. After adding the optimized blocking solution and incubating for a period of time, the serum sample and secondary antibody at the optimal dilution were added. After adding the optimal color development solution, the action time was set to 5 min, 10 min and 15 min, and the action temperature was selected at 22°C (normal temperature) and 37°C, respectively, and a two-factor crossover experiment was performed. After adding the stop solution, the OD450nm or OD630nm value was read. Two parallel controls were set for each well, and the rest were operated according to the indirect ELISA method. The optimal color development time and color development temperature were screened according to the size of the A/B and A/N values.

2.2.11 Optimization of washing steps

After diluting CF at the optimal concentration with the optimal coating buffer, the ELISA plate was coated according to the optimal coating time. After adding the optimized blocking solution and incubating for a period of time, the serum sample and secondary antibody at the optimal dilution were added, and the color was developed according to the optimized conditions. After termination, the OD450nm or OD630nm value was read, and two parallel controls were set for each well. The washing steps in the whole operation process were set as follows: wash 3 times continuously, and pat dry the last time; wash 3 times, each time with an interval of 1 min, and pat dry the last time; wash 3 times (3 min/time), and pat dry the last time. The rest was operated according to the indirect ELISA method, and the best washing method was screened according to the size of the A/B and A/N values.

2.3 Determination of positive and negative critical values

）和标准差（SD），根据和SD计算临界值，当检测临床样本时，根据临床样本OD值（S或

）（有重复孔的样本要计算平均值），标准阳性血清OD值（P）和标准阴性血清OD值（N），按照以下公式进行计算：According to the optimized results in 2.2, 200 sera with MAP-negative bacterial cultures were tested, and the average OD of all sera was calculated after reading the values (

) and standard deviation (SD), and the critical value is calculated based on the OD value (S or

) (the average value should be calculated for samples with duplicate wells), the standard positive serum OD value (P) and the standard negative serum OD value (N) are calculated according to the following formula:

S/P (%) = 100 ×

+3×SD的值作为MAP阳性临界值，以

+2×SD的值作为MAP阴性临界值，若值在两者之间，判为MAP抗体可疑，应重新检测。by

The value of +3×SD was used as the positive cutoff value of MAP.

The value of +2×SD is used as the MAP negative critical value. If the value is between the two, it is judged as suspicious for MAP antibodies and should be retested.

2.4 Evaluation of the effect of the established indirect ELISA method

2.4.1 Specificity experiments

(1) Cross-reaction detection

+3×SD的值，其他10种疾病阳性血清的S/P（%）值不高于

+2×SD的值，则无交叉反应，特异性良好。Ten positive sera of cattle diseases commonly seen in clinical practice were selected, including 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 (FMD), bovine Mycoplasma agalactiae positive serum (bovine agalactiae), bovine viral diarrhea positive serum (BVDV), bovine Escherichia coli (28a) positive serum (E. coli 28a), bovine Escherichia coli (empty vector) positive serum (E. coli empty), and Mycoplasma bovis positive serum (bovine Mycoplasma). The optimized ELISA method was used for detection. Each serum was repeated in 4 wells. The experiment was repeated twice. The results were judged based on the S/P (%) value. If the S/P (%) value of the MAP positive serum was not less than

+3×SD, the S/P (%) values of the positive sera of the other 10 diseases were not higher than

If the value is +2×SD, there is no cross-reaction and the specificity is good.

(2) 200 negative serum samples tested

200 sera with MAP-negative bacterial culture were selected and tested according to the optimized indirect ELISA procedure. Each serum was repeated twice, and the positive and negative results were judged according to the S/P (%) value, and the positive detection rate was calculated.

2.4.2 Sensitivity test

(1) Positive quality control serum test

The MAP-positive serum was diluted with PBST according to seven gradients of 1:100, 1:200, 1:500, 1:1,000, 1:1,500, 1:3,000, and 1:5,000. The other steps were detected according to the optimized indirect ELISA method. After the microplate reader read the value, the sensitivity was calculated according to the S/P (%) value.

(2) 50 positive serum samples tested

Fifty sera that were MAP-positive in bacterial culture were selected and tested according to the optimized indirect ELISA procedure. Each serum was repeated twice, and the positive and negative results were judged based on the S/P (%) value, and the positive detection rate was calculated.

2.4.3 Repeatability Experiment

(1) Intra-batch reproducibility test

和SD，根据公式SD/

×100%计算批内变异系数（C×V%），确定批内重复性。Four ELISA plates coated with CF were randomly selected from the same batch of prepared plates, and 20 serum samples (including 6 MAP strong positive serum samples, 8 MAP weak positive serum samples and 6 MAP negative serum samples) were tested according to the optimized method. The average value of each serum sample (repeated 3 wells) on each plate was compared with the average value of the serum sample on other plates, and the total value of each serum sample in the four plates was calculated.

and SD, according to the formula SD/

×100% to calculate the intra-batch coefficient of variation (C×V%) and determine the intra-batch repeatability.

(2) Batch reproducibility experiment

和SD，根据公式SD/

×100%计算批间变异系数（C×V%），确定批间重复性。Randomly select 3 ELISA plates coated with CF prepared in different batches, and test the above 20 serum samples according to the optimized method. Take the average value of each serum (repeated 3 wells) on each plate and compare it with the average value of the serum on other plates, and calculate the total value of each serum in the three plates.

and SD, according to the formula SD/

×100% was used to calculate the inter-batch coefficient of variation (C×V%) and determine the inter-batch repeatability.

2.4.4 Compliance experiment

After the CF-coated ELISA plate was blocked, it was assembled into a kit together with 13 MAP-positive sera and 19 MAP-negative sera, secondary antibody, TMB colorimetric solution, stop solution, washing solution and diluent. Three representative regions, namely Shanghai, Xinjiang and Hefei, were selected for the coincidence experiment. The experimental operations were performed by different people in each region. The positive and negative results were judged according to the S/P (%) value, and the positive coincidence rate was calculated.

2.4.5 Comparative test

250 MAP sera with known positive and negative results after bacterial culture were selected and tested using the assembled detection kit and IDEXX's Mycobacterium paratuberculosis antibody detection kit respectively. The experimental results were summarized and compared.

2.4.6 Shelf life experiment

Take out 6 boxes of the assembled test kit and store them at 2-8℃ for 12 months. Take out one box at 1, 2, 3, 6, 9 and 12 months for related experiments of each part and compare them with the newly assembled test kit. First, check whether the volume and appearance color of each solution have changed, whether precipitate has been produced, and whether the ELISA plate is well blocked. Select the 20 sera in 2.3.3, and conduct compliance, specificity and sensitivity experiments on the ELISA plate. Determine the stability of the test kit based on the experimental results.

2.4.7 Testing of clinical serum samples

A total of 1900 clinical serum samples were selected for ELISA testing (collected or sent locally), including 585 bovine sera from Urumqi, 1015 bovine sera from Shuangyashan, 123 bovine sera from Changchun, 58 bovine sera from Shanghai, 10 bovine sera from Zhejiang, 44 bovine sera from Henan, and 65 bovine sera from Hefei. These 1900 sera came from different provinces and cities, covering the Northeast, Northwest, East China and Central Plains, and can represent the prevalence of MAP in my country to a certain extent.

3 Results

3.1 Optimization of various conditions during indirect ELISA detection

3.1.1 Screening of coating buffer

PBS (0.01 mol/L), CBS (0.05 mol/L) and Tris-HCl (20 mmol/L) were used as coating buffers to dilute CF to 50 μg/mL. The experiment was completed according to the operating steps of indirect ELISA. The values of A/B and A/N were analyzed after the microplate reader read the values. The results showed (see Table 8) that when the coating buffer was Tris-HCl (20 mmol/L), the values of A/B and A/N were the best. Therefore, Tris-HCl (20 mmol/L) was selected as the coating buffer in subsequent experiments.

3.1.2 Preliminary optimization of antigen coating concentration

CF was diluted to 10 concentration gradients in the range of 5 μg/mL-200 μg/mL with the selected optimal coating buffer, and then coated on the ELISA plate for indirect ELISA detection. The A/B and A/N values were analyzed after the ELISA instrument read the values. The results showed (see Figure 6) that when the coating concentration was 30 μg/mL, 40 μg/mL and 50 μg/mL, the A/B and A/N values were high. Therefore, these three concentrations were initially selected for subsequent experiments.

3.1.3 Optimization of coating time

The CF coating time was set to 1 h at 37℃, 2 h at 37℃, 12 h at 4℃ overnight, 1 h at 37℃ and then 4℃ overnight, and 2 h at 37℃ and then 4℃ overnight. According to the indirect ELISA operation steps, the A/B and A/N values were analyzed after the microplate reader read the values. The results showed (see Table 9) that when the coating time was 4℃ overnight, the A/B and A/N values were higher. Therefore, the best coating time was to choose the coating antigen and incubate at 4℃ overnight.

3.1.4 Preliminary screening of blocking solution

Fifteen materials including 5% skim milk and 1% PEG-2000 were initially selected as blocking solutions. After blocking at 37°C for 2 h, the samples were tested according to the indirect ELISA procedure. The A/B and A/N values were analyzed after reading the values with an enzyme reader. The results showed (see Figure 7) that when the blocking solution was 1% v/v skim milk + 5% v/v porcine serum, both the A/B and A/N values were higher. Therefore, skim milk plus porcine serum was initially selected as the component of the blocking solution for subsequent optimization.

3.1.5 Adsorption of nonspecific factors in serum antibodies

CF of three bacteria, Msg, M. Phlei and BCG, were selected as antigens that can cross-react with other mycobacteria in serum antibodies and were mixed with equal volumes of diluted serum. The mixture was allowed to interact at 4°C overnight (12h), 37°C for 30 min, and 37°C for 2h, respectively. The test was then carried out according to the operating procedures of indirect ELISA. The A/B and A/N values were analyzed after the microplate reader read the values. The results showed (Table 10) that, considering the A/B and A/N values, the CF of M. Phlei had a better effect on absorbing and removing nonspecific factors in serum antibodies. However, the interaction time and temperature had little effect on the results. Considering that time should be saved as much as possible in the actual experimental operation, the CF of M. Phlei was selected to react with the diluted serum antibodies at 37°C for 30 min.

3.1.6 Optimization of coating concentration, blocking solution, blocking time and serum dilution multiple

According to the previous experimental results, four factors, namely, coating concentration, blocking solution, blocking time and serum dilution multiple, were selected with three levels for L ₉ (3 ⁴ ) orthogonal experimental design (as shown in Table 11). The antigen coating concentration was selected as 30 μg/mL, 40 μg/mL and 50 μg/mL; the blocking solution was selected as 1% v/v skim milk + 1% v/v pig serum (blocking solution 1), 1% skim milk + 3% pig serum (blocking solution 2) and 1% v/v skim milk + 5% v/v pig serum (blocking solution 3); the blocking time was selected as 37℃ 30 min, 37℃ 1h and 37℃ 2 h respectively; the serum dilution multiple was selected as 20 times, 50 times and 100 times, and two parallel controls were set for each well. Then, the detection was carried out according to the operating steps of indirect ELISA, and the OD 450nm value was read after the experiment was completed. The experimental results were statistically analyzed, as shown in Table 12. The results of variance analysis showed that the coating concentration and serum dilution multiple had extremely significant effects on the A/N value (α=0.01). The coating concentration, blocking solution and serum dilution multiple had significant effects on the A/N value (α=0.05). The blocking time had no significant effect on the results. As can be seen from Figure 8, the differences mainly came from 30 μg/mL and 40 μg/mL in the coating concentration, 1 and 3 in the blocking solution, and 20-fold and 100-fold dilutions in the serum dilution multiple. Therefore, it can be determined that the optimal coating concentration is 50 μg/mL, the blocking solution is 1% v/v skim milk + 3% v/v pig serum, the blocking time is 37℃ 1h, and the serum dilution multiple is 50-fold dilution.

3.1.7 Screening of different secondary antibodies and optimization of secondary antibody dilution multiples

The five antibodies were diluted 5,000, 10,000, 20,000, 50,000, and 100,000 times, respectively, and tested according to the indirect ELISA detection method. The A/B and A/N values were analyzed after the enzyme marker read the values. The results showed (see Figure 9 and Figure 10) that when the secondary antibody was secondary antibody 2, that is, HRP-labeled rabbit anti-bovine (Fc-specific) IgG antibody, the A/B and A/N values were significantly better than other secondary antibodies. At the same time, when the HRP-labeled rabbit anti-bovine (Fc-specific) IgG antibody was used as the secondary antibody, the A/B and A/N values were the best when the dilution factor was 10,000. Therefore, the HRP-labeled rabbit anti-bovine (Fc-specific) IgG antibody was selected as the secondary antibody, and the dilution factor was 10,000.

3.1.8 Optimization of serum samples and secondary antibody action time

The action time of serum samples and secondary antibodies was set to 37°C for 30 min, 45 min and 60 min, respectively, for a two-factor crossover test. After completing the test according to the indirect ELISA operation method, the OD450nm value of the microplate reader was read, and the A/B and A/N values were analyzed. As shown in Figure 11, when the serum sample was incubated for 45 min and the secondary antibody was incubated for 45 min, the A/N value was significantly better than the other time combinations. At the same time, the A/B value was also higher. Considering the A/B and A/N values comprehensively and taking into account the need to save as much time as possible during the experimental operation, the serum sample and the secondary antibody were incubated for 45 min as the optimal incubation time.

3.1.9 Screening of colorimetric and stop solutions

TMB produced by 5 different companies was selected as the colorimetric solution, 2 M sulfuric acid and 0.05% hydrofluoric acid were selected as the stop solution, two parallel controls were set up in each well, and the rest were operated according to the indirect ELISA method. The A/B and A/N values were analyzed after the microplate reader read the values. The results showed (see Figure 12) that when using amresco's TMB colorimetric solution and 0.05% hydrofluoric acid for termination, the OD630nm readings, A/B and A/N values were significantly better than other combinations.

3.1.10 Optimization of color development time and color development temperature

A two-factor crossover experiment was performed with color development times of 5 min, 10 min and 15 min and color development temperatures of 22°C (normal temperature) and 37°C. Two parallel controls were set up in each well, and the rest were operated according to the indirect ELISA method. The A/B and A/N values were analyzed after the microplate reader read the values. The results showed (see Table 13) that when the color development time was 10 min, the A/B and A/N values were significantly higher than those at other times, and the A/B and A/N values at 37°C were significantly better than those at 22°C. Although the A value was higher when the color development time was 15 minutes, the B value and N value were also relatively high at the same time. Therefore, considering all factors, 37°C for 10 min was selected as the optimal color development condition.

3.1.11 Optimization of washing steps

The washing steps in the ELISA operation process were set to the following three methods: continuous washing for 3 times, patting dry at the last time; washing 3 times (1 min/time), patting dry at the last time; washing 3 times (3 min/time), patting dry at the last time. The rest was operated according to the indirect ELISA method. After reading the A/B and A/N values, it was found that there was no significant difference between the groups. Considering the time cost and convenience of operation, continuous washing for 3 times and patting dry at the last time was selected as the washing method.

3.2 Determination of positive and negative critical values

）为0.286，标准差（SD）为0.0695。以

+3×SD的值为MAP阳性临界值，即0.5，以

+2×SD的值为MAP阴性临界值，即0.425。因此，当临床样本检测完成后，根据临床样本OD630nm读值（S或

）（有重复孔的样本要计算平均值），标准阳性血清OD630nm值（P）和标准阴性血清OD630nm值（N），按照以下公式进行计算：According to the optimized results in 2.2, 200 sera with MAP-negative bacterial cultures were tested. The OD630nm readings were used to calculate the OD mean and standard deviation of all sera. A graph was drawn based on the mean and standard deviation (see Figure 13). As shown in the figure, it can be analyzed that the distribution of the data tends to be normal distribution. The mean value of this group of data (

) is 0.286, and the standard deviation (SD) is 0.0695.

The value of +3×SD is the positive critical value of MAP, which is 0.5.

The value of +2×SD is the MAP negative critical value, which is 0.425. Therefore, after the clinical sample test is completed, according to the clinical sample OD630nm reading (S or

) (the average value should be calculated for samples with duplicate wells), the standard positive serum OD630nm value (P) and the standard negative serum OD630nm value (N) are calculated according to the following formula:

S/P (%) = 100 ×

When the S/P value is greater 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 greater than 42.5% and less than 50%, the clinical sample is judged to be suspicious and should be retested.

3.3 Evaluation of the effect of indirect ELISA method

3.3.1 Specificity experiment

(1) Cross-reaction detection

Positive sera for 10 common diseases in cattle were selected and tested using the optimized ELISA method. The S/P (%) values were analyzed after reading with an enzyme labeler. The results showed (see Figure 14) that the S/P (%) value of MAP-positive sera was greater than 50%, and the S/P (%) values of the positive sera for the other 10 diseases were less than 42.5%, demonstrating that the detection method has good specificity.

(2) 200 negative serum samples tested

200 sera with negative MAP in bacterial culture were selected and tested according to the optimized indirect ELISA operation procedure. Two replicates were performed for each serum and the S/P value of the test results was calculated. Among the 200 sera, 9 sera had S/P (%) values greater than 50%, which were positive sera, and the S/P (%) values of the remaining sera were all less than 42.5%. Therefore, the specificity of this method was 95.5% (9/200).

3.3.2 Sensitivity test

(1) Positive quality control serum test

The MAP-positive serum was diluted seven times with PBST, and the other steps were detected according to the optimized indirect ELISA method. After the microplate reader read the value, the S/P (%) value was calculated. The results showed (see Table 14) that when the positive serum was diluted to 1,000 times, the S/P (%) value was still greater than 50%, which was positive, proving that the detection method of this experiment has high sensitivity.

(2) 50 positive serum samples tested

50 sera with MAP positive bacterial culture were selected and tested according to the optimized indirect ELISA operation procedure. Each serum was repeated twice and the S/P value of the test results was calculated. Among the 100 sera, 46 sera had S/P (%) values greater than 50%, which were positive sera, and the S/P (%) values of the remaining sera were all less than 42.5%. Therefore, the sensitivity of this method was 92.0% (46/50).

3.3.3 Repeatability experiment

(1) Intra-batch reproducibility test

和SD，根据公式SD/

×100%计算出批内变异系数（C×V%）如表15所示，C×V%在1.04%~9.97%之间，均小于10%，说明本方法批内重复性良好。Calculate the total serum volume of each serum sample in the 4 plates

and SD, according to the formula SD/

The intra-batch coefficient of variation (C×V%) was calculated by 100% as shown in Table 15. The C×V% was between 1.04% and 9.97%, all less than 10%, indicating that this method had good intra-batch repeatability.

(2) Batch reproducibility experiment

和SD，根据公式SD/

×100%计算批间变异系数（C×V%），如表16，C×V%在3.16%~9.44%之间，均小于10%，说明本方法批间重复性良好。Calculate the total serum volume of each serum sample in the three plates.

and SD, according to the formula SD/

×100% was used to calculate the inter-batch coefficient of variation (C×V%). As shown in Table 16, C×V% was between 3.16% and 9.44%, all less than 10%, indicating that this method had good inter-batch repeatability.

3.3.4 Compliance experiment

The results of the three regions of Shanghai, Xinjiang and Hefei were consistent with the results of the experiment, with 13 MAP-positive sera and 19 MAP-negative sera, indicating that the experiment had good repeatability.

3.3.5 Comparative test

250 serum samples were tested with the kit assembled by this method and the Mycobacterium paratuberculosis antibody detection kit of IDEXX, and the compliance rate was calculated. The results are shown in Table 17. Therefore, the compliance rate (%) of the kit assembled by this method and the Mycobacterium paratuberculosis antibody detection kit of IDEXX = (47 + 189) / 250 × 100% = 94.4%.

3.3.6 Shelf life test

Six boxes of the assembled kit were extracted and stored at 2-8°C for 12 months. One box was extracted at 1, 2, 3, 6, 9, and 12 months for related experiments in each part, and compared with the newly assembled kit. First, the volume of each solution was tested. There was no obvious change in the dosage and appearance color of all reagents, no precipitation was produced, and the ELISA plate was well sealed. The ELISA plate was tested for the coincidence rate, and the positive and negative of the 20 sera were completely consistent, and there was no cross reaction with the positive sera of the 10 other diseases mentioned above. The specific sensitivity was completely consistent with the newly assembled kit, indicating that the kit has good stability and can be stably stored for 12 months.

3,4 Detection of clinical serum samples

1,900 clinical serum samples were collected from different provinces and cities. These serum samples were distributed throughout the Northeast, Northwest, East China and Central Plains, and can represent the prevalence of MAP in my country to a certain extent. The test results showed that among the 1,900 serum samples, 96 were MAP-positive, with a positive rate of 5.05%.

3.5 Initial Assembly of the Kit

Claims (6)

1. a bovine paratuberculosis (Paratuberculosis, PTB) indirect ELISA detection kit, is characterized in that, comprises the culture filtrate coating of Mycobacterium avium subsp.paratuberculosis (Mycobacterium avium subsp.paratuberculosis, MAP) in the kit It also includes the culture filtrate of Mycobacterium phlei used for the adsorption of non-specific factors in serum antibodies, coating buffer, sample diluent, blocking solution, concentrated washing solution, HRP-labeled rabbit Anti-bovine IgG antibody, chromogenic solution and stop solution; wherein the culture filtrate of Mycobacterium avium subsp. paratuberculosis is prepared by the following method: (1) Inoculate Mycobacterium avium subsp. paratuberculosis containing 10 ⁹ CFU/mL into a homemade culture medium containing 0.05% v/v Tween-80, 0.2% v/v glycerol, 5mM asparagine and 10% v/v In the 7H9 medium of ADC, culture at 37°C and 160rpm for 10 weeks, and collect the bacterial liquid; wherein, the self-made ADC is prepared by the following method: NaCl 8.5g, D-Dextrose 20.0g, catalase 0.03g , dilute to 1L, filter and sterilize with a 0.22μm filter; (2) The collected bacterial solution is centrifuged, and the supernatant is filtered through a filter membrane with a pore size of 0.22 μm to obtain a culture filtrate. 2. the bovine paratuberculosis indirect ELISA detection kit as claimed in claim 1, is characterized in that, the concentration of culture filtrate coating microplate plate is 50 μ g/mL. 3. The indirect ELISA detection kit for bovine paratuberculosis as claimed in claim 1, characterized in that, the centrifugation described in step (2) refers to centrifugation at 10000rpm at 4°C for 30min. 4. bovine paratuberculosis indirect ELISA detection kit as claimed in claim 1, is characterized in that, described coating buffer is 20mmol/L Tris-HCl damping fluid, and sample diluent is PBST, and described blocking solution It is 1% v/v skimmed milk + 5% v/v pig serum, the concentrated washing solution is 25×PBST, the color developing solution is TMB color developing solution, and the stop solution is 0.05% v/v v hydrofluoric acid. 5. The use of the bovine paratuberculosis indirect ELISA detection kit according to any one of claims 1-4 in the preparation of a reagent for detecting or diagnosing bovine paratuberculosis. 6. The use according to claim 5, wherein the reagent is an indirect ELISA detection reagent.

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