Kit for detecting antibody against foot-and-mouth disease virus nonstructural protein 3ABC by tube type chemiluminescence immunoassay technique and application thereof

Abstract

The present invention provides a kit for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody based on tube-type chemiluminescent immunoassay technology and its application, belonging to the field of in vitro detection technology. The kit provided by the present invention includes magnetic particle-coupled 3ABC fusion protein, enzyme-labeled 3A antibody, standard product, diluent, concentrated washing solution and luminescent substrate. The kit of the present invention can detect specific antibodies of 3ABC in porcine, bovine and ovine sera, and realizes quantitative, automated, highly sensitive and highly repeatable detection, and has obvious advantages compared with ELISA detection kits.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro detection, and particularly relates to a kit for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody by tube type chemiluminescent immunoassay technology. Background Art

[0002] Foot-and-mouth disease (FMD) is a highly contagious disease common to cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV). The affected groups include important economic livestock such as pigs, cattle, sheep, deer, camels and wild animals of more than 70 species in more than 20 families. FMDV can be transmitted through direct contact between animals or indirect contact with pollutants and air. After infection, the morbidity rate is 100%. It is a global infectious disease and is listed as one of the legally reportable diseases by the World Organization for Animal Health (OIE). Due to its infectious characteristics and prevention and control difficulties, since the first report of foot-and-mouth disease by humans in 1514, there are still many regions where it is prevalent worldwide. FMDV has 7 different serotypes, namely A, O, C, Asia1, SAT1, SAT2 and SAT3 (South Africa type, SAT). According to relevant reports in recent years, except for serotype C which has not been reported, the other 6 serotypes are prevalent globally, and the prevention and control situation remains severe.

[0003] At present, the technologies for detecting FMDV include virus isolation and identification, virus neutralization test, real-time fluorescence PCR, enzyme-linked immunosorbent assay (ELISA), etc. Virus isolation and identification and neutralization test both require live virus operations, with harsh experimental conditions and high safety protection level requirements, so they are less used in clinical applications; real-time fluorescence quantitative PCR has high requirements for samples and laboratories, and is prone to contamination and false positives.

[0004] Most countries take inactivated vaccines as the main prevention and control measures. Since the inactivated vaccines do not contain or contain a small amount of non-structural protein (NSP), the infection or immunization status of FMD can be identified by detecting NSP antibodies. Among them, 3ABC antibody is generally regarded as the gold standard for detecting FMD infection. The ELISA detection kits reported currently are relatively simple to operate and have low requirements for samples, so they are the most widely used. However, their disadvantages are that they take a long time, cannot accurately quantify the detection, and are difficult to automate.

[0005] Chemiluminescence immunoassay (CLIA) technology is a diagnostic technique that combines a chemiluminescence system with immunological methods. By amplifying and converting immune signals into chemiluminescence signals, the content of corresponding substances is detected by measuring the luminescence intensity. The first-generation CLIA technology conducts reactions on a luminescence plate. Except that the substrate is changed to a chemiluminescence system, the other steps are basically the same as those of ELISA. Therefore, there are also problems such as a large amount of laborious detection and high difficulty in automation. The second-generation CLIA technology uses nanomaterials as the solid-phase reaction carrier, enabling the reaction to proceed in a system closer to the liquid phase in reaction tubes, that is, the tube-type chemiluminescence method. It reduces the influence of steric hindrance, making the antigen-antibody reaction more sufficient, thus achieving automated, highly sensitive detection. Although this method has been used for the detection of hepatitis B three items, tumor markers, etc. in human medicine, and veterinarians have also started to use this method to detect antibodies against foot-and-mouth disease serotypes O, A, and Asia I, due to the differences in the detection substances, its detection system cannot be universal, and there is no report on the detection of foot-and-mouth disease non-structural protein 3ABC antibody by this technology. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a kit for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody based on tube-type chemiluminescence immunoassay technology and its application, which can accurately quantify the specific antibody of 3ABC in animal serum and achieve the purposes of automated, highly sensitive, and highly repeatable detection.

[0007] The present invention provides a kit for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody based on tube-type chemiluminescence immunoassay technology, comprising a magnetic particle-coupled 3ABC fusion protein, an enzyme-labeled antibody, a diluent, a standard, a standard diluent, a concentrated washing solution, and a luminescence substrate;

[0008] The antibody in the enzyme-labeled antibody is a 3A monoclonal antibody.

[0009] Preferably, the 3A monoclonal antibody is secreted by the hybridoma cell line 3A24.

[0010] Preferably, the standard is a standard antiserum.

[0011] Preferably, the diluent is a 0.1M MOPS buffer solution with a pH of 7;

[0012] The MOPS buffer solution contains BSA with a mass concentration of 0.1-2%, Tween-20 with a volume concentration of 0.01-0.5%, ProClin300 with a volume concentration of 0.01-0.5%, MgCl2 with a concentration of 0.01-0.5M, NaCl with a concentration of 0.01-0.5M, hydrolyzed milk protein with a mass concentration of 0.05-3%, and PEG 6000 with a mass concentration of 0.1-1%.

[0013] Preferably, the standard diluent is a PBS buffer solution with a pH value of 7 and a concentration of 0.1M;

[0014] The PBS buffer solution contains BSA with a mass concentration of 0.1% - 2%, PEG with a mass concentration of 0.1 - 1%, trehalose with a mass concentration of 0.5 - 5%, gelatin with a mass concentration of 0.1 - 0.5%, and ProClin300 with a volume concentration of 0.01 - 0.5%.

[0015] Preferably, the concentrated washing solution is a PBS buffer solution with a pH value of 7 containing Tween - 20 with a volume concentration of 0.1% - 0.5% or a Tris buffer solution with a pH value of 8 and a concentration of 0.05M containing Tween - 20 with a volume concentration of 0.1% - 0.5%.

[0016] Preferably, the enzyme of the enzyme - labeled antibody is horseradish peroxidase or alkaline phosphatase;

[0017] The chemiluminescent substrate corresponds to the enzyme in the enzyme - labeled antibody.

[0018] The present invention provides the application of the kit in differentiating animals naturally infected with foot - and - mouth disease virus from vaccinated animals.

[0019] Preferably, the method for differentiating inactivated vaccine - vaccinated animals from naturally infected animals includes the following steps:

[0020] Mix the serum of the sample to be tested with the working solution of magnetic particle - conjugated 3ABC fusion protein and the working solution of enzyme - labeled antibody, incubate, perform magnetic separation, wash the solid phase, then incubate with the chemiluminescent substrate, read the chemiluminescent value, substitute the chemiluminescent value into the standard curve to calculate the content of 3ABC antibody, and determine whether the sample to be tested is an inactivated vaccine - vaccinated animal or a naturally infected animal based on the content of 3ABC antibody: when the antibody content ≥ 200U, it is positive for non - structural protein antibody and judged as a foot - and - mouth disease virus - infected sample; when the antibody content < 200U, it is negative for non - structural protein antibody and judged as an inactivated vaccine - vaccinated sample or an unvaccinated sample.

[0021] The present invention provides a kit for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody based on tube-type chemiluminescent immunoassay technology, which includes magnetic particle-coupled 3ABC fusion protein, enzyme-labeled antibody, diluent, standard, standard diluent, concentrated washing solution and luminescent substrate; the antibody in the enzyme-labeled antibody is 3A monoclonal antibody. In the present invention, the magnetic particle-coupled 3ABC fusion protein is used to make the reaction proceed in a more liquid-phase environment, reducing the influence of steric hindrance, making the antigen-antibody reaction more sufficient, and achieving higher sensitivity than plate-type chemiluminescence; at the same time, the immune reaction is carried out in a test tube, and with the supporting automated chemiluminescent immunoassay analyzer and standard curve, accurate quantification and fully automated detection can be realized; the enzyme-labeled 3A monoclonal antibody can competitively bind to the 3ABC antibody in the serum to be detected and the 3ABC fusion protein coupled to the magnetic particles, with a fast detection speed, and the detection can be completed in 23 minutes; at the same time, through the prepared standard, standard diluent and diluent, each component of the kit remains stable during the trial period, ensuring good detection repeatability.

[0022] At the same time, the kit provided by the present invention can quantitatively detect foot-and-mouth disease virus non-structural protein 3ABC antibody through an automated chemiluminescent immunoassay analyzer. Compared with the currently used detection methods and kits, it can achieve automated, highly sensitive and highly repeatable detection. It has fewer detection steps, is easy to use and has simple operation, and is suitable for the differential diagnosis of foot-and-mouth disease virus-infected animals and immunized animals.

[0023] The present invention provides the application of the kit in differentiating inactivated vaccine-immunized animals and naturally infected animals. Based on the detection of 3ABC antibody by the kit of the present invention, it has the characteristics of high sensitivity and high repeatability detection. At the same time, based on the fact that the non-structural protein 3ABC in the inactivated vaccine contains little or extremely low content, a negative sample detected can be accurately judged as an inactivated vaccine-immunized sample, while naturally infected FMDV contains a large amount of non-structural protein 3ABC and a large amount of corresponding antibodies in the serum. Therefore, when the test sample is positive, it is judged as a naturally infected sample. It can be seen that the application provided by the present invention has the characteristics of convenient detection, simple operation and fast detection, and can be used for clinical differential diagnosis of naturally infected animals and vaccine-immunized animals of foot-and-mouth disease virus. Description of the Drawings

[0024] Figure 1 It is the standard curve of the tube-type chemiluminescent antibody detection kit for foot-and-mouth disease virus non-structural protein 3ABC;

[0025] Figure 2 It is the detection results of positive sera of different pathogen antibodies using this kit. Detailed Embodiments

[0026] The present invention provides a kit for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody based on tube-type chemiluminescent immunoassay technology, comprising magnetic particle-coupled 3ABC fusion protein, enzyme-labeled antibody, diluent, standard, standard diluent, concentrated washing solution and luminescent substrate; the antibody in the enzyme-labeled antibody is 3A monoclonal antibody.

[0027] In the present invention, the kit comprises magnetic particle-coupled 3ABC fusion protein. The particle size of the magnetic particles in the magnetic particle-coupled 3ABC fusion protein is preferably 0.1 - 5 μm, more preferably 0.5 - 4 μm, and even more preferably 3 μm. The magnetic particles have Fe3O4 as the core, and the surface functional groups are hydroxyl, carboxyl, amino, epoxy or sulfonyl groups. In the present invention, the magnetic particles are obtained by purchasing from JSR Corporation. The recombinant expression of the 3ABC fusion protein is preferably prepared with reference to the prior art (Cao Yimei, Liu Zaixin, Lu Zengjun, et al. High expression of foot-and-mouth disease virus non-structural protein gene 3ABC in Escherichia coli [J]. Acta Veterinaria et Zootechnica Sinica, 2004, 35(1): 115 - 118; Cao Yimei, Lu Zengjun, Liu Zaixin, et al. Purification, renaturation and activity detection of FMDV 3ABC non-structural protein expressed by Escherichia coli [J]. Chinese Journal of Veterinary Science and Technology, 2003, 33(8): 22 - 25.). The amino acid sequence of the 3ABC fusion protein is as shown in SEQ ID NO: 1. The preparation method of the magnetic particle-coupled 3ABC fusion protein preferably comprises the following steps (a multiplex tube-type chemiluminescent detection kit for foot-and-mouth disease O, A, AsiaI type antibodies, patent number: ZL201910646270.2). The results of the examples of the present invention show that when detecting 3ABC antibody by coupling 3ABC fusion protein and 3AB with magnetic particles respectively, the results show that 3ABC fusion protein as an antigen has higher detection sensitivity.

[0028] In the present invention, the kit includes an enzyme-labeled antibody. The antibody in the enzyme-labeled antibody is a 3A monoclonal antibody. The 3A monoclonal antibody is preferably secreted by the hybridoma cell line 3A24. The hybridoma cell line 3A24 is a known hybridoma cell line previously reported by the applicant. For details, see the prior art (Fu Y, Li P, Cao Y, Wang N, Sun P, Shi Q, et al. Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine. PLoS ONE. 2017:12(1):0170560.). The enzyme of the enzyme-labeled antibody can be any enzyme well-known in the art, such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP). The present invention has no special limitation on the preparation method of the enzyme-labeled antibody, and any enzyme-labeling method well-known in the art can be used. It has been verified in the examples of the present invention that using the 3A monoclonal antibody as the detection antibody has higher detection sensitivity than the 3B monoclonal antibody.

[0029] In the present invention, the kit includes a diluent. The diluent is used to dilute the magnetic particle-conjugated 3ABC fusion protein and the enzyme-labeled antibody to their respective working concentrations to form a working solution. The diluent is preferably a MOPS buffer solution with a pH value of 7 and a concentration of 0.05 - 0.2 M; the MOPS buffer solution preferably contains BSA with a mass concentration of 0.1% - 2%, Tween-20 with a volume concentration of 0.01% - 0.5%, ProClin300 with a volume concentration of 0.01% - 0.5%, MgCl2 with a concentration of 0.01 - 0.5 M, NaCl with a concentration of 0.01 - 0.5 M, hydrolyzed milk protein with a mass concentration of 0.05% - 3%, and PEG 6000 with a mass concentration of 0.1 - 1%. More preferably, it contains BSA with a mass concentration of 1%, Tween-20 with a volume concentration of 0.1%, ProClin300 with a volume concentration of 0.1%, MgCl2 with a concentration of 0.2 M, NaCl with a concentration of 0.2 M, hydrolyzed milk protein with a mass concentration of 0.1%, and PEG with a mass concentration of 0.5%.

[0030] In the present invention, the standard product is preferably the serum of animals infected with foot-and-mouth disease virus. The serum of animals infected with foot-and-mouth disease virus is collected from cattle 1 to 3 months after being infected with foot-and-mouth disease virus. After detection, both the non-structural protein antibody and the structural protein antibody are positive. After separation, purification, and aseptic treatment, a preservative is added for preparation. The method of separation and purification is preferably centrifuging the serum at 8000 r / min for 30 min and purifying it with an affinity chromatography column. The preparation method of the standard product is preferably calibrating the standard positive serum with a foot-and-mouth disease virus non-structural protein 3ABC blocking ELISA kit to obtain standard products with 10 gradient antibody contents, and defining their antibody contents as 0U, 2.5U, 5U, 10U, 20U, 40U, 80U, 160U, 320U, and 640U according to the size of their inhibition rates. The unit of the antibody content is U, and 1U is defined by the method of detecting the critical point serum with a foot-and-mouth disease virus non-structural protein 3ABC blocking ELISA antibody detection kit (purchased from Lanzhou Veterinary Research Institute Bio-Tech Co., Ltd.).

[0031] In the present invention, the kit includes a standard product diluent. The standard product diluent is a PBS buffer solution with a pH value of 7 and a concentration of 0.05 - 0.2M; the PBS buffer solution contains BSA with a mass concentration of 0.1% - 2%, PEG with a mass concentration of 0.1 - 1%, trehalose with a mass concentration of 0.5 - 5%, gelatin with a mass concentration of 0.1 - 0.5%, and ProClin300 with a volume concentration of 0.01 - 0.5%. More preferably, it contains BSA with a mass concentration of 0.1%, PEG with a mass concentration of 0.5%, trehalose with a mass concentration of 2%, gelatin with a mass concentration of 0.2%, and ProClin300 with a volume concentration of 0.1%.

[0032] In the present invention, the kit includes a concentrated washing solution. The concentrated washing solution is a PBS buffer solution with a pH value of 7 containing Tween-20 with a volume concentration of 0.1% - 0.5% or a Tris buffer solution with a pH value of 8 and a concentration of 0.05M containing Tween-20 with a volume concentration of 0.1% - 0.5%. More preferably, it is a PBS buffer solution with a pH value of 7 containing Tween-20 with a volume concentration of 0.2% - 0.4% or a Tris buffer solution with a pH value of 8 and a concentration of 0.05M containing Tween-20 with a volume concentration of 0.2% - 0.4%. More preferably, it is a PBS buffer solution with a pH value of 7 containing Tween-20 with a volume concentration of 0.3% or a Tris buffer solution with a pH value of 8 and a concentration of 0.05M containing Tween-20 with a volume concentration of 0.3%. When the concentrated washing solution is used, it is preferably diluted 10 - 30 times, more preferably 15 - 25 times, and most preferably 25 times.

[0033] In the present invention, the kit includes a luminescent substrate. The luminescent substrate corresponds to horseradish peroxidase, phosphatase, or alkaline phosphatase in the enzyme-labeled antibody.

[0034] In the present invention, the concentration of the working solution of the magnetic particle-coupled 3ABC fusion protein in the kit is preferably 0.2 - 0.5 mg / ml, more preferably 0.3 - 0.4 mg / ml, and most preferably 0.35 mg / ml. The concentration of the enzyme-labeled antibody working solution is preferably 1:500 - 1:5000, more preferably 1:2000 - 1:3000, and most preferably 1:2500. The working concentration of the washing solution is diluted 25 times.

[0035] In the present invention, the kit replaces the 96-well plate with magnetic particles coupled with foot-and-mouth disease 3ABC fusion protein, enabling the reaction to be carried out in a moving reaction tube, achieving full automation of detection, and simultaneously realizing accurate quantification, high sensitivity, and high repeatability of detection. The detection specificity of the kit is relatively high.

[0036] The present invention provides the application of the kit in differentiating animals naturally infected with foot-and-mouth disease virus from animals immunized with inactivated vaccines.

[0037] In the present invention, the method for differentiating animals immunized with inactivated vaccines and naturally infected animals preferably includes the following steps:

[0038] Mix and incubate the serum of the sample to be tested with the working solution of magnetic particle-coupled 3ABC fusion protein and the working solution of enzyme-labeled antibody, perform magnetic separation, wash the solid phase and then incubate with the luminescent substrate, read the luminescence value, substitute the luminescence value into the standard curve, calculate the content of 3ABC antibody, and determine whether the sample to be tested is immunized with inactivated vaccine or naturally infected according to the content of 3ABC antibody: when the antibody content ≥ 200 U, it is positive for non-structural protein antibody and judged as a foot-and-mouth disease virus-infected sample; when the antibody content < 200 U, it is negative for non-structural protein antibody and judged as an inactivated vaccine-immunized sample or a non-immunized sample.

[0039] In the present invention, the standard curve is preferably shown as Formula I:

[0040] y = 14102.97 + (1360648.205 - 14102.97) / (1 + (x / 0.9373) 1.0821 ) Formula I

[0041] where x is the antibody content and y is the relative luminescence intensity. The antibody content of the sample to be tested can be calculated by substituting the luminescence intensity of the test sample.

[0042] The following is a detailed description of a kit for detecting foot-and-mouth disease non-structural protein 3ABC antibody based on tube-type chemiluminescence immunoassay technology and its application provided by the present invention in combination with examples, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] Preparation method of a kit for detecting foot-and-mouth disease non-structural protein 3ABC antibody based on tube type chemiluminescence immunoassay technology

[0045] 1. Refer to the following literature for the expression, purification and renaturation of non-structural protein 3ABC fusion protein.

[0046] Cao Yimei, Liu Zaixin, Lu Zengjun, et al. Expression of foot-and-mouth disease virus non-structural protein gene 3ABC in Escherichia coli [J]. Acta Veterinaria et Zootechnica Sinica, 2004, 35(1): 115-118.

[0047] Cao Yimei, Lu Zengjun, Liu Zaixin, et al. Purification, renaturation and activity detection of FMDV 3ABC non-structural protein expressed by Escherichia coli [J]. Chinese Journal of Veterinary Science and Technology, 2003, 33(8): 22-25.

[0048] 1.1 Identification results of non-structural protein 3ABC protein

[0049] After the 3ABC fusion protein undergoes purification processes such as inclusion body purification and affinity chromatography, and is analyzed by SDS-PAGE electrophoresis, the purity of the target protein reaches over 90%. The electrophoresis shows that the molecular weight of the 3ABC fusion protein is 49 kDa, which is consistent with the expected size.

[0050] 2. Preparation and identification of 3A monoclonal antibody, refer to the following literature:

[0051] Fu Y, Li P, Cao Y, Wang N, Sun P, Shi Q, et al. Development of a Blocking ELISA Using a Monoclonal Antibody to a Dominant Epitope in Non-Structural Protein 3A of Foot-and-Mouth Disease Virus, as a Matching Test for a Negative-Marker Vaccine. PLoS ONE. 2017:12(1):0170560.

[0052] Identification results of purity and content of 3A monoclonal antibody

[0053] The purity of the purified monoclonal antibody can reach over 95% detected by SDS-PAGE electrophoresis. The content of the 3A monoclonal antibody is 1.951 mg / ml. A total of 50 mg is prepared, dispensed into 1 ml / tube, and stored in a -70°C ultra-low temperature refrigerator for standby.

[0054] 3. Preparation method of magnetic particle-coupled 3ABC fusion protein working solution

[0055] Take 1 mg of magnetic beads, dilute them to 10 mg / ml with 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.0) respectively, adsorb the magnetic beads fully with a magnet, discard the supernatant, then add 100 μL of 0.1 M MES (pH 5.0), mix the magnetic beads evenly, and repeat the washing operation once.

[0056] Take 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) out of -20 °C and restore them to room temperature. Weigh EDC and NHS. Dissolve them with 0.1 M MES (pH 5.0) respectively to prepare a solution of 10 mg / ml.

[0057] Add 10 μl of NHS solution to the magnetic beads respectively, then add 10 μl of EDC solution, mix evenly at room temperature and react for 30 min. Wash the magnetic beads: adsorb the magnetic beads fully with a magnet, discard the supernatant, then add 100 μL of 0.1 M MES (pH 5.0), and mix the magnetic beads evenly.

[0058] Add 30 μg of foot-and-mouth disease 3ABC protein to the above-activated magnetic beads respectively, mix the magnetic beads evenly. Incubate at room temperature with shaking for 3 h. Block by adding 10 μl of 10% BSA solution, and incubate at room temperature with shaking for 3 h. Wash 3 times with the washing solution (1× washing solution), disperse with 4 ml of preservation solution to form a magnetic particle working solution, and store at 4 °C.

[0059] 4. Preparation method of enzyme-labeled antibody working solution

[0060] 4.1 AP enzyme activation: Prepare sodium periodate (NaIO4) at 15 mg / ml and AP at 10 mg / ml, react at a ratio of m:m of 1.5:1, shake and mix evenly, react at 37 °C for 30 min; add ethylene glycol (1%) with the same volume as the mixture, react at 4 °C for 45 min, and the AP activation is completed and ready for use.

[0061] 4.2 AP-labeled monoclonal antibody

[0062] Dialyze the 3ABC protein overnight with 0.5 M carbonate buffer at pH 9.6, add it to the above-activated AP mixture, with a ratio of AP:Ab of 2:1 (m:m), and dialyze the mixture in 0.5 M carbonate buffer at pH 9.6 overnight at 4 °C.

[0063] Add 80 μl / mg AP (2 mg / ml) of NaBH4 to the above mixture respectively, react at 4 °C for 2 h, use an ultrafiltration tube to centrifuge at 5000 r / min for 10 min, change the solution to concentrate the labeled product, dissolve it with PBS containing 50% glycerol, store at -20 °C, and dilute it 1:50000 with the enzyme-labeled antibody diluent to form the enzyme-labeled antibody working solution when in use, and store at 4 °C for standby.

[0064] 5. Preparation methods of reference standards and quality control products

[0065] 5.1 Preparation method of high-concentration serum of 3ABC antibody

[0066] Cows were collected 1 month after foot-and-mouth disease virus infection. After detection, both non-structural protein antibodies and structural protein antibodies were positive. After aseptic treatment, preservatives were added for preparation and stored at -20°C or below for later use.

[0067] 5.2 Purification method of high-concentration standard serum of 3ABC antibody

[0068] The pipeline of the AKTA protein purifier was rinsed with pure water 3 times, and a Protein A column for protein purification was connected. After equilibration with pure water until all instrument parameters were stable, 0.02M phosphate buffer (PB) was used for equilibration until all instrument parameters were stable. The sample was injected at 1 ml / 5 ml. Impurities were eluted with 0.02M phosphate buffer (PB), and then the sample was eluted with 0.1M citrate buffer (SSC). The sample was collected when the UV was above 200, the pH value was adjusted to 7.0, and the absorbance at 260 nm and 280 nm was detected with a UV spectrophotometer to calculate the antibody concentration for later use.

[0069] 5.3 Preparation and calibration of reference standards

[0070] The antibody prepared in 5.2 was serially diluted with reference standard diluent. The foot-and-mouth disease virus non-structural protein 3ABC blocking ELISA antibody detection kit (purchased from Lanzhou Veterinary Research Institute Biotechnology Co., Ltd.) was used for detection. The detection results are shown in Table 1. When the dilution ratio was 1:64, the blocking ELISA detection result became negative. Therefore, the reference standards at 1:32 and 1:64 were used as the critical sera for ELISA detection. The detection results of tube chemiluminescence are shown in Table 2. When detecting sera at 1:64, 1:128, and 1:256, the ratios of the detection results to the sera of the previous dilution factor were 11.27, 7.85, and 3.66 respectively, with good sensitivity. The judgment criterion for blocking ELISA was that "if the PI value of the serum sample was not less than 50%, it was judged as positive for non-structural protein antibodies; if it was less than 50%, it was judged as negative for non-structural protein antibodies". Since the sensitivity of the CLIA method was relatively high, we defined the antibody content of the serum diluted at 1:64 (i.e., the serum with a PI value of 44% in ELISA detection) as 10 active units, i.e., 10 U. Then, the antibody contents of reference standards 1 - 10 were as shown in Table 2, which were 0 U, 2.5 U, 5 U, 10 U, 20 U, 40 U, 80 U, 160 U, 320 U, and 640 U respectively. Among them, 0 U, 2.5 U, 10 U, 40 U, 160 U, and 640 U were used as calibration standards 1 - 6.

[0071] Table 1 Results of reference standard 3ABC antibody 1 (blocking ELISA)

[0072]

[0073]

[0074] Table 2 Results of Standard Product 3ABC Antibody 1 (CLIA)

[0075]

[0076] 6. Preparation of Standard Curve

[0077] The antibody contents of the foot-and-mouth disease non-structural protein 3ABC antibody standard products are 0U, 2.5U, 5U, 10U, 20U, 40U, 80U, 160U, 320U, and 640U respectively. Take no less than 300 μl of the above standard products with different concentrations and place them in 1.5 ml conical bottom centrifuge tubes. Place them on a special sample rack and put them into the sample chamber of the automatic chemiluminescence analyzer. Set the parameters of the automatic chemiluminescence analyzer as required and start the detection. The detection reaction steps are as follows (fully automatic for the instrument):

[0078] (1) Take 50 μl of the sample to be tested, standard product, and negative / positive serum and add them into the reaction cup;

[0079] (2) Add 15 μl of magnetic particle working solution and 50 μl of enzyme-labeled antibody working solution, and incubate at 37 °C for 20 minutes;

[0080] (3) Wash 4 times and add 100 μl of chemiluminescence substrate, and incubate for 3 minutes;

[0081] (4) Read the luminescence value.

[0082] (5) Standard curve making: Use the chemiluminescence analyzer to detect the chemiluminescence value. Take the antibody concentration as the abscissa and the luminescence value (RLU) as the ordinate to draw a four-parameter standard curve y = a + (b - a) / (1 + (x / c) d ) and the standard curve is as Figure 1 shown.

[0083] 7. Composition and Assembly of a Tube-Type Chemiluminescence Antibody Detection Kit for Foot-and-Mouth Disease Non-Structural Protein 3ABC

[0084] Take the magnetic particle working solution prepared above, with a concentration of 0.35 mg / ml.

[0085] Enzyme-labeled antibody solution, with a concentration of 1:2500.

[0086] Dispense them into the reagent boat magnetic bead bottle and enzyme-labeled antibody bottle according to different specifications, and place them on the reagent boat rack to form a "chemiluminescence reaction system for foot-and-mouth disease non-structural protein 3ABC antibody detection";

[0087] Prepare the concentrated solution as 25×PBST;

[0088] The luminescent substrate (Wuhan Hanhai New Enzyme Biotechnology Co., Ltd., HH3605) is aliquoted into 100 ml per bottle;

[0089] The negative serum and the standard product 5 with an antibody content of 20 U are aliquoted as quality control product 1 and quality control product 2.

[0090] Assemble the tube-type chemiluminescent antibody detection kit for foot-and-mouth disease non-structural protein 3ABC according to the corresponding specification requirements

[0091] Example 2

[0092] Detection steps of a kit for detecting foot-and-mouth disease non-structural protein 3ABC antibody based on tube-type chemiluminescent immunoassay technology

[0093] 1. Preparation before detection

[0094] 1.1 Sample requirements

[0095] After collecting the serum, prepare the serum for detection. Before centrifugation, it is necessary to ensure that the serum sample is completely coagulated. After separating the serum, place it in a sterile serum tube. If it is to be detected within one week, it can be stored at 2°C - 8°C. If it is to be detected more than one week later, it should be stored frozen at -15°C or below.

[0096] 1.2 Resuspension of magnetic particle reagent

[0097] Before the first installation of the instrument, it is necessary to mix the magnetic particle reagent to resuspend the magnetic particles that have precipitated during transportation. After the first loading of the magnetic particle reagent, no further mixing is required.

[0098] 1.3 Solution preparation

[0099] Washing solution (1× washing solution): Dilute the 25× concentrated washing solution provided in this kit with deionized water or distilled water at a ratio of 1:25;

[0100] 1.4 Instrument preparation

[0101] Read the instrument instruction manual, set the instrument parameters according to the determined conditions, place the relevant reagents, samples, and required consumables, and start the detection. (For the specific setting method, refer to the instrument instruction manual).

[0102] 2. Standard curve calibration

[0103] Take calibration standards 1 - 6 for foot-and-mouth disease, with antibody contents of 0 U, 2.5 U, 10 U, 40 U, 160 U, and 640 U respectively, and calibrate the standard curve according to the instrument instruction manual. When the R2 of the calibrated standard curve is ≥ 0.99, the quality control product can be detected.

[0104] 1.7 Quality control

[0105] When necessary, the quality control product shall be detected. The detected value must be within the reference range before the sample detection can continue. The quality control product needs to be detected under the following circumstances:

[0106] a. After each change in the kit batch number or calibration;

[0107] b. It needs to be detected at least once within 24 hours.

[0108] 2 Sample Detection

[0109] a. Take 50 μl of the sample to be detected, standard product, and positive and negative sera, and add them to the reaction cup;

[0110] b. Add 15 μl of the magnetic particle working solution and 50 μl of the enzyme-labeled antibody working solution, and incubate at 37 °C for 20 minutes;

[0111] c. Wash 4 times, add 100 μl of the chemiluminescent substrate, and incubate for 3 minutes;

[0112] d. Read the luminescence value and calculate the concentration.

[0113] 3 Results

[0114] After the detection is completed, check the content of the foot-and-mouth disease virus non-structural protein 3ABC antibody in the sample according to the instrument instructions, and judge the positive and negative of the foot-and-mouth disease virus non-structural protein 3ABC antibody in the sample.

[0115] 4 Basis for Result Judgment

[0116] If the antibody activity content ≥ 200, it is judged as positive for the foot-and-mouth disease virus non-structural protein 3ABC antibody; if the antibody activity content < 200, it is judged as negative for the foot-and-mouth disease virus non-structural protein 3ABC antibody.

[0117] Example 3

[0118] Detection Characteristics of a Kit for Detecting Foot-and-Mouth Disease Non-Structural Protein 3ABC Antibody Based on Tube-Type Chemiluminescent Immunoassay Technology

[0119] Sensitivity and Sensitivity of the Kit

[0120] 1.1 Gradient dilute the positive serum and detect it by tube-type CLIA and ELISA respectively. The detection results are shown in Table 3. ELISA detection shows that OD (negative sample) / OD (original P++) is 10.85 times, and OD (negative sample) / OD (1:256) is 1.18 times; while the CLIA detection results are 210.61 times and 3.66 times respectively. Compared with ELISA, the difference in positive and negative detection results of CLIA detection is larger and the sensitivity is better.

[0121] Table 3 Sensitivity Detection Results

[0122]

[0123] 1.2 The 34 sera with positive background were tested using this kit and blocking ELISA respectively. The test results are shown in Table 4. The results of the 35 sera tested by this kit were all positive, with a 100% consistency with the background; 3 of the 35 sera were negative by ELISA, with a consistency with the background of 91.4%; but the PI values of the three sera were 42.29%, 43.30%, and 49.67%, respectively, which are close to the critical value of "PI ≥ 50% is judged as positive for foot-and-mouth disease non-structural protein 3ABC, and less than 50% is judged as negative for foot-and-mouth disease non-structural protein 3ABC". It can be seen that this kit has a high sensitivity.

[0124] Table 4 Test results of 35 positive serum samples

[0125]

[0126]

[0127]

[0128] 2. Specificity of the kit

[0129] 2.1 Test results before and after foot-and-mouth disease vaccine immunization

[0130] The purpose of this test kit is to detect antibodies to the non-structural protein 3ABC of foot-and-mouth disease virus, so as to distinguish animals infected with foot-and-mouth disease virus from animals immunized with the vaccine. Therefore, we immunized 50 cattle with negative foot-and-mouth disease antibodies. The vaccine was "foot-and-mouth disease type O and type A bivalent inactivated vaccine (O / MYA98 / BY / 2010 strain + Re-A / WH / 09 strain)", purchased from "China Agricultural Weite Technology Co., Ltd.". The 50 pigs were immunized three times, and the sera before immunization and after 3 immunizations were collected and tested using this test kit. The test results are shown in Table 5. It can be seen that the serum antibody content after 3 immunizations detected by this test kit is slightly higher than that before immunization. However, compared with the positive 3ABC antibody in Table 4, the antibody content is very low. According to the judgment criteria of this test kit, the serum test results before immunization and after 3 immunizations were all judged to be negative. This test kit has high specificity.

[0131] Table 5 Test results before and after immunization of 50 pigs with foot-and-mouth disease type O and type A bivalent inactivated vaccines

[0132]

[0133]

[0134]

[0135] 2.2 Other pathogen detection results

[0136] Nine related pathogen antibody positive samples were separately selected, including foot-and-mouth disease infection samples, lumpy skin disease virus of cattle, classical swine fever virus, sheep pox virus, Seneca virus, porcine reproductive and respiratory syndrome virus, foot-and-mouth disease type O immunity, foot-and-mouth disease type A immunity, and African swine fever virus. The kit was used for detection, and the results are as Figure 1 shown. When detecting the control serum sample, the content of 3ABC antibody was the highest, and the content of 3ABC antibody in the other 8 samples was very low and all were negative. Therefore, the specificity of this kit is very good.

[0137] 3. Detection repeatability

[0138] Three samples (1 strongly positive, 1 suspicious, and 1 negative) were selected and detected with two batches of the kit, and each batch was detected 5 times respectively. The detection results are shown in Table 6. The CV(%) values of Sample 1, Sample 2, and Sample 3 are 1.35%, 3.42%, and 8.57% respectively. When detecting the negative sample, due to the overall low value, the detection CV is on the high side. However, overall, the repeatability of this kit is good.

[0139] Table 6 Detection results of repeatability test

[0140]

[0141]

[0142] In the kit described in the present invention, the 96-well plate is replaced by magnetic particles conjugated with foot-and-mouth disease 3ABC fusion protein, so that the reaction is carried out in a moving reaction tube, and the full automation of detection can be realized. At the same time, accurate quantification, high sensitivity, and high repeatability detection can be achieved, and the results are expressed in activity units (U). The calculation results and determination methods are applicable to actual use, meet the actual requirements, and are beneficial to the unification and comparison of the detection results of various diagnostic methods.

[0143] Example 4

[0144] Comparison of experimental results of 3A and 3B monoclonal antibodies as detection antibodies

[0145] In the initial stage of establishing the method, 3A and 3B monoclonal antibodies (Li Yongliang, Tian Meina, Lu Zengjun, etc. Preparation and identification of monoclonal antibodies against non-structural protein 3B of foot-and-mouth disease virus [J]. Jiangsu Journal of Agricultural Sciences, 2009, 25(2): 296-300.) were labeled with alkaline phosphatase and paired with different antigens respectively to screen the optimal antigen and antibody. When using 3ABC protein as the antigen to conjugate magnetic particles, 3A and 3B monoclonal antibodies were respectively labeled with alkaline phosphatase as detection antibodies to establish a competitive method for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody. The experiment and results are as follows.

[0146] 1. Experimental method:

[0147] The competitive method was established. The 3ABC protein-coupled magnetic beads and the alkaline phosphatase-labeled 3A and 3B monoclonal antibodies were the same as those described in Patent Example 1.

[0148] Test procedure: ① 25 μl of the sample, 25 μl of the magnetic particles, and 150 μl of the enzyme-labeled antibody were incubated at 37 °C for 20 min.

[0149] ② Washed 4 times with the washing solution, 100 μl of the chemiluminescent substrate was added, and incubated at 37 °C for 3 min, and the luminescence intensity was detected.

[0150] Test results

[0151] 2.1 Detection of negative and positive sera

[0152] The standard negative and positive sera were detected. The results are shown in Table 7. Because of the established competitive method, the test results were negatively correlated with the antibody content in the sample. Therefore, the larger the N / P, the better the test effect. When the 3B monoclonal antibody was used as the detection antibody, the non-specific reaction was strong, and the N / P value was lower than that when the 3A monoclonal antibody was used as the detection antibody.

[0153] Table 7 Results of detecting negative and positive controls with 3A and 3B monoclonal antibodies as detection antibodies

[0154]

[0155] 2.2 Results of detecting clinical samples

[0156] Seventeen field samples were detected, including 6 negative samples and 1 positive sample. The test results are shown in Table 2. Among the negative samples, the N5 sample had the lowest test results by both methods. Therefore, the N5 / P value was calculated to compare the test effects of the two methods for detecting field samples. The test results are shown in Table 8. When the 3A-AP was used as the enzyme-labeled antibody, the N / P of the detected field samples was higher than that when the 3A-AP was used as the enzyme-labeled antibody.

[0157] Table 8 Results of detecting clinical samples with 3A and 3B monoclonal antibodies as detection antibodies

[0158]

[0159]

[0160] In summary, in the preliminary screening test, the 3A-AP as the enzyme-labeled antibody had better results in detecting negative and positive controls and clinical samples. Therefore, the 3A monoclonal antibody was selected as the detection antibody for subsequent method establishment and optimization.

[0161] Example 5

[0162] Comparison of 3AB and 3ABC proteins

[0163] Using 3AB, 3ABC proteins and 3A-AP as enzyme-labeled antibody pairs respectively, a competitive method was established. N1 / P was calculated respectively to compare the detection results using the two proteins.

[0164] Table 9 Detection results of negative and positive controls using 3AB and 3ABC as antigens respectively

[0165]

[0166] Table 10 Detection results of clinical samples using 3AB and 3ABC as antigens respectively

[0167]

[0168]

[0169] As shown in Table 9, when detecting negative and positive control sera, the N1 detection results of the two methods were lower than N2. When using 3ABC as the antigen to detect the positive control, N1 / P was 3.72 and 11.06, which was higher than the detection results using 3AB as the antigen. As shown in Table 10, when detecting clinical samples, when 3ABC was the antigen, V2 / P was calculated, with the highest value of 34.53; when 3AB was the antigen, V1 / P was calculated, with the highest value of 6.47. The detection results using 3ABC as the antigen were better.

[0170] In summary, when using 3ABC protein as the antigen for detection, the detection results of negative and positive controls and clinical samples are better. Therefore, 3ABC protein is selected as the antigen for subsequent method establishment and optimization.

[0171] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A kit for detecting foot-and-mouth disease virus non-structural protein 3ABC antibody based on tube type chemiluminescent immunoassay technology, characterized in that, It consists of the following components: magnetic particle-coupled 3ABC fusion protein, enzyme-labeled antibody, diluent, standard, standard diluent, concentrated washing solution, and luminescent substrate; The antibody in the enzyme-labeled antibody is a 3A monoclonal antibody; The 3A monoclonal antibody is secreted by the hybridoma cell line 3A24; The standard is foot-and-mouth disease virus-infected animal serum gradient-diluted with a standard diluent, and the foot-and-mouth disease infection antibody contents are 0U, 2.5U, 5U, 10U, 20U, 40U, 80U, 160U, 320U, and 640U respectively; the foot-and-mouth disease virus-infected animal serum is collected from cattle 1 to 3 months after being infected with the foot-and-mouth disease virus; The standard diluent is a PBS buffer solution with a concentration of 0.05 - 0.2M and a pH value of 7; The PBS buffer solution contains BSA with a mass concentration of 0.1% - 2%, PEG with a mass concentration of 0.1% - 1%, trehalose with a mass concentration of 0.5% - 5%, gelatin with a mass concentration of 0.1% - 0.5%, and ProClin300 with a volume concentration of 0.01% - 0.5%; For the preparation method of the magnetic particle-coupled 3ABC fusion protein, take 1mg of magnetic beads, dilute them to 10mg / ml with 0.1M MES, fully adsorb the magnetic beads with a magnet, discard the supernatant, then add 100μL of 0.1M MES, mix the magnetic beads evenly, and repeat the washing operation once; Take out EDC and NHS from -20°C and restore them to room temperature; Weigh EDC and NHS; dissolve them separately with 0.1M MES to prepare a 10mg / ml solution; Add 10μl of NHS solution to the magnetic beads, then add 10μl of EDC solution, and mix evenly at room temperature for 30 minutes; Fully adsorb the magnetic beads with a magnet, discard the supernatant, then add 100μL of 0.1M MES, mix the magnetic beads evenly to obtain activated magnetic beads; Add 30μg of foot-and-mouth disease virus 3ABC fusion protein to be coated to the activated magnetic beads, mix the magnetic beads evenly; shake and coat at room temperature for 3 hours; add 10μl of 10% BSA solution, shake and block at room temperature for 3 hours; wash 3 times with the washing solution to obtain the magnetic particle-coupled 3ABC fusion protein; The diluent is a MOPS buffer solution with a pH value of 7 and a concentration of 0.05 - 0.2M; the MOPS buffer solution contains BSA with a mass concentration of 0.1% - 2%, Tween-20 with a volume concentration of 0.01% - 0.5%, ProClin300 with a volume concentration of 0.01% - 0.5%, 0.01 - 0.5M of MgCl2, 0.01 - 0.5M of NaCl, hydrolyzed milk protein with a mass concentration of 0.05% - 3%, and PEG 6000 with a mass concentration of 0.1% - 1%; the diluent is used to dilute the magnetic particle-coupled 3ABC fusion protein and the enzyme-labeled antibody to their respective working concentrations to form a working solution; the concentration of the magnetic particle-coupled 3ABC fusion protein working solution is 0.2 - 0.5mg / ml; the concentration of the enzyme-labeled antibody working solution is 1:500 - 1:5000; The concentrated washing solution is a PBS buffer solution with a pH value of 7 containing 0.1% to 0.5% (v / v) Tween-20 or a Tris buffer solution with a pH value of 8 and 0.05 M containing 0.1% to 0.5% (v / v) Tween-20; when in use, the concentrated washing solution is diluted 10 to 30 times; The enzyme in the enzyme-labeled antibody is alkaline phosphatase; the luminescent substrate corresponds to the enzyme in the enzyme-labeled antibody.

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