CN108303543B - A detection kit for swine fever E2 protein antibody and its detection method - Google Patents

Abstract

The invention discloses a swine fever E2 protein antibody detection kit and a detection method thereof, wherein the kit adopts an antigen coated by magnetic particles, a protein marked by a luminescent marker and an antibody in a sample to be detected to perform specific reaction so as to determine the content of the antibody in the sample to be detected. The kit disclosed by the invention realizes the effects of quantitative detection of the swine fever E2 protein antibody, high sensitivity, wide detection range, rapidness, good repeatability, full-automatic operation and high-throughput detection.

Description

A swine fever E2 protein antibody detection kit and detection method thereof

Technical Field

The present invention relates to the technical field of classical swine fever E2 protein antibody detection, and in particular to a classical swine fever E2 protein antibody detection kit and a detection method using the kit for detection.

Background Art

Classical swine fever, also known as Classical Swine Fever (CSF), is a highly lethal, contagious disease of pigs caused by Classical swine fever virus (CSFV). It is one of the major infectious diseases closely watched by the Food and Agriculture Organization of the United Nations and governments of various countries. According to the 2005 edition of the Terrestrial Animal Health Code formulated by the OIE, CSF is listed as one of the legally reportable diseases and is listed as a "Class I animal disease" in my country, causing huge economic losses to my country's pig industry.

my country has adopted comprehensive epidemic prevention measures such as immunization, quarantine and elimination of infected pigs to promote the establishment of areas free of prescribed animal diseases. The attenuated swine fever rabbit vaccine and swine fever E2 subunit vaccine are important means for the prevention and control of swine fever in my country. The monitoring of antibody levels to formulate a reasonable and effective immunization program is the guarantee for improving the level of group immunity. Over the years, due to the great demand of scientific research and the market, people have been committed to the development of swine fever antibody detection kits. The methods currently established and applied include ELISA method, virus neutralization test method, immunofluorescence method, colloidal gold test strips and other methods.

Classical swine fever E2 protein is the main neutralizing antigen protein of classical swine fever. Its antibody is the main target for monitoring antibody levels after vaccine immunization and detecting antibody levels after virus infection. The antibody level is directly related to its immune or infection status, so there is a real need for quantitative detection of classical swine fever E2 protein antibodies. Existing testing institutions include veterinary stations, vaccine companies, farms, etc., which have a large number of samples and also have a demand for high-throughput classical swine fever E2 protein antibody detection kits and instruments. Existing methods such as ELISA and colloidal gold are difficult to meet.

The established and applied methods for detecting antibodies to the E2 protein of classical swine fever include ELISA, virus neutralization test, immunofluorescence, colloidal gold test strips, etc. The ELISA method is currently the most commonly used method for antibodies to the E2 protein of classical swine fever. The OD value measured by ELISA ranges from 0.1 to 3.5, which limits the measured antibody range to a narrow range and can only be semi-quantitative. The reaction time is usually about 2 hours, which is a long time. The virus neutralization test determines the antibody titer through antigen-antibody reaction between classical swine fever E2 protein and antibody on cells, which can more comprehensively reflect the level of neutralizing antibodies. The whole process of virus titration, antibody neutralization, and result determination takes 5-7 days. The whole process can only be operated and determined manually, and the repeatability is poor. The immunofluorescence method is usually also operated on cells, and has the disadvantages of low sensitivity and poor repeatability. The colloidal gold method can quickly obtain results, but it can only be qualitative and difficult to quantify, which limits the scope of application.

Patent CN104237513A (published on December 24, 2014) discloses a thyroid peroxidase antibody magnetic microparticle chemiluminescent immunoquantitative detection kit, including TPO-Ab calibrator, TPO-Ab diluent, magnetic microparticle suspension coupled with streptavidin, biotin-labeled TPO-Ab antigen, mouse anti-human enzyme-labeled conjugate, TPO-Ab quality control, chemiluminescent solution A and B, 20-fold concentrated washing solution and reaction tube. The kit quantitatively determines the content of thyroid peroxidase antibody, achieving high sensitivity, specificity, precision and good stability. The kit is widely used in human antibodies, but has not been reported in animal antibodies.

At present, the semi-quantitative ELISA method is mainly used for the detection of swine fever E2 antibodies, which cannot meet the quantitative requirements. This is mainly due to the lack of swine fever E2 antibody standards and quantitative standards, and the lack of accurate and reproducible detection methods. Unlike veterinary medicine, in the field of human medicine, markers of various diseases, such as the above-mentioned thyroid peroxidase antibodies, have been fully studied, and universal national standards, international standards or industry standards have been established. Quantification is simple and easy, so products produced by different manufacturers have been standardized and the test results are consistent. In the veterinary field, most of the current swine fever E2 antibody detection market is occupied by imported ELISA qualitative detection reagents, and the test results of products from various manufacturers vary greatly, which is not conducive to the development of animal husbandry. It is urgent to establish quantitative standards and accurate, reliable and reproducible detection methods.

Summary of the invention

In order to solve the problems of long detection time, difficulty in quantification, poor repeatability, and difficulty in automation in existing veterinary detection technologies, the purpose of the present invention is to provide a swine fever E2 protein antibody detection kit, which can quantitatively detect the content of swine fever E2 protein antibodies. To this end, the present invention also provides a method for detection using the kit, and the detection method of the present invention has high sensitivity, quantitative detection, wide detection range, short detection time, and good repeatability.

In order to achieve the above object, the technical solution adopted by the present invention is:

The first aspect of the present invention provides a classical swine fever E2 protein antibody detection kit, comprising a solution of classical swine fever E2 protein coupled or indirectly coupled to magnetic particles, a protein solution labeled with a luminescent marker, and a luminescent substrate.

The coupling includes condensation of carboxyl groups of magnetic particles and amino groups of proteins to form amides, cross-linking of amino groups of magnetic particles and amino groups of proteins through glutaraldehyde to form five-carbon bridges, and covalent coupling of toluenesulfonyl magnetic particles and amino groups of proteins.

Wherein, the indirect coupling includes coupling mediated by the following means: streptavidin-biotin mediated coupling, anti-FITC antibody-FITC coupling.

The method for preparing the solution of classical swine fever E2 protein coupled to magnetic particles comprises the following steps:

S1, taking a solution containing magnetic particles, washing the magnetic particles with a buffer solution, and suspending the magnetic particles in the buffer solution;

S2, adding purified classical swine fever E2 protein;

S3, adding a cross-linking agent or a catalyst and oscillating the reaction;

S4, the magnetic particles are adsorbed by a magnet, washed, and suspended in a solution containing a blocking agent.

The ratio of the magnetic particle solution to the classical swine fever E2 protein is 10 mg:0.64-4 nmol.

The method for preparing the solution of the swine fever E2 protein indirectly coupled to magnetic particles comprises the following steps:

1) Magnetic particles bind to avidin

S1, taking a solution containing magnetic particles, washing the magnetic particles with a buffer solution, and suspending the magnetic particles in the buffer solution;

S2, addition of avidin;

S3, adding a cross-linking agent or a catalyst and oscillating the reaction;

S4, the magnetic particles are adsorbed by a magnet, washed, and suspended in a solution containing a blocking agent to form a magnetic particle-avidin complex;

2) Classical Swine Fever E2 protein binds biotin

S1, take swine fever E2 protein and dialysis purification;

S2, add biotin and blocking agent in sequence and react;

S3, dialyze to remove unbound biotin and obtain classical swine fever E2 protein-biotin;

3) The magnetic particles-avidin are mixed with the classical swine fever E2 protein-biotin complex, and the magnetic particles and classical swine fever E2 protein are connected through the binding force between avidin and biotin.

The method for preparing the protein solution labeled with the luminescent marker comprises the following steps:

S1, taking the protein that specifically binds to the classical swine fever E2 protein or the classical swine fever virus antibody and dialysis purification;

S2, adding luminescent markers and reacting;

S3, adding blocking agent and reacting;

S4, dialysis to separate unbound luminescent marker.

The classical swine fever E2 protein is selected from a combination of one or more of the full-length classical swine fever virus E2 protein, a natural E2 protein fragment, a recombinantly expressed full-length classical swine fever E2 protein, a recombinantly expressed E2 protein fragment, an _E2 protein polypeptide, and an E2 protein chemical synthesis product. The magnetic particles are particles with _Fe3O4 as the core, a polymer coating on the surface, and hydroxyl, carboxyl, sulfonyl or amino active groups introduced.

Wherein, the diameter of the magnetic particles is 0.1-5 μm.

Preferably, the diameter of the magnetic particles is 1-3 μm, and the diameter CV of the magnetic particles is less than 3%.

Wherein, the luminescent marker is selected from any one of acridinium ester, alkaline phosphatase and peroxidase.

Wherein, the protein labeled with the luminescent marker is selected from one or more combinations of classical swine fever virus antigen, monoclonal antibody, polyclonal antibody, genetically engineered antibody, anti-swine IgG antibody, and anti-swine IgM antibody.

Wherein, the luminescent substrate corresponds to the luminescent marker one by one.

Preferably, when the luminescent marker is acridinium ester, the luminescent substrate consists of a first luminescent substrate and a second luminescent substrate, the first luminescent substrate is a solution containing 0.1 mol/L nitric acid and 0.1% hydrogen peroxide, and the second luminescent substrate is a solution containing 2% Tween-20 and 0.25 mol/L NaOH; when the luminescent marker is alkaline phosphatase, the luminescent substrate is a substrate solution based on adamantane; when the luminescent marker is peroxidase, the luminescent substrate consists of a first luminescent substrate and a second luminescent substrate, the first luminescent substrate is a solution containing 0.5 g/L luminol and 0.1 g/L p-iodophenol, and the second luminescent substrate is a 0.625 g/L urea peroxide solution.

The kit further includes a diluent, a quality control product, a calibrator and a cleaning solution, and the diluent is a combination of one or more of a buffer, bovine serum albumin, a blocking agent, a monoclonal antibody and a polyclonal antibody.

In order to establish a reliable quantitative standard, the present invention adopts calibration of calibrators to ensure the repeatability of the test. The calibrators are traced back to the classic virus neutralization test, and the concentration of the calibrators is determined according to the serum antibody titer obtained from the virus neutralization test. Among them, the calibrators are divided into 8 concentration gradients, which are 0, 3.13, 6.25, 12.5, 25, 50, 100, and 200 U/mL.

Among them, the quality control products are low-value quality control products and high-value quality control products for classical swine fever E2 protein antibodies, the quality control range of the low-value quality control products is 24-36U/mL, and the quality control range of the high-value quality control products is 80-120U/mL; the cleaning solution is 0.05mol/L tris(hydroxymethylaminomethane) (Tris) buffer with a pH of 8.0 or 0.01mol/L phosphate (PBS) buffer with a pH of 7.0, and the Tris buffer and PBS buffer contain 0.1% Tween-20 respectively.

The second aspect of the present invention provides a method for detecting antibodies to the classical swine fever E2 protein, using the above-mentioned detection kit, comprising the following steps:

S1, sequentially adding 10-100 μL of the sample to be tested or the calibrator, and the solution of the classical swine fever E2 protein coupled or indirectly coupled magnetic particles into the reaction container;

S2, react at 35-39°C for 10-20 minutes;

S3, adsorb with a magnet, remove the supernatant, add 200-500 μL of cleaning solution for washing, and discard the cleaning solution;

S4, add 100-200 μL of protein solution labeled with luminescent marker to S3;

S5, repeating steps S2 and S3;

S6, add luminescent substrate to S5 and react at 35-39°C for 0.5-10 minutes;

S7, detecting the luminescence value with a chemiluminometer, drawing a calibration curve, and calculating the concentration of the antibody in the test serum according to the calibration curve.

The samples to be tested include blood samples, body fluid samples and tissue samples.

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

1. The kit of the present invention uses magnetic particles with a diameter of 0.1-5 μm as the coating carrier. The magnetic particles are spherical, magnetic, and have the characteristics of large surface area, and can coat more classical swine fever E2 proteins; the magnetic particles can be suspended in the liquid, so they can fully and comprehensively contact and react with the reactants, while the coating carriers of the existing classical swine fever E2 protein antibody ELISA detection methods, virus neutralization tests, immunofluorescence and other methods are the surface of the enzyme labeling plate or the surface of the cell plate, and the proteins coated or contained are limited. Therefore, compared with ELISA, virus neutralization test, immunofluorescence and other methods:

1) There are more coated proteins and a wide detection range;

2) The detection method of the present invention is a liquid phase reaction with sufficient contact and a short reaction time, usually 5 to 10 minutes.

2. The detection method of the present invention uses a one-to-one correspondence between the color developing solution and the luminescent marker. The luminescent marker used in the detection method of the present invention can be one of the following: acridinium ester and its derivatives, alkaline phosphatase (AP), peroxidase (HRP). Acridinium ester and its derivatives are chemiluminescent agents directly labeled on the protein, and emit light through the action of the starting luminescent reagent (NaOH- _H2O2 ); protein labeling alkaline phosphatase and _peroxidase is enzymatic luminescence, the substrate of alkaline phosphatase is a solution prepared by adamantane and its derivatives, and the substrate of peroxidase is a solution prepared by luminol and its derivatives. The ELISA color developing substrate of the current swine fever E2 protein antibody detection method is usually TMB, and the color development sensitivity and intensity are much lower than chemiluminescence.

3. The detection method of the present invention is easy to implement full-automatic operation of sample addition, reaction, cleaning, separation and detection because the magnetic particles can be easily cleaned and separated under the action of a magnet. It is easy to implement rapid, high-throughput and full-automatic detection. The entire process is precisely controlled by the detection instrument, and the detection results have good repeatability. However, existing detection methods such as ELISA, virus neutralization test, immunofluorescence, colloidal gold and other detection methods are difficult to implement full-automatic operation and have poor repeatability.

4. The present invention adopts a highly uniform liquid phase reaction, and cooperates with calibration products and quality control products to control the accuracy of the test, so that the repeatability is greatly improved, and the comparability of the test results between different batches of tests is improved, which is more suitable for monitoring after vaccine immunization. However, the current swine fever antibody detection method is limited by its principle and operation, and the test results of different batches of tests are difficult to repeat.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

FIG1 is an optimal dosage curve of classical swine fever virus E2 protein for carboxyl magnetic particles of the present invention;

FIG2 is an optimal dosage curve of classical swine fever virus E2 protein of tosyl magnetic particles of the present invention;

FIG3 is a calibration curve in Example 1 of the present invention;

FIG4 is a calibration curve in Embodiment 2 of the present invention;

FIG5 is a calibration curve in Embodiment 3 of the present invention;

FIG. 6 is a calibration curve in the fourth embodiment of the present invention.

DETAILED DESCRIPTION

The classical swine fever virus E2 protein or its fragment that can be used in the present invention is not particularly limited, and can include the full length or fragment of the natural or recombinant classical swine fever virus E2 protein. Preferably, the full length sequence of the classical swine fever virus E2 protein can be shown in SEQ ID NO.: 1, which contains 373 amino acids and a molecular weight of 46.6KD; the fragment sequence of the classical swine fever virus E2 protein can be shown in SEQ ID NO.: 2, which contains 152 amino acids and a molecular weight of 19KD; the polypeptide sequence of the classical swine fever virus E2 protein can be shown in SEQ ID NO.: 3, which contains 43 amino acids and a molecular weight of 5KD.

Those skilled in the art can purify the polypeptide using standard protein purification techniques. Substantially pure polypeptides can produce a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed using an amino acid sequence. The protein or fragment thereof of the present invention can be a recombinant, natural, or synthetic protein or fragment thereof. The protein or fragment thereof of the present invention can be a naturally purified product, or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plant).

Magnetic particles refer to particles with a magnetic core inside and a polymer coating outside. The coating contains active groups that can be coupled with proteins, peptides, etc., without affecting the immune activity of proteins and peptides; the magnetic core enables the particles to move and aggregate in a directional manner under the action of an external magnetic field, and can be evenly dispersed in the solution after leaving the magnetic field, thereby taking into account the liquid phase reaction of antigen-antibody and the separation of antigen-antibody complexes from unreacted substances.

There is no particular limitation on the magnetic particles that can be used in the present invention, and they can be any magnetic particles that have a magnetic core and a polymer attached to the surface. The core of the magnetic particles that can be used in the present invention is iron oxide (Fe3O4); the polymers that can be used on the surface of the magnetic particles of the present invention include polystyrene, acrylic resin, polymethyl methacrylate, etc. The size of the magnetic particles of the present invention is preferably 0.1-5μm, more preferably 1-3μm. The magnetic particles that can be used in the present invention are usually in the form of a particle group solution. Usually, in the particle group solution, the particle size and shape are highly uniform, and the particle size CV <3%.

The magnetic particles that can be used in the present invention may also contain multiple active groups, so that proteins and polypeptides can be bound to the surface of the magnetic particles by chemical cross-linking. Preferably, the active groups include hydroxyl, carboxyl, sulfonyl or amino active groups. Magnetic particles containing active groups can be prepared by conventional techniques in the art or directly commercially available. For example, magnetic particles containing carboxyl groups purchased from JSR Corporation of Japan, item number: MagnosphereTM MS300/Caboxyl; or magnetic particles containing toluenesulfonyl groups purchased from JSR Corporation of Japan, item number: MagnosphereTM MS300/Tosyl.

In the present invention, the Classical Swine Fever Virus E2 protein and the magnetic particles can be coupled directly or indirectly. For example, the direct coupling includes condensation of the carboxyl group of the magnetic particles with the amino group of the protein to form an amide, cross-linking of the amino group of the magnetic particles with the amino group of the protein through glutaraldehyde to form a five-carbon bridge, and covalent coupling of the tosyl magnetic particles with the amino group of the protein. The indirect coupling includes coupling mediated by the following methods: streptavidin-biotin mediated coupling, anti-FITC antibody-FITC coupling. The preferred method is: streptavidin is coated on the magnetic particles, biotin is coupled to the porcine reproductive and respiratory syndrome virus, and the porcine reproductive and respiratory syndrome virus and the magnetic particles are combined through the streptavidin-biotin action; anti-FITC antibodies are coated on the magnetic particles, FITC is cross-linked on the porcine reproductive and respiratory syndrome virus, and the porcine reproductive and respiratory syndrome virus and the magnetic particles are combined through the anti-FITC antibody-FITC interaction.

A swine fever E2 protein antibody detection kit comprises a solution of swine fever E2 protein coupled or indirectly coupled to magnetic particles, a protein solution labeled with a luminescent marker, a quality control product, a calibrator, a cleaning solution, and a luminescent substrate.

A method for detecting antibodies to the swine fever E2 protein, using the above-mentioned detection kit, comprises the following steps:

S1, sequentially adding 10-100 μL of the serum to be tested or the calibrator, and the solution of the classical swine fever E2 protein coupled or indirectly coupled magnetic particles into the reaction container;

S2, react at 35-39°C for 10-20 minutes;

S3, adsorb with a magnet, remove the supernatant, add 200-500 μL of cleaning solution for washing, and discard the cleaning solution;

S4, add 100-200 μL of protein solution labeled with luminescent marker to S3;

S5, repeating steps S2 and S3;

S6, add luminescent substrate to S5 and react at 35-39°C for 0.5-10 minutes;

S7, detecting the luminescence value with a chemiluminometer, drawing a calibration curve, and calculating the concentration of the antibody in the test serum according to the calibration curve.

Taking carboxyl magnetic particles and toluenesulfonyl magnetic particles as examples, an experiment on the optimal dosage of magnetic particles and classical swine fever E2 protein was conducted.

1. Experiment on the optimal dosage of carboxyl magnetic particles and classical swine fever virus E2 protein

Three kinds of classical swine fever virus E2 proteins were used, among which the addition amounts of the full-length classical swine fever virus E2 proteins were 15.7, 31.3, 62.5, 125, 250, and 500 μg, and the corresponding amounts of substances were 0.34, 0.67, 1.34, 2.68, 5.36, and 10.73 nmol; the addition amounts of the classical swine fever virus E2 protein fragments were 6.3, 12.5, 25, 50, 100, and 200 μg, and the corresponding amounts of substances were 0.33, 0.66, 1.32, 2.63, 5.26, and 10.53 nmol; and the addition amounts of the classical swine fever virus E2 protein polypeptides were 1.6, 3.2, 6.3, 12.5, 25, and 50 μg, and the corresponding amounts of substances were 0.32, 0.64, 1.26, 2.5, 5, and 10 nmol.

The above-mentioned classical swine fever virus E2 viral protein and carboxyl magnetic particles were used to prepare magnetic suspensions coated with classical swine fever virus E2 protein, and the kits containing the magnetic suspensions were used to detect classical swine fever virus E2 antibody-positive serum. The results are shown in Table 1. Figure 1 is a curve showing the corresponding amount of coating protein and its luminescence value.

The results showed that as the amount of protein increased, the rate of increase in the luminescence value gradually slowed down, indicating that the magnetic particles were close to saturation in binding the protein; while as the amount of protein continued to increase, the luminescence value decreased, indicating that the protein was excessive and more protein self-cross-linking occurred.

Therefore, for carboxyl magnetic particles, the most suitable amount of full-length, fragments and polypeptides of classical swine fever virus E2 protein is 0.64-4nmol/10mg magnetic particles. Although the number of amino acids and molecular weight of the three proteins are very different, the optimal amount of coating protein is not much different when calculated by the amount of substance.

Table 1

2. Experiment on the optimal dosage of tosyl magnetic particles and classical swine fever virus E2 protein

Three kinds of classical swine fever virus E2 proteins were used, among which the addition amounts of the full-length classical swine fever virus E2 proteins were 15.7, 31.3, 62.5, 125, 250, and 500 μg, and the corresponding amounts of substances were 0.34, 0.67, 1.34, 2.68, 5.36, and 10.73 nmol; the addition amounts of the classical swine fever virus E2 protein fragments were 6.3, 12.5, 25, 50, 100, and 200 μg, and the corresponding amounts of substances were 0.33, 0.66, 1.32, 2.63, 5.26, and 10.53 nmol; and the addition amounts of the classical swine fever virus E2 protein polypeptides were 1.6, 3.2, 6.3, 12.5, 25, and 50 μg, and the corresponding amounts of substances were 0.32, 0.64, 1.26, 2.5, 5, and 10 nmol.

The above-mentioned classical swine fever virus E2 virus protein and tosyl magnetic particles were used to prepare magnetic suspensions coated with classical swine fever virus E2 protein, and the kits containing the magnetic suspensions were used to detect classical swine fever virus E2 antibody-positive serum. The results are shown in Table 2. Figure 2 is a curve showing the corresponding amount of coating protein and its luminescence value.

The results show that as the amount of protein increases, the rate of increase of the luminescence value gradually slows down, indicating that the magnetic particles are close to saturation in binding to the protein. Therefore, for tosyl magnetic particles, the most suitable amount of the full-length, fragment, and polypeptide of the classical swine fever virus E2 protein is 0.64nmol/10mg magnetic particles or more. Although the number of amino acids and molecular weight of the three proteins vary greatly, the optimal amount of coating protein calculated by the amount of substance is not much different. Considering the cost factor, the amount of protein should not be too much. Combined with the results of carboxyl magnetic particles, the optimal dosage range of the three proteins is determined to be 0.64-4nmol/10mg magnetic particles.

Table 2

Embodiment 1

A kit for detecting antibodies to classical swine fever E2 protein comprises a magnetic suspension coated with classical swine fever E2 protein, an alkaline phosphatase-labeled classical swine fever E2 protein antibody solution, a diluent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate.

Preparation of magnetic suspension coated with classical swine fever E2 protein:

(1) Take 1 mL of a solution containing magnetic microparticles (purchased from JSR Corporation, Japan, product number: Magnosphere ^TM MS300/Caboxyl) at a concentration of 10 mg/mL, wash it twice with 0.1 mol/L 2-morpholineethanesulfonic acid (MES) buffer at pH 5.0, and finally suspend it in 1 mL of 0.1 mol/L MES buffer at pH 5.0;

(2) Add 125 μg of purified classical swine fever E2 antigen (purchased from Qingdao Yibang Bioengineering Co., Ltd.);

(3) Weigh 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and dissolve it in 0.1 mol/L MES buffer (pH 5.0) to make the concentration of EDC 10 mg/mL;

(4) Take 100 μL of the EDC solution in (3) and add it to (2), and shake the reaction at 37°C for 2 hours;

(5) Adsorb on a magnet, remove the supernatant, wash three times with 0.01 mol/L PBS solution (pH 7.4) (containing 0.1% Tween-20), and finally suspend in 0.01 mol/L PBS solution (pH 7.4) (containing 1% BSA), and add 0.1% ProClin ^TM 300 (purchased from Sigma, catalog number: 48914-U).

The magnetic particles are magnetic particles containing carboxyl groups.

Preparation of alkaline phosphatase labeled classical swine fever E2 protein antibody solution:

(1) Take 1 mg of alkaline phosphatase and dilute it to 10 mg/mL with 0.05 mol/L carbonate buffer (pH 9.5) (CB buffer);

(2) Weigh sodium periodate (NaIO ₄ ) and dissolve it in 0.05 mol/L CB buffer (pH 9.5) to make the concentration of NaIO ₄ 12.5 mg/mL;

(3) Take 100 μL of the NaIO ₄ solution in (2), add it to (1), shake and mix, and react at 2°C in the dark for 1 hour;

(4) Take 10 μL of ethylene glycol and add 1 mL of 0.05 mol/L CB buffer with a pH of 9.5 to obtain an ethylene glycol solution;

(5) Take 100 μL of the ethylene glycol solution in (4) and add it to (3), and react at 6°C in the dark for 1 hour;

(6) 1 mg of classical swine fever virus monoclonal antibody was added to (5), mixed and dialyzed with 0.05 mol/L CB buffer (pH 9.5) at 2°C in the dark for 20 hours;

(7) Weigh sodium borohydride (NaBH ₄ ) and dissolve it in pure water to prepare a 2 mg/mL NaBH ₄ solution;

(8) Take 10 μL of the NaBH ₄ solution in (7) and add it to (6). React at 2°C in the dark for 2 hours.

(9) purifying and separating unbound alkaline phosphatase and classical swine fever virus monoclonal antibody through molecular sieve;

(10) The antibody solution purified in (9) was diluted with 0.05 M 3-morpholinepropanesulfonic acid (MOPS) buffer at pH 7.0 containing 1% BSA for later use.

The dilution concentration is 0.1-0.5μg/mL.

The calibrator is a buffered solution containing known concentrations of antibodies against the Classical Swine Fever E2 protein.

Preparation of calibrators:

(1) The strong positive serum of classical swine fever E2 protein antibody obtained after booster immunization with classical swine fever vaccine was heat-inactivated at 60°C for 1 hour;

(2) filtering the strongly positive serum inactivated in (1) through a 0.2 μm microfiltration membrane and adding 0.1% ProClin ^TM 300;

(3) The serum in (2) was calibrated and diluted to a certain concentration to obtain a series of calibrators of 0, 3.13, 6.25, 12.5, 25, 50, 100, and 200 U/mL.

The quality control product is a calibrated swine serum positive for swine fever virus antibodies. It is divided into low-value quality control products and high-value quality control products. The quality control range of low-value quality control products is 24-36U/mL, and the quality control range of high-value quality control products is 80-120U/mL. Quality control products are used to control the effectiveness of the test. Quality control products are tested regularly. If they exceed the quality control range, they must be recalibrated with calibrators.

Preparation of quality control products:

More than 10 sera positive for swine fever virus antibodies were selected, heat-inactivated at 60°C for 1 hour, mixed and filtered through a 0.2 μm microfiltration membrane, and 0.1% ProClin ^TM 300 was added.

The diluent is a PBS buffer containing 1% BSA, pH 7.4, and a concentration of 0.01 mol/L; the cleaning solution is a 0.05 mol/L Tris buffer with a pH of 8.0 and containing 0.1% Tween-20; the luminescent substrate is a solution based on adamantane and its derivatives, and the luminescent substrate in this example is Lumi-Phos 530, purchased from Lumigen, item number: P-5000.

A method for detecting antibodies to swine fever E2 protein comprises the following steps:

S1. Take several reaction tubes and add 10 μL of serum or calibrator to be tested, 100 μL of diluent and 25 μL of magnetic suspension coated with classical swine fever E2 protein in sequence;

S2, react at 37°C for 10 minutes;

S3, magnet adsorption, remove the supernatant, add 300 μL of cleaning solution to each reaction tube, repeat the washing 3 times, and discard the cleaning solution;

S4, add 100 μL of alkaline phosphatase-labeled classical swine fever E2 protein antibody solution to S3;

S5, react at 37°C for 10 minutes;

S6, repeat the cleaning steps in S3;

Add 100 μL of luminescent substrate to S7 and S6;

S8, react at 37°C for 0.5 min;

S9. Use a chemiluminescence instrument to detect the luminescence value, draw a calibration curve, and calculate the concentration of antibodies in the test serum based on the calibration curve.

The logarithm of the concentration value was taken as the X-axis, and the Logit of the luminescence value was taken as the Y-axis. Linear fitting was performed to draw a calibration curve.

Sn: luminescence value of calibrator (except calibrator zero value) or sample;

S0: Luminescence value of the calibrator zero value.

Table 3 shows the luminescence values corresponding to the calibration samples of different concentrations, and Figure 3 shows the calibration curve.

Table 3

1. Sensitivity test

The kit in this embodiment and the swine fever E2 protein antibody detection kit (enzyme-linked immunosorbent assay, hereinafter referred to as ELISA kit) produced by a well-known foreign manufacturer were used to simultaneously detect swine fever E2 protein antibody positive serum of different dilution multiples, wherein the kit in this embodiment performed 10 repeated tests on each blood sample and calculated the coefficient of variation (CV% = standard deviation of 10 test results/arithmetic mean). The results show that the kit in this embodiment is quantitatively accurate. Taking the lowest concentration of CV% <20% as the sensitivity, the sensitivity in this embodiment is <4.14U/mL, which is slightly better than the ELISA kit.

Table 4 is a comparison of the sensitivity of the kit of Example 1 and the ELISA kit

2. Repeatability Experiment

Three sera positive for swine fever E2 protein antibodies were tested with this kit. The tests were divided into two batches per day, and two tests were performed on each of the three sera in each batch. The two batches of tests were separated by at least 2 hours, and the tests were performed continuously for 5 days. Twenty test data were obtained for each serum, and the coefficient of variation of its concentration was calculated. The results are shown in Table 5. The results show that the test results of the three serum samples have good repeatability.

Table 5

3. Compliance Rate Experiment

This kit and ELISA kit were used to test multiple pig sera at the same time, and the test results are shown in Table 6. The results showed that the positive coincidence rate of this kit and ELISA kit was 96.4%, the negative coincidence rate was 90.4%, and the overall coincidence rate was 87.7%.

Table 6

4. Using the kit of this embodiment for immune monitoring of swine fever vaccine

In this embodiment, a concentration <10U/mL is judged as negative, and a concentration ≥10U/mL is judged as positive; an Elisa kit blocking rate <0.30 is judged as negative, 0.30-0.40 is judged as suspicious, and a blocking rate ≥0.40 is judged as positive.

After immunization with swine fever vaccine, 4 pigs were randomly selected, and blood samples were drawn on days 1, 7, 14, and 21 after immunization to detect swine fever E2 protein antibodies, and non-immunized pigs were used as controls. The test results are shown in Table 7. The results show that except for 2#, swine fever antibodies turned from negative to positive on days 14 and 21 after immunization, and the concentration gradually increased; while the antibody content of non-immunized pigs did not change significantly; except for 1#, the ELISA kit test result of the blood sample 14 days after immunization was suspicious, while the test result of this kit was positive, the test results of the other blood samples were consistent with the two reagents.

Table 7

5. Specificity Experiment

The kit of this embodiment was used to detect a variety of related virus antibody strong positive serum, including foot-and-mouth disease virus type O (FMDV-O), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), bovine viral diarrhea virus (BVDV). The test results were all lower than 10U/mL, all negative, and no cross reaction was found.

Table 8

Related viruses FMDV-O PRV PRRSV PCV2 BVDV Concentration (U/mL) 3.26 3.3 3.08 4.16 6.73

Embodiment 2

A kit for detecting antibodies to classical swine fever E2 protein comprises a magnetic suspension coated with classical swine fever E2 protein, an acridinium ester-labeled goat anti-swine IgG antibody solution (purchased from Beijing Solebow Technology Co., Ltd.), a diluent, a calibrator, a quality control product, a cleaning solution, a first luminescent substrate and a second luminescent substrate.

Preparation of magnetic suspension coated with classical swine fever E2 protein:

(1) Take 1 mL of a solution containing magnetic particles at a concentration of 10 mg/mL, wash it twice with 0.1 mol/L boric acid buffer at pH 9.5, and finally suspend it in 1 mL of 0.1 mol/L boric acid buffer at pH 9.5;

(2) Add 60 μg of purified classical swine fever virus recombinant antigen and vortex to mix;

(3) Add 0.5 mL of 0.1 mol/L boric acid buffer (containing 3 mol/L ammonium sulfate) at pH 9.5 and shake at 37°C for 20 hours;

(4) Add 0.5 mL of 10% BSA aqueous solution, vortex to mix, and shake at 37°C for 12 hours;

(5) Adsorb on a magnet, remove the supernatant, wash three times with 0.01 mol/L PBS solution at pH 7.4 (containing 0.1% Tween-20), and finally suspend in 0.01 mol/L PBS solution at pH 7.4 (containing 1% BSA), and add 0.1% ProClin ^™ 300.

The magnetic particles are magnetic particles containing tosyl groups.

Preparation of acridinium ester labeled goat anti-swine IgG antibody solution:

(1) Take 1 mg of goat anti-swine IgG antibody and dialyze it against 0.05 mol/L CB buffer (pH 9.5) at 8°C overnight;

(2) adding an acridinium ester solution containing 0.2 mg of acridinium ester to (1) and reacting at room temperature in the dark for 2 hours;

(3) Add 100 μL of 0.1 g/mL lysine solution and react at room temperature in the dark for 2 hours;

(4) dialyze with 0.05 mol/L CB buffer (pH 9.5) at 8°C in the dark for 24 h;

(5) The antibody solution in (4) was diluted with a MOPS buffer containing 1% BSA and pH 7.0, 0.05 mol/L for later use.

The preparation method of the calibrator is the same as that of Example 1.

The preparation method of the quality control product is the same as that of Example 1.

The diluent is a PBS buffer solution containing 1% BSA, pH 7.4, and a concentration of 0.01 mol/L; the cleaning solution is a 0.01 mol/L PBS buffer solution with a pH of 7.0 and containing 0.1% Tween-20; the first luminescent substrate is a solution containing 0.1 mol/L nitric acid and 0.1% hydrogen peroxide, and the second luminescent substrate is a solution containing 2% Tween-20 and 0.25 mol/L NaOH.

A method for detecting antibodies to swine fever E2 protein comprises the following steps:

S1. Take several reaction tubes and add 20 μL of serum or calibrator to be tested, 100 μL of diluent and 20 μL of magnetic suspension coated with classical swine fever E2 protein in sequence;

S2, react at 35°C for 15 minutes;

S3, magnet adsorption, remove the supernatant, add 200 μL of cleaning solution to each reaction tube, repeat the washing 3 times, and discard the cleaning solution;

S4, add 150 μL of acridinium ester-labeled goat anti-swine IgG antibody solution to S3;

S5, react at 35°C for 15 minutes;

S6, repeat the cleaning step in step S3;

100 μL of the first luminescent substrate and 100 μL of the second luminescent substrate were added to S7 and S6 respectively;

S8. Use a chemiluminescence instrument to detect the luminescence value, draw a calibration curve, and calculate the concentration of the antibody in the test serum based on the calibration curve.

Table 9 shows the luminescence values corresponding to different calibration sample concentrations, and Figure 4 shows the calibration curve drawn.

1. Sensitivity test

The kit of this embodiment and the ELISA kit were used to simultaneously detect sera positive for swine fever E2 protein antibodies at different dilution multiples, wherein the kit was used to perform 10 repeated tests on each blood sample, and the coefficient of variation was calculated, and the results are shown in Table 10. The results show that the kit of this embodiment is accurate in quantitative determination, and the sensitivity is <1.4U/mL, which is better than the ELISA kit.

Table 10 is a comparison of the sensitivity of the kit of this embodiment and the ELISA kit

2. Repeatability Experiment

Three sera positive for swine fever E2 protein antibodies were taken and tested with the kit of this example. The tests were divided into two batches per day, and two tests were performed on each of the three sera in each batch. The two batches of tests were separated by at least 2 hours, and the tests were performed continuously for 5 days. 20 test data were obtained for each serum, and the coefficient of variation of its concentration was calculated. The results are shown in Table 11. The results show that the test results of the three serum samples have good repeatability.

Table 11

3. Compliance Rate Experiment

The kit of this embodiment and the ELISA kit simultaneously tested multiple pig sera, and the results are shown in Table 12. The positive coincidence rate of the kit of this embodiment and the ELISA kit was 94.8%, the negative coincidence rate was 92.3%, and the overall coincidence rate was 88%.

Table 12

4. Using the kit of this embodiment for immune monitoring of swine fever vaccine

In this embodiment, a concentration <10U/mL is judged as negative, and a concentration ≥10U/mL is judged as positive; an Elisa kit blocking rate <0.30 is judged as negative, 0.30-0.40 is judged as suspicious, and a blocking rate ≥0.40 is judged as positive.

After immunization with swine fever vaccine, 4 pigs were randomly selected, and blood samples were drawn 1, 7, 14, and 21 days after immunization to detect swine fever E2 protein antibodies, and non-immunized pigs were used as controls. The results are shown in Table 13. The results show that except for 2#, swine fever antibodies turned from negative to positive 14 days and 21 days after immunization, and the concentration gradually increased; while the antibody content of non-immunized pigs did not change significantly; except for 1#, the ELISA kit test result of the blood sample 14 days after immunization was suspicious, while the test result of this kit was positive, the test results of the other blood samples were consistent with the two reagents.

Table 13

5. Specificity Experiment

The kit of this embodiment was used to detect a variety of related virus antibody strong positive serum, including foot-and-mouth disease virus type O (FMDV-O), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), bovine viral diarrhea virus (BVDV). The test results were all lower than 10U/mL, all negative, and no cross reaction was found.

Table 14

Related viruses FMDV-O PRV PRRSV PCV2 BVDV Concentration (U/mL) 4.08 4.17 4.71 3.53 7.46

Embodiment 3

A kit for detecting antibodies to classical swine fever E2 protein comprises a magnetic suspension coated with classical swine fever E2 protein, a solution of classical swine fever virus E2 antigen labeled with horseradish peroxidase, a diluent, a calibrator, a quality control product, a cleaning solution, a first luminescent substrate and a second luminescent substrate.

Preparation of magnetic suspension coated with classical swine fever E2 protein:

(1) Take 1 mL of a solution containing magnetic particles at a concentration of 10 mg/mL, wash it twice with 0.01 mol/L phosphate-buffered saline (PBS) buffer at pH 7.4, and finally suspend it in 1 mL of 0.01 mol/L PBS buffer at pH 7.4;

(2) Add 0.1 mL of 25% (v/v) glutaraldehyde solution and shake at 37°C for 2 hours;

(3) Wash three times with 1 mL of 0.01 mol/L PBS buffer (pH 7.4);

(4) Add 50 μg of purified classical swine fever virus E2 recombinant antigen and react at 37°C with shaking for 20 hours;

(5) Add 0.5 mL of 10% bovine serum albumin (BSA) aqueous solution, vortex to mix, and shake at 37°C for 2 hours;

(6) Wash three times with 0.01 mol/L PBS solution (pH 7.4) (containing 0.1% Tween-20), and finally suspend in 0.01 mol/L PBS solution (pH 7.4) (containing 1% BSA), and add 0.1% ProClinTM300.

The magnetic particles are magnetic particles containing amino groups.

Preparation of horseradish peroxidase labeled classical swine fever virus E2 antigen:

(1) Take 1 mg of horseradish peroxidase and dilute it to 10 mg/mL with 0.05 mol/L CB buffer (pH 9.5);

(2) Weigh _NaIO4 and dissolve it in 0.05 mol/L CB buffer (pH 9.5) to make the concentration of NaIO4 12.5 mg/mL;

(3) Take 100 μL of the NaIO ₄ solution in (2), add it to (1), shake and mix, and react at 8°C in the dark for 1 hour;

(4) Take 10 μL of ethylene glycol and add 1 mL of 0.05 mol/L CB buffer with a pH of 9.5 to obtain an ethylene glycol solution;

(5) Take 1 mL of the ethylene glycol solution in (4) and add it to (3), and react at 2°C in the dark for 1 hour;

(6) 1 mg of classical swine fever virus E2 antigen was added to (5), mixed and dialyzed with 0.05 mol/L CB buffer (pH 9.5) at 8°C in the dark for 24 hours;

(7) Weigh NaBH ₄ and dissolve it in pure water to prepare a 2 mg/mL NaBH ₄ solution;

(8) Take 10 μL of the NaBH ₄ solution in (7) and add it to (6). React at 8°C in the dark for 2 hours.

(9) Purification by molecular sieve;

(10) The antigen solution purified in (9) was diluted with a MOPS buffer (pH 7.0, 0.05 mol/L) containing 1% BSA for later use.

The preparation method of the calibrator is the same as that of Example 1.

The preparation method of the quality control product is the same as that of Example 1.

The diluent is a PBS buffer solution containing 1% BSA, pH 7.4, and a concentration of 0.01 mol/L; the cleaning solution is a 0.01 mol/L pH 7.0 PBS buffer solution containing 0.1% Tween-20; the first luminescent substrate is a solution containing 0.5 g/L luminol and 0.1 g/L p-iodine phenol, and the second luminescent substrate is a 0.625 g/L urea peroxide solution.

A method for detecting antibodies to swine fever E2 protein comprises the following steps:

S1. Take several reaction tubes and add 50 μL of serum or calibrator to be tested, 100 μL of diluent and 50 μL of magnetic suspension coated with classical swine fever E2 protein in sequence;

S2, react at 39°C for 20 minutes;

S3, magnet adsorption, remove the supernatant, add 500 μL of cleaning solution to each reaction tube, repeat the washing 3 times, and discard the cleaning solution;

S4, adding 200 μL of horseradish peroxidase-labeled classical swine fever virus E2 antigen solution to S3;

S5, react at 39°C for 20 minutes;

S6, repeat the cleaning steps in S3;

50 μL of the first luminescent substrate and 50 μL of the second luminescent substrate were added to S7 and S6 respectively;

S8, react at 35°C for 5 minutes;

S9. Use a chemiluminescence instrument to detect the luminescence value, draw a calibration curve, and calculate the concentration of antibodies in the test serum based on the calibration curve.

Table 15 shows the luminescence values corresponding to different concentrations of the calibrator, and Figure 5 shows the calibration curve.

Table 15

1. Sensitivity test

The kit of this embodiment and the ELISA kit were used to simultaneously detect sera positive for swine fever E2 protein antibodies at different dilution multiples, wherein the kit was used to perform 10 repeated tests on each blood sample, and the coefficient of variation was calculated, and the results are shown in Table 16. The results show that the kit is accurate in quantitative determination, and the sensitivity is <1.18U/mL, which is better than the ELISA kit.

Table 16

2. Repeatability Experiment

Three sera positive for swine fever E2 protein antibodies were taken and tested with the kit of this example. The tests were divided into two batches per day, and two tests were performed on each of the three sera in each batch. The two batches of tests were separated by at least 2 hours, and the tests were performed continuously for 5 days. 20 test data were obtained for each serum, and the coefficient of variation of its concentration was calculated. The results are shown in Table 17. The results show that the test results of the three serum samples have good repeatability.

Table 17

3. Compliance Rate Experiment

The kit of this embodiment and the ELISA kit simultaneously tested multiple pig sera, and the test results are shown in Table 18. The positive coincidence rate of the kit of this embodiment and the ELISA kit was 92.7%, the negative coincidence rate was 94.6%, and the overall coincidence rate was 88%.

Table 18

4. The kit of this embodiment is used for swine fever vaccine immune monitoring

In this embodiment, a concentration <10U/mL is judged as negative, and a concentration ≥10U/mL is judged as positive; an Elisa kit blocking rate <0.30 is judged as negative, 0.30-0.40 is judged as suspicious, and a blocking rate ≥0.40 is judged as positive.

After immunization with swine fever vaccine, 4 pigs were randomly selected, and blood samples were drawn 1, 7, 14, and 21 days after immunization to test swine fever antibodies, and non-immunized pigs were used as controls. The results showed that except for 2#, swine fever antibodies turned from negative to positive 14 and 21 days after immunization, and the concentration gradually increased; while the antibody content of non-immunized pigs did not change significantly; except for 1#, the ELISA kit test result of the blood sample 14 days after immunization was suspicious, while the test result of this kit was positive, the test results of the other blood samples were consistent with the two reagents.

Table 19

5. Specificity Experiment

The kit of this embodiment was used to detect a variety of related virus antibody strong positive serum, including foot-and-mouth disease virus type O (FMDV-O), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), bovine viral diarrhea virus (BVDV). The test results were all lower than 10U/mL, all negative, and no cross reaction was found.

Table 20

Related viruses FMDV-O PRV PRRSV PCV2 BVDV Concentration (U/mL) 1.89 2.41 1.98 2.49 7.26

Embodiment 4

A kit for detecting classical swine fever E2 protein antibodies comprises a magnetic suspension coated with classical swine fever E2 protein, classical swine fever E2 protein antibodies labeled with alkaline phosphatase, biotinylated antigens, calibrators, quality control products, cleaning fluid, and luminescent substrates.

Preparation of magnetic suspension coated with classical swine fever E2 protein:

(1) 1 mL of a solution containing magnetic particles at a concentration of 10 mg/mL was taken, washed twice with 0.1 mol/L 1-morpholineethanesulfonic acid (MES) buffer at pH 5.0, and finally suspended in 1 mL of 0.1 mol/L MES buffer at pH 5.0;

(2) Add 350 μg of streptavidin;

(3) Weigh EDC and dissolve it in 0.1 mol/L MES buffer (pH 5.0) to make the concentration of EDC 10 mg/mL;

(4) Take 100 μL of the EDC solution in (3) and add it to (2), and shake the reaction at 37°C for 2 hours;

(5) Adsorb on a magnet, remove the supernatant, wash three times with 0.01 mol/L PBS solution (pH 7.4) (containing 0.1% Tween-20), and finally suspend in 0.01 mol/L PBS solution (pH 7.4) (containing 1% BSA), and add 0.1% ProClin ^™ 300.

The magnetic particles are magnetic particles containing carboxyl groups.

Preparation of alkaline phosphatase labeled classical swine fever E2 protein antibody solution:

(1) Take 1 mg of alkaline phosphatase and dilute it to 10 mg/mL with 0.05 mol/L CB buffer (pH 9.5);

(2) Weigh NaIO ₄ and dissolve it in 0.05 mol/L CB buffer with a pH of 9.5 to make the concentration of NaIO ₄ 12.5 mg/mL;

(3) Take 100 μL of the NaIO ₄ solution in (2), add it to (1), shake and mix, and react at 8°C in the dark for 1 hour;

(4) Take 10 μL of ethylene glycol and add 1 mL of 0.05 mol/L CB buffer with a pH of 9.5 to obtain an ethylene glycol solution;

(5) Take 1 mL of the ethylene glycol solution in (4) and add it to (3), and react at 6°C in the dark for 1 hour;

(6) 1 mg of monoclonal antibody against classical swine fever virus E2 protein was added to (5), mixed and dialyzed with 0.05 mol/L CB buffer (pH 9.5) at 2-8°C in the dark for 24 hours;

(7) Weigh NaBH ₄ and dissolve it in pure water to prepare a 2 mg/mL NaBH ₄ solution;

(8) Take 10 μL of the NaBH ₄ solution in (7) and add it to (6). React at 2°C in the dark for 2 hours.

(9) Purification by molecular sieve;

(10) The antibody solution purified in (9) was diluted with 0.05 mol/L MOPS buffer at pH 7.0 containing 1% BSA for later use.

Preparation of biotin antigen:

(1) Take 1 mg of classical swine fever virus E2 antigen and dialyze it against 0.01 mol/L PBS buffer (pH 7.4) at 8°C overnight;

(2) dissolving preactivated biotin in pure water to prepare a 50 mmol/L biotin solution;

(3) Take 20 μL of the biotin solution in (2) and add it to (1) and react at room temperature for 1 hour;

(4) Add 100 μL of 0.1 g/mL lysine solution to (3) and react at room temperature for 1 hour;

(5) The solution in (4) was dialyzed against 0.01 mol/L PBS buffer (pH 7.4) at 5°C for 24 hours.

(6) Dilute the solution in (5) with 0.05 mol/L MOPS buffer (pH 7.0) containing 1% BSA for later use.

The preparation method of the calibrator is the same as that of Example 1.

The preparation method of the quality control product is the same as that of Example 1.

The washing solution is 0.05 mol/L pH 8.0 Tris buffer containing 0.1% Tween-20; the luminescent substrate is a solution based on adamantane and its derivatives. The luminescent substrate in this example is Lumi-Phos 530, purchased from Lumigen, product number: P-5000.

A method for detecting antibodies to swine fever E2 protein comprises the following steps:

S1. Take several reaction tubes and add 100 μL of serum or calibrator to be tested, 100 μL of biotinylated antigen and 25 μL of magnetic suspension coated with classical swine fever E2 protein in sequence;

S2, react at 37°C for 10 minutes;

S3, magnet adsorption, remove the supernatant, add 500 μL of cleaning solution to each reaction tube, repeat the washing 3 times, and discard the cleaning solution;

S4, add 200 μL of alkaline phosphatase-labeled classical swine fever E2 protein antibody solution to S3;

S5, react at 37°C for 10 minutes;

S6, repeat the cleaning steps in S3;

Add 100 μL of luminescent substrate to S7 and S6;

S8, react at 39°C for 10 minutes;

S9. Use a chemiluminescence instrument to detect the luminescence value, draw a calibration curve, and calculate the concentration of antibodies in the test serum based on the calibration curve.

Table 21 shows the luminescence values corresponding to different concentrations of the calibrator, and Figure 6 shows the drawn calibration curve.

Table 21

1. Sensitivity test

The kit of this embodiment and the ELISA kit were used to simultaneously detect sera positive for swine fever E2 protein antibodies at different dilution multiples, wherein the kit of this embodiment performed 10 repeated tests on each blood sample, and the coefficient of variation was calculated, and the results are shown in Table 22. The results show that the kit is accurate in quantitative determination, and the sensitivity is <2.18U/mL, which is better than the ELISA kit.

Table 22

2. Repeatability Experiment

Three sera positive for swine fever E2 protein antibodies were taken and tested with the kit of this example. The tests were divided into two batches per day, and two tests were performed on each of the three sera in each batch. The two batches of tests were separated by at least 2 hours, and the tests were performed continuously for 5 days. 20 test data were obtained for each serum, and the coefficient of variation of its concentration was calculated. The results are shown in Table 23. The results show that the test results of the three serum samples have good repeatability.

Table 23

3. Compliance Rate Experiment

The kit of this embodiment and the ELISA kit simultaneously tested multiple pig sera, and the results are shown in Table 24. The positive coincidence rate of the kit of this embodiment and the ELISA kit was 92%, the negative coincidence rate was 95.1%, and the overall coincidence rate was 87.6%.

Table 24

4. The kit of this embodiment is used for swine fever vaccine immune monitoring

In this embodiment, a concentration <10U/mL is judged as negative, and a concentration ≥10U/mL is judged as positive; an Elisa kit blocking rate <0.30 is judged as negative, 0.30-0.40 is judged as suspicious, and a blocking rate ≥0.40 is judged as positive.

After immunization with swine fever vaccine, 4 pigs were randomly selected, and blood samples were drawn 1, 7, 14, and 21 days after immunization to test swine fever antibodies, and non-immunized pigs were used as controls. The results showed that except for 2#, swine fever antibodies turned from negative to positive 14 and 21 days after immunization, and the concentration gradually increased; while the antibody content of non-immunized pigs did not change significantly; except for 1#, the ELISA kit test result of the blood sample 14 days after immunization was suspicious, while the test result of this kit was positive, the test results of the other blood samples were consistent with the two reagents.

Table 25

5. Specificity Experiment

The kit of this embodiment was used to detect a variety of related virus antibody strong positive serum, including foot-and-mouth disease virus type O (FMDV-O), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), bovine viral diarrhea virus (BVDV). The test results were all lower than 10U/mL, all negative, and no cross reaction was found.

Table 26

Related viruses FMDV-O PRV PRRSV PCV2 BVDV Concentration (U/mL) 3.63 3.93 3.25 3.39 6.65

The present invention adopts magnetic microparticles with a specific particle size as coating carriers, and adopts a specific ratio of classical swine fever E2 protein to magnetic microparticles for mixing and reacting, thereby obtaining classical swine fever E2 protein-coupled magnetic microparticles with homogeneous coating and stable structure, and also saves coating protein raw materials. The coated protein is sufficient, the detection range is wide, the sensitivity is high, the reaction time is very short (only 5-10 minutes), and it has the excellent characteristics of high throughput, automation, and repeatability.

The above-mentioned specific implementation methods are only exemplary, and are intended to better enable those skilled in the art to understand this patent, and should not be understood as limiting the scope of this patent; any equivalent changes or modifications made based on the spirit disclosed in this patent fall within the scope of this patent.

Sequence Listing

<110> Shanghai Mingjie Biotechnology Co., Ltd.

<120> A swine fever E2 protein antibody detection kit and its detection method

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<170> PatentIn version 3.5

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AVSPTTLRTE VVKTFKRERP FPHRVDCVTT IV 152

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Claims (6)

1. The swine fever E2 protein antibody detection kit is characterized by comprising a solution of swine fever E2 protein coupled or indirectly coupled magnetic particles, a protein solution marked by a luminescent marker and a luminescent substrate; wherein the coupling comprises the steps of forming amide through condensation of carboxyl groups of the magnetic particles and amino groups of the protein, forming five-carbon bridge through glutaraldehyde crosslinking of the amino groups of the magnetic particles and amino groups of the protein, and connecting the magnetic particles of tosyl and amino groups of the protein through covalent coupling;

The swine fever E2 protein is one of full length, fragment or polypeptide of swine fever virus E2 protein;

the magnetic particles are Fe ₃ O ₄ As a core, particles whose surfaces are coated with a polymer coating and into which carboxyl or tosyl groups are introduced; the size of the magnetic particles is 1-3 mu m;

the optimal dosage range of the swine fever E2 protein is 0.64-4nmol/10mg magnetic particles;

the kit also comprises a diluent, a quality control product, a calibrator and a cleaning solution, wherein the diluent is selected from one or more of buffer solution, bovine serum albumin, a blocker, a monoclonal antibody and a polyclonal antibody;

the quality control products are a swine fever E2 protein antibody low-value quality control product and a high-value quality control product, the quality control range of the low-value quality control product is 24-36U/mL, and the quality control range of the high-value quality control product is 80-120U/mL; the washing liquid is a Tris buffer solution with the concentration of 0.05mol/LpH of 8.0 or a phosphate buffer solution with the concentration of 0.01mol/L of 7.0 (PBS), and the Tris buffer solution and the PBS buffer solution respectively contain 0.1% of Tween-20.

2. The swine fever E2 protein antibody detection kit of claim 1, wherein the solution preparation method of the swine fever E2 protein-coupled magnetic particles comprises the following steps:

S1, taking a solution containing magnetic particles, cleaning the magnetic particles by using a buffer solution, and suspending the magnetic particles in the buffer solution;

s2, adding purified swine fever E2 protein;

s3, adding a cross-linking agent or a catalyst, and carrying out oscillation reaction;

s4, the magnetic particles are adsorbed by the magnet, washed and suspended in a solution containing the blocking agent.

3. The swine fever E2 protein antibody detection kit of claim 1, wherein the method for preparing a solution of swine fever E2 protein indirectly coupled magnetic particles comprises the steps of:

1) Magnetic microparticle-bound avidin

S1, taking a solution containing magnetic particles, cleaning the magnetic particles by using a buffer solution, and suspending the magnetic particles in the buffer solution;

s2, adding avidin;

s3, adding a cross-linking agent or a catalyst, and carrying out oscillation reaction;

s4, the magnetic particles are adsorbed by the magnet, washed and suspended in a solution containing a blocking agent to form a magnetic particle-avidin complex;

2) Swine fever E2 protein binding biotin

S1, taking swine fever E2 protein and dialyzing;

s2, adding biotin for reaction;

s3, adding a sealing agent to react;

s3, dialyzing to remove unbound biotin to obtain swine fever E2 protein-biotin;

3) The magnetic particles-avidin are mixed with the swine fever E2 protein-biotin complex, and the magnetic particles are connected with the swine fever E2 protein through the binding force of the avidin and the biotin.

4. The swine fever E2 protein antibody detection kit of claim 1, wherein the preparation method of the luminescent marker-labeled protein solution comprises the following steps:

s1, taking protein specifically combined with a swine fever E2 protein or a swine fever E2 virus antibody, and dialyzing;

s2, adding a luminous marker, and reacting;

s3, adding a sealing agent to react;

s4, dialyzing to separate unbound luminescent marker.

5. The swine fever E2 protein antibody detection kit of claim 1, wherein the luminescent label is selected from any one of acridinium ester, alkaline phosphatase, peroxidase, and the luminescent label-labeled protein is selected from one or more combinations of swine fever virus E2 antigen, monoclonal antibody, polyclonal antibody, genetically engineered antibody, anti-pig IgG antibody, anti-pig IgM antibody.

6. The swine fever E2 protein antibody detection kit of claim 1, wherein the luminescent substrates are in one-to-one correspondence with luminescent markers.

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