CN108303542B

CN108303542B - Pig breeding and respiratory syndrome antibody detection kit and detection method thereof

Info

Publication number: CN108303542B
Application number: CN201710019500.3A
Authority: CN
Inventors: 宋启超; 左青山; 纪良心; 刘聪; 李炎晖
Original assignee: Shanghai Mingjie Biotechnology Co ltd
Current assignee: Shanghai Mingjie Biotechnology Co ltd
Priority date: 2017-01-11
Filing date: 2017-01-11
Publication date: 2023-06-09
Anticipated expiration: 2037-01-11
Also published as: CN108303542A

Abstract

The invention discloses a kit for detecting antibodies of porcine reproductive and respiratory syndrome and a detection method thereof. The kit disclosed by the invention realizes quantitative detection of the antibodies of the porcine reproductive and respiratory syndrome, and has the effects of high sensitivity, wide detection range, rapidness, good repeatability, full-automatic operation and high-throughput detection.

Description

Pig breeding and respiratory syndrome antibody detection kit and detection method thereof

Technical Field

The invention relates to the technical field of detection of antibodies of porcine reproductive and respiratory syndrome, in particular to a detection kit of the antibodies of porcine reproductive and respiratory syndrome and a detection method for detecting the antibodies by adopting the kit.

Background

Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV), and mainly causes reproductive disorders such as abortion, premature birth, stillbirth, etc. in pregnant sows, and respiratory symptoms in piglets and fattening pigs. The infectious nature of the disease is extremely strong, once infected, the sick pigs can last at high temperature for more than 41 ℃ and die acutely; pregnant sow abortion yield is up to more than 30%; piglets are 100% ill and are accompanied by mortality risks of more than 50%, so that serious economic losses are caused to pig industry in China.

PRRSV belongs to the genus arterivirus of the family arterividae, and is divided into 2 types, european (type i) and american (type ii). The current popularity of China is mainly type II strains. The virus particle contains a nucleocapsid, the nucleocapsid consists of a positive strand RNA genome and a nucleocapsid protein (N), and a layer of envelope is wrapped outside the nucleocapsid; the envelope of the virus has 6 viral structural proteins: E. m, GP2, GP3, GP4 and GP5. The three proteins M, N and GP5 are the major structural proteins of PRRS virus.

In China, immunization is a main strategy for preventing and treating porcine reproductive and respiratory syndrome virus. The virus antigen protein of the porcine reproductive and respiratory syndrome virus plays an important role, is also a necessary structural protein, and can generate protective neutralizing antibodies after immunization. The antibody titer of the porcine reproductive and respiratory syndrome virus after immunization has obvious correlation with the virus attack protection, so that a reasonable and effective immunization program is formulated through monitoring the antibody level, and the guarantee of improving the immunity level of the population is provided.

The antigen protein of the porcine reproductive and respiratory syndrome is a main neutral antigen protein, and the antibody is a main target of monitoring the antibody level after vaccine immunization and detecting the antibody level after virus infection, and the antibody level is directly related to the immune or infection condition, so the real requirement of quantitative detection of the porcine reproductive and respiratory syndrome antibody exists. The existing detection institutions have a large number of samples in veterinary stations, vaccine companies, farms and the like, and also have demands for large-flux pig breeding and respiratory syndrome antibody detection kits and instruments. The existing ELISA, virus neutralization test, colloidal gold and other methods are difficult to meet.

The existing established and applied methods for detecting the antibodies of the porcine reproductive and respiratory syndrome comprise ELISA methods, virus neutralization tests, immunofluorescence methods, colloidal gold test strips and the like, wherein the ELISA methods are the most common methods for detecting the antibodies of the porcine reproductive and respiratory syndrome, the range of OD values measured by ELISA is 0.1-3.5, the measured antibody range is limited to be narrower, only semi-quantitative can be realized, and the reaction time is usually about 2 hours and longer; the virus neutralization test judges the antibody titer by carrying out antigen-antibody reaction on cells of the porcine reproductive and respiratory syndrome virus and the antibody, can more comprehensively reflect the height of the neutralizing antibody, takes 5-7 days in the whole process of virus titration, antibody neutralization, result judgment and the like, can only be manually operated and judged in the whole process, and has poor repeatability; immunofluorescence is also usually performed on cells, and has the disadvantages of low sensitivity, poor repeatability and the like; the colloidal gold method can rapidly obtain results, but can only be used for qualitative and difficult quantitative determination, and limits the application range.

Patent CN104237513A (publication date 2014.12.24) discloses a magnetic particle chemiluminescence immune quantitative detection kit for a thyroid peroxidase antibody, which comprises a TPO-Ab calibrator, a TPO-Ab diluent, a streptavidin-coupled magnetic particle suspension, a biotin-labeled TPO-Ab antigen, a mouse anti-human enzyme-labeled conjugate, a TPO-Ab quality control product, chemiluminescent liquid A and B, 20-time concentrated washing liquid and a reaction tube, wherein the kit is used for quantitatively measuring the content of the thyroid peroxidase antibody, and has the effects of high sensitivity, specificity, precision and good stability. The kit is widely applied to human antibodies, but has not been reported on animal antibodies.

At present, semi-quantitative ELISA method is mainly adopted for detecting the antigen and antibody of the porcine reproductive and respiratory syndrome virus, and the semi-quantitative ELISA method can not meet the quantitative requirement, which is mainly caused by the lack of standard substances and quantitative standards of the antigen and antibody of the porcine reproductive and respiratory syndrome virus, and the lack of accurate and repeatable detection method. Different from veterinary medicine, in the field of human medicine, markers of various diseases, such as the thyroid peroxidase antibody, have been fully researched, and general national standards, international standards or industry standards are established, so that quantification is simple and easy, and products produced by different manufacturers are standardized, and the consistency of detection results is good. In the veterinary field, most of the current pig breeding and respiratory syndrome virus antigen and antibody detection market is occupied by imported ELISA qualitative detection reagents, and the product detection results of all factories are quite different, so that the development of livestock breeding industry is not facilitated, and a detection method with quantitative standard establishment, accuracy, reliability and good repeatability is needed.

Disclosure of Invention

The invention aims to solve the problems of long detection time, difficult quantification, poor repeatability, difficult automation and the like in the existing veterinary detection technology, and provides a kit for detecting antibodies of porcine reproductive and respiratory syndrome, which can quantitatively detect the content of the antibodies of porcine reproductive and respiratory syndrome. Therefore, the invention also provides a detection method by using the kit, and the detection method has the advantages of high sensitivity, quantitative detection, wide detection range, short detection time and good repeatability.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a kit for detecting antibodies to porcine reproductive and respiratory syndrome comprising a solution of magnetic particles coupled to or indirectly coupled to an antigen protein of porcine reproductive and respiratory syndrome, a solution of a protein labeled with a luminescent marker and a luminescent substrate.

Wherein the coupling comprises that the carboxyl of the magnetic particles is condensed with the amino of the protein to form amide, the amino of the magnetic particles is crosslinked with the amino of the protein to form five-carbon bridge through glutaraldehyde, and the tosyl magnetic particles are connected with the amino of the protein in a covalent coupling way.

Wherein said indirect coupling comprises a coupling mediated by: streptavidin-biotin mediated coupling, anti-FITC antibody-FITC coupling.

The preparation method of the solution of the porcine reproductive and respiratory syndrome antigen 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 porcine reproductive and respiratory syndrome antigen 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.

Wherein the proportion of the solution of the magnetic particles and the antigen protein of the porcine reproductive and respiratory syndrome is 10mg:0.6-9.5nmol.

Preferably, the ratio of the solution of the magnetic particles to the antigen protein of porcine reproductive and respiratory syndrome is 10mg:2-8nmol.

The preparation method of the solution of the indirect coupling magnetic particles of the antigen protein of the porcine reproductive and respiratory syndrome comprises the following steps:

1) Magnetic microparticle-bound avidin

s2, adding avidin;

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) Porcine reproductive and respiratory syndrome antigen protein binding biotin

S1, taking antigen protein of porcine reproductive and respiratory syndrome, dialyzing and purifying;

s2, sequentially adding biotin and a blocking agent for reaction;

s3, dialyzing to remove unbound biotin, and obtaining the antigen protein-biotin of the porcine reproductive and respiratory syndrome.

3) The magnetic particles-avidin are mixed with the porcine reproductive and respiratory syndrome antigen protein-biotin complex, and the magnetic particles are connected with the porcine reproductive and respiratory syndrome antigen protein through the binding force of the avidin and the biotin.

The preparation method of the protein solution marked by the luminescent marker comprises the following steps:

s1, taking a protein which is specifically combined with antigen protein of porcine reproductive and respiratory syndrome or an antibody of porcine reproductive and respiratory syndrome, dialyzing and purifying;

s2, adding a luminous marker, and reacting;

s3, adding a sealing agent to react;

s4, dialyzing to separate unbound luminescent marker.

Wherein the antigen protein of the porcine reproductive and respiratory syndrome can be one or more of GP2a, GP3, GP4, GP5 and 5a, N, M, E proteins of the porcine reproductive and respiratory syndrome virus, and is one or more of antigen protein full length, natural antigen protein fragments, recombinant expressed porcine reproductive and respiratory syndrome antigen protein full length, recombinant expressed antigen protein fragments, antigen protein polypeptide and antigen protein chemical composition, and the magnetic particles are prepared by using Fe ₃ O ₄ As a core, the surface is coated with a polymer coating and particles of hydroxyl, carboxyl, sulfonyl or amino reactive groups are 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 < 3%.

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

Wherein the protein marked by the luminous marker is selected from any one or a combination of a plurality of porcine reproductive and respiratory syndrome antigens, monoclonal antibodies, polyclonal antibodies, genetic engineering antibodies, anti-porcine IgG antibodies and anti-porcine IgM antibodies.

Wherein the luminescent substrates are in one-to-one correspondence with the luminescent markers.

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

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

In order to establish a reliable quantitative standard, the invention adopts calibration materials for calibration, thereby ensuring the repeatability of the test. And tracing the calibrator to a classical virus neutralization test, and determining the concentration of the calibrator according to the serum antibody titer obtained by the virus neutralization test. Wherein, the calibrator is divided into 8 concentration gradients, which are 0,3.13,6.25, 12.5, 25, 50, 100 and 200U/mL in sequence.

The quality control products are low-value quality control products and high-value quality control products of the antibodies of the porcine reproductive and respiratory syndrome, wherein the quality control range of the low-value quality control products is 20-30U/mL, and the quality control range of the high-value quality control products is 100-150U/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.

In a second aspect, the invention provides a method for detecting antibodies of porcine reproductive and respiratory syndrome, which adopts the detection kit and comprises the following steps:

s1, sequentially adding 10-100 mu L of a sample to be detected or a calibrator or a solution of magnetic particles coupled or indirectly coupled with antigen protein of porcine reproductive and respiratory syndrome into a reaction container;

S2, reacting for 10-20 minutes at the temperature of 35-39 ℃;

s3, adsorbing by using a magnet, sucking the supernatant, adding 200-500 mu L of cleaning liquid for washing, and discarding the cleaning liquid;

s4, adding 100-200 mu L of a luminous marker-labeled protein solution into the S3;

s5, repeating the steps of S2 and S3;

s6, adding a luminescent substrate into the S5, and reacting for 0.5-10 minutes at the temperature of 35-39 ℃;

s7, detecting a luminescence value by using a chemiluminescent instrument, drawing a calibration curve, and calculating the concentration of the antibody in the serum to be detected according to the calibration curve.

The sample to be tested includes a blood sample, a body fluid sample and a tissue sample.

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

1. the kit adopts the magnetic particles with the diameter of 0.1-5 mu m as a coating carrier, the magnetic particles are spheres and are magnetic, and the kit has the characteristic of large surface area and can coat more porcine reproductive and respiratory syndrome antigen proteins; the magnetic particles can be suspended in liquid, so that the magnetic particles can fully and omnidirectionally contact and react with reactants, and the coating carrier of the existing methods such as ELISA detection method for antibodies of porcine reproductive and respiratory syndrome, virus neutralization test, immunofluorescence and the like is the surface of an ELISA plate or the surface of a cell plate, and the coated or contained protein is limited. Therefore, compared with ELISA, virus neutralization test, immunofluorescence and other methods, the detection method of the invention has the advantages that:

1) The coated proteins are more, and the detection range is wide;

2) The detection method of the invention is a fully contacted liquid phase reaction, and the reaction time is short, usually 5-10 minutes.

2. The detection method of the invention uses the one-to-one correspondence of the luminescent substrate and the luminescent marker. The luminescent marker adopted by the detection method can be one of the following: acridinium ester and its derivatives, alkaline Phosphatase (AP), peroxidase (HRP). Acridinium esters and their derivatives are chemiluminescent agents which are directly labeled on proteins by activating the luminescent agent (NaOH-H ₂ O ₂ ) Acts to emit light; the protein-labeled alkaline phosphatase and the peroxidase are enzymatically luminescent, the substrate of the alkaline phosphatase is a solution prepared from adamantane and derivatives thereof, and the substrate of the peroxidase is a solution prepared from luminol and derivatives thereof. The ELISA chromogenic substrate of the existing detection method for the antibodies of the porcine reproductive and respiratory syndrome is usually TMB, and the chromogenic sensitivity and intensity are far lower than those of chemiluminescence.

3. The detection method of the invention is convenient for cleaning and separating the magnetic particles under the action of the magnet, is easy to realize full-automatic operations of sample adding, reaction, cleaning, separation and detection, is easy to realize rapid, high-flux and full-automatic detection, and has good repeatability of detection results due to accurate control of the whole process by a detection instrument; however, the existing detection methods such as ELISA, virus neutralization test, immunofluorescence, colloidal gold and the like are difficult to realize full-automatic operation, and have poor repeatability.

4. The invention adopts a highly uniform liquid phase reaction, and is matched with the accuracy of a calibrator and a quality control product control test, so that the repeatability is greatly improved, the comparability of detection results among different batches of tests is improved, and the invention is more suitable for monitoring after vaccine immunization. The existing detection method of the porcine reproductive and respiratory syndrome virus antibody is limited by the principle and operation, and the detection results of different batches of tests are difficult to repeat.

Drawings

The invention is described in further detail below with reference to the attached drawings and detailed description:

FIG. 1 is a graph showing the amounts of antigen used in the carboxyl magnetic particles of the present invention for PRRSV;

FIG. 2 is a graph showing the amount of antigen used by the tosyl magnetic particles of the present invention for porcine reproductive and respiratory syndrome virus;

FIG. 3 is a calibration curve in accordance with a first embodiment of the present invention;

FIG. 4 is a calibration curve in a second embodiment of the invention;

FIG. 5 is a calibration curve in embodiment three of the present invention;

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

Detailed Description

The porcine reproductive and respiratory syndrome virus antigen protein or fragment thereof useful in the present invention is not particularly limited and may include the full length of the native or recombinant gE protein or fragment thereof. Preferably, the full-length sequence of GP5 protein of porcine reproductive and respiratory syndrome virus is shown as SEQ ID NO. 1, which contains 200 amino acids and has a molecular weight of 25kD; the GP5 protein fragment sequence of the porcine reproductive and respiratory syndrome virus is shown as SEQ ID NO. 2, contains 102 amino acids and has a molecular weight of 12.5kD; the GP5 protein polypeptide sequence of the porcine reproductive and respiratory syndrome virus is shown as SEQ ID NO. 3, which contains 42 amino acids and has a molecular weight of 5kD; or adopting a porcine reproductive and respiratory syndrome virus N protein sequence shown as SEQ ID NO. 4, wherein the N protein sequence contains 123 amino acids and has a molecular weight of 15kD; the M protein sequence of the porcine reproductive and respiratory syndrome virus is shown as SEQ ID NO. 5, and the M protein contains 174 amino acids and has a molecular weight of 15kD.

The person skilled in the art is able to purify the polypeptides using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the polypeptide can also be further analyzed by amino acid sequence. The protein or fragment thereof of the present invention may be a recombinant, natural, synthetic protein or fragment thereof. The proteins or fragments thereof of the invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.

The magnetic particles are particles having a magnetic core inside and a polymer coated outside. The coating layer contains active groups, can be coupled with proteins, polypeptides and the like, and does not influence the immunocompetence of the proteins and the polypeptides; the magnetic core makes the particles directionally move and aggregate under the action of an external magnetic field, and can be uniformly dispersed in the solution after leaving the magnetic field, so that the liquid phase reaction of antigen and antibody and the separation of antigen and antibody complex and unreacted substances are considered.

The magnetic particles usable in the present invention are not particularly limited, and may be any magnetic particles having a magnetic core and a polymer attached to the surface thereof. The core of the magnetic particles useful in the present invention is iron oxide (Fe 3O 4); polymers useful for the magnetic particle surfaces of the present invention include polystyrene, acrylic resins, polymethyl methacrylate, and the like. The size of the magnetic particles of the present invention is preferably 0.1 to 5. Mu.m, more preferably 1 to 3. Mu.m. The magnetic particles useful in the present invention are typically present in the form of a particle population solution in which the particle size and shape are highly uniform, typically with a particle diameter CV <3%.

The magnetic particles useful in the present invention may also contain a plurality of reactive groups to bind proteins, polypeptides to the surface of the magnetic particles by chemical cross-linking. Preferably, the reactive group comprises a hydroxyl, carboxyl, sulfonyl or amino reactive group. The magnetic particles containing reactive groups may be prepared by techniques conventional in the art or are directly commercially available. For example, available from JSR corporation, japan, cat#: magnetic particles containing carboxyl groups of magnospere MS 300/Cabox; or purchased from JSR corporation, japan, cat: magnospere MS300/Tosyl magnetic particles containing Tosyl groups.

The porcine reproductive and respiratory syndrome virus antigen and the magnetic particles can be directly or indirectly coupled. For example, the direct coupling includes the formation of amide by condensation of carboxyl groups of the magnetic particles with amino groups of the protein, the formation of five carbon bridges by glutaraldehyde crosslinking of amino groups of the magnetic particles with amino groups of the protein, and covalent coupling of tosyl magnetic particles with amino groups of the protein. The indirect coupling includes coupling mediated by: streptavidin-biotin mediated coupling, anti-FITC antibody-FITC coupling. The preferred mode is: streptavidin is coated on the magnetic particles, biotin is coupled on the porcine reproductive and respiratory syndrome virus, and the porcine reproductive and respiratory syndrome virus and the magnetic particles are combined through the action of streptavidin-biotin; the anti-FITC antibody is coated on the magnetic particles, FITC is crosslinked 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 kit for detecting antibodies of porcine reproductive and respiratory syndrome comprises a solution of antigen protein coupling or indirect coupling magnetic particles of porcine reproductive and respiratory syndrome, a protein solution marked by a luminescent marker and a luminescent substrate.

The detection method of the antibody of the porcine reproductive and respiratory syndrome adopts the detection kit and comprises the following steps:

s2, reacting for 10-20 minutes at the temperature of 35-39 ℃;

s5, repeating the steps of S2 and S3;

Taking carboxyl magnetic particles and tosyl magnetic particles as examples, the most applicable amount experiment of the magnetic particles and antigen proteins of porcine reproductive and respiratory syndrome is carried out.

1. Carboxyl magnetic particles, pig breeding and respiratory syndrome antigen protein optimum amount experiment

Adopting 5 porcine reproductive and respiratory syndrome antigen proteins, wherein the total length of the porcine reproductive and respiratory syndrome virus GP5 protein is added in an amount of 10, 20, 50, 100, 200 and 500 mug, and the corresponding substances are added in an amount of 0.4, 0.8, 2, 4, 8 and 20nmol; nmol; the addition amount of GP5 protein fragments of porcine reproductive and respiratory syndrome virus is 5, 10, 25, 50, 100 and 250 mug, and the corresponding amount of substances is 0.4, 0.8, 2, 4, 8 and 20nmol; the addition amount of the GP5 protein polypeptide of the porcine reproductive and respiratory syndrome virus is 2, 4, 10, 20, 40 and 100 mug, and the corresponding amount of substances is 0.4, 0.8, 2, 4, 8 and 20nmol; the total length of N protein of the porcine reproductive and respiratory syndrome virus is added in the amount of 5, 10, 25, 50, 100 and 250 mug, and the corresponding substances are added in the amount of 0.33, 0.67, 1.67, 3.33, 6.67 and 16.7nmol; the addition amount of M protein fragments of porcine reproductive and respiratory syndrome virus is 5, 10, 25, 50, 100 and 250 mug, the addition amount of corresponding substances is 0.45, 0.91, 2.27, 4.55, 9.09 and 22.7nmol, and the addition amount of carboxyl magnetic particles is 10mg.

The porcine reproductive and respiratory syndrome virus protein and carboxyl magnetic particles are respectively used for preparing magnetic suspension liquid for coating the porcine reproductive and respiratory syndrome antigen protein, a kit containing the magnetic suspension liquid is respectively used for detecting the porcine reproductive and respiratory syndrome antibody positive serum, the results are shown in table 1, and the graph of the coating protein dosage and the luminescence value thereof is shown in fig. 1.

The result shows that the luminous value gradually increases gradually along with the increase of the protein consumption, which indicates that the magnetic particle binding protein is close to saturation; the amount of protein is continuously increased, the luminescence value is reduced, and the excessive protein is indicated, so that more protein self-crosslinking occurs.

Therefore, for carboxyl magnetic particles, the optimal dosage of the full length, fragments and polypeptides of the GP5 protein of the porcine reproductive and respiratory syndrome is 0.8-8nmol/10mg of the magnetic particles; the optimal dosage of the total length of the N protein is 0.67-6.67nmol/10mg magnetic particles; the most suitable amount of M protein fragment is 0.91-9.09nmol/10mg magnetic particles. The amount of the optimal coating protein calculated according to the amount of substances is not very different, and the amount of the optimal coating protein is in the range of 0.6-9.5nmol/10mg of magnetic particles although the amount of amino acids and the molecular weight of the five proteins are very different.

TABLE 1

2. Tosyl magnetic particles, pig breeding and respiratory syndrome antigen protein optimum amount experiment

Adopting 5 porcine reproductive and respiratory syndrome antigen proteins, wherein the total length of the porcine reproductive and respiratory syndrome virus GP5 protein is added in an amount of 10, 20, 50, 100, 200 and 500 mug, and the corresponding substances are added in an amount of 0.4, 0.8, 2, 4, 8 and 20nmol; nmol; the addition amount of GP5 protein fragments of porcine reproductive and respiratory syndrome virus is 5, 10, 25, 50, 100 and 250 mug, and the corresponding amount of substances is 0.4, 0.8, 2, 4, 8 and 20nmol; the addition amount of the GP5 protein polypeptide of the porcine reproductive and respiratory syndrome virus is 2, 4, 10, 20, 40 and 100 mug, and the corresponding amount of substances is 0.4, 0.8, 2, 4, 8 and 20nmol; the total length of N protein of the porcine reproductive and respiratory syndrome virus is added in the amount of 5, 10, 25, 50, 100 and 250 mug, and the corresponding substances are added in the amount of 0.33, 0.67, 1.67, 3.33, 6.67 and 16.7nmol; the addition amount of M protein fragments of porcine reproductive and respiratory syndrome virus is 5, 10, 25, 50, 100 and 250 mug, the addition amount of corresponding substances is 0.45, 0.91, 2.27, 4.55, 9.09 and 22.7nmol, and the addition amount of tosyl magnetic particles is 10mg.

The porcine reproductive and respiratory syndrome virus protein and the tosyl magnetic particles are respectively used for preparing magnetic suspension liquid for coating the porcine reproductive and respiratory syndrome antigen protein, a kit containing the magnetic suspension liquid is respectively used for detecting the porcine reproductive and respiratory syndrome antibody positive serum, the results are shown in table 2, and the graph of the amount of the coating protein and the luminescence value thereof is shown in fig. 2.

The results showed that the luminescence value increased gradually as the amount of protein increased, indicating that the magnetic particle-bound protein was near saturation. Therefore, for the tosyl magnetic particles, the optimal dosage of the full length, the fragment and the polypeptide of the GP5 protein of the porcine reproductive and respiratory syndrome is more than 0.8nmol/10mg of the magnetic particles; the optimal dosage of the total length of the N protein is more than 0.67nmol/10mg magnetic particles; the most suitable amount of M protein fragment is more than 0.91nmol/10mg magnetic particles. The optimal dosage of the five proteins is not quite different according to the amount of substances, the cost factor is considered, the dosage of the proteins is not excessive, and the optimal dosage range of the five proteins is 0.65-9.5nmol/10mg magnetic particles is determined according to the result of combining the carboxyl magnetic particles.

TABLE 2

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Example 1

A kit for detecting antibodies of porcine reproductive and respiratory syndrome comprises a magnetic suspension for coating antigen proteins of porcine reproductive and respiratory syndrome, an alkaline phosphatase-labeled antibody solution of porcine reproductive and respiratory syndrome, a diluent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate.

Preparation of magnetic suspension coated with antigen protein of porcine reproductive and respiratory syndrome:

(1) 1mL of magnetic particles (available from JSR corporation, japan, cat# Magnospere) ^TM MS 300/Cabox), at a concentration of 10mg/mL, was washed 2 times with 0.1 mol/L2-morpholinoethanesulfonic acid (MES) buffer at pH 5.0, and finally suspended in 1mL 0.1mol/L MES buffer at pH 5.0;

(2) Purified porcine reproductive and respiratory syndrome antigen protein (purchased from Qingdao Yibang bioengineering Co., ltd.) was added at 30. Mu.g;

(3) 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) was weighed and dissolved in 0.1mol/L MES buffer pH 5.0 to give an EDC concentration of 10mg/mL;

(4) Adding 100 mu L of EDC solution in the step (3) into the step (2), and carrying out oscillation reaction for 2 hours at 37 ℃;

(5) Magnet adsorption, removing supernatant, washing with 0.01mol/L PBS solution (containing 0.1% Tween-20) with pH 7.4 for 3 times, suspending in 0.01mol/L PBS solution (containing 1% BSA) with pH 7.4, and adding 0.1% ProClin ^TM 300 (purchased from Sigma Co., ltd., product number: 48914-U).

The magnetic particles are magnetic particles containing carboxyl groups.

Preparation of alkaline phosphatase-labeled porcine reproductive and respiratory syndrome antibody solution:

(1) 1mg of alkaline phosphatase was diluted to 10mg/mL with 0.05mol/L of carbonate buffer (CB buffer) having a pH of 9.5;

(2) Weighing sodium periodate (NaIO) ₄ ) And dissolving with 0.05mol/L CB buffer with pH of 9.5 to make NaIO ₄ Is 12.5mg/mL;

(3) Taking NaIO in (2) ₄ Adding 100 mu L of the solution into the step (1), shaking and uniformly mixing, and carrying out light-shielding reaction for 1 hour at the temperature of 2 ℃;

(4) 10. Mu.L of ethylene glycol was taken and 1mL of 0.05mol/L CB buffer at pH 9.5 was added to obtain an ethylene glycol solution;

(5) Adding 100 mu L of the ethylene glycol solution in the step (4) into the step (3), and carrying out light-shielding reaction at the temperature of 6 ℃ for 1 hour;

(6) Adding 0.5mg of monoclonal antibody of porcine reproductive and respiratory syndrome into the step (5), uniformly mixing, and dialyzing for 20 hours at 2 ℃ in a dark place by using 0.05mol/L CB buffer with the pH of 9.5;

(7) Weighing sodium borohydride (NaBH) ₄ ) Dissolving in pure water to prepare 2mg/mL NaBH ₄ A solution;

(8) Taking NaBH of (7) ₄ 10 mu L of the solution is added into the step (6) and is reacted for 2 hours at 2 ℃ in a dark place;

(9) Purifying and separating unbound alkaline phosphatase and porcine reproductive and respiratory syndrome monoclonal antibody by molecular sieve;

(10) The antibody solution purified in (9) was diluted with 3-morpholinopropanesulfonic acid (MOPS) buffer containing 1% BSA at pH 7.0.05M for use.

The dilution concentration is 0.1-0.5 mug/mL.

The calibrator is a calibrated buffer containing known concentrations of antibodies to porcine reproductive and respiratory syndrome.

Preparation of a calibrator:

(1) Heat-inactivating the strong positive serum of the porcine reproductive and respiratory syndrome antibody obtained after the porcine reproductive and respiratory syndrome virus vaccine is subjected to the intensified immunity at 60 ℃ for 1 hour;

(2) Filtering the strong positive serum after inactivation in (1) by a microfiltration membrane of 0.2 mu m, adding 0.1% ProClin ^TM 300；

(3) Calibrating the serum in the step (2), and diluting the serum according to a certain concentration to obtain 0,3.13,6.25, 12.5, 25, 50, 100 and 200U/mL serial calibration products.

The quality control product is marked pig serum positive for the antibodies of the porcine reproductive and respiratory syndrome. The method is divided into a low-value quality control product and a high-value quality control product, wherein the quality control range of the low-value quality control product is 20-30U/mL, and the quality control range of the high-value quality control product is 100-150U/mL. The quality control product is used for controlling the validity of the test, the quality control product is detected periodically, and if the quality control product exceeds the quality control range, the calibration product is needed to be used for rescaling.

And (3) preparation of a quality control product: selecting more than 10 parts of pig breeding and respiratory syndrome antibody positive serum, heat inactivating at 60deg.C for 1 hr, mixing, filtering with 0.2 μm microfiltration membrane, adding 0.1% ProClin ^TM 300, obtaining the quality control product.

The dilution was PBS buffer containing 1% BSA, pH 7.4, and concentration 0.01 mol/L; the cleaning solution is Tris buffer solution containing 0.1% Tween-20 and having a pH of 8.0 and 0.05 mol/L; the luminescent substrate is a solution based on adamantane and its derivatives, in this case Lumi-Phos 530, available from Lumigen under the trade designation: p-5000.

A detection method of antibodies of porcine reproductive and respiratory syndrome comprises the following steps:

s1, taking a plurality of reaction tubes, and sequentially adding 10 mu L of serum or calibrator to be detected, 100 mu L of diluent and 25 mu L of magnetic suspension for coating antigen proteins of porcine reproductive and respiratory syndrome;

s2, reacting for 10 minutes at 37 ℃;

s3, carrying out magnet adsorption, sucking out supernatant, adding 300 mu L of cleaning liquid into each reaction tube, repeatedly cleaning for 3 times, and discarding the cleaning liquid;

s4, adding 100 mu L of alkaline phosphatase-labeled porcine reproductive and respiratory syndrome antibody solution into the S3;

s5, reacting for 10 minutes at 37 ℃;

s6, repeating the cleaning step in the step S3;

s7, adding 100 mu L of luminous substrate into S6;

s8, reacting for 5 minutes at 37 ℃;

s9, detecting a luminescence value by using a chemiluminescent instrument, drawing a calibration curve, and calculating the concentration of the antibody in the serum to be detected according to the calibration curve.

Taking the logarithm of the concentration value as an X axis, taking the Logit of the luminescence value as a Y axis, performing linear fitting, and drawing a calibration curve.

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

s0: a luminescent value of zero for the calibrator.

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

TABLE 3 Table 3

1. Sensitivity experiment

Pig breeding and respiratory syndrome antibody positive serum of different dilution factors was simultaneously detected by using the kit of this example and a pig breeding and respiratory syndrome antibody detection kit (enzyme-linked immunosorbent assay, hereinafter abbreviated as ELISA kit) manufactured by foreign well-known manufacturers, wherein the kit of this example performs 10 repeated detections for each blood sample, and a variation coefficient (CV% =standard deviation/arithmetic mean of 10 test results) was calculated. The results prove that the kit in the embodiment has accurate quantification. The lowest concentration of CV% <20% was used as the sensitivity, which in this example is <2.69U/mL, superior to ELISA kit.

In the embodiment, the concentration is less than 10U/mL and is judged to be negative, and the concentration is more than or equal to 10U/mL and is judged to be positive; the Elisa kit has S/P of less than 0.40 judged as negative and S/P of more than or equal to 0.40 judged as positive.

Table 4 shows the sensitivity of the kit of example I and ELISA kit

2. Repeatability experiments

3 cases of pig breeding and respiratory syndrome antibody positive serum are taken, detected by the kit, 2 batches of serum are detected every day, 2 tests are respectively carried out on 3 batches of serum, and the two batches of serum are continuously detected for 5 days at least at intervals of 2 hours. Each serum was subjected to 20 pieces of measurement data, and the coefficient of variation in concentration was calculated, and the results are shown in Table 5. The results demonstrate that 3 serum test results are good in reproducibility.

TABLE 5

3. Compliance rate experiment

The kit and ELISA kit detect a plurality of pig serum at the same time, and the detection results are shown in Table 6. The results show that the positive coincidence rate of the kit and the ELISA kit is 93.5%, the negative coincidence rate is 94.6%, and the overall coincidence rate is 93.7%.

TABLE 6

4. The kit of this example was used for immunomonitoring of porcine reproductive and respiratory syndrome virus vaccines

After immunization with porcine reproductive and respiratory syndrome virus vaccine, 4 animals were randomly sampled, blood samples were taken 1, 7, 14, 21 days after immunization to detect porcine reproductive and respiratory syndrome antibodies, and non-immunized pigs were used as controls, with the detection results shown in table 7. The results show that the porcine reproductive and respiratory syndrome virus antibodies change from negative to positive and the concentration gradually increases 14 days and 21 days after immunization; the antibody content of the non-immune pigs is not obviously changed; the detection results of the two reagents are consistent.

TABLE 7

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5. Specificity experiments

The kit of this example was used to detect various relevant viral antibody strong positive sera including Classical Swine Fever Virus (CSFV), foot and mouth disease virus type O (FMDV-O), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV 2), bovine Viral Diarrhea Virus (BVDV). The detection results are all lower than 10U/mL, are all negative, and have no cross reaction.

TABLE 8

Related viruses	CSFV	FMD-O	PRV	PCV2	BVDV
						Concentration (U/mL)	4.34	3.37	3.71	2.67	2.81

Example two

A kit for detecting antibodies of porcine reproductive and respiratory syndrome comprises a magnetic suspension for coating antigen proteins of porcine reproductive and respiratory syndrome, an acridinium ester-marked goat anti-pig IgG antibody (purchased from Beijing Soy Bao technology Co., ltd.) solution, a diluent, a calibrator, a quality control product, a cleaning solution, a first luminescent substrate and a second luminescent substrate.

(1) Taking 1mL of a solution containing magnetic particles, wherein the concentration is 10mg/mL, washing the solution 2 times by using 0.1mol/L boric acid buffer solution with the pH of 9.5, and finally suspending the solution in 1mL boric acid buffer solution with the pH of 9.5;

(2) Adding purified antigen protein of porcine reproductive and respiratory syndrome 40 mug, and mixing by vortex;

(3) Adding 0.1mol/L boric acid buffer solution (containing 3mol/L ammonium sulfate) with pH of 9.5, 0.5mL, and oscillating at 37 ℃ for 20 hours;

(4) 0.5mL of 10% BSA aqueous solution is added, vortex mixed evenly and shake reacted for 12 hours at 37 ℃;

(5) Magnet adsorption, removing supernatant, washing with 0.01mol/L PBS solution (containing 0.1% Tween-20) with pH7.4 for 3 times, suspending in 0.01mol/L PBS solution (containing 1% BSA) with pH7.4, and adding 0.1% ProClin ^TM 300。

The magnetic particles are magnetic particles containing tosyl groups.

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

(1) 1mg of goat anti-pig IgG antibody was taken and dialyzed overnight at 8℃with 0.05mol/L of CB buffer at pH 9.5;

(2) Adding an acridinium ester solution containing 0.2mg of acridinium ester into the reaction kettle (1), and reacting for 2 hours at normal temperature in a dark place;

(3) Adding 100 mu L of 0.1g/mL lysine solution, and reacting for 2 hours at normal temperature in a dark place;

(4) Dialyzing against light at 8deg.C for 24 hr with 0.05mol/L CB buffer at pH 9.5;

(5) The antibody solution in (4) was diluted with MOPS buffer containing 1% BSA at pH 7.0.05 mol/L for use.

The preparation method of the calibrator was the same as in example one.

The preparation method of the quality control product is the same as that of the first embodiment.

The dilution was PBS buffer containing 1% BSA, pH7.4, and concentration 0.01 mol/L; the washing liquid is PBS buffer solution containing 0.1% Tween-20 and having pH of 7.0 and 0.01 mol/L; the first luminescent substrate is a solution containing 0.1mol/L nitric acid and 0.1% hydrogen peroxide, and the second luminescent substrate is a solution containing 2% Tween-20 and 0.25mol/L NaOH.

s1, taking a plurality of reaction tubes, and sequentially adding 20 mu L of serum or calibrator to be detected, 100 mu L of diluent and 20 mu L of magnetic suspension for coating antigen proteins of porcine reproductive and respiratory syndrome;

s2, reacting for 15 minutes at 35 ℃;

s3, carrying out magnet adsorption, sucking out supernatant, adding 200 mu L of cleaning liquid into each reaction tube, repeatedly cleaning for 3 times, and discarding the cleaning liquid;

s4, adding 150 mu L of acridinium ester marked goat anti-pig IgG antibody solution into the S3;

s5, reacting for 15 minutes at 35 ℃;

s6, repeating the cleaning step in the step S3;

s7, sequentially adding 100 mu L of a first luminescent substrate and 100 mu L of a second luminescent substrate into S6;

s8, detecting a luminescence value by using a chemiluminescent instrument, drawing a calibration curve, and calculating the concentration of the antibody in the serum to be detected according to the calibration curve.

Table 9 shows luminescence values corresponding to different calibrator concentrations, and FIG. 4 shows calibration curves.

1. Sensitivity experiment

The kit and ELISA kit of the example were used to simultaneously detect positive serum of porcine reproductive and respiratory syndrome antibodies at different dilution factors, wherein the kit repeatedly detects 10 blood samples each, and the coefficient of variation was calculated, and the results are shown in Table 10. The results prove that the kit of the embodiment has accurate quantification, and the sensitivity is less than 1.51U/mL and is superior to ELISA kits.

Table 10 shows the sensitivity of the kit of this example compared with ELISA kit

2. Repeatability experiments

3 cases of pig breeding and respiratory syndrome antibody positive serum are taken, detected by the kit of the embodiment, 2 batches of serum are detected every day, 2 tests are respectively carried out on 3 batches of serum, and the two batches of serum are continuously detected for 5 days at least at intervals of 2 hours. Each serum was subjected to 20 pieces of measurement data, and the coefficient of variation in concentration was calculated, and the results are shown in Table 11. The results demonstrate that 3 serum test results are good in reproducibility.

TABLE 11

3. Compliance rate experiment

The test kit and ELISA test kit of this example detect multiple pig serum at the same time, and the results are shown in Table 12. The positive compliance rate of the kit and the ELISA kit of the embodiment is 94.6%, the negative compliance rate is 94.6%, and the overall compliance rate is 94.6%.

Table 12

After immunization with porcine reproductive and respiratory syndrome vaccine, 4 animals were randomly sampled, blood samples were taken 1, 7, 14, 21 days after immunization to detect porcine reproductive and respiratory syndrome antibodies, and non-immunized pigs were used as controls, with the results shown in table 13. The results show that the porcine reproductive and respiratory syndrome virus antibodies change from negative to positive and the concentration gradually increases 14 days and 21 days after immunization; the antibody content of the non-immune pigs is not obviously changed; the detection results of the two reagents are consistent.

TABLE 13

5. Specificity experiments

TABLE 14

Related viruses	CSFV	FMD-O	PRV	PCV2	BVDV
						Concentration (U/mL)	5.6	5.62	5.47	4.98	4.55

Example III

A kit for detecting antibodies of porcine reproductive and respiratory syndrome comprises a magnetic suspension for coating antigen proteins of porcine reproductive and respiratory syndrome, a horseradish peroxidase-labeled antigen solution of porcine reproductive and respiratory syndrome, a diluent, a calibrator, a quality control product, a cleaning solution, a first luminescent substrate and a second luminescent substrate.

(1) 1mL of a solution containing magnetic particles with the concentration of 10mg/mL is taken, washed 2 times with 0.01mol/L of Phosphate Buffer Solution (PBS) with the pH of 7.4, and finally suspended in 1mL of PBS buffer solution with the pH of 0.01mol/L of 7.4;

(2) 0.1mL of a 25% (v/v) glutaraldehyde solution was added and reacted at 37℃for 2 hours with shaking;

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

(4) Adding 30 mug of purified porcine reproductive and respiratory syndrome antigen recombinant antigen, and carrying out shaking reaction for 20 hours at 37 ℃;

(5) Adding 0.5mL 10% Bovine Serum Albumin (BSA) aqueous solution, mixing uniformly by vortex, and carrying out oscillation reaction for 2 hours at 37 ℃;

(6) Washed 3 times with 0.01mol/L PBS solution (containing 0.1% Tween-20) pH7.4, finally suspended in 0.01mol/L PBS solution (containing 1% BSA) pH7.4, and 0.1% ProClin 300 was added.

The magnetic particles are magnetic particles containing amino groups.

Preparation of horseradish peroxidase-labeled porcine reproductive and respiratory syndrome antigen:

(1) Taking 1mg of horseradish peroxidase, and diluting to 10mg/mL with 0.05mol/L CB buffer with pH of 9.5;

(2) Weigh NaIO ₄ And dissolving with 0.05mol/L CB buffer with pH of 9.5 to make NaIO ₄ Is 12.5mg/mL;

(3) Taking NaIO in (2) ₄ 100 mu L of the solution is added into the (1), and is mixed evenly by shakingLight-shielding reaction is carried out for 1 hour at 8 ℃;

(5) Adding 1mL of the ethylene glycol solution in the step (4) into the step (3), and carrying out light-shielding reaction for 1 hour at the temperature of 2 ℃;

(6) Adding 1mg of antigen of porcine reproductive and respiratory syndrome into the step (5), uniformly mixing, and dialyzing for 22 hours at 8 ℃ in a dark place by using 0.05mol/L CB buffer with the pH of 9.5;

(7) Weigh NaIO ₄ Dissolving in pure water to prepare 2mg/mL NaBH ₄ A solution;

(8) Taking NaBH of (7) ₄ 10 mu L of the solution is added into the step (6) and is reacted for 2 hours at 8 ℃ in a dark place;

(9) Purifying by molecular sieve;

(10) The antigen solution purified in (9) was diluted with MOPS buffer containing 1% BSA at pH 7.0.05 mol/L for use.

The preparation method of the calibrator was the same as in example one.

The dilution was PBS buffer containing 1% BSA, pH 7.4, and concentration 0.01 mol/L; the washing solution is PBS buffer solution containing 0.1% Tween-20 and 0.01mol/L pH 7.0; the first luminescent substrate is a solution of 0.5g/L luminol and 0.1g/L p-iodophenol, and the second luminescent substrate is a solution of 0.625g/L carbamide peroxide.

s1, taking a plurality of reaction tubes, and sequentially adding 100 mu L of serum or calibrator to be detected, 100 mu L of diluent and 50 mu L of magnetic suspension for coating antigen proteins of porcine reproductive and respiratory syndrome;

s2, reacting for 20 minutes at 39 ℃;

s3, carrying out magnet adsorption, sucking out supernatant, adding 500 mu L of cleaning liquid into each reaction tube, repeatedly cleaning for 3 times, and discarding the cleaning liquid;

s4, adding 200 mu L of horseradish peroxidase-labeled antigen solution of porcine reproductive and respiratory syndrome into the S3;

S5, reacting for 20 minutes at 39 ℃;

s6, repeating the cleaning step in the step S3;

s7, sequentially adding 50 mu L of a first luminescent substrate and 50 mu L of a second luminescent substrate into S6;

s8, reacting for 0.5 minutes at 35 ℃;

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

1. Sensitivity experiment

The kit and ELISA kit of the example were used to simultaneously detect positive serum of porcine reproductive and respiratory syndrome antibodies at different dilution factors, 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 result proves that the kit has accurate quantification, and the sensitivity is less than 0.80U/mL and is superior to ELISA kit.

Table 16

2. Repeatability experiments

3 cases of pig breeding and respiratory syndrome antibody positive serum are taken, detected by the kit of the embodiment, 2 batches of serum are detected every day, 2 tests are respectively carried out on 3 batches of serum, and the two batches of serum are continuously detected for 5 days at least at intervals of 2 hours. Each serum was subjected to 20 pieces of measurement data, and the coefficient of variation in concentration was calculated, and the results are shown in Table 17. The results demonstrate that 3 serum test results are good in reproducibility.

TABLE 17

3. Compliance rate experiment

The test results of the test kit and ELISA test kit of this example were shown in Table 18. The positive compliance rate of the kit and the ELISA kit of the embodiment is 94.2%, the negative compliance rate is 93.4%, and the overall compliance rate is 93.9%.

TABLE 18

4. The kit of the embodiment is used for pig reproduction and respiratory syndrome virus vaccine immunity monitoring

After immunization with porcine reproductive and respiratory syndrome virus vaccine, 4 animals were randomly extracted, blood samples were taken 1, 7, 14, 21 days after immunization to detect porcine reproductive and respiratory syndrome virus antibodies, and non-immunized pigs were used as controls. The results show that the porcine reproductive and respiratory syndrome virus antibodies change from negative to positive and the concentration gradually increases 14 days and 21 days after immunization; the antibody content of the non-immune pigs is not obviously changed; the detection results of the two reagents are consistent.

TABLE 19

5. Specificity experiments

Table 20

Related viruses	CSFV	FMD-O	PRV	PCV2	BVDV
						Concentration (U/mL)	2.26	2.22	2.25	1.71	1.53

Example IV

A kit for detecting an antibody of porcine reproductive and respiratory syndrome comprises a magnetic suspension for coating antigen proteins of porcine reproductive and respiratory syndrome, an alkaline phosphatase-labeled antibody of porcine reproductive and respiratory syndrome, a biotinylated antigen, a calibrator, a quality control material, a cleaning solution and a luminescent substrate.

(1) 1mL of a solution containing magnetic particles at a concentration of 10mg/mL was washed 2 times with 0.1mol/L of 1-morpholinoethanesulfonic acid (MES) buffer at pH 5.0, and finally suspended in 1mL of 0.1mol/L MES buffer at pH 5.0;

(2) Adding 50-500 mug of streptavidin;

(3) EDC is weighed and dissolved by using 0.1mol/L MES buffer solution with pH of 5.0, so that the concentration of EDC is 10mg/mL;

(5) Magnet adsorption, removing supernatant, washing with 0.01mol/L PBS solution (containing 0.1% Tween-20) with pH 7.4 for 3 times, suspending in 0.01mol/L PBS solution (containing 1% BSA) with pH 7.4, and adding 0.1% ProClin ^TM 300。

The magnetic particles are magnetic particles containing carboxyl groups.

(1) 1mg of alkaline phosphatase is taken and diluted to 10mg/mL with 0.05mol/L of CB buffer with pH of 9.5;

(2) Weigh NaIO ₄ And dissolved with 0.05mol/L CB buffer with pH of 9.5 to make NaIO4 concentration be 12.5mg/mL;

(3) Taking NaIO in (2) ₄ Adding 100 mu L of the solution into the step (1), shaking and uniformly mixing, and carrying out light-shielding reaction for 1 hour at 8 ℃;

(5) Adding 1mL of the ethylene glycol solution in the step (4) into the step (3), and carrying out light-shielding reaction for 1 hour at the temperature of 6 ℃;

(6) Adding 1mg of the monoclonal antibody of the foot-and-mouth disease virus O into the step (5), uniformly mixing, and dialyzing for 24 hours at 2 ℃ in a dark place by using 0.05mol/L CB buffer with the pH of 9.5;

(7) Weighing NaBH ₄ Dissolving in pure water to prepare 2mg/mL NaBH ₄ A solution;

(9) Purifying by molecular sieve;

(10) The antibody solution purified in (9) was diluted with MOPS buffer containing 1% BSA and having a pH of 7.0.05 mol/L for use.

Preparation of biotin antigen:

(1) 1mg of porcine reproductive and respiratory syndrome virus antigen is taken and dialyzed overnight at 8 ℃ with 0.01mol/L PBS buffer with pH of 7.4;

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

(3) Adding 20 mu L of the biotin solution in the step (2) into the step (1), and reacting for 1 hour at normal temperature;

(4) Adding 100 mu L of 0.1g/mL lysine solution into the solution (3), and reacting for 1 hour at normal temperature;

(5) The solution of (4) was dialyzed against PBS buffer having a pH of 7.4 at 0.01mol/L at 5℃for 24 hours.

(6) The solution in (5) was diluted with MOPS buffer containing 1% BSA at pH 7.00.05mol/L for use.

The preparation method of the calibrator was the same as in example one.

The cleaning solution is Tris buffer solution containing 0.1% Tween-20 and having the concentration of 0.05mol/L and the pH of 8.0; the luminescent substrate is a solution based on adamantane and derivatives thereof. The luminescent substrate in this example is Lumi-Phos 530, available from Lumigen under the name: p-5000.

s1, taking a plurality of reaction tubes, and sequentially adding 20 mu L of serum or calibrator to be detected, 100 mu L of biotinylated antigen and 25 mu L of magnetic suspension coated with porcine reproductive and respiratory syndrome antigen protein;

s2, reacting for 10 minutes at 37 ℃;

s4, adding 200 mu L of alkaline phosphatase-labeled porcine reproductive and respiratory syndrome antibody solution into the S3;

S5, reacting for 10 minutes at 37 ℃;

s6, repeating the cleaning step in the step S3;

s7, adding 100 mu L of luminous substrate into S6;

s8, reacting for 10 minutes at 39 ℃;

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

Table 21

1. Sensitivity experiment

The kit and ELISA kit of this example were used to simultaneously detect pig breeding and respiratory syndrome antibody positive serum at different dilution factors, wherein the kit of this example performed 10 repeated tests on each blood sample, and the coefficient of variation was calculated, and the results are shown in Table 22. The result proves that the reagent has accurate quantification, and the sensitivity is less than 3.02U/mL and is superior to ELISA kit.

Table 22

2. Repeatability experiments

3 cases of pig breeding and respiratory syndrome antibody positive serum are taken, detected by the kit of the embodiment, 2 batches of serum are detected every day, 2 tests are respectively carried out on 3 batches of serum, and the two batches of serum are continuously detected for 5 days at least at intervals of 2 hours. Each serum was subjected to 20 pieces of measurement data, and the coefficient of variation in concentration was calculated, and the results are shown in Table 23. The results demonstrate that 3 serum test results are good in reproducibility.

Table 23

3. Compliance rate experiment

The test kit and ELISA test kit of this example detect multiple pig serum at the same time, and the results are shown in Table 24. The positive compliance rate of the kit and the ELISA kit of the embodiment is 92.3%, the negative compliance rate is 92.7%, and the overall compliance rate is 92.4%.

Table 24

Table 25

5. Specificity experiments

The kit of this example was used to detect various relevant viral antibody strong positive sera including Classical Swine Fever Virus (CSFV), foot-and-mouth disease virus type O (FMDV-O), porcine pseudorabies virus (PRV), porcine circovirus (PCV 2), bovine Viral Diarrhea Virus (BVDV). The detection results are all lower than 10U/mL, are all negative, and have no cross reaction.

Table 26

Related viruses	CSFV	FMD-O	PRV	PCV2	BVDV
						Concentration (U/mL)	2.96	3.48	2.12	2.79	2.19

The invention adopts the magnetic particles with specific particle size as the coating carrier, and adopts the specific ratio of the porcine reproductive and respiratory syndrome virus protein to the magnetic particles to carry out mixing and reaction, thus obtaining the porcine reproductive and respiratory syndrome virus antigen coupled magnetic particles with homogeneous coating and stable structure, saving the raw materials of the coating protein, having the advantages of full coated protein, wider detection range, higher sensitivity, very short reaction time (only 5-10 minutes), high flux, automation and repeatability.

The foregoing detailed description is given by way of example only, and is not intended to limit the scope of the claims to the exact form disclosed, as defined by the claims and their equivalents; any equivalent alterations or modifications made in accordance with the spirit of the disclosure fall within the scope of the disclosure.

Sequence listing

<110> Shanghai Seagakuai Biotech Co., ltd

<120> kit for detecting antibodies of porcine reproductive and respiratory syndrome and detection method thereof

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MGSSLDDFCY DSTAPQKVLL AFSITYTPVM IYALKVSRGR LLGLLHLLIF LNCTFTFGYM 60

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GFHPIAANDN HAFVVRRPGS TTVNGTLVPG LKSLVLGGRK AVKQGVVNLV KYAK 174

Claims

1. The kit for detecting the antibody of the porcine reproductive and respiratory syndrome is characterized by comprising a solution of antigen protein coupling or indirect coupling magnetic particles of the porcine reproductive and respiratory syndrome, a protein solution marked by a luminescent marker and a luminescent substrate;

The antigen protein of the porcine reproductive and respiratory syndrome is selected from one of the whole length, GP5 protein fragment, GP5 protein polypeptide, N protein and M protein of the porcine reproductive and respiratory syndrome virus GP5 protein, and the magnetic particles are Fe ₃ O ₄ As a core, particles with surfaces coated with polymer and introduced with carboxyl and tosyl groups;

the diameter of the magnetic particles is 1-3 mu m, and the diameter CV of the magnetic particles is less than 3%;

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 low-value quality control products and high-value quality control products of the antibodies of the porcine reproductive and respiratory syndrome, the quality control range of the low-value quality control products is 20-30U/mL, and the quality control range of the high-value quality control products is 100-150U/mL; the cleaning solution is a Tris buffer solution with the concentration of 0.05mol/LpH of 8.0 or a Phosphate (PBS) buffer solution with the concentration of 0.01mol/LpH of 7.0, and the Tris buffer solution and the PBS buffer solution respectively contain 0.1% of Tween-20;

the optimal dosage of the full length, fragments and polypeptides of the GP5 protein of the porcine reproductive and respiratory syndrome is 0.8nmol/10mg magnetic particles; the optimal amount of the total length of the N protein is 0.67nmol/10mg of magnetic particles; the most suitable amount of M protein fragment is 0.91nmol/10mg magnetic particles.

2. The kit for detecting antibodies to porcine reproductive and respiratory syndrome according to claim 1, wherein the method for preparing a solution of antigen protein-coupled magnetic particles for porcine reproductive and respiratory syndrome comprises the steps of:

3. The kit for detecting antibodies to porcine reproductive and respiratory syndrome according to claim 1, wherein the method for preparing a solution of magnetic particles indirectly coupled to antigen proteins of porcine reproductive and respiratory syndrome comprises the steps of:

1) Magnetic microparticle-bound avidin

s2, adding avidin;

2) Porcine reproductive and respiratory syndrome antigen protein binding biotin

S1, taking antigen protein of porcine reproductive and respiratory syndrome, and dialyzing;

s2, adding biotin for reaction;

s3, adding a sealing agent to react;

s3, dialyzing to remove unbound biotin to obtain porcine reproductive and respiratory syndrome antigen protein-biotin;

3) The magnetic particles-avidin are mixed with the porcine reproductive and respiratory syndrome antigen protein-biotin complex, and the magnetic particles and the porcine reproductive and respiratory syndrome antigen protein are connected through the binding force of the avidin and the biotin.

4. The kit for detecting antibodies to porcine reproductive and respiratory syndrome according to claim 1, wherein the method for preparing a protein solution labeled with a luminescent marker comprises the steps of:

s1, dialyzing a protein specifically combined with antigen protein or antibody of porcine reproductive and respiratory syndrome;

s2, adding a luminous marker, and reacting;

s3, adding a sealing agent to react;

s4, dialyzing to separate unbound luminescent marker.

5. The kit for detecting the porcine reproductive and respiratory syndrome antibody according to claim 1, wherein the luminescent marker is selected from any one of acridinium ester, alkaline phosphatase and peroxidase, and the protein marked by the luminescent marker is selected from one or more combinations of porcine reproductive and respiratory syndrome antigen, monoclonal antibody, polyclonal antibody, genetic engineering antibody, anti-porcine IgG antibody and anti-porcine IgM antibody.

6. The kit for detecting antibodies to porcine reproductive and respiratory syndrome according to claim 1, wherein the luminescent substrates and the luminescent markers are in one-to-one correspondence.