CN108303540B - Porcine pseudorabies gE antibody detection kit and detection method thereof - Google Patents
Porcine pseudorabies gE antibody detection kit and detection method thereof Download PDFInfo
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Abstract
The invention discloses a porcine pseudorabies gE antibody detection kit and a detection method thereof. The kit provided by the invention realizes qualitative detection of the porcine pseudorabies gE antibody, and has the effects of high sensitivity, wide detection range, rapidness, good repeatability, full-automatic operation and high-throughput detection.
Description
Technical Field
The invention relates to the technical field of pig pseudorabies gE antibody detection, in particular to a pig pseudorabies gE antibody detection kit and a detection method adopting the kit for detection.
Background
Porcine Pseudorabies (PR) is an acute infectious disease caused by Pseudorabies virus (Pseudorabies Virus, PRV). Adult pigs are often infected with stealth, pregnant sows feel aborted, stillbirth and mummy; pseudorabies virus congenital or early infection causes neurological symptoms and high mortality of the piglets; for nursery and fattening pigs, pseudorabies virus is an important pathogen of respiratory epidemic disease syndrome (PRDC), causing respiratory symptoms and growth retardation thereof. The disease is widely existed in China and is one of important infectious diseases. Once an animal is infected, the virus can be in a long-term latent infection state in vivo, can survive for years in vivo and even for life, and is difficult to clear. Under certain conditions, viruses may also be reactivated to expel the virus from transmission. Therefore, the decontamination and eradication of the disease is important.
In China, immunization is a main strategy for preventing and treating pseudorabies. The vast majority of porcine pseudorabies vaccines used in the current market are attenuated vaccines lacking the gE gene and TK gene. After vaccine immunization, antibodies against the gE protein cannot be detected in the animal body, and the wild strain contains the gE gene, so that whether the serum sample contains the gE antibodies can be detected to judge whether the animal body is infected by the wild strain.
The currently established and applied detection method of the porcine pseudorabies gE antibody comprises ELISA method, latex agglutination experiment, colloidal gold test strip and other methods. The ELISA method is the most common method for the porcine pseudorabies gE antibody, the OD value measured by ELISA is in the range of 0.1-3.5, the sensitivity is low, the reaction time is usually about 2 hours, and the reaction time is longer; the latex agglutination experiment is judged by whether the pseudorabies antigen and the pseudorabies antibody can generate agglutination, the sensitivity and the accuracy are not very high, and the application is less; the colloidal gold method can rapidly obtain results, but has low sensitivity, and limits the application range.
Disclosure of Invention
The invention aims to solve the problems of long detection time, low sensitivity, poor repeatability, difficult automation and the like in the existing veterinary detection technology, and provides a porcine pseudorabies gE antibody detection kit which can rapidly and sensitively detect the porcine pseudorabies gE antibody. Therefore, the invention also provides a detection method by using the kit, and the detection method has high sensitivity, 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 porcine pseudorabies gE antibody detection kit comprising a solution of porcine pseudorabies gE protein coupled or indirectly coupled magnetic particles, a protein solution 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 pseudorabies gE 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 pseudorabies gE 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 ratio of the solution of the magnetic particles to the porcine pseudorabies gE protein is 10mg:0.5-5.5nmol.
Preferably, the ratio of the solution of the magnetic particles to the porcine pseudorabies gE protein is 10mg:1-3nmol.
The preparation method of the solution of the porcine pseudorabies gE protein indirect coupling magnetic particles comprises the following steps:
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) Porcine pseudorabies gE protein-binding biotin
S1, taking porcine pseudorabies gE protein, and dialyzing and purifying;
s2, sequentially adding biotin and a blocking agent for reaction;
s3, dialyzing to remove unbound biotin, and obtaining the porcine pseudorabies gE protein-biotin.
3) The magnetic particles-avidin are mixed with the porcine pseudorabies gE protein-biotin complex, and the magnetic particles are connected with the porcine pseudorabies gE protein through the binding force of the avidin and the biotin.
Wherein the magnetic particle core is ferric oxide.
The preparation method of the protein solution marked by the luminescent marker comprises the following steps:
S1, taking a protein specifically combined with a porcine pseudorabies gE protein or a porcine pseudorabies gE antibody, 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 porcine pseudorabies gE protein is selected from one or a combination of a plurality of porcine pseudorabies gE protein full length, natural porcine pseudorabies gE protein fragment, recombinant expressed porcine pseudorabies gE protein full length, recombinant expressed porcine pseudorabies gE protein fragment, porcine pseudorabies gE protein polypeptide and porcine pseudorabies gE 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 mode of indirect coupling of the porcine pseudorabies gE protein with the magnetic particles is any one of the following modes: the porcine pseudorabies gE antibody is coupled with the magnetic particles, and the porcine pseudorabies gE protein is captured on the antibodies on the magnetic particles through incubation to form a porcine pseudorabies gE protein and magnetic particle combination; streptavidin is coated on the magnetic particles, biotin is coupled on the porcine pseudorabies gE protein, and the porcine pseudorabies gE protein 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 pseudorabies gE protein, and the porcine pseudorabies gE protein and the magnetic particles are combined through anti-FITC antibody-FITC interaction.
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 one or a combination of a plurality of porcine pseudorabies gE antigen, monoclonal antibody, polyclonal antibody, genetic engineering antibody, anti-porcine IgG antibody and anti-porcine IgM antibody.
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 blocking agent, monoclonal antibodies, polyclonal antibodies and a reaction tube.
Wherein the quality control products are a pig pseudorabies gE antibody quality control product 1 and a quality control product 2; the cleaning solution comprises the following components: 0.05mol/L of Tris buffer with pH of 8.0 or 0.01mol/L of phosphate buffer with pH of 7.0, wherein each of the Tris buffer and the PBS buffer contains 0.1% of Tween-20.
Wherein, the number of the calibrator is 2, and the calibrator is a negative and positive serum diluent of the porcine pseudorabies gE antibody respectively.
In a second aspect of the invention, there is provided a method of detecting a porcine pseudorabies gE antibody, using the above detection kit, comprising the steps of:
s1, sequentially adding 10-100 mu L of a sample to be detected or a calibrator, and a solution of porcine pseudorabies gE protein coupling or indirect coupling magnetic particles 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.
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 the coating carrier, the magnetic particles are spheres, are magnetic, have the characteristic of large surface area, and can coat more porcine pseudorabies gE 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 of the porcine pseudorabies gE antibody is the surface of an ELISA plate, and the coated or contained protein is limited. Therefore, compared with ELISA 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 color development liquid and the luminous 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 current detection method of the porcine pseudorabies gE antibody 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, latex agglutination experiments and colloidal gold are difficult to realize full-automatic operation, and have poor repeatability.
4. The invention adopts a highly uniform liquid phase reaction, and combines the accuracy of the control test of the calibrator and the quality control product, so that the repeatability is greatly improved, and the comparability of detection results among different tests is improved. The existing detection method of the porcine pseudorabies gE antibody is limited by the principle and operation, and the detection results of different tests are difficult to repeat.
Drawings
FIG. 1 is a graph showing the amount of porcine pseudorabies gE protein used in the present invention with the carboxyl magnetic particles;
FIG. 2 is a graph of the amount of porcine pseudorabies gE protein used with the tosyl magnetic particles of the present invention.
Detailed Description
The whole length of the porcine pseudorabies gE protein contains 579 amino acids, has a molecular weight of 69kD, and has a protein sequence shown in SEQ ID NO. 1. The porcine pseudorabies gE 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 fragment sequence of the porcine pseudorabies gE protein can be included as shown in SEQ ID NO. 2, which contains 324 amino acids and has a molecular weight of 38kD; or the polypeptide sequence of the porcine pseudorabies gE protein is shown as SEQ ID NO. 3, and the porcine pseudorabies gE protein contains 46 amino acids and has the molecular weight of 5.5kD.
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 pseudorabies gE protein 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 pseudorabies gE protein, and the porcine pseudorabies gE protein 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 pseudorabies gE protein, and the porcine pseudorabies gE protein and the magnetic particles are combined through anti-FITC antibody-FITC interaction.
The porcine pseudorabies gE antibody detection kit comprises a magnetic suspension for coating porcine pseudorabies gE protein, a protein solution marked by a luminescent marker and a luminescent substrate.
The detection method of the porcine pseudorabies gE antibody 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 magnetic suspension coated with porcine pseudorabies gE protein 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.
Taking carboxyl magnetic particles and tosyl magnetic particles as examples, the best applicable amount experiment of the magnetic particles and the porcine pseudorabies gE protein is carried out.
1. Carboxyl magnetic particle and porcine pseudorabies gE protein optimal application amount experiment
3 porcine pseudorabies gE proteins are adopted, wherein the total length of the porcine pseudorabies gE proteins is added in the amounts of 10, 20, 50, 100, 200 and 500 mug, and the corresponding substances are added in the amounts of 0.14, 0.29, 0.72, 1.45, 2.90 and 7.25nmol; the addition amount of the porcine pseudorabies gE protein fragment is 10, 20, 50, 100, 200 and 500 mug, and the corresponding amount of substances is 0.26, 0.53, 1.32, 2.63, 5.26 and 13.16nmol; the addition amount of the porcine pseudorabies gE protein polypeptide is 1, 2, 5, 10, 20 and 50 mug, and the corresponding amount of substances is 0.18, 0.36, 0.91, 1.82, 3.64 and 9.09nmol; the amount of carboxyl magnetic particles added was 10mg.
The porcine pseudorabies gE protein and the carboxyl magnetic particles are respectively used for preparing magnetic suspension liquid for coating the porcine pseudorabies gE protein, a kit containing the magnetic suspension liquid is respectively used for detecting the porcine pseudorabies gE antibody positive serum, the results are shown in table 1, and fig. 1 is a curve of the coating protein dosage corresponding to the luminescence value.
The result shows that when the protein dosage is below 0.5nmol/10mg magnetic beads, the luminescence value rises rapidly with the increase of the protein dosage; the luminescence value increases slowly above 0.5nmol/10mg magnetic beads, indicating that the magnetic bead binding protein is near saturation; when the amount of the protein exceeds 5nnom/10mg of the magnetic beads, the luminescence value is reduced along with the increase of the amount of the protein, which indicates that the protein starts to have more self-crosslinking.
Therefore, for carboxyl magnetic particles, the optimal amount of porcine pseudorabies gE protein is 0.5-5nmol/10mg of magnetic particles; although the three proteins (full length, fragment, polypeptide) have large differences in amino acid number and molecular weight, the optimal coating protein amounts are not very different, calculated on the amount of the substance.
TABLE 1
2. Tosyl magnetic particles and pig pseudorabies gE protein optimal application amount experiment
3 porcine pseudorabies gE proteins are adopted, wherein the total length of the porcine pseudorabies gE proteins is added in the amounts of 10, 20, 50, 100, 200 and 500 mug, and the corresponding substances are added in the amounts of 0.14, 0.29, 0.72, 1.45, 2.90 and 7.25nmol; the addition amount of the porcine pseudorabies gE protein fragment is 10, 20, 50, 100, 200 and 500 mug, and the corresponding amount of substances is 0.26, 0.53, 1.32, 2.63, 5.26 and 13.16nmol; the addition amount of the porcine pseudorabies gE protein polypeptide is 1, 2, 5, 10, 20 and 50 mug, and the corresponding amount of substances is 0.18, 0.36, 0.91, 1.82, 3.64 and 9.09nmol; the amount of the tosyl magnetic particles added was 10mg.
The porcine pseudorabies gE protein and the carboxyl magnetic particles are respectively used for preparing magnetic suspension liquid for coating the porcine pseudorabies gE protein, a kit containing the magnetic suspension liquid is respectively used for detecting the porcine pseudorabies gE antibody positive serum, the results are shown in table 2, and fig. 2 is a curve of the coating protein dosage corresponding to the luminescence value.
The result shows that when the protein dosage is below 0.5nmol/10mg magnetic beads, the luminescence value rises rapidly with the increase of the protein dosage; the luminescence value increases slowly above 0.5nmol/10mg magnetic beads, indicating that the magnetic bead binding protein is near saturation; when the protein dosage exceeds 5nnom/10mg of magnetic beads, the luminescence value is reduced along with the increase of the protein dosage, which indicates that the protein begins to have more self-crosslinking, and the protein dosage is not more than 5nmol/10mg of magnetic beads in consideration of cost factors.
Therefore, for the tosyl magnetic particles, the optimal amount of porcine pseudorabies gE protein is 0.5-5nmol/10mg magnetic particles; although the three proteins (full length, fragment, polypeptide) have large differences in amino acid number and molecular weight, the optimal coating protein amounts are not very different, calculated on the amount of the substance.
TABLE 2
Example 1
A kit for detecting a porcine pseudorabies gE antibody comprises a magnetic suspension liquid coated with porcine pseudorabies gE protein, an alkaline phosphatase-marked porcine pseudorabies gE antibody solution, a diluent, a calibrator, a quality control product, a cleaning liquid and a luminous substrate.
Preparation of a magnetic suspension coated with porcine pseudorabies gE protein:
(1) 1mL of magnetic particles (available from JSR corporation, japan, cat# Magnospere) TM MS 300/Cabox) at a concentration of 10mg/mL, washed 2 times with 0.1 mol/L2-morpholinoethanesulfonic acid (MES) buffer pH 5.0, and finally suspended in 1mL 0.1mol/L MES buffer pH 5.0;
(2) Purified porcine pseudorabies gE protein (purchased from Yiminox biotechnology Co., hangzhou) was added at 80 μg;
(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 pseudorabies gE 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 porcine pseudorabies gE monoclonal antibody 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 pseudorabies gE 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 calibration material is divided into a calibration material 1 and a calibration material 2. Calibrator 1 is a porcine pseudorabies gE antibody negative serum diluent and calibrator 2 is a porcine pseudorabies gE antibody positive serum diluent.
Preparation of a calibrator:
(1) Heat-inactivating the pig pseudorabies gE antibody negative serum and the pig pseudorabies gE antibody positive serum at 60 ℃ for 1 hour;
(2) Filtering the inactivated serum obtained in step (1) through a microfiltration membrane with a diameter of 0.2 μm, and adding 0.1% ProClin TM 300；
(3) The serum in (2) was subjected to calibration and diluted with a dilution containing 1% BSA.
The quality control product is marked pig pseudorabies gE antibody positive pig serum. The method is divided into a quality control product 1 and a quality control product 2. The S/CO (ratio of the luminous value to the critical value) of the quality control product 1 is between 1.2 and 1.8, and the S/CO of the quality control product 2 is between 0.6 and 0.9. 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:
(1) Selecting more than 10 parts of pig pseudorabies gE antibody positive serum, heat-inactivating at 60 ℃ for 1 hour, mixing, filtering with a microfiltration membrane of 0.2 μm, adding 0.1% ProClin TM 300。
(2) And (3) regulating the mixed positive serum to a proper concentration, so that the S/CO of the quality control product 1 is between 1.2 and 1.8, and the S/CO of the quality control product 2 is between 0.6 and 0.9.
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 a porcine pseudorabies gE antibody 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 coated with porcine pseudorabies gE protein;
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 pseudorabies gE 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 the luminescence value by using a chemiluminescent instrument.
And calculating the ratio (S/CO) of the sample luminescence value to the critical value.
Critical value = calibrator 1 average luminescence value x 0.8+ calibrator 2 average luminescence value x 0.2
And the sample S/CO >1.00 is judged to be negative to the pseudorabies virus gE antibody, and no further detection is needed.
And the sample S/CO is less than or equal to 1.00, and the sample S/CO is judged to be positive to the pseudorabies virus gE antibody.
All the samples with positive primary detection are retested for 2 times by using the kit, and if the results of 2 times are negative, the samples are judged to be negative; if at least one of the two results is positive, the sample is judged to be positive in retest.
2. Sensitivity experiment
Porcine pseudorabies gE antibody detection kits (enzyme-linked immunosorbent assay, hereinafter abbreviated as ELISA kits) produced by foreign well-known manufacturers and the kit in the embodiment are used for simultaneously detecting porcine pseudorabies gE antibody positive serum with different dilution factors, wherein the kit in the embodiment carries out 10 repeated detections on each blood sample, and a variation coefficient (CV% = standard deviation/arithmetic average of 10 test results) is calculated. The sensitivity was superior to the ELISA kit in this example with the maximum dilution of CV% < 20%.
The ELISA kit is judged to be negative when the S/N is more than or equal to 0.70, suspicious when the S/N is less than or equal to 0.60, and positive when the S/N is less than or equal to 0.60.
Table 3 shows sensitivity comparison of the kit of example I and ELISA kit
3. Repeatability experiments
2 cases of pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody positive serum are taken, the pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody negative serum are detected by the kit, 2 cases of pig pseudorabies virus gE antibody positive serum and 3 cases of pig pseudorabies virus gE antibody negative serum are detected every day, 2 cases of pig pseudorabies virus gE antibody positive serum are detected every day, and the test is carried out for 5 days continuously every day at least 2 hours. Each serum was subjected to 20 test data, and the coefficient of variation was calculated, and the results are shown in Table 4. The results demonstrate that 3 serum test results are good in reproducibility. TABLE 4 Table 4
4. 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 5. The result shows that the positive coincidence rate of the reagent and ELISA kit is 96.2%, the negative coincidence rate is 96.7%, and the total coincidence rate is 95.9%.
TABLE 5
5. Specificity experiments
The kit of this example was used to detect various relevant virus antibody strong positive sera including Classical Swine Fever Virus (CSFV), foot-and-mouth disease virus type O (FMDV-O), porcine pseudorabies gE deficiency virus (PRV, gE-), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV 2), bovine Viral Diarrhea Virus (BVDV). The detection results S/CO are all greater than 1.0, are all negative, and have no cross reaction.
TABLE 6
| Related viruses | CSFV | FMDV-O | PRV，gE- | PRRSV | PCV2 | BVDV |
| S/CO | 1.61 | 1.48 | 1.72 | 1.73 | 1.97 | 1.75 |
Example two
A kit for detecting a porcine pseudorabies gE antibody comprises a magnetic suspension for coating a porcine pseudorabies gE protein, an acridinium ester-marked goat anti-porcine 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.
Preparation of a magnetic suspension coated with porcine pseudorabies gE protein:
(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 90 mug of purified porcine pseudorabies gE antigen, and uniformly 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, pH 7.4, 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.
A detection method of a porcine pseudorabies gE antibody comprises the following steps:
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 coated with porcine pseudorabies gE protein;
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.
And calculating the ratio (S/CO) of the sample luminescence value to the critical value.
Critical value = calibrator 1 average luminescence value x 0.1+ calibrator 2 average luminescence value x 0.9
The sample S/CO <1.00 is judged to be negative to the pseudorabies virus gE antibody, and no further detection is needed.
And the sample S/CO is more than or equal to 1.00, and the sample S/CO is judged to be positive to the pseudorabies virus gE antibody.
All the samples which are initially detected to be positive are retested for 2 times by using the reagent, and if the results of the 2 times are negative, the samples are judged to be negative; if at least one of the two results is positive, the sample is judged to be positive in retest.
2. Sensitivity experiment
The pig pseudorabies gE antibody positive serum with different dilution factors was simultaneously detected by using the kit and ELISA kit of the embodiment, wherein 10 repeated detections are carried out on each blood sample by the reagent, and the variation coefficient is calculated, and the result is shown in Table 7. The results demonstrate that the sensitivity of this example is better than the ELISA kit.
The ELISA kit is judged to be negative when the S/N is more than or equal to 0.70, suspicious when the S/N is less than or equal to 0.60, and positive when the S/N is less than or equal to 0.60.
Table 7 shows the sensitivity of the kit of this example compared with ELISA kit
3. Repeatability experiments
2 cases of pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody positive serum are taken, and detected by the kit of the embodiment, 2 cases of pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody negative serum are detected every day, and the two cases of pig pseudorabies virus gE antibody positive serum are detected every day for 5 days at least 2 hours. Each serum was subjected to 20 test data, and the coefficient of variation was calculated, and the results are shown in Table 8. The results demonstrate that 3 serum test results are good in reproducibility.
TABLE 8
4. Compliance rate experiment
The results of the simultaneous detection of multiple pig sera by the kit and ELISA kit of this example are shown in Table 9. The positive compliance rate of the kit and the ELISA kit of the embodiment is 96.2%, the negative compliance rate is 94.1%, and the overall compliance rate is 94.0%.
TABLE 9
5. Specificity experiments
The kit of this example was used to detect various relevant virus antibody strong positive sera including Classical Swine Fever Virus (CSFV), foot-and-mouth disease virus type O (FMDV-O), porcine pseudorabies gE deficiency virus (PRV, gE-), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV 2), bovine Viral Diarrhea Virus (BVDV). The detection results S/CO are all lower than 1.0, are all negative, and have no cross reaction.
Table 10
| Related viruses | CSFV | FMDV-O | PRV，gE- | PRRSV | PCV2 | BVDV |
| S/CO | 0.12 | 0.19 | 0.17 | 0.10 | 0.20 | 0.13 |
Example III
A kit for detecting a porcine pseudorabies gE antibody comprises a magnetic suspension liquid coated with porcine pseudorabies gE protein, a horseradish peroxidase-labeled porcine pseudorabies gE antigen solution, a diluent, a calibrator, a quality control product, a cleaning liquid, a first luminescent substrate and a second luminescent substrate.
Preparation of a magnetic suspension coated with porcine pseudorabies gE protein:
(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 pseudorabies gE 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 pseudorabies gE 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 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 ℃;
(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 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 porcine pseudorabies gE antigen 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) Weighing 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 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 preparation method of the quality control product is the same as that of the first embodiment.
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.
A detection method of a porcine pseudorabies gE antibody comprises the following steps:
s1, taking a plurality of reaction tubes, and sequentially adding 50 mu L of serum or calibrator to be detected, 100 mu L of diluent and 50 mu L of magnetic suspension coated with porcine pseudorabies gE protein;
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 porcine pseudorabies gE antigen solution 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 10 minutes at 35 ℃;
s9, detecting the luminescence value by using a chemiluminescent instrument.
And calculating the ratio (S/CO) of the sample luminescence value to the critical value.
Critical value = calibrator 1 average luminescence value x 0.1+ calibrator 2 average luminescence value x 0.9
The sample S/CO <1.00 is judged to be negative to the pseudorabies virus gE antibody, and no further detection is needed.
And the sample S/CO is more than or equal to 1.00, and the sample S/CO is judged to be positive to the pseudorabies virus gE antibody.
All the samples which are initially detected to be positive are retested for 2 times by using the reagent, and if the results of the 2 times are negative, the samples are judged to be negative; if at least one of the two results is positive, the sample is judged to be positive in retest.
1. Sensitivity experiment
The pig pseudorabies gE antibody positive serum with different dilution factors was simultaneously detected by using the kit and ELISA kit of the embodiment, wherein 10 repeated detections are carried out on each blood sample by the reagent, and the variation coefficient is calculated, and the results are shown in Table 11. The result proves that the sensitivity of the reagent is better than that of ELISA kit.
The ELISA kit is judged to be negative when the S/N is more than or equal to 0.70, suspicious when the S/N is less than or equal to 0.60, and positive when the S/N is less than or equal to 0.60.
TABLE 11
2. Repeatability experiments
2 cases of pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody positive serum are taken, and detected by the kit of the embodiment, 2 cases of pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody negative serum are detected every day, and the two cases of pig pseudorabies virus gE antibody positive serum are detected every day for 5 days at least 2 hours. Each serum was subjected to 20 test data, and the coefficient of variation was calculated, and the results are shown in Table 12. The results demonstrate that 3 serum test results are good in reproducibility.
Table 12
3. Compliance rate experiment
The test results of the test kit and ELISA test kit of this example were shown in Table 13. The positive compliance rate of the kit and the ELISA kit of the embodiment is 94.9%, the negative compliance rate is 95.1%, and the overall compliance rate is 94.4%.
TABLE 13
5. Specificity experiments
The kit of this example was used to detect various relevant virus antibody strong positive sera including Classical Swine Fever Virus (CSFV), foot and mouth disease virus type O (FMDV-O), porcine pseudorabies gE deficiency virus (PRV, gE-), porcine pseudorabies virus gD (PRV-gD), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV 2), bovine Viral Diarrhea Virus (BVDV). The detection results S/CO are all lower than 1.0, are all negative, and have no cross reaction.
TABLE 14
| Related viruses | CSFV | FMDV-O | PRV，gE- | PRRSV | PCV2 | BVDV |
| S/CO | 0.13 | 0.08 | 0.09 | 0.08 | 0.11 | 0.07 |
Example IV
A kit for detecting a porcine pseudorabies gE antibody comprises a magnetic suspension coated with porcine pseudorabies gE protein, an alkaline phosphatase-marked porcine pseudorabies gE antibody, a biotinylated antigen, a calibrator, a quality control product, a cleaning solution and a luminescent substrate.
Preparation of a magnetic suspension coated with porcine pseudorabies gE protein:
(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 350 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;
(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% to the solution
ProClin TM 300。
The magnetic particles are magnetic particles containing carboxyl groups.
Preparation of alkaline phosphatase-labeled porcine pseudorabies gE antibody solution:
(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 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 8 ℃;
(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 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 porcine pseudorabies virus gE monoclonal antibody into the step (5), uniformly mixing, and dialyzing for 24 hours at 8 ℃ 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;
(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 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 pseudorabies virus gE 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 2-8℃for 20-24 hours.
(6) The solution in (5) 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 wash solution was 0.05M Tris buffer pH8.0 containing 0.1% Tween-20; 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.
A detection method of a porcine pseudorabies gE antibody comprises the following steps:
s1, taking a plurality of reaction tubes, and sequentially adding 100 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 pseudorabies gE protein;
s2, reacting for 10 minutes at 37 ℃;
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 alkaline phosphatase-labeled porcine pseudorabies gE 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 0.5 minutes at 39 ℃;
s9, detecting the luminescence value by using a chemiluminescent instrument.
And calculating the ratio (S/CO) of the sample luminescence value to the critical value.
Critical value = calibrator 1 average luminescence value x 0.1+ calibrator 2 average luminescence value x 0.9
The sample S/CO <1.00 is judged to be negative to the pseudorabies virus gE antibody, and no further detection is needed.
And the sample S/CO is more than or equal to 1.00, and the sample S/CO is judged to be positive to the pseudorabies virus gE antibody.
All the samples which are initially detected to be positive are retested for 2 times by using the reagent, and if the results of the 2 times are negative, the samples are judged to be negative; if at least one of the two results is positive, the sample is judged to be positive in retest.
1. Sensitivity experiment
The pig pseudorabies gE antibody positive serum was simultaneously detected at different dilution factors by using the kit and ELISA kit of this example, wherein the kit of this example carries out 10 repeated detections for each blood sample, and the coefficient of variation was calculated, and the results are shown in Table 15. The result proves that the sensitivity of the reagent is better than that of ELISA kit.
The ELISA kit is judged to be negative when the S/N is more than or equal to 0.70, suspicious when the S/N is less than or equal to 0.60, and positive when the S/N is less than or equal to 0.60.
TABLE 15
2. Repeatability experiments
2 cases of pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody positive serum are taken, and detected by the kit of the embodiment, 2 cases of pig pseudorabies virus gE antibody positive serum and 1 case of pig pseudorabies virus gE antibody negative serum are detected every day, and the two cases of pig pseudorabies virus gE antibody positive serum are detected every day for 5 days at least 2 hours. Each serum was subjected to 20 test data, and the coefficient of variation was calculated, and the results are shown in Table 16. The results demonstrate that 3 serum test results are good in reproducibility.
Table 16
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 17. The positive compliance rate of the kit and the ELISA kit of the embodiment is 94.9%, the negative compliance rate is 94.6%, and the overall compliance rate is 94.0%.
TABLE 17
4. Specificity experiments
The kit of this example was used to detect various relevant virus antibody strong positive sera including Classical Swine Fever Virus (CSFV), foot and mouth disease virus type O (FMDV-O), porcine pseudorabies gE deficiency virus (PRV, gE-), porcine pseudorabies virus gD (PRV-gD), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), porcine circovirus type 2 (PCV 2), bovine Viral Diarrhea Virus (BVDV). The detection results S/CO are all lower than 1.0, are all negative, and have no cross reaction.
TABLE 18
| Related viruses | CSFV | FMDV-O | PRV，gE- | PRRSV | PCV2 | BVDV |
| S/CO | 0.14 | 0.24 | 0.14 | 0.23 | 0.15 | 0.21 |
The invention adopts magnetic particles with specific particle size as a coating carrier, and adopts the specific ratio of the porcine pseudorabies gE protein to the magnetic particles to carry out mixing and reaction, thus obtaining the porcine pseudorabies gE antibody coupled magnetic particles with homogeneous coating and stable structure, saving the raw materials of the coating protein, having the advantages of full coating 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> porcine pseudorabies gE antibody detection kit and detection method thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 579
<212> PRT
<213> artificial sequence
<400> 1
MRPFLLRAAQ LLALLALALS TEAPSLSAET TPGPVTEVPS PSAEVWDDLS TEADDDDLNG60
DLDGDDRRAG FGSALASLRE APPAHLVNVS EGANFTLDAR GDGAVLAGIW TFLPVRGCDA120
VAVTTVCFET ACHPDLVLGR ACVPEAPEMG IGDYLPPEVP RLRREPPIVT PERWSPHLSV180
LRATPNDTGL YTLHDASGPR AVFFVAVGDR PPAPADPVGP ARHEPRFHAL GFHSQLFSPG240
DTFDLMPRVV SDMGDSRENF TATLDWYYAR APPRCLLYYV YEPCIYHPRA PECLRPVDPA300
CSFTSPARAR LVARRAYASC SPLLGDRWLT ACPFDAFGEE VHTNATADES GLYVLVMTHN360
GHVATWDYTL VATAAEYVTV IKELTAPARA PGTPWGPGGG DDAIYVDGVT TPAPPARPWN420
PYGRTTPGRL FVLALGSFVM TCVVGGAVWL CVLCSRRRAA SRPFRVPTRA RTHMLSPVYT480
SLPTHEDYYD GDDDDDEEAG VIRRRPASPG GDSGYEGPYA SLDPEDEFSS DEDDGLYVRP540
EEAPRSGFDV WFRDPEKPEV TNGPNYGVTA NRLLMSRPA579
<210> 2
<211> 324
<212> PRT
<213> artificial sequence
<400> 2
PNDTGLYTLH DASGPRAVFF VAVGDRPPAP ADPVGPARHE PRFHALGFHS QLFSPGDTFD60
LMPRVVSDMG DSRENFTATL DWYYARAPPR CLLYYVYEPC IYHPRAPECL RPVDPACSFT120
SPARARLVAR RAYASCSPLL GDRWLTACPF DAFGEEVHTN ATADESGLYV LVMTHNGHVA180
TWDYTLVATA AEYVTVIKEL TAPARAPGTP WGPGGGDDAI YVDGVTTPAP PARPWNPYGR240
TTPGRLFVLA LGSFVMTCVV GGAVWLCVLC SRRRAASRPF RVPTRARTHM LSPVYTSLPT300
HEDYYDGDDD DDEEAGVIRR RPAS 324
<210> 3
<211> 46
<212> PRT
<213> artificial sequence
<400> 3
ADESGLYVLV MTHNGHVATW DYTLVATAAE YVTVIKELTA PARAPG46
Claims (1)
1. The kit for detecting the porcine pseudorabies gE antibody is characterized by comprising a magnetic suspension coated with porcine pseudorabies gE protein, an alkaline phosphatase-marked porcine pseudorabies gE antibody solution, a diluent, a calibrator, a quality control product, a cleaning solution and a luminous substrate;
preparation of a magnetic suspension coated with porcine pseudorabies gE protein:
(1) 1mL of a solution containing magnetic particles with the concentration of 10mg/mL is taken, washed 2 times with 0.1mol/L of 2-morpholinoethanesulfonic acid buffer with the pH of 5.0, and finally suspended in 1mL of MES buffer with the pH of 5.0 at 0.1 mol/L;
(2) Adding purified porcine pseudorabies gE protein with the total length of 80 mug;
(3) 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide 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 with pH of 7.4 for 3 times, suspending in 0.01mol/L PBS solution with pH of 7.4, and adding 0.1% ProClin TM 300；
The magnetic particles are magnetic particles containing carboxyl groups, and are purchased from JSR corporation, japan, product number: magnospere MS 300/Cabox;
preparation of alkaline phosphatase-labeled porcine pseudorabies gE antibody solution:
(i) 1mg of alkaline phosphatase is taken and diluted to 10mg/mL with 0.05mol/L of carbonate buffer with pH of 9.5;
(ii) Weighing sodium periodate and dissolving with 0.05mol/L CB buffer solution with pH of 9.5 to make NaIO 4 Is 12.5mg/mL;
(iii) Taking NaIO from (ii) 4 Adding 100 mu L of the solution into the (i), shaking and uniformly mixing, and carrying out light-shielding reaction for 1 hour at the temperature of 2 ℃;
(iv) 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;
(v) Adding 100 mu L of the ethylene glycol solution in the step (iv) into the step (iii), and carrying out light-shielding reaction at the temperature of 6 ℃ for 1 hour;
(vi) Adding 0.5mg of porcine pseudorabies gE monoclonal antibody into (v), 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;
(vii) Weighing sodium borohydride, dissolving in pure water, and preparing 2mg/mL NaBH 4 A solution;
(viii) Taking NaBH of (vii) 4 10 μl of the solution was added to (vi), and the reaction was carried out at 2deg.C for 2 hours in the absence of light;
(ix) Purifying and separating unbound alkaline phosphatase and porcine pseudorabies gE monoclonal antibody by molecular sieve;
(x) Diluting the purified antibody solution in (ix) with 3-morpholinopropanesulfonic acid buffer containing 1% BSA and having a pH of 7.00.05M to a concentration of 0.1-0.5 μg/mL;
the calibration material is divided into a calibration material 1 and a calibration material 2; the calibrator 1 is a porcine pseudorabies gE antibody negative serum diluent, and the calibrator 2 is a porcine pseudorabies gE antibody positive serum diluent;
the preparation of the calibrator comprises the following steps:
(a) Heat-inactivating the pig pseudorabies gE antibody negative serum and the pig pseudorabies gE antibody positive serum at 60 ℃ for 1 hour;
(b) Filtering the serum inactivated in (a) with a microfiltration membrane of 0.2 μm, adding 0.1% ProClin TM 300；
(c) Calibrating the serum in (b), and diluting with a dilution solution containing 1% BSA;
the quality control product is marked pig pseudorabies gE antibody positive pig serum; the method comprises a quality control product 1 and a quality control product 2, wherein the S/CO of the quality control product 1 is between 1.2 and 1.8, and the S/CO of the quality control product 2 is between 0.6 and 0.9;
the preparation of the quality control product comprises the following steps:
selecting more than 10 parts of pig pseudorabies gE antibody positive serum, heat-inactivating for 1 hour at 60 ℃, mixing, filtering by a microfiltration membrane of 0.2 mu m, adding 0.1% ProClin TM 300；
(II) regulating the mixed positive serum to a proper concentration, so that the S/CO of the quality control product 1 is between 1.2 and 1.8, and the S/CO of the quality control product 2 is between 0.6 and 0.9;
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 was Lumi-Phos 530, available from Lumigen, cat: p-5000.
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| CN201710019644.9A CN108303540B (en) | 2017-01-11 | 2017-01-11 | Porcine pseudorabies gE antibody detection kit and detection method thereof |
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| CN201710019644.9A CN108303540B (en) | 2017-01-11 | 2017-01-11 | Porcine pseudorabies gE antibody detection kit and detection method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109307772B (en) * | 2018-10-12 | 2021-12-24 | 华南农业大学 | Double fluorescent microsphere immunological detection method for antibodies of pseudorabies virus gE and gB IgG |
| CN112326971A (en) * | 2020-10-20 | 2021-02-05 | 成都海默云因医学检验实验室有限公司 | Novel method and kit for NMDAR antibody quantitative detection |
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| CN102608315A (en) * | 2012-02-21 | 2012-07-25 | 武汉科前动物生物制品有限责任公司 | Enzyme linked immunosorbent assay kit for porcine pseudorabies virus gE protein antibody and application thereof |
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| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
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| CN101452001A (en) * | 2007-12-06 | 2009-06-10 | 王颖 | Quantitative determination RBP4 kit by chemiluminescence magnetic enzymoimmune method |
| CN102608315A (en) * | 2012-02-21 | 2012-07-25 | 武汉科前动物生物制品有限责任公司 | Enzyme linked immunosorbent assay kit for porcine pseudorabies virus gE protein antibody and application thereof |
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