CN108303543B - Swine fever E2 protein antibody detection kit and detection method thereof - Google Patents
Swine fever E2 protein antibody detection kit and detection method thereof Download PDFInfo
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- CN108303543B CN108303543B CN201710019498.XA CN201710019498A CN108303543B CN 108303543 B CN108303543 B CN 108303543B CN 201710019498 A CN201710019498 A CN 201710019498A CN 108303543 B CN108303543 B CN 108303543B
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses a swine fever E2 protein antibody detection kit and a detection method thereof, wherein the kit adopts an antigen coated by magnetic particles, a protein marked by a luminescent marker and an antibody in a sample to be detected to perform specific reaction so as to determine the content of the antibody in the sample to be detected. The kit disclosed by the invention realizes the effects of quantitative detection of the swine fever E2 protein antibody, high sensitivity, wide detection range, rapidness, good repeatability, full-automatic operation and high-throughput detection.
Description
Technical Field
The invention relates to the technical field of swine fever E2 protein antibody detection, in particular to a swine fever E2 protein antibody detection kit and a detection method adopting the kit for detection.
Background
Classical swine fever (Classical Swine Fever, CSF), a highly lethal and contagious disease in pigs caused by swine fever virus (Classical swine fever virus, CSFV). Is one of the main infectious diseases closely concerned by the world grain and agriculture organizations and the governments of various countries. CSF is classified as one of the legally reported epidemic diseases according to 2005 edition of the "land animal health act" formulated by OIE, and is classified as "one kind of animal epidemic disease" in our country, which brings huge economic loss to the pig industry in our country.
The method adopts the comprehensive epidemic prevention measures of immunization, quarantine and elimination of pigs with toxicity, promotes the establishment of random animal epidemic disease areas, and is an important means for preventing and controlling swine fever in China. The establishment of a reasonable and effective immunization program through the monitoring of the antibody level is the guarantee of improving the immunity level of the population. For many years, due to the wide demands of scientific research and market, development of a swine fever antibody detection kit has been pursued. The methods established and applied at present are ELISA method, virus neutralization test method, immunofluorescence method, colloidal gold test strip and the like.
The swine fever E2 protein is a main neutral antigen protein of swine fever, and the antibody is a main target of monitoring of antibody level after vaccine immunization and detecting of antibody level after virus infection, and the antibody level is directly related to the immune or infection condition, so that the real requirement of quantitative detection of the swine fever E2 protein 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 a high-flux swine fever E2 protein antibody detection kit and instrument. The existing ELISA, colloidal gold and other methods are difficult to meet.
The detection method of the swine fever E2 protein antibody established and applied at present comprises ELISA method, virus neutralization test, immunofluorescence method, colloidal gold test strip and other methods. The ELISA method is the most commonly used method for the swine fever E2 protein antibody, the range of the OD value measured by ELISA is 0.1-3.5, the measured antibody range is limited to be narrower, only half quantification can be realized, the reaction time is usually about 2 hours, and the reaction time is longer; the virus neutralization test judges the antibody titer by carrying out antigen-antibody reaction on cells by using the swine fever E2 protein 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, a semi-quantitative ELISA method is mainly adopted for detecting the swine fever E2 antibody, and the quantitative requirement cannot be met, which is mainly caused by the lack of a swine fever E2 antibody standard substance and a quantitative standard and a detection method with no accurate repeatability. 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 swine fever E2 antibody detection market is occupied by imported ELISA qualitative detection reagents, and the detection results of products of various factories are quite different, so that the development of livestock breeding industry is not facilitated, and a detection method with quantitative standard, accuracy, reliability and good repeatability is needed to be established.
Disclosure of Invention
In order to solve the problems of long detection time, difficult quantification, poor repeatability, difficult automation and the like in the existing veterinary detection technology, the invention aims to provide a swine fever E2 protein antibody detection kit which can quantitatively detect the content of a swine fever E2 protein antibody. 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 swine fever E2 protein antibody detection kit comprising a solution of swine fever E2 protein coupled or indirectly coupled magnetic particles, a luminescent marker-labeled protein solution 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 swine fever E2 protein coupled magnetic particles comprises the following steps:
s1, taking a solution containing magnetic particles, cleaning the magnetic particles by using a buffer solution, and suspending the magnetic particles in the buffer solution;
s2, adding purified swine fever E2 protein;
s3, adding a cross-linking agent or a catalyst, and carrying out oscillation reaction;
s4, the magnetic particles are adsorbed by the magnet, washed and suspended in a solution containing the blocking agent.
Wherein, the ratio of the solution of the magnetic particles to the swine fever E2 protein is 10mg:0.64-4nmol.
The preparation method of the solution of the swine fever E2 protein indirectly coupled 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) Swine fever E2 protein binding biotin
S1, taking swine fever E2 protein, and dialyzing and purifying;
s2, sequentially adding biotin and a blocking agent for reaction;
s3, dialyzing to remove unbound biotin to obtain swine fever E2 protein-biotin;
3) The magnetic particles-avidin are mixed with the swine fever E2 protein-biotin complex, and the magnetic particles are connected with the swine fever E2 protein through the binding force of the avidin and the biotin.
The preparation method of the protein solution marked by the luminescent marker comprises the following steps:
s1, taking a protein specifically combined with a swine fever E2 protein or a swine fever virus 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 hog cholera E2 protein is selected from one or more of hog cholera virus E2 protein full length, natural E2 protein fragment, recombining expressed hog cholera E2 protein full length, recombining expressed E2 protein fragment, E2 protein polypeptide and E2 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 one or a combination of more of a swine fever virus antigen, a monoclonal antibody, a polyclonal antibody, a genetic engineering antibody, an anti-pig IgG antibody and an anti-pig 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 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 a swine fever E2 protein antibody 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 24-36U/mL, and the quality control range of the high-value quality control product is 80-120U/mL; the washing liquid is a Tris buffer solution with the pH value of 0.05mol/L and 8.0 or a phosphate buffer solution with the pH value of 0.01mol/L and 7.0, and the Tris buffer solution and the PBS buffer solution respectively contain 0.1% of Tween-20.
In a second aspect of the present invention, a method for detecting a swine fever E2 protein antibody is provided, and the detection kit comprises the following steps:
S1, sequentially adding 10-100 mu L of a sample to be detected or a calibrator, and a solution of swine fever E2 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, 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 the coating carrier, the magnetic particles are spheres, are magnetic, have the characteristic of large surface area and can coat more swine fever E2 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 swine fever E2 protein antibody, 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 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. In the existing method for detecting the swine fever E2 protein antibody, an ELISA chromogenic substrate 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 swine fever antibody detection method is limited by the principle and operation, and the test detection results of different batches 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 optimal amount of E2 protein of classical swine fever virus in the carboxyl magnetic particles of the present invention;
FIG. 2 is a graph showing the amount of E2 protein of classical swine fever virus in which the tosyl magnetic particles of the present invention are optimal;
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 classical swine fever virus E2 protein or fragment thereof which can be used in the present invention is not particularly limited and may include the full length of the native or recombinant classical swine fever virus E2 protein or fragment thereof. Preferably, the full-length sequence of the E2 protein of the swine fever virus can be shown as SEQ ID NO. 1, which contains 373 amino acids and has a molecular weight of 46.6KD; the sequence of the E2 protein fragment of the swine fever virus is shown as SEQ ID NO. 2, and the fragment contains 152 amino acids and has the molecular weight of 19KD; the polypeptide sequence of the E2 protein of the swine fever virus is shown as SEQ ID NO. 3, and the polypeptide contains 43 amino acids and has a molecular weight of 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.
In the invention, the E2 protein of the swine fever virus 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 swine fever E2 protein antibody detection kit comprises a solution of swine fever E2 protein coupled or indirectly coupled magnetic particles, a protein solution marked by a luminescent marker, a quality control product, a calibrator, a cleaning solution and a luminescent substrate.
The detection method of the swine fever E2 protein antibody adopts the detection kit, and comprises the following steps:
s1, sequentially adding 10-100 mu L of serum to be tested or a calibrator, and solution of swine fever E2 protein coupled or indirectly coupled 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, drawing a calibration curve, and calculating the concentration of the antibody in the serum to be detected according to the calibration curve.
Taking carboxyl magnetic particles and tosyl magnetic particles as examples, the best applicable amount experiment of the magnetic particles and the swine fever E2 protein is carried out.
1. Carboxyl magnetic particles and most applicable amount experiment of hog cholera virus E2 protein
3 classical swine fever virus E2 proteins are adopted, wherein the addition amount of the whole classical swine fever virus E2 protein is 15.7, 31.3, 62.5, 125, 250 and 500 mug, and the corresponding amount of substances is 0.34, 0.67, 1.34, 2.68, 5.36 and 10.73nmol; the addition amount of E2 protein fragments of the swine fever virus is 6.3, 12.5, 25, 50, 100 and 200 mug, and the corresponding amount of substances is 0.33, 0.66, 1.32, 2.63, 5.26 and 10.53nmol; the addition amount of the E2 protein polypeptide of the swine fever virus is 1.6, 3.2, 6.3, 12.5, 25 and 50 mug, and the corresponding amount of the corresponding substances is 0.32, 0.64, 1.26, 2.5, 5 and 10nmol.
The above-mentioned swine fever virus E2 virus protein and carboxyl magnetic particles are respectively used for preparing magnetic suspensions coated with swine fever virus E2 protein, and the kit containing the magnetic suspensions is respectively used for detecting swine fever virus E2 antibody positive serum, and 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 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 most suitable amount of the full length, fragments and polypeptides of the E2 protein of the swine fever virus is 0.64-4nmol/10mg of magnetic particles. The three proteins have very different amino acid numbers and molecular weights, but the optimal coating protein dosage is not very different according to the mass amount.
TABLE 1
2. Tosyl magnetic particles, swine fever virus E2 protein best applicable amount experiment
3 classical swine fever virus E2 proteins are adopted, wherein the addition amount of the whole classical swine fever virus E2 protein is 15.7, 31.3, 62.5, 125, 250 and 500 mug, and the corresponding amount of substances is 0.34, 0.67, 1.34, 2.68, 5.36 and 10.73nmol; the addition amount of E2 protein fragments of the swine fever virus is 6.3, 12.5, 25, 50, 100 and 200 mug, and the corresponding amount of substances is 0.33, 0.66, 1.32, 2.63, 5.26 and 10.53nmol; the addition amount of the E2 protein polypeptide of the swine fever virus is 1.6, 3.2, 6.3, 12.5, 25 and 50 mug, and the corresponding amount of the corresponding substances is 0.32, 0.64, 1.26, 2.5, 5 and 10nmol.
The above-mentioned swine fever virus E2 virus protein and tosyl magnetic particles are respectively used for preparing magnetic suspensions coated with swine fever virus E2 protein, and the kit containing the magnetic suspensions is respectively used for detecting swine fever virus E2 antibody positive serum, and the results are shown in Table 2, and FIG. 2 is a curve of the coating protein dosage corresponding to the luminescence value.
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 most suitable amount of the full length, fragment and polypeptide of the E2 protein of the swine fever virus is more than 0.64nmol/10mg magnetic particles. Although the three proteins have large differences in amino acid number and molecular weight, the optimal coating protein dosage is not very different according to the amount of substances, the cost factor is considered, the protein dosage is not too large, and the optimal dosage range of the three proteins is 0.64-4nmol/10mg magnetic particles is determined according to the result of combining carboxyl magnetic particles.
TABLE 2
Example 1
A kit for detecting a swine fever E2 protein antibody comprises a magnetic suspension coated with swine fever E2 protein, an alkaline phosphatase-labeled swine fever E2 protein antibody solution, a diluent, a calibrator, a quality control product, a cleaning solution and a luminescent substrate.
Preparation of magnetic suspension coated with classical swine fever E2 protein:
(1) 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 swine fever E2 antigen (purchased from Qingdao Yibang bioengineering Co., ltd.) was added at 125. 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 swine fever E2 protein 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 1mg of swine fever virus 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 swine fever virus 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 a known concentration of antibodies to the swine fever E2 protein.
Preparation of a calibrator:
(1) Heat-inactivating the hog cholera E2 protein antibody strong positive serum obtained after the hog cholera 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 swine fever virus antibody positive swine serum. 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 24-36U/mL, and the quality control range of the high-value quality control product is 80-120U/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 hog cholera virus 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。
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 swine fever E2 protein 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 swine fever E2 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 swine fever E2 protein 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 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
The kit in this example and a swine fever E2 protein antibody detection kit (enzyme-linked immunosorbent assay, hereinafter abbreviated as ELISA kit) manufactured by foreign well-known manufacturers are used to detect swine fever E2 protein antibody positive serum at different dilution factors simultaneously, wherein the kit in this example performs 10 repeated detections on each blood sample, and calculates a variation coefficient (standard deviation/arithmetic mean of CV% =10 test results). 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 was <4.14U/mL, slightly better than ELISA kit.
Table 4 shows the sensitivity of the kit of example I and ELISA kit
2. Repeatability experiments
3 cases of swine fever E2 protein antibody positive serum are taken, the kit is used for detection, 2 batches of the serum are detected every day, 2 tests are respectively carried out on 3 batches of the serum, and the two batches of the 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 96.4%, the negative coincidence rate is 90.4%, and the overall coincidence rate is 87.7%.
TABLE 6
4. The kit of the embodiment is used for carrying out the immune monitoring of the swine fever vaccine
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 blocking rate less than 0.30, determined as negative, 0.30-0.40 as suspicious, and blocking rate greater than or equal to 0.40 as positive.
After immunization with swine fever vaccine, 4 animals were randomly extracted, blood samples were taken 1, 7, 14, and 21 days after immunization to detect swine fever E2 protein antibodies, and non-immune pigs were used as a control, and the detection results are shown in Table 7. The results show that, except for 2#, the swine fever antibody changes 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; except that the ELISA kit for the blood samples at 14 days after the immunization of the No. 1 is suspicious, and the detection result of the ELISA kit for the blood samples is positive, the detection results of two other reagents of the blood samples are consistent.
TABLE 7
5. Specificity experiments
The kit of this example was used to detect various relevant virus antibodies, strong positive sera, including foot-and-mouth disease virus type O (FMDV-O), porcine pseudorabies virus (PRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), 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 | FMDV-O | PRV | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 3.26 | 3.3 | 3.08 | 4.16 | 6.73 |
Example two
A kit for detecting a swine fever E2 protein antibody comprises a magnetic suspension coated with the swine fever E2 protein, an acridinium ester-labeled 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.
Preparation of magnetic suspension coated with classical swine fever E2 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 60 mug of purified swine fever virus recombinant 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, 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.
A detection method of a swine fever E2 protein 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 swine fever E2 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, 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 the swine fever E2 protein antibody positive serum 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 1.4U/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 swine fever E2 protein antibody positive serum are taken, detected by the kit of the embodiment, 2 batches of the serum are detected every day, 2 tests are respectively carried out on 3 batches of the serum, and the two batches of the 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 94.8%, the negative compliance rate is 92.3%, and the overall compliance rate is 88%.
Table 12
4. The kit of the embodiment is used for carrying out the immune monitoring of the swine fever vaccine
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 blocking rate less than 0.30, determined as negative, 0.30-0.40 as suspicious, and blocking rate greater than or equal to 0.40 as positive.
After immunization with swine fever vaccine, 4 animals were randomly drawn, blood samples were drawn 1, 7, 14, 21 days after immunization to detect swine fever E2 protein antibodies, and non-immunized pigs were used as controls, and the results are shown in Table 13. The results show that, except for 2#, the swine fever antibody changes 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; except that the ELISA kit for the blood samples at 14 days after the immunization of the No. 1 is suspicious, and the detection result of the ELISA kit for the blood samples is positive, the detection results of two other reagents of the blood samples are consistent.
TABLE 13
5. Specificity experiments
The kit of this example was used to detect various relevant virus antibodies, strong positive sera, including foot-and-mouth disease virus type O (FMDV-O), porcine pseudorabies virus (PRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), 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 14
| Related viruses | FMDV-O | PRV | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 4.08 | 4.17 | 4.71 | 3.53 | 7.46 |
Example III
A kit for detecting a swine fever E2 protein antibody comprises a magnetic suspension coated with swine fever E2 protein, a swine fever virus E2 antigen solution marked by horseradish peroxidase, a diluent, a calibrator, a quality control product, a cleaning solution, a first luminescent substrate and a second luminescent substrate.
Preparation of magnetic suspension coated with classical swine fever E2 protein:
(1) 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 50 mug of purified swine fever virus E2 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 classical swine fever virus E2 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 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 ℃;
(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 the swine fever virus E2 antigen 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 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 containing 0.5g/L luminol and 0.1g/L p-iodophenol, and the second luminescent substrate is a solution of carbamide peroxide and 0.625 g/L.
A detection method of a swine fever E2 protein 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 swine fever E2 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 swine fever virus E2 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 5 minutes at 35 ℃;
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.
Table 15 shows the luminescence values corresponding to different concentrations of the calibrator, and FIG. 5 shows the calibration curve.
TABLE 15
1. Sensitivity experiment
The kit and ELISA kit of the example were used to simultaneously detect the swine fever E2 protein antibody positive serum 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 16. The result proves that the kit has accurate quantification, and the sensitivity is less than 1.18U/mL and is superior to ELISA kit.
Table 16
2. Repeatability experiments
3 cases of swine fever E2 protein antibody positive serum are taken, detected by the kit of the embodiment, 2 batches of the serum are detected every day, 2 tests are respectively carried out on 3 batches of the serum, and the two batches of the 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 92.7%, the negative compliance rate is 94.6%, and the overall compliance rate is 88%.
TABLE 18
4. The kit of the embodiment is used for swine fever vaccine immune monitoring
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 blocking rate less than 0.30, determined as negative, 0.30-0.40 as suspicious, and blocking rate greater than or equal to 0.40 as positive.
After immunization with swine fever vaccine, 4 animals were randomly drawn, blood samples were drawn 1, 7, 14, 21 days after immunization to detect swine fever antibodies, and non-immunized pigs were used as controls. The results show that, except for 2#, the swine fever antibody changes 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; except that the ELISA kit for the blood samples at 14 days after the immunization of the No. 1 is suspicious, and the detection result of the ELISA kit for the blood samples is positive, the detection results of two other reagents of the blood samples are consistent.
TABLE 19
5. Specificity experiments
The kit of this example was used to detect various relevant virus antibodies, strong positive sera, including foot-and-mouth disease virus type O (FMDV-O), porcine pseudorabies virus (PRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), 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 20
| Related viruses | FMDV-O | PRV | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 1.89 | 2.41 | 1.98 | 2.49 | 7.26 |
Example IV
A kit for detecting a swine fever E2 protein antibody comprises a magnetic suspension coated with swine fever E2 protein, an alkaline phosphatase-labeled swine fever E2 protein antibody, a biotinylated antigen, a calibrator, a quality control product, a cleaning solution and a luminescent substrate.
Preparation of magnetic suspension coated with classical swine fever E2 protein:
(1) 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% ProClin TM 300。
The magnetic particles are magnetic particles containing carboxyl groups.
Preparation of alkaline phosphatase-labeled swine fever E2 protein 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 swine fever virus E2 protein monoclonal antibody into the step (5), uniformly mixing, and dialyzing for 24 hours at 2-8 ℃ in a dark place by using 0.05mol/L CB buffer with 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 the swine fever virus E2 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 7.0.05 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 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.
A detection method of a swine fever E2 protein 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 swine fever E2 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 swine fever E2 protein 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 ℃;
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.
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 the swine fever E2 protein antibody positive serum at different dilutions, 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 kit has accurate quantification, and the sensitivity is less than 2.18U/mL and is superior to ELISA kit.
Table 22
2. Repeatability experiments
3 cases of swine fever E2 protein antibody positive serum are taken, detected by the kit of the embodiment, 2 batches of the serum are detected every day, 2 tests are respectively carried out on 3 batches of the serum, and the two batches of the 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%, the negative compliance rate is 95.1%, and the overall compliance rate is 87.6%.
Table 24
4. The kit of the embodiment is used for swine fever vaccine immune monitoring
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 blocking rate less than 0.30, determined as negative, 0.30-0.40 as suspicious, and blocking rate greater than or equal to 0.40 as positive.
After immunization with swine fever vaccine, 4 animals were randomly drawn, blood samples were drawn 1, 7, 14, 21 days after immunization to detect swine fever antibodies, and non-immunized pigs were used as controls. The results show that, except for 2#, the swine fever antibody changes 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; except that the ELISA kit for the blood samples at 14 days after the immunization of the No. 1 is suspicious, and the detection result of the ELISA kit for the blood samples is positive, the detection results of two other reagents of the blood samples are consistent.
Table 25
5. Specificity experiments
The kit of this example was used to detect various relevant virus antibodies, strong positive sera, including foot-and-mouth disease virus type O (FMDV-O), porcine pseudorabies virus (PRV), porcine Reproductive and Respiratory Syndrome Virus (PRRSV), 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 26
| Related viruses | FMDV-O | PRV | PRRSV | PCV2 | BVDV |
| Concentration (U/mL) | 3.63 | 3.93 | 3.25 | 3.39 | 6.65 |
The invention adopts magnetic particles with specific particle size as coating carrier, and adopts specific ratio of swine fever E2 protein to magnetic particles to mix and react, thus obtaining swine fever E2 protein coupled magnetic particles with homogeneous coating and stable structure, saving coating protein raw materials, having sufficient coating protein, wider detection range, higher sensitivity, very short reaction time (only 5-10 minutes), and having the excellent characteristics of 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 swine fever E2 protein antibody and detection method thereof
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 373
<212> PRT
<213> artificial sequence
<400> 1
RLSCKEDYRY AISSTNEIGP LGAEGLTTTW REYSHGLQLD DGTVRAICTA GSFKVIALNV 60
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NGSAFYLVCP IGWTGVIECT AVSPTTLRTE VVKTFKRERP FPHRVDCVTT IVEKEDMFYC 180
KWGGNWTCVK GNPVTYMGGQ VKQCKWCGFD FKEPDGLPHY PIGKCILANE TGYRVVDSTD 240
CNRDGVVIST EGEHECLIGN TTVKVHALDG RLGPMPCRPK EIVSSAGPVR KTSCTFNYTK 300
TLKNKYYEPR DSYFQQYMLK GEYQYWFDLD ATDHHTDYFA EFVVLVVVAL LGGRYVLWLI 360
VTYIVLTEQL AAG 373
<210> 2
<211> 152
<212> PRT
<213> artificial sequence
<400> 2
LGAEGLTTTW REYSHGLQLD DGTVRAICTA GSFKVIALNV VSRRYLASLH KRALPTSVTF 60
ELLFDGTSPA IEEMGDDFGF GLCPFDTTPV VKGKYNTTLL NGSAFYLVCP IGWTGVIECT 120
AVSPTTLRTE VVKTFKRERP FPHRVDCVTT IV 152
<210> 3
<211> 43
<212> PRT
<213> artificial sequence
<400> 3
KRALPTSVTF ELLFDGTSPA IEEMGDDFGF GLC 43
Claims (6)
1. The swine fever E2 protein antibody detection kit is characterized by comprising a solution of swine fever E2 protein coupled or indirectly coupled magnetic particles, a protein solution marked by a luminescent marker and a luminescent substrate; wherein the coupling comprises the steps of forming amide through condensation of carboxyl groups of the magnetic particles and amino groups of the protein, forming five-carbon bridge through glutaraldehyde crosslinking of the amino groups of the magnetic particles and amino groups of the protein, and connecting the magnetic particles of tosyl and amino groups of the protein through covalent coupling;
The swine fever E2 protein is one of full length, fragment or polypeptide of swine fever virus E2 protein;
the magnetic particles are Fe 3 O 4 As a core, particles whose surfaces are coated with a polymer coating and into which carboxyl or tosyl groups are introduced; the size of the magnetic particles is 1-3 mu m;
the optimal dosage range of the swine fever E2 protein is 0.64-4nmol/10mg magnetic particles;
the kit also comprises a diluent, a quality control product, a calibrator and a cleaning solution, wherein the diluent is selected from one or more of buffer solution, bovine serum albumin, a blocker, a monoclonal antibody and a polyclonal antibody;
the quality control products are a swine fever E2 protein antibody low-value quality control product and a high-value quality control product, the quality control range of the low-value quality control product is 24-36U/mL, and the quality control range of the high-value quality control product is 80-120U/mL; the washing liquid is a Tris buffer solution with the concentration of 0.05mol/LpH of 8.0 or a phosphate buffer solution with the concentration of 0.01mol/L of 7.0 (PBS), and the Tris buffer solution and the PBS buffer solution respectively contain 0.1% of Tween-20.
2. The swine fever E2 protein antibody detection kit of claim 1, wherein the solution preparation method of the swine fever E2 protein-coupled magnetic particles comprises the following steps:
S1, taking a solution containing magnetic particles, cleaning the magnetic particles by using a buffer solution, and suspending the magnetic particles in the buffer solution;
s2, adding purified swine fever E2 protein;
s3, adding a cross-linking agent or a catalyst, and carrying out oscillation reaction;
s4, the magnetic particles are adsorbed by the magnet, washed and suspended in a solution containing the blocking agent.
3. The swine fever E2 protein antibody detection kit of claim 1, wherein the method for preparing a solution of swine fever E2 protein indirectly coupled magnetic particles comprises the steps of:
1) Magnetic microparticle-bound avidin
S1, taking a solution containing magnetic particles, cleaning the magnetic particles by using a buffer solution, and suspending the magnetic particles in the buffer solution;
s2, adding avidin;
s3, adding a cross-linking agent or a catalyst, and carrying out oscillation reaction;
s4, the magnetic particles are adsorbed by the magnet, washed and suspended in a solution containing a blocking agent to form a magnetic particle-avidin complex;
2) Swine fever E2 protein binding biotin
S1, taking swine fever E2 protein and dialyzing;
s2, adding biotin for reaction;
s3, adding a sealing agent to react;
s3, dialyzing to remove unbound biotin to obtain swine fever E2 protein-biotin;
3) The magnetic particles-avidin are mixed with the swine fever E2 protein-biotin complex, and the magnetic particles are connected with the swine fever E2 protein through the binding force of the avidin and the biotin.
4. The swine fever E2 protein antibody detection kit of claim 1, wherein the preparation method of the luminescent marker-labeled protein solution comprises the following steps:
s1, taking protein specifically combined with a swine fever E2 protein or a swine fever E2 virus antibody, and dialyzing;
s2, adding a luminous marker, and reacting;
s3, adding a sealing agent to react;
s4, dialyzing to separate unbound luminescent marker.
5. The swine fever E2 protein antibody detection kit of claim 1, wherein the luminescent label is selected from any one of acridinium ester, alkaline phosphatase, peroxidase, and the luminescent label-labeled protein is selected from one or more combinations of swine fever virus E2 antigen, monoclonal antibody, polyclonal antibody, genetically engineered antibody, anti-pig IgG antibody, anti-pig IgM antibody.
6. The swine fever E2 protein antibody detection kit of claim 1, wherein the luminescent substrates are in one-to-one correspondence with luminescent markers.
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| 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 |
| CN116068175A (en) * | 2022-09-07 | 2023-05-05 | 中国农业科学院兰州兽医研究所 | Swine fever virus tubular chemiluminescent antibody detection kit based on E2 protein dimer and application thereof |
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