CN109374886B - Infectious bovine rhinotracheitis virus antibody detection kit and application thereof - Google Patents
Infectious bovine rhinotracheitis virus antibody detection kit and application thereof Download PDFInfo
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Abstract
The detection kit consists of a recombinant gD antigen coated enzyme label plate, a negative control, a positive control, an IBR specific monoclonal antibody marked by horseradish peroxidase, a sample diluent, a washing solution, a substrate solution and a stop solution. By using the kit, whether the cattle are infected with the infectious bovine rhinotracheitis can be detected in a short time, and a corresponding management strategy is formulated. Aiming at the complex situation of the current animal epidemic disease prevention and control, the method has very wide market prospect and can play an important role in basic level detection and government supervision.
Description
Technical Field
The invention belongs to the technical field of rapid detection of animal epidemic diseases, and relates to a bovine infectious rhinotracheitis virus antibody detection kit and application thereof.
Background
Infectious Bovine Rhinotracheitis (IBR) is a viral infectious disease caused by infecting domestic cattle and wild cattle with bovine herpes virus type 1 (BHV-1), which is classified as a B-type disease by the world animal health organization, and is classified as a second-type animal epidemic disease by the Ministry of agriculture of China. The disease is widely distributed around the world and has a low mortality rate. Many infected cattle experience subclinical symptoms, often due to bacterial secondary infections leading to more severe respiratory diseases and mixed infections, and are therefore very important for the early diagnosis and detection of IBR infection.
The industry standard NY/T575 & 2002 infectious bovine rhinotracheitis diagnostic technology of the department of agriculture lists 3 IBR detection methods: the method comprises the steps of virus separation and identification, a trace serum neutralization test and enzyme-linked immunoassay, wherein the first two methods are complicated in operation, long in time consumption, more in required reagents and equipment and difficult to develop in a grassroots pasture; the enzyme-linked immunoassay generally takes approximately 4 hours and requires preparation of various solutions and preparation components, which are difficult to perform in basic veterinary laboratories.
Furthermore, the enzyme-linked immunosorbent assay described in the standard is an indirect enzyme-linked immunosorbent assay using rabbit polyclonal antibodies, and a virus antigen is used to coat the ELISA plate, so that a certain risk of virus elimination exists, and cross contamination may also occur due to improper treatment of experimental wastes. If the kit for detecting the infectious bovine rhinotracheitis is developed, the current IBR detection situation can be greatly improved, and the kit is convenient, rapid, free of toxicity scattering risk, time-saving and labor-saving.
Disclosure of Invention
The invention aims to provide a kit for detecting infectious bovine rhinotracheitis virus antibodies, which can detect whether a cattle group is infected with infectious bovine rhinotracheitis virus within 1 hour.
The detection kit provided by the invention does not need expensive equipment, is simple and convenient to operate, does not need special training, has high detection sensitivity, and is very suitable for detection application in primary veterinarian laboratories and farms.
The kit for detecting the antibody of the infectious bovine rhinotracheitis virus is based on the detection principle of an enzyme-linked immunosorbent assay (ELISA) blocking method and comprises a recombinant gD antigen coated ELISA plate, a negative control, a positive control, a horseradish peroxidase-labeled IBR specific monoclonal antibody, a sample diluent, a washing solution, a substrate solution and a stop solution.
The recombinant gD antigen coated ELISA plate is coated with recombinant infectious bovine rhinotracheitis gD protein, the recombinant gD protein is obtained by expressing a truncated gD sequence listed as SEQ ID No.1 in an Escherichia coli system, and a gene sequence for expressing the protein is listed as SEQ ID No. 2.
The negative control is imported fetal bovine serum and is confirmed to be negative by the method listed in NY/T575-2002 infectious bovine rhinotracheitis diagnostic technology.
The positive control is standard bovine serum positive for infectious bovine rhinotracheitis antibody.
The horseradish peroxidase-labeled IBR specific monoclonal antibody is prepared by using horseradish peroxidase-labeled IBR specific murine monoclonal antibody, wherein the IBR specific murine monoclonal antibody is obtained by immunizing a mouse with recombinant gD protein and then screening, and has specific reaction on the infectious bovine rhinotracheitis virus.
The sample dilution was 0.02mol/L PBS, pH 7.2.
The washing solution is 0.01mol/L PBS (pH7.2) containing 0.05% Tween-20.
The substrate solution is a commercial single-component TMB chromogenic substrate solution purchased from the market.
The stop solution is 2mol/L sulfuric acid solution.
The invention also aims to provide a preparation method of the detection kit, which specifically comprises the following steps:
(1) preparing recombinant gD protein;
(2) preparing a gD protein specific monoclonal antibody;
(3) preparing each component of the detection kit;
(4) and (5) assembling the detection kit.
In addition, the invention also relates to a detection method for detecting the infectious bovine rhinotracheitis virus antibody in the bovine serum by using the infectious bovine rhinotracheitis virus antibody detection kit, which comprises the following steps:
in the first step, a serum sample is collected and isolated.
And secondly, diluting the sample by using a sample diluent according to the ratio of 1:40 for later use. If the food is not used immediately, the food is stored at 4 ℃ within a short time (1-4 hours), and is stored at-20 ℃ for a long time (more than or equal to 4 hours).
And thirdly, detecting the treated sample by using the infectious bovine rhinotracheitis virus antibody detection kit, and finally judging the result.
Compared with the traditional etiology analysis method and the conventional ELISA method, the method has the following beneficial effects:
1) the operation is simple, and special instruments and equipment are not needed. From the perspective of reagents, instruments and equipment, the method does not need organic reagents, the detection sample can be directly detected after being diluted, the operation difficulty is reduced, and the method is very suitable for popularization and application in basic-level detection laboratories.
2) The time consumption is short, and the detection efficiency is high. The whole detection time is shorter, and the method is time-saving and labor-saving compared with the standard recommendation method of the ministry of agriculture.
3) The detection does not need to involve external viruses, does not have the risk of virus dispersion, and is favorable for epidemic disease prevention and control and cross contamination control of cattle farms and basic veterinary laboratories.
4) The detection is sensitive and the accuracy is high. The blocking method principle can be used for effectively monitoring the change of the virus antibody, and is different from the general indirect ELISA.
Compared with the traditional technology and a standard method, the infectious bovine rhinotracheitis virus antibody detection kit provided by the invention has the advantages of obvious advantages and obvious technical improvement, particularly has very wide market prospect aiming at the current situation that the current infectious bovine rhinotracheitis prevention and control form is complex, and can play an important role in basic level detection and government supervision.
Drawings
FIG. 1 is a diagram showing PCR amplification of the gD gene.
FIG. 2 shows the identification and purification of gD recombinant protein.
Detailed Description
The invention is further described below in conjunction with specific embodiments. Advantages and features of the present invention will become more apparent as the description proceeds, but the examples are merely exemplary in nature and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention. The following reagents and experimental materials were all derived from commercial products unless otherwise specified.
Example 1: preparation of recombinant gD protein
(1) Preparation and purification of recombinant gD antigen
According to the genetic information of the infectious bovine rhinotracheitis virus in Genbank NC 001847, DNAStar is utilized to analyze signal peptide, antigenicity, hydrophilicity and the like of an amino acid sequence of gD protein, remove a signal peptide sequence, screen an antigenic region, finally intercept 2 protein sequences, carry out tandem combination, synthesize a gD gene sequence which is a SEQ ID No.2 sequence, and design a pair of primers, wherein an upstream primer sequence is a SEQ ID No.3 sequence, and a downstream sequence is a SEQ ID No.4 sequence.
The PCR reaction was carried out using the synthetic gene as a template. The PCR reaction system was 50. mu.L. In a sterilized 0.2mL EP tube, the following reagents were added: template DNA 0.5. mu.L, 10XEx TaqTN Buffer (Mg)2+free) 5. mu.L, dNTP mix (2.5 mM each) 2. mu.L, MgCl2 4 μ L (25 mM), 0.5 μ L of upstream primer (20 pM/. mu.L), 0.5 μ L of downstream primer (20 pM/. mu.L), TaKaRa Ex TaqTM0.25. mu.L (5 u/. mu.L) of sterilized ultrapure water 37.25. mu.L. After the PCR reaction was completed, the PCR product was mixed with 10 Xbromophenol blue loading buffer and subjected to 1.5% agarose gel electrophoresis (EB 0.5. mu.g/mL). The electrophoresis was terminated, observed under an ultraviolet lamp, photographed with a gel imaging system and analyzed. As shown in fig. 1.
And (2) recovering a purified product by using a commercial recovery kit (Tiangen biochemistry), mixing the gD gene fragment subjected to double enzyme digestion with a plasmid vector pET-32a (+) in a ratio of 3:1, carrying out viscous end directional connection with T4 DNA ligase at the temperature of 16 ℃, then transforming into BL21(DE3) competent cells, screening positive colonies, sending to HaiJeri for sequencing identification, and carrying out Blast comparative analysis on a sequencing result and a Genbank database to ensure that the sequence is correct.
Inoculating gD recombinant protein expression genetic engineering bacteria into LB liquid culture medium containing 100 microgram/ml ampicillin, shaking and culturing at 37 ℃ and 180r/min to the middle of logarithmic phase (OD 600nm is 0.6-0.8), adding IPTG to the final concentration of 1.0mM, continuing induction culture for 1-5 hours, washing 1 time with PBS of pH7.2, cracking the thallus suspension with lysozyme and ultrasonic, centrifuging at 4 ℃ and 12000r/min for 20min, and separating soluble protein from inclusion body. The lysis supernatant and the precipitate were separately subjected to SDS-PAGE, and the solubility of the recombinant protein was analyzed. The recombinant gD protein was isolated and purified by His-Tag affinity column (Merck product), and stored at-20 ℃ or below for further use. The gD purified protein is shown in FIG. 2.
(2) Specific detection of recombinant gD proteins
And transferring the purified recombinant gD protein to an NC membrane through SDS-PAGE electrophoresis, and carrying out Western Blotting detection. A1: 100 dilution of IBRV positive bovine serum was used as a primary antibody, incubated at 37 ℃ for 2h, and washed 3 times with TBST for 10 min/time. Incubation with HRP rabbit anti-bovine secondary antibody diluted 1:5000 times in TBST at 37 deg.C for 1h, and washing with TBST for 3 times, 10 min/time. DAB color development, deionized water termination reaction.
Example 2: preparation and purification of gD protein specific monoclonal antibody
The prepared recombinant gD antigen is emulsified with Freund's adjuvant in an equal amount according to the amount of 50 mu g, and then a plurality of Balb/C mice are immunized by subcutaneous multi-point injection. The adjuvant is Freund's complete adjuvant in the first immunization, and the subsequent immunization adjuvant is Freund's incomplete adjuvant. The immunization interval was 2 weeks, and the serum titer was measured by direct ELISA after 3 immunizations. And (3) screening mice with high serum titer and small cross reaction, and screening hybridoma cell strains secreting specific monoclonal antibodies by cell fusion according to a conventional method.
The antigens are selected to be inactivated bovine infectious rhinotracheitis virus, bovine viral diarrhea virus, bovine foot and mouth disease virus and the like and recombinant gD protein antigens, negative serum is used as reference control, monoclonal antibody cell strains which are reactive to used cross-reactive substances are excluded by screening, cell strains which are only simultaneously reactive with the bovine infectious rhinotracheitis virus and the recombinant gD protein are reserved, and finally 13 positive cell strains are obtained, wherein the results are as follows:
culturing 5A1-1A11 and 7C10-1B1 with highest titer in an enlarged manner, inducing ascites by an in vivo method, collecting, precipitating with saturated ammonium sulfate, purifying with Protein G column, measuring Protein concentration, and freezing.
Example 3: identification of IBR-specific monoclonal antibodies
The prepared monoclonal antibody is identified by using a commercial IgG subtype identification test paper box, and the result shows that 5A1-1A11 and 7C10-1B1 are respectively IgG2a and IgG2B subtypes, and the specific operation is as follows:
1) the prepared purified monoclonal antibody is diluted with PBS solution at 1:10000, 1:15000, 1:20000, 1:25000, 1:30000, 1:35000 and 1: 40000.
2) The gD protein coated plate (0.1. mu.g/ml) was washed 1 time with 1 Xwashing solution 300. mu.l/well, and the washing solution was discarded. The corresponding diluted monoclonal antibody was added in 50. mu.l/well and 5 replicate wells were made for each monoclonal antibody, and mixed by shaking for 1 minute. After incubation at 37 ℃ for 30 minutes, the reaction plate was removed, the reaction solution was discarded, 300. mu.l of 1 Xwashing solution was added to each well, and after washing 3 times, the plate was spin-dried.
3) IgG1, IgG2b and IgG3 were diluted 1:1000 in PBS and IgG2a and IgM were diluted 1:5000 in PBS. Diluted IgG1, IgG2b, IgG3, IgG2a and IgM antibodies were added to each monoclonal antibody in sequence, 100. mu.l of each well was added, and incubation was performed at 37 ℃ for 2 hours.
4) Diluting HRP-labeled goat anti-rabbit IgG antibody with PBS at a ratio of 1:5000, adding 100 μ l of the diluted antibody into each well, incubating at 37 ℃ for 30 minutes, washing 3 times according to the method in the previous step, and spin-drying.
5) The substrate solution was immediately added to an ELISA reaction plate at 100. mu.l/well, and after 10 minutes of color development at room temperature in the absence of light, the reaction was terminated by adding 50. mu.l of stop solution per well.
6) After the reaction was terminated, the OD450nm value was measured by a microplate reader within 10 minutes.
7) Comparing the OD450nm values of IgG1, IgG2b, IgG3, IgG2a and IgM antibody wells added to each monoclonal antibody, the subclass corresponding to the highest OD450nm was the subclass of the monoclonal antibody.
Example 4: horse radish peroxidase labeled IBR specific monoclonal antibody
The method for labeling the horseradish peroxidase and the IBR specific monoclonal antibody by utilizing the glutaraldehyde method comprises the following specific steps:
(1) horseradish peroxidase 25mg was weighed into a 1.25% glutaraldehyde solution and allowed to stand overnight at room temperature.
(2) The enzyme solution after the reaction is eluted by normal saline through a Sephadex G-25 chromatographic column. The flow rate was controlled at 1ml/min and the brown effluent was collected. If the volume is more than 5ml, the solution is concentrated to 5ml by PEG. Placed in a 25ml small beaker and stirred slowly.
(3) The murine monoclonal antibodies 5A1-1A11 and 7C10-1B1 to be labeled were each diluted to 5ml with 12.5mg of physiological saline, and added dropwise to the enzyme solution with stirring.
(4) Stirring was continued for 3 hours using 0.25ml of 1M carbonate buffer pH 9.5.
(5) 0.25ml of 0.2M lysine was added thereto, and the mixture was left at room temperature for 2 hours after mixing.
(6) An equal volume of saturated ammonium sulfate was added dropwise with stirring and left at 4 ℃ for 1 hour.
(7) Centrifuge at 3000rpm for half an hour, and discard the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate and finally dissolved in a small amount of 0.15M PBS pH 7.2.
(8) Putting the solution into a dialysis bag, dialyzing 0.15M PB buffer saline with pH7.4, removing ammonium ions (detected by a naphthalene reagent), centrifuging at 10000rpm for 30 minutes to remove precipitates, and subpackaging and freezing for storage, wherein the supernatant is the enzyme-labeled monoclonal antibody.
Finally, 2 labeled antibodies were obtained, designated HRP-1A11 and HRP-1B 1.
Example 5: screening for blocking antibodies
And (3) coating the recombinant gD protein on an enzyme label plate according to the concentration of 1 mu g/ml, adding a diluted known sample (the result is confirmed by the detection of the animal epidemic disease prevention control center in Beijing), then respectively adding 2 strains of HRP-labeled antibodies to carry out ELISA detection, and screening and determining the preferred HRP-labeled antibody for the kit according to the detection result. The specific operation is as follows:
(1) coating of ELISA plate: the recombinant gD protein was diluted to 1. mu.g/mL with 0.05M carbonate buffer (pH 9.6), the plate was coated with 100. mu.L of ELISA per well, overnight at 4 ℃, then washed with PBST for 3 times, 60s each time, then blocked with 200. mu.L of 1% gelatin (1 g gelatin dissolved in 100mL PBS, pH 7.2-7.4, filter sterilized), and blocked at 37 ℃ for 2 hours. Then the plate is washed for 1 time and then is dried for standby.
(2) And (3) ELISA detection: adding 100 μ L of sample to be detected into the well, incubating at 37 deg.C for 30min, discarding the liquid in the well, and washing the plate for 3 times. Adding 100 mu L of HRP-labeled monoclonal antibody into each hole, continuously incubating at 37 ℃ for 30min, discarding liquid in the holes, washing the plate for 3 times, adding TMB substrate, incubating at 37 ℃ for 10min and stopping the reaction by using 50 mu L of sulfuric acid of 2 mol/L. And reading the absorbance value of 450nm by using a microplate reader.
The results of the detection of 2 different antibodies are given in the following table:
according to the result, when the HRP-1A11 is used for detecting the import negative serum, the OD value is about 1.0, and the OD value is higher and accords with the actual sample; when HRP-1B1 is used for detecting the import negative serum, the OD value is 0.706, which is lower, and the OD value of the sample 1 is also the same, the N/P value detected by the whole sample is larger, and the HRP-1A11 is selected as a subsequent preferred enzyme labeled antibody for a reagent kit component preparation.
Example 6: preparation of components of infectious bovine rhinotracheitis virus antibody detection kit
(1) Coating of ELISA plate: the coating process is the same as the coating process in the operation step of example 5, and after the coating process is dried, the coating process is sealed in vacuum by using an aluminum foil bag for standby.
(2) Preparation of negative control: import fetal calf serum is determined to be negative by the method listed in NY/T575 & 2002 infectious bovine rhinotracheitis diagnostic technology, and is subpackaged into plastic bottles with 1mL per bottle after 0.03 percent of sodium azide is added.
(3) Positive control: serum purchased from the institute of veterinary medicine in China was diluted 200-fold, added with 0.03% sodium azide, and dispensed into plastic bottles with 1mL each.
(4) Horseradish peroxidase-labeled IBR specific monoclonal antibodies: diluting HRP-1A11 to 1:3000 times with PBS, adding 0.04% sodium azide, and packaging into brown bottles with 10mL each;
(5) sample diluent: 0.02mol/L PBS, and subpackaging into 50mL per bottle;
(6) washing liquid: 0.2mol/L PBS, pH7.4, containing 1% Tween-20, 60mL each bottle, when using, need to dilute 20 times with deionized water;
(7) substrate solution: the single-component TMB substrate liquid and commercial products are subpackaged into brown bottles, wherein each bottle is 7 mL;
(8) stopping liquid: 2mol/L sulfuric acid solution, prepared by self, and subpackaged into plastic bottles, 7mL per bottle.
Example 7: assembly and detection steps of infectious bovine rhinotracheitis virus antibody detection kit
Preparing each kit component according to the embodiment 6, sticking labels, respectively inserting the kit components on foam trays carved with hole sites, putting the kit components into an external kit box, putting an operation instruction and a quality control report into the external kit box, and sealing and storing the external kit box at 2-8 ℃.
The detection steps of the kit for detecting the infectious bovine rhinotracheitis virus antibody are as follows:
(1) the kit and the sample to be detected are restored to the room temperature (19-25 ℃), for example, the kit and the sample to be detected are placed in the room temperature environment for 45 min;
(2) diluting a sample to be tested: taking 10 mu L of a sample by using a micropipettor, adding 390 mu L of sample diluent, and uniformly mixing for later use, wherein if the diluted sample cannot be immediately detected, the sample is preserved at 4 ℃ within a short time (1-4 hours), and is preserved at-20 ℃ for no more than 1 month for a long time (more than or equal to 4 hours);
(3) plus sample/negative positive control: taking a proper enzyme label plate strip as required, adding a sample/negative and positive control, making double holes parallel by 100 mu L per hole, and then incubating for 30min in a dark place at 37 ℃;
(4) plate washing for 3 times, and patting dry. Adding 100 mu L enzyme labeled monoclonal antibody, and incubating for 15min at 37 ℃ in the dark;
(5) plate washing for 3 times, and patting dry. Adding 100 μ L substrate solution, incubating at 37 deg.C in dark for 15 min;
(6) taking out, adding 50 mu L of stop solution to stop reaction, reading the absorbance value of 450nm by an enzyme-labeling instrument, and judging the detection result of the sample according to the positive and negative control OD values.
(7) Result judgment mode: the blocking rate was calculated using the following formula
Blocking rate = (negative control mean-sample mean OD value)/negative control mean 100%
If the blocking rate is more than or equal to 45 percent, the product is positive; suspicious in 40-45% and negative in less than or equal to 40%.
Example 8: stability experiment of infectious bovine rhinotracheitis virus antibody detection kit
Preparing 3 batches of the infectious bovine rhinotracheitis virus antibody detection kit, detecting a standard negative control and a standard positive control, repeatedly detecting each sample for 2 times, and judging the stability of the detection kit according to the detection result.
Statistics shows that the negative and positive control intra-batch variation coefficient of the infectious bovine rhinotracheitis virus antibody detection kit is 3.2-6.3%, the inter-batch variation coefficient is 4.3-5.5%, and the stability is good.
Example 9: sensitivity experiment of infectious bovine rhinotracheitis virus antibody detection kit
The purchased standard positive serum (China veterinary medicine institute) was diluted to 1:10, 1:50, 1:100, 1:200, 1:400, and 1:800 with a sample diluent, and then detected using an infectious bovine rhinotracheitis virus antibody detection kit. The results are given in the table below.
As can be seen from the table, when diluted to 1:400, the blocking rate was 34.8%, and less than 40% was negative. Namely, the kit has higher sensitivity to the positive standard serum with the lowest detection dilution factor of 1: 200-1: 400.
Example 10: specificity experiment of bovine infectious rhinotracheitis virus antibody detection kit
Common bovine disease virus antibody positive standard serum, such as A-type bovine foot and mouth disease virus positive standard serum, bovine viral diarrhea virus positive standard serum and calf diarrhea virus positive standard serum, is diluted by 100 times by using sample diluent respectively and then is detected by using a self-made kit, the result shows that the blocking rate is less than 10 percent and the blocking rate is negative, which shows that the self-made bovine infectious rhinotracheitis virus antibody detection kit has no cross reaction with the common bovine disease positive serum and has good specificity.
Example 11: contrast of self-made kit with imported like products
358 parts of serum clinically collected from cattle farms in different regions throughout the country were detected using a homemade bovine infectious rhinotracheitis virus antibody detection kit and a gB antibody blocking kit of american IDEXX, and the results are tabulated below.
According to results, all the self-made kits are detected in 358 samples and 312 negative results detected by the American IDEXX kit, and the coincidence rate is 100%; in 44 positive results detected by the American IDEXX kit, 44 positive results are detected by the self-made kit, and the coincidence rate is 100 percent; and the self-made kit detects 2 suspicious samples detected by the American imported kit to be positive, and the result shows that the sensitivity of the self-made kit is higher than that of the American kit. In combination, the self-made and imported kits have high positive and negative coincidence rates, the overall coincidence rate is 99.44%, and the kit has a good application prospect.
Example 12: detection of artificially infected animal by self-made kit
Using IBR virus to artificially infect 3 cattle which are negative by serum screening and pathogen screening, using a self-made kit and an American IDEXX kit to synchronously detect the IBR virus antibody in the cattle serum at 1, 3, 5, 7, 9, 12 and 15 days after infection respectively, and the results are shown in the following table:
according to results, the self-made kit detects 1 part of positive on the fifth day after artificial infection, and the American kit is all negative; 7-15 days after artificial infection, the detection results of the self-made kit are all positive, while the detection results of the American kit are all positive after 12 days after artificial infection, so that the self-made kit can be preliminarily judged to be more sensitive than the detection of the American IDEXX kit, and the method is more suitable for clinical screening of the IBR virus antibody general investigation.
Sequence listing
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Claims (5)
1. The infectious bovine rhinotracheitis virus antibody detection kit is characterized in that recombinant gD protein in the detection kit is obtained by expressing a truncated gD sequence with an amino acid sequence of SEQ ID No.1 in an escherichia coli system, and a gene sequence of the recombinant gD protein is a sequence with SEQ ID No. 2.
2. The infectious bovine rhinotracheitis virus antibody detection kit of claim 1, wherein the primer sequence expressed by the recombinant gD protein clone is: the sequence of the upstream primer is SEQ ID No.3, and the sequence of the downstream primer is SEQ ID No. 4.
3. The infectious bovine rhinotracheitis virus antibody detection kit of claim 1, wherein the detection kit is composed of a recombinant gD antigen coated enzyme label plate, a negative control, a positive control, a horseradish peroxidase-labeled IBR specific monoclonal antibody, a sample diluent, a washing solution, a substrate solution and a stop solution, wherein:
the recombinant gD antigen coated ELISA plate is coated with recombinant infectious bovine rhinotracheitis gD protein;
the negative control is imported fetal calf serum and is confirmed to be negative by the method listed in NY/T575-2002 infectious bovine rhinotracheitis diagnostic technique;
the positive control is standard bovine serum positive for infectious bovine rhinotracheitis antibody;
the horseradish peroxidase-labeled IBR specific monoclonal antibody is prepared by using horseradish peroxidase-labeled IBR specific mouse monoclonal antibody, wherein the IBR specific mouse monoclonal antibody is obtained by screening a recombinant gD protein immunized mouse and has specific reaction aiming at infectious bovine rhinotracheitis virus;
the sample diluent is 0.02mol/L PBS (pH7.2);
the washing solution is 0.01mol/L PBS (pH7.2) containing 0.05% Tween-20; the substrate solution is a commercial single-component TMB chromogenic substrate solution purchased from the market;
the stop solution is 2mol/L sulfuric acid solution.
4. The infectious bovine rhinotracheitis virus antibody detection kit of claim 1, wherein the kit is used for detecting infectious bovine rhinotracheitis antibodies in bovine serum.
5. The infectious bovine rhinotracheitis virus antibody detection kit of claim 1, wherein the kit detection process comprises the following steps:
(1) collecting and separating a serum sample;
(2) diluting a sample for later use by using a sample diluent according to a ratio of 1:40, if the sample is not used immediately, storing the sample at 4 ℃ for 1-4 hours, and storing the sample at-20 ℃ for more than or equal to 4 hours;
(3) and detecting the treated sample by using the infectious bovine rhinotracheitis virus antibody detection kit, and judging the result.
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| CN113607942A (en) * | 2021-07-30 | 2021-11-05 | 河北科技师范学院 | Establishment method of indirect ELISA (enzyme-Linked immuno sorbent assay) for detecting IBR (infectious bronchitis Virus) based on gL protein |
| CN116003580B (en) * | 2022-09-15 | 2024-08-23 | 北京纳百生物科技有限公司 | Infectious bovine rhinotracheitis virus gD monoclonal antibody, detection kit and application |
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