CN213875707U - Porcine pseudorabies virus gB & gD antibody detection kit - Google Patents
Porcine pseudorabies virus gB & gD antibody detection kit Download PDFInfo
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Abstract
The utility model discloses a pig pseudorabies virus gB & gD antibody detection kit in one aspect, this kit can detect pig pseudorabies virus gB antigen antibody and gD antigen antibody simultaneously, this kit has a box body (1), be equipped with antigen coated plate storage tank (2), quality control article storage tank (3), positive control liquid pore groove (4), negative control liquid pore groove (5), goat anti pig IgG enzyme labeled antibody pore groove (6), concentrated washing liquid pore groove (7), sample diluent pore groove (8), color development liquid pore groove (9), termination liquid pore groove (10) and operation instruction storage tank (11) in box body (1); an antigen coating reaction plate is placed in the antigen coating plate storage tank (2), a reagent bottle filled with corresponding solution is placed in each hole tank (3-10), and an operation instruction is placed in the operation instruction storage tank (11). The utility model discloses a kit can realize detecting PRV-gB protein antibody and PRV-gD protein antibody simultaneously. In addition the utility model discloses a kit stability is good, sensitivity is high, the specificity is strong, can be applicable to extensive clinical detection.
Description
Technical Field
The utility model belongs to virus epidemic disease diagnostic technique and animal quarantine field, concretely relates to pig pseudorabies virus gB & gD antibody detection kit.
Background
Porcine Pseudorabies is an acute infectious disease of pigs caused by porcine Pseudorabies virus (PRV). The disease is fulminant in pigs. Can cause abortion and stillbirth of pregnant sows, sterility of boars, mass death of newborn piglets, dyspnea and growth retardation of fattening pigs and the like, and is one of serious infectious diseases harming the global pig industry.
The pseudorabies virus belongs to Herpesviridae (Herpesviridae) and porcine herpesvirus, and the virus particle is circular, the diameter is 150-180 nm, and the diameter of the nucleocapsid is 105-110 nm. The outermost layer of the virion is the viral envelope, which is a lipid bilayer structure derived from the host cell. The surface of the capsular sac is provided with fiber protrusions which are radially arranged and have the length of about 8-10 nm. The pseudorabies virus genome is a linear double-stranded DNA molecule with the size of about 150kb and the average G + C content of up to 74 percent, has the structural characteristics of a typical herpesvirus genome and consists of a unique long segment, a unique short segment, terminal repetitive sequences (TR) and internal repetitive sequences (IR) which are positioned on both sides of US. It has now been found that 11 PRV glycoproteins, wherein gB, gC and gD are all proteins that stimulate the body to produce neutralizing antibodies, and the antibodies produced have the ability to neutralize PRV in vivo, in vitro or in the presence or absence of complement, and therefore gB, gC and gD are the preferred glycoproteins for the development of PRV subunit vaccines and for the evaluation of the immune efficacy of vaccines, with gB and gD being particularly preferred.
However, all of the currently marketed kits for evaluating the immune effect of PRV can only detect antibodies produced by gB protein, but cannot evaluate antibodies produced by gD protein, so there may be some false negative detection for the evaluation of the actual situation after PRV vaccine immunization.
SUMMERY OF THE UTILITY MODEL
In order to make up for the deficiencies of the prior art, one of the objectives of the present invention is to provide a kit for simultaneously evaluating antibodies against PRV-gB protein and PRV-gD protein, so as to make up for the deficiencies of the prior art kits.
Therefore, the utility model provides a pig pseudorabies virus gB & gD antibody detection kit in one aspect, the kit can detect pig pseudorabies virus gB antigen antibody and gD antigen antibody simultaneously, the kit have a box body (1), be equipped with antigen coating plate storage tank (2) in the box body (1), quality control article storage tank (3), positive control liquid hole groove (4), negative control liquid hole groove (5), goat anti-pig IgG enzyme-labeled antibody hole groove (6), concentrated washing liquid hole groove (7), sample diluent liquid hole groove (8), color development liquid hole groove (9), termination liquid hole groove (10) and operation instruction storage tank (11); an antigen coating reaction plate is placed in the antigen coating plate storage tank (2), a reagent bottle filled with corresponding solution is placed in each hole tank (3-10), and an operation instruction is placed in the operation instruction storage tank (11).
Preferably, the antigen coated plate of the utility model is an ELISA plate coated with PRV-gB protein and PRV-gD protein at the same time, the coating concentration of the PRV-gB protein and the PRV-gD protein on the antigen coated plate is 100 ng/hole/100 mu L, and each kit contains 2 or 5 antigen coated plates.
Preferably, the sample diluent of the present invention is a 1 x PBST solution containing 2% BSA, and each kit contains 80 mL/vial or 200 mL/vial.
Preferably, the concentrated wash solution of the invention is a 25 x PBST solution, diluted to a 1 x PBST solution prior to use, each kit containing 30 mL/bottle or 60 mL/bottle.
Preferably, the goat anti-pig IgG enzyme-labeled antibody of the utility model is obtained by using a sample diluent to dilute commercial goat anti-pig IgG enzyme-labeled antibody 20000 times, and each kit contains 25 mL/bottle or 60 mL/bottle.
Preferably, the color developing solution of the present invention is a TMB single component solution, and each kit contains 25 mL/bottle or 60 mL/bottle.
Preferably, the stop solution of the present invention is 2MH2SO4Solutions, each kit contained 12 mL/vial or 30 mL/vial.
Preferably, the positive control of the invention is positive serum with PRV antibody OD450nm value between 0.9-1.5; the negative control is negative serum with PRV antibody OD450nm value less than 0.2; each kit positive control and negative control contained 1 mL/tube or 3 mL/tube.
Preferably, the quality control product of the utility model comprises a quality control product 1, a quality control product 2 and a quality control product 3, wherein the quality control product 1 is strong positive serum with an S/P value of 1.5-2.0; the quality control product 2 is weak positive serum with an S/P value of 0.5-1.0; the quality control product 3 is negative serum with an S/P value less than 0.35; each of the kit quality control 1, quality control 2 and quality control 3 contains 1 mL/tube or 3 mL/tube.
The utility model discloses a kit can realize detecting PRV-gB protein antibody and PRV-gD protein antibody simultaneously, and the antibody titer that detects all within the detection range of ELIASA, does benefit to the evaluation to whole PRV antibody titer to omit the antibody of PRV-gD protein, thereby cause the existence of possible false negative. Additionally, the utility model discloses a quality control article (quality control article 1, quality control article 2, quality control 3) are introduced to the kit, not only have the correction of negative, positive control to the detection at every turn of kit, still have the correction of 3 quality control articles (strong yang, weak yang and negative) to ensure that the detection at every turn of kit all is accurate and controllable, further strengthen the quality control and the accuracy control of kit. Furthermore, the utility model discloses a kit stability is good, sensitivity is high, the specificity is strong, can be applicable to extensive clinical detection.
Drawings
FIG. 1 is a schematic diagram of a kit.
Fig. 2 shows the specific placement position of the quality control material in the quality control material storage tank (3).
Detailed Description
The following detailed description of the preferred embodiments of the present invention will be provided in conjunction with the accompanying drawings, so as to enable those skilled in the art to more easily understand the advantages and features of the present invention, and thereby define the scope of the invention more clearly and clearly. The experimental procedures not described in detail in the examples are generally carried out according to the routine procedures in the art or according to the conditions recommended by the manufacturers. The reagents and drugs mentioned in the examples are all common commercial products unless otherwise specified.
Example 1 preparation of PRV-gD and PRV-gB proteins
The PRV-gD protein used in the utility model is derived from the invention patent with the patent number of 201710551363.8, and the specific preparation method refers to the relevant examples of the patent.
The PRV-gB protein used in the utility model is derived from an invention patent with the patent number of 201910577700.X, and the specific preparation method refers to the relevant examples of the patent.
Example 2 preparation and use of porcine pseudorabies gB & gD antibody detection kit
1 preparation of antigen-coated plates
1.1 sources and standards of enzyme-linked reaction plates CoSTAR microplate available from Corning, USA, with a format of 8 wells × 12 rows.
1.2 coating PRV-gB protein and PRV-gD protein are respectively diluted to 2 mu g/mL by coating buffer solution (0.15 g of sodium carbonate and 0.293 g of sodium bicarbonate are weighed, pH value is adjusted to 9.6 after dissolution, then double distilled water is added to 100mL, the mixture is uniformly mixed, a 0.22 mu m filter membrane is used for filtration and sterilization, quantitative subpackaging is carried out), the diluted PRV-gB protein and the PRV-gD protein are mixed in equal volume, the mixture is uniformly mixed, an ELISA plate, 100 mu L/hole is added, and the mixture is incubated in a 37 ℃ wet box (factory production, air humidity is kept between 65 and 70 percent) for 1 hour and then kept overnight at 4 ℃ (12 to 14 hours). Taking out, discarding the liquid in the hole, washing with washing solution for 3 times, 300 μ L/hole, standing for 30 seconds each time, and patting dry.
1.3 blocking the enzyme-labeled plate, adding a blocking solution (0.27 g of monopotassium phosphate, 0.2g of potassium chloride, 3.58g of disodium hydrogen phosphate dodecahydrate and 8.0g of sodium chloride are weighed, dissolved in 800mL of double distilled water, 0.5mL of Tween-20, ProClin 300 with the final concentration of 0.01% (m/V) and BSA with the final concentration of 1% (m/V) are added, after dissolution, the pH value is adjusted to 7.2, then the double distilled water is added to 1L, mixing is carried out uniformly, a filter membrane with the diameter of 0.22 mu m is used for filtration and sterilization, quantitative subpackaging) for 300 mu L/hole, and incubation is carried out for 120 minutes in a wet box with the temperature of 37 ℃ (factory production, and the air humidity is kept between 65 and 70 percent). The blocking solution was removed and discarded, and the cells were washed with washing solution 3 times at 300. mu.L/well and allowed to stand for 30 seconds each time.
1.4 drying the ELISA plate, putting the ELISA plate on a frame, and drying the ELISA plate for 30 minutes at 37 ℃.
1.5 bagging, filling the enzyme label plate and the drying agent into an aluminum foil bag, and sealing after vacuum.
2 preparation of Positive control
2.1 animals for preparation: 3 piglets of 3 days old are screened by PEDV, CSFV, FMDV, PRV, PCV2, PRRSV and TGEV antibodies corresponding to blood collected by sows in the first 7 days of delivery, and healthy piglets born by all negative sows are selected;
2.2 preparation of immunogen: taking a commercial porcine pseudorabies attenuated vaccine, and diluting according to a specification;
2.3 immunization program: performing neck intramuscular multipoint injection of 1mL of recombinant vaccine on 3 pigs, performing secondary immunization 14 days later, wherein the method and the dosage are the same as those of the first immunization, collecting blood every 7 days after the secondary immunization, and detecting a serum PRV antibody by adopting a neutralization method;
2.4 potency assay: adopting a neutralization method to measure the serum titer, and selecting pigs with PRV (porcine reproductive and respiratory syndrome) neutralizing antibodies not less than 1:100 for preparing positive serum;
2.5 Positive serum preparation: carrying out carotid bleeding on experimental pigs meeting the conditions, storing blood of each pig in a sterilized triangular flask, transferring separated serum into a centrifugal bottle, centrifuging for 5 minutes at 3000r/min, taking supernatant, mixing the supernatant uniformly, filtering and sterilizing by a 0.22-micron filter membrane, and quantitatively subpackaging by 1 mL/tube;
2.6 Positive control preparation: diluting qualified positive serum into 1:100, 1:200, 1:400, 1:800, 1:1600 and 1:3200 by sample diluent, detecting each dilution with the manufactured kit, selecting the highest dilution multiple with OD450nm value not less than 1.1, diluting the positive serum by the sample diluent according to the dilution multiple, mixing uniformly, filtering and sterilizing by using a 0.22 mu m filter membrane, and quantitatively subpackaging with 1 mL/tube or 3 mL/tube.
2.7 test of Positive control Positive serum prepared 2.6 was tested using the kit of the present invention, wherein OD450nm should be between 0.9-1.5. However, according to the requirement of 2.6, the OD450nm value of the positive control is preferably between 1.1 and 1.2 in combination with the actual detection requirement of the kit.
Preparation of negative control
3.1 animals for preparation: 3 piglets of 3 days old are screened by PEDV, CSFV, FMDV, PRV, PCV2, PRRSV and TGEV antibodies corresponding to blood collected by sows at 7 days before delivery, and healthy piglets born by all negative sows are selected.
3.2 serum preparation and packaging: and (3) carrying out carotid artery exsanguination and death on the pig, placing the blood into a sterilized and clean triangular flask, transferring the separated serum into a centrifugal bottle, centrifuging for 5 minutes at 3000r/min, taking the supernatant, mixing the supernatant uniformly, filtering and sterilizing by using a 0.22-micron filter membrane, and quantitatively subpackaging by 1 mL/tube.
3.3 negative control preparation: diluting the negative serum by 100 times with the sample diluent, mixing well, filtering with 0.22 μm filter membrane for sterilization, and quantitatively subpackaging with 1 mL/tube or 3 mL/tube.
3.4 testing of negative controls: the negative serum prepared by 3.3 is detected by using the kit prepared by the utility model, and the OD450nm value is less than 0.2. However, in combination with the actual detection requirements of the kit, preferably, the OD450nm values of the negative controls are all less than 0.1.
4 preparation of sample Diluent 0.27g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 3.58g of disodium hydrogen phosphate dodecahydrate and 8.0g of sodium chloride are weighed and dissolved in 800mL of double distilled water, 0.05% of Tween-20 (V/V), 0.1% of preservative ProClin 300 sigma (V/V) and 2% of BSA (m/V) are added to the solution to be 1L, the mixture is uniformly mixed, filtered and sterilized by a 0.22 mu m filter membrane, and quantitatively packaged.
5 preparation of concentrated washing solution (25X) 6.8g of monopotassium phosphate, 5g of potassium chloride, 89.5g of disodium hydrogen phosphate dodecahydrate, and 198.7g of sodium chloride were weighed, dissolved in 800mL of double distilled water, 1.25% of Tween-20 (V/V) and a preservative ProClin 300(V/V) at a final concentration of 0.1% were added to 1L of double distilled water, mixed well, filtered through a 0.22 μm filter membrane for sterilization, and quantitatively dispensed.
Preparation of 6 goat anti-pig IgG enzyme-labeled antibody
6.1 sources were purchased from Saimer Feishale science (China) Co.
6.2 preparation of goat anti-pig IgG enzyme-labeled antibody sample diluent horse radish peroxidase-labeled goat anti-pig IgG is diluted at 1:20,000(V/V), phenol red with final concentration of 0.01g/L and 0.22 μm filter membrane are added for filtration sterilization, and the mixture is quantitatively packaged.
Preparation and inspection of 7 color developing solution
7.1 sources were purchased from Biotech, Inc. of Solebao, Beijing.
7.2 preparing single-component TMB color developing solution or other general color developing solution, and quantitatively packaging.
Preparation and inspection of 8 stopping solution
8.1 from the national drug group.
8.2 preparation of stop solution 21.74mL of concentrated sulfuric acid was slowly added to a beaker containing 178.26mL of double distilled water, stirred and mixed well, and then quantitatively dispensed.
9 preparation and inspection of quality control product
9.1 quality control 1 is prepared from positive serum, the positive serum qualified in the test is diluted into 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and the like by sample diluent, each dilution is detected by using the manufactured kit (containing positive control and negative control), the highest dilution multiple with the S/P value of 1.5-2.0 is selected, the positive serum is diluted by the sample diluent according to the dilution multiple, the positive serum is uniformly mixed, filtered and sterilized by a 0.22 mu m filter membrane, and quantitatively subpackaged by 1 mL/tube or 3 mL/tube.
9.2 quality control 2 is prepared from positive serum, the positive serum qualified in the test is diluted into 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 and the like by sample diluent, each dilution is detected by using the manufactured kit (containing positive control and negative control), the highest dilution multiple with the S/P value of 0.5-1.0 is selected, the positive serum is diluted by the sample diluent according to the dilution multiple, the positive serum is uniformly mixed, filtered and sterilized by a 0.22 mu m filter membrane, and quantitatively subpackaged by 1 mL/tube or 3 mL/tube.
9.3 preparation of quality control 3 derived from negative serum, test qualified negative serum was diluted with sample diluent 1:5, 1:10, 1:20, 1:40, 1:80, etc., each dilution was tested with the manufactured kit (containing positive control, negative control), the highest dilution factor with S/P value less than 0.35 was selected, the positive serum was diluted with sample diluent by this dilution factor, mixed well, sterilized by filtration with 0.22 μm filter membrane, and quantitatively dispensed at 1 mL/tube or 3 mL/tube.
10 kit Assembly
10.1 the components of each qualified kit are assembled according to the following table
10.2 the kit is packaged in a proper external packing box and is labeled, and the label comprises information such as identification name, batch number, production date, validity period, production unit and the like.
11 methods and determinations
11.1 methods of use
11.1.1 preparation of the Material
11.1.1.1 the kit to be detected comprises an antigen coated plate, a positive control, a negative control, a sample diluent, a concentrated washing solution, a goat anti-pig IgG enzyme-labeled antibody, a developing solution, a stop solution, a quality control product 1, a quality control product 2 and a quality control product 3.
11.1.1.2 microplate reader, pipettor, timer, and serum to be tested.
11.1.2 preparation of reagents
11.1.2.1 before the reagents are ready for use, all reagents and samples are returned to room temperature (15-25 deg.C) and the reagents are mixed by gentle rotation or shaking.
11.1.2.2 washing solution preparation 1 part of concentrated washing solution is added into 24 parts of double distilled water and mixed evenly. The prepared washing liquid is used up within 3 days.
11.1.2.3 dilution of serum to be tested the serum to be tested was diluted 1:100(V/V) with the sample diluent.
11.1.3 test
11.1.3.1 sample application
Taking a detachable coating plate according to the number of samples to be detected, horizontally placing the detachable coating plate on a table top, adding 100 mu L/hole of diluted serum to be detected, simultaneously setting 2 holes for positive control and negative control, and 1 hole for quality control 1, quality control 2, quality control 3 and blank control; the positions of addition of the control sample and the sample to be tested on the coated plate are shown in the following figure.
(Note: P is a positive control, N is a negative control, and the position of the sample addition can be adjusted according to the number of the samples to be detected)
11.1.3.2 incubation: incubating in 37 ℃ incubator for 30 minutes;
11.1.3.3 washing: throwing off liquid in the holes, adding washing liquid, washing for 3-5 times at a concentration of 300 mu L/hole, standing for 30 seconds each time, throwing off the liquid in the holes, and beating to dry;
11.1.3.4 secondary antibody incubation: adding goat anti-pig IgG enzyme-labeled antibodies into corresponding holes respectively, and putting the holes at 100 mu L/hole into a 37 ℃ incubator for incubation for 30 minutes;
11.1.3.5 washing: throwing off liquid in the holes, adding washing liquid, washing for 3-5 times at a concentration of 300 mu L/hole, standing for 30 seconds each time, throwing off the liquid in the holes, and beating to dry;
11.1.3.6 color development: adding color development liquid with the concentration of 100 mu L/hole, and placing the mixture in an incubator at 37 ℃ for incubation for 10 minutes in a dark place;
11.1.3.7 terminating: adding stop solution, 50 mu L/hole, slightly shaking and mixing uniformly;
11.1.3.8 reading: after the stop solution is added, immediately placing the coated plate in an enzyme-linked immunosorbent assay, and reading the OD450nm value under the wavelength of 450 nm;
11.1.3.9S/P value calculation: calculating the S/P value according to the following calculation formula:
11.1.3.10 test validity judgment: the reading of OD450nm of each hole of the positive control hole is more than 0.5, the maximum difference value among the holes is less than 0.3, the reading of OD450nm of each hole of the negative control hole is less than 0.3, the reading of blank control OD450nm is less than 0.1, the S/P value of the quality control product 1 is between 1.5 and 2.0, the S/P value of the quality control product 2 is between 0.5 and 1.0, and the S/P value of the quality control product 3 is less than 0.35;
11.1.3.11 judging the result: when the S/P value is more than 0.399, the test result is positive; when the S/P value is less than or equal to 0.399, the result is judged to be negative.
12 notes on
The 12.1 kit should be transported and stored at 2-8 ℃.
12.2 during storage, all the panels must be sealed with a sealing film to prevent moisture damage to the coated panels.
12.3 the description is carefully read.
12.4 the substrate solution is not exposed to strong light and oxides. All reagents are taken out and then are not required to be added back into the bottle.
12.5 not to use expired components or mix different batches of reagents.
12.6 when the concentrated washing solution was diluted, if crystals were observed, it was dissolved at 37 ℃ and then used.
12.7 Care was taken about the loading and washing process to ensure the accuracy of the assay. The liquid cannot be sucked by mouth.
12.8 the serum to be tested is not used for detection when it is rotten.
12.9 the inspection vessel must be cleaned and handled to avoid contact with metallic objects.
12.10 should be performed strictly in accordance with the kit instructions, strictly following the times and temperatures specified for the individual operating procedures.
13 storage and effective period of 2-8 ℃, and the effective period is 12 months.
14 specification (1)2 plates (192 holes/box) (2)5 plates (480 holes/box).
Example 4 Performance evaluation of porcine pseudorabies gB & gD antibody detection kit
5 batches of kits were prepared according to example 3, and performance evaluations were performed using these 5 batches of kits.
1 specificity test 3 batches of kits were randomly selected, 7 specific quality control serum samples were used to perform specificity test on the kits, and the specific results are shown in the following table:
2 repeatability test
2.1 Intra-batch reproducibility test the selected clinical samples were tested for intra-and inter-batch reproducibility using 5-batch kits. The in-batch repeatability is that 3 quality control samples are respectively detected by 5 batches of kits according to the specification, each sample is repeatedly detected for 4 times, the average value, the standard deviation and the variation coefficient of each serum are respectively calculated, the in-batch variation coefficient is calculated after the result is measured, the result shows that the in-batch variation coefficient of the detection result of the 5 batches of kits is less than 10 percent, the in-batch variation coefficient meets the requirement, and the specific data are shown in the following table.
2.2 batch-to-batch repeatability test 1 box is randomly extracted from each batch of kit, 3 quality control samples are respectively detected according to the instruction, each sample is repeatedly detected for 4 times, the average value, the standard deviation and the variation coefficient of the S/P value of each serum are respectively calculated, and the batch variation coefficient is calculated after the result is measured. The results show that the coefficient of variation of each sample is less than 10%, and the specific data meet the requirements, which are shown in the following table.
3 sensitivity test 5 dilutions of sensitive quality control sera (i.e., positive sera in example 3) were tested separately using 1:100, 1:200, 1:400, 1:800 and 1: 1600. The results are shown in the following table:
4, performing aseptic inspection on the positive and negative controls, the sample diluent, the concentrated washing solution, the goat anti-pig IgG enzyme labeled antibody, the quality control product 1, the quality control product 2 and the quality control product 3 in the kit according to the appendix of the existing Chinese veterinary pharmacopoeia. The results are shown in the following table.
5 clinical sample detection, a batch of the kit is selected for carrying out PRV-gB & PRV-gD antibody detection on a clinically collected serum sample, and the result shows that the comprehensive coincidence rate of the kit and an imported kit is 96.6%, and the specific result is shown in the following table.
The above only is the embodiment of the present invention, not limiting the patent scope of the present invention, all the equivalent structures or equivalent processes that are used in the specification and the attached drawings or directly or indirectly applied to other related technical fields are included in the patent protection scope of the present invention.
Claims (9)
1. A detection kit for porcine pseudorabies virus gB & gD antibodies is characterized in that the kit can detect a porcine pseudorabies virus gB antigen antibody and a porcine pseudorabies virus gD antigen antibody simultaneously, and comprises a kit body (1), wherein an antigen coating plate storage tank (2), a quality control product storage tank (3), a positive control liquid pore tank (4), a negative control liquid pore tank (5), a goat anti-porcine IgG enzyme labeled antibody pore tank (6), a concentrated washing liquid pore tank (7), a sample diluent pore tank (8), a developing liquid pore tank (9), a stopping liquid pore tank (10) and an operation instruction storage tank (11) are arranged in the kit body (1); an antigen coating reaction plate is placed in the antigen coating plate storage tank (2), a reagent bottle filled with corresponding solution is placed in each hole tank (3-10), and an operation instruction is placed in the operation instruction storage tank (11).
2. The kit according to claim 1, wherein the antigen-coated plate is an ELISA plate coated with PRV-gB protein and PRV-gD protein at the same time, the coating concentration of the PRV-gB protein and the PRV-gD protein on the antigen-coated plate is 100 ng/well/100 μ L, and each kit contains 2 or 5 antigen-coated plates.
3. The kit of claim 1, wherein the sample diluent is a 1 x PBST solution containing 2% BSA, and each kit contains 80 mL/vial or 200 mL/vial.
4. The kit of claim 1, wherein the concentrated wash solution is a 25 x PBST solution diluted to a 1 x PBST solution prior to use, each kit containing 30 mL/vial or 60 mL/vial.
5. The kit of claim 1, wherein the goat anti-pig IgG enzyme-labeled antibody is obtained by 20000-fold dilution of a commercial goat anti-pig IgG enzyme-labeled antibody with a sample diluent, and each kit contains 25 mL/bottle or 60 mL/bottle.
6. The kit according to claim 1, wherein the color developing solution is a TMB single-component solution, and each kit contains 25 mL/bottle or 60 mL/bottle.
7. The kit of claim 1, wherein the stop solution is 2M H2SO4Solutions, each kit contained 12 mL/vial or 30 mL/vial.
8. The kit according to claim 1, wherein the positive control is positive serum with a PRV antibody OD450nm value of 0.9-1.5; the negative control is negative serum with PRV antibody OD450nm value less than 0.2; each kit positive control and negative control contained 1 mL/tube or 3 mL/tube.
9. The kit according to claim 1, wherein the quality control product comprises a quality control product 1, a quality control product 2 and a quality control product 3, and the quality control product 1 is strong positive serum with an S/P value of 1.5-2.0; the quality control product 2 is weak positive serum with an S/P value of 0.5-1.0; the quality control product 3 is negative serum with an S/P value less than 0.35; each of the kit quality control 1, quality control 2 and quality control 3 contains 1 mL/tube or 3 mL/tube.
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