CN110554201A - Kit for detecting porcine pseudorabies virus antibody - Google Patents
Kit for detecting porcine pseudorabies virus antibody Download PDFInfo
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- CN110554201A CN110554201A CN201910885694.4A CN201910885694A CN110554201A CN 110554201 A CN110554201 A CN 110554201A CN 201910885694 A CN201910885694 A CN 201910885694A CN 110554201 A CN110554201 A CN 110554201A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/032—Pseudorabies virus, i.e. Aujetzky virus
Abstract
The invention discloses a kit for detecting a porcine pseudorabies virus antibody, and belongs to the technical field of biological detection. The kit for detecting the porcine pseudorabies virus antibody comprises two parts of reaction liquid and a reaction device; the kit can be used for rapidly analyzing the PRV-gE/gB antibody in serum, plasma, peripheral blood or venous blood, and has the advantages of high sensitivity, strong specificity, good repeatability and the like; the method has good stability, linearity and accuracy, can be used for distinguishing wild virus infection and monitoring the level of vaccine antibodies, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting a porcine pseudorabies virus antibody.
Background
Pseudorabies Virus (PR) is an acute infectious disease of a wide variety of domestic and wild animals caused by Pseudorabies Virus (PRV) infection. The pig is the most main storage host and infection source of the pseudorabies, and the pseudorabies is epidemic in the pig, can cause abortion and stillbirth of pregnant sows, sterility of boars, diarrhea, nervous symptoms and mass death of newborn piglets, dyspnea and growth retardation of fattening pigs and the like, and is one of major infectious diseases harming the global pig industry. PRV decontamination is achieved in succession in European and American countries, UK, Denmark, the Netherlands, Belgium and the United states. Since 2011 in China, the PRV gE positive rate is increased year by year, and the prevention and control strength is higher and higher. China already starts a PRV purification plan, and the PRV purification is required in a national core breeding farm in 2020.
In the interaction between PRV and host, the gB structural protein is very conservative, has good immunogenicity and very clear epitope, and can be used as an ideal antigen for serological detection. The gE protein is a very important glycoprotein of PRV, plays an important role in determining PRV virulence, neurotropic property and the like, but is not a protein necessary for PRV proliferation and is often used as a deletion candidate gene. When the PRV antibody is detected, two antibodies gE and gB need to be monitored simultaneously, PRV gE antibody detection can effectively distinguish wild virus infection, and vaccine antibody level can be monitored while distinguishing wild virus infection by combining with gB antibody detection. Because gB and gE are simultaneously positive when the wild virus is infected, if the wild virus is not infected, after the gE gene deletion vaccine is immunized, the gE is negative, and the gB is positive. If only the gB antibody is detected, the high level of the gB antibody in the detection result is probably caused by wild virus infection; if only gE is detected to be positive, which indicates wild virus infection, a gB antibody level is also detected to determine an immunization scheme in order to protect susceptible swineries.
The PRV differential diagnosis method comprises PCR, ELISA, IPMA, immunochromatography and the like, and the PCR and the IPMA are difficult to popularize and detect in situ due to complex operation and need of professional knowledge and special equipment. Monoclonal antibodies have now become a good tool for the study of various diseases. Monoclonal antibodies, because of their strong specificity, high homogeneity, easy standardization and mass production, have been widely used in the research of various complex phenomena in immunology, oncology, genetics and other disciplines, as well as in technical practices such as diagnosis, therapy, purification of biological materials, and many diagnostic applications. The colloidal gold immunochromatography has the advantages of rapidness, simplicity and convenience, but also has the defects of low sensitivity, poor accuracy and repeatability, difficulty in quantification and the like. In recent years, with the development of POCT technology, fluorescence immunochromatography technology has a great deal of development, the technology overcomes the defects of GICA, retains the advantages of rapidness, simplicity and convenience, and has good application due to the advantages of high sensitivity and high specificity.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a kit for detecting a porcine pseudorabies virus antibody, which can be used for quickly analyzing a PRV antibody in peripheral blood or venous blood and has the advantages of high sensitivity, strong specificity and good repeatability.
In order to achieve the purpose, the invention adopts the technical scheme that:
A kit for detecting a porcine pseudorabies virus antibody comprises a reaction solution and a reaction device; the reaction device comprises a box body (1) with an open top, a buckle cover (2) is buckled on the open top of the box body, a test paper plate (3) is installed in the buckle cover (2), the buckle cover (2) is obliquely arranged, the low end of the buckle cover (2) is connected to the box body (1) through an elastic sheet (4), a reaction key (5) is arranged at the high end of the buckle cover (2), and a sample adding hole (6) is formed in the buckle cover (2) at a position close to the reaction key (5); the test paper board (3) comprises a PVC bottom lining board (7), a nitrocellulose membrane (8), a water absorption pad (9), a sample processing pad (14) and the nitrocellulose membrane (8), the water absorption pad (9) and the sample processing pad (14) are both adhered to the PVC bottom lining plate (7), the nitrocellulose membrane (8) is positioned in the middle, the sample processing pad (14) and the water absorption pad (9) are respectively lapped at the high end and the low end of the nitrocellulose membrane (8), the middle part of the nitrocellulose membrane (8) is provided with a detection line (L-T1) coated with porcine pseudorabies virus gE recombinant protein, a detection line (L-T2) coated with porcine pseudorabies virus gB recombinant protein and a quality control line (L-C) coated with mouse anti-sheep IgG secondary antibody in parallel, and the buckle cover (2) is provided with an observation window (13) which is used for directly facing the middle part of the cellulose membrane (8) by nitric acid; a reagent groove (10) is arranged in the box body (1), the reagent groove (10) is positioned right below one end, attached with a sample processing pad (14), of the PVC bottom lining plate (7), reaction liquid is contained in the reagent groove (10), a sealing film (11) is packaged on an opening of the reagent groove (10), and a hook (12) capable of puncturing the sealing film (11) when a reaction key is pressed down is connected to the bottom surface of the PVC bottom lining plate (7).
On the basis of the scheme, the sample processing pad (14) comprises a whole blood filter membrane processed by the processing fluid and a glass fiber membrane processed by the erythrocyte monoclonal antibody processing fluid, wherein the glass fiber membrane is arranged on the outer layer, and the whole blood filter membrane is arranged on the inner layer; and sealing and drying the glass fiber membrane and the whole blood filter membrane at the room temperature under the condition that the relative humidity is not higher than 30%.
On the basis of the scheme, the treatment solution is Tri-Cl buffer solution with 0.02M, pH7.0-8.0 and containing 0.1-1% Tween-20, 0.1-5% BSA and 0.5-2% blocking agent; the erythrocyte monoclonal antibody treating solution is Tri-Cl buffer solution which contains 0.1-1% Tween-20, 0.1-5% BSA and 0.5-5mg/mL anti-Hb monoclonal antibody and has the pH value of 0.02M and 7.0-8.0.
On the basis of the scheme, the detection line L-T1, the detection line L-T2 and the quality control line L-C are formed by a film spraying instrument by parallel scribing on a nitrocellulose membrane at the spraying speed of 1-2 mu L/cm and the interval of 0.5cm, and solutions used by the film spraying instrument are a porcine pseudorabies virus gE recombinant protein solution, a porcine pseudorabies virus gB recombinant protein solution and a mouse anti-sheep IgG secondary antibody solution respectively; the porcine pseudorabies virus gE recombinant protein solution, the porcine pseudorabies virus gB recombinant protein solution and the mouse anti-sheep IgG secondary antibody solution are 1-2mg/mL coating solutions prepared from the porcine pseudorabies virus gE recombinant protein, the porcine pseudorabies virus gB recombinant protein and the mouse anti-sheep IgG secondary antibody respectively by using Tri-Cl buffer solutions with the pH value of 0.02M and the pH value of 7.0, and the cellulose nitrate membrane after line spraying is dried and sealed for storage under the condition that the relative humidity at room temperature is not higher than 30%.
On the basis of the scheme, the preparation method of the reaction liquid in the reagent tank comprises the following steps:
Step 1) centrifuging fluorescent microspheres at 4000rpm for 30min, collecting precipitates, re-suspending the precipitates by using 0.01M borate buffer solution with the pH value of 5.0, adjusting the concentration of the microspheres to OD 450 to be 0.2, respectively adding 200 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the concentration of 10mg/mL and 80 mu L of N-hydroxysuccinimide with the concentration of 30mg/mL into each milliliter of the fluorescent microsphere re-suspension, uniformly shaking the mixture, and activating the mixture at room temperature for 45min to obtain a primary product;
Step 2) centrifuging the primary product at 4000rpm for 30min, resuspending the precipitate with 0.05M CBS solution with pH9.5, adjusting the concentration of the microsphere to OD 450nm to 0.2, dialyzing the goat anti-pig IgG antibody with 0.025M carbonate buffer solution with pH9.5 overnight, adding the goat anti-pig IgG antibody into the fluorescent microsphere resuspension solution according to the proportion of 600 plus 800 mu g/mg (namely adding 600 plus 800 mu g of goat anti-pig IgG antibody into each mg of fluorescent microspheres), stirring overnight at 4 ℃, adding NaBH 3 CN to the final concentration of 5mM, and reacting for 1.5h to obtain a medium-grade product;
And step 3) adding equal volume of blocking solution into the intermediate product, blocking for 1h at 37 ℃, washing for 3 times with Tri-Cl buffer solution with the concentration of 0.1M and the pH value of 7.8, washing for 20min each time, finally, resuspending with 100 mu L of sample buffer solution to prepare the fluorescent microsphere labeled goat anti-pig IgG antibody reaction solution, and storing for later use at 4 ℃.
On the basis of the scheme, the blocking solution is Tri-Cl buffer solution which contains 2-5% BSA and is 0.1M and has the pH value of 7.0-8.0; the sample buffer solution is Tri-Cl buffer solution which contains 1-2% BSA, 0.1-0.5% PEG2000, 2-5% trehalose and is 0.02M and pH7.0-8.0.
The use process of the kit comprises the following steps: dropping a certain amount of sample to be detected (such as 10 muL of serum or 20 muL of whole blood) into the sample adding hole, adsorbing the sample by the sample processing pad, pressing down a reaction key, piercing a sealing film of the reagent tank by a barb, immersing the sample processing pad into the reagent tank, allowing a target antibody in the sample to perform specific reaction with a reaction solution in the reagent tank to generate an antigen-antibody complex containing a fluorescent marker, allowing the complex to perform forward chromatography on the nitrocellulose membrane under capillary action, allowing the complex to react with an antigen at the detection line and an antibody at the quality control line, and judging the level of the PRV-gE/gB antibody in the sample according to a fluorescence signal of the reaction area.
the invention has the beneficial effects that: the sample to be detected is specially pretreated by a sample treatment pad before reaction, can be used for quickly analyzing PRV-gE/gB antibodies in serum, plasma, peripheral blood, venous blood and the like, has the advantages of quickness, simplicity and convenience of a colloidal gold immunochromatography and high sensitivity, high specificity of an ELISA technology, and has the advantages of quickness, convenience, accuracy, strong anti-interference capability and the like; the unique fluorescent antibody reagent is independently designed, the defects of unstable reagent performance, poor accuracy and repeatability and the like caused by the traditional dry coating are overcome, the stability of the product is greatly improved, the detection result is more accurate and reliable, the reagent can be used for distinguishing wild virus infection and monitoring the level of the vaccine antibody, and the reagent has a good application prospect.
Drawings
FIG. 1 is a schematic structural diagram of a kit according to the present invention;
Fig. 2 is a schematic structural view of a test sheet according to the present invention.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
example 1
The kit for detecting the porcine pseudorabies virus antibody is shown in figures 1 and 2 and comprises a reaction solution and a reaction device; the reaction device comprises a box body 1 with an opening at the top, a buckle cover 2 is buckled on the opening of the box body, a test paper board 3 is arranged in the buckle cover 2, the buckle cover 2 is obliquely arranged, the lower end of the buckle cover 2 is connected to the box body 1 through an elastic sheet 4, a reaction key 5 is arranged at the high end of the buckle cover 2, and a sample adding hole 6 is formed in the buckle cover 2 at a position close to the reaction key 5; the test paper board 3 comprises a PVC bottom lining board 7, a nitrocellulose membrane 8, a water absorption pad 9 and a sample processing pad 14, wherein the nitrocellulose membrane 8, the water absorption pad 9 and the sample processing pad 14 are all adhered to the PVC bottom lining board 7, the nitrocellulose membrane 8 is positioned in the middle, the sample processing pad 14 and the water absorption pad 9 are respectively lapped at the high end and the low end of the nitrocellulose membrane 8, the middle of the nitrocellulose membrane 8 is parallelly provided with a detection line L-T1 coated with porcine pseudorabies virus gE recombinant protein, a detection line L-T2 coated with porcine pseudorabies virus gB recombinant protein and a quality control line L-C coated with mouse anti-sheep IgG secondary antibody, and the buckle closure 2 is provided with an observation window 13 which is opposite to the middle of the nitrocellulose membrane 8; the reagent box comprises a box body 1, a reagent groove 10 is arranged in the box body 1, the reagent groove 10 is positioned right below one end, attached with a sample processing pad 14, of a PVC bottom lining plate 7, reaction liquid is contained in the reagent groove 10, a sealing film 11 is packaged on an opening of the reagent groove 10, and a hook 12 capable of puncturing the sealing film 11 when a reaction key is pressed down is connected to the bottom surface of the PVC bottom lining plate 7.
the sample processing pad (14) comprises a whole blood filter membrane processed by the processing fluid and a glass fiber membrane processed by the erythrocyte monoclonal antibody processing fluid, the glass fiber membrane is arranged on the outer layer, and the whole blood filter membrane is arranged on the inner layer; and sealing and drying the glass fiber membrane and the whole blood filter membrane at the room temperature under the condition that the relative humidity is not higher than 30%.
The treatment solution is Tri-Cl buffer solution which contains 0.1-1% Tween-20, 0.1-5% BSA and 0.5-2% blocking agent and has the pH value of 7.0-8.0; the erythrocyte monoclonal antibody treating solution is Tri-Cl buffer solution which contains 0.1-1% Tween-20, 0.1-5% BSA and 0.5-5mg/mL anti-Hb monoclonal antibody and has the pH value of 0.02M and 7.0-8.0.
the detection line L-T1, the detection line L-T2 and the quality control line L-C are formed by a film spraying instrument by parallel scribing on a nitrocellulose membrane at the spraying speed of 1-2 mu L/cm and the interval of 0.5cm, and solutions used by the film spraying instrument are a porcine pseudorabies virus gE recombinant protein solution, a porcine pseudorabies virus gB recombinant protein solution and a mouse anti-sheep IgG secondary antibody solution respectively; the porcine pseudorabies virus gE recombinant protein solution, the porcine pseudorabies virus gB recombinant protein solution and the mouse anti-sheep IgG secondary antibody solution are 1-2mg/mL coating solutions prepared from the porcine pseudorabies virus gE recombinant protein, the porcine pseudorabies virus gB recombinant protein and the mouse anti-sheep IgG secondary antibody respectively by using Tri-Cl buffer solutions with the pH value of 0.02M and the pH value of 7.0, and the cellulose nitrate membrane after line spraying is dried and sealed for storage under the condition that the relative humidity at room temperature is not higher than 30%.
The preparation method of the reaction liquid in the reagent tank comprises the following steps:
Step 1) centrifuging fluorescent microspheres at 4000rpm for 30min, collecting precipitates, resuspending the precipitates by using 0.01M borate buffer solution with the pH of 5.0, adjusting the concentration of the microspheres to OD 450 ═ 0.2, respectively adding 200 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the concentration of 10mg/mL and 80 mu L of N-hydroxysuccinimide with the concentration of 30mg/mL into each milliliter of the fluorescent microsphere resuspension solution, shaking and uniformly mixing the mixture, and activating the mixture at room temperature for 45min to obtain a primary product;
Step 2) centrifuging the primary product at 4000rpm for 30min, resuspending the precipitate with 0.05M CBS solution with pH9.5, adjusting the concentration of the microsphere to OD 450nm to 0.2, dialyzing the goat anti-pig IgG antibody with 0.025M carbonate buffer solution with pH9.5 overnight, adding the goat anti-pig IgG antibody into the fluorescent microsphere resuspension solution according to the proportion of 600 plus 800 mu g/mg (namely adding 600 plus 800 mu g of goat anti-pig IgG antibody into each mg of fluorescent microspheres), stirring overnight at 4 ℃, adding NaBH 3 CN to the final concentration of 5mM, and reacting for 1.5h to obtain a medium-grade product;
and step 3) adding equal volume of blocking solution into the intermediate product, blocking for 1h at 37 ℃, washing for 3 times with 0.1M Tris-Cl buffer solution with pH7.8 for 20min each time, finally, resuspending with 100 mu L of sample buffer solution to prepare fluorescent microsphere labeled goat anti-pig IgG antibody reaction solution, and storing for later use at 4 ℃.
The confining liquid is Tri-Cl buffer solution which contains 2-5% BSA and is 0.1M and pH7.0-8.0; the sample buffer solution is Tri-Cl buffer solution which contains 1-2% BSA, 0.1-0.5% PEG2000, 2-5% trehalose and is 0.02M and pH7.0-8.0.
When the kit is used for detecting PRVgE and PRVgB, the detection result of a fluorescence immunoassay analyzer shows that the linear fitting relationship is good in the range of 0.5-100IU/mL, and r is greater than 0.9900; and (3) repeatedly testing the negative serum for 20 times, calculating the T/C mean value, and substituting the mean value into a dose-reaction curve to obtain that the lowest detection limits of the kit for detecting PRVgE and PRVgB are both 0.45 IU/mL. The PRVgE and PRVgB calibrators were diluted to 1IU/mL and 2IU/mL, and the measurements were repeated 10 times with the kit, respectively, to calculate the coefficient of variation, with CV values of both concentrations being less than 10% (6.5% and 4.7% for gE, respectively, and 5.01% and 5.6% for gB, respectively).
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. The person skilled in the art can apply it to the detection of other molecules, cells, microorganisms. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (6)
1. A kit for detecting porcine pseudorabies virus antibody is characterized in that: comprises two parts of reaction liquid and a reaction device; the reaction device comprises a box body (1) with an open top, a buckle cover (2) is buckled on the open top of the box body, a test paper plate (3) is installed in the buckle cover (2), the buckle cover (2) is obliquely arranged, the low end of the buckle cover (2) is connected to the box body (1) through an elastic sheet (4), a reaction key (5) is arranged at the high end of the buckle cover (2), and a sample adding hole (6) is formed in the buckle cover (2) at a position close to the reaction key (5); the test paper board (3) comprises a PVC bottom lining board (7), a nitrocellulose membrane (8), a water absorption pad (9), a sample processing pad (14) and the nitrocellulose membrane (8), the water absorption pad (9) and the sample processing pad (14) are both adhered to the PVC bottom lining plate (7), the nitrocellulose membrane (8) is positioned in the middle, the sample processing pad (14) and the water absorption pad (9) are respectively lapped at the high end and the low end of the nitrocellulose membrane (8), the middle part of the nitrocellulose membrane (8) is provided with a detection line (L-T1) coated with porcine pseudorabies virus gE recombinant protein, a detection line (L-T2) coated with porcine pseudorabies virus gB recombinant protein and a quality control line (L-C) coated with mouse anti-sheep IgG secondary antibody in parallel, and the buckle cover (2) is provided with an observation window (13) which is used for directly facing the middle part of the cellulose membrane (8) by nitric acid; a reagent groove (10) is arranged in the box body (1), the reagent groove (10) is positioned right below one end, attached with a sample processing pad (14), of the PVC bottom lining plate (7), reaction liquid is contained in the reagent groove (10), a sealing film (11) is packaged on an opening of the reagent groove (10), and a hook (12) capable of puncturing the sealing film (11) when a reaction key is pressed down is connected to the bottom surface of the PVC bottom lining plate (7).
2. The kit for detecting porcine pseudorabies virus antibody according to claim 1, characterized in that: the sample processing pad (14) comprises a whole blood filter membrane processed by the processing fluid and a glass fiber membrane processed by the erythrocyte monoclonal antibody processing fluid, the glass fiber membrane is arranged on the outer layer, and the whole blood filter membrane is arranged on the inner layer; and sealing and drying the glass fiber membrane and the whole blood filter membrane at the room temperature under the condition that the relative humidity is not higher than 30%.
3. The kit for detecting porcine pseudorabies virus antibody according to claim 2, characterized in that: the treatment solution is Tri-Cl buffer solution which contains 0.1-1% Tween-20, 0.1-5% BSA and 0.5-2% blocking agent and has the pH value of 7.0-8.0; the erythrocyte monoclonal antibody treating solution is Tri-Cl buffer solution which contains 0.1-1% Tween-20, 0.1-5% BSA and 0.5-5mg/mL anti-Hb monoclonal antibody and has the pH value of 0.02M and 7.0-8.0.
4. The detection kit for porcine pseudorabies virus antibody according to claim 1, characterized in that: the detection line L-T1, the detection line L-T2 and the quality control line L-C are formed by a film spraying instrument by parallel scribing on a nitrocellulose membrane at the spraying speed of 1-2 mu L/cm and the interval of 0.5cm, and solutions used by the film spraying instrument are a porcine pseudorabies virus gE recombinant protein solution, a porcine pseudorabies virus gB recombinant protein solution and a mouse anti-sheep IgG secondary antibody solution respectively; the porcine pseudorabies virus gE recombinant protein solution, the porcine pseudorabies virus gB recombinant protein solution and the mouse anti-sheep IgG secondary antibody solution are 1-2mg/mL coating solutions prepared from the porcine pseudorabies virus gE recombinant protein, the porcine pseudorabies virus gB recombinant protein and the mouse anti-sheep IgG secondary antibody respectively by using Tri-Cl buffer solutions with the pH value of 0.02M and the pH value of 7.0, and the cellulose nitrate membrane after line spraying is dried and sealed for storage under the condition that the relative humidity at room temperature is not higher than 30%.
5. The detection kit for porcine pseudorabies virus antibody according to any one of claims 1 to 4, characterized in that: the preparation method of the reaction liquid in the reagent tank comprises the following steps:
Step 1) centrifuging fluorescent microspheres at 4000rpm for 30min, collecting precipitates, re-suspending the precipitates by using 0.01M borate buffer solution with the pH value of 5.0, adjusting the concentration of the microspheres to OD 450 to be 0.2, respectively adding 200 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide with the concentration of 10mg/mL and 80 mu L of N-hydroxysuccinimide with the concentration of 30mg/mL into each milliliter of the fluorescent microsphere re-suspension, uniformly shaking the mixture, and activating the mixture at room temperature for 45min to obtain a primary product;
step 2) centrifuging the primary product at 4000rpm for 30min, resuspending the precipitate with 0.05M CBS solution with pH9.5, adjusting the concentration of the microsphere to OD 450nm to 0.2, dialyzing the goat anti-pig IgG antibody with 0.025M carbonate buffer solution with pH9.5 overnight, adding the goat anti-pig IgG antibody into the fluorescent microsphere resuspension solution according to the proportion of 600 plus 800 mu g/mg (namely adding 600 plus 800 mu g of goat anti-pig IgG antibody into each mg of fluorescent microspheres), stirring overnight at 4 ℃, adding NaBH 3 CN to the final concentration of 5mM, and reacting for 1.5h to obtain a medium-grade product;
And step 3) adding equal volume of blocking solution into the intermediate product, blocking for 1h at 37 ℃, washing for 3 times with Tri-Cl buffer solution with the concentration of 0.1M and the pH value of 7.8, washing for 20min each time, finally, resuspending with 100 mu L of sample buffer solution to prepare the fluorescent microsphere labeled goat anti-pig IgG antibody reaction solution, and storing for later use at 4 ℃.
6. the detection kit for porcine pseudorabies virus antibody according to claim 5, characterized in that: the confining liquid is Tri-Cl buffer solution which contains 2-5% BSA and is 0.1M and pH7.0-8.0; the sample buffer solution is Tri-Cl buffer solution which contains 1-2% BSA, 0.1-0.5% PEG2000, 2-5% trehalose and is 0.02M and pH7.0-8.0.
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| CN111537727A (en) * | 2020-06-04 | 2020-08-14 | 湖南康润药业股份有限公司 | Test strip for combined detection of influenza and COVID-19 and application thereof |
| CN111781378A (en) * | 2020-07-13 | 2020-10-16 | 祁寒松 | Colloidal gold test strip for pathogen and antibody detection and differential diagnosis thereof |
| CN112098658A (en) * | 2020-09-16 | 2020-12-18 | 中国海洋大学 | Rapid diagnosis test paper for infection state and immune state of paralichthys olivaceus rhabdovirus disease |
| CN115951049A (en) * | 2022-08-02 | 2023-04-11 | 大连交通大学 | Quantum dot fluorescent microsphere labeled double-flux immunochromatography detection card and application thereof |
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2019
- 2019-09-19 CN CN201910885694.4A patent/CN110554201A/en not_active Withdrawn
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| CN111781378A (en) * | 2020-07-13 | 2020-10-16 | 祁寒松 | Colloidal gold test strip for pathogen and antibody detection and differential diagnosis thereof |
| CN111781378B (en) * | 2020-07-13 | 2023-04-14 | 祁寒松 | Colloidal gold test strip for pathogen and antibody detection and differential diagnosis thereof |
| CN112098658A (en) * | 2020-09-16 | 2020-12-18 | 中国海洋大学 | Rapid diagnosis test paper for infection state and immune state of paralichthys olivaceus rhabdovirus disease |
| CN112098658B (en) * | 2020-09-16 | 2024-04-05 | 中国海洋大学 | Rapid diagnosis test paper for Rhabdoviral disease infection state and immune state |
| CN115951049A (en) * | 2022-08-02 | 2023-04-11 | 大连交通大学 | Quantum dot fluorescent microsphere labeled double-flux immunochromatography detection card and application thereof |
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Application publication date: 20191210 |
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