CN114878819A

CN114878819A - Rapid quantitative detection method for infectious bovine rhinotracheitis virus

Info

Publication number: CN114878819A
Application number: CN202210811780.2A
Authority: CN
Inventors: 刘文晓; 李永清; 段景龙; 程晶; 朱如楠; 江波
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2022-07-12
Filing date: 2022-07-12
Publication date: 2022-08-09
Anticipated expiration: 2042-07-12
Also published as: CN114878819B

Abstract

The invention relates to detection of infectious bovine rhinotracheitis virus, in particular to a rapid quantitative detection method of infectious bovine rhinotracheitis virus. The invention provides a fluorescent microsphere immunochromatographic test strip for detecting IBRV, which comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, wherein the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and stuck on the bottom plate; the detection line is arranged on the nitrocellulose membrane and is coated with an IBRV monoclonal antibody, and the IBRV monoclonal antibody is secreted by a hybridoma cell with the preservation number of CGMCC No. 45040. The test strip can be used for qualitatively or quantitatively detecting IBRV in eye and nose secretion, semen and vaginal secretion of infected cattle, has the advantages of high sensitivity, strong specificity, good stability, high accuracy and high detection speed, and can complete the whole detection within 15 min.

Description

Rapid quantitative detection method for infectious bovine rhinotracheitis virus

Technical Field

The invention relates to detection of infectious bovine rhinotracheitis virus, in particular to a rapid quantitative detection method of infectious bovine rhinotracheitis virus.

Background

Infectious Bovine Rhinotracheitis Virus (IBRV), also known as Bovine herpes virus type I (BHV-1), belongs to the family of herpesviridae, is an important pathogen of cattle and can cause Infectious Bovine Rhinotracheitis (IBR). Infectious bovine rhinotracheitis is an infectious disease with strong heat, acute and latent properties. Since Miller first reported the disease in the United states in 1995, IBR has assumed a global epidemic situation, which causes enormous economic losses to the world cattle industry. The main hazard of the IBRV is that after the virus invades a host, the virus can potentially infect trigeminal ganglia of the host, and when the body is weak or stimulated by stressors, the virus can be activated, proliferated and released into the environment, so that the disease is spread in cattle, and great challenge is brought to the prevention and control of the disease. In view of its harmfulness, the world animal health Organization (OIE) ranks it as a B-type infectious disease, and China also ranks it as a second-type infectious disease. Pathogen diagnosis is an essential means to assess immune efficacy, monitor cattle health and the prevalence of infectious diseases.

In order to effectively prevent and control infectious bovine rhinotracheitis, various IBRV diagnosis technologies are developed, and mainly can be summarized into etiology diagnosis and serological detection. Serological detection techniques are used to detect IBRV antibodies, including neutralization assays and ELISA, among others. The IBRV pathogen diagnosis method mainly comprises virus separation and real-time fluorescence PCR method, and the method has high specificity, but has the defects of complex operation, need of certain detection equipment, time and operation by high-level inspection personnel and the like, and cannot carry out rapid diagnosis in a dairy farm. The immunochromatography technology has the characteristics of simplicity and convenience in operation, rapidness, sensitivity, specificity and the like, is very suitable for being used in farms and quarantine departments, and is widely applied to detection of clinical samples.

The immunochromatography technology marked by fluorescent microspheres is a novel membrane detection technology based on antigen-antibody specific immunoreaction. The technology takes strip-shaped fiber chromatography materials fixed with a detection line (coated antibody or antigen) and a quality control line (anti-antibody) as a stationary phase, a sample liquid as a mobile phase, fluorescent microspheres for marking the antibody or the antigen to be fixed on a binding pad, and an analyte moves on the chromatography strip through capillary action. For macromolecular antigens with multiple antigenic determinants, such as proteins, viruses, pathogenic bacteria and the like, a double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescence labeling antibody under the action of a mobile phase, and then is combined with a coating antibody to form a double-antibody sandwich when reaching a detection line. At present, no report related to an immunochromatographic test strip marked by fluorescent microspheres for detecting IBRV is seen.

Disclosure of Invention

The invention aims to solve the technical problem of realizing the rapid qualitative and quantitative detection of the infectious bovine rhinotracheitis virus.

In order to solve the technical problem, the invention provides a fluorescent microsphere immunochromatographic test strip for detecting IBRV, which comprises a bottom plate, and a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped and stuck on the bottom plate, wherein the binding pad is coated with an IBRV polyclonal antibody marked by fluorescent microspheres; the detection line is arranged on the nitrocellulose membrane and is coated with an IBRV monoclonal antibody, the name of the monoclonal antibody is 1C2, and the monoclonal antibody is secreted by hybridoma with the preservation number of CGMCC number 45040.

In the test strip, in the IBRV polyclonal antibody marked by the fluorescent microspheres, the mass ratio of the fluorescent microspheres to the IBRV polyclonal antibody can be 8: 1-15: 1.

In the test strip, the coating concentration of the IBRV monoclonal antibody can be 450-550 mu g/mL.

In the test paper strip, the combination pad is also coated with chicken IgY marked by fluorescent microspheres; and the nitrocellulose membrane is also provided with a quality control line which is spaced from the detection line by a certain distance, and the quality control line is coated with goat anti-chicken IgY antibody.

In the test strip, in the chicken IgY marked by the fluorescent microspheres, the mass ratio of the fluorescent microspheres to the chicken IgY is 8: 1-15: 1; the coating concentration of the goat anti-chicken IgY antibody is 500-650 mu g/mL.

The invention also provides a method for preparing any test strip, which is characterized by comprising the following steps:

(1) coating the IBRV polyclonal antibody marked by the fluorescent microspheres on a glass cellulose membrane to obtain a binding pad;

(2) carrying out scribing and coating on a nitrocellulose membrane by using an IBRV monoclonal antibody 1C2 to obtain a detection line;

(3) and (3) overlapping and adhering the sample pad, the combined pad obtained in the step (1), the nitrocellulose membrane obtained in the step (2) and the water absorption pad on the bottom plate in sequence along the chromatography direction.

In the method, in the IBRV polyclonal antibody marked by the fluorescent microspheres, the mass ratio of the fluorescent microspheres to the IBRV polyclonal antibody can be 8: 1-15: 1.

In the method, the coating concentration of the IBRV monoclonal antibody is 450-550 mu g/mL.

In the method, the step (1) further comprises coating chicken IgY marked by fluorescent microspheres on the glass cellulose membrane; and (2) scribing and coating the goat anti-chicken IgY antibody on the nitrocellulose membrane at a certain distance from the detection line to obtain a quality control line.

In the method, in the chicken IgY marked by the fluorescent microspheres, the mass ratio of the fluorescent microspheres to the chicken IgY is 8: 1-15: 1; the coating concentration of the goat anti-chicken IgY antibody is 500-650 mu g/mL.

The fluorescent microsphere immunochromatographic test strip disclosed by the invention not only can be used for quickly detecting the IBRV in a sample, but also can be used for quantifying the IBRV. The detection principle of the test strip is as follows: dropwise adding a sample liquid on a sample pad, enabling the sample liquid to flow towards a water absorption pad under the capillary action, dissolving the IBRV polyclonal antibody marked by the fluorescent microspheres on the combination pad, and if the sample liquid contains IBRV, enabling the IBRV and the IBRV polyclonal antibody marked by the fluorescent microspheres to be specifically combined to form a compound A (fluorescent microspheres-polyclonal antibody-IBRV); the compound A flows towards the absorbent pad along with the sample liquid, and when the sample liquid flows to a detection line (T line) on the nitrocellulose membrane, the compound A is specifically combined with the IBRV monoclonal antibody 1C2 on the T line to form a compound B (fluorescent microsphere-polyclonal antibody-IBRV-monoclonal antibody 1C 2), the compound B is gathered on the T line, and a fluorescent strip is generated under ultraviolet light; when the sample liquid flows to a quality control line (C line) on the nitrocellulose membrane, free fluorescent microsphere-labeled chicken IgY dissolved in the sample liquid and the goat anti-chicken IgY antibody on the C line are specifically combined to form a compound C (fluorescent microsphere-chicken IgY-goat anti-chicken IgY), and the compound C is gathered on the C line to generate a fluorescent strip under ultraviolet light.

Judging whether the sample contains IBRV according to the existence of the fluorescence band, and if the T line has no band and the C line has a band, judging that the sample is IBRV negative; if the T line and the C line both have strips, judging that the IBRV is positive; if the C line has no stripe, the C line is judged to be invalid. If IBRV in a sample is required to be quantified, the test strip after color development is placed into a fluorescence immunochromatography analyzer for scanning, and the ratio (T/C value) of a T-line fluorescence signal and a C-line fluorescence signal is read. And substituting the T/C value of the sample into a standard curve, and calculating the IBRV content in the sample. Experiments prove that when the TCID of the IBRV is used ₅₀ Is 10 ² ~10 ⁶ And the corresponding T/C value presents a good exponential relation, and the regression equation is that y =0.0044e ^0.7427x ，R ² =0.8823, correlation is good.

The fluorescent microsphere immunochromatographic test strip has the following advantages:

(1) the method can be used for quantifying: the test strip can accurately quantify the IBRV in a sample, realizes convenient and quick quantitative detection of the IBRV, and widens the quantitative detection of the IBRV from a laboratory to a farm and a quarantine site;

(2) the sensitivity is high: the test strip has a minimum detection limit of 8.58 x 10 aiming at IBRV ¹ TCID ₅₀ ；

(3) The specificity is strong: the test strip only reacts with the IBRV specifically and does not react with other viruses and bacteria;

(4) the stability is good: after the test strip is stored for 12 months at 4 ℃ or room temperature (22-25 ℃), the sensitivity and specificity of IBRV are not obviously changed;

(5) the accuracy is high: aiming at 50 bovine nasal swab samples, vaginal swab samples and semen stored in the subject group, the total coincidence rate of the test strip and the detection result of the cell virus separation method reaches 100%.

(6) The detection speed is high: the test strip provided by the invention is used for qualitatively or quantitatively detecting the IBRV in a sample within 15 min.

The deposit information of the hybridoma cells secreting the IBRV monoclonal antibody is as follows:

biological material (strain): 1C2

And (3) classification and naming: hybridoma cell

The preservation date is as follows: 15/02/2022

The preservation number is: CGMCC number 45040

The preservation organization: china general microbiological culture Collection center

Address: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

Drawings

FIG. 1 shows SDS-PAGE of purified 1B6 monoclonal antibody. 1 is a protein molecule marker, 2-8 is an eluent, 9 is a flow-through peak, and 10 is a washing impurity peak.

FIG. 2 shows SDS-PAGE of purified 1C2 monoclonal antibody. 1 is a protein molecule marker, 2-8 is an eluent, 9 is a flow-through peak, and 10 is a washing impurity peak.

FIG. 3 shows SDS-PAGE of purified 2C4 monoclonal antibody. 1 and 7 are protein molecule marker, 2 is ascites stock solution, 3 is flow through peak, 4-5 are washing peak, 6 and 8 are concentrated 2C4 monoclonal antibody solution.

FIG. 4 shows SDS-PAGE of purified 3F9 monoclonal antibody. 1 is a protein molecule marker, 2-7 is an eluent, 8 is a flow-through peak, and 9 is a washing impurity peak.

FIG. 5 shows the result of Western blot detection of immunogenicity of monoclonal antibodies.

FIG. 6 shows the effect of fluorescent microsphere labeling of antibodies. A is monoclonal antibody 1B 6-fluorescent microsphere compound, B is monoclonal antibody 1C 2-fluorescent microsphere compound, C is monoclonal antibody 3F 9-fluorescent microsphere compound, D is monoclonal antibody 2C 4-fluorescent microsphere compound, and E is IBRV multi-antibody-fluorescent microsphere compound.

FIG. 7 is a schematic diagram of a fluorescent microsphere immunochromatographic test strip structure and a detection method. 1-4 are a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad in sequence.

FIG. 8 shows the assembly of the shell of the fluorescent microsphere immunochromatographic test strip.

FIG. 9 shows the specificity evaluation of the fluorescent microsphere immunochromatographic test strip for IBRV detection. 1-5 are the detection results of cell cultures of infectious bovine rhinotracheitis virus Bartha Nu/67, bovine viral diarrhea/mucosal virus ISO39, bovine coronavirus J1109, bovine rotavirus J0721 and escherichia coli DH5 alpha in sequence.

FIG. 10 shows the sensitivity evaluation of the fluorescent microsphere immunochromatographic test strip for IBRV detection. 1 is the detection result of a negative sample (PBS), and 2-8 are 8.58X 10 in sequence ⁶ TCID ₅₀ 、8.58×10 ⁵ TCID ₅₀ 、8.58×10 ⁴ TCID ₅₀ 、8.58×10 ³ TCID ₅₀ 、8.58×10 ² TCID ₅₀ 、8.58×10 ¹ TCID ₅₀ 、8.58TCID ₅₀ The detection result of the IBRV virus liquid of (1).

Detailed Description

The present invention is described in detail below with reference to examples, it being understood that the following examples are only illustrative and illustrative of the present invention and do not limit the scope of the present invention in any way.

Virus: bovine infectious rhinotracheitis virus (IBRV) Bartha Nu/67 standard strain is provided by livestock and poultry epidemic disease research center of agriculture and forestry academy of sciences in Beijing, and is a known Bovine herpes virus type I strain, which is disclosed in the documents Xu J, Zhang X, ZHou S, Shen J, Yang D, Wu J, Li X, Li M, Huang X, Sealy JE, Iqbal M, Li Y. A DNA aptamer affinity inhibition of infection of Bovine herpesvirus 1 by blocking virus entry Sci Rep.2017 Sep 18, (7) (1) 11796, doi: 10.1038/S41598-017 10070-1, PMID: 28924154, ID: PMC 3556041. Bovine viral diarrhea/mucosal virus ISO39, bovine coronavirus J1109 and bovine rotavirus J0721 are provided by livestock and poultry epidemic disease research center of agroforestry academy of sciences of Beijing. The above viruses were also stored in the laboratory and the applicant stated that they could be released to the public for validation experiments within twenty years from the filing date.

Animals: SPF-grade female BALB/c mice and SPF rabbits of 6-8 weeks old are purchased from Beijing Wintonlifa laboratory animal technology, Inc.

Cell: MDBK cells, High Five cells, SP2/0 myeloma cells and Escherichia coli DH5 alpha are provided by livestock and poultry epidemic disease research center of agriculture and forestry academy of sciences of Beijing, and the cells can be obtained commercially.

Reagent: HAT medium was purchased from Sigma-Aldrich under product number H0262. HT medium was purchased from Sigma-Aldrich under product number H0137. DMEM medium, Fetal Bovine Serum (FBS) was purchased from Gibco. PEG6000, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride and potassium chloride were purchased from Beijing Sorleibao technologies, Inc. N-hydroxysuccinimide (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma.

Consumable material: the sample pad, the glass cellulose membrane, the nitrocellulose membrane, the water absorption pad, the PVC base plate and the test strip shell are all purchased from Shanghai gold-labeled Biotech Co.

PBS: 2.9g of disodium hydrogen phosphate (containing 12 crystal waters), 0.2g of potassium dihydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride were weighed, and 1000ml of purified water was added thereto and dissolved by stirring.

The nasal swab, vaginal swab and semen samples of 50 cattle used for the coincidence detection in example 3 were provided by the livestock and poultry epidemic research center of agroforestry academy of sciences of Beijing.

Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, and may be of laboratory pure grade. Unless otherwise specified, the experimental methods and conditions used in the following examples are all conventional in the art, and reference may be made to relevant experimental manuals, well-known literature, or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1 acquisition of monoclonal and polyclonal IBRV antibodies

1. Purification of IBRV virus

MDBK cells grown in a monolayer were prepared, and IBRV (Bartha Nu/67 standard strain) was added at a MOI =1 ratio for 1h, followed by addition of DMEM medium containing 2% (v/v) FBS, and incubation at 37 ℃ for 2-3 days. When the cell CPE (cytopathic effect) reaches about 80%, the cell CPE is repeatedly frozen and thawed three times in a refrigerator at minus 80 ℃, and supernatant (virus solution) is collected by centrifugation. The virus liquid is concentrated and purified by adopting a sucrose gradient density centrifugation method, and the method comprises the following steps:

(1) cut a dialysis bag MD25 (Beijing Solebao technologies Co., Ltd.) of appropriate length, and place the bag in 500 mL of washing solution (2% NaCO) ₃ +1mmol/L EDTA), boiling for 10 min.

(2) After washing the dialysis bag with deionized water, the bag was placed in 500 mL of 1mmol/L EDTA.2Na (pH 8.0) and boiled for 10 min.

(3) After cooling, the dialysis bag is washed by deionized water, then is immersed in 50% ethanol, and is placed at 4 ℃ for standby.

(4) And cleaning the inside and the outside of the pretreated dialysis bag by using deionized water, and then placing the dialysis bag in a proper groove. Adding virus liquid into the dialysis bag, and clamping the two ends of the dialysis bag by using a clamp.

(5) And covering the dialysis bag with a proper amount of PEG6000 until the virus liquid is concentrated to about 20-30 mL.

(6) The virus solution was aspirated, the interior of the dialysis bag was washed with 5mL of PBS, and the virus adsorbed on the inner wall of the dialysis bag was eluted and incorporated into the concentrated virus solution.

(7) The concentrated virus solution was centrifuged at 5000 r/min (4500 Xg) for 30min and the supernatant was collected.

(8) Preparing a sucrose gradient tube, sequentially adding 5mL of sucrose solutions with the concentrations of 20%, 40% and 60% into a high-speed centrifuge tube from top to bottom by using a long needle, and finally slowly adding the supernatant collected in the previous step.

(9) The virus bands were carefully aspirated at 35000 r/min (54200 Xg) for 2h using a syringe where the virus bands were separated by 40% sucrose and 60% sucrose. Dissolving the precipitate with 10mL PBS to obtain purified IBRV virus liquid, subpackaging and storing at-70 ℃ for later use.

Preparation of recombinant gD protein

10mL of recombinant baculovirus rBac-gD was inoculated with 100 mL of High Five cells in logarithmic growth phase, and the cells were placed in a shaker at 27 ℃ and cultured in suspension at 140 rpm. rBac-gD is a recombinant baculovirus prepared in the laboratory at the early stage, and contains a recombinant shuttle particle Bacmid-gD capable of expressing recombinant gD protein. The preparation method of rBac-gD is described in the Chinese invention patent with the application number of 2021104031189, the application date of 2021, 14/04, entitled "blocking ELISA kit for detecting neutralizing antibody of infectious bovine rhinotracheitis virus and application thereof". The entire contents of this patent are hereby incorporated by reference.

30 mL of recombinant baculovirus rBac-gD is inoculated with 300 mL of High Five cells, and the cells are placed in a shaking table at 27 ℃ and subjected to suspension culture at 140 rpm for 72 h. The cell culture was centrifuged at 12000 rpm for 30min, and the supernatant was collected and subjected to protein purification using a His-nickel column (Qiagen, 70971). At 4 deg.C, with 5 column volumes (about 50 mL) of ddH ₂ O low flow rate displaced 20% ethanol from the His-nickel column. 0.2M NiCl over 2 column volumes (about 20 mL) ₂ Until the reading is stable. ddH over 5 column volumes ₂ The O washes away unbound Ni ions. The His-nickel column was equilibrated with 5 column volumes of lysis buffer (20 mM Tris, 0.5M NaCl, pH 8.0). Circulating the cell culture supernatant through His-nickel column at 4 deg.C overnight, and collecting the sample before loading, and collecting the effluent. The sample was washed with lysine buffer containing 20mM imidazole until the OD280 absorbance was stabilized, and the sample was retained. The column was washed with lysis buffer containing 50 mM imidazole until the OD280 absorbance stabilized, and the sample was retained, and PMSF was added to a final concentration of 1%. The column was washed with lysis buffer containing 400 mM imidazole until the OD280 absorbance stabilized, and the sample was retained, followed by addition of PMSF to a final concentration of 1%. ddH was added in 5 column volumes in sequence ₂ O, EDTA solution (0.5M), ddH ₂ O, NaOH solution (0.5M), ddH ₂ The Ni column was treated with O and 20% ethanol.

The purification results of the proteins were examined by SDS-PAGE. The sample corresponding to the single band was placed in a 50mL protein concentration tube, DTT was added to a final concentration of 1mM, and centrifuged at 2900 rpm at 4 ℃ until the solution was left at about 2mL to obtain a concentrated recombinant gD protein solution.

Obtaining monoclonal antibodies

(1) Immunization of animals

After purified IBRV virus solution (Bartha Nu/67 standard strain) and equal volume of Freund's complete adjuvant (Sigma-Aldrich, F5881) are mixed and emulsified completely, the SPF-grade female BALB/c mice of 6-8 weeks are immunized for the first time, and the immunizing dose is 50 mug/mouse. 2 weeks after the first immunization, after fully emulsifying an IBRV virus solution (Bartha Nu/67 standard strain) with the same volume with Freund's incomplete adjuvant (Sigma-Aldrich, F5506), the 2 nd, 3 rd and 4 th immunizations are carried out, wherein the immunizing dose is 50 mug/mouse, and each immunization is separated by 2 weeks.

(2) Determination of serum titre

And (3) establishing an indirect ELISA method for screening the hybridomas by using the positive serum of the mice immunized for the 3 rd time, and calibrating the optimal antigen coating concentration and the optimal positive serum dilution by using a chessboard method. The indirect ELISA method was as follows:

coating: the concentrated recombinant gD protein solution is respectively diluted into 5 mu g/ml, 2.5 mu g/ml, 1.25 mu g/ml, 0.625 mu g/ml, 0.3125 mu g/ml and 0.1562 mu g/ml, an ELISA plate is added according to 100 mu L of each well, PBST is added in the last line of the ELISA plate as an antigen negative control, and after 1h incubation at 37 ℃, the solution is kept overnight at 4 ℃.

And (3) sealing: after washing the plate 5 times with a plate washer, 300. mu.L of 5% skim milk formulated with PBST was added to each well and blocked for 2h at 37 ℃.

Incubating primary antibody: PBST is used for diluting positive serum and negative serum of the mice after 3 rd immunization; after washing the plate, diluted serum was added at 100. mu.L/well and incubated at 37 ℃ for 1 h.

Hatching a secondary antibody: HRP-labeled goat anti-mouse IgG (Sigma-Aldrich, A9309) was diluted 1:10000 times with PBST, and diluted secondary antibody was added after washing the plate, 100. mu.L/well, and incubated at 37 ℃ for 1 h.

Color development: after washing the plate, TMB developing solution (Solarbio, PR 1210) was added at 100. mu.L/well, and the reaction was stopped by adding 50. mu.L of 2M sulfuric acid after 15min at 37 ℃ in the dark. Reading the absorbance (OD) at a wavelength of 450 nm _450nm ）。

Selection of OD _450nm The antigen concentration and serum dilution corresponding to the wells with the value closest to 1.0 are the optimal antigen coating concentration and the optimal positive serum dilution. And (3) carrying out blood sampling on mouse eyeballs one week after 4 th immunization, and measuring the serum titer of the mouse by adopting the optimal antigen coating concentration and the optimal positive serum dilution according to the indirect ELISA method. Two mice with the highest titer are selected, and peritoneal boosting immunization is carried out 3 days before cell fusion, wherein the immunization dose is 50 mug/mouse.

(3) Cell fusion and screening

Spleen of the BALB/c mice after the boosting immunization is taken under a sterile environment to prepare a spleen cell suspension, the spleen cells of the mice are fused with SP2/0 myeloma cells according to the cell number ratio of 5:1, the fused spleen cells are paved on 6 96-well cell culture plates, HAT culture medium (Sigma-Aldrich, H0262) containing 20% fetal bovine serum is used for selective culture, and HT culture medium (Sigma-Aldrich, H0137) is used for gradual change for 7-12 days. On the 10 th day after the fusion, the hybridoma cells were preliminarily screened by the indirect ELISA method using IBRV as a coating antigen, and the coating amount of IBRV was 500 ng/well. The assay was repeated every 2 days and wells positive for IBRV were screened. The fused cells in all the wells with strong positive detection results in 6 plates are transferred to 48-well plates for amplification culture, and 5 wells with high ELISA value are selected for first subcloning. Subcloning was performed 2 more times according to the same method until the positive rate was 100%. 4 hybridoma cell strains which can stably secrete IBRV monoclonal antibody (IBRV monoclonal antibody) are obtained, and the serial numbers are 1B6, 1C2, 2C4 and 3F9 respectively.

(4) Ascites production and monoclonal antibody purification

Injecting 1mL of autoclaved liquid paraffin into the abdominal cavity of an SPF female BALB/c mouse of 6-8 weeks old for sensitization, and injecting 1.0 multiplied by 10 into the abdominal cavity after 7 days for sensitization ⁶ And (3) the hybridoma cells with good growth vigor. The abdominal cavity of the mouse is observed every day, and when the mouse is expanded to be inconvenient to move, the ascites is collected. Monoclonal antibody purification: mixing Binding Buffer (Thermo Fisher, cat # 21019) with the same volume as ascites, centrifuging at 10000g for 20min, collecting supernatant, and filtering with 0.22 μm filter membrane. After filtration, the mixture was purified using a Protein A/G purification column (Thermo Fisher, cat # TF 266548), and the filtered supernatant was subjected to column chromatography using a Binding Buffer 5 times the column volume, and the liquid was retained after column chromatography. The column was washed with 10 column volumes of Binding Buffer, eluted through 5 column volumes of Elution Buffer (Thermo Fisher, cat # 21027), and the OD280 values were monitored during the Elution, and when the number of changes, the liquid was collected and 100. mu.l of PBS was added per ml of liquid. The purification results of IBRV monoclonal antibody were examined by SDS-PAGE (FIGS. 1-4).

Immunogenicity testing of monoclonal antibodies

And mixing the purified recombinant gD protein solution and the protein loading buffer solution uniformly, boiling, performing SDS-PAGE, and transferring the protein on the gel to a PVDF membrane by a conventional wet transfer method. Sealing the PVDF membrane by using 5% skim milk at room temperature for 2 h; adding purified IBRV monoclonal antibody (diluted by PBST according to a ratio of 1: 1000), incubating for 2h at room temperature, washing the membrane with PBST for 5 times, and each time for 5 min; adding goat anti-mouse IgG (Sigma, A9309) labeled with HRP (diluted 1:10000 using PBST), incubating at 25 ℃ for 2h, washing the membrane 5 times with PBST, 5min each; chemiluminescence and photography. As shown in FIG. 5, the purified IBRV monoclonal antibodies 1B6, 1C2, 2C4 and 3F9 can specifically react with the recombinant gD protein.

Preparation of polyclonal antibodies

After mixing the purified IBRV virus solution with equal volume of Freund's complete adjuvant (Sigma-Aldrich, F5881) and fully emulsifying, the SPF rabbits were immunized for the first time at an immunizing dose of 0.5 mg/mouse. 2 weeks after the initial immunization, purified IBRV virus fluid was mixed and emulsified well with an equal volume of incomplete Freund's adjuvant (Sigma-Aldrich, F5506), and 2, 3 and 4 immunizations were performed, 1mg each at each immunization interval of 2 weeks. Three rabbits are immunized in total, blood is collected after four times of immunization, and serum is separated to obtain IBRV polyclonal antibody (IBRV polyclonal antibody). 10mL of rabbit serum was taken from each group and antibody purification was performed using Protein A/G purification column (Thermo Fisher, cat # TF 266548). The method comprises the following steps: mixing the serum with binding buffer (Thermo Fisher, Cat: 21019) in equal volume, centrifuging at 10000rpm at 4 deg.C for 30min, and filtering with 0.45um filter membrane; at 4 deg.C, 5 timesColumn volume binding buffer (about 50 mL) displaced ddH containing 2% sodium azide ₂ O, the sample was applied to the column, washed with binding buffer in 5 column volumes, eluted with Elution buffer (Thermo Fisher, cat # 21027) in 5 column volumes, and the collected eluate was added to 100. mu.L of PBS until the value of OD280 was substantially unchanged. Using successively 5 column volumes of Elution buffer, 5 column volumes of ddH containing 2% sodium azide ₂ And O, treating the column. The purification results of the IBRV polyclonal antibody were checked by SDS-PAGE and the antibody concentration was determined using a nucleic acid protein concentration analyzer (Nanodrop, Denovix DS-11 +).

Example 2 preparation of fluorescent microsphere immunochromatographic test strip for detecting IBRV

1. Fluorescent microsphere labeling of antibodies

The purified IBRV monoclonal antibody, IBRV polyclonal antibody and chicken IgY (Nanjing Han Rui Bio, T111101M) in example 1 were labeled with fluorescent microspheres, respectively, as follows:

(a) mu.l of fluorescent microspheres (FT 0100CA, 10mL, Zusanweikon, Ltd.) was added to 900. mu.l of labeling buffer and mixed well, centrifuged at 16000rpm for 10min, and the supernatant was discarded.

(b) Adding 1mL of labeling buffer solution to resuspend the fluorescent microspheres, centrifuging at 16000rpm for 10min, and discarding the supernatant.

(c) Weighing NHS and EDC, and dissolving with labeling buffer solution (prepared at present); adding 10 mul of NHS solution (20 mg/ml) into the washed fluorescent microspheres, quickly and uniformly mixing, then adding 5 mul of EDC solution (20 mg/ml) into the fluorescent microspheres, and quickly and uniformly mixing; incubate at room temperature for 20 min.

(d) Centrifuging the activated fluorescent microspheres at 17000rpm for 20min, discarding the supernatant, repeating the centrifugation once, discarding the supernatant, and adding 1000. mu.l of a labeling buffer solution to resuspend the fluorescent microspheres.

(e) And (d) putting 0.1mg of antibody into a 2mL centrifuge tube, adding 1mL of the fluorescent microsphere suspension (containing 1mg of fluorescent microsphere) obtained in the step (d), quickly mixing uniformly, and incubating at room temperature for 2 hours to obtain the antibody-fluorescent microsphere compound.

(f) Add 100. mu.l blocking solution to the antibody-fluorescent microsphere complex and incubate for 1h at room temperature.

(g) Centrifuging and discarding the supernatant. 1000. mu.l of resuspension solution was added to resuspend the antibody-fluorescent microsphere complex.

(h) Repeating step (g) once.

(i) Centrifuging and discarding the supernatant. And (3) resuspending the antibody-fluorescent microsphere complex by using 1000 mul of resuspension solution to obtain the antibody-fluorescent microsphere complex.

The formula of the solution used in the fluorescent microsphere labeling experiment is as follows:

labeling buffer solution: 9.762g of MES were weighed out and dissolved in 1L of distilled water to adjust the pH to 6.0.

Sealing liquid: 20mg of BSA was weighed and dissolved well in 1mL of ethanolamine solution with a final concentration of 100 mM.

Resuspending: 1.21g Tris and 5g BSA were weighed, 5mL Tween-20 was measured and dissolved in 1L distilled water.

The obtained antibody-fluorescent microsphere complexes were sprayed onto glass cellulose membranes, respectively, using a gold spraying instrument, and the labeling effect of the fluorescent microspheres was examined under ultraviolet light (fig. 6).

Coating combined with cushion

And carrying out ultrasonic treatment on the obtained antibody-fluorescent microsphere compound and the chicken IgY-fluorescent microsphere compound for 15-20 min. And (3) fully and uniformly mixing the antibody-fluorescent microsphere compound (80-120 mu g/mL) after ultrasonic treatment and the chicken IgY-fluorescent microsphere compound (80-120 mu g/mL) according to the volume ratio of 3:1, and uniformly coating the mixture on a glass cellulose membrane. And (3) placing the glass cellulose membrane coated with the antibody-fluorescent microsphere composite at 37 ℃ and drying for 16h to obtain the binding pad combined with the antibody-fluorescent microsphere composite.

Coating of film

Respectively diluting unlabeled monoclonal antibody 1B6, monoclonal antibody 1C2, monoclonal antibody 2C4, monoclonal antibody 3F9 and IBRV polyclonal antibody to 0.5 mg/mL. Anti-goat IgY (Nanjing HanRui Bio, T111201M) was diluted to 0.5 mg/mL. Unlabeled antibody and goat anti chicken IgY were coated on a nitrocellulose membrane (NC membrane) using a three-dimensional planimeter (shanghai gold-labeled biotechnology limited, HM 3030). The method specifically comprises the following steps: the diluted monoclonal antibody or polyclonal antibody is streaked on an NC membrane by a gush amount of 1 mu L/cm to obtain a detection line (T line), and the diluted goat anti-chicken IgY is streaked on the NC membrane by a gush amount of 1 mu L/cm to obtain a quality control line (C line). The scribed NC film was dried at 40 ℃ for 16 h.

Test strip assembly

According to the test strip structure shown in fig. 7, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad are sequentially adhered to a PVC base plate, and adjacent components are overlapped by 2 mm to form a large plate. Then, the large plate was cut into test strips of 4 mm width by a slitter (Shanghai gold-labeled Biotech Co., Ltd., ZQ 2402), and a plastic test strip case (Shanghai gold-labeled Biotech Co., Ltd.) was mounted thereon and sealed and packaged in an aluminum foil bag. And a sample hole and an observation window are arranged on the test strip shell, a sample pad is arranged below the sample hole, and the positions of the detection line and the quality control line on the nitrocellulose membrane are arranged below the observation window.

Antibody pairing screening

Combining the combination pad combined with different antibody-fluorescent microsphere complexes with NC films coated with different antibodies, and assembling the test strip according to the method. The antibody combinations are shown in table 1.

TABLE 1 combination of antibodies on each test strip

Test paper strip number	Antibodies on conjugate pads	Antibodies on the T-line of NC membranes
			1	IBRV polyclonal antibody	1B6
2	IBRV polyclonal antibody	1C2
			3	IBRV polyclonal antibody	3F9
4	IBRV polyclonal antibody	2C4
			5	IBRV polyclonal antibody	IBRV polyclonal antibody
6	1C2	1C2
			7	1C2	1B6
8	1C2	3F9
			9	1C2	2C4
10	1C2	IBRV polyclonal antibody
			11	1B6	1C2
12	1B6	1B6
			13	1B6	3F9
14	1B6	2C4
			15	1B6	IBRV polyclonal antibody
16	3F9	1C2
			17	3F9	1B6
18	3F9	3F9
			19	3F9	2C4
20	3F9	IBRV polyclonal antibody
			21	2C4	1C2
22	2C4	1B6
			23	2C4	3F9
24	2C4	2C4
			25	2C4	IBRV polyclonal antibody

Antibody pairing assays were performed by the double antibody sandwich method. The method comprises the following specific steps: mixing 8.58X 10 ⁷ The IBRV virus solution of TCID50 was diluted at a ratio of 1:10, 80. mu.l of the diluted virus was dropped onto the sample pad of each test strip, and the results were observed under UV light within 10 min. Sample dilutions (PBS) were used as negative controls. If the T line has no strip and the C line has a strip, the judgment is negative; if the T line and the C line both have strips, judging the test result to be positive; if the C line has no stripe, the C line is judged to be invalid.

The results show that only the T line of the test strip No. 2 coated with the IBRV multi-anti-fluorescent microsphere compound on the binding pad and the 1C2 monoclonal antibody on the T line has clear bands, and the T lines of the other test strips have no bands or have light bands. Therefore, the test strip No. 2 can accurately detect the IBRV virus. The following experiments were performed using test strips No. 2.

Qualitative detection method of test strip

And adding 40 mu l of sample solution to be detected into the sample hole of the No. 2 test strip, then adding equal volume of sample diluent (PBS), reacting for 10min, irradiating by adopting an ultraviolet lamp, observing the color development condition of the strip, and then judging the result.

And (4) result judgment standard:

positive: if the C line and the T line are both developed, the sample solution to be detected contains IBRV virus;

negative: if the C line is colored and the T line is not colored, the sample solution to be detected does not contain IBRV virus;

and (3) suspicious: if the C line is colored, and the T line is hidden or light, the result is uncertain, and the re-detection is needed;

and (4) invalidation: if the C line is not colored, the detection is invalid.

Quantitative detection method of test strip

Infection of half of the Tissue Cells (TCID) ₅₀ ) Is the classical method recommended by OIE for determining the content of infectious bovine infectious rhinotracheitis virus.

Establishing a standard curve: get 10 ⁷ TCID ₅₀ 、10 ⁶ TCID ₅₀ 、10 ⁵ TCID ₅₀ 、10 ⁴ TCID ₅₀ 、10 ³ TCID ₅₀ 、10 ² TCID ₅₀ 、10 ¹ TCID ₅₀ 、10 ⁰ TCID ₅₀ The IBRV virus solution (1 part) is respectively detected by the No. 2 test strip, and each sample is repeatedly detected for 10 times. The method specifically comprises the following steps: adding 40 μ l of IBRV virus solution into the sample hole of test strip No. 2, adding equal volume of sample diluent (PBS), reacting for 10min, irradiating with ultraviolet lamp, and reading the ratio of T-line fluorescence signal to C-line fluorescence signal (T/C value) with fluorescence immunochromatographic analyzer (model FIC-H1). The average value of the T/C values was used as the CUT-OFF value to establish a standard curve. According to the correlation between the concentration of the virus to be detected in the detection system and the T/C value read by the fluorescence immunochromatographic analyzer under a certain condition, the lg (TCID) of the virus is used ₅₀ ) The T/C value read by the fluorescence immunochromatography analyzer is plotted on the abscissa and the ordinate, and a standard curve is established. The results show that when TCID ₅₀ Is 10 ² ~10 ⁶ And the corresponding T/C value presents a good exponential relation, and the regression equation is that y =0.0044e ^0.7427x ，R ² =0.8823, correlation is good (fig. 7).

Sample detection: adding 40 mu l of sample solution to be detected into a sample hole of the No. 2 test strip, then adding equal volume of sample diluent (PBS), reacting for 10min, irradiating by using an ultraviolet lamp, placing the test strip after color development into a fluorescence immunochromatography analyzer for scanning, and reading the T/C value. And (4) bringing the T/C value of the sample liquid to be detected into a standard curve, and calculating the IBRV content in the sample liquid to be detected.

TABLE 2 result determination criteria for quantitative determination methods

T/C value	Viral infectivity (TCID 50)	Determination of results
			T/C<0.02	TCID50<200	±/-
0.02<T/C<0.03	200<TCID50<1000	+
			0.03<T/C<0.3	1000<TCID50<100000	+
T/C>0.3	TCID50>100000	+

Example 3 Performance verification of fluorescent microsphere immunochromatographic test strip for detection of IBRV

1. Specificity detection

Using the preparation of example 2Test paper strip No. 2 for infectious bovine rhinotracheitis virus Bartha Nu/67 (8.58X 10) ⁶ TCID ₅₀ ) Bovine viral diarrhea/mucosal virus ISO39 (6.32 × 10) ⁶ TCID ₅₀ ) Bovine coronavirus J1109 (2.19X 10) ⁶ TCID ₅₀ ) Bovine rotavirus J0721 (7.59X 10) ⁶ TCID ₅₀ ) And E.coli DH 5. alpha. were specifically detected. And adding 40 mu l of virus solution to be detected into a sample hole of the test strip, then adding equal volume of sample diluent (PBS), reacting for 10min, irradiating by using an ultraviolet lamp, and observing the color development condition of the strip.

The result is shown in fig. 9, the fluorescent microsphere immunochromatographic test strip of the present invention can only perform specific reaction with the infectious bovine rhinotracheitis virus, has no reaction with other viruses and bacteria, and has strong specificity.

Sensitivity detection

Mixing 8.58X 10 ⁷ The TCID50 IBRV virus was diluted 10-fold and 80. mu.l of each gradient was sampled and tested using test strip No. 2 prepared in example 2. The method specifically comprises the following steps: adding 80 mul of sample into a sample hole of the test strip, reacting for 10min, irradiating by using an ultraviolet lamp, putting the test strip after color development into a fluorescence immunochromatography analyzer to read the ratio (T/C value) of a T-line fluorescence signal and a C-line fluorescence signal, and judging the result according to the judgment standard shown in table 2 in example 2. As shown in Table 3 and FIG. 10, the lowest detection limit of the fluorescent microsphere immunochromatographic test strip of the present invention was 8.58X 10 ¹ TCID ₅₀ And the sensitivity is high.

TABLE 3 detection results of standard samples with immunochromatographic test strips labeled with fluorescent microspheres

IBRV concentration	T/C value	Determination of results	Qualitative determination
				8.58×10 ⁶ TCID50	0.668	+	+
8.58×10 ⁵ TCID50	0.514	+	+
				8.58×10 ⁴ TCID50	0.295	+	+
8.58×10 ³ TCID50	0.175	+	+
				8.58×10 ² TCID50	0.093	+	±
8.58×10 ¹ TCID50	0.022	+	-
				8.58TCID50	0.019	-	-
NC	0.017	-	-

NC indicates a negative sample (PBS), "+" indicates positive IBRV, "-" indicates negative IBRV and "+" indicates suspicious.

Stability detection

The test paper strip No. 2 prepared in example 2 was stored at 4 ℃ and room temperature (22-25 ℃) and taken out at 1 month, 3 months, 6 months, 9 months, 12 months after storage, respectively, at a concentration of 8.58X 10 ² TCID ₅₀ The IBRV virus solution can be used for detecting the sensitivity of test strips with different storage periods at different temperatures, and the nasal swab of a healthy cow is used for detecting the specificity of the test strips with different storage periods at different temperatures. And (3) detecting by adopting a qualitative detection method. The results are shown in tables 4 and 5, and the fluorescent microsphere immunochromatographic test strip has no obvious change in sensitivity and specificity and good stability after being stored at 4 ℃ and room temperature for 12 months.

Table 4 evaluation of stability of test strips at room temperature

Storage time at 22 to 25 ℃ (month)	1	2	3	4	5	6	7	8	9	10	11	12
													Sensitivity of the composition	+	+	+	+	+	+	+	+	+	+	±	-
Specificity of	-	-	-	-	-	-	-	-	-	-	-	-

In the table, "+" indicates positive IBRV, "-" indicates negative IBRV, and "+ -" indicates suspicious.

TABLE 5 evaluation of test strips for stability at 4 deg.C

Storage time at 4 ℃ (moon)	1	2	3	4	5	6	7	8	9	10	11	12
													Sensitivity of the composition	+	+	+	+	+	+	+	+	+	+	+	+
Specificity of	-	-	-	-	-	-	-	-	-	-	-	-

In the table, "+" indicates positive IBRV and "-" indicates negative IBRV.

Coincidence rate detection

Taking 50 cattle nose swab samples, vagina swab samples and semen samples with clear backgrounds stored in the subject group, and respectively using the test strip No. 2 prepared in the embodiment 2 and a conventional cell virus separation method to perform a parallel comparison test so as to determine the coincidence rate of the test strip detection method and the cell virus separation method. The test strip detection method is the same as the quantitative detection method of the test strip in the embodiment 2. The cell virus isolation method is described in Wang Tao.infectious rhinotracheitis virus isolation and identification and monoclonal antibody preparation [ D ]. Heilongjiang eight university of agricultural reclamation, 2008.

The results are shown in table 6, where 30 parts of the sample confirmed to be positive by the cell virus isolation method and 20 parts of the sample confirmed to be negative by the cell virus isolation method were detected using the fluorescent microsphere immunochromatographic strip of the present invention, the test strip after color development was placed in a fluorescence immunochromatographic analyzer to read the ratio of the T-line fluorescence signal to the C-line fluorescence signal (T/C value), and the T/C value was substituted into the standard curve (y =0.0044 e) ^0.7427x ) And calculating the IBRV content in the sample liquid. The coincidence rate is 100%.

TABLE 6 comparison of test results of the test strip of the present invention with cellular virus isolation results

Sample (I) Numbering	Cell virus Result of separation	The test strip of the invention detects Results (T/C value)	Viral content （lgTCID ₅₀ )	Sample (I) Numbering	Cell virus Separation knotFruit	The test strip of the invention detects Results (T/C value)	Viral content （lgTCID ₅₀ )
								1	+	0.870	7.12	26	+	0.611	6.64
2	+	0.759	6.93	27	+	0.521	6.43
								3	+	0.360	5.93	28	+	0.726	6.87
4	+	0.590	6.60	29	+	0.720	6.86
								5	+	0.280	5.59	30	+	0.680	6.79
6	+	0.292	5.65	31	-	0.013	0.00
								7	+	0.548	6.50	32	-	0.012	0.00
8	+	0.660	6.75	33	-	0.009	0.00
								9	+	0.640	6.71	34	-	0.015	0.00
10	+	0.434	6.18	35	-	0.016	0.00
								11	+	0.254	5.46	36	-	0.019	0.00
12	+	0.296	5.67	37	-	0.010	0.00
								13	+	0.515	6.41	38	-	0.017	0.00
14	+	0.175	4.96	39	-	0.018	0.00
								15	+	0.490	6.35	40	-	0.013	0.00
16	+	0.584	6.58	41	-	0.013	0.00
								17	+	0.260	5.49	42	-	0.019	0.00
18	+	0.476	6.31	43	-	0.014	0.00
								19	+	0.710	6.84	44	-	0.015	0.00
20	+	0.720	6.86	45	-	0.012	0.00
								21	+	0.610	6.64	46	-	0.019	0.00
22	+	0.570	6.55	47	-	0.017	0.00
								23	+	0.680	6.79	48	-	0.012	0.00
24	+	0.390	6.04	49	-	0.013	0.00
								25	+	0.410	6.11	50	-	0.015	0.00

Claims

1. The fluorescent microsphere immunochromatographic test strip for detecting the IBRV comprises a bottom plate, and a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially lapped and stuck on the bottom plate, and is characterized in that the combination pad is coated with an IBRV polyclonal antibody marked by fluorescent microspheres; the detection line is arranged on the nitrocellulose membrane and is coated with an IBRV monoclonal antibody, and the IBRV monoclonal antibody is secreted by a hybridoma cell with the preservation number of CGMCC No. 45040.

2. The test strip of claim 1, wherein in the fluorescent microsphere-labeled IBRV polyclonal antibody, the mass ratio of the fluorescent microsphere to the IBRV polyclonal antibody is 8: 1-15: 1.

3. The test strip of claim 1, wherein the IBRV monoclonal antibody is coated at a concentration of 450-550 μ g/mL.

4. The test strip of claim 1, wherein the conjugate pad is further coated with chicken IgY labeled with fluorescent microspheres; and the nitrocellulose membrane is also provided with a quality control line which is spaced from the detection line by a certain distance, and the quality control line is coated with goat anti-chicken IgY antibody.

5. The test strip of claim 4, wherein in the fluorescent microsphere-labeled chicken IgY, the mass ratio of the fluorescent microsphere to the chicken IgY is 8: 1-15: 1; the coating concentration of the goat anti-chicken IgY antibody is 500-650 mu g/mL.

6. A method for preparing the test strip of any one of claims 1-5, comprising the steps of:

(2) carrying out streak coating on a nitrocellulose membrane by using the IBRV monoclonal antibody described in claim 1 to obtain a detection line;

7. The method of claim 6, wherein the mass ratio of the fluorescent microspheres to the IBRV polyclonal antibody in the fluorescent microsphere labeled IBRV polyclonal antibody is 8: 1-15: 1.

8. The method of claim 6, wherein the IBRV monoclonal antibody is coated at a concentration of 450-550 μ g/mL.

9. The method according to claim 6, wherein the step (1) further comprises coating fluorescent microsphere-labeled chicken IgY on the glass cellulose membrane; and (2) scribing and coating the goat anti-chicken IgY antibody on the nitrocellulose membrane at a certain distance from the detection line to obtain a quality control line.

10. The method according to claim 9, wherein in the chicken IgY marked by the fluorescent microspheres, the mass ratio of the fluorescent microspheres to the chicken IgY is 8: 1-15: 1; the coating concentration of the goat anti-chicken IgY antibody is 500-650 mu g/mL.