CN111505289A - Peste des petits ruminants detection kit - Google Patents
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- CN111505289A CN111505289A CN202010510242.0A CN202010510242A CN111505289A CN 111505289 A CN111505289 A CN 111505289A CN 202010510242 A CN202010510242 A CN 202010510242A CN 111505289 A CN111505289 A CN 111505289A
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- petits ruminants
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
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- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
The invention relates to a peste des petits ruminants detection kit, and an important component used in the detection kit, namely a monoclonal antibody, is an antibody aiming at peste des petits ruminants virus H protein, and has the advantages of good specificity, strong sensitivity, high titer and the like. The peste des petits ruminants virus monoclonal antibody and different matched reagents are assembled to form an immunological detection kit which can be used for developing and producing peste des petits ruminants virus or detecting the antibody of the peste des petits ruminants virus, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunoassay (ELISA) test paper strips. The kit can be used for detecting whether peste des petits ruminants virus or peste des petits ruminants virus antibody exists in a sample, identifying whether the virus to be detected is peste des petits ruminants virus, identifying whether an animal to be detected is infected with peste des petits ruminants virus or identifying whether serum contains peste des petits ruminants virus antibody and the like. The kit disclosed by the invention is sensitive and specific, is quick, convenient, economical and practical, is easy to popularize and can be applied to large-scale monitoring systems and field detection.
Description
Technical Field
The invention relates to a peste des petits ruminants detection kit. Belongs to the field of biological products for animals.
Background
Peste des petits ruminants (PPR) is commonly called as 'sheep plague', also known as peste des petits ruminants pseudocattle plague (pseudocardipole), pulmonary enteritis (pneumoenteritis), and stomatitis-pneumonitis complex disease (stomatitis-pneumonitis complex), is an acute viral infectious disease caused by peste des petits ruminants virus, and is characterized by fever, stomatitis, diarrhea, and pneumonia. Is a must report animal epidemic disease regulated by the world animal health Organization (OIE), seriously harms the small ruminants and livestock, and according to the FAO, about 62.5 percent of the small ruminants are threatened by the plague all over the world. Animal husbandry is the backbone industry of rural economy in China, and the number of sheep raised per year reaches 5.6 hundred million. The peste des petits ruminants are fulminating infectious diseases seriously harming the sheep raising industry in China, are introduced into the Tibetan Ali region in China for the first time in 2007, outbreak of 250 peste ruminants in 261 counties in 22 provinces in China in sequence in 2013 + 2014, the morbidity is 100 percent, the mortality is more than 80 percent, and huge economic losses are caused due to the lack of prevention and control technologies and products. Until now, no mature diagnostic reagent for Peste des petits ruminants has been reported and registered in China, and the import diagnostic reagent is basically relied on.
In the face of the severe situation of the prevalence and spread of PPR, combined technical attack is carried out aiming at the key technical problems of PPR pathogen diagnosis and detection technology and the like, a breakthrough is made in the technology, monoclonal antibody hybridoma cells are obtained, and an antigen and antibody kit which is economical, practical, strong in specificity and high in sensitivity is developed on the basis of the monoclonal antibody hybridoma cells, so that the problem of the lack of the current peste des petits ruminants detection technology is improved, and remarkable economic and social benefits are brought to the sheep raising industry in China.
Disclosure of Invention
According to the needs and deficiencies in the art, the invention aims to provide a monoclonal antibody of peste des petits ruminants virus H protein, which has good sensitivity, strong specificity and high titer, and is assembled with related reagents into a detection kit for detecting peste des petits ruminants virus antigens or peste des petits ruminants virus antibodies.
Technical scheme of the invention
1. The peste des petits ruminants detection kit is characterized in that the kit is assembled by monoclonal antibodies of peste des petits ruminants virus H protein and matched reagents thereof;
the monoclonal antibody of the Peste des petits ruminants virus H protein is secreted by a hybridoma cell strain, the cell strain is named as a Peste des petits ruminants virus H protein monoclonal antibody hybridoma cell strain, and the Peste des-petits ruminants virus H protein monoclonal antibody hybridoma cell strain is delivered to the national institute of microbiology of Chongy-West Lu No.1, No. 3 of China academy of sciences, China Committee for culture Collection of microorganisms (CGMCC No. 19663) in the Kyoho-Naja-area, Beijing, 04.08.
2. The kit for detecting the peste des petits ruminants is characterized in that the monoclonal antibody of the peste des petits ruminants virus H protein, an enzyme label plate and a matched reagent are assembled to be used for detecting the peste des petits ruminants virus.
3. The kit for detecting the peste des petits ruminants is characterized in that the monoclonal antibody of the peste des petits ruminants virus H protein, the enzyme label plate and a matched reagent are assembled to be used for detecting the peste des petits ruminants virus antibody.
4. The peste des petits ruminants detection kit is characterized in that a monoclonal antibody of the peste des petits ruminants virus H protein is coated on a gold-labeled fiber mat after being labeled by colloidal gold, a detection zone is coated with rabbit anti-peste des petits ruminants virus polyclonal antibody, and during detection, when peste des petits ruminants virus exists in a sample, an antigen-antibody complex is formed with the monoclonal antibody, and then the antigen-antibody complex is combined with the rabbit anti-peste des ruminants virus polyclonal antibody in the detection zone and develops color.
5. The kit for detecting the peste des petits ruminants is characterized in that the monoclonal antibody of the peste des petits ruminants virus H protein is coated on a gold-labeled fiber mat after being labeled by colloidal gold, a detection area is coated with a peste des ruminants virus purified antigen, and a control area is coated with a goat anti-mouse secondary antibody. When the peste des petits ruminants virus antibody exists in the sample, the peste des petits ruminants virus antibody can inhibit the combination of the monoclonal antibody marked by the colloidal gold and the purified antigen of the peste des petits ruminants virus, and the color is lightened or disappears.
The invention is implemented by the following steps.
1. Cultivation of H protein monoclonal antibody hybridoma cell strain of peste des petits ruminants virus
The invention relates to a strategy for screening monoclonal antibodies by using a detection antigen, which is a strategy for immunizing a mouse by using prokaryotic expression peste des petits ruminants virus H protein, purifying the protein to be used as the immune antigen, immunizing the mouse, fusing splenocytes of the immune mouse and myeloma cells of the mouse, and then using a peste des petits ruminants virus Vero cell culture for differential centrifugal concentration and inactivation. And then a H protein monoclonal antibody aiming at the peste des petits ruminants virus and a hybridoma cell secreting the monoclonal antibody are screened out through further subcloning, the cell is named as a peste des petits ruminants virus H protein monoclonal antibody hybridoma cell strain and is delivered to China general microbiological culture Collection center of China institute of microbiology, institute No.1, Ministry of sciences, North Dynasty, No. 3, of the republic of Beijing, Chaozhou, 04 and 08 days in 2020, with the preservation number of CGMCC No. 19663.
2. Preparation of monoclonal antibodies
The monoclonal antibody of the peste des petits ruminants virus H protein secreted by the hybridoma cell strain is obtained by purifying through a certain process, and the monoclonal antibody can be specifically combined with the peste des petits ruminants virus H protein.
3. Application of monoclonal antibody of peste des petits ruminants virus H protein
(1) A test kit for detecting peste des petits ruminants virus and/or antibody: the peste des petits ruminants virus H protein monoclonal antibody, an enzyme label plate and a matched reagent are prepared and assembled into an enzyme linked immunosorbent assay kit for detecting peste des petits ruminants virus and/or antibody.
1) Peste des petits ruminants virus antigen enzyme-linked immunoassay kit
The antigen of peste des petits ruminants virus in the specimen is detected by using a double antibody sandwich method. The kit is characterized in that a polyethylene microporous plate strip of the kit is pre-coated with a polyclonal antibody of goat anti-peste des petits ruminants virus protein, when the cracked peste des petits ruminants virus is added into micropores, the pre-coated polyclonal antibody can capture the peste des petits ruminants virus, then an enzyme-labeled monoclonal antibody is added to be combined with the peste des petits ruminants virus, and then the result is judged according to the display degree of an enzyme catalysis substrate. When the specimen does not contain antigens of Peste des petits ruminants virus or is not Peste des petits ruminants virus, the substrate does not develop color. The sample detectable by the kit comprises animal excrement, oral nasal cavity secretion, complete virus or split virus cultured by chick embryos, and the like.
2) Peste des petits ruminants virus antibody enzyme-linked immunoassay kit
Peste des petits ruminants virus antigen-specific antibodies in serum samples were detected using a competition method. The antigen purified by peste des petits ruminants virus is pre-coated on a polyethylene micropore plate of the kit. If the added serum sample can obviously inhibit the combination of the enzyme-labeled monoclonal antibody and the antigen, the sample contains the specific antibody of the peste des petits ruminants virus antigen. When the specimen does not contain the peste des petits ruminants virus antibody or the peste des petits ruminants virus antibody, the combination of the enzyme-labeled monoclonal antibody and the antigen is not inhibited, and the color development is darker, otherwise, the color development is lighter.
(2) Colloidal gold kit for the detection of peste des petits ruminants virus and/or antibodies:
the immune colloidal gold kit is a new generation diagnostic reagent developed by adopting a colloidal gold immunochromatography technology, is simple and reliable to operate, does not need instruments or equipment, is self-provided with quality control contrast, does not need any additional reagent, has a clear display result, is quick to react, and only needs a plurality of minutes for the whole operation time.
1) And (3) virus detection: and coating the colloidal gold-labeled peste des petits ruminants virus monoclonal antibody on a gold-labeled fiber mat, and coating rabbit anti-peste des petits ruminants virus polyclonal antibody on a test strip detection area to assemble the colloidal kit for detecting the peste des petits ruminants virus. During detection, when peste des petits ruminants virus exists in a sample, the peste des petits ruminants virus is combined with the colloidal gold-labeled monoclonal antibody of the peste des petits ruminants virus to form an antigen-antibody complex, and then the antigen-antibody complex is combined with the polyclonal antibody of the detection area to develop color.
The colloidal gold kit gold standard method for detecting the peste des petits ruminants virus respectively coats a polyclonal antibody of rabbit anti-peste des petits ruminants virus on a detection area of a nitrocellulose membrane of a colloidal gold test strip, and coats a second goat anti-mouse antibody on a control area. When in detection, the peste des petits ruminants virus in the sample and the colloidal gold labeled peste des petits ruminants virus monoclonal antibody form an antigen-antibody complex, the complex moves along a membrane due to the chromatography, is combined with the polyclonal antibody coated in the detection area to form a complex, and is combined with a second antibody coated on a control line to form a second antibody complex. If the sample is positive, a red line can be formed in the detection area and the control area respectively; if the sample is negative, only one red line is formed in the control area. The kit can be used for detecting whether excrement, oral and nasal secretion and excrement of the small ruminants contain the small ruminants virus.
2) Antibody detection: and coating the colloidal gold-labeled peste des petits ruminants virus monoclonal antibody on a gold-labeled fiber mat, coating the peste des petits ruminants virus purified antigen in the detection area, and coating the goat anti-mouse secondary antibody in the control area to form a colloidal kit for detecting the virus antibody. During detection, when the peste des petits ruminants virus antibody exists in the sample, the peste des petits ruminants virus antibody inhibits the combination of the colloidal gold-labeled peste des petits ruminants virus monoclonal antibody and the peste des petits ruminants virus purified antigen, a detection area is colorless or obviously lightened in color, and a control area combines a secondary antibody and the colloidal gold-labeled peste des petits ruminants virus monoclonal antibody to develop color.
The colloidal gold kit gold-labeling method for detecting the peste des petits ruminants virus antibody respectively coats the peste des petits ruminants virus purified antigen in the detection area on the nitrocellulose membrane of the colloidal gold test strip, the goat anti-mouse secondary antibody is coated in the contrast area, and during detection, the antibody in the sample and the colloidal gold-labeled peste des petits ruminants virus monoclonal antibody are combined with the peste des petits ruminants virus purified antigen coated in the detection area in a competitive manner. If the added sample can obviously inhibit the combination of the colloidal gold labeled peste des petits ruminants virus monoclonal antibody and the purified antigen, the specific antibody of the peste des petits ruminants virus is contained in the standard product. When the specimen does not contain peste des petits ruminants virus antibodies or does not contain peste des petits ruminants virus antibodies, the combination cannot be inhibited, and the darker the detection line, the lighter the detection line. If the detected sample is positive, only a red line is formed in the control area; if the sample is negative, a red line can be formed in each of the detection zone and the control zone. The kit can be used for detecting whether serum of small ruminants such as sheep and the like contains the small ruminants virus antibody.
Novel technical effects of the invention
The invention relates to a peste des petits ruminants detection kit, wherein a monoclonal antibody which is a main component in the detection kit is an antibody aiming at peste des petits ruminants virus H protein, and the kit has the advantages of good specificity, strong sensitivity, high titer and the like. The peste des petits ruminants virus monoclonal antibody and different matched reagents are assembled to form an immunological detection kit which can be used for developing and producing peste des petits ruminants virus or detecting the antibody of the peste des petits ruminants virus, such as enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunoassay (ELISA) test paper strips. The kit can be used for detecting whether peste des petits ruminants virus or peste des petits ruminants virus antibody exists in a sample, identifying whether the virus to be detected is peste des petits ruminants virus, identifying whether an animal to be detected is infected with peste des petits ruminants virus or identifying whether serum contains peste des petits ruminants virus antibody and the like. The kit disclosed by the invention is sensitive and specific, is quick, convenient, economical and practical, is easy to popularize and can be applied to large-scale monitoring systems and field detection.
Drawings
FIG. 1 shows the expression and purification of Peste des petits ruminants virus H protein 1.Marker, 2.PPR-H SQ, 3.PPR-H precipitation, 4.PPR-H first urea elution supernatant, 6.PPR-H second urea elution supernatant, and 8PPR-H third urea elution supernatant.
FIG. 2 is a schematic diagram showing the determination of the detection result of the peste des petits ruminants virus colloidal gold test strip
FIG. 3 is a schematic diagram showing the determination of the detection result of the peste des petits ruminants virus antibody colloidal gold test strip
Detailed Description
The following examples are provided to further understand the present invention, and are not intended to limit the scope of the present invention, and any methods and products similar or equivalent to the present invention, which can be obtained by combining the features of the present invention with other prior art, while falling within the scope of the present invention.
The values of concentrations, temperatures and other variables of the reagents of the invention are merely illustrative of the application of the invention and are not to be construed as limiting the invention.
Sources of Experimental materials of the invention
Biological material
The immune antigen is Peste DEs petits ruminants virus H protein, and by referring to the published nucleotide sequence of Peste DEs petits ruminants virus H protein antigen of NCBI, a region with a concentrated N-terminal antigenic determinant is selected by the laboratory, a sequence (sequence 1) is artificially synthesized after codon optimization, and is cloned to a pET28a (+) expression vector, transformed into Escherichia coli Transetta DE3 for expression, and an expression product is purified to be used as the immune antigen.
The detection antigen is a concentrated antigen of the peste des petits ruminants virus, the selected virus strain is Clone 9 vaccine strain, which is called vaccine strain for short and is provided by the China veterinary microorganism strain preservation center (the virus strain is delivered to the China general microorganism preservation center of microorganism research institute of China academy of sciences No. 3, Naja West Lu 1, Beijing, etc. in 2016, 12 months and 15 days, and the preservation numbers are CGMCC No.13388 respectively). The vaccine strain is selected mainly for the consideration of biological safety risk, has good representativeness and is well proved in the detection effect.
Examples
The following examples are intended to further illustrate the invention and are not to be construed as limiting the invention.
Example 1 preparation of Peste des petits ruminants Virus H protein monoclonal antibody hybridoma cells
1. Expression and purification of peste des petits ruminants virus H protein
Referring to the published nucleotide sequence of the H protein antigen of the peste DEs petits ruminants virus published by NCBI, a region with more concentrated N-terminal antigenic determinants is selected by a laboratory, a sequence (sequence 1) is artificially synthesized after codon optimization, and is directionally cloned to a target site of a pET28a (+) expression vector by double enzyme digestion, and then escherichia coli Transetta DE3 is transformed for prokaryotic expression. The expression mode of the H protein is the expression of an inclusion body, after the inclusion body is purified, washed and purified, 4 percent urea is redissolved, and then gradient dialysis is carried out to obtain the soluble H protein. After concentration determination, the antibody is used for immunizing mice.
2. Peste des petits ruminants virus propagation and purification
Vero cells with good growth inoculated by small ruminant animal disease virus Clone 9 vaccine strain and 5% CO2After culturing at 37 ℃ for 2-3 days, the virus is harvested when about 50% of cells are diseased. Freeze thawing twice, performing ultrasonic intermittent lysis for 3min, purifying virus by differential centrifugation, namely centrifuging at 8000r/min for 5min, taking supernatant, centrifuging at 30000r/min for 3h at high speed, and collecting precipitate to obtain peste des petits ruminants virus concentrated solution. And (3) redissolving by using a TE solution containing 40% of sucrose, shaking for 1h at the temperature of 2-8 ℃, fully redissolving, centrifuging at a high speed of 30000r/min for 3h, and collecting precipitates to obtain the virus purification solution.
3. Preparation of polyclonal antibodies
After being diluted properly, the peste des petits ruminants virus purified liquid is mixed with Seppic 1313 adjuvant in equal volume, homogenized and emulsified at high speed to form immune antigen, and the rabbit is immunized by adopting a method combining back subcutaneous multipoint injection and leg muscle immunization. The first immunization adopts subcutaneous multipoint immunization at the back, and the immunization is strengthened by intramuscular injection of the front leg after 2 weeks. One week thereafter, the immunization was performed with a retrogressive intramuscular injection. Blood sampling detection is carried out 10-13 days after the last immunization, the titer of the polyclonal antibody is determined by adopting an agar diffusion test, the agar diffusion titer reaches more than 1:4, and then blood can be collected from the heart to extract serum, and the blood can be used for preparing a kit.
4. Preparation of monoclonal antibody hybridoma
And (2) immunizing the mice, namely mixing and emulsifying the H protein expression antigen purified solution and a quick antibody adjuvant, performing multipoint injection on four limbs of the mice, wherein the immunization dose is 0.1 ml/mouse each time, performing secondary immunization by using the same dose 21 days after the first immunization, performing immunization once every 1 week, collecting blood after the third immunization, detecting the antibody titer by using an E L ISA plate coated with the H protein expression purified antigen, and taking the mice with the titer of more than 1:20000 for cell fusion.
Cell fusion: the H protein expression antigen purified solution without adjuvant is injected into the abdominal cavity to strengthen the immunity of the mice. And taking mouse spleen cells and SP20 cells for fusion 3-5 days after immunization. Grinding spleen to obtain spleen cell suspension, respectively counting cells with well-cultured mouse myeloma cells, mixing the spleen cell suspension and the well-cultured mouse myeloma cells in proportion after adjusting to proper concentration, slowly adding the spleen cell suspension into a cell mixed solution through PEG 1500 to perform cell fusion, re-suspending the fused cells in a DMEM (DMEM) culture medium containing 1% HAT and 20% FBS, and paving 3-4 cells in a 96-hole cell plate for each mouse. At 5% CO2The carbon dioxide incubator (2) is subjected to static culture at 37 ℃. On day 4 post-fusion, medium was changed half-way with DMEM medium containing 1% HAT and 20% FBS; on day 8 post-fusion, medium was replaced half-way with DMEM medium containing 1% HT and 20% FBS.
And (3) cell screening, namely, observing cells of a cell plate hole by hole on the 10 th day after fusion, taking a hole containing the cells, sucking a supernatant for screening detection, wherein the screening method comprises the steps of coating the purified virus liquid of the peste des petits ruminants virus Clone 9 strain, carrying out E L SIA detection on the cell supernatant, carrying out secondary cloning and subcloning on the detected positive hole, sampling after each cloning, carrying out E L SIA detection, and carrying out E L ISA detection until the titer of an antibody secreted by the cell strain is kept stable.
And (4) screening results: obtaining a hybridoma cell capable of stably secreting monoclonal antibodies aiming at the H protein of peste des petits ruminants virus. After expanded culture, suspending the cell with frozen cell stock solution, subpackaging the suspension in a freezing tube, marking the tube, putting the tube into liquid nitrogen for freezing, and naming the cell as a Peste des petits ruminants virus H protein monoclonal antibody hybridoma cell strain, wherein the cell strain is delivered to China general microbiological culture Collection center of microbiological research institute of China academy of sciences, No. 3, West Lu No.1, Beijing, Injaward, the North Cheng, on the year 2020 and 08 months, and the preservation number is CGMCC No. 19663.
5. Preparation and purification of monoclonal antibodies
Taking hybridoma cell strain which is stored by liquid nitrogen and stably secretes monoclonal antibody, thawing, centrifuging, and re-suspending and inoculating to 48-hole cell plate with DMEM culture medium containing 20% FBS (fetal bovine serum) and 5% CO2The carbon dioxide incubator (2) is subjected to static culture at 37 ℃. After good growth, the cells were transferred to a 24-well plate and then passed through a 45cm cell2And (5) carrying out enlarged culture on the cell bottle. Collecting cells in the cell bottle, adjusting the cell concentration after counting the cells, injecting the cells into the abdominal cavity of the mouse, and sucking ascites from the abdominal cavity of the mouse after 10-14 times.
The ascites fluid was centrifuged at 10000r/min for 10min, the supernatant was collected, precipitated with 50% ammonium sulfate, centrifuged to remove the supernatant, and the precipitate was resuspended in 20mM phosphate buffer (pH 7.2). And purifying and desalting and degreasing by using a Sephadex G25 column to obtain the purified monoclonal antibody.
6. Determination of the potency of monoclonal antibodies
Because the standard serum and the standard antigen of the peste des petits ruminants virus do not exist at present, the invention adopts the purified antigen liquid of the peste des petits ruminants virus Clone 9 strain as the standard antigen, and the ELISA plate is coated by the concentration of 10 mu g/m L and is used for measuring the titer of the monoclonal antibody, and the result shows that the titer of the monoclonal antibody of the peste des petits ruminants virus H protein is 211。
Example 2 Peste des petits ruminants virus antigen enzyme-linked immunosorbent assay kit
1. Preparation of ELISA plates
The polyclonal antibody of rabbit anti-peste des petits ruminants virus is diluted moderately by using a coating solution (carbonate buffer solution, pH 9.6), then a plate is coated, each sample well is coated for 12-24 h at the temperature of 2-8 ℃, washing is carried out twice by using a washing solution (PBS (0.01mol// L, pH 7.4 and containing 0.1% Tween-20)), then PBS (0.01mol// L and pH 7.4) containing 3% BSA is added for blocking for 2h at the temperature of 37 ℃, a sample diluent of 5% sucrose is covered, dried, evacuated and packaged, and the sample is stored at the temperature of 2-8 ℃.
2. Preparation of matched reagent
(1) Sample treatment liquid
Lysate A (L B-A) 6% CHAPS containing 2% Tween20 and 1% Tween 80.
Lysate B (L B-B), a 100mM solution of PMSF in isopropanol, was diluted to working final concentration (2mM) with PBS (0.01mol// L, pH 7.4) at the time of use.
Sample dilution 0.01 mol/L in PBS, pH 7.4.
(2) Enzyme labeling reagent
The preparation method comprises the following steps of weighing 5mg HRP, completely dissolving the HRP in pure water to obtain brown solution, and adding freshly prepared NaIO (0.06 mol/L)4Stirring the solution at room temperature in the dark for 30min, wherein the solution turns from brown to dark green, adding 1ml of new 0.08 mol/L ethylene glycol solution [ ethylene glycol: water (V/V) ═ 9: 11%]And intermittently shaking the mixture for 30min in the dark at room temperature, adding 1ml of purified monoclonal antibody solution containing 5mg, stirring the mixture for 2-3 h in the dark at room temperature, adding 0.2ml of freshly prepared sodium borohydride solution (5mg/ml), uniformly mixing the mixture, standing the mixture for 2h at 2-8 ℃, filling the prepared enzyme-labeled monoclonal antibody solution into a dialysis bag, dialyzing the mixture in PBS (0.01 mol/L, pH 7.2) overnight to obtain the HRP-labeled peste des petits ruminants monoclonal antibody, adding isovolumetric glycerol, subpackaging and labeling the mixture, and storing the mixture at the temperature of below 15 ℃ below zero for later use.
(3) Positive control peste des petits ruminants virus of appropriate titer was used as positive control.
(4) Negative control: lysate a was used as a negative control.
(5) The concentrated washing solution (20 ×) was 0.2 mol/L in PBS, pH 7.4, containing 2% Tween-20.
(6) Display solution commercially available color development A and B solutions (available from U.S. KP L).
(7) Stop solution 1 mol/L H2SO4And (3) solution.
3. Detection step
(1) Preparation before detection
20ml of the concentrated washing solution is diluted with deionized water to the working concentration, namely 400ml for standby. Numbering samples to be detected, marking corresponding detection holes, and setting a negative control hole, a positive control hole and a blank control hole for each detection.
(2) Sample treatment and application
When the sample is liquid, such as blood, tissue fluid or secretion, L B-A of 100 mu L and L B-B of 4 mu L are uniformly mixed, then 100 mu L of the sample to be detected is added, and after the mixture is fully mixed, 100 mu L of the sample to be detected is added into the reaction hole.
When the sample is solid (such as tissue block, feces, etc.) or cotton swab, 1m L L B-A and 40 mu L L B-B are mixed uniformly, then 1m L sample diluent is added, 0.1-0.2 g of crushed solid sample or cotton swab is placed in the solution, after 30min of intermittent shaking at room temperature, 8000r/min is centrifuged for 3min, 100 mu L supernatant is added into the reaction hole.
100 μ L control solution was added to each of the negative and positive control wells, and no solution was added to the blank control wells.
(3) And (3) incubation: sealing, placing on a micro-oscillator, oscillating at medium speed and room temperature for 60min or reacting at 37 ℃ for 30min, and then removing the reaction solution.
(4) Washing plate, adding 350 μ L washing solution into each well, standing for 3min, and washing repeatedly for 3 times, or washing with plate washing machine for 3 times.
(5) Adding enzyme and adding enzyme labeling reagent into corresponding holes respectively.
(6) Sealing, placing on a micro-oscillator, oscillating at medium speed and room temperature for 60min or reacting at 37 ℃ for 30min, and then removing the reaction solution. And (4) repeating the step.
(7) And (3) developing, namely mixing the developing solution A and the developing solution B in equal proportion, adding 100 mu L of freshly prepared developing solution into each hole, and developing for 10min at room temperature in a dark place.
(8) And measuring the absorbance, adding 100 mu L stop solution into each hole to stop the reaction, and measuring the OD value of each hole by adopting a dual wavelength of 450nm (detection wavelength)/630 nm (reference wavelength).
(9) Determination of results
And when the light absorption value of the negative control hole is less than or equal to 0.1 and the light absorption value of the positive control hole is more than or equal to 0.5, the test is established, otherwise, the test needs to be performed again.
Calculating the value of a negative control hole by using a critical value, wherein the value is + 0.15;
OD value is not less than ODNegative of+0.15, judging the sample peste des petits ruminants antigen positive;
OD value < ODNegative of+0.15, judge the sample peste des petits ruminants antigen negative.
The kit can sensitively and specifically detect complete or split virus antigens in samples such as animal excrement, oral and nasal cavity secretion, virus culture and the like.
Example 3 Peste des petits ruminants virus antibody ELISA kit
1. Preparation of ELISA plates
The purified peste des petits ruminants virus H protein Escherichia coli expression antigen is moderately diluted by using a coating solution (carbonate buffer solution, pH 9.6) to be coated, each sample well is coated for 12-24H at the temperature of 2-8 ℃, washing is carried out twice by using a washing solution (PBS (0.01 mol/L, pH 7.4 and containing 0.1% Tween-20)), then PBS (0.01 mol/L and pH 7.4) containing 3% BSA is added for blocking for 2H at the temperature of 37 ℃, the sample diluent is covered by a sample diluent containing 5% sucrose, the sample diluent is dried, evacuated and packaged, and the sample is stored at the temperature of 2-8 ℃.
2. Arrangement of other Components of the kit
(1) Sample dilutions 0.01 mol/L in PBS, pH 7.4, containing 0.05% Tween-20 and 5% horse serum.
(3) Monoclonal antibody competition solution: the purified monoclonal antibody is diluted to an appropriate concentration using a sample diluent.
(3) Enzyme labeling reagent: diluting the goat anti-mouse enzyme-labeled secondary antibody with a diluent with a proper concentration to obtain the enzyme-labeled reagent.
(4) Control sample: positive sera against peste des petits ruminants were used at appropriate titers as positive control samples. Calf serum was used as a negative control sample.
(5) The concentrated washing solution (20 ×) was 0.2 mol/L in PBS, pH 7.4, containing 2% Tween-20.
(6) Display solution commercially available color development A and B solutions (available from U.S. KP L).
(7) Stop solution 1 mol/L H2SO4And (3) solution.
3. Detection step
(1) Preparation before detection
And (3) diluting the 20m L concentrated washing solution to a working concentration by using deionized water, namely 400m L for later use, numbering samples to be detected, and marking corresponding detection holes, wherein a negative control hole and a positive control hole are required to be arranged for each detection.
(2) Sample treatment and application
Taking 60 mu L of serum to be detected, adding 60 mu L of monoclonal antibody competition liquid, fully mixing, taking 100 mu L, adding into a reaction hole, and if the titer of the serum to be detected needs to be measured, diluting the serum to be detected by using a sample diluent according to a certain proportion and multiple ratio, and then detecting.
Respectively taking 60 mu L of the negative control sample and the positive control sample, respectively adding the monoclonal antibody competition solution of 60 mu L, uniformly mixing, respectively adding 100 mu L of the mixture into the negative control sample and the positive control sample,
(3) and (3) incubation: sealing, placing on a micro-oscillator, oscillating at medium speed and room temperature for 60min or reacting at 37 ℃ for 30min, and then removing the reaction solution.
(4) Washing plate, adding 350 μ L washing solution into each well, standing for 3min, and washing repeatedly for 3 times, or washing with plate washing machine for 3 times.
(5) Add enzyme 100. mu. L enzyme-labeled reagent was added to each well.
(6) Sealing, placing on a micro-oscillator, oscillating at medium speed and room temperature for 60min or reacting at 37 ℃ for 30min, and then removing the reaction solution. And (4) repeating the step.
(7) And (3) developing, namely mixing the developing solution A and the developing solution B in equal proportion, adding 100 mu L of freshly prepared developing solution into each hole, and developing for 10min at room temperature in a dark place.
(8) And measuring the absorbance, adding 100 mu L stop solution into each hole to stop the reaction, and measuring the OD value of each hole by adopting a dual wavelength of 450nm (detection wavelength)/630 nm (reference wavelength).
(9) Determination of results
And when the light absorption value of the negative control hole is more than or equal to 0.5 and the light absorption value of the positive control hole is less than or equal to 0.1, the test is established, otherwise, the test needs to be performed again.
Critical value: OD value of 50% negative control wells;
OD value > 50%. ODNegative ofJudging that the sample peste des petits ruminants antibody is negative;
OD value less than or equal to 50% ODNegative ofAnd judging the sample peste des petits ruminants antibody to be positive.
The kit can sensitively and specifically detect the small ruminant animal serum small ruminant animal plague antibody.
Example 4 colloidal gold kit gold labeling method for detecting Peste des petits ruminants Virus
The kit coats rabbit anti-Peste des petits ruminants polyclonal antibodies in a detection area (T) and coats rabbit anti-mouse secondary antibodies in a control area (C) on a nitrocellulose membrane respectively. When in detection, the peste des petits ruminants virus in the sample and a monoclonal antibody (Au-McAb) of the colloidal gold labeled H protein form an antigen-antibody complex (Au-MAb-Ag), the complex moves along a membrane due to chromatography, is combined with peste des petits ruminants polyclonal antibody (PAb) coated by a detection line to form a complex (Au-MAb-Ag-PAb), and is combined with rabbit anti-mouse secondary antibody coated by a control line to form a complex (Au-MAb-Ag-secondary antibody or Au-MAb-secondary antibody). If the sample is positive, a red line can be formed in the detection area and the control area respectively; if the sample is negative, only one red line is formed in the control area.
1. Preparation of colloidal gold
Adding 1ml of 1% HAuCl4 solution into a reagent bottle containing 100m L deionized water, heating to boil, quickly adding 5m L trisodium citrate solution, continuing heating until the solution is transparent wine red, cooling, and storing at 2-8 ℃ in a dark place for later use.
2. Preparation of colloidal gold conjugate pad
Marking the Peste des petits ruminants virus H protein monoclonal antibody by using the prepared colloidal gold, uniformly spraying the colloidal gold on a strip-shaped glass fiber membrane, immediately putting the glass fiber membrane on a freeze dryer for freeze drying after the glass fiber membrane is completely soaked, taking out the glass fiber membrane, adding a drying agent, and sealing and storing the glass fiber membrane for later use.
3. Preparation of the reaction film
And respectively taking a rabbit anti-mouse secondary antibody and a goat anti-peste des petits ruminants polyclonal antibody as a control line and a detection line on the nitrocellulose membrane by using a film spraying machine according to a set program, placing the control line and the detection line in a drying box for drying, taking out, adding a drying agent, and sealing and storing for later use.
4. Preparation of sample dilutions
To 1000ml of PBS (0.01 mol/L, pH 7.4) was added 1ml of Tween-20, and the mixture was thoroughly mixed, sterilized by filtration through a filter and dispensed.
5. Preparation of test paper strip
And sequentially adhering the sample pad, the colloidal gold conjugate pad, the reaction membrane and the water absorption pad on the adhesive backboard. The bonded adhesive backing was cut into strips of 4mm wide using a Bio-dot cutter according to a predetermined procedure. And (3) putting the test paper strips qualified in the inspection into a cleaned plastic shell, compacting, putting into an aluminum foil bag, putting into a drying agent, sealing by using a continuous sealing machine, and storing at room temperature.
The temperature is controlled not to exceed 30 ℃ and the humidity is controlled not to exceed 30% in the whole process of assembling, subpackaging and sealing.
6. Composition, action and use of kit
The kit comprises: 30 test strips, 30 sample diluents, 30 to sampling cotton swabs and 1 part of a specification are used for detecting whether the excrement, the oral and nasal secretion and the excrement of the small ruminants contain the small ruminants virus.
7. Methods of use and result determination
(1) Sample preparation
Dipping excrement and oral and nasal secretion by using a sampling cotton swab, inserting the sampling cotton swab into a sample diluent tube, fully mixing uniformly, and standing for later use. If the sample can not be detected on site, the sample should be stored below-15 ℃, and the collection and transportation of the sample are carried out according to the national regulations.
(2) Procedure for the preparation of the
Taking the test strip out of the sealed packaging bag, and placing the test strip on a horizontal desktop; sucking the diluted sample by a dropper, dropwise adding 4 drops of the sample into the sample hole of the test strip, standing for 20min, and observing the result.
(3) Determination of results
Positive: parallel purple or red lines appear in the C and T regions, indicating that peste des petits ruminants virus is detected in the sample.
Negative: a purple or red line appeared only in the C region, indicating that peste des petits ruminants virus was not detected in the sample.
And (4) invalidation: no line appears in the area C of the test paper, which indicates that the detection fails, and the detection is carried out by replacing the area C with a new one.
The test paper is a new generation diagnostic reagent developed by adopting a colloidal gold immunochromatographic technique, and detectable samples comprise animal excrement, oral and nasal secretion, and peste des petits ruminants complete virus or split virus existing in cell culture.
Example 5 colloidal gold kit for detecting Peste des petits ruminants Virus antibody
The kit coats the purified antigen of the peste des petits ruminants in a detection area (T) on a nitrocellulose membrane respectively, and coats a rabbit anti-mouse secondary antibody in a control area (C). During detection, the antibody of the peste des petits ruminants virus in the sample and the monoclonal antibody (Au-McAb) of the colloidal gold labeled H protein are combined with the peste des petits ruminants virus purified antigen coated in the detection area in a competitive mode. If the sample can obviously inhibit the combination of the colloidal gold labeled monoclonal antibody and the peste des petits ruminants virus purified antigen, the sample is shown to contain the specific antibody of the peste des petits ruminants virus. When the sample does not contain antibodies to peste des petits ruminants virus, the binding is not inhibited, and the darker the detection zone, and conversely the lighter the color. If the sample is positive, only a red line is formed in the control area; if the sample is negative, a red line can be formed in each of the detection zone and the control zone.
1. Preparation of colloidal gold
Adding 1ml of 1% HAuCl4 solution into a reagent bottle containing 100m L deionized water, heating to boil, quickly adding 5m L trisodium citrate solution, continuing heating until the solution is transparent wine red, cooling, and storing at 2-8 ℃ in a dark place for later use.
2. Preparation of colloidal gold conjugate pad
Marking the Peste des petits ruminants virus H protein monoclonal antibody by using the prepared colloidal gold, uniformly spraying the colloidal gold on a strip-shaped glass fiber membrane, immediately putting the glass fiber membrane on a freeze dryer for freeze drying after the glass fiber membrane is completely soaked, taking out the glass fiber membrane, adding a drying agent, and sealing and storing the glass fiber membrane for later use.
3. Preparation of the reaction film
And respectively taking a rabbit anti-mouse secondary antibody and a peste des petits ruminants virus antigen as a control line and a detection line on the nitrocellulose membrane by using a film spraying machine according to a set program, placing the control line and the detection line in a drying box for drying, taking out, adding a drying agent, and sealing and storing for later use.
4. Preparation of sample dilutions
1ml of Tween-20 was added to 1000m L of PBS (0.01 mol/L, pH 7.4), mixed well, sterilized by filtration through a filter and dispensed.
5. Preparation of test paper strip
The sample pad, the colloidal gold conjugate pad, the reaction membrane and the water absorption pad are sequentially stuck on the adhesive backboard as shown in the figure. The bonded adhesive backing was cut into strips of 4mm wide using a Bio-dot cutter according to a predetermined procedure. And (3) putting the test paper strips qualified in the inspection into a cleaned plastic shell, compacting, putting into an aluminum foil bag, putting into a drying agent, sealing by using a continuous sealing machine, and storing at room temperature.
The temperature is controlled not to exceed 30 ℃ and the humidity is controlled not to exceed 30% in the whole process of assembling, subpackaging and sealing.
6. Composition, action and use of kit
The kit comprises: 30 test strips, 30 sample diluents and 1 part of an instruction manual are used for detecting whether the excrement, the oral and nasal secretion and the excrement of the small ruminants contain the small ruminants virus or not.
7. Methods of use and result determination
(1) Sample preparation
Blood samples were collected and serum was isolated for use. If the serum sample can not be detected on site, the serum sample should be stored below-15 ℃, and the collection and transportation of the sample are carried out according to the national regulations.
(2) Procedure for the preparation of the
Taking the test strip out of the sealed packaging bag, and placing the test strip on a horizontal desktop; sucking the diluted sample by a dropper, dropwise adding 4 drops of the sample into the sample hole of the test strip, standing for 20min, and observing the result.
(3) Determination of results
Positive: a purple or red line appeared only in the C region, indicating that antibodies to Peste des petits ruminants virus were detected in the sample.
Negative: parallel purple or red lines appear in the C and T regions, indicating that no peste des petits ruminants virus antibodies were detected in the sample.
And (4) invalidation: no line appears in the area C of the test paper, which indicates that the detection fails, and the detection is carried out by replacing the area C with a new one.
The test paper is a new generation diagnostic reagent developed by adopting a colloidal gold immunochromatographic technique, and a detectable sample comprises a peste des petits ruminants virus antibody in serum of peste ruminants.
Sequence listing
<110> Beijing Zhonghai Biotechnology Ltd
<120> Peste des petits ruminants detection kit
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>541
<212>DNA
<213> Peste-des-pets-ruminants virus H protein nucleotide sequence)
<400>1
ggatcctgga gatccgatgc cagggatccg agcaccgatc taggtattgg ccacttttta 60
agagtcttcg agattggact ggtaagagat ctcgggctgg gtccccctgt ttttcatatg 120
accaactatc tcacagtgaa catgagtgat gactatcgga gatgtctttt agcggtaggg 180
gagttaaagt tgacagccct atgcagctca tctgagactg tgacactggg cgagagagga 240
gttccaaaga gggagcctct tgtagttgtg atactcaacc tagctgggcc cactctaggg 300
ggcgaactat acagtgtctt gcctacctct gatctcatgg tggagaaact ctatttatct 360
tcacatagag ggattatcaa agatgacgag gccaattggg tagtgccgtc taccgatgtt 420
cgtgatcttc aaaacaaagg agaatgtttg gtggaggcat gcaagactcg acctccttca 480
ttttgcaatg gcacaggatc aggcccgtgg tcagagggga gaatccctgc ttactaagct 540
t 541
Claims (5)
1. The peste des petits ruminants detection kit is characterized in that the kit is assembled by monoclonal antibodies of peste des petits ruminants virus H protein and matched reagents thereof;
the monoclonal antibody of the Peste des petits ruminants virus H protein is secreted by a hybridoma cell strain, the cell strain is named as a Peste des petits ruminants virus H protein monoclonal antibody hybridoma cell strain, and the Peste des-petits ruminants virus H protein monoclonal antibody hybridoma cell strain is delivered to the national institute of microbiology of Chongy-West Lu No.1, No. 3 of China academy of sciences, China Committee for culture Collection of microorganisms (CGMCC No. 19663) in the Kyoho-Naja-area, Beijing, 04.08.
2. The Peste des petits ruminants detection kit of claim 1, wherein the Peste des petits ruminants virus H protein monoclonal antibody, the ELISA plate and a reagent matched with the ELISA plate are assembled to detect Peste des petits ruminants virus.
3. The Peste des petits ruminants detection kit of claim 1, wherein the Peste des petits ruminants virus H protein monoclonal antibody, the ELISA plate and its matching reagent are assembled to detect Peste des petits ruminants virus antibody.
4. The Peste des petits ruminants detection kit of claim 1, wherein the monoclonal antibody of the Peste des petits ruminants virus H protein is coated on a gold-labeled fiber mat after being labeled with colloidal gold, the detection zone is coated with rabbit anti-Peste des petits ruminants virus polyclonal antibody, during detection, when Peste des petits ruminants virus exists in a sample, an antigen-antibody complex is formed with the monoclonal antibody, and then the antigen-antibody complex is combined with the rabbit anti-Peste des ruminants virus polyclonal antibody in the detection zone and develops color.
5. The kit for detecting Peste des petits ruminants according to claim 1, wherein the monoclonal antibody of the Peste des petits ruminants virus H protein is coated on a gold-labeled fiber mat after being labeled with colloidal gold, the detection zone is coated with a Peste des ruminants virus purified antigen, and the control zone is coated with a goat anti-mouse secondary antibody. When the peste des petits ruminants virus antibody exists in the sample, the peste des petits ruminants virus antibody can inhibit the combination of the monoclonal antibody marked by the colloidal gold and the purified antigen of the peste des petits ruminants virus, and the color is lightened or disappears.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115785288A (en) * | 2022-12-23 | 2023-03-14 | 北京标驰泽惠生物科技有限公司 | Peste des petits ruminants virus H-N epitope fusion protein and application thereof |
| CN116144601A (en) * | 2022-08-23 | 2023-05-23 | 中国农业科学院兰州兽医研究所 | Hybridoma cell strain secreting anti-peste des petits ruminants virus H protein extracellular region monoclonal antibody and application |
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| CN115785288A (en) * | 2022-12-23 | 2023-03-14 | 北京标驰泽惠生物科技有限公司 | Peste des petits ruminants virus H-N epitope fusion protein and application thereof |
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