CN112521495B - Preparation and application of PPR-N protein monoclonal antibody of peste des petits ruminants virus - Google Patents
Preparation and application of PPR-N protein monoclonal antibody of peste des petits ruminants virus Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- 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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- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
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Abstract
The invention relates to a PPR-N protein monoclonal antibody preparation of peste des petits ruminants virus and application thereof. The large-scale production of the PPR-N protein monoclonal antibody is realized by researching the components of the culture medium and the content of the added serum and optimizing each parameter of the suspension culture. The method can greatly reduce the culture cost, increase the number of cells in unit volume and improve the titer of the monoclonal antibody. Through the purified concentrated solution, the concentration of the monoclonal antibody can be improved by 8 times, and the original sensitivity and specificity can be kept unchanged, so that the monoclonal antibody can be used for immunological detection of peste des petits ruminants virus antibodies. The PPR-N improved competitive ELISA kit for peste des petits ruminants prepared by the invention can distinguish negative and positive controls more obviously, has larger difference between the negative and positive controls, and has better sensitivity and specificity compared with the traditional competitive ELISA method. By comparison, the traditional competitive ELISA method cannot distinguish negative and positive limits, and the improved competitive ELISA method can accurately distinguish the negative and positive limits.
Description
Technical Field
The invention relates to a preparation method and application of PPR-N protein monoclonal antibody of peste des petits ruminants virus. Belongs to the field of biological products for animals.
Background
Peste des petits ruminants (PPR) is commonly called 'sheep plague', also called as pneumocoritis (pneumocoentitis), is a virulent infectious disease caused by Peste des petits ruminants virus (PPRV), mainly infected animals include small ruminants such as oviridae, antelope horn and the like, the morbidity reaches more than 90%, the main symptoms are expressed as high fever, oral cavity intima inflammation, diarrhea, pneumonia and the like, and the mortality rate in high-risk areas can reach 100%. According to the FAO (2019), 60% of the global small ruminant breeding industry is threatened by the disease. The second industry in China is the breeding industry, and is an integral part of an economic system, and the average sheep raising amount per year can reach 5.6-6 hundred million. The disease is introduced into the Tibetan Ali area in China for the first time in 2007, and then a collective outbreak epidemic phenomenon appears in several years, the morbidity reaches 100%, and the mortality can reach more than 80%. Due to the imperfection of the large-scale culture system and the shortage of biological products in China, huge economic loss is caused. The disease is an animal epidemic disease which is regulated by the world animal health Organization (OIE) and must be reported in time. 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.
Disclosure of Invention
The invention aims at key technical problems of peste des petits ruminants pathogenic microorganism diagnosis and detection technology, antigen antibody production and purification and the like, and particularly makes a substantial breakthrough on pathogenic microorganism identification and separation and monoclonal antibody large-scale production technology, obtains monoclonal antibody hybridoma cells, performs low serum culture medium suspension culture domestication on the cells, can be put into large-scale production enterprises for production, and has lower production cost than a marketable culture medium. The peste des petits ruminants antibody improved competitive ELISA kit which is economical, practical, mild in specificity and high in sensitivity is further developed aiming at the hybridoma cells capable of secreting the monoclonal antibody, the problem of lack of peste des petits ruminants detection technology is facilitated, and remarkable economic and social benefits are brought to the sheep industry in China. Therefore, the present invention aims to provide a monoclonal antibody of PPR-N protein of peste des petits ruminants virus with strong sensitivity, mild specificity and high titer according to the needs and deficiencies in the field, and the monoclonal antibody can be applied to the detection of PPR-N antibody.
The technical scheme of the invention is as follows:
1. a PPR-N protein monoclonal antibody of Peste des petits ruminants virus is characterized in that the PPR-N protein monoclonal antibody of Peste des petits ruminants virus is secreted by a hybridoma cell strain, the cell strain is named as a PPR-N (P-1-5) hybridoma cell strain of the N protein monoclonal antibody of Peste des petits ruminants virus, and the PPR-N is delivered to the microbial strain preservation management center CGMCC No.21413 of China Committee for microorganism culture Collection, china institute of microbiology, no. 1 institute 3, naja, china national academy of sciences, north Yang, beijing, 12 months and 14 days in 2020;
the PPR-N protein monoclonal antibody contains a sequence 1, which is a main antigen region sequence of the peste des petits ruminants N protein gene optimized according to the codon characteristics of escherichia coli.
2. The invention relates to a large-scale production method of PPR-N protein monoclonal antibody of peste des petits ruminants virus, which is characterized in that a hybridoma cell strain PPR-N (P-1-5) of the PPR-N protein monoclonal antibody of peste des petits ruminants virus adapts to a 10% serum DMEM culture medium; according to the transfer amount of 50 ten thousand cells/mL, transferring a cell bottle to a shake flask, transferring the shake flask to a 10L seeding tank cell bioreactor, and then transferring the shake flask to a 50L cell bioreactor for amplification culture step by step, after the culture is finished, sampling a sampling hole and counting the cells, wherein the counting result is 600-650 ten thousand cells/mL.
3. The invention relates to an application of PPR-N protein monoclonal antibody of peste des petits ruminants virus in an improved competitive ELISA kit, which is characterized in that the PPR-N protein monoclonal antibody of peste des petits ruminants virus, an enzyme label plate and a matched reagent are assembled to be used for detecting the peste des petits ruminants virus.
4. The invention relates to a Peste des petits ruminants virus antibody improvement competition ELISA kit and a matched reagent thereof, which are characterized in that the Peste des petits ruminants virus antibody improvement competition ELISA kit uses an improvement competition ELISA method to detect Peste des petits ruminants virus specific antibodies in serum samples;
the improved competitive ELISA method is characterized in that a polyethylene micropore plate of a kit is pre-coated with a peste des petits ruminants recombinant PPR-N protein purified antigen, sample serum is specifically combined with the antigen firstly, and then is competitively combined with a monoclonal antibody, so that the sensitivity and the accuracy of detection are enhanced.
The invention achieves the new technical effect
The invention relates to a PPR-N protein monoclonal antibody preparation of peste des petits ruminants virus and application thereof. The large-scale production of the monoclonal antibody is realized by researching the components of the culture medium and the content of the added serum and optimizing each parameter of the suspension culture. The method can greatly reduce the culture cost, increase the number of cells in unit volume and improve the titer of the monoclonal antibody. Through the purified concentrated solution, the concentration of the monoclonal antibody can be improved by 8 times, the original sensitivity and specificity can be kept unchanged, and the monoclonal antibody can be used for immunological detection of peste des petits ruminants virus antibodies. The PPR-N protein monoclonal antibody of the peste des petits ruminants virus produced in large scale is used, the reaction time and the sample adding sequence are optimized, the PPR-N improved competitive ELISA kit for peste des petits ruminants is prepared, negative and positive controls can be distinguished more obviously, the difference between the negative and positive controls is larger, and compared with the traditional competitive ELISA method, the PPR-N improved competitive ELISA kit has better sensitivity and specificity. Through test data comparison, the traditional competitive ELISA method cannot distinguish negative and positive limits, and the improved competitive ELISA method can successfully and accurately distinguish the negative and positive limits.
The invention relates to microbial resource information
The immune antigen used in the invention is PPR-N protein of peste DEs petits ruminants virus, and by referring to the published nucleotide sequence of the N protein antigen of peste DEs petits ruminants virus of NCBI, the laboratory selects the region in which the antigenic determinants at the N end are concentrated, artificially synthesizes the sequence after codon optimization, clones the sequence to a pET28a (+) expression vector, converts Escherichia coli Transetta DE3 to express, and purifies the expression product to be used as the immune antigen.
The detection antigen is a ruminant peste des petits ruminants virus concentrated antigen, and the selected virus strain is Clone9 vaccine strain provided by China veterinary microorganism strain preservation center.
Detailed Description
1. Cultivation of monoclonal antibody hybridoma cells
According to the published nucleotide sequence of the peste des petits ruminants virus N protein antigen of NCBI, a region with a concentrated N-terminal antigenic determinant is selected, a sequence (sequence 1) is artificially synthesized after codon optimization, and is cloned to a pET28a (+) expression vector, and escherichia coli is transformed for prokaryotic expression.
Inoculating Vero cells which grow well to the peste des petits ruminants virus Clone9 vaccine strain, culturing for multiple generations, and purifying the virus by using Purose 6Fast Flow filler, namely concentrated purified solution of the peste des petits ruminants virus. Immunizing Balb/c mice by using the recombinant PPR-N protein as an antigen, taking a spleen cell suspension and SP20 cells to fuse according to a certain proportion when the antibody titer reaches more than 1 and 20000, then plating, and 5 percent of CO 2 And (3) standing and culturing at 37 ℃. Half of the fluid change was performed on days 4 and 8 after the fusion. At day 12 after fusion, the supernatants from wells with clusters appearing at the bottom of the wells were selected for screening. And performing propagation and subcloning on the strong positive hole, performing three times of subcloning, and then freezing and storing in liquid nitrogen. The Peste des petits ruminants virus N protein monoclonal antibody hybridoma cell strain PPR-N (P-1-5) is named as Peste des petits ruminants virus N protein monoclonal antibody hybridoma cell strain PPR-N (P-1-5), and has been delivered to the microbial research institute of China academy of sciences, china Committee for culture Collection of microorganisms (CGMCC No. 21413) of Naciotu des Hebei Lu No. 1, beijing, ing-Yang district, 12 months and 14 days in 2020.
2. The invention relates to a preparation method of PPR-N protein monoclonal antibody of peste des petits ruminants virus, which is characterized in that a hybridoma cell strain PPR-N (P-1-5) of the PPR-N protein monoclonal antibody of the peste des petits ruminants virus adapts to a 10% serum DMEM culture medium; according to the transfer amount of the cells of 50 ten thousand/mL, the cell flask is transferred to a shake flask, and the shake flask is transferred to a 10L cell bioreactor. After 72h of culture, the seeding tank was transferred to a 50L cell bioreactor. The liquid loading of a 50L reactor is about 50 percent; the rotating speed is controlled to be 70-100 r/min; the pH value is controlled within the range of 7.0-7.2; the DO value is controlled to be below 40%; CO 2 2 The early stage of the value is controlled to be about 4 to 5 percent; controlling the temperature to be (37 +/-1) ° C; the tank pressure is controlled to be about 0.1 MPa; after 56h of culture, supplementary energy substances (carbon source, nitrogen source, amino acid, growth factor and the like) are fed at a feeding speed of 200-300 mL/h. The culture period is 72-80 h. Culture knotAfter that, the sampling hole samples and the cells are counted, and the counting result is 600 to 650 ten thousand/mL. The set of culture system realizes the large-scale production of the monoclonal antibody. And monoclonal antibody purification was performed using a fully automated chromatographic purification system. The method can greatly reduce the culture cost, increase the number of cells in unit volume and improve the titer and purity of the monoclonal antibody.
Examples
The following examples are presented to provide a better understanding of the present invention, and are not intended to limit the scope of the present invention to the best mode and the content of the present invention, and any method and product similar to the present invention or similar thereto by combining the present invention with other features of the prior art will fall within the scope of the present invention.
Example 1 Scale Pilot production of monoclonal antibodies
1. Preparation of monoclonal antibody hybridoma cells
According to the published nucleotide sequence of the peste des petits ruminants virus N protein antigen of NCBI, a region with a concentrated N-terminal antigenic determinant is selected, a sequence (sequence 1) is artificially synthesized after codon optimization, and is cloned to a pET28a (+) expression vector, and escherichia coli is transformed for prokaryotic expression.
Inoculating Vero cells which grow well to the peste des petits ruminants virus Clone9 vaccine strain, culturing for multiple generations, and purifying the virus by using Purose 6Fast Flow filler, namely concentrated purified solution of the peste des petits ruminants virus. Immunizing Balb/c mice by using the recombinant PPR-N protein as an antigen, taking a spleen cell suspension and SP20 cells to fuse according to a certain proportion when the antibody titer reaches more than 1 and 20000, then plating, and 5 percent of CO 2 And standing and culturing at 37 ℃. Half of the fluid change was performed on days 4 and 8 after the fusion. On day 12 after fusion, the supernatants from wells with clusters appearing at the bottom of the wells were selected for screening. And performing propagation and subcloning on the strong positive hole, performing three times of subcloning, and then freezing and storing in liquid nitrogen. The Peste des petits ruminants virus N protein monoclonal antibody hybridoma cell strain PPR-N (P-1-5) is delivered to Beijing city Kogyang district No. 1 Homeh No. 3 Chinese family in 12.14.2020China general microbiological culture Collection center (CGMCC) No.21413 of the institute of microbiology and research.
Sequence 1: and the main antigen region sequence of the peste des petits ruminants N protein gene is optimized according to the codon characteristics of escherichia coli.
2. Reviving and expanding propagation passage of peste des petits ruminants fused cell
Taking a Peste des petits ruminants fused cell from liquid nitrogen, resuscitating and culturing in a 6-well cell plate by using a DMEM medium containing 20% serum, and performing CO 2 The incubator is kept still for 3 days. Transfer to small cell bottle for subculture.
3. Low-serum domestication and culture medium optimization of peste des petits ruminants fused cells
Performing gradient serum-lowering acclimation on cells in good growth state, acclimating according to the addition ratio of 20%, 17%, 14% and 10% serum, culturing for 3 generations in each serum ratio to make the cells slowly adapt to low serum culture medium, and gradually adapting to DMEM culture medium containing 10% serum after 10-12 subcultures. Adding 0.5% glucose, 0.1% glutamine, 0.08% L-cysteine and 0.2% bovine serum albumin to a low serum medium, subculturing for 3-4 times, observing with an electron microscope, and obtaining a full cell morphology and a good growth state. (the novel modified Medium is hereinafter referred to as 10% modified DMEM medium)
4. Peste des petits ruminants fused cell expansion culture
Using 10% modified DMEM medium, small cell flasks transferred to medium cell flasks, CO 2 The incubator is kept still for 3 days. Transferring a medium cell bottle to a shake flask according to the transfer amount of 40 ten thousand cells/mL, wherein the liquid loading of the shake flask is about 20%, and culturing for 2-3 d in a shaking incubator. After shaking the flask to shake culture for a sufficient amount, transferring to a tank for seed liquid suspension culture.
5. Peste des petits ruminants fused cell 10L seed tank suspension culture
Use of 10% modified DMEM Medium, according to cellsTransfer of several 40 ten thousand/mL flasks to 10L cell bioreactors (seed tanks). The control parameters of the bioreactor include liquid loading amount, rotation speed, pH value, DO value and CO 2 Value, temperature, etc., the liquid loading of the 10L reactor is between 30 and 40 percent; the rotating speed is controlled to be 80-90 r/min; the pH value is controlled within the range of 7.0-7.2; the DO value is slowly controlled to be below 50%; CO 2 2 The value is controlled to be about 5 percent, and the temperature is controlled to be (37 +/-0.5) DEG C. The culture time is 72h. After the culture is finished, sampling holes are used for sampling and counting cells, and the counting result is 400-450 ten thousand/mL.
6. Peste des petits ruminants fused cell 50L large-scale pilot production
The seeding tank was transferred to a 50L cell bioreactor using 10% modified DMEM medium in a transfer amount of 50 ten thousand cells/mL. The control parameters of the bioreactor include liquid loading amount, rotation speed, pH value, DO value and CO 2 The value, the temperature, the tank pressure, the material supplement and the like, wherein the liquid loading of a 50L reactor is about 50 percent; the rotating speed is controlled to be 70-100 r/min; the pH value is controlled within the range of 7.0-7.2; the DO value is controlled to be below 40%; CO 2 2 The early stage of the value is controlled to be about 4 to 5 percent; controlling the temperature to be (37 +/-1) ° C; the tank pressure is controlled to be about 0.1 MPa; after the cultivation for 56 hours, supplementary energy substances (carbon source, nitrogen source, amino acid, growth factor and the like) are fed in at a feeding speed of 200-300 mL/h. The culture period is 72-80 h. After the culture is finished, sampling holes are used for sampling and counting cells, and the counting result is 600-650 ten thousand/mL.
Taking out the cell sap in a sterile environment, centrifuging, collecting supernatant, and purifying.
Example 2 purification and detection of monoclonal antibodies
Using a purification column having a diameter of 350mM and a fully automatic purification system, water for injection, a sample, an equilibrium solution [ PB solution (20 mM, pH 7.2) ], an elution solution [ PB solution (20 mM, pH 7.2) + NaCl solution (0.5M, pH 7.2) ], a 20% ethanol solution, etc. were prepared (each of the above solutions was purified using a 0.45. Mu.L filter). Adding a filler (protein G) into the purification column, exhausting after covering, pressurizing after no bubble exists in the column, manually compressing and emptying liquid after the filler is precipitated, and successfully filling the column. Purifying according to a full-automatic operation system, wherein system parameters and an operation process are as follows: controlling the column pressure to be 3-5 MPa; the column was washed 3 times with water for injection; the equilibrium solution was passed through the column for 3 column volumes; loading a sample (cell supernatant and a PB solution (20 mM, pH7.2) which are mixed uniformly in equal volumes); the equilibrium solution was passed through the column for 3 column volumes; loading the elution solution to a column, and collecting a purified concentrated solution of an elution peak; the column was washed 3 times with water for injection; the column was washed 3 times with 20% ethanol solution.
Because standard serum and standard antigen of peste des petits ruminants virus do not exist at present, the research adopts peste des petits ruminants virus Clone9 strain purified antigen liquid as standard antigen, an enzyme label plate is coated by 10 mug/mL concentration and is used for measuring the titer of a monoclonal antibody, indirect ELISA antibody value detection is carried out on supernatant and the purified liquid, and the dilution gradient of a detection result near an OD value of 1.0 is as follows: the dilution multiple of the supernatant is 512 times; the dilution ratio of the purified solution is 4000 times. The antibody concentration of the purified solution was increased 8-fold compared to the supernatant.
Example 3 Peste des petits ruminants PPR-N protein improvement competition ELISA antibody detection kit
The kit is more precisely improved on the basis of the traditional competitive ELISA kit, and the sample of the traditional competitive kit and the monoclonal antibody are simultaneously added into a coating plate for competitive epitope reaction, so that the negative and positive are distinguished through color reaction. However, the conventional competition method has the following problems: the detection method of the improved competitive ELISA is successfully developed through continuous efforts and attempts of a team due to the phenomena of over-strong specific binding, low sensitivity, unstable positioning under competitive pressure and the like. The improved method has the advantages that the sample reacts with the coating antigen preferentially, after the reaction is carried out for a period of time, the monoclonal antibody is added for competitive epitope reaction, the method can effectively reduce competitive pressure, reduce specific reaction and increase sensitivity, the sample can be better combined with the antigen, and the negative and positive limit range is wider.
1. Preparation of ELISA plates
The Escherichia coli expressing PPR-N protein antigen after the purification of the cationic filler is proportionally diluted by using a coating solution (carbonate buffer solution, pH = 9.4-9.6) to coat a plate, each sample well is coated with 100 mu L of the PPR-N protein antigen at 4-8 ℃ for 18-24 h, the plate is washed for 3 times by using a washing solution (0.01 mol/L PBS, pH =7.4 and containing 0.2 percent Tween-20), and then a 5 percent skim milk powder-containing PBS (0.01 mol/L, pH = 7.4) blocking solution is added to stand and block for 1-2 h at 37 ℃. The sample was covered with a 2.5% sucrose-containing dilution and then dried. Standing, oven drying for 8 hr, evacuating, packaging, and storing at 4 deg.C.
2. Arrangement of other Components of the kit
(1) Sample diluent: 0.01mol/l PBS, pH =7.4, containing 0.15% sucrose and 5% horse serum.
(2) Monoclonal antibody competition solution: protein G pad purified monoclonal antibody was diluted 512 fold using sample diluent.
(3) Enzyme labeling reagent: and (3) diluting the rabbit anti-mouse enzyme-labeled secondary antibody by using a diluent by 1.
(4) Control sample: positive sera against Peste des petits ruminants at appropriate titers were used as positive control samples. Fetal bovine serum was used as a negative control sample.
(5) The preparation of the standard positive serum comprises the steps of concentrating the antigen of the PPRV by using an ultrafiltration centrifugal tube, selecting a general immune complex micro-hydrogel adjuvant (Saedo) and the concentrated antigen to carry out low-speed homogenate emulsification to prepare an inactivated vaccine, and immunizing a goat by adopting a neck muscle multi-point injection method. The immunization is carried out 14 days after the first immunization, and the immunization interval is 7-8 days after the first immunization. Blood sampling detection is carried out 7-9 days after the last immunization, the antibody value of the goat serum is determined by adopting an indirect ELISA detection test, and the detection antibody value is more than 1.
(6) Wash concentrated (20 ×): 0.2mol/L PBS, pH =7.4, 2% Tween-20.
(7) Display liquid: commercially available color developing solutions A and B.
(8) Stopping liquid: 1mol/L H 2 SO 4 And (3) solution.
3. Detection step
(1) Preparation before detection
30ml 10-fold concentrated washing solution was taken and diluted with deionized water to 300mL working solution for use. Numbering samples to be detected, marking corresponding detection holes, and setting 2 groups of negative and positive control holes for each detection.
(2) Sample treatment and application
30 mu L of serum to be detected is taken, 30 mu L of diluent is added, and after full mixing, 50 mu L of the serum is added into a reaction hole. If the titer of the serum to be detected needs to be determined, the sample diluent is diluted by a certain proportion of multiple ratio of the serum to be detected and then the detection is carried out.
50 μ L of each of the negative and positive control samples was added to the negative and positive control wells, respectively.
(3) And (3) incubation: sealing, standing at 37 deg.C for 30min, and adding 50 μ L of monoclonal antibody competition solution. Sealing, standing at 37 deg.C, reacting for 20min, and removing the reaction solution.
(4) Washing the plate: adding 300 μ L of washing solution into each well, standing at room temperature for 4min, and repeatedly washing for 3 times.
(5) Adding a secondary antibody: 100 microliter of diluted enzyme labeling reagent is added into the corresponding holes respectively.
(6) Sealing, standing at 37 deg.C for 30min, and removing the reaction solution. And (5) repeating the step (4).
(7) Color development: mixing the color development solution A and the color development solution B in equal proportion, adding 100 mu L of freshly prepared color development solution into each hole, and placing the mixture in an incubator at 37 ℃ for color development in a dark place for 10min.
(8) And measuring the light absorption value, adding 40 mu L of stop solution into each hole to stop the reaction (the stop time is not more than 15 minutes), and measuring the OD value of each hole by using a single wavelength of 450 nm.
(9) Determination of results
OD value (OD) of PPRV-N negative serum n (ii) a OD value (OD) of PPRV-N positive serum c (ii) a Control OD value of enzyme-labeled antibody p (ii) a OD value of sample s ；
Positive serum inhibition ratio PI (c/p) = [ (1-OD) c /OD p )×100]％
Negative serum inhibition rate PI (n/p) = [ (1-OD) n /OD p )×100]％
Positive serum inhibition ratio PI (s/p) = [ (1-OD) s /OD p )×100]％
Judging the detection result according to the inhibition rate, wherein PI (c/p) > 60% and PI (n/p) < 40%, and the test result is effective; otherwise, the test is carried out again.
And (3) judging: PI (s/p) > 50, and the result is positive; PI (s/p) is less than or equal to 50, and the result is negative.
The kit can sensitively and specifically detect the small ruminant animal serum small ruminant animal plague antibody.
Example 4 detection method of Peste des petits ruminants fusion traditional competitive ELISA kit
1. Preparation of ELISA plates and configuration of other kit components (same as preparation method in example 4).
2. Detection step
(1) Preparation before detection
20mL of the concentrated washing solution was diluted with deionized water to the working concentration, i.e., 400mL for use. Numbering samples to be detected, marking corresponding detection holes, and setting negative and positive control holes for each detection.
(2) Sample treatment and application
60 mu L of serum to be detected is taken, 60 mu L of monoclonal antibody competition liquid is added, and after full mixing, 100 mu L of monoclonal antibody competition liquid is added into a reaction hole. If the titer of the serum to be detected needs to be determined, the sample diluent is used for detecting after the serum to be detected is diluted according to a certain proportion and multiple ratio.
Respectively taking 60 mu L of the negative control sample and the positive control sample, respectively adding 60 mu L of the monoclonal antibody competition solution, uniformly mixing, respectively adding 100 mu L of the monoclonal antibody competition solution 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 the plate, adding enzyme-labeled secondary antibody, developing and the like (the same procedure as in example 4).
(5) And measuring the light absorption value, adding 100 mu L of stop solution into each hole to terminate the reaction, and measuring the OD value of each hole by adopting a dual wavelength of 450nm (detection wavelength)/630 nm (reference wavelength).
(6) 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%. OD Negative of Judging that the sample peste des petits ruminants antibody is negative;
OD value less than or equal to 50% OD Negative of And 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 5 comparison of the present invention with conventional competitive ELISA versus detection assay
46 serum samples were collected from the sheep farm and tested and compared using OIE recommended standard competitive ELISA kit, traditional competitive ELISA kit and modified competitive ELISA kit. The sample detection and sample adding sequence is as follows: A1-B1 are blank controls, C1-D1 are negative controls, and E1-F1 are positive controls; the 2 nd column is the repeated detection holes of samples No. 1 to No. 44 and No. 1 to No. 44 from E7.
Results of 44 samples detected by OIE recommended standard competition ELISA kit
The OD value of the sample is detected and the negative and positive are judged (detected according to the operation flow of the kit) by an OIE recommended standard competitive ELISA kit, and the result shows that: 18 positive samples and 26 negative samples have obvious difference range of negative and positive data; the 2-time detection is carried out synchronously, the repetition rate reaches 100%, and the stability of the standard competitive ELISA kit is good (see table 1).
TABLE 1 Standard Competition ELISA kit test results
Note: underlined is a positive sample
2. Results of 44 samples detected by traditional competitive ELISA kit
The OD value detection and the positive and negative judgment of the sample are carried out by the traditional competitive ELISA kit (the detection is carried out according to the operation flow of the embodiment 4), and the comparison with the result of the OIE recommended standard competitive ELISA kit shows that: 2 positive samples and 42 negative samples fail to be successfully judged in negative and positive limit, and the same samples have larger numerical difference after repeated tests, so that the traditional competitive ELISA kit cannot be used for detection (see Table 2).
TABLE 2 detection results of conventional competitive ELISA kits
Note: samples positive are underlined
3. Result of 44 samples detected by improved competitive ELISA kit
The OD value of the sample is detected and the positive and negative are judged by the improved competitive ELISA kit (the detection is carried out according to the operation flow of the example 4), and the comparison with the result of the OIE recommended standard competitive ELISA kit shows that: 17 positive samples and 27 negative samples are successfully subjected to negative and positive limit judgment, and the coincidence rate can reach over 90%. And the improved competitive ELISA kit is successfully developed and can be put into production to form a commercial industry (see Table 3) by repeated tests with smaller numerical difference of the same sample.
TABLE 3 improved competitive ELISA kit test results
Note: underlined is a positive sample
4. The comparison of test data of three different kits can clearly find that the traditional competition method cannot overcome the problem that the antigen with stronger specificity is combined with the monoclonal antibody, the sensitivity cannot be improved, the negative and positive boundaries are fuzzy, and the detection data of the same repeated sample has larger deviation; the improved competition method successfully overcomes the specificity problem, improves the sensitivity, has clear positive and negative limits and well embodies the coincidence rate and the repeatability. The improved competition method and the traditional competition method have the advantages that the obtained test results are different on the basis of the same antigen, antibody and reagent. The reaction sequence, time and space of the antigen and the antibody are optimized, and the defects of strong specific binding force, high primary antibody sensitivity and the like are reduced by using an improved competition system. Test data prove that the detection quality and efficiency can be greatly improved by changing the detection process. Based on the detection data of the standard competition method, the conformity rate of the improved competition method is up to more than 90 percent, the repetition rate is up to more than 98 percent, the detection data deviation of the same sample is extremely small, and the standard accords with the commercial production standard.
Sequence listing
<110> Beijing Zhonghai Biotechnology Ltd
<120> preparation and application of PPR-N protein monoclonal antibody of peste des petits ruminants virus
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 544
<212> DNA
<213> sequence of main antigenic region of peste des petits ruminants virus N protein gene optimized according to codon characteristics of Escherichia coli (2 Ambysoma laterale x Ambysoma jeffersonia)
<400> 1
ggatcccgtc tggcatgtaa ggaggattac cgttacgcaa tcagcagcac caacgaaatc 60
ggtctgctgg gtgccggtgg tctgactacc acctggaaag aatacagcca cgacctgcag 120
ctgaacgacg gcactgtcaa agctatctgc gtggctggtt ctttcaaagt cactgcgctg 180
aacgtagtgt cccgtcgtta cctggcttct ctgcacaaag gtgcgctgct gacttctgta 240
accttcgaac tgctgttcga tggtaccaac ccatccaccg aagaaatggg cgacgacttc 300
ggtttcggcc tgtgtccttt cgacacttct ccggttgtga aaggcaaata taacaccacc 360
ctgctgaacg gctccgcctt ttatctggtg tgcccgattg gctggactgg cgttattgaa 420
tgcactgcgg tttccccgac cacgctgcgt accgaagttg taaagacctt tcgccgcgag 480
aaaccgtttc cgcatcgcat ggattgcgtt accacgacgg ttgagaatga agatctgtaa 540
gctt 544
Claims (3)
1. The PPR-N protein monoclonal antibody of the peste des petits ruminants virus is characterized in that the PPR-N protein monoclonal antibody of the peste des petits ruminants virus is secreted by a hybridoma cell strain, the cell strain is named as a PPR-N (P-1-5) hybridoma cell strain of the PPR-N protein monoclonal antibody of the peste des petits ruminants virus, and the preservation number of the PPR-N protein monoclonal antibody is CGMCC No.21413.
2. A large-scale production method of PPR-N monoclonal antibody of Peste des petits ruminants virus according to claim 1, wherein the PPR-N monoclonal antibody hybridoma cell strain PPR-N (P-1-5) is adapted to 10% serum DMEM medium; according to the transfer amount of 50 ten thousand cells/mL, transferring a cell bottle to a shake flask, transferring the shake flask to a 10L seeding tank cell bioreactor, and then transferring the shake flask to a 50L cell bioreactor for amplification culture step by step, after the culture is finished, sampling a sampling hole and counting the cells, wherein the counting result is 600-650 ten thousand cells/mL.
3. The use of PPR-N protein monoclonal antibody of Peste des petits ruminants virus of claim 1 in the preparation of improved competitive ELISA kit, characterized in that the PPR-N protein monoclonal antibody of Peste des petits ruminants virus, the ELISA plate and its kit are assembled into PPR-N protein improved competitive ELISA kit.
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