CN109580942B - Application of QX type IBV cell adapted strain MJ in preparation of whole virus antigen ELISA detection kit and kit - Google Patents
Application of QX type IBV cell adapted strain MJ in preparation of whole virus antigen ELISA detection kit and kit Download PDFInfo
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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
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Abstract
The invention provides application of a QX type IBV cell adapted strain MJ in preparation of a whole virus antigen ELISA detection kit and the kit, belonging to the technical field of virus antibody detection. In the application of the invention, the preservation number of the adopted QX type IBV cell adaptive strain MJ is CGMCC No.14681, the cell adaptive strain MJ does not contain other virus antigens, only specifically binds with IBV positive serum and does not have cross reaction with other virus positive serum, and the cell adaptive strain MJ is effectively proliferated in Vero cells, thereby effectively solving the problems of difficult purification and high non-specificity of IBV whole virus. The invention also provides a whole virus antigen indirect ELISA detection kit constructed based on the cell adaptive strain MJ, which has high detection sensitivity and strong specificity, can be used for detecting the early infection of the QX type IBV, and can also be used for detecting the early antibody level of the chicken flock immunized by the attenuated vaccine.
Description
Technical Field
The invention relates to the technical field of virus antibody detection, in particular to application of a QX type IBV cell adapted strain MJ in preparation of a whole virus antigen ELISA detection kit and a kit.
Background
Infectious Bronchitis (IB) is an acute, highly contagious respiratory infectious disease caused by Infectious Bronchitis Virus (IBV). The disease is widely prevalent worldwide and is one of the major viral infectious diseases seriously harming the poultry industry. Because IBV is easy to have variation, new variant strains are caused to continuously appear, and the cross protection among different strains is weak, so that the IBV often explodes in immune and non-immune poultry groups, huge economic loss is caused, and the healthy development of poultry industry in China is severely restricted.
Currently, the laboratory diagnostic methods for IBV mainly include: virus isolation identification, chick embryo neutralization assay, agar-agar amplification assay, hemagglutination inhibition assay (HI), indirect immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). The conventional virus separation and identification and chick embryo neutralization test has complicated operation and long test period. The sensitivity of the agar-agar gel electrophoresis is low. In the hemagglutination inhibition test, since IBV does not have the capability of naturally agglutinating erythrocytes, hemagglutination antigen of IBV needs to be treated by lecithin enzyme C, and meanwhile, in order to reduce the non-specificity in serum, the serum needs to be pretreated by kaolin, so the test operation is complicated and has instability. Although the IFA test can effectively detect the viral antigen in the pathological material, the antibody of the fluorescent label has higher cost, high requirement on experimental instruments and no universality.
ELISA detection is used as a serological detection method, has high sensitivity, low cost and convenient operation, and is suitable for large-scale detection. At present, one or more structural proteins or antigen epitopes of recombinant expression IBV are mainly used in the market, such as an indirect ELISA kit for prokaryotic expression of IBV antibody coated by IBV N protein by poultry institute team of Shandong province academy of agricultural sciences in 2010, an nsp5ELISA antibody detection kit constructed by prokaryotic expression of IBV non-structural protein nsp5 by Zhou Jieyang team of Zhejiang university in 2016, and an immune magnetic microsphere-ELISA method for detection of IBV antibody based on IBV tandem antigen S-M-N invented by Wang Hongning team of Sichuan university in 2017. Although these methods avoid the problem of great difficulty in purifying IBV virus, prokaryotic expression of viral protein antigen easily affects the natural structure of protein and thus the antigen-antibody binding efficiency. In addition, because the spike protein of IBV is easy to fall off in the ultracentrifugation process, the purification difficulty of the virus is higher, the ELISA detection method coated by the whole virus antigen has the problem of lower sensitivity, and the impure protein content in the whole virus allantoic fluid which is not purified is higher, thereby also influencing the detection specificity. Although the indirect ELISA kit for detecting the IBV antibody is prepared by purifying the IBV whole virus through the prepared ELISA plate coated by the IBV monoclonal antibody by the rural hospital of Jiangsu province in 2012 to remove the foreign protein in the allantoic fluid, the method has high production cost and relatively complex operation. Therefore, the IBV holovirus antigen ELISA detection kit with simple production process, good sensitivity and high specificity is still lacked in the market at present.
Disclosure of Invention
In view of the above, the invention provides an application of the cell adaptive strain MJ of the QX-type IBV in the preparation of the whole virus antigen ELISA detection kit in order to solve the problem of low specificity of the existing ELISA detection kit for IBV, and the whole virus antigen indirect ELISA detection kit with high sensitivity and strong specificity can be prepared by utilizing the characteristics of high specificity, no other virus antigens and simple purification of the cell adaptive strain MJ.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a QX type IBV cell adapted strain MJ in preparation of a whole virus antigen ELISA detection kit, wherein the preservation number of the QX type IBV cell adapted strain MJ is CGMCC No. 14681.
The invention also provides a QX type IBV whole virus antigen indirect ELISA detection kit, which comprises the following components:
a detection plate coated with an inactivated cell adaptive strain MJ; the preservation number of the cell adaptive strain MJ is CGMCC No. 14681;
coating buffer solution;
antibody diluent;
washing liquid;
standard positive sera;
a standard negative serum;
enzyme-labeled secondary antibody;
a color developing solution;
and (4) stopping the solution.
Preferably, the coating buffer is a carbonate buffer with a pH value of 9.6;
the antibody diluent is a PBS buffer solution of skim milk with the mass volume concentration of 1-5%;
the washing solution is PBS buffer solution with the volume concentration of 0.05-0.2% Tween-20;
the standard positive serum is SPF chicken serum resisting QX IBV strain and M41 strain;
the standard negative serum is 7-day-old negative SPF chicken serum;
the enzyme-labeled secondary antibody is a goat anti-chicken IgG antibody marked by HRP;
the color development liquid is TMB substrate color development liquid;
the stop solution is a 2mol/L sulfuric acid solution.
Preferably, the inactivation method of the cell adaptive strain MJ comprises a heating inactivation method, a BEI inactivation method or a beta-propiolactone inactivation method.
Preferably, the coating concentration of the inactivated cell adapted strain MJ is 100-300 mug/mL.
Preferably, the preparation method of the detection plate of the coating inactivated cell adaptive strain MJ comprises the following steps:
(1) preparation of inactivated whole virus antigen: inoculating the cell adapted strain MJ to Vero cells, and collecting supernatant after culturing; inactivating the obtained supernatant to obtain an inactivated cell adaptive strain MJ;
(2) adding the inactivated cell adapted strain MJ into a hole of the detection plate according to 100 mu L/hole, incubating for 2h at 35-38 ℃, discarding the rest inactivated cell adapted strain MJ, washing for 2-4 times by using a washing solution, adding a confining solution, incubating for 1h at 37 ℃, and washing for 2-4 times by using a washing solution, thereby obtaining the detection plate coated with the inactivated cell adapted strain MJ.
Preferably, the QX type IBV strain is an IBYZ isolate strain, and the preservation number is CGMCC No. 14682.
Preferably, the method for preparing the standard positive serum comprises the following steps:
the M41 strain and the IBYZ isolate are 104 EID 50/HALFImmunizing SPF chicks of 1 day of age at the concentration, repeating the steps for re-immunization at the age of 14 days, and separating the serum of the SPF chicks at the age of 30 days to obtain standard positive serum.
Preferably, the dilution of the standard positive serum and the standard negative serum is independently 1: 50-400.
The invention also provides a using method of the whole virus antigen indirect ELISA detection kit in the technical scheme, which comprises the following steps:
(1) diluting serum to be detected with an antibody diluent 1: 50-400, adding the diluted serum to be detected into a detection plate coated with an inactivated cell adaptive strain MJ, setting standard positive serum and standard negative serum as controls, performing first incubation for 0.5-1.5 h at 35-38 ℃, and performing first washing for 3-6 times;
(2) diluting the enzyme-labeled secondary antibody with an antibody diluent, adding the diluted enzyme-labeled secondary antibody into a washed detection plate, performing secondary incubation at 35-38 ℃ for 0.5-1.5 h, and performing secondary washing for 3-6 times;
(3) adding a developing solution into the detection plate, incubating for 10-25 min at 35-38 ℃ in the dark, and adding a stop solution;
(4) determination of OD of solution after termination450nmCalculate S/P values from the reading:
S/P value (OD serum to be examined)450nmMean-negative control OD450nmMean)/(positive control OD450nmMean-negative control OD450nmMean value);
if the S/P value of the serum sample to be detected is more than 0.2, the antibody is judged to be positive, otherwise, the antibody is judged to be negative.
The invention provides application of a QX type IBV cell adapted strain MJ in preparation of a whole virus antigen ELISA detection kit, wherein the preservation number of the QX type IBV cell adapted strain MJ is CGMCC No. 14681. The cell adaptive strain MJ provided by the inventor does not contain other virus antigens, only specifically binds with IBV positive serum and does not have cross reaction with other virus positive serum, can be effectively proliferated in Vero cells, and the problems of difficult purification of IBV whole virus and high non-specificity can be solved by taking the cell adaptive strain MJ as a whole virus antigen preparation kit.
The invention provides a whole virus antigen indirect ELISA detection kit constructed by a cell adaptive strain MJ based on QX type IBV, which comprises a detection plate coated with an inactivated cell adaptive strain MJ, a coating buffer solution, an antibody diluent, a washing solution, standard positive serum, standard negative serum, an enzyme-labeled secondary antibody, a developing solution and a stop solution. The invention adopts the cell adaptive strain MJ as the whole virus coating antigen for the first time, has high detection sensitivity, strong specificity and good reproducibility, can be used for detecting the early infection of the QX type IBV, and can also be used for detecting the early antibody level of the chicken flock immunized by the attenuated vaccine. Compared with the existing IDEXX kit, the kit has better detection sensitivity, stronger specificity and low cost, and is suitable for batch sample detection.
Biological material preservation instructions
The cell adapted strain MJ of the QX type Infectious Bronchitis Virus (IBV) is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (CGMCC, China, microbial institute of China academy of sciences 3, North West Lu No.1, North Cheng, the sunny region, Beijing) at 2017, 8 and 31 days, the preservation number is CGMCC No.14681, the strain is classified and named as infectious bronchitis Virus, and the Latin is named as InfectS Bronchis Virus.
The IBYZ isolate was deposited in the general microbiological center of China Committee for culture Collection of microorganisms (CGMCC, China, institute of microbiology of China academy of sciences, China, No. 3, West Lu 1, North Cheng, south China) at 31.8.7.7.8.7.8.4.82, with the accession number of CGMCC No.14682, which was named as Infectious Bronchitis Virus (IBV) by classification, and Latin was named as Infectous Bronchitis Virus.
Drawings
FIG. 1 is a ROC curve plotted in example 6;
FIG. 2 is the number of samples detected by different detection kits in example 8 at different immunization times;
FIG. 3 shows the detection rates of positive samples at different immunization times by different detection kits in example 8.
Detailed Description
The invention provides application of a QX type IBV cell adapted strain MJ in preparation of a whole virus antigen ELISA detection kit, wherein the preservation number of the QX type IBV cell adapted strain MJ is CGMCC No. 14681. In the invention, the cell adaptive strain MJ of the QX-type IBV is applied to the preparation of the kit as a whole virus antigen, and only contains the IBV antigen, so that cross reaction with other viruses is avoided, the interference of foreign proteins is less, the purification operation is simple, and the specificity is high.
The invention also provides application of the QX type IBV cell adapted strain MJ in the technical scheme in preparation of a whole virus antigen ELISA detection kit. The QX type IBV cell adapted strain MJ is used as an antigen in an ELISA kit, and compared with an IBV detection kit using one or more structural proteins or antigen epitopes, the kit coated by the whole virus antigen has higher detection sensitivity and wider applicable detection range.
Specifically, the invention also provides a QX type IBV whole virus antigen indirect ELISA detection kit, which comprises the following components:
a detection plate coated with the inactivated cell adaptive strain MJ in the technical scheme;
coating buffer solution;
antibody diluent;
washing liquid;
standard positive sera;
a standard negative serum;
enzyme-labeled secondary antibody;
a color developing solution;
and (4) stopping the solution.
In the detection plate coated with the inactivated cell adaptive strain MJ, the coating concentration of the inactivated cell adaptive strain MJ is preferably 100-300 mug/mL, and more preferably 220-240 mug/mL. In the present invention, the inactivation method of the cell adapted strain MJ includes, but is not limited to, a warming inactivation method, a BEI inactivation method or a beta-propiolactone inactivation method; more preferably, the beta-propiolactone 1:1000 is used for inactivating the cell adaptive strain MJ, and the P/N value (positive average value P/negative average value N of the inactivator) is higher, so that the operation is easier and safer.
In the present invention, the test plate of the coating inactivated cell adaptive strain MJ is preferably prepared as follows:
(1) preparation of inactivated whole virus antigen: inoculating the cell adaptive strain MJ in the technical scheme to Vero cells, and collecting supernatant after culturing; inactivating the obtained supernatant to obtain an inactivated cell adaptive strain MJ;
(2) adding the inactivated cell adapted strain MJ into a hole of the detection plate according to 100 mu L/hole, incubating for 2h at 35-38 ℃, discarding the rest inactivated cell adapted strain MJ, washing for 2-4 times by using a washing solution, adding a confining solution, incubating for 1h at 37 ℃, and washing for 2-4 times by using a washing solution, thereby obtaining the detection plate coated with the inactivated cell adapted strain MJ.
The virus allantoic fluid of the cell adapted strain MJ is preferably selected according to the proportion of 0.5-2 x 107Vero cells are inoculated to the virus copy number/bottle, the Vero cells are cultured in serum-free DMEM medium, and the cultured supernatant is collected. In the invention, the culture temperature is preferably 35-38 ℃, and more preferably 37 ℃; the culture time is preferably 32-40 h, and more preferably 36 h. In the invention, the method for collecting the supernatant is preferably centrifugal separation, the centrifugal rate is preferably 2600-3000 Xg, more preferably 2850 Xg, and the centrifugal time is preferably 8-15 min, more preferably 10 min.
After the supernatant is obtained by separation, the supernatant is inactivated in the invention, and the inactivation mode is as described above and is not described herein again.
According to the invention, the inactivated supernatant is preferably purified by adding polyethylene glycol 6000 and sodium chloride, wherein the final concentration of the polyethylene glycol 6000 in the supernatant is preferably 6-10%, and more preferably 8%; the sodium chloride is preferably added to the supernatant to achieve a final concentration of 0.3-0.8 mol/L, and more preferably 0.5 mol/L. After polyethylene glycol 6000 and sodium chloride are added for purification, the invention centrifuges the purified supernatant, discards the supernatant, and takes the precipitate for heavy suspension to obtain the inactivated cell adaptive strain MJ. In the present invention, the centrifugation conditions are preferably: 7000 to 10000 Xg centrifugation for 25 to 40min, more preferably 8000 Xg centrifugation for 30 min. In the present invention, the solution for resuspending the pellet is preferably PBS buffer. The purification steps of the cell adaptive strain MJ are simple, the cost is low, and the repeatability is good.
After obtaining the inactivated cell adapted strain MJ, diluting the inactivated cell adapted strain MJ to a coating concentration, adding the diluted cell adapted strain MJ into a hole of a detection plate according to 100 mu L/hole, incubating for 2h at 35-38 ℃ for coating, and washing the detection plate for 2-4 times by using a washing solution after the reaction is finished; and adding a confining liquid, incubating for 1h at 37 ℃, and washing for 2-4 times by using a washing liquid to obtain the detection plate coated with the inactivated cell adaptive strain MJ. In the specific embodiment of the invention, the antigen coating condition of 2 hours of action at 37 ℃ is adopted, and the P/N value is obviously higher than that of the antigen coating condition of 1 hour of incubation at 35-38 ℃ and overnight coating condition at 4 ℃.
In the present invention, the blocking solution is preferably a PBS buffer solution of skim milk with a mass volume concentration of 1% to 5%, and more preferably a PBS buffer solution of skim milk with a mass volume of 5%. The specific implementation mode of the invention shows that compared with BSA solution with the mass concentration of 1%, BSA solution with the mass concentration of 2% and calf serum with the volume concentration of 10% as blocking solutions, the P/N value of the buffer solution of the PBS buffer solution with the mass volume concentration of 1-5% of the skim milk is obviously higher than that of the other buffer solutions.
In the present invention, the coating buffer is preferably a carbonate buffer at ph 9.6; specifically, the pH9.6 carbonate buffer (1000mL) was an aqueous solution of 1.59g of sodium carbonate and 2.93g of sodium bicarbonate. The coating buffer of the present invention is used to dilute the antigen.
In the present invention, the antibody diluent is preferably a PBS buffer solution of skim milk with a mass volume concentration of 1% to 5%, more preferably a PBS buffer solution of 2% skim milk. The specific implementation mode of the invention shows that when the mass concentration of the skim milk reaches 5%, the positive serum value is reduced remarkably, so that the antibody diluent at the concentration of the invention is selected as the optimal antibody diluent. The antibody diluent is used for diluting standard positive serum, standard negative serum, serum to be detected and enzyme-labeled secondary antibody.
In the invention, the washing solution is preferably PBS buffer solution with the volume concentration of 0.05-0.2% Tween-20, and is more preferably PBS buffer solution with the volume concentration of 0.05% Tween-20. In the present invention, the washing solution is used for washing in each step of ELISA detection.
In the present invention, the standard positive serum is preferably SPF chicken serum against QX type IBV strain and M41 strain; the QX type IBV strain is more preferably an IBYZ isolate strain, and the preservation number is CGMCC No. 14682. The IBYZ isolate is a molecular clone strain of a QX type virulent strain, and because most QX type IBV strains in the market have the problem of antigen impurity, other virus strains can be mixed or viruses cannot be purified. The IBYZ isolate only contains QX type IBV antigen, and the specificity of the prepared standard serum is better guaranteed.
In the present invention, the standard positive serum is preferably prepared by: the strain against QX IBV and M41 is expressed as 104EID50The concentration of each individual is obtained by immunizing SPF chicks of 1 day old, immunizing again in the same way at 14 days old, and collecting blood and separating serum when the chicken is 30 days old.
In the invention, the standard negative serum is preferably 7-day-old negative SPF chicken serum, and the specific preparation method is preferably as follows: collecting healthy SPF chicks of 7 days old, and separating blood serum to obtain the final product.
In the invention, the dilution of the standard positive serum and the standard negative serum is preferably 1: 50-400 independently, and more preferably 1:200 independently.
In the invention, the enzyme-labeled secondary antibody is preferably an HRP-labeled goat anti-chicken IgG antibody. In the present invention, the color-developing solution is preferably a TMB substrate color-developing solution. In the present invention, the stop solution is preferably a 2mol/L sulfuric acid solution. And (3) catalyzing TMB (horse radish peroxidase) (HRP) in the enzyme-labeled secondary antibody for color development, adding a stop solution, stopping catalytic reaction, and measuring the OD (optical density) value of a color development solution by using an enzyme-labeling instrument and the like to realize quantitative detection of ELISA. The enzyme-labeled secondary antibody, the developing solution and the stop solution are not specially limited in source, and can be purchased from commercial products or prepared by self.
When the whole virus antigen indirect ELISA detection kit provided by the invention is used for detection, no cross reaction occurs with newcastle disease virus, avian influenza virus and infectious bursal disease virus, and the specificity is high; the detection sensitivity is 99.58%, and the repeatability in the same batch and among batches is good; compared with the commercial IDEXX kit, the IDEXX kit has obvious difference of positive detection rate, and the IDEXX kit has lower detection rate for short-term (14d and 21d) after QX type IBV attenuated vaccine immunization, but the whole virus antigen indirect ELISA detection kit can effectively detect the antibody level in the chicken after the IBV attenuated vaccine immunization for 14d, which shows that the ELISA detection kit has higher sensitivity and can be suitable for detecting early infected chicken.
The invention also provides a using method of the whole virus antigen indirect ELISA detection kit in the technical scheme, which comprises the following steps:
(1) diluting serum to be detected with an antibody diluent 1: 50-400, adding the diluted serum to be detected into a detection plate coated with an inactivated cell adaptive strain MJ, setting standard positive serum and standard negative serum as controls, performing first incubation for 0.5-1.5 h at 35-38 ℃, and performing first washing for 3-6 times;
(2) diluting the enzyme-labeled secondary antibody with an antibody diluent, adding the diluted enzyme-labeled secondary antibody into a washed detection plate, performing secondary incubation at 35-38 ℃ for 0.5-1.5 h, and performing secondary washing for 3-6 times;
(3) adding a developing solution into the detection plate, incubating for 10-25 min at 35-38 ℃ in the dark, and adding a stop solution;
(4) determination of OD of solution after termination450nmCalculate S/P values from the reading:
S/P value (OD serum to be examined)450nmMean-negative control OD450nmMean)/(positive control OD450nmMean-negative control OD450nmMean value);
if the S/P value of the serum sample to be detected is more than 0.2, the antibody is judged to be positive, otherwise, the antibody is judged to be negative.
Diluting serum to be detected by using an antibody diluent at a ratio of 1: 50-400, adding the diluted serum to be detected into a detection plate coated with the inactivated cell adaptive strain MJ, setting standard positive serum and standard negative serum as controls, performing primary incubation at 35-38 ℃ for 0.5-1.5 h, and performing primary washing for 3-6 times. In the present invention, the test sample includes a biological isolated sample such as serum or plasma, which may contain the antibody against the test virus. The dilution of the serum to be tested is preferably 1:200, and the dilutions of the standard positive serum and the standard negative serum are the same as the dilution of the serum to be tested.
The invention sets the standard positive serum and the standard negative serum to ensure that the detection result is accurate and credible. The amount of the liquid added to each reaction hole of the diluted serum to be detected, the standard positive serum and the standard negative serum is consistent. According to the invention, the addition amount of the diluted serum to be detected in each hole is preferably 50-200 mu L, and more preferably 100 mu L.
In the present invention, the temperature of the first incubation is preferably 37 ℃, and the time of the first incubation is preferably 1 h. In the invention, the number of times of the first washing is preferably 4-5, and the temperature of the first washing is preferably 18-30 ℃, and more preferably 20-25 ℃; in the present invention, the interval time between the multiple washing steps in the first washing step is preferably 0 to 5min (the same applies below).
After the first washing is finished, diluting the enzyme-labeled secondary antibody with an antibody diluent, adding the diluted enzyme-labeled secondary antibody into a washed detection plate, performing secondary incubation at 35-38 ℃ for 0.5-1.5 h, and performing secondary washing for 3-6 times. In the invention, the dilution degree of the enzyme-labeled secondary antibody is preferably 5000-20000, more preferably 1:10000, and in the invention, the addition amount of the diluted enzyme-labeled secondary antibody is preferably 100 mu L. In the present invention, the second incubation time is preferably 1h, and the second incubation temperature is preferably 37 ℃; the second washing is the same as the first washing, and is not described herein again.
After the second washing is finished, adding a developing solution into the hole of the detection plate after the second washing, incubating for 10-25 min at 35-38 ℃ in a dark place, and adding a stop solution to obtain the developed detection plate. In the present invention, the amount of the color-developing solution added is preferably 100. mu.L/well. In the present invention, the incubation temperature in the dark is preferably 37 ℃, and the incubation time in the dark is preferably 15 min.
And observing or detecting the obtained developed detection plate to obtain a qualitative result. The OD of each hole in the chromogenic detection plate is measured450nmLower reading valueAnd calculating an S/P value:
S/P value (OD serum to be examined)450nmMean-negative control OD450nmMean)/(positive control OD450nmMean-negative control OD450nmMean value);
if the S/P value of the serum sample to be detected is more than 0.2, the antibody is judged to be positive, otherwise, the antibody is judged to be negative.
The technical solutions of the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The QX type IBV whole virus antigen indirect ELISA detection kit comprises the following components:
a detection plate coated with an inactivated cell adapted strain MJ (biological preservation number CGMCC No. 14681);
the coating buffer solution: carbonate buffer at pH 9.6;
the antibody diluent is: PBS buffer solution of skimmed milk with mass volume concentration of 2%;
the washing solution comprises: PBS Buffer (PBST) at a volume concentration of 0.05% Tween-20;
the standard positive sera: SPF chicken serum resisting IBYZ isolate (preservation number is CGMCC No.14682) and M41 strain;
the standard negative sera: 7-day-old negative SPF chicken serum;
the enzyme-labeled secondary antibody: HRP-labeled goat anti-chicken IgG antibody;
the color developing liquid: TMB substrate developing solution;
the stop solution is a 2mol/L sulfuric acid solution.
(1) Preparation of inactivated QX-type IBV cell adapted Strain MJ
Taking virus allantoic fluid of QX type IBV cell adapted strain MJ, determining virus copy number in the allantoic fluid by q-PCR, and selecting 1 × 107The Vero cells are inoculated in a virus copy number/bottle, serum-free DMEM is added for culture for 36 hours at 37 ℃, and cell supernatant is collected. Centrifuging at 2850 Xg for 10min, removing cell precipitate to obtain virus supernatant, and adding beta-propiolactone and virus supernatant at a volume ratio of 1:1000Adding beta-propiolactone, and standing at 4 deg.C for 24 hr. And (3) putting the inactivated virus supernatant into water bath at 37 ℃ for 2h, and hydrolyzing the residual inactivating agent beta-propiolactone.
Adding polyethylene glycol 6000 into the inactivated virus supernatant to a final concentration of 8%, adding NaCl to a final concentration of 0.5mol/L, centrifuging at 8000 Xg for 30min, discarding the supernatant, and suspending the precipitate with a certain amount of PBS to obtain an inactivated cell adapted strain MJ solution; the total protein content of the inactivated adapted cell strain MJ solution was measured and stored at-70 ℃ for further use.
(2) Preparation of assay plate coated with inactivated cell-adapted Strain MJ
The buffer solution was coated with carbonate (0.05 mol/LNa) at pH9.62CO3And NaHCO3) Diluting the antigen 1:4, coating a 96-well enzyme label plate according to 100 mu L/well, coating for 2h at 37 ℃, and washing for 3 times by PBST;
100 μ L of blocking solution, which is PBS buffer solution of skim milk with a mass volume concentration of 5%, was added to each well. Sealing at 37 deg.C for 2h, and washing with PBST for 3 times.
(3) Preparation of Standard Positive serum and Standard negative serum
The M41 strain and IBYZ isolate (preservation number is CGMCC No.14682) are mixed with each other by 104EID50The SPF chicks of 1 day old are simultaneously immunized by dropping nose and eyes, the SPF chicks of 14 days old are immunized in the same way, blood is collected for separation and serum is collected for standby use at the age of 30 days old, and the serum is standard positive serum.
Collecting blood of healthy SPF chicks of 7 days old, separating and collecting serum for later use, wherein the serum is standard negative serum.
Comparative example 1
The procedure was the same as in example 1 except that it was not inactivated.
Comparative example 2
The operation is the same as that of example 1 except that the heating inactivation method is adopted for inactivation;
heating inactivation method: the viral supernatant was placed in a 56 ℃ water bath for 30 min.
Comparative example 3
The operation was the same as in example 1 except that the inactivation was carried out by the BEI inactivation method;
BEI inactivation method: mixing BEA4.1g and 100mL of 0.2mol/L NaOH solution, and carrying out water bath at 37 ℃ for 1h to obtain a BEI solution with the mass concentration of 2%. Adding the supernatant into BEI solution with the mass concentration of 2% according to the volume ratio of 100:1, inactivating at room temperature for 28h, and adding sodium thiosulfate with the mass concentration of 2% according to the volume ratio of 50:1 to terminate.
Comparative example 4
The procedure is as in example 1 except that beta-propiolactone is added at a ratio of 1:2000 for inactivation.
Comparative example 5
The procedure is as in example 1 except that beta-propiolactone is added at a ratio of 1:4000 for inactivation.
Example 2
Application method of QX type IBV whole virus antigen indirect ELISA detection kit
(1) Diluting the serum to be detected by using an antibody diluent 1:200, adding 100 mu L/hole primary antibody, simultaneously adding a standard positive serum and a negative serum control, acting for 1h at 37 ℃, and washing the plate for 5 times by using 300 mu L/hole PBST;
(2) diluting an HRP-labeled goat anti-chicken IgG secondary antibody by using an antibody diluent 1:10000, adding 100 mu L/hole, acting at 37 ℃ for 1h, and washing the plate by using 300 mu L/hole PBST for 5 times;
(3) color development: adding 100 μ L/hole TMB color development solution, and keeping away from light at 37 deg.C for 15 min;
(4) and (4) terminating: adding 50 mu L/hole stop solution;
(5) and (4) judging a result:
to enzyme labeling instrument OD450nmThe S/P value was calculated:
S/P value (OD serum to be examined)450nmMean-negative control OD450nmMean)/(positive control OD450nmMean-negative control OD450nmMean value).
Example 3
The inactivated virus supernatants prepared in example 1 and comparative examples 1-4 were diluted with a carbonate buffer solution of pH9.6 at a volume ratio of 1:40, added to a 96-well microplate, coated at 4 ℃ for 24h, reacted with standard positive serum and standard negative serum, and tested according to the method of example 2, with the results shown in Table 1.
TABLE 1 comparison of ELISA readings for different antigen inactivation methods
From the detection results (Table 1), after inactivation at 56 ℃, the ELISA positive reading value is lowest, the BEI inactivation positive serum reading value is highest, and the P/N value is highest although the positive reading value after the beta-propiolactone 1:1000 inactivation is reduced. The invention selects the beta-propiolactone 1:1000 inactivation method to prepare the envelope antigen for ELISA detection, and has the advantages of simple operation and best safety.
Example 4 optimization of ELISA assay conditions
(1) Determination of optimal coating concentration of antigen and optimal serum dilution
The optimal concentration for antigen-antibody response was optimized using a square matrix assay. The coating antigen was diluted 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 with carbonate coating buffer (pH 9.6), and the positive and negative sera were diluted 1:50, 1:100, 1:200, 1:400 with antibody diluent. After the ELISA reaction, OD was measured450nmAbsorbance, calculating P/N value (positive serum OD)450nmMean/negative serum OD450nmMean value).
As a result: as shown in Table 2, after the purified antigen is diluted, the initial concentration of the square matrix test is 938 mug/mL, and the OD values of the positive serum and the negative serum show a certain descending trend along with the increasing dilution of the antigen and the antibody, which indicates that a certain nonspecific reading value exists in the negative serum.
On the basis that the P/N value is more than or equal to 2.1, the positive serum reading value and the negative serum reading value are comprehensively considered, and the positive reading value OD is selected450nmThe test well was about 1.0, that is, the dilution of the antigen was 1:40, the antigen concentration was 234.5. mu.g/mL, and the dilutions of the positive serum and the negative serum were 1: 200.
TABLE 2 Square matrix test for different antigen, antibody dilutions
(2) Determination of optimal conditions for coating antigen
Antigen is coated under 3 different conditions of 1h at 37 ℃, 2h at 37 ℃ and overnight at 4 ℃ by using the optimal coating amount of the antigen, and the rest conditions refer to an ELISA basic program, and positive serum and negative serum with the serum dilution of 1:200 are selected to perform ELISA reaction, so that the optimal coating condition of the antigen is determined.
As a result: as shown in Table 3, OD was coated at 37 ℃ for 2h450nmThe positive readings were highest and the P/N values were also significantly higher than the remaining two groups. Therefore, 2h at 37 ℃ was chosen as the optimal coating condition for the antigen.
TABLE 3 ELISA readings analysis of different antigen coating conditions
(3) Determination of confining liquid
Coating the antigen to be detected at 37 ℃ for 2h by using the optimal coating concentration, washing by PBST for 3 times, adding PBS (phosphate buffer solution) diluted 5% skimmed milk, 1% BSA, 2% BSA and 10% calf serum into different sealing solutions of 200 mu L/hole, sealing for 2.5h at 37 ℃, and carrying out ELISA (enzyme-linked immunosorbent assay) detection under the condition that the rest conditions are unchanged to determine the type of the sealing solution.
As a result: as shown in table 4, 5% skim milk, 1% BSA, 2% BSA, and 10% calf serum were selected as blocking solutions for ELISA assay, and analysis of P/N values revealed that the P/N value of 5% skim milk was significantly higher than that of the other groups, and thus 5% skim milk was selected as blocking solution for ELISA assay.
TABLE 4 analysis of ELISA readings for different types of blocking solutions
(4) Determination of the closing time
After the ELISA plate was coated with the optimal antigen coating concentration and coating conditions, PBST was washed 5 times, PBS containing 5% skim milk at 200. mu.L/well was added as a blocking solution, and the plate was blocked overnight at 37 ℃ for 1 hour, 37 ℃ for 2 hours, and 4 ℃, and detection was performed using standard positive serum and negative serum, and ELISA readings were measured to determine the optimal conditions for blocking, with other test conditions being unchanged.
As a result: after the ELISA plate is sealed by 5% skim milk sealing solution under different conditions, the reading values are shown in Table 5, and under the condition that the positive reading value difference is not obvious, the P/N value is maximum when the ELISA plate is sealed for 1h at 37 ℃, so that the condition is selected as the optimal sealing condition for ELISA detection.
TABLE 5ELISA reading analysis of different blocking conditions
(5) Selection of antibody dilutions
On the basis of determining a sealing solution and sealing conditions, PBS with the concentration of 1% skim milk, 2% skim milk and 5% skim milk is prepared as an antibody diluent of a primary antibody and a secondary antibody respectively, other conditions are not changed, and a P/N value is calculated to determine an optimal antibody diluent.
As a result: as shown in table 6, since the P/N value was the largest when 5% skim milk was used as the antibody diluent, but the positive serum value was significantly decreased, 2% skim milk was selected as the optimal antibody diluent, and the P/N value was 27.
TABLE 6ELISA readings analysis of different antibody dilutions
(6) Determination of optimal reaction time of primary antiserum
After an enzyme label plate is coated by the optimal antigen coating concentration and the optimal coating conditions, PBST is washed for 3 times, 5% skim milk is added as a sealing liquid to seal for 1h at 37 ℃, PBST is washed for 3 times and dried, positive serum and negative serum diluted by 1:200 are added to be placed at 37 ℃ and act for 30min, 60min and 120min respectively, other acting conditions are unchanged, ELISA reading values are obtained through detection, and the optimal primary antibody reaction time is determined after the P/N value is calculated and analyzed.
As a result: as shown in Table 7, although the mean value of P/N is the largest when the serum acts for 30min, the positive serum reading value is obviously lower than that of 60min and 120 min; and when the drug acts for 120min, the P/N value is obviously reduced, and the non-specific reaction is increased. Therefore, the optimal reaction conditions for the selection of primary antisera were 60min at 37 ℃ taking into account the positive serum readings and the P/N values.
TABLE 7 analysis of ELISA readings for different duration of action
(7) Determination of enzyme-labeled Secondary antibody dilution
Diluting the goat anti-chicken antibody marked by HRP according to the proportion of 1:5000, 1:8000, 1:10000 and 1:20000 respectively, acting at 37 ℃ for 1h, washing by PBST for 5 times, adding TMB substrate color development liquid and stop solution, carrying out ELISA detection, calculating P/N value, carrying out comprehensive analysis, and determining the optimal dilution of the secondary antibody.
As a result: the results after the action of the secondary antibodies with different concentrations are shown in Table 8, the reading values of the positive sera of the 1:8000 and 1:10000 dilution groups are not greatly different, and the P/N value of the 1:10000 dilution group is slightly higher, so that the optimal dilution of the secondary antibody is selected to be 1:10000.
TABLE 8 ELISA readings analysis of different secondary antibody concentrations
(8) Selection of enzyme-labeled secondary antibody action time
The time of action of the enzyme-labeled secondary antibody is selected after the dilution concentration of the enzyme-labeled secondary antibody is determined. 100 μ L of 1:10000 diluted enzyme-labeled secondary antibody is added into each well, and the mixture is acted at 37 ℃ for 30min, 45min, 60min and 90min respectively. At itUnder the condition of constant conditions, ELISA detection is carried out, and OD is analyzed450nmReading value and P/N value.
As a result: as shown in Table 9, the P/N readings were high at 30min and 45min, but the positive readings were low. Therefore, the optimal action time of the secondary antibody is selected to be 60min by comprehensively considering the positive serum reading value and the P/N value, and the P/N value is 29.883 at the moment.
TABLE 9 analysis of ELISA readings for different secondary antibody action times
(9) Determination of the reaction time of the TMB substrate
TMB was selected as a substrate reaction solution, and the reaction solution was developed at 37 ℃ for 5, 7, 10, 15 and 20min in the absence of light. And other reaction conditions are unchanged, ELISA detection is carried out, negative and positive serum reading values and P/N values are comparatively analyzed, and the optimal substrate reaction time is determined.
As a result: by comprehensively considering the positive and negative serum readings and the P/N value, the positive serum reading is higher when TMB acts for 15min, the P/N value is 28.565, and the time is selected as the substrate reaction time of the TMB.
TABLE 10 ELISA readings analysis of different TMB action times
EXAMPLE 5 specificity test
The established ELISA detection method is used for respectively carrying out 1:200 dilution on Newcastle Disease Virus (NDV) positive serum, avian influenza virus H5 subtype positive serum, avian influenza virus H7 subtype positive serum, avian influenza virus H9 subtype positive serum and Infectious Bursal Disease Virus (IBDV) positive serum (provided by infectious disease laboratories of Yangzhou university college of veterinary medicine), meanwhile, the IBV positive serum and the IBV negative serum are set as controls, and according to the use method shown in the embodiment 2, the ELISA detection kit shown in the embodiment 1 is used for detecting the serum samples, so that the specificity of the IBV ELISA is determined.
As a result: through detection, positive serum readings of strains AIV-H5Re-8, AIV-H5Re-10, AIV-H7, AIV-H9, NDV and IBDV are 0.0413, 0.0455, 0.109, 0.05, 0.02 and 0.028 respectively, and S/P values are all less than 0.2; therefore, the IBV ELISA detection method does not generate cross reaction with the serum and has good specificity.
TABLE 11 specificity test for sera from different kinds of viruses
Example 6 determination of ELISA detection cut-off values
The detection was carried out by ELISA detection method shown in example 2 using standard positive serum and standard negative serum as control and OD was measured for different serotypes of IBV positive serum 236 parts and negative serum 82 parts (among which serum of partial chickens in the post-immunization fowl field and serum obtained after immunization of the unit animal experiment) of known background450The value is obtained. And calculating S/P according to the measured OD value, wherein the formula is as follows: S/P ═ sample OD450nmMean-negative control OD450nmMean)/(positive control OD450nmMean-negative control OD450nmMean value). And judging the sample to be detected as positive if the S/P value is larger than the critical value, and judging the sample to be detected as negative if the S/P value is smaller than or equal to the critical value.
The Sensitivity (Sensitivity) and Specificity (Specificity) of the indirect ELISA detection method for obtaining the detection of the QX type IBV antibody were analyzed by plotting an ROC curve using SPSS software. After data processing and analysis, the S/P value corresponding to the maximum john index is selected and set as the Cut-off value of positive and negative serum.
As a result: the detection of IBV ELISA was carried out on 318 clinical samples of known background, using standard positive and negative sera as reference, and by analysis of a ROC curve drawn by SPSS software (as shown in FIG. 1), the sensitivity of IBV ELISA was 99.58% and the specificity was 100% at a maximum of about 0.9958, and the S/P value was about 0.2, so the cut-off value was set at 0.2.
TABLE 12 ROC analysis of partial sensitivity, specificity values and jordan index values
Example 7 repeatability test
1. In-batch repeatability test
According to the kit composition shown in example 1, the same batch of antigen-coated ELISA plates were prepared, 10 sera were detected separately, the experiment was repeated 4 times, and OD of each sample was calculated separately450nmMean (Mean), Standard Deviation (SD) and Coefficient of Variation (CV), determining the range of coefficient of variation for the same batch of antigens. Results the OD of 10 samples tested for the same antigen batch is shown in the table below450nmThe coefficient of variation of the reading values is between 2% and 14%, which indicates that the repeatability of the antigen in batches is better.
TABLE 13 results of the in-batch repeat test
2. Batch to batch repeatability test
3 inactivated antigens of different batches are prepared according to the ELISA detection kit shown in example 1, enzyme-linked immunosorbent assay (ELISA) plates are coated at different times, 10 parts of serum are used for detection under the same test conditions, each part of serum is repeated for 3 times in parallel, and the detection result is subjected to statistical analysis.
The result shows that the variation coefficient range of the repeated test among batches of the ELISA plates coated by the antigens prepared in different batches is between 5 and 13 percent after detection, and the ELISA plates show good repeatability.
TABLE 14 results of the repeated tests between batches
Example 8 comparison of IBV ELISA kit with import IDEXX kit
Selecting QX type IBV isolates from different sources as 104A total of 266 SPF chicks of 1 day old were inoculated by EID50, 67 SPF chicks of PBS immunization group as negative control were inoculated, and serum was collected every 7 days after 14 days old to collect 333 sera of known background.
The serum against the known background was detected using the QX IBV ELISA kit and the imported IDEXX kit (available from Aldrich, Henry, Inc., Beijing) shown in example 1. The results are shown in Table 15, and out of 333 sera, 266 positive sera and 67 negative sera were detected from the QX type IBV ELISA kit. 48 parts of positive serum and 285 parts of negative serum are detected by an imported IDEXX kit. The detection rates of the two kits are different greatly.
As shown in FIG. 2 and FIG. 3, the comparative analysis of the immune background of the control animal experiment shows that the IDEXX kit has a low detection rate for the QX type IBV attenuated vaccine immunization in a short period (14d and 21d), while the QX type IBV ELISA kit can effectively detect the antibody level in the chicken after the IBV attenuated vaccine immunization of 14 d.
TABLE 15 comparison of the results of the detection with ELISA kit of IDEXX
In conclusion, the cell adaptive strain MJ provided by the invention is used as an ELISA detection kit prepared by coating a whole virus antigen, has high detection sensitivity and good specificity, is easy to purify, and can be suitable for detecting early-stage IBV infection or early-stage antibodies after immunization.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The application of an inactivated cell adapted strain MJ of QX-type IBV in the preparation of a whole virus antigen ELISA detection kit is disclosed, wherein the preservation number of the cell adapted strain MJ of the QX-type IBV is CGMCC No. 14681;
the inactivation method of the QX type IBV cell adaptive strain MJ is a beta-propiolactone inactivation method;
adding beta-propiolactone according to the volume ratio of the beta-propiolactone to the virus supernatant of 1: 1000.
2. A QX type IBV whole virus antigen indirect ELISA detection kit comprises the following components:
a detection plate coated with an inactivated cell adaptive strain MJ; the preservation number of the cell adaptive strain MJ is CGMCC No. 14681; the inactivation method of the QX type IBV cell adaptive strain MJ is a beta-propiolactone inactivation method; adding beta-propiolactone according to the volume ratio of the beta-propiolactone to the virus supernatant of 1: 1000;
coating buffer solution;
antibody diluent;
washing liquid;
standard positive sera;
a standard negative serum;
enzyme-labeled secondary antibody;
a color developing solution;
and (4) stopping the solution.
3. The whole virus antigen indirect ELISA detection kit of claim 2, wherein,
the coating buffer solution is a carbonate buffer solution with the pH value of 9.6;
the antibody diluent is a PBS buffer solution of skim milk with the mass volume concentration of 1-5%;
the washing solution is PBS buffer solution with the volume concentration of 0.05-0.2% Tween-20;
the standard positive serum is SPF chicken serum resisting QX IBV strain and M41 strain;
the standard negative serum is 7-day-old negative SPF chicken serum;
the enzyme-labeled secondary antibody is a goat anti-chicken IgG antibody marked by HRP;
the color development liquid is TMB substrate color development liquid;
the stop solution is a 2mol/L sulfuric acid solution.
4. The whole virus antigen indirect ELISA detection kit of claim 2, wherein the coating concentration of the inactivated cell adapted strain MJ is 100-300 μ g/mL.
5. The kit for the indirect ELISA detection of the whole virus antigen according to any one of claims 2 to 4, wherein the preparation method of the detection plate of the inactivated coated cell adapted strain MJ comprises the following steps:
(1) preparation of inactivated whole virus antigen: inoculating the cell adapted strain MJ to Vero cells, and collecting supernatant after culturing; inactivating the obtained supernatant to obtain an inactivated cell adaptive strain MJ;
(2) adding the inactivated cell adapted strain MJ into a hole of the detection plate according to 100 mu L/hole, incubating for 2h at 35-38 ℃, discarding the rest inactivated cell adapted strain MJ, washing for 2-4 times by using a washing solution, adding a confining solution, incubating for 1h at 37 ℃, and washing for 2-4 times by using a washing solution, thereby obtaining the detection plate coated with the inactivated cell adapted strain MJ.
6. The whole virus antigen indirect ELISA detection kit of claim 3, wherein the QX type IBV strain is IBYZ isolate with a preservation number of CGMCC No. 14682.
7. The whole virus antigen indirect ELISA detection kit of claim 6, wherein the standard positive serum is prepared by the method comprising:
the M41 strain and the IBYZ isolate are 104Immunizing SPF chicks of 1 day old at the EID 50/mouse concentration, repeating the steps for re-immunization at the age of 14 days, and separating the serum of the SPF chicks at the age of 30 days to obtain standard positive serum.
8. The indirect ELISA detection kit for whole virus antigen according to claim 3, 6 or 7 wherein the dilutions of the standard positive serum and the standard negative serum are independently 1: 50-400.
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