CN113552336A - Detection method for BRV (Breast cancer) serum neutralizing antibody titer level based on IFA (IFA) - Google Patents
Detection method for BRV (Breast cancer) serum neutralizing antibody titer level based on IFA (IFA) Download PDFInfo
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Abstract
The invention discloses a detection method of BRV serum neutralizing antibody titer level based on IFA, belonging to the technical field of biology. The method provided by the invention is based on the IFA detection neutralizing antibody method, two DMEM maintaining solutions containing pancreatin with different concentrations are adopted to carry out two-stage 2-fold dilution on a clinical serum sample to prepare a serum sample to be detected for incubation with BRV neutralizing virus, so that the influence of neutralization of the pancreatin by the clinical serum sample on the growth of BRV in cells can be effectively avoided, and the BRV serum neutralizing antibody titer level can be accurately detected.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a BRV serum neutralizing antibody titer level detection method based on IFA, in particular to a BRV serum neutralizing antibody titer level rapid and accurate detection method based on IFA.
Background
Bovine Rotavirus (BRV) is a rotavirus of reoviridae, a non-enveloped double-stranded RNA virus with the diameter of about 60-80 nanometers, is a symmetrical icosahedron, and can be observed as a typical 'wheel shape' under a mirror. The diarrhea caused by the virus brings huge economic loss to the dairy cow breeding industry, which is reflected in that no special-effect medicine is available for infecting calves and high death rate is often caused.
At present, the prevention of BRV infection is mainly based on virus vaccines (such as bovine rotavirus and bovine coronavirus inactivated vaccines disclosed in patent document CN 110124027A), wherein antibody-negative experimental animals need to be screened and the immune potency of the vaccines needs to be evaluated to evaluate the quality of the vaccines in the vaccine development process, so that the virus serum neutralizing antibody titer level needs to be detected (neutralizing antibody detection method) and a virus immune-challenge test needs to be performed (immune-challenge method). However, since the inoculation and passage of BRV requires the reliance on pancreatin, but the clinical serum sample itself can neutralize pancreatin and thus inhibit the growth of BRV in cells and affect the production of cell lesions induced by BRV (even fail to produce cell lesions), it is difficult to accurately detect the level of BRV neutralizing antibody titers in the clinical serum sample in the detection of levels of BRV neutralizing antibody titers (e.g., serum neutralization assay, micro-serum neutralization assay, or ELISA). On the other hand, in the neutralizing antibody detection method and the immune toxicity attacking method in the BRV vaccine efficacy detection, healthy calves with negative antibodies (namely the serum neutralizing antibody titer is less than or equal to 1:4) need to be selected for testing, since BRV is most susceptible to calves of 1-7 days old, it is best to select calves of 1-7 days old which are negative for antibodies as experimental animals, however, when judging whether the calves to be selected are negative for antibodies, typically, cytopathic methods for measuring the level of serum neutralizing antibody titers of BRV (e.g., serum neutralization assays) require a duration of about 7 days to achieve the end result, therefore, even if healthy calves (within 7 days of age) are screened which are negative for BRV serum neutralizing antibodies, the optimal time is exceeded when the calves are used for experiments, therefore, healthy calves of 7 days or more (for example, patent document CN110124027A uses healthy calves of 2 months) are commonly used as experimental animals in the prior art. In view of the above, a method for rapidly and accurately detecting the titer level of neutralizing antibodies against BRV serum is needed to guide the development and production of BRV vaccines.
Disclosure of Invention
In view of one or more of the problems in the prior art, the present invention provides a method for detecting IFA-based levels of neutralizing antibody titers in BRV serum, comprising the steps of:
s1: preparing serial two-fold diluted serum samples to be detected; and
s2: respectively inoculating mixed liquor of equivalent BRV neutralizing poison and the serial two-fold-ratio diluted serum samples to be detected prepared in the step S1 into MA104 single-layer cells, and carrying out fluorescence microscope observation after fixing by using a fixing solution, adding a primary antibody and a secondary antibody;
wherein the operation of preparing the serial two-fold-ratio diluted serum sample to be detected in the step S1 comprises the following steps: diluting a clinical serum sample to 1:16 dilution according to a 2-fold ratio by using a DMEM maintaining solution containing 136-144 mu g/ml of pancreatin, and then diluting the clinical serum sample to 2-fold ratio by using a DMEM maintaining solution containing 4-6 mu g/ml of pancreatin.
In the above detection method, the MA104 monolayer cells are plated in the wells of the cell plate in step S2, and a DMEM maintenance solution containing pancreatin is added in advance to the wells of the cell plate plated with the MA104 monolayer cells.
In the detection method, the concentration of pancreatin in a DMEM maintenance solution containing pancreatin previously added to the wells of the cell plate plated with the MA104 monolayer cells is 4-6 [ mu ] g/ml.
In the above detection method, the operation of preparing the cell plate on which the MA104 monolayer cells are spread specifically is: the density of 100 to 200 μ L is 1X 105～3×105MA104 cell suspension/ml was plated into wells of a multi-well plate and placed at 37. + -. 0.5 ℃ in 5% CO2Culturing in an incubator, when the cells grow into a monolayer, washing the cell surface layer by using DMEM maintaining liquid containing 4-6 mu g/ml pancreatin, and then adding 80-120 mu L, preferably 100 mu L of DMEM maintaining liquid containing 4-6 mu g/ml pancreatin into each hole to obtain the cell plate paved with MA104 monolayer cells。
In the above detection method, step S2 specifically includes the following steps:
(1) inoculation: respectively incubating the equivalent BRV neutralizing poison and the serial two-fold-ratio diluted serum samples to be detected prepared in the step S1 for 55-65 min at 37 +/-0.5 ℃, respectively inoculating the incubated mixed liquid into the holes of the cell plate paved with MA104 single-layer cells, and culturing for 46-50 h again to obtain a first treated cell plate;
(2) fixing the fixing liquid: using pre-cooled acetone solution as a fixing solution to fix cells in the holes of the first treated cell plate in the step (1) to obtain a second treated cell plate;
(3) adding a primary antibody: adding BRV standard positive serum diluted by PBS according to the proportion of 1: 800-1: 1200 into the holes of the second treated cell plate in the step (2) as primary antibody, incubating at 37 +/-0.5 ℃ for 55-65 min, discarding, and washing by PBS to obtain a third treated cell plate;
(4) adding a secondary antibody: adding goat anti-mouse IGg-FITC diluted by PBS according to the proportion of 1: 800-1: 1200 into the holes of the third processed cell plate in the step (3) as a secondary antibody, incubating at 37 +/-0.5 ℃ for 55-65 min, discarding, washing by PBS, adding PBS into each hole, and storing in a dark place to obtain a fourth processed cell plate;
(5) and (3) observation by a fluorescence microscope: and (5) observing each hole in the fourth cell processing plate obtained in the step (4) under a fluorescence inverted microscope, and taking the dilution of the serum sample to be detected corresponding to the hole with specific fluorescence of only 1-2 cells in the hole as the titer level of the BRV serum neutralizing antibody.
In the detection method, the BRV standard positive serum is guinea pig serum obtained after the inactivated vaccine of bovine rotavirus is immunized.
In the detection method, the amount of the inoculated mixture after incubation in the step (1) is 80 to 120. mu.L, preferably 100. mu.L per well. The fixing solution in the step (2) is pre-cooled 80% acetone solution, and the adding amount of the fixing solution is 80-120 mu L per hole, preferably 100 mu L per hole; the adding amount of the primary antibody in the step (3) is 40-60 mu L per hole, and preferably 50 mu L per hole; and (4) adding the secondary antibody in an amount of 40-60 mu L per hole, preferably 50 mu L per hole.
In the detection method, the virus titer of the BRV neutralizing virus is 200TCID50。
In the above detection method, the clinical serum sample is derived from one or more of the following: cattle, pigs, rabbits, guinea pigs.
Based on the detection method of BRV serum neutralization antibody titer level based on IFA provided by the technical scheme, the characteristic that the BRV needs to depend on pancreatin during growth is combined on the basis of the traditional IFA method for detecting serum neutralization antibody titer level, two DMEM maintenance solutions containing pancreatin with different concentrations are adopted to carry out two-stage 2-fold dilution on a clinical serum sample to obtain a serum sample to be detected for incubation with BRV neutralization virus, wherein the first stage is to dilute the clinical serum sample to 1:16 dilution by using the DMEM maintenance solution containing 136-144 mug/ml pancreatin, and then the second stage is to dilute the clinical serum sample to 2-fold dilution (namely 2-fold dilution after 1:16 dilution) by using the DMEM maintenance solution containing 4-6 mug/ml pancreatin a manner of diluting with 2-fold ratio, and the serum sample to be detected is obtained by the two-stage 2-fold dilution series, so that the relative proportion of the pancreatin the serum sample to be detected and the pancreatin the serum can be effectively improved, the influence on the growth of BRV in cells caused by the neutralization of clinical serum samples and pancreatin is avoided, and the accuracy of detecting the titer level of the BRV serum neutralizing antibody is further improved. On the other hand, the method provided by the invention is based on an IFA (indirect immunofluorescence) detection principle, has the characteristics of short detection period, strong specificity, easy discrimination and objectivity of detection results (the detection results are observed through a fluorescence microscope) and the like, and the IFA is used for inoculating BRV live virus into cells and marking full virus particles with complete structures, so that the method provided by the invention also has the advantages of rapidness (the detection results can be obtained in about 2 days) and more representativeness, and can be used for screening antibody negative healthy susceptible calves within 7 days of age so as to be used as antibody negative experimental animals in BRV vaccine efficacy evaluation.
In conclusion, the method provided by the invention can effectively and quickly evaluate the immune efficacy of the BRV vaccine, can provide antibody-negative healthy susceptible calves within 7 days of age as experimental animals for a neutralizing antibody detection test and an immune attack test in the evaluation of the BRV vaccine efficacy, and has important significance for BRV vaccine production and vaccine quality monitoring.
Drawings
FIG. 1 is a gel electrophoresis image of PCR products identifying BRV;
FIG. 2 is a fluorescence microscopic image of clinical serum samples of the test group and the control group at different dilutions;
FIG. 3 is a fluorescence microscopic observation image of a guinea pig serum diluted at a ratio of 1:1000 after immunizing a bovine rotavirus inactivated vaccine as a primary antibody, and a rabbit anti-guinea pig IgG-FITC fluorescent antibody diluted at a ratio of 1:1000 as a secondary antibody;
FIG. 4 is a fluorescence microscopic image of clinical serum samples at different dilutions and different incubation times.
Detailed Description
Because the clinical serum sample can generate a neutralizing effect on pancreatin, the growth of BRV (pancreatic cell necrosis factor) which depends on pancreatin in the existing BRV serum neutralizing antibody titer level detection method (such as a serum neutralizing test) can be influenced, and further the cell lesion induced by the BRV can be influenced, so that the detection result of the BRV serum neutralizing antibody titer level is inaccurate; in addition, the existing detection methods for the titer level of neutralizing antibodies against BRV serum (such as serum neutralization test) take a long time (generally about 7 days) and cannot screen healthy susceptible calves within 7 days of age that are negative for antibodies in time. In view of the above problems, the present invention provides a method for detecting the neutralizing antibody titer level of BRV serum based on the indirect Immunofluorescence (IFA) principle, which combines the characteristics of BRV growth that depends on pancreatin with two DMEM maintenance solutions containing pancreatin at different concentrations to obtain a serum sample to be detected by diluting a clinical serum sample at two-stage double ratio (e.g. 2-fold ratio), based on the prior steps of detecting the neutralizing antibody titer level of serum by IFA (the step of detecting the neutralizing antibody titer level of serum by IFA generally comprises the steps of incubating equal volumes of a virus solution and the serum sample to be detected, fixing by a fixing solution, adding a primary antibody, adding a secondary antibody and observing by a fluorescence microscope, for example, the method for detecting the neutralizing antibody of serum by IFA method disclosed in patent documents CN112362880A, CN112630443A, etc.) and can effectively avoid the neutralizing effect of pancreatin the pancreatin and the detection result by the clinical serum sample itself, thereby the neutralizing antibody titer level of the BRV serum can be accurately detected. On the other hand, the method provided by the invention has the characteristic of short period (the detection result can be obtained in about 2 days), so that healthy susceptible calves within 7 days of antibody negative age can be screened in time to be used as antibody negative experimental animals in the BRV vaccine efficacy detection. In addition, the detection result of the method provided by the invention is obtained by observing through a fluorescence microscope, and is more objective and accurate compared with a method for observing cytopathic effect, and the influence on the growth of BRV in cells caused by the neutralization effect of a clinical serum sample on pancreatin can be further reduced due to the short time required by IFA detection, and the accuracy of the detection result is improved together with the technical means for preparing the serum sample to be detected by adopting two-stage dilution.
The present invention is illustrated in detail by the following specific examples. The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, which are helpful for understanding the invention, but should not be taken as limiting the content of the invention.
The methods used in the following examples are conventional methods unless otherwise specified. The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The primers used in the following examples were synthesized by the prior art.
Example 1: BRV virus collection, identification and culture
1.1, collecting the excrement of suspicious BRV infected calves from the city of inner Mongolia and Haote, repeatedly dissolving the excrement by using 1ml of PBS, repeatedly freezing and thawing the mixture for three times at 3000r/min, centrifuging the mixture for 10min, taking supernate as a virus sample, using a nucleic acid extraction kit (Tiangen) to extract double-stranded RNA in the virus sample, then reversely transcribing the RNA into cDNA, and identifying the cDNA by using a BRV target gene primer (the length of an amplified target band is 1062bp) designed by DNAman. Wherein the sequence of the BRV target gene primer is as follows:
BRV-F：5’GGCTTTAATTGCGAGAATTTCC 3’(SEQ ID NO:1)；
BRV-R：5’GGTCTCATCATTCAACTCTAAT 3’(SEQ ID NO:2)；
1.2, carrying out PCR amplification by using the primer and taking the reverse transcribed cDNA as a template, wherein the specific amplification system is as follows: mu.l template, 0.5. mu.l BRV forward primer (BRV-F), 0.5. mu.l BRV reverse primer (BRV-R), EXTap enzyme 13. mu.l, ddH2O is 9 mu l; the PCR cycling conditions were: pre-denaturation at 94 ℃ for 4min, then 30 cycles of 94 ℃ for 45s, 55 ℃ for 45s, and 72 ℃ for 30s, and finally extension at 72 ℃ for 10 min. The amplification products were subjected to agarose gel electrophoresis, and the results are shown in FIG. 1, wherein lane 2 is a cDNA template obtained by reverse transcription of RNA extracted from a virus sample, and lanes 3, 4 and 5 are negative controls.
As can be seen from the results shown in FIG. 1, the band of interest in lane 2 is about 1000bp, and the band of interest is recovered by gel, and the sequencing result shows that the sequence is consistent with the BRV sequence and the length is 1062bp, which proves that the collected virus sample contains the BRV virus.
1.3, filtering a virus sample with a positive BRV (blast furnace Virus) detection result through a 0.22-micrometer filter membrane, inoculating the virus sample to MA104 cells according to a dose of 1ml, harvesting virus liquid after the cytopathic effect reaches 80%, continuously carrying out passage according to the same method until the MA104 cells are found to have typical pathological changes (cells are shrunk and rounded after pathological changes, cell boundaries are unclear, typical cauliflower-like forms are presented, finally diseased cells completely fall off), and harvesting the virus liquid after the cytopathic effect reaches 80%, namely the BRV virus liquid.
Example 2: preparation of BRV neutralizing poison
2.1 preparation of MA104 monolayer cells (laboratory preservation of Jinyubain biopharmaceutical Co., Ltd., commercially available) into 2X 10 cells by digestion and passage5Cell suspension/ml, 150. mu.L of cell suspension plated in wells of a 96-well plate at 37 ℃ with 5% CO2After the cells were cultured in the incubator for 24 hours to form a monolayer, the cell surface layer was washed with a DMEM-containing solution containing 5. mu.g/ml of pancreatic enzymes (Gibco), and then 100. mu.L of a DMEM-containing solution containing 5. mu.g/ml of pancreatic enzymes was added to each well to obtain a cell plate on which MA104 monolayer cells were plated.
2.2 BRV Virus solution obtained in example 1 was serially diluted 10-fold with a DMEM-containing solution containing 5. mu.g/ml of pancreatin and then diluted 10-fold-1～10-8Inoculating each diluted titer of virus solution into the wells of the treated monolayer cell plate of step 2.1, and adding 100 μ L of virus solution into each well to serve as test wells; meanwhile, a cell control hole is set, and DMEM maintenance liquid containing 5 mu g/ml pancreatin is added into the control hole, but BRV virus liquid is not added. Cells in both test and control wells were incubated at 37 ℃ in 5% CO2After culturing in an incubator for 7 days, cytopathic conditions in the test wells and the control wells were observed and recorded, and the results are shown in Table 1 below, where TCID of BRV was calculated according to Reed-Muench50。
TABLE 1 statistics of cytopathic conditions in test and control wells
TCID of BRV was calculated according to Reed-Muench based on cytopathic condition in the test and control wells as shown in Table 1 above50The calculation method is as follows:
distance ratio (percentage higher than 50% -50%)/(percentage higher than 50% -percentage lower than 50%) i.e.: (75-50)/(75-0) ═ 0.333
Lg TCID50The difference between distance ratio × logarithm of dilution + logarithm of dilution higher than 50% disease rate ═ 0.333 × (-1) + (-7) ═ 7.33, and the final TCID of the BRV virus was found from the above results50＝10-7.33/0.1ml＝10-8.33Perml, as BRV virus stock solution, i.e. 0.1ml virus stock solution contains 107.33A TCID50I.e. 107.33TCID50/0.1ml。
200TCID50Neutralizing poison (IFA method for detecting neutralizing antibody titer water in serumNeutralizing titer conventionally used at ordinary times) preparation:
to prepare 200ml of 200TCID50Neutralizing the venom, diluting the BRV virus stock solution 105After doubling, 187ul ml of the solution is added into 200ml of the maintenance solution, thus obtaining the product.
Example 3: determination of neutralization reaction conditions in IFA-neutralizing antibody detection method for BRV
3.1 determination of pancreatin concentration in maintenance liquid during clinical serum sample dilution
BRV neutralizing antibody negative serum samples (gibco fetal bovine serum) were serially diluted 2-fold to 1:128 dilutions using DMEM maintenance solutions containing 20. mu.g/ml, 40. mu.g/ml, 60. mu.g/ml, 80. mu.g/ml, 100. mu.g/ml, 120. mu.g/ml, 140. mu.g/ml, 160. mu.g/ml, 180. mu.g/ml pancreatin, respectively, to obtain serial dilutions of serum samples obtained from dilutions of DMEM maintenance solutions containing different concentrations of pancreatin. Serum samples of each dilution were mixed with an equal amount of 200TCID50After 1h of neutralization with BRV neutralizing poison (prepared in example 2), 100. mu.l of the neutralized mixture was inoculated into wells of a cell plate plated with MA104 monolayer cells (prepared in step 2.1 of example 2), and a normal cell-inoculating control was set up and cultured again for 5 to 7 days. The cytopathic effect and the cell reticulum (cell damage distinguished from the cytopathic effect due to the increasing concentration of pancreatin) were observed, and the results are shown in table 2 below.
Table 2: relationship between different pancreatin concentrations and cytopathic and cellular trawl titers
As can be seen from the results of Table 2 above, the cytopathic titer was maintained at 1:8 after the serum samples were diluted with a DMEM maintaining solution at a pancreatin concentration of 140. mu.g/ml, and the pathological titer of the cells does not change any more with the increase of the pancreatin content in the later period, so that the adding amount of the pancreatin concentration of 140 mug/ml reaches the limit value which can cause the cells to be pathological, and under the concentration condition, the cell net pulling titer is at least 1:32, which indicates that the pancreatin concentration exceeds the limit value born by the cells under the condition of dilution of 1:32, therefore, serum samples are continuously diluted by 2 times to 1:16 dilution, 140 mu g/ml pancreatin concentration is selected as a proper concentration, the serum sample is neutralized, so that the influence of the serum sample on the growth of BRV is eliminated, and meanwhile, cells are not damaged (namely, the condition of cell net pulling is not caused). While the pancreatin concentrations of 160. mu.g/ml and 180. mu.g/ml already exceed the limit values which can cause cell lesions, which can lead to cell damage (cell netting) caused by too high a pancreatin concentration, the pancreatin concentrations of 120. mu.g/ml and below obviously do not reach the limit values which can cause cell lesions, i.e. the growth influence of BRV caused by the neutralization of pancreatin by the serum sample itself can not be completely eliminated.
In the same experiment, serial 2-fold dilutions of BRV neutralizing antibody negative serum samples were also performed using DMEM maintenance solutions containing 135. mu.g/ml, 136. mu.g/ml, 137. mu.g/ml, 138. mu.g/ml, 139. mu.g/ml, 141. mu.g/ml, 142. mu.g/ml, 143. mu.g/ml, 144. mu.g/ml, 145. mu.g/ml pancreatin, respectively, to observe the relationship between different pancreatin concentrations and cytopathic and cell-trawl titers. As a result, it was observed that when serum samples were serially diluted 2-fold to 1:16 dilution with a DMEM maintenance solution containing the above pancreatin enzymes 136. mu.g/ml, 137. mu.g/ml, 138. mu.g/ml, 139. mu.g/ml, 141. mu.g/ml, 142. mu.g/ml, 143. mu.g/ml, and 144. mu.g/ml, the effect of the serum samples on BRV growth could be effectively eliminated without significantly damaging the cells (the lesion titer was maintained at 1:8, and the cell-trawl titer was maintained at 1: 32). Therefore, in the method provided by the invention, DMEM maintenance liquid containing 136-144 mu g/ml pancreatin is determined to be used when the serum sample is continuously diluted to 1:16 dilution in a 2-fold ratio.
Determination of pancreatin concentration on serum samples diluted after 3.2, 1:16 dilution
Serum samples diluted 1:16 in 3.1 with a DMEM maintaining solution containing 140. mu.g/ml of pancreatic enzymes were serially diluted 2-fold to 1:256 dilutions using DMEM maintaining solutions containing 5. mu.g/ml, 10. mu.g/ml, and 15. mu.g/ml of pancreatic enzymes, respectively, to obtain serial dilution serum samples. Each dilution of serum sample was separately mixed with an equal amount of 200TCID50After 1h neutralization of BRV neutralizing poison (prepared in example 2), neutralization was carried outThe resulting mixture (100. mu.l) was inoculated into wells of a cell plate plated with MA104 monolayer cells (prepared in step 2.1 of example 2), and a normal cell-inoculation control was set up and cultured again for 5 to 7 days. The cytopathic effect and its cell network pulling condition were observed, and the results are shown in table 3 below.
Table 3: relationship between different pancreatin concentrations and cytopathic and cellular trawl titers
From the results of Table 3 above, it can be seen that, when the serum sample diluted to 1:16 dilution in 3.1 was further diluted with the DMEM maintenance solution containing 5. mu.g/ml of pancreatin, the cells were not subjected to cell-spreading, whereas when the serum sample diluted to 1:16 dilution was diluted with the DMEM maintenance solutions containing 10. mu.g/ml and 15. mu.g/ml of pancreatin, the cells were subjected to cell-spreading at 1:128 and 1:64 titers, respectively, and therefore, the serum sample diluted to 1:16 dilution was subjected to continuous 2-fold dilution with the DMEM maintenance solution containing 5. mu.g/ml of pancreatin, and the cell-spreading was not caused.
In the same experiment, serial 2-fold dilutions of BRV neutralizing antibody negative serum samples were also performed using DMEM maintenance solutions containing 4. mu.g/ml, 4.5. mu.g/ml, 5.5. mu.g/ml, 6. mu.g/ml pancreatin, respectively, to observe the relationship between different pancreatin concentrations and cytopathic and cell trawl titers. As a result, it was observed that when the serum samples diluted 1:16 were continuously diluted 2-fold to 1:256 in a DMEM-maintained solution containing the above pancreatin concentrations (4. mu.g/ml, 4.5. mu.g/ml, 5.5. mu.g/ml, 6. mu.g/ml), the cytopathic titers were all 1:256, and no cell spreading occurred in any of the cells. Therefore, in the method provided by the invention, a DMEM maintaining solution containing 4-6 mu g/ml pancreatin is determined to be used when the serum sample with the dilution ratio of 1:16 is continuously diluted by 2 times.
In summary, in the method for detecting the neutralizing antibody titer level of the BRV serum based on IFA according to the present invention, when a clinical serum sample is diluted, in order to avoid the influence of the neutralizing effect of the serum sample on pancreatin on the detection result, the clinical serum sample is diluted to 1:16 by using a DMEM maintenance solution containing 136 to 144 μ g/ml pancreatin, and then a series of 2-fold dilution (for example, 2-fold dilution to 1:256 dilution) is diluted by using a DMEM maintenance solution containing 4 to 6 μ g/m pancreatin, so as to obtain serum samples to be detected under each dilution condition.
3.3 determination of working concentration of BRV Positive serum as Primary antibody and Rabbit anti-Guinea pig IgG-FITC as Secondary antibody in IFA neutralizing antibody detection method for BRV
(1) Cell plates plated with a monolayer of MA104 cells were prepared according to example 2, step 2.1. Clinical serum samples (gibco BRV negative fetal bovine serum) were first diluted to 1:16 dilution using DMEM maintenance solution containing 140. mu.g/ml pancreatin, after which diluted 1:16 dilution serum samples were diluted to 1:256 using DMEM maintenance solution containing 5. mu.g/ml pancreatin, and each dilution serum sample was mixed with an equal volume of 200TCID50BRV neutralizing poison (prepared in example 2) was neutralized, incubated at 37 ℃ for 1h, and 100. mu.l of the neutralized solution was inoculated into wells of a cell plate plated with MA104 monolayer cells, while a normal cell-inoculation control and a blank control were set, and cultured again for 48 h.
(2) The cells of the test group, the control group and the blank control group were fixed with pre-cooled solution containing 80% acetone, 100. mu.l of each well was left at 4 ℃ for 1 hour, the fixing solution was discarded, after air-drying, the cell surface was washed with PBS, and the PBS solution was discarded.
(3) anti-BRV positive serum (guinea pig serum after immunizing bovine rotavirus inactivated vaccine, wherein the bovine rotavirus inactivated vaccine was prepared according to the preparation method of the bovine rotavirus inactivated vaccine disclosed in CN110124027A using BRV virus solution obtained in example 1) as a primary antibody, PBS was used to perform continuous dilution at multiple ratios according to 1:50, 1:100, 1:200, 1:500, 1:1000 and 1:2000, respectively, primary antibody was added to the fixed wells of step (2) by the fixative at 50. mu.L per well, incubated at 37 ℃ for 1h, discarded, washed with PBS, 250. mu.L per well was added, left to stand for 5min, patted dry, and washed repeatedly for 3 times.
(4) Goat anti-mouse IGg-FITC (Eibo anti-goat anti-guinea pig IgG H & L (Alexa flow 488)) as a secondary antibody was diluted in PBS at 1:100, 1:200, 1:500, 1:1000, and 1:2000 in serial multiples, 50. mu.L of primary antibody was added to each well to which primary antibody had been added in step (3), incubated at 37 ℃ for 1 hour, discarded, washed with PBS, 250. mu.L of primary antibody was added to each well, allowed to stand for 5min, patted dry, washed 5 times again, then 50. mu.L of PBS was added to each well and stored in the dark, and observed under a fluorescence inverted microscope.
As shown in FIG. 2, it was found that the serum of immunized guinea pigs was diluted at a ratio of 1:1000 to obtain primary antibodies, and the IgG-FITC fluorescent antibodies of rabbit anti-guinea pigs were diluted at a ratio of 1:1000 to obtain secondary antibodies, which were observed under a fluorescence microscope (fluorescence observation results at a dilution of 1: 8), and showed excellent fluorescent coloration effects. In the same experiment, the serum of immunized guinea pig diluted according to the ratio of 1:800 and 1:1200 is used as a primary antibody, and the IgG-FITC fluorescent antibody of rabbit anti-guinea pig diluted according to the ratio of 1:800 and 1:1200 is used as a secondary antibody, and the observation result of a fluorescence microscope shows that the result with better fluorescence developing effect can be obtained. Therefore, in the method provided by the invention, the immune guinea pig serum diluted according to the ratio of 1: 800-1: 1200 is used as a primary antibody, and the rabbit anti-guinea pig IgG-FITC fluorescent antibody diluted according to the ratio of 1: 800-1: 1200 is used as a secondary antibody.
3.4 determination of cell culture time in IFA neutralizing antibody detection method of BRV
(1) Cell plates plated with a monolayer of MA104 cells were prepared according to example 2, step 2.1. Equal volume of 200TCID50After incubation of BRV-neutralized (prepared in example 2) and diluted serum samples (1:2, 1:4 and 1:8 dilutions) at 37 ℃ for 1h, 100. mu.l of neutralized liquid was inoculated into wells of a cell plate plated with MA104 monolayers, while setting up a normal cell inoculation control and a blank control at 37 ℃ with 5% CO2After 24h, 48h and 72h of culture in an incubator, cell plates are taken out one by one according to the steps (2), (3) and (4) in the step 3.3 for fixation, primary antibody and secondary antibody are added, and observation is carried out under a fluorescence inverted microscope.
As a result, as shown in FIG. 3, it was found that the fluorescence was small at the incubation time of 24 hours, and the evaluation result was affected. Fluorescence was very significant at 48h incubation time and hardly dropped off after cell treatment, while fluorescence was very high in cells at 72h incubation timeAfter the treatment of the fluorescent staining process, the cells are more exfoliated, so 48 hours are selected as the proper culture time of immunofluorescence. In the same experiment, 5% CO was also present at 37 deg.C2The culture in the incubator is carried out for 46h, 47h, 49h and 50h, and the results are similar to the 48h culture, namely, the fluorescence is very obvious when observed by a fluorescence inverted microscope, and the cells are hardly shed after being treated. Therefore, the cell culture time after the neutralization solution for inoculating the BRV and the serum sample in the method provided by the invention is determined to be 46-50 h.
Example 4: establishment of BRV serum neutralizing antibody titer level detection method based on IFA
The method for detecting the neutralizing antibody titer level of the BRV serum based on IFA established in the example aims to detect the neutralizing antibody titer level of the BRV serum, can detect the neutralizing antibody titer level of the BRV in clinical serum samples from any species, such as bovine serum, pig serum, rabbit serum, guinea pig serum, human serum and the like, and specifically comprises the following steps taking a bovine clinical serum sample (gibco BRV negative fetal bovine serum) as an example in the following steps.
(1) Cell plates plated with a monolayer of MA104 cells were prepared according to example 2, step 2.1. Equal volume of 200TCID50BRV neutralizing Virus (prepared in example 2) and serum samples to be tested at various dilutions (obtained by dilution according to example 3, diluted to 1:256 dilutions, called test set), or by equal volume of 200TCID50BRV neutralizing poison (prepared in example 2) and control serum samples to be tested at each dilution (bovine clinical serum samples were serially diluted 2-fold to 1:256 dilution using DMEM maintenance medium containing 5. mu.g/ml pancreatin, referred to as control group) and incubated at 37 ℃ for 1h, then 100. mu.l of the neutralized mixture was inoculated into wells of a cell plate plated with MA104 monolayer cells, and a normal cell-inoculation control and a blank control were set up and incubated for another 48 h.
(2) The cells of the test group, the control group and the blank control group were fixed with pre-cooled solution containing 80% acetone, 100. mu.l of each well was left at 4 ℃ for 1 hour, the fixing solution was discarded, after air-drying, the cell surface was washed with PBS, and the PBS solution was discarded.
(3) The anti-BRV positive serum is used as a primary antibody, PBS is used for dilution according to the ratio of 1:1000, the primary antibody is added into the fixed hole of the fixing solution in the step (2) according to the volume of 50 mu L of each hole, the hole is incubated for 1h at the temperature of 37 ℃, discarded, washed by PBS, 250 mu L of each hole is added, the hole is placed for 5min, patted dry, and the washing is repeated for 3 times.
(4) And (3) using goat anti-mouse IGg-FITC as a secondary antibody, diluting the secondary antibody by using PBS according to a ratio of 1:1000, adding 50 mu L of the secondary antibody into each hole in the step (3), incubating the secondary antibody at 37 ℃ for 1h, discarding the secondary antibody, washing the secondary antibody by using PBS, adding 250 mu L of the secondary antibody into each hole, standing the secondary antibody for 5min, patting the secondary antibody dry, repeatedly washing the secondary antibody for 5 times, adding 50 mu L of PBS into each hole, storing the secondary antibody in a dark place, and observing the secondary antibody under a fluorescence inverted microscope, wherein the result is shown in figure 4.
And (4) IFA result judgment: when the clinical serum sample does not contain BRV neutralizing antibodies, BRV neutralizing viruses incubated with the serum sample to be detected at any dilution are not neutralized, further BRV can infect cell nucleus or cell cytoplasm of MA104 cells, specific fluorescence (green) appears under a fluorescence inverted microscope, and the clinical serum sample is negative under a normal condition (the neutralizing virus titer is 200 TCID)50) When the serum sample has specific fluorescence under the dilution of 1:2, the serum sample is considered as a negative serum sample; when the clinical serum sample contains BRV neutralizing antibodies, the BRV neutralizing antibodies incubated with the serum sample to be detected are neutralized by the BRV neutralizing antibodies in the serum sample, and the more the BRV is neutralized with the reduction of the dilution factor, the less the BRV capable of infecting the cell nucleus or cell cytoplasm of MA104 cells is, the less the specific fluorescence quantity appears under a fluorescence inverted microscope, when the specific fluorescence appears or does not appear just in 1-2 cells, the BRV neutralizing antibodies in the serum sample to be detected at the dilution factor can just neutralize the BRV viruses in the BRV neutralizing viruses, the BRV neutralizing antibody titer level in the clinical serum sample can be detected, and when the BRV neutralizing virus titer is 200TCID50When the method is used, the dilution value of the serum sample to be detected in a hole corresponding to the condition that only 1-2 cells generate specific fluorescence or do not generate specific fluorescence is observed under a fluorescence inverted microscope, and the dilution value is the neutralizing antibody titer value of the clinical serum sample; when in clinical serum samplesWhen the content of the neutralizing antibody for BRV is high enough, all the BRV virus in the BRV neutralizing virus is neutralized, so that the BRV cannot infect the nucleus or cytoplasm of MA104 cells, and specific fluorescence cannot occur under a fluorescence inverted microscope.
Referring to FIG. 4, panel A shows the observation results under the fluorescence inverted microscope of the control group, and panel B shows the observation results under the fluorescence inverted microscope of the test group. Under the condition that the detected clinical serum sample is known to be BRV negative fetal calf serum, the clinical serum sample of the control group still does not generate a large amount of fluorescence when the dilution is 1:16, and the detection result of the level of BRV neutralizing antibodies in the clinical serum sample is obviously higher (because the growth of BRV is inhibited); when the dilution of the clinical serum sample of the test group is 1:2, only 2 cells in the hole have fluorescence, the fluorescence quantity of the next dilution (1:4) obviously has a sharp increase phenomenon, and the cells with fluorescence are more and more along with the increasing dilution times, which shows that the test result of the test group is negative of the clinical sample, and the method is consistent with the actual situation, and the method is proved to have accurate and reliable test results. Therefore, compared with the dilution method of the clinical serum sample of a control group, the dilution method of the clinical serum sample in the test group can avoid the problem that the clinical serum sample can inhibit the growth of BRV due to the neutralization effect of pancreatin and finally the detection result of the level of the BRV neutralizing antibody in the serum sample is obviously high, and proves that the detection of the level of the BRV neutralizing antibody in the serum sample by the method provided by the invention is more accurate and reliable, and the method provided by the invention can be used for rapidly detecting the serum sample with the negative BRV antibody and providing ideal healthy susceptible calves with the negative BRV antibody within 7 days of age for the detection of the effectiveness of the BRV vaccine.
Example 5: practical application of BRV (Breast cancer) serum neutralizing antibody titer level detection method based on IFA (IFA)
The example illustrates the application of the detection method of the neutralizing antibody titer level of BRV serum based on IFA in the detection of the immune efficacy of the rotavirus vaccine, which is provided by the invention, in the serum sample of a healthy calf, wherein the immune rotavirus inactivated vaccine (prepared by using the BRV virus liquid prepared in example 1 according to the preparation method of the rotavirus inactivated vaccine disclosed in CN110124027A, the immune mode is that 2.0ml of the vaccine is injected into the neck muscle, the immunity is strengthened in the same way after 21 days, and the neutralizing antibody level is determined by taking cardiac blood collection after 21 days of secondary immunization), the specific operation is as described in example 4, wherein the serum sample to be detected in the test group is obtained according to the dilution method in example 3 and is diluted to 1:256 degrees; control group the serum sample to be tested was obtained by serial 2-fold dilution of the serum sample to 1:256 dilution using a DMEM maintenance solution containing 5 μ g/ml pancreatin.
The results of fluorescence microscope observation of a test group and a control group show that the BRV neutralizing antibody titer of a serum sample obtained by detection of the test group is 1:32, while the BRV neutralizing antibody titer of a serum sample obtained by detection of the control group is more than or equal to 1:64, and obviously, the result of the BRV neutralizing antibody titer of the serum sample obtained by the method of the control group is generally higher, which is the result of inhibition on the growth of BRV due to the neutralizing effect of the serum sample on pancreatin, and the result can result in overestimation of the immune efficacy of BRV in the immune efficacy evaluation of the BRV vaccine, is unfavorable for quality monitoring in vaccine production and practical application of the vaccine, and can have the potential risk that the immune protection effect is not good and the epidemic situation cannot be effectively prevented and controlled.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Claims (10)
1. A method for detecting IFA-based levels of neutralizing antibody titers in BRV serum, comprising the steps of:
s1: preparing serial two-fold diluted serum samples to be detected; and
s2: respectively inoculating mixed liquor of equivalent BRV neutralizing poison and the serial two-fold-ratio diluted serum samples to be detected prepared in the step S1 into MA104 single-layer cells, and carrying out fluorescence microscope observation after fixing by using a fixing solution, adding a primary antibody and a secondary antibody;
wherein the operation of preparing the serial two-fold-ratio diluted serum sample to be detected in the step S1 comprises the following steps: diluting a clinical serum sample to 1:16 dilution according to a 2-fold ratio by using a DMEM maintaining solution containing 136-144 mu g/ml of pancreatin, and then diluting the clinical serum sample to 2-fold ratio by using a DMEM maintaining solution containing 4-6 mu g/ml of pancreatin.
2. The method according to claim 1, wherein the MA104 monolayer cells are plated in wells of a cell plate in step S2, and a DMEM maintenance solution containing pancreatin is added in advance to the wells of the cell plate plated with the MA104 monolayer cells.
3. The method according to claim 2, wherein the concentration of pancreatin in a DMEM-containing solution containing pancreatin added in advance to the wells of the cell plate plated with MA104 monolayer cells is 4 to 6 μ g/ml.
4. The detection method according to claim 2 or 3, wherein the preparation of the cell plate plated with MA104 monolayers is carried out in particular by: the density of 100 to 200 μ L is 1X 105～3×105MA104 cell suspension/ml was plated into wells of a multi-well plate and placed at 37. + -. 0.5 ℃ in 5% CO2Culturing in an incubator, when cells grow into a monolayer, washing the cell surface layer by using DMEM maintaining liquid containing 4-6 mug/ml pancreatin, and then adding 80-120 muL, preferably 100 muL of DMEM maintaining liquid containing 4-6 mug/ml pancreatin into each hole to obtain the cell plate paved with MA104 monolayer cells.
5. The detection method according to any one of claims 1 to 4, wherein step S2 specifically comprises the steps of:
(1) inoculation: respectively incubating the equivalent BRV neutralizing poison and the serial two-fold-ratio diluted serum samples to be detected prepared in the step S1 for 55-65 min at 37 +/-0.5 ℃, respectively inoculating the incubated mixed liquid into the holes of the cell plate paved with MA104 single-layer cells, and culturing for 46-50 h again to obtain a first treated cell plate;
(2) fixing the fixing liquid: using pre-cooled acetone solution as a fixing solution to fix cells in the holes of the first treated cell plate in the step (1) to obtain a second treated cell plate;
(3) adding a primary antibody: adding BRV standard positive serum diluted by PBS according to the proportion of 1: 800-1: 1200 into the holes of the second treated cell plate in the step (2) as primary antibody, incubating at 37 +/-0.5 ℃ for 55-65 min, discarding, and washing by PBS to obtain a third treated cell plate;
(4) adding a secondary antibody: adding goat anti-mouse IGg-FITC diluted by PBS according to the proportion of 1: 800-1: 1200 into the holes of the third processed cell plate in the step (3) as a secondary antibody, incubating at 37 +/-0.5 ℃ for 55-65 min, discarding, washing by PBS, adding PBS into each hole, and storing in a dark place to obtain a fourth processed cell plate;
(5) and (3) observation by a fluorescence microscope: and (5) observing each hole in the fourth cell processing plate obtained in the step (4) under a fluorescence inverted microscope, and taking the dilution of the serum sample to be detected corresponding to the hole with specific fluorescence of only 1-2 cells in the hole as the titer level of the BRV serum neutralizing antibody.
6. The assay of claim 5 wherein the BRV standard positive serum is guinea pig serum after immunization with a bovine rotavirus inactivated vaccine.
7. The detection method according to claim 5 or 6, wherein the amount of the inoculated mixture after the incubation in the step (1) is 80 to 120. mu.L, preferably 100. mu.L, per well.
8. The detection method according to any one of claims 5 to 7, wherein:
the fixing solution in the step (2) is pre-cooled 80% acetone solution, and the adding amount of the fixing solution is 80-120 mu L per hole, preferably 100 mu L per hole;
the adding amount of the primary antibody in the step (3) is 40-60 mu L per hole, and preferably 50 mu L per hole;
and (4) adding the secondary antibody in an amount of 40-60 mu L per hole, preferably 50 mu L per hole.
9. The assay of any one of claims 1-8, wherein the viral titer of the BRV neutralizing virus is 200TCID50。
10. The assay of any one of claims 1-9, wherein the clinical serum sample is derived from one or more of: cattle, pigs, rabbits, guinea pigs.
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