CN112611865A - Detection method of IPMA (ionic polymer A) neutralizing antibody of PCV2 - Google Patents
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Abstract
The invention discloses a detection method of an IPMA (Ionic Polymer electrolyte analog) neutralizing antibody of PCV2, belonging to the technical field of biology. According to the detection method of the IPMA neutralizing antibody of PCV2, the cell reaction plate can be prepared in advance and can be stored for a long time at the temperature of minus 20 ℃; the reaction results were observed by a fluorescence microscope. The IPMA detection has the characteristics of strong specificity, high sensitivity and simple operation; and the IPMA method utilizes live virus inoculation, uses whole virus, has more complete antigen structure and more accurate and representative result.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection method of an IPMA (ionic liquid chromatography) neutralizing antibody of PCV 2.
Background
Porcine circovirus type 2 (PCV 2) is the main pathogen causing Postweaning Multisystemic Wasting Syndrome (PMWS) of piglets, and after PCV2 infection, immunosuppression is caused in pigs, often is concurrent or secondary to a plurality of pathogens of pigs, so that the control difficulty is increased. PCV2 related diseases (PCVAD) is a complex, multifactorial group of diseases including PMWS, Porcine Dermatitis and Nephropathy Syndrome (PDNS), porcine respiratory syndrome (PRDC), and others. At present, the diagnostic criteria, pathogenic mechanisms, and defense studies against the virus itself and the PCVAD are not well understood. Based on the method, the establishment of a rapid and accurate diagnosis method and the research of the infection mechanism of the method have important significance for the comprehensive prevention and control of PCV 2.
Therefore, it is a problem to be solved by those skilled in the art to provide a method for detecting PCV2 IPMA neutralizing antibody.
Disclosure of Invention
In view of the above, the present invention provides a detection method of neutralizing antibody of PCV2 by IPMA (immunoperoxidase cell monolayer assay).
In order to achieve the purpose, the invention adopts the following technical scheme:
an IPMA neutralizing antibody detection method of PCV2 comprises the following specific steps:
(1) PK15 cells were digested and centrifuged to prepare a gel containing 1.0X 105cell/mL 100. mu.L per well of cell suspension was plated onto a 96-well plate and placed at 37 deg.C5％CO2Culturing for 12h in an incubator, incubating PCV2 virus liquid and clinical serum with the same volume for 1h at 37 ℃ after cells are completely attached to the wall, then inoculating the cells onto PK15 cells in a 96-well plate, culturing for 48h, and taking out the 96-well plate after the cells are overgrown; setting a normal virus cell control hole at the same time;
(2) fixing with pre-cooled anhydrous ethanol, adding pre-cooled anhydrous ethanol, and standing at-20 deg.C for 30min, wherein each well is 50 μ L; removing absolute ethyl alcohol, washing PBST, and patting to dry;
(3) adding 5% skimmed milk powder, sealing, placing at 37 deg.C in incubator for 2 hr, and sealing at 200 μ L per hole; PBST cleaning and patting dry;
(4) adding PCV2 positive antibody, diluting with PBS according to a ratio of 1:400, adding 50 mu L of the PCV in each hole, and placing the PCV in an incubator at 37 ℃ for 1 h; PBST cleaning and patting dry;
(5) adding goat anti-pig IgG-HRP, diluting with PBS at a ratio of 1:1000, adding 50 μ L into each well, standing at 37 deg.C in incubator for 1h, and washing with PBST; finally, 50 μ L of PBST was added to each well, DAB was developed for 5min, the reaction was stopped, and observation was performed in an inverted fluorescence microscope.
Further, the preparation method of the PCV2 virus solution comprises the following steps: grinding clinically collected pathological tissues, adding 1ml of LPBS (multi-layered double hydroxide) for dissolving according to 1mg of tissue samples, repeatedly freezing and thawing for 3 times, and taking supernatant; centrifugation was carried out at 12000rpm for 5min, and the filtrate was filtered through a 0.22 μm filter to obtain PCV2 virus solution.
Further, the preparation method of the clinical serum comprises the following steps: and centrifuging the collected blood, and collecting serum for later clinical serum detection.
According to the technical scheme, compared with the prior art, the invention discloses the IPMA neutralizing antibody detection method of PCV2, the cell reaction plate can be prepared in advance, and can be stored for a long time at the temperature of 20 ℃ below zero; the reaction results were observed by a fluorescence microscope. The IPMA detection has the characteristics of strong specificity, high sensitivity and simple operation; and the IPMA method utilizes live virus inoculation, uses whole virus, has more complete antigen structure and more accurate and representative result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the result of PCR amplification according to the present invention;
wherein, M, DL 2000; 1, PCV2 amplification product; 2, negative control;
FIG. 2 is a graph showing the color development result of IPMA detection of PCV2 neutralizing antibody according to the present invention;
where A is the neutralizing antibody assay and B is the normal cytotoxic cell control.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Blood samples and pathological tissues such as suspected PCV2 infected lymph nodes and the like are collected in a pig farm, the collected blood is centrifuged, and serum is collected for later clinical serum detection. The diseased tissue is ground and tested for PCV2 virus and the virus is isolated. The cell line is PK15 cell and PCV2 positive antibody (stored by a laboratory).
Example 1 PCV2 Virus isolation and identification
According to the PCV2 gene sequence in GenBank (sequence number: FJ667592.1), a primer is designed, the amplification length is 600bp, and the specific primer sequence is as follows:
PCV2-F：5’-gatctcaaggacaacggagt-3’；SEQ ID NO.1；
PCV2-R：5’-catatggaaattcagggcatgg-3’；SEQ ID NO.2。
clinically collected pathological tissues such as lymph nodes and the like are ground, 1mL of PBS is added into 1mg of tissue samples for dissolving, freeze thawing is repeated for 3 times, supernatant is taken, then centrifugation is carried out at 12000rpm for 5min, and the filtrate is filtered by a 0.22 mu m filter, so as to obtain PCV2 virus solution. Inoculating PK15 cells with 1mL of virus solution, repeatedly freezing and thawing for 3 times after virus is proliferated for 48h, collecting the virus solution, centrifuging at 12000rpm for 10min, taking the supernatant, and extracting PCV2DNA according to the biological DNA extraction kit.
PCR amplification was performed in 25. mu.L: DNA template 1. mu.L, PCV 2-F0.5. mu.L, PCV 2-R0.5. mu.L, Ex Taq enzyme 12.5. mu.L, double distilled water 10.5. mu.L. Mixing and putting the mixture into a PCR instrument for amplification, wherein the reaction conditions are as follows: pre-denaturation at 98 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 50s, for 30 cycles; then, the extension is carried out for 10min at 72 ℃, and the product is stored at 4 ℃ after the extension is finished. And subjected to agarose gel electrophoresis, and the results are shown in FIG. 1.
FIG. 1 shows that the target fragment size is about 600bp, and the sequencing result of Baobao biology is consistent with the expected result; indicating that the disease contains PCV 2.
Example 2 establishment of a method for detecting clinically neutralizing antibodies
PK15 cells were digested and centrifuged to prepare a gel containing 1.0X 105cell/mL cell suspension 100. mu.L per well plated on 96-well plate, placed at 37 ℃ 5% CO2Culturing for 12h in an incubator, incubating PCV2 virus liquid (50 mu L) and clinical serum with the same volume for 1h at 37 ℃ after cells are completely attached to the wall, then inoculating the cells onto PK15 cells in a 96-well plate, culturing for 48h, and taking out the 96-well plate after the cells are overgrown; setting a normal virus cell control hole at the same time; fixing with pre-cooled anhydrous ethanol, adding pre-cooled anhydrous ethanol, and standing at-20 deg.C for 30min, wherein each well is 50 μ L. Then taking out, discarding absolute ethyl alcohol, PBST cleaning for 5 times, patting to dry, adding 5% skimmed milk powder, sealing, each well 200 μ L, and standing at 37 deg.C in incubator for 2 h. PBST was washed 5 times, dried, and added with PCV2 positive antibody (PCV 2 positive antibody obtained by clinical separation according to the detection method of IDEXX kit), diluted with PBS at a ratio of 1:400, added at 50. mu.L per well, and left at 37 ℃ in an incubator for 1 hour. PBST was washed 5 times, patted dry, goat anti-porcine IgG-HRP was added, diluted with PBS at a ratio of 1:1000, 50. mu.L was added to each well, incubated at 37 ℃ for 1h, PBST was washed 5 times, finally, 50. mu.L of PBST was added to each well, DAB was developed for 5min, the reaction was stopped, and observed on an inverted fluorescence microscope.
Judging the IPMA result: PCV2 was not neutralized by serum and infected nuclei or cytoplasm stained reddish brown, i.e., negative (FIG. 2B), while neutralized and uninfected nuclei or cytoplasm were not stained, i.e., positive (FIG. 2A).
Example 3IPMA reaction conditions
1)PCV2 TCID50Measurement of
Performing amplification culture on PK15 cells, then inoculating PCV2 virus solution with proper metering, harvesting viruses after 48h, repeatedly freezing and thawing for 3 times, and centrifuging at 12,000rpm for 10min to obtain a large amount of PCV2 virus solution. Digesting PK15 cells grown to monolayer with 0.25% pancreatin, adding DMEM containing 10% fetal calf serum, repeatedly blowing, counting cells, and diluting to 1.0 × 105one/mL, 100. mu.L per well was added to a 96 well cell culture plate. After the cells are attached, PCV2 virus solution is diluted by DMEM containing 2% fetal calf serum in a 10-fold gradient, and then 10 cells are taken-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8Each gradient was seeded into 96 well cell culture plates, 8 wells per gradient, 100 μ L per well, with negative controls. Put in 5% CO at 37 DEG C2The incubator is used for 48 hours, and the growth condition of the cells is observed every day. Finally, the virus TCID was calculated by determining the number of wells containing PCV2 positive cells CPE per dilution using IPMA (Table 1)50。TCID50The calculation of (b) is carried out according to the Reed-Muench two-degree method.
TABLE 1
TCID calculation by Reed-Muench two-law50The calculation method is as follows:
distance ratio (percentage above 50% rate of illness-50%)/(percentage above 50% rate of illness-percentage below 50% rate of illness) — (55.5-50)/(55.5-8.3) — 0.1
lgTCID50Distance ratio x difference between log of dilutions + log of dilutions above 50% disease rate-0.1 × (-1) + (-5) ═ 5.1
TCID is obtained from the data50＝10-5.1/0.1ml＝10-6.1/ml
The meaning is as follows: diluting the virus 105.1Inoculation with 100. mu.l resulted in 50% of the cells being diseased.
2) Viral inoculation amount and fixation time
Respectively using 1000 TCIDs 50100 TCIDs5010 TCIDs 501 TCID500.1 TCID50The virus of (4) was inoculated to PK15 cells and an unvaccinated control was set. Positive serum and negative serum are respectively used as primary antibody for detection by utilizing an IPMA detection method, and the optimal virus inoculation amount is determined by observing results. Inoculating PCV2 virus (optimal virus inoculation amount) into PK15 cell bottles growing to about 60% -70%, spreading the cells on 4 96 pore plates respectively after the cells grow full, and carrying out inoculation at 37 ℃ with 5% CO2After 12, 24, 36 and 48 hours of culture in the incubator, the culture medium is taken out one by one and fixed. Then, the IPMA detection method is used for detecting positive serum and negative serum respectively as primary antibody, and the fixed time is determined by observing the result.
As a result, it was found that the number of TCIDs was 10050The immobilized IPMA cell reaction plate is optimally prepared after the PK15 cell is inoculated by the virus and cultured for 48 hours.
3) Serum dilution concentration, goat anti-porcine IgG-HRP working concentration
Porcine serum infected with PCV2 was treated as 1: 50. and (3) carrying out continuous multiple dilution at the ratio of 1:100, 1:200, 1:400 and the like, adding the diluted solution serving as a primary antibody into the prepared cell reaction plate, respectively carrying out detection by using an IPMA antibody detection method, and determining the optimal serum dilution concentration according to the observation result.
Taking out the prepared cell reaction plate, adding 1: positive and negative sera were diluted 400-fold and then added to 1:500, 1: 1000. goat anti-porcine IgG-HRP diluted by 2 times at the ratio of 1:2000, 1:4000 and the like, and the optimal secondary antibody dilution concentration is determined by observing the experimental result.
As a result, the serum to be detected starts from 1:50, the antibody titer can be detected by continuously diluting 2 times according to the strength of the antibody, the work concentration of goat anti-pig IgG-HRP is 1:1000 optimally, and DAB color development is carried out for 5 min.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
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<120> detection method of IPMA neutralizing antibody of PCV2
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gatctcaagg acaacggagt 20
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Claims (3)
1. The detection method of the IPMA neutralizing antibody of PCV2 is characterized by comprising the following specific steps:
(1) PK15 cells were digested and centrifuged to prepare a gel containing 1.0X 105cell/mL cell suspension 100. mu.L per well plated on 96-well plate, placed at 37 ℃ 5% CO2Culturing in an incubator for 12h, after the cells are completely attached to the wall, incubating equal volume of PCV2 virus solution and clinical serum for 1h at 37 ℃, and then inoculating the cells to 96 holesCulturing PK15 cells in the plate for 48h, and taking out a 96-well plate after the cells grow full; setting a normal virus cell control hole at the same time;
(2) fixing with pre-cooled anhydrous ethanol, adding pre-cooled anhydrous ethanol, and standing at-20 deg.C for 30min, wherein each well is 50 μ L; removing absolute ethyl alcohol, washing PBST, and patting to dry;
(3) adding 5% skimmed milk powder, sealing, placing at 37 deg.C in incubator for 2 hr, and sealing at 200 μ L per hole; PBST cleaning and patting dry;
(4) adding PCV2 positive antibody, diluting with PBS according to a ratio of 1:400, adding 50 mu L of the PCV in each hole, and placing the PCV in an incubator at 37 ℃ for 1 h; PBST cleaning and patting dry;
(5) adding goat anti-pig IgG-HRP, diluting with PBS at a ratio of 1:1000, adding 50 μ L into each well, standing at 37 deg.C in incubator for 1h, and washing with PBST; finally, 50 mu L of PBST is added into each hole, DAB color development is carried out for 5min, the reaction is stopped, and observation is carried out in an inverted fluorescence microscope.
2. The method for detecting IPMA neutralizing antibodies of PCV2 according to claim 1, wherein the PCV2 virus fluid is prepared by the following steps: grinding clinically collected pathological tissues, adding 1mL of PBS (phosphate buffer solution) into 1mg of tissue samples for dissolving, repeatedly freezing and thawing for 3 times, and taking supernatant; centrifugation was carried out at 12000rpm for 5min, and the filtrate was filtered through a 0.22 μm filter to obtain PCV2 virus solution.
3. The method for detecting IPMA neutralizing antibodies of PCV2 according to claim 1, wherein the clinical serum is prepared by the following method: and centrifuging the collected blood, and collecting serum for later clinical serum detection.
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| US20100184016A1 (en) * | 2007-06-22 | 2010-07-22 | David Jacques Gerard Lefebvre | Methods and compositions in the treatment of porcine circoviral infection |
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