CN112362880A

CN112362880A - IFA (IFA neutralizing antibody) detection method for PRRSV (porcine reproductive and respiratory syndrome Virus)

Info

Publication number: CN112362880A
Application number: CN202011240685.9A
Authority: CN
Inventors: 王选年; 李鹏; 冯春花; 王利平; 高小静; 李红; 孙国鹏; 岳锋; 朱艳平; 张艳芳; 郭东光; 齐永华; 潘鹏涛
Original assignee: Xinxiang University
Current assignee: Xinxiang University
Priority date: 2020-11-09
Filing date: 2020-11-09
Publication date: 2021-02-12

Abstract

The invention discloses an IFA (IFA neutralizing antibody) detection method of PRRSV (porcine reproductive and respiratory syndrome Virus), belonging to the technical field of molecular biological detection. The invention discloses a PRRSV IFA neutralizing antibody detection method, which comprises the steps of inoculating PRRSV virus liquid and clinical serum with equal volume to Marc145 cells, fixing, adding a first antibody, adding a second antibody and observing results. The method of the invention utilizes the interaction of the whole virus and the antibody to infect the target cell, and truly reflects the interaction condition of the virus and the neutralizing antibody.

Description

IFA (IFA neutralizing antibody) detection method for PRRSV (porcine reproductive and respiratory syndrome Virus)

Technical Field

The invention relates to the technical field of biology, in particular to a PRRSV IFA neutralizing antibody detection method.

Background

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a positive-stranded enveloped RNA virus. The virion consists of a nucleocapsid core constructed from nucleocapsid proteins (encoded by open reading frame 7 or ORF 7) bound to viral RNA. The nucleocapsid is surrounded by a lipid envelope, which contains six structural proteins: glycoproteins GP2(ORF2a), GP3(ORF3), GP4(ORF4) and GP5(ORF5), and non-glycosylated proteins M (ORF6) and E (ORF2 b). GP5 and M are considered to be the most abundant proteins in the envelope, while the content of other envelope proteins is lower. Therefore, the antibody response after PRRSV infection is very complex, and the total antibody response can be divided into a Neutralizing Antibody (NA) and a non-neutralizing antibody (NNA), and studies show that the neutralizing antibody titer reaches 1:8 can effectively eliminate PRRSV viremia.

Various methods have been established to detect PRRSV specific antibodies, such as enzyme linked immunosorbent assays (ELISA) and immunofluorescence and immunochromatographic strip-based assays. PRRSV-specific neutralizing antibodies (nabs) typically appear 28 days after vaccination (dpi), but non-protective antibodies generated within the first week after infection may be more useful for early detection of PRRSV infection. These early antibodies include non-neutralizing antibodies specific for prrsv protein or structural proteins such as nsps, N protein and certain non-structural proteins (nsp1, nsp2 and nsp7) have been shown to be highly immunogenic.

Most commercial ELISA kits currently detecting PRRSV specific antibodies (e.g., IDEXX Herd Chek PRRS ELISA) employ anti-N antibodies as serological markers of PRRSV infection or improved live virus (MLV) immune status. Although commercial tests such as ELISA are very sensitive to determine the presence of PRRSV specific antibodies in serum samples, ELISA is not suitable for quantitative analysis of antibody levels: the OD values obtained from ELISA usually vary within a small range (0.1 to 2). Furthermore, most ELISA kits use a single recombinant PRRSV structural antigen (typically PRRSV-N protein) that is prokaryotically expressed as the coating antigen. Thus, such systems cannot assess PRRSV-specific antibody responses against other PRRSV envelope proteins or non-structural proteins (nsps). It should be noted here that since great effort is required to express and purify multiple ELISA plate coating antigens, a system using multiple PRRSV antigens may not be developed. Furthermore, due to the general nature of expression of ELISA envelope antigens in e.coli, false positive or false negative results are often reported. Therefore, it is an urgent problem to be solved by those skilled in the art to provide a method for detecting PRRSV IFA neutralizing antibody for accurate detection and quantification of PRRSV specific antibodies.

Disclosure of Invention

In view of the above, the present invention provides a method for detecting IFA neutralizing antibody of PRRSV, which is used for accurately detecting and quantifying PRRSV specific antibody.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting IFA neutralizing antibody of PRRSV comprises the following steps:

(1) plate paving: digesting and centrifuging Marc145 cells to prepare cell suspension, spreading the cell suspension on a 96-well plate, culturing in a 37 ℃ 5% CO2 culture box, incubating the PRRSV virus solution and clinical serum with the same volume at 37 ℃ for 1h after the cells are completely attached to the wall, inoculating the cell suspension on the Marc145 cells, adding a DMEM culture medium with fetal bovine serum content of 2%, culturing, and placing the cell suspension in a CO₂CulturingContinuously culturing for 48h in the box, observing the growth condition of the cells, and taking out the 96-well plate after the cells grow full; meanwhile, setting a non-virus-inoculated normal cell control hole;

(2) fixing: washing the taken 96-well plate with PBS for 2-3 times, and fixing with precooled absolute ethyl alcohol at-20 deg.C for 30min to obtain a cell reaction plate;

(3) adding a primary antibody: washing the cell reaction plate with PBS for 2-3 times, adding 100 μ l PRRSV standard positive serum diluted with 0.01M PBS1:400, and reacting at 37 deg.C for 1 h;

(4) adding a secondary antibody: adding 100 μ l goat anti-porcine IgG-FITC fluorescent antibody diluted 200 times with 0.01M PBS (pH7.4), and allowing reaction at 37 deg.C for 1 hr;

(5) and (4) observing results: the color development was observed under a fluorescence microscope after washing with PBS.

Further, the preparation method of the PRRSV virus liquid comprises the following steps: and (3) washing the pathogenic tissue of the PRRSV with PBS, shearing, adding 1mL of PBS into 1g of tissue sample, and repeatedly freezing and thawing for three times to prepare the pathogenic grinding fluid.

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 method for detecting the IFA neutralizing antibody of the PRRSV disclosed by the invention has the advantages that the target cells are infected after the interaction of the whole virus and the antibody, and the interaction condition of the virus and the neutralizing antibody is truly reflected.

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 PCR amplification results of each pathogenic tissue according to the present invention;

wherein, M: DL 2000Marker; 1: lymph nodes; 2: a liver; 3: spleen; 4: the lung; 5: blank control (template is ddH)₂O)；

FIG. 2 is a graph showing the color development result of detecting PRRSV neutralizing antibodies by IFA according to the present invention;

wherein, A is a neutralizing antibody test, and B is a normal virus cell control;

FIG. 3 is a graph showing the results of IFA detection of different PRRSV inoculum sizes according to the present invention;

wherein A, B, C, D, E represents 1000, 100, 10, 1, 0.1 TCIDs respectively₅₀Virus inoculation;

FIG. 4 is a graph showing the results of detecting the titer of neutralizing antibodies in serum by IFA according to the present invention;

wherein A, B, C, D, E, F respectively represents the serum dilution times of 1:4, 1: 8. 1: 16. 1: 32. 1: 64. 1: 128.

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.

Collecting a blood sample and pathological tissues from a peripheral pig farm, centrifuging the collected blood, and collecting serum for later clinical serum detection; grinding pathological tissues, detecting PRRSV and separating viruses; the cell line is Marc145 cell (African green monkey kidney cell), and various virus antibodies are preserved by the laboratory.

Example 1 isolation and identification of PRRSV

The pathological tissue (lymph, liver, spleen and lung) suspected of PRRSV is washed by PBS, 1mL of PBS is added according to 1g of tissue sample after being cut into pieces, freezing and thawing are carried out repeatedly for three times to prepare the pathological grinding fluid (used as PRRSV virus fluid), the RNA extraction kit is used for extracting PRRSV RNA from the pathological tissue, and then the RNA is reversely transcribed into cDNA.

DNAMAN software is utilized to design PRRSV target gene primers, and specific primer sequences are as follows:

PRRSV-F：5’-GGCCAGCCAGTCAATCAG-3’；SEQ ID NO.1；

PRRSV-R：5’-GGCAAACTAAACTCCACAGTG-3’；SEQ ID NO.2。

PCR amplification is carried out by using the designed primers and the extracted lymph, liver, spleen and lung cDNA of the PRRSV as templates, wherein the system is 25 mu L, and 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; extending for 10min at 72 ℃, and storing at 4 ℃ after finishing. The PCR products were subjected to agarose gel electrophoresis, and the results are shown in FIG. 1. The result shows that a specific target band appears when the PCR amplification is carried out by taking cDNA extracted from pathological materials as a template, the length is about 537bp, and the size is consistent with the expected size; the disease contains PRRSV.

Example 2 establishment of a Stable IFA neutralizing antibody detection method

Marc145 cells are prepared to contain 1.0 x 10 after digestion and centrifugation⁵cell/ml cell suspension 100. mu.l/well in 96-well plates, placed at 37 ℃ 5% CO₂Culturing for 12h in an incubator, incubating PRRSV virus solution (50 mu l) and clinical serum with the same volume at 37 ℃ for 1h when the cells are completely attached to the wall, then inoculating the cells onto Marc145 cells in a 96-well plate, culturing for 48h, taking out the 96-well plate after the cells are overgrown, and fixing by precooled absolute ethyl alcohol. Adding PRRSV standard positive serum, incubating at 37 ℃ for 1h, adding goat anti-pig IgG-FITC, incubating at 37 ℃ for 1h, observing under a fluorescence microscope, and determining that the generated specific green fluorescence is negative, otherwise, the generated specific green fluorescence is positive. The serum identified as positive is diluted in multiple proportions, and the above operation is repeated to obtain the antibody titer.

(1) Plate paving: marc145 cells are prepared to contain 1.0 x 10 after digestion and centrifugation⁵cell/ml cell suspension 100. mu.l/well in 96-well plates, placed at 37 ℃ 5% CO₂Culturing in incubator for 12h, after cells are completely attached to the wall, incubating equal volume of PRRSV virus solution and clinical serum at 37 deg.C for 1h, inoculating to Marc145 cells, adding DMEM culture medium containing 2% fetal calf serum, culturing, and placing in CO₂Continuously culturing for 48h in the incubator, observing cell growth condition, and waiting for cell growthTaking out the 96-well plate after the plate is full; setting a normal virus cell control hole at the same time;

(2) fixing: washing the 96-well plate with PBS for 2-3 times, and fixing with precooled anhydrous ethanol at-20 deg.C for 30min, or storing for a long time, i.e. the cell reaction plate is successfully prepared;

(3) adding a primary antibody: washing 96-well plate with PBS for 2-3 times, adding 100 μ l PRRSV standard positive serum (IDEXX) diluted with 0.01M PBS1:400, and standing at 37 deg.C for 1.0 h;

(4) adding a secondary antibody: adding 100 μ l goat anti-pig IgG-FITC fluorescent antibody (Solebao scientific Co., Ltd.) diluted 200 times with 0.01M PBS (pH7.4), and reacting at 37 deg.C for 1 hr;

(5) and (4) observing results: the color development was observed under a fluorescent microscope after washing with PBS, and the results are shown in FIG. 2.

Example 3IFA reaction conditions

1) TCID of PRRSV₅₀Measurement of

(1) The Marc145 cell suspension was plated on a 96-well plate at 100. mu.L per well to achieve a cell mass of 2-3X 10⁵Culturing for 12h per mL until the cells are completely attached to the wall;

(2) continuously diluting PRRSV virus liquid in penicillin bottle or centrifuge tube by 10 times^-1-10^-10；

(3) Inoculating the diluted virus to a 96-well plate with cells growing into a single layer, wherein each dilution is inoculated to a longitudinal row of 8 wells, and each well is inoculated with 100 mu L;

(4) leaving two longitudinal rows without virus inoculation, and setting normal cell control (100 μ L of maintenance solution per well, the maintenance solution is DMEM culture medium with fetal bovine serum content of 2%);

(5) culturing for 48h, taking out and fixing after the cells are full, and placing at-20 ℃ for later use;

(6) the IFA method is used for detection, and the number of holes with pathological Changes (CPE) is observed and recorded, and the result is shown in table 1; (7) TCID₅₀Calculated according to the Reed-Muench two-law method.

TABLE 1 TCID₅₀Statistics of measurement results

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

lg TCID₅₀Distance ratio x difference between log of dilutions + log of dilutions above 50% disease rate-0.1 × (-1) + (-4) ═ 4.1.

TCID is obtained from the data₅₀＝10^-4.1/0.1ml＝10^-5.1/ml

The meaning is as follows: diluting the virus 10^4.1Inoculation 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 TCID₅₀Marc145 cells were inoculated with the virus of (1) and an unvaccinated control was set. The IFA detection method is used for detecting positive serum and negative serum respectively serving as primary antibodies, and the optimal virus inoculation amount is determined by observing results, and the result is shown in figure 3.

The results in FIG. 3 show that: 100 TCIDs₅₀The virus of (3) has the best inoculation amount and strong signal.

Inoculating PRRSV (optimal virus inoculation amount) into Marc145 cell bottles with the length of about 60% -70%, spreading the cells on 4 96-well plates respectively after the cells are fully grown, and carrying out 5% CO treatment at 37 DEG C₂After 12, 24, 36 and 48 hours of culture in the incubator, the culture medium is taken out one by one and fixed. Then, positive serum and negative serum are respectively used as primary antibody for detection by using an IFA detection method, and the fixed time is determined by observing the result.

As a result, it was found that the number of TCIDs was 100₅₀The virus is inoculated with Marc145 cells, cultured for 48 hours and fixed to prepare an IFA cell reaction plate optimally.

3) Serum dilution concentration, goat anti-porcine IgG-FITC working concentration

The serum of PRRSV infected pig is mixed according to the proportion of 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 IFA 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 the 1:100, 1: 200. goat anti-porcine IgG-FITC diluted by 1:300 and 1:400 times, 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 continuous 2-fold dilution on the basis of the strength of the antibody, and the goat anti-porcine IgG-FITC working concentration is optimal to be 1: 200.

Example 4 specificity and sensitivity of IFA

Known porcine PRRSV positive serum, negative healthy porcine serum, porcine pseudorabies virus (PRV) positive serum, porcine circovirus type 2 (PCV2) positive serum and Classical Swine Fever Virus (CSFV) positive serum are respectively used for incubation and detection. The specificity and sensitivity of the IFA detection method are determined by the results.

The result shows that the PRRSV only has specific neutralization reaction with PRRSV positive serum, and has no cross reaction with other virus positive serum, which indicates that the IFA detection method has specificity. The IFA neutralizing antibody detection method is proved to have good sensitivity.

Example 6 Intra-batch Interbatch repeatability and shelf life of IFA

And (3) detecting a plurality of neutralizing antibodies prepared in the same batch and different batches, wherein the detection results are basically consistent, and the IFA detection method is good in repeatability.

Example 7IFA clinical neutralizing antibody detection

Neutralizing antibody detection was performed on sera from both pig farms using the established IFA neutralizing antibody detection method for PRRSV, and the results are shown in table 3. Carrying out neutralizing antibody detection on 51 parts of serum of a No. 1 pig farm, wherein 20 parts of serum are positive, 27 parts of serum are negative, and 4 parts of serum are suspected to be positive; neutralization antibody tests were performed on 67 sera from pig farm 2, with 22 positive, 39 negative, and 6 suspected positives.

TABLE 3 results of detection of clinically neutralizing antibodies

Example 9 clinical serum neutralizing antibodies and potency assay for PRRSV

Detecting the positive serum of the antibody by using the IFA neutralizing antibody detection method, carrying out titer detection on the neutralizing antibody, continuously diluting the serum in multiple proportions of 1:2, 1:4, 1:8 and 1:16 to 1:4096 in a 96-hole culture plate, adding 50 mu l of diluted PRRSV virus solution into each hole, mixing uniformly, and then placing at 37 ℃ and 5% CO₂The incubator is used for 1 hour. Meanwhile, negative and positive serum controls, a virus control and a normal cell control are set.

The steps of the IFA neutralizing antibody detection method were then repeated to detect serum neutralizing antibody titers.

Taking one positive serum as an example, the neutralizing antibody titer of the serum is detected, and fig. 4 is a result graph of serum dilution times of 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128, so that the neutralizing antibody titer of the serum is 1: 32.

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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Claims

1. A method for detecting IFA neutralizing antibody of PRRSV is characterized by comprising the following specific steps:

(1) plate paving: marc145 cells are digested and centrifuged to prepare cell suspension, the cell suspension is paved on a 96-well plate and placed at 37 ℃ with 5% CO₂Culturing in incubator until cells are completely attached to the wall, incubating PRRSV virus solution and clinical serum with equal volume at 37 deg.C for 1h, inoculating to Marc145 cells, adding DMEM culture medium containing 2% fetal calf serum, culturing, and placing in CO₂Continuously culturing for 48h in the incubator, observing the growth condition of the cells, and taking out the 96-well plate after the cells grow full; meanwhile, setting a non-virus-inoculated normal cell control hole;

2. The method for detecting IFA neutralizing antibody of PRRSV of claim 1, wherein the PRRSV virus solution is prepared by the following steps: and (3) washing the pathogenic tissue of the PRRSV with PBS, shearing, adding 1mL of PBS into 1g of tissue sample, and repeatedly freezing and thawing for three times to prepare the pathogenic grinding fluid.

3. The method for detecting IFA neutralizing antibody of PRRSV of claim 1, wherein the clinical serum is prepared as follows: and centrifuging the collected blood, and collecting serum for later clinical serum detection.