CN102297969A - Preparation method of immunofluorescent reagent for differential diagnosis of traditional American type and variant porcine reproductive and respiratory syndrome - Google Patents
Preparation method of immunofluorescent reagent for differential diagnosis of traditional American type and variant porcine reproductive and respiratory syndrome Download PDFInfo
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Abstract
The invention discloses a preparation method of an immunofluorescent reagent for differential diagnosis of traditional American type and variant porcine reproductive and respiratory syndrome (PRRS), comprising the following steps: using a monoclonal antibody Mab-B37 which can only identify the traditional American type PRRSV strain and a monoclonal antibody which can identify all American type PRRSV strains including the traditional American type and variant PRRSV strains; and applying the characteristic of the monoclonal antibody which can identify all American type PRRSV strains, respectively marking FITC, selecting different fluorescent monoclonal antibodies to establish an immunofluorescent diagnostic method for distinguishing the traditional American type PRRS and the variant PRRS. According to the immunofluorescent diagnostic method, the detection time is 40 min, the coincidence is 92.5 % compared with virus isolation, and the coincidence is 92.2 % compared with RT-PCR detection kit. The invention shows that the detection result of the method is accurate and reliable, and the immunofluorescent diagnostic method can be used for the differential diagnosis of traditional American type PRRS and the variant PRRS, and has good clinical application value.
Description
Technical field
The present invention relates to the reagent of a kind of antidiastole tradition american type and variant porcine reproductive and respiration syndrome, the preparation method of the immunofluorescence reagent of particularly a kind of antidiastole tradition american type and variant porcine reproductive and respiration syndrome.
Background technology
Porcine reproductive and respiratory syndrome (is commonly called as pig blue-ear disease, Porcine Reproductive and Respiratory Syndrome, PRRS), be by porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) causing, is the pig infectious disease of cardinal symptom with sow breeding difficulty and piglet breathing problem.Be divided into two genotype of american type and Europe class.That China is since nineteen ninety-six popular is american type PRRSV.Variant porcine reproductive and respiration syndrome are to be popular in China since 2006, a kind of acute high lethal eqpidemic disease that causes by american type porcine reproductive and respiratory syndrome virus variant, mainly show as the high heat of pig (more than 41 ℃), high incidence, high mortality, low cure rate, all responsive to the pig of any age in days.Existing immunology fast diagnosis method can not be distinguished conventional P RRSV and variant PRRSV; Have only RT-PCR kit and PCR kit for fluorescence quantitative to can be used for the antidiastole of conventional P RRSV and variant PRRSV, but complex operation, Test Condition Requirements height are not suitable for veterinary clinic and the basic unit quick diagnosis to PRRS.
Summary of the invention
The object of the invention is to overcome the difference of prior art and a kind of quick, easy, preparation method of being easy to the immunofluorescence reagent of antidiastole tradition american type that basic unit applies and variant porcine reproductive and respiration syndrome is provided.
The present invention is achieved by the following technical solution:
The preparation method of the immunofluorescence reagent of a kind of antidiastole tradition american type and variant porcine reproductive and respiration syndrome, with a kind of hybridoma Mab-B47 strain that only can discern traditional american type PRRSV, be deposited in CCTCC(China typical culture collection center), April 15 2011 preservation day, deposit number: CCTCC C201122, can discern the hybridoma cell strain of all american type PRRSV strains with a strain, recover respectively, enlarged culture, and prepare corresponding ascites, ascites is separated, getting supernatant is the ascites monoclonal antibody, measures its IFA and tires, and the IFA of ascites monoclonal antibody tires 1 * 10
3More than ,-40 ℃ of preservations are standby after the packing;
Behind two kinds of ascites monoclonal antibody measurings mensuration protein concentrations, protein concentration should be greater than 10 mgmL
-1, be diluted to 10 mgmL with the pH9.2-PH9.5 carbonate buffer solution respectively
-1After, pack into separately in the bag filter, according to the total amount of protein to be marked, get the fluorescein of requirement, the consumption of fluorescein its measurement unit of 1/20(suitable and protein content is the weight meter), dissolve with the pH9.2-PH9.5 carbonate buffer solution, its amount stirs during dissolving gently for 10 times of protein liquid volume to be marked, avoids bubble, then bag filter is placed the fluorescein lysate, 4 ℃ of black outs stir mark 24 h on the stirrer.
With above-mentioned two kinds the fluorescence monoclonal antibody of mark add Sephadex G50 post respectively, with sterilization PBS wash-out, collection has the first peak of fluorescence, the mensuration first peak is respectively collected the fluorescence of liquid and is tired, positive pipe is merged, be the immunofluorescence diagnostic reagent of differentiating traditional american type PRRS and anomaly PRRS ,-40 ℃ of preservations.
Adopt the trial-production of this method preparation to have characteristics such as specificity is good, susceptibility is high, easy and simple to handle, quick, can accurately diagnose the sick pig of doubtful PRRS.
The characteristic of described immunofluorescence diagnostic reagent is:
(1) Mab-B47 and can discern all american type PRRSV strains the hybridoma cell strain fluorescence antibody tire should 〉=1:32.
(2) the Mab-B47 fluorescence antibody only reacts with traditional american type PRRSV, with normal Marc-145 cell, pig 2 type PCV-II, pig parvoviral, the equal no cross reaction of PRV.The hybridoma cell strain fluorescence antibody that can discern all american type PRRSV strains can react with all american type PRRSV, with normal Marc-145 cell, pig 2 type PCV-II, pig parvoviral, the equal no cross reaction of PRV.
(3) fluorescence antibody storage life under-20 ℃ of conditions is 6 months.
(4) coincidence rate of fluorescence antibody DFA and IFA detection is more than 95%.
(5) the total coincidence rate of DFA and VI is more than 90%, and the total coincidence rate between DFA and the RT-PCR is more than 90%.
(6) be 30-60min the detection time of diagnostic reagent.
Two kinds of ascites MONOCLONAL ANTIBODIES SPECIFIC FOR methods are specially gets the hybridoma cell strain that hybridoma Mab-B47 and a strain can be discerned all american type PRRSV strains, recovery is after enlarged culture respectively, lumbar injection Balb/c mouse in 10～12 age in week, induce every mouse 1 * 10 in advance with sterilized liquid paraffin
6Individual cell, when treating that mouse web portion obviously expands, ascites is collected in drainage, in centrifugal 15 min of 10 000r/min, gets supernatant and is the ascites monoclonal antibody, measures its IFA and tires, and-40 ℃ of preservations are standby after the packing.
Described stirrer is a magnetic stirring apparatus.
The immunofluorescence diagnostic reagent of traditional american type PRRS of discriminating of the present invention and anomaly PRRS can be applicable to detect contamination cell and Clinical differential diagnosis.
The described hybridoma cell strain that can discern all american type PRRSV strains that comprise traditional american type and anomaly is Mab-C65, Mab-D4.
The best applications of immunofluorescence diagnostic reagent of the present invention in Clinical differential diagnosis is: organizing freezing microtome section thickness is 0.1-0.3mm, adopts the methanol/acetone fixation (promptly earlier behind 100% refrigerated methanol effect 5min, again through 100% freezing acetone effect 5min; Perhaps use the fixing fixing means of ice methyl alcohol again with the ice acetone fixed earlier), antibody diluent is 1%BSA-PBS, fluorescence antibody doubly dilutes through 1:32 and is optimum reaction condition.
In sum, the present invention has following advantage compared to existing technology:
Adopt and only can discern the monoclonal antibody Mab-B47 of traditional american type PRRSV strain, and the monoclonal antibody that can discern all american type PRRSV strains that comprise traditional american type and anomaly; Use monoclonal antibody and can discern the characteristic of dissimilar PRRSV strains, difference flag F ITC, select different fluorescence monoclonal antibodies and set up the immunofluorescence diagnostic method that to distinguish traditional american type PRRS and anomaly PRRS, be 40min the detection time of this method, separate coincidence rate with virus and reach 92.5%, reach 92.2% with the coincidence rate of RT-PCR detection kit.Use this diagnostic method 87 parts of clinical suspected cases and 12 parts of artificial challenge's cases are detected, show that this method test result accurately, reliably, can be used for the antidiastole of traditional american type and anomaly PRRS, have the good clinical using value.
For effect of the present invention better is described, the applicant also carries out Preliminary Applications to the fluorescence monoclonal antibody in differentiating artificial challenge's case and clinical case, below be applicable cases:
1: the collection of artificial challenge pig and pathological material of disease
12 of 28 ages in days, the healthy piglets of PRRSV negative antibody are divided into 3 groups, 4/group at random.Use variant PRRSV-FJ0605 the 7th generation viral cultures (10 for first group
4.0TCID
50/ mL) through collunarium, the inoculation of intramuscular injection approach, dosage is the 2mL/ head; Second group with traditional America strain PRRSV-FZ(10
4.5TCID
50/ mL) through collunarium, the inoculation of intramuscular injection approach, dosage is the 2mL/ head; The 3rd group for not attacking malicious normal healthy controls pig.Clinical response is observed in three groups of isolated rearings every day, and 8 d compel for every group to kill 2 behind virus inoculation, 14d cuts open and kills 2, gather lung, hilar lymph node, making frozen section and tissue homogenate are used for the coincidence rate that virus is separated, immunofluorescence detects, RT-PCR detects and compare.
The DFA testing result of 2 artificial challenge's porcine tissue samples
Variant is attacked in the poison group, and hilar lymph node and lungs recall rate are 100%, and fluorescence signal is very strong; Tradition America pig is attacked the poison group and detect positive antigen in lungs and hilar lymph node, but a little less than fluorescence signal attacks the poison group than variant; Each tissue sample testing result of normal healthy controls pig is all negative.
The coincidence rate of 3 artificial challenge pig DFA, RT-PCR, three kinds of detection methods of VI relatively
Gather the tissues such as lungs, lymph node, kidney of every first tap poison pig respectively, make homogenate and in the Marc145 cell, carry out virus and separate, when waiting cytopathy to occur, carry out immunofluorescence with the PRRSV monoclonal antibody to verify; Get and attack malicious haslet homogenate, extract nucleic acid, detect according to PRRSV RT-PCR differential diagnosis kit.As a result, variant attacks that 4 swine diseases poison of poison group separate and RT-PCR is all positive, and the coincidence rate between DFA, RT-PCR, the VI three is 100%.It is all positive that 4 pig RT-PCR of poison group are attacked in tradition America strain, all is positive and have only 3 first taps poison pig VI and DFA to detect.Coincidence rate between DFA and the RT-PCR is 75%, and the coincidence rate of DFA and VI is 100%.
4 artificial challenges compare with total coincidence rate of making a definite diagnosis pathological material of disease DFA detection and VI and RT-PCR detection
This research has been carried out VI and DFA coincidence rate statistics to 12 parts of artificial challenge's pathological material of diseases and 15 parts of clinical definite pathological material of diseases altogether.9 parts of anomaly PRRS, viral separation, RT-PCR and DFA detect all positive.9 parts of traditional PRRS, virus is separated 7 parts of number positive, negative 2 parts; DFA detects 8 parts of number positive, negative 1 part; It all is positive that RT-PCR detects.It all is negative that three kinds of methods of 9 parts of negative pathological material of diseases detect.The positive coincidence rate of DFA and VI is 94%(16/17), negative match-rate is 91%(10/11), total coincidence rate of DFA and VI is 92.5%.Positive coincidence rate between DFA and the RT-PCR is 94%(17/18), negative match-rate is 90%(9/10), the total coincidence rate between DFA and the RT-PCR is 92.2%.
Clinical application effect is estimated: use kit 87 parts of clinical suspected cases and 12 parts of artificial challenge's cases are detected, more than 90%, be 30-60min detection time to the coincidence rate of this kit and VI or RT-PCR all as a result.Show that this kit test findings accurately, reliably, can be used for the antidiastole of traditional american type and anomaly PRRS, have the good clinical using value.
Anti-american type PRRSV hybridoma Mab-B47 strain is deposited in CCTCC(China typical culture collection center, the address: Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University), and April 15 2011 preservation day, deposit number: CCTCC C201122.
Embodiment
The present invention is further described below in conjunction with specific embodiment.
Embodiment 1
The hybridoma Mab-B47 strain of anti-PRRSV, has following characteristic: the only traditional american type PRRSV reaction of this cell line monoclonal antibody specific, with Marc-145 cell, pig 2 type PCV-II, pig parvoviral, the equal no cross reaction of PRV, the IFA of the antibody of hybridoma supernatant and ascites tires respectively 2
4With 10
3More than.Mab-B47 is deposited in CCTCC(China typical culture collection center), April 15 2011 preservation day, deposit number: CCTCC C201122.
The preparation method that the hybridoma Mab-B47 strain of above-mentioned anti-PRRSV of the present invention, Mab-C65 use in conjunction are diagnosed in the immunofluorescence of traditional american type PRRS and anomaly PRRS:
(1) take out hybridoma Mab-B47 strain, Mab-C65 strain from liquid nitrogen, recovery is after enlarged culture, 10～12 week of lumbar injection Balb/c mouse in age (inducing with sterilized liquid paraffin in advance), every mouse 1 * 10
6Individual cell when treating that mouse web portion obviously expands, is collected ascites with No. 12 syringe needle drainages, in centrifugal 15 min of 10 000r/min, gets supernatant and is the ascites monoclonal antibody, measures its ELISA and IFA respectively and tires, and-40 ℃ of preservations are standby after the packing.
(2) fluorescence labeling of monoclonal antibody
Behind Mab-B47, Mab-C65 ascites monoclonal antibody measuring protein concentration, be diluted to finite concentration with the pH9.2-PH9.5 carbonate buffer solution after, in the bag filter of packing into.According to the total amount of IgG to be marked, get the fluorescein (quite with protein content 1/20) of requirement, be dissolved in the beaker with the pH9.2-PH9.5 carbonate buffer solution, it is measured is 10 times of protein liquid volume to be marked, stirs gently during dissolving, avoids bubble.Bag filter is placed beaker, and 4 ℃ of black outs stir mark 24 h on the magnetic stirring apparatus; Bag filter is taken out in 0.01 MolL
-14 ℃ of dialysis 4 h～5 h among the pH7.2 PBS, liquid is changed for several times in the centre, fully desalination.
(3) fluorescence antibody purifying
Use Sephadex G50 post and remove free FITC.The FITC labelled antibody of the above-mentioned desalination of having dialysed is added Sephadex G50 post,, collects the first peak that fluorescence is arranged, measure first peak and respectively collect the fluorescence of liquid and tire, positive pipe is merged, be labelled antibody with sterilization PBS wash-out, be stored in-40 ℃ standby.
The characteristic of described immunofluorescence diagnostic reagent is:
(1) Mab-B47 and can discern all american type PRRSV strains the hybridoma cell strain fluorescence antibody tire should 〉=1:32.
(2) the Mab-B47 fluorescence antibody only reacts with traditional american type PRRSV, with normal Marc-145 cell, pig 2 type PCV-II, pig parvoviral, the equal no cross reaction of PRV.The hybridoma cell strain fluorescence antibody that can discern all american type PRRSV strains can react with all american type PRRSV, with normal Marc-145 cell, pig 2 type PCV-II, pig parvoviral, the equal no cross reaction of PRV.
(3) fluorescence antibody storage life under-20 ℃ of conditions is 6 months.
(4) coincidence rate of fluorescence antibody DFA and IFA detection is more than 95%.
(5) the total coincidence rate of DFA and VI is more than 90%, and the total coincidence rate between DFA and the RT-PCR is more than 90%.
(6) be 30-60min the detection time of diagnostic reagent.
The following describes the immunofluorescence diagnostic reagent in the running program that detects the contamination cell.
(1): the preparation of PRRSV infection cell
With the Marc-145 cellular incubation in 96 porocyte plates, be cultured to cell monolayer under 37 ℃ of 5%CO2 conditions after, inoculate traditional american type and variant PRRSV strain respectively, inoculum concentration is 100 TCID
50When cytopathy reaches the 30 % left and right sides, abandon supernatant, PBS washing 1 time, every hole adds cold methanol 100 μ L, fixes 30 min for 4 ℃, dry, PBS washing 1 time ,-40 ℃ are frozen standby.Establish the negative contrast of normal Marc-145 cell simultaneously.
(2) DFA detects
Get the contamination cell plates, after embathing with the PBS hole, every hole adds a certain amount of fluorescent-labeled antibody working fluid with the 1%BSA-PBS dilution, places in the wet box, and 37 ℃ of senses are done to take out behind the 30min.Outwell the fluorescence antibody dye liquor gently and blot with filter paper, wash several times with PBS 200 μ L/ holes, last every Kong Jiakang fluorescence decay mountant (50% glycerine-PBS), take pictures by observations under the fluorescence inverted microscope.With the normal Marc-145 cell hole of establishing virus inoculation not as negative control.
(3) fluorescence antibody titration
After fluorescence antibody carried out 2 multiple proportions gradient dilutions with the PBS that contains 1%BSA, the Marc145 cell that adds PRRSV virus strain infection respectively carried out fluorescent antibody staining, establishes the negative cells contrast of not contamination simultaneously.The greatest dilution of clear bright specificity fluorescent (++) to occur, be tiring of this fluorescence antibody.As a result, tiring of fluorescence antibody is 1:64～1:128.Be the specificity intensity of assurance fluorescence antibody, and can eliminate the non-specific fluorescence reaction of normal tissue cell, real work concentration is 1:32.
(4) fluorescence antibody specific assay
In 96 porocyte culture plates, carry out.After the suitable dilution of fluorescence antibody, respectively with infection cells such as PRRSV, PRV, PPV, CSFV, TGEV, PCV2, MPV, NDRV and normal cell effect 30min after, with physiological saline washing 3 times, every hole adds 50% glycerine-PBS 50ul, and inverted fluorescence microscope is observed down.The result shows: PRRS fluorescence antibody and PRRSV infection cell are positive, and present special bright green fluorescence in endochylema, and the fluorescent characteristic of DFA is identical with IFA; And other virus infected cell and the equal no cross reaction of normal Marc145 cell.The fluorescence antibody that tentatively shows preparation does not have specificity with other multiple swine disease poison antigen and intersects, and specificity is preferably arranged, and can be used for the evaluation of PRRSV infection cell.
(5) coincidence rate of DFA and IFA detection contamination cell relatively
Get B47 and C65 fluorescence antibody respectively and in PRRSV infection cell and normal control cell, carry out direct immuno fluorescence test (DFA), carry out corresponding IFA test with identical monoclonal antibody simultaneously, and carry out 3 times and repeat.As a result, DFA detects characteristic and conforms to IFA, carries out revision test at the Marc145 cell of contaminating, and the coincidence rate of method is 100% in two, shows the use in conjunction of fluorescent-labeled antibody, can be used for the evaluation of all kinds of PRRSV infection cells.
(6) storage life of fluorescence antibody is measured
Get under-20 ℃ of conditions and preserve fluorescence antibody, carry out a DFA test every month, observe antibody titer and change, measuring fluorescence antibody storage life under-20 ℃ of conditions is 6 months.
The following describes the running program of immunofluorescence diagnostic reagent in clinical diagnosis.
One: the optimization of PRRSV DFA reaction conditions
(1) preparation of frozen section
Doubtful PRRS pathological material of disease is taked in the section of making pathological material of disease, cuts soya bean size lungs and hilar lymph node piece of tissue, be positioned over and organize on the holdfast and freezing, treat that piece of tissue is mounted on the microtome after freezing, adjust the position after, cut the tissue slice of 0.1 ~ 0.3mm, stick on the microslide.
(2) selection of fixing agent and fixing means
The hilus pulumonis lymph node section of preparation health pig and PRRS infected pigs adopts following 4 kinds of fixing meanss to fix with ice methyl alcohol and ice acetone respectively: (1) 100% freezing methyl alcohol, effect 10min; The freezing acetone mixed liquor of (2) 50% freezing methyl alcohol/50%, effect 10min; (3) 100% freezing methyl alcohol, effect 5min, after shifting out, freezing acetone, effect 5min; Perhaps ice methyl alcohol effect 5min(4 again with ice acetone effect 5min earlier) 100% freezing acetone, effect 10min.The section that fixes adds the fluorescence monoclonal antibody with the 1%BSA-PBS dilution, behind 37 ℃ of effect 30min, fully washs with PBS, adds glycerine-PBS and carries out fluorescence microscope after the mounting.Compare different fixing agent and fixing means Color and non-specific staining situation, thereby determine the optimal fixation condition.The result, adopt (2) method promptly: 100% freezing methyl alcohol, effect 5min, after shifting out, 100% freezing acetone, act on 5min or after icing acetone fixed, use the fixing means of icing the methyl alcohol effect more earlier, good, the specific fluorescence dyeing nothing but of section DFA detection background, histocyte complete form, positive fluorescence focus get a clear view.And other three groups of fixing meanss have certain non-specific fluorescence dyeing, cause false-positive judgement easily.
(3) selection of the best dilution of fluorescence antibody
Except purpose antigen, also may have group antigen in the histotomy, can combine with the corresponding antibodies beyond the specific antigen in the tissue, this just needs to seal the site that cross reaction is arranged in energy and the tissue with dilution at the trial.Adopt PBS, 0.5%BSA-PBS, 1%BSA-PBS, 2%BSA-PBS that fluorescence antibody is diluted to best effort concentration respectively, respectively the DFA fluorescent dye is carried out in the hilus pulumonis lymph node section of health pig and PRRS infected pigs, determine the best dilution of fluorescence antibody.As a result, fluorescence antibody dilutes with PBS, and the non-specific false positive fluorescent dye of section is more, is difficult for distinguishing with the positive of positive focus; After the PBS dilution that contains variable concentrations bovine serum albumin(BSA) (BSA), non-specific fluorescence reduces along with the raising of BSA concentration, but the susceptibility of fluorescence antibody slightly descends along with the raising of BSA concentration, so during real work, select the dilution of 1%BSA-PBS as fluorescence antibody.
By repetition test, finally determine to organize freezing microtome section to adopt the methanol/acetone fixation, antibody diluent is 1%BSA-PBS, fluorescence antibody doubly dilutes through 1:32 and is optimum reaction condition.
Embodiment 2
Embodiment 2 and embodiment 1 are roughly the same, and the hybridoma cell strain Mab-D4 that can discern all american type PRRSV strains that comprise traditional american type and anomaly exactly replaces Mab-C65.
Claims (6)
1. the preparation method of the immunofluorescence reagent of antidiastole tradition american type and variant porcine reproductive and respiration syndrome, it is characterized in that: with a kind of hybridoma Mab-B47 strain that only can discern traditional american type PRRSV, can discern the hybridoma cell strain of all american type PRRSV strains with a strain, recover respectively, enlarged culture, and prepare corresponding ascites, ascites is separated, getting supernatant is the ascites monoclonal antibody, measures its IFA ascites monoclonal antibody IFA that tires and tires 1 * 10
3More than ,-40 ℃ of preservations are standby after the packing;
Behind two kinds of ascites monoclonal antibody measurings mensuration protein concentrations, protein content should be greater than 10 mgmL
-1, be diluted to 10 mgmL with the pH9.2-PH9.5 carbonate buffer solution respectively
-1After, pack into separately in the bag filter,, get the fluorescein of requirement according to the total amount of protein to be marked, the consumption of fluorescein quite and protein content 1/20, its measurement unit is the weight meter, and with the dissolving of pH9.2-PH9.5 carbonate buffer solution, its amount is 10 times of protein liquid volume to be marked, stir gently during dissolving, avoid bubble, then bag filter is placed the fluorescein lysate, 4 ℃ of black outs stir mark 24 h on the stirrer;
Above-mentioned two kinds of fluorescence monoclonal antibodies that mark is good are added Sephadex G50 post respectively, with sterilization PBS wash-out, collection has the first peak of fluorescence, the mensuration first peak is respectively collected the fluorescence of liquid and is tired, positive pipe is merged, be the immunofluorescence diagnostic reagent of differentiating traditional american type PRRS and anomaly PRRS ,-40 ℃ of preservations.
2. the preparation method of the immunofluorescence reagent of antidiastole according to claim 1 tradition american type and variant porcine reproductive and respiration syndrome, it is characterized in that: the technical indicator of described immunofluorescence diagnostic reagent is: (1) Mab-B47 and the tiring of hybridoma cell strain fluorescence antibody that can discern all american type PRRSV strains are answered 〉=1:32;
(2) the Mab-B47 fluorescence antibody only reacts with traditional american type PRRSV, with normal Marc-145 cell, pig 2 type PCV-II, pig parvoviral, the equal no cross reaction of PRV, the hybridoma cell strain fluorescence antibody that can discern all american type PRRSV strains can react with all american type PRRSV, with normal Marc-145 cell, pig 2 type PCV-II, pig parvoviral, the equal no cross reaction of PRV;
(3) fluorescence antibody storage life under-20 ℃ of conditions is 6 months;
(4) coincidence rate of fluorescence antibody DFA and IFA detection is more than 95%;
(5) the total coincidence rate of DFA and VI is more than 90%, and the total coincidence rate between DFA and the RT-PCR is more than 90%;
(6) be 30-60min the detection time of diagnostic reagent.
3. the preparation method of the immunofluorescence reagent of antidiastole tradition american type according to claim 2 and variant porcine reproductive and respiration syndrome, it is characterized in that: two kinds of ascites MONOCLONAL ANTIBODIES SPECIFIC FOR methods are specially gets the hybridoma cell strain that hybridoma Mab-B47 and a strain can be discerned all american type PRRSV strains, recovery is after enlarged culture respectively, lumbar injection Balb/c mouse in 10～12 age in week, induce every mouse 1 * 10 in advance with sterilized liquid paraffin
6Individual cell, when treating that mouse web portion obviously expands, ascites is collected in drainage, in centrifugal 15 min of 10 000r/min, gets supernatant and is the ascites monoclonal antibody, measures its IFA and tires, and-40 ℃ of preservations are standby after the packing.
4. the preparation method of the immunofluorescence reagent of antidiastole tradition american type according to claim 3 and variant porcine reproductive and respiration syndrome, it is characterized in that: described stirrer is a magnetic stirring apparatus.
5. according to the preparation method of the immunofluorescence reagent of any one described antidiastole tradition american type of claim 1 to 4 and variant porcine reproductive and respiration syndrome, it is characterized in that: the described hybridoma cell strain that can discern all american type PRRSV strains that comprise traditional american type and anomaly is Mab-C65, Mab-D4.
6. the preparation method of the immunofluorescence reagent of antidiastole tradition american type according to claim 5 and variant porcine reproductive and respiration syndrome, it is characterized in that: the best applications of described immunofluorescence diagnostic reagent in Clinical differential diagnosis is: organizing freezing microtome section thickness is 0.1-0.3mm, adopt the methanol/acetone fixation (promptly earlier behind 100% refrigerated methanol effect 5min, again through 100% freezing acetone effect 5min; Perhaps use the fixing fixing means of ice methyl alcohol again with the ice acetone fixed earlier), antibody diluent is 1%BSA-PBS, fluorescence antibody doubly dilutes through 1:32 and is optimum reaction condition.
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