CN103941013B - Actinobacillus pleuropneumoniae endotoxin Rapid detection test strip - Google Patents
Actinobacillus pleuropneumoniae endotoxin Rapid detection test strip Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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Abstract
The present invention relates to the utensil that a boar bacterial disease endotoxin detects reagent display, particularly relate to a kind of actinobacillus pleuropneumoniae endotoxin (ApxIV) Rapid detection test strip, test strips is containing supporting layer, reaction reagent carrier absorption layer, supporting layer is not for absorbing water strip of foil, reaction reagent carrier absorption layer is pasted on supporting layer, is followed successively by fibrage from sample test end, ApxIV gold mark monoclonal antibody or how anti-fibrage, cellulose rete, handle end is absorbent material layer; match monoclonal antibody or many anti-or monoclonal antibody solution with ApxIV respectively on fibrage, spray detection trace " <b>|</bGreatT.Gr eaT.GT ",? " <b>/</bGreatT.Gr eaT.GT "? or " <b> \ </b> ", use sheep (rabbit) anti-mouse or pig IgG resist or on cellulose rete, spray contrast trace " <b>|</bGreatT.Gr eaT.GT " with SPA solution respectively more,? " <b>/</bGreatT.Gr eaT.GT "? or " <b> \ </b> ".This test strips is special, responsive, directly perceived, accurate, easy, quick, can apply in relevant departments such as its feeding, meat packing and quarantines.
Description
one, technical field
The present invention is the detection reagent displaying appliance that one relates to actinobacillus pleuropneumoniae endotoxin (ApxIV), particularly relates to the test strip that one can detect actinobacillus pleuropneumoniae endotoxin (ApxIV).
two, technical background
Pig pleuropneumonia is the breathing problem of a kind of acute, hot, the hyperinfection of the pig caused by actinobacillus pleuropneumoniae (APP).Actinobacillus pleuropneumoniae is the member of Pasteurella section Actinobacillus, is the little coccobacillus of a kind of Gram-negative, and what have is very thin shaft-like or thread, shows as multiform state property and the two poles of the earth coloring.There is pod membrane, have pili, without gemma.Clinically with acute hemorrhagic cellulosic pleuropneumonia and chronic fibro disposition necrotizing pleuropneumonia for feature.This disease is distributed in world many countries and area, is a kind of worldwide disease, at present in the fashion trend risen.China, in this disease of alleged occurrence in 1987, causes great economic loss to Large-scale pig farm.The change of weather and the arriving of autumn and winter season easily cause the Occurrence & epidemic of this disease, should attract great attention, take effective measures, and avoid the Occurrence & epidemic of this disease cold season.
The major storage host of the pig that carries disease germs of sick pig, subclinical infection to be the sow of the major source of infection, particularly subclinical infection of this disease be cause of disease, plays an important role in the propagation of this bacterium.Although this disease can not infect piglet by vertical transmission, by directly contacting through respiratory tract infection, have influence on the safety of piglet.By air borne, another pig house can be passed to from a morbidity pig house great-jump-forward.Also can by contact by infect indirectlies such as the vehicle of pathogen contamination, instrument, apparatus and keeper's flowings.Rodent and birds also can propagate this disease.The pig of various age, sex has neurological susceptibility, and wherein more with the morbidity of the pig in 6 ~ 16 week age, the highest with the pig incidence of disease at 3 monthly ages, can reach 80% ~ 100%, mortality ratio is generally 50%.
Pig pleuropneumonia is one of principal disease of current harm countries in the world pig industry.Current APP finds that there is 15 kinds of serotypes altogether, and the popular predominant serotype of every country is different.Between different serotypes and the virulence of the different strains of same serotype all there are differences, pathogenic also have power point, thus cause the Diagnosis and Treat of this disease very difficult.Research in recent years shows, the factor relevant to pathogen virulence has toxin, pod membrane, outer membrane protein, urase etc., wherein toxin is main virulence factor, also be main immunogene, can be used as diagnostic antigen and set up Serology test, can also be used for diagnosis and weigh immune state, therefore toxin study has become the study hotspot of this disease.APP has 4 kinds of toxin, i.e. toxin
, II,
and the toxin IV found recently, they all belong to RTX (Repeatsinthestructural, RTX) toxin family.In recent years find, do not have the APP of One serotype can secrete 4 kinds of toxin, but all serotype of APP can secrete ApxlV toxin protein in infection animal body, and the bacterium of in vitro culture can not secrete this toxin simultaneously.Therefore, ApxlV toxin detected in animal body, just can prove the infection of APP, ApxlV toxin protein is setting up the larger theory value of tool in diagnostic method and application prospect, can be used for distinguishing the infection of diagnosis APP and non-infection.At present the diagnostic method of above-mentioned disease is mainly contained following several.
(1) microscopy.Get nasal cavity and bronchial secretion or pulmonary lesion material smear, Gram’s staining, microexamination, as seen gram-negative little coccobacillus or the bacillus exilis of the dense dye in polymorphic the two poles of the earth, can do further qualification.
(2) Antigen isolation and identification.Asepticly get pathological material of disease, with 5% sheep blood agar, dull and stereotyped and staphylococcus aureus intersects streak inoculation, cultivate 24 hours for 37 DEG C under 5% ~ 10% gas concentration lwevel condition, around staphylococcus aureus, have satellite colony, then carry out the biochemical identification such as urase and mannose fermentation.And carry out the inspections such as CAMP (CAMP) test, hemolytic mensuration and serotype.
(3) serodiagnosis.Fluorescent antibody technics can be used for the inspection of specific antigen; Enzyme linked immunosorbent assay (ELISA) and complement fixation test (CFT) can be used for detecting antibody, find the pig of subclinical infection.Indirect hemagglutination test (IHA), the indirect hemagglutination test of APP is used for detection and the somatotype of APP.Latex agglutination test is a kind of simple and rapid detection method, and it both may be used for the diagnosis of APP, also can be used for somatotype.Carry out the serodiagnosis of APP with enzyme linked immunosorbent assay technology, type specificity ELISA and species specificity ELISA can be divided into.The former can only make detection to the APP of One serotype, and the latter can make detection to the APP of all serotype.
(4) PCR (PCR) technology: round pcr has the advantage such as high sensitivity, high specific, multiple PCR method has been widely used qualification in APP or ApxIV and epidemiology survey.
Above-mentioned detection method needs professional in laboratory operation, complex operation, and detection is wasted time and energy; And need expensive instrument and equipment, as PCR instrument, microplate reader etc., for layman, above-mentioned detection method has been difficult to.Although the special sensitivity of said method, cannot realize field quick detection or diagnosis.The present invention, research a kind of easy fast, real-time online Test paper, to control with to eliminate this disease significant.
three, summary of the invention
The object of the invention is to overcome in prior art the shortcoming detecting swine disease cause of disease and exist, there is provided a kind of special, responsive, easy quick detection actinobacillus pleuropneumoniae endotoxic method, develop the test strip detecting actinobacillus pleuropneumoniae endotoxin (ApxIV).
Technical scheme of the present invention is: the test strip providing a kind of actinobacillus pleuropneumoniae endotoxin (ApxIV), this test strips contains supporting layer and adsorbed layer, supporting layer is the lamella do not absorbed water, adsorbed layer is attached on supporting layer, adsorbed layer is followed successively by the absorbent material layer of sample adsorption fibrage, golden labeling antibody fibrage, cellulose rete and handle end from test lead, cellulose rete is provided with and detects trace and contrast trace; Gold labeling antibody fibrage is adsorbed with the monoclonal antibody of the anti-ApxIV of nanometer grade gold particle marker, detects the trace pairing monoclonal antibody of anti-ApxIV and prints, contrasts the polyclonal antibody of trace sheep or rabbit anti-mouse IgG; Or golden labeling antibody fibrage is adsorbed with the polyclonal antibody of the anti-ApxIV of nanometer grade gold particle marker, detects the trace monoclonal antibody of anti-ApxIV and prepare, contrast trace staphylococcal protein A (SPA) or the how anti-preparation of anti-pig IgG.
Namely the pairing monoclonal antibody preparation detecting the anti-ApxIV of trace is prepared with the pairing monoclonal antibody solution of ApxIV; Detect the trace polyclonal antibody preparation of anti-ApxIV and be the polyclonal antibody preparation using ApxIV.
Supporting layer the hard plastic slip do not absorbed water or cardboard bar are made; Test lead sample adsorption fibrage glass wool is made; Gold labeling antibody fibrage glass wool and golden labeling antibody are made, and golden labeling antibody can be monoclonal antibody or polyclonal antibody.
Cellulose rete nitrocellulose filter or pure cellulose film or carboxylated cellulose film or polyvinylidene fluoride PVDF cellulose membrane are made.
Absorbent material layer thieving paper is made.
Detect trace and contrast trace be orthoscopic or oblique line formula, on cellulose rete containing one detect trace and one contrast trace, detect trace and contrast trace spread pattern be "
||", "
//", "
?" in any one.
Containing layer protective layer above test strips adsorbed layer; protective seam is attached on adsorbed layer; test lead sample adsorption fibrage, golden labeling antibody fibrage and absorbent material layer are coated with diaphragm; the diaphragm that test lead sample adsorption fibrage is corresponding with golden labeling antibody fibrage intersection is printed with sample mark line, and this mark line deflection test lead sample adsorption fibrage side place is about 0.5cm place.
As required, select above-mentioned golden labeling antibody fibrage, detect a kind of form in trace and contrast trace spread pattern.
Positive beneficial effect of the present invention:
1. detection specificity is strong, susceptibility is high: test strip of the present invention is made based on nanometer grade gold particle marker high-affinity monoclonal antibody specific or specific polyclonal antibody, formed without covalent bond between gold grain and antibody molecule in gold labeling antibody, the two is combined by the Van der Waals force between the charges of different polarity, gold grain does not affect specificity and the adhesion of monoclonal antibody or polyclonal antibody, and has higher mark rate.Test strip of the present invention has higher specificity and susceptibility, nanogram level actinobacillus pleuropneumoniae endotoxin can be detected.
2. easy and simple to handle, quick: without the need to additional any Other Instruments and reagent when using ELISA test strip of the present invention, only its test lead need to be inserted in sample liquid to be checked about 30 seconds, then can judge testing result in 1-5 minute.
3. testing result is directly perceived, accurately: whether test strips of the present invention show henna detection line and control line as the foundation judging positive and negative findings, namely only a brownish red control line C is shown at the control line marking place of cellulose membrane, and at detection line marking place without the display of brownish red band, represent that detected ApxIV is negative findings; Show a brownish red control line C at the control line marking place of cellulose membrane, in detection, trace place shows a brownish red band T, then represent that detected ApxIV is positive findings.No matter positive findings or negative findings control line C all should show, and when control line C does not show, illustrate that test strips lost efficacy.
4. testing cost reduces: use test strip of the present invention, do not need Other Instruments and reagent, save instrument, equipment and additive reagent expense; Layman also can detect by real-time online at any time, without the need to paying expert diagnosis Laboratory Fee and correlative charges thereof, can reduce the input of testing cost greatly, reducing testing cost.
5. usable range is wide: test strip of the present invention is simple to operate, i.e. " foolproof " operation, and easy to carry, easily preserve, the needs of not commensurate and different levels personnel can be met, comprise specialty chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, livestock products processing, intensive culture to individual cultivation etc., there is wide market outlook and social benefit.
four, accompanying drawing illustrates:
The side-looking structural representation of the test strip of a kind of actinobacillus pleuropneumoniae endotoxin (ApxIV) of Fig. 1
The plan structure schematic diagram of the test strip of a kind of actinobacillus pleuropneumoniae endotoxin (ApxIV) of Fig. 2
five, embodiment:
Following examples, only in order to further illustrate the present invention, do not limit content of the present invention.The preparation of the test strip of actinobacillus pleuropneumoniae endotoxin (ApxIV), needs monoclonal antibody and the polyclonal antibody of preparing anti-ApxIV, for the preparation of detection trace and golden labeling antibody fibrage; Need preparation sheep or rabbit anti-mouse igg antibody, sheep or the anti-pig IgG antibody of rabbit, for the preparation of contrast trace simultaneously.
1. the preparation of sheep (rabbit) against murine or anti-pig IgG antibody:
Extract the IgG in mouse or Swine serum with saturated ammonium sulfate method, get 1 part of serum and add 2 parts of PBS liquid (pH7.2) mixings, add the mixing of equal-volume saturated ammonium sulfate liquid, put 2h in 4 DEG C of refrigerators, at 4 DEG C, the centrifugal 15min of 10000r/min, abandon supernatant; With appropriate PBS liquid (pH7.2) dissolution precipitation, adding saturated ammonium sulfate liquid to its ultimate density is 33%, put 2h in 4 DEG C of refrigerators, at 4 DEG C, centrifugal 15min under 10000r/min condition, abandon supernatant, with a small amount of PBS liquid (pH7.2) dissolution precipitation, to put in 4 DEG C of refrigerators with PBS liquid (pH7.2) dialyzed overnight, change liquid 2 ~ 3 times, at 4 DEG C, centrifugal 15min under 10000r/min condition, collect supernatant, measure its protein concentration with ultraviolet spectrophotometer.With 50 μ g ~ 100 μ g(IgG)/kg body weight is through subcutaneous or intramuscular injection negative antibody Healthy Sheep or rabbit 3 ~ 4 times, final immunization is after 20 days, venous blood collection, its serum antibody titer is measured at more than 1:2000 with ELISA, Culling heart blood or arteria carotis bloodletting, collect its hyper-immune serum, and its extracting method of IgG(extracting sheep (rabbit) anti-mouse or pig with saturated ammonium sulfate method is identical with said extracted mice serum IgG, no longer repeat), for the preparation of the contrast trace of test strips of the present invention.
The preparation of 2.ApxIV monoclonal antibody (Mi):
Every only with 50 μ g ~ 100 μ gApxIV antigen immune Balb/c system mouse three times, every minor tick 15 ~ 30d; 3 ~ 4d after third time booster immunization, by the bloodletting of immune mouse eyeball, draws neck lethal, with 75% alcohol-pickled 5 ~ 10min, asepticly gets its spleen, shreds and through 100 order nylon net filters, the centrifugal 10min of 1000r/min, collection splenocyte; By 1 × 10
8individual splenocyte and 2 ~ 5 × 10
7individual NS0 myeloma cell's mixing, 1000r/min is centrifugal, and 10min abandons supernatant, centrifuge tube containing sedimentation cell is placed in the water of 37 DEG C, and slowly add 0.7 ~ 1ml40% ~ 50%PEG4000(pH8.5 ~ 9.0) effect 1min, then slowly add serum-free 1640 nutrient culture media 15ml, to stop the effect of PEG, 37 DEG C of water-bath 5 ~ 10min, 1000r/min is centrifugal, and 10min abandons supernatant, is resuspended in by cell precipitation in HAT Selective agar medium, and adds
96 well culture plates (100 μ l/ hole, μ l ~ 200), are placed in 37 DEG C of 5%CO
2cultivate in incubator.After cultivating 7 ~ 10d, with the pathogen specific antigen bag of the purifying of 5 μ g ~ 10 μ g/ml by 96 hole ELISA Plate, detect the culture supernatant of hybridoma with enzyme linked immunosorbent assay (ELISA), picking strong positive cell clone (OD
450>=0.5), carry out the limiting dilution assay cloning of continuous three times, obtain positive hybridoma cell strain, its chromosome number is 92 ~ 98, the monoclonal antibody of its secretion, can specific recognition ApxIV, and not with other toxin generation cross reaction, affinity constant reaches 10
9 ~ 10, light chain subtype is к or λ, and heavy chain subgroup is IgG
1, IgG
2a, IgG
2b, IgG
3; The pairing monoclonal antibody obtained, for making gold mark monoclonal antibody body glass wool or detecting trace.
3. the preparation of gold mark monoclonal antibody glass wool:
Utilize reduction of sodium citrate legal system for nanometer grade gold particle: 0.5 ~ 2% citric acid three sodium solution namely adding 2 ~ 4ml in 0.01 ~ 0.05% aqueous solution of chloraurate of 50 ~ 100ml boiling, obtain the nanometer grade gold particle of diameter about 15nm.With the K of 0.1mol/L
2cO
3adjust pH to 8.5 ~ 9.5 of gold grain solution, with the mark of 1:1000 ~ 1300 than adding in the aurosol of pH8.5 ~ 9.5 by monoclonal antibody to be marked, after mark 10min, adding 20%PEG10000 to ultimate density is 0.05%, 4 DEG C, the centrifugal 20min of 1500 ~ 3000r/min, remove unconjugated gold grain particle, 4 DEG C, the centrifugal 1h of 15000r/min, abandon supernatant, after obtaining golden labeling antibody potpourri, with propylene glucosan S-400 column chromatography, separation and purification gold labeling antibody, the golden labeling antibody of acquisition.By the golden labeling antibody that 1:100 ~ 500 are diluted, be adsorbed in processed glass cotton, 4 DEG C of low-temperature vacuum dryings, preparation gold mark monoclonal antibody glass wool.
The preparation of 4.ApxIV polyclonal antibody (Ci):
The preparation of ApxIV polyclonal antibody (Ci).Adopt ApxIV antigen repeatedly immunity inoculation negative antibody health pig.Final immunization posterior vein blood sampling in 20 days, measure its serum antibody titer at more than 1:2000 with ELISA, Culling heart blood or arteria carotis bloodletting, collect its hyper-immune serum, IgG antibody (method is identical with the extraction of mice serum IgG, no longer repeats) in serum is extracted with saturated ammonium sulfate method.
Gold mark resists the preparation with the how anti-glass wool of gold mark, identical with the preparation method of gold mark monoclonal antibody glass wool, no longer repeats.Refer to the content 3 in embodiment.
5. test strip Cleaning Principle of the present invention
After test strip test lead of the present invention inserts measuring samples solution, solution to be checked drives ApxIV to be checked to enter golden labeling antibody fibrage by siphon, and spread along nitrocellulose filter to handle end together with golden labeling antibody (Mi or Ci) wherein, final infiltration handle end absorbent material layer, in diffusion process, golden labeling antibody can be combined with corresponding ApxIV to be checked, ApxIV in conjunction with golden labeling antibody cellulose membrane can be detected the pairing monoclonal antibody of trace or how anti-interception, when containing tested ApxIV in sample liquid, then there is a henna detection line; Sheep or rabbit against murine or anti-pig IgG then can with corresponding gold mark monoclonal antibody or many anti-bindings, there is 1 brownish red control line.When not containing ApxIV in measuring samples liquid, test strips only demonstrates a brownish red control line; When cellulose membrane not having control line show, then show that test strips lost efficacy.
6. the detection method of operating of test strip of the present invention
(1) detect the process of sample: get disease pig pathological tissues, 1:1 ~ 5 add physiological saline and shred with scissors, and leachate is measuring samples, sick pig whole blood or serum are measuring samples after adding the dilution of physiological saline 1:1 ~ 5.
(2) operation is detected: test strip sample end of the present invention inserted in measuring samples liquid, insertion depth is no more than mark line 9, takes out test strips after about 30 seconds, horizontal positioned about 1 ~ 5 minute, simultaneously observations.
(3) result judges: if only demonstrate a brownish red control line C on test strip cellulose membrane, represents that testing result is negative, illustrates in test sample not containing ApxIV; If control line C appears in the cellulose membrane in test strip, detect trace place and occur a detection line, represent that testing result is positive, namely in measuring samples, contain ApxIV; If cellulose membrane does not have control line C show, then show that test strips lost efficacy.
Embodiment one: the test strip of actinobacillus pleuropneumoniae endotoxin (ApxIV)
See Fig. 1 and Fig. 2, in figure, 1 is supporting layer, make by hard plastic strip of foil, 2 is the sample adsorption fibrage of test lead, make with glass wool, 3 is golden labeling antibody fibrage, be adsorbed with the glass wool of the monoclonal antibody of the anti-ApxIV of nanometer grade gold particle marker, its gold mark monoclonal antibody glass wool is prepared according to the preparation method described in above-mentioned embodiment 3, 4 is cellulose rete, employing nitrocellulose filter is made, 5 is absorbent material layer, make with thieving paper, will numbering 2, 3, 4, 5 each layers are pasted onto hard plastic strip of foil 1 from left end test lead to the right side, intersection crosses one another overlap each other.On cellulose nitrate rete 4,6 is the detection trace T printed with the pairing monoclonal antibody solution of anti-ApxIV, 7 is the contrast trace C printed with sheep or rabbit anti-mouse igg solution, detects trace and contrast trace is orthoscopic or oblique line formula, the array configuration that the arrangement of two kinds of trace bands is formed be "
||", "
//", "
?" in any one.8-1 covers the white diaphragm above test lead sample adsorption fibrage 2 and golden labeling antibody fibrage 3; 0.5cm place, sample adsorption fibrage 2 side is partial in the corresponding diaphragm 8-1 position of 2 and 3 intersections and is printed on mark line 9; the right-hand member of 9 is printed on arrow and max printed words, absorbent material layer 5(handle end) on be coated with other color (as yellow) diaphragm 8-2.
The preparation of testing sample solution and detection operation steps, identical with the detection method of operating in embodiment 6, no longer repeat.
Embodiment two: the test strip of actinobacillus pleuropneumoniae endotoxin (ApxIV), substantially identical with embodiment one, difference is:
Gold labeling antibody fibrage 3 is made with the glass wool of the polyclonal antibody being adsorbed with the anti-ApxIV that gold grain marks, and prepares its gold mark polyclonal antibody glass wool according to the preparation method described in above-mentioned embodiment 3; On cellulose nitrate rete 4,6 is the detection trace T printed with the monoclonal antibody solution of anti-ApxIV, 7 is print contrast trace C with the IgG solution of sheep or the anti-pig of rabbit, and the array configuration that two kinds of trace band arrangements are formed is " || ", " // ", " \ any one in \ ".It is all identical with the method for operating in embodiment 6 that other comprises detection sample preparation, method of operating and result judgement etc., no longer repeats.
Claims (7)
1. one kind is detected the test strip of actinobacillus pleuropneumoniae endotoxin ApxIV, this test strips contains supporting layer and adsorbed layer, supporting layer is the lamella do not absorbed water, adsorbed layer is attached on supporting layer, adsorbed layer is followed successively by sample adsorption fibrage from test lead, gold labeling antibody fibrage, the absorbent material layer of cellulose rete and handle end, on cellulose rete, preparation has detection trace and contrast trace, it is characterized in that golden labeling antibody fibrage is adsorbed with the anti-ApxIV monoclonal antibody of nanometer grade gold particle marker, the pairing monoclonal antibody or the polyclonal antibody that detect the anti-ApxIV of trace are printed, contrast the trace polyclonal antibody of sheep or rabbit anti-mouse IgG or staphylococcal protein A are printed, or golden labeling antibody fibrage is adsorbed with the polyclonal antibody of the anti-ApxIV of nanometer grade gold particle marker, detects the trace monoclonal antibody of anti-ApxIV and prepare, contrast trace staphylococcal protein A or the how anti-preparation of anti-pig IgG,
Wherein, the preparation method of ApxIV monoclonal antibody is as follows:
With 50 μ g ~ 100 μ gApxIV antigen immune Balb/c system mouse three times, every minor tick 15 ~ 30d; 3 ~ 4d after third time booster immunization, by the bloodletting of immune mouse eyeball, draws neck lethal, with 75% alcohol-pickled 5 ~ 10min, gets its spleen under aseptic condition, to shred and through 100 order nylon net filters, the centrifugal 10min of 1000r/min, collection splenocyte; By 1 × 10
8individual splenocyte and 2 ~ 5 × 10
7individual NS0 myeloma cell's mixing, 1000r/min is centrifugal, and 10min abandons supernatant, centrifuge tube containing sedimentation cell is placed in the water of 37 DEG C, and slowly add 0.7 ~ 1ml40% ~ 50%PEG4000 effect 1min, the pH of described PEG4000 is 8.5 ~ 9.0, then serum-free 1640 nutrient culture media 15ml is slowly added, to stop the effect of PEG, 37 DEG C of water-bath 5 ~ 10min, 1000r/min is centrifugal, and 10min abandons supernatant, is resuspended in by cell precipitation in HAT Selective agar medium, and adds 96 well culture plates, 100 μ l/ hole, μ l ~ 200, are placed in 37 DEG C of 5%CO
2cultivate in incubator; After cultivating 7 ~ 10d, with the pathogen specific antigen bag of the purifying of 5 μ g ~ 10 μ g/ml by 96 hole ELISA Plate, detect the culture supernatant of hybridoma with enzyme linked immunosorbent assay, picking OD
450the strong positive cell clone of>=0.5, carries out the limiting dilution assay cloning of continuous three times, obtains positive hybridoma cell strain, its chromosome number is 92 ~ 98, monoclonal antibody of its secretion can specific recognition ApxIV, and not with other toxin generation cross reaction, affinity constant reaches 10
9 ~ 10, light chain subtype is к or λ, and heavy chain subgroup is IgG
1, IgG
2a, IgG
2bor IgG
3.
2. test strips according to claim 1, is characterized in that namely the pairing monoclonal antibody preparation detecting the anti-ApxIV of trace is prepared with the pairing monoclonal antibody solution of anti-ApxIV.
3. test strips according to claim 1, is characterized in that supporting layer the hard plastic slip do not absorbed water or cardboard bar are made; Test lead sample adsorption fibrage glass wool is made; Gold labeling antibody fibrage glass wool and golden labeling antibody are made, and golden labeling antibody is monoclonal antibody or polyclonal antibody.
4. test strips according to claim 1, is characterized in that cellulose rete nitrocellulose filter or pure cellulose film or carboxylated cellulose film are made.
5. test strips according to claim 1, is characterized in that absorbent material layer thieving paper is made.
6. test strips according to claim 1, it is characterized in that detecting trace and contrasting trace is orthoscopic or oblique line formula, on cellulose rete containing one detect trace and one contrast trace, detect trace and contrast trace spread pattern be " || ", " // ", “ " in any one.
7. test strips according to claim 1; it is characterized in that being coated with diaphragm on test lead sample adsorption fibrage, golden labeling antibody fibrage and absorbent material layer; the diaphragm that test lead sample adsorption fibrage is corresponding with golden labeling antibody fibrage intersection is printed with sample mark line, and this mark line deflection test lead sample adsorption fibrage side is about 0.5cm place.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004052925A2 (en) * | 2002-12-09 | 2004-06-24 | Imperial College Innovations Limited | Actinobacillus pleuropneumoniae virulence genes |
| CN101265457A (en) * | 2007-07-20 | 2008-09-17 | 华中农业大学 | Vaccine for differentiating porcine actinobacillus pleuropneumonia serum 7-type double-gene deletion mutant of vaccine immunity and virus infection animal and application thereof |
| CN101339192A (en) * | 2008-08-28 | 2009-01-07 | 河南省农业科学院 | Test paper for one-step detection for pig virus diarrhoea disease pathogen |
| CN101949934A (en) * | 2010-09-04 | 2011-01-19 | 扬州大学 | Early infection detection kit of monoclonal antibody-mediated pig pleuropneumoniae |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004052925A2 (en) * | 2002-12-09 | 2004-06-24 | Imperial College Innovations Limited | Actinobacillus pleuropneumoniae virulence genes |
| CN101265457A (en) * | 2007-07-20 | 2008-09-17 | 华中农业大学 | Vaccine for differentiating porcine actinobacillus pleuropneumonia serum 7-type double-gene deletion mutant of vaccine immunity and virus infection animal and application thereof |
| CN101339192A (en) * | 2008-08-28 | 2009-01-07 | 河南省农业科学院 | Test paper for one-step detection for pig virus diarrhoea disease pathogen |
| CN101949934A (en) * | 2010-09-04 | 2011-01-19 | 扬州大学 | Early infection detection kit of monoclonal antibody-mediated pig pleuropneumoniae |
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