CN103941015A - Rapid detection test paper strip for porcine actinobacillus pleuropneumoniae endotoxin antibody - Google Patents
Rapid detection test paper strip for porcine actinobacillus pleuropneumoniae endotoxin antibody Download PDFInfo
- Publication number
- CN103941015A CN103941015A CN201310083890.2A CN201310083890A CN103941015A CN 103941015 A CN103941015 A CN 103941015A CN 201310083890 A CN201310083890 A CN 201310083890A CN 103941015 A CN103941015 A CN 103941015A
- Authority
- CN
- China
- Prior art keywords
- layer
- spa
- trace
- antibody
- apxiv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to an appliance for displaying a detection reagent of a porcine actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody, and in particular relates to a rapid detection test paper strip for the porcine actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody. The test paper strip contains a support layer and a reaction reagent carrier adsorption layer; the support layer is a non-water-absorbing thin sheet strip, and the reaction reagent carrier adsorption layer is pasted on the support layer; a fiber layer, a gold-labeled porcine actinobacillus pleuropneumoniae endotoxin (ApxIV) or SPA fiber layer, and a cellulose membrane layer are successively arranged from a sample test end, and a handle end adopts a water-absorbing material layer; a detection imprint of '|', '/' or '\' is printed on the fiber layer with an anti-pig IgG polyclonal antibody or an SPA or ApxIV; a contrast imprint of '|', '/' or '\' is printed on the cellulose membrane layer with an ApxIV monoclonal antibody or polyclonal antibody solution or an anti-SPA polyclonal antibody solution or an anti-pig IgG polyclonal antibody solution. The detection test paper strip is strong in specificity, high in sensitivity, low in cost, and simple and fast in operation, allows detection results to be vivid, intuitive and accurate in display, has no need of instruments and equipment and professional detection personnel, can greatly reduce the labor intensity, shortens the detection time, can perform on-site detection in feedlots, meat packing plants, entry-exit inspection and quarantine bureaus and other places, and is easily popularized and applied.
Description
one, technical field
The present invention is the detection reagent displaying appliance that one relates to actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody, particularly relates to one and can detect the test strip of actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody.
two, technical background
Pig pleuropneumonia is the breathing problem of a kind of acute, hot, the hyperinfection of the pig that 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 being of having 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 taking acute hemorrhagic fiber disposition pleuropneumonia and chronic fiber disposition gangrenosum acne pleuropneumonia as feature.This disease is distributed in world many countries and area, is a kind of worldwide disease, is at present the fashion trend of rising.China, in this disease of alleged occurrence in 1987, causes great economic loss to large scale of pig farm field.The variation of weather and the arriving of autumn and winter season easily cause the generation of this disease and popular, should attract great attention, and take effective measures, and avoid the generation of this disease cold season and popular.
The pig that carries disease germs of sick pig, subclinical infection is that the sow of the main infection sources, particularly subclinical infection of this disease is the main reservoir host of cause of disease, in the propagation of this bacterium, plays an important role.Although this disease can not infect piglet by vertical transmission, can, by directly contacting through respiratory tract infection, have influence on the safety of piglet.By air borne, can pass to another pig house from a morbidity pig house great-jump-forward.Also can by contact by the vehicle of pathogen contamination, instrument, apparatus and keeper flow etc. infect indirectly.Rodent and birds also can be propagated this disease.The pig of various ages, sex has neurological susceptibility, wherein more with the pig morbidity in 6～16 week age, the highest with the pig incidence of disease at 3 monthly ages, can reach 80%～100%, and mortality ratio is generally 50%.
Pig pleuropneumonia is one of principal disease of current harm countries in the world pig industry.At present APP finds that there is 15 kinds of serotypes altogether, and the popular advantage 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, thereby cause this disease diagnosis and treatment 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 this sick study hotspot.APP has 4 kinds of toxin, i.e. toxin
, II,
and the toxin IV of recent findings, they all belong to RTX (Repeatsinthe structural, 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 be secreted 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 being set up larger theory value and the application prospect of tool aspect diagnostic method, can be used for infection and the non-infection of difference diagnosis APP.At present the detection method of above-mentioned disease and antibody is mainly contained following several.
(1) microscope inspection.Get nasal cavity and bronchial secretion or pulmonary lesion material smear, Gram’s staining, microexamination, as see dense gram-negative little coccobacillus or the bacillus exilis dying in polymorphic the two poles of the earth, can do further qualification.
(2) pathogen separation qualification.The aseptic pathological material of disease of getting, intersect streak inoculation with 5% Blood In Sheep agar plate and staphylococcus aureus, under 5%～10% gas concentration lwevel condition, cultivate 24 hours for 37 DEG C, around staphylococcus aureus, have satellite colony, then carry out the biochemical identification such as urase and mannose fermentation.And carry out that CAMP (CAMP) test, hemolytic are measured and the inspection such as serotype.
(3) serodiagnosis.Fluorescent antibody technics can be used for the inspection of specific antigen or antibody; Enzyme linked immunosorbent assay (ELISA) can be used for detecting antibody with complement fixation test (CFT), finds the pig of subclinical infection.Indirect hemagglutination test (IHA), the indirect hemagglutination test of APP is for detection and the somatotype of APP.Latex agglutination test is a kind of simple and rapid detection method, and it both can, for the diagnosis of APP, also can be used for APP antibody test.Carry out the serodiagnosis of APP and antibody with enzyme linked immunosorbent assay technology, can be divided into type specificity ELISA and species specificity ELISA.The former can only make detection to the APP of One serotype, and the latter can make detection to the APP of all serotypes.
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 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 controlling and to eliminate this disease significant.
three, summary of the invention
The object of the invention is the shortcoming that detects the existence of actinobacillus pleuropneumoniae endotoxin ApxIV antibody in prior art in order to overcome, a kind of method of special, responsive, easy fast detecting actinobacillus pleuropneumoniae endotoxin antibody is provided, develops the test strip that detects actinobacillus pleuropneumoniae endotoxin ApxIV antibody.
Technical scheme of the present invention is: the test strip that a kind of actinobacillus pleuropneumoniae endotoxin ApxIV antibody is provided, this test strips contains supporting layer and adsorbed layer, supporting layer is the lamella not absorbing water, adsorbed layer is attached on supporting layer, adsorbed layer is followed successively by the absorbent material layer of sample adsorbing fiber layer, golden labeling antibody fibrage, cellulose rete and handle end from test lead, be provided with and detect trace and contrast trace on cellulose rete; Gold mark ApxIV or the absorption of SPA fibrage have ApxIV or the SPA of nanometer grade gold particle marker, detect polyclonal antibody or the staphylococcal protein A (SPA) of trace sheep or the anti-pig IgG of rabbit, or expressing protein Apx
print; The anti-Apx of contrast trace
monoclonal antibody or polyclonal antibody print, or with anti-Staphylococcus aureus A albumen (SPA) or the how anti-preparation of anti-pig IgG.
Detect polyclonal antibody or the staphylococcal protein A (SPA) of trace sheep or the anti-pig IgG of rabbit and print i.e. polyclonal antibody or the preparation of staphylococcal protein A solution with anti-pig IgG; Detect trace expressing protein Apx
printing is uses expressing protein Apx
solution preparation.
Supporting layer is made with the hard plastic slip or the cardboard bar that do not absorb water; Test lead sample adsorbing fiber layer is made with glass wool; Gold mark ApxIV or SPA fibrage mark ApxIV with glass wool and gold or SPA makes.
Cellulose rete is made with nitrocellulose filter or pure cellulose film or carboxylation cellulose membrane or polyvinylidene fluoride PVDF cellulose membrane.
Absorbent material layer is made with thieving paper.
Detect trace and contrast trace is orthoscopic or oblique line formula, on cellulose rete, contain one and detect trace and one contrast trace, the spread pattern that detects trace and contrast trace be "
||", "
//", "
?" in any.
Above test strips adsorbed layer, contain layer protective layer; protective seam is attached on adsorbed layer; on test lead sample adsorbing fiber layer, gold mark ApxIV or SPA fibrage and absorbent material layer, be coated with diaphragm; on the test lead sample adsorbing fiber layer diaphragm corresponding with gold mark ApxIV or SPA fibrage intersection, be printed with sample mark line, this mark line deflection test lead sample adsorbing fiber layer one about 0.5cm place of side place.
As required, select above-mentioned gold mark ApxIV or SPA 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 as basis taking nanometer grade gold particle marker high-affinity expressing protein ApxIV or SPA, in gold mark albumen, between gold grain and protein molecular, form without covalent bond, the two combines by the Van der Waals force between the charges of different polarity, gold grain does not affect specificity and the adhesion of gold mark albumen, and has higher mark rate.Test strip of the present invention has higher specificity and susceptibility, nanogram level actinobacillus pleuropneumoniae endotoxin ApxIV antibody can be detected.
2. easy and simple to handle, quick: to use when ELISA test strip of the present invention without additional any Other Instruments and reagent, its test lead need be inserted in sample liquid to be checked about 30 seconds, then in 1-5 minute, can judge testing result.
3. testing result is directly perceived, accurate: whether test strips of the present invention is to show that henna detection line and control line are as the foundation of judging positive and negative findings, only show a brownish red control line C at the control line marking place of cellulose membrane, and show without brownish red band at detection line marking place, represent the detected negative result of ApxIV antibody; Control line marking place at cellulose membrane shows a brownish red control line C, detecting a brownish red band T of trace place demonstration, represents the detected positive result of ApxIV antibody.No matter positive findings or negative findings control line C all should show, in the time that 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, saved instrument, equipment and additive reagent expense; Layman is real-time online detection at any time also, without paying expert diagnosis Laboratory Fee and correlative charges thereof, can reduce greatly the input of testing cost, reduces 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 preservation, can meet the not needs of commensurate and different levels personnel, comprise that professional chemical examination, customs quarantine control, health and epidemic prevention, quality monitoring, livestock products processing, intensive culture arrive individual cultivation etc., have wide market outlook and social benefit.
four, brief description of the drawings:
The side-looking structural representation of the test strip of a kind of actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody of Fig. 1
The plan structure schematic diagram of the test strip of a kind of actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody 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) antibody, need to prepare ApxIV expressing protein, the anti-pig IgG antibody of sheep or rabbit is for the preparation of detecting trace, need to prepare monoclonal antibody and the polyclonal antibody of anti-ApxIV, and the polyclonal antibody of anti-SPA is for the preparation of contrast trace simultaneously.
1. the preparation of the anti-pig IgG antibody of sheep (rabbit):
Extract the IgG in pig serum with saturated ammonium sulfate method, get 1 part of serum and add 2 parts of PBS liquid (pH 7.2) and mix, add equal-volume saturated ammonium sulfate liquid and mix, 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, centrifugal 15min under 4 DEG C, 10000r/min condition, abandons supernatant, with a small amount of PBS liquid (pH7.2) dissolution precipitation, put in 4 DEG C of refrigerators with PBS liquid (pH7.2) dialyzed overnight, change liquid 2～3 times, centrifugal 15min under 4 DEG C, 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, last immunity is after 20 days, venous blood collection, measure its serum antibody titer more than 1:2000 with ELISA, heart blood sampling or arteria carotis bloodletting, collect its hyper-immune serum, and its extracting method of IgG(that extracts the anti-pig of sheep (rabbit) with saturated ammonium sulfate method is identical with said extracted pig serum IgG, no longer repeat), for the preparation of the detection trace of test strips of the present invention.
2. the preparation of ApxIV monoclonal antibody (Mi):
Every use 50 μ g～100 μ g ApxIV antigen immune Balb/c are mouse three times, every minor tick 15～30d; 3～4d after booster immunization for the third time, by the bloodletting of immune mouse eyeball, draws neck lethal, and with 75% alcohol-pickled 5～10min, aseptic its spleen of getting, shreds and through 100 order nylon net filters, the centrifugal 10min of 1000r/min, collects splenocyte; By 1 × 10
8individual splenocyte and 2～5 × 10
7individual NS0 myeloma cell mixes, 1000r/min is centrifugal, and 10min abandons supernatant, the centrifuge tube that contains sedimentation cell is placed in to the water of 37 DEG C, and slowly add 0.7～1ml, 40%～50% PEG4000(pH 8.5～9.0) 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, cell precipitation is resuspended in to HAT and selects in nutrient culture media, and add
96 well culture plates (100 μ l/ hole, μ l～200), are placed in 37 DEG C of 5% CO
2in incubator, cultivate.Cultivate after 7～10d, with the coated 96 hole ELISA Plate of pathogen specific antigen of the purifying of 5 μ g～10 μ g/ml, 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, and 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, heavy chain hypotype is IgG
1, IgG
2a, IgG
2b, IgG
3; The pairing monoclonal antibody obtaining, for making gold mark monoclonal antibody body glass wool or detecting trace.
3. the preparation of gold mark ApxIV or SPA glass wool:
Utilize sodium citrate reduction method for preparing nanometer level gold grain: in 0.01～0.05% aqueous solution of chloraurate of 50～100ml boiling, add 0.5～2% citric acid three sodium solution of 2～4ml, obtain the nanometer grade gold particle of diameter 15nm left and right.With the K of 0.1mol/L
2cO
3adjust pH to 8.5～9.5 of gold grain solution, mark with 1:1000～1300 adds in the aurosol of pH8.5～9.5 than by ApxIV to be marked or SPA, after mark 10min, add 20% PEG10000 to ultimate density be 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, obtain after golden labeling antibody potpourri, with propylene glucosan S-400 column chromatography, separation and purification gold labeling antibody, gold mark ApxIV or the SPA of acquisition.By the gold mark thing of 1:100～500 dilution, be adsorbed in processed glass cotton 4 DEG C of low-temperature vacuum dryings, preparation gold mark ApxIV or SPA glass wool.
4. the preparation of ApxIV polyclonal antibody (Ci):
The preparation of ApxIV polyclonal antibody (Ci).Adopt repeatedly immunity inoculation negative antibody health pig of ApxIV antigen.Last immunity posterior vein blood sampling in 20 days, measure its serum antibody titer more than 1:2000 with ELISA, heart blood sampling or arteria carotis bloodletting, collect its hyper-immune serum, extract IgG antibody (method is identical with the extraction of pig serum IgG, no longer repeats) in serum with saturated ammonium sulfate method.
5. test strip of the present invention detects principle
When test strip test lead of the present invention inserts after sample solution to be checked, solution to be checked drives ApxIV antibody to be checked to enter gold mark ApxIV or SPA fibrage by siphon, and spread to handle end along nitrocellulose filter with together with gold mark thing (ApxIV or SPA) wherein, the final handle end absorbent material layer that infiltrates, in diffusion process, gold mark thing can be combined with corresponding ApxIV antibody to be checked, bond can be detected the anti-pig IgG of trace or SPA or ApxIV and be tackled on cellulose membrane, in the time containing tested ApxIV antibody in sample liquid, there is a henna detection line, ApxIV monoclonal antibody or the how how anti-and corresponding gold of anti-or anti-SPA are marked things and are combined, and occur 1 brownish red control line.In the time not containing ApxIV antibody in sample liquid to be checked, test strips only demonstrates a brownish red control line; In the time not having control line to show on cellulose membrane, show that test strips lost efficacy.
6. the detection method of operating of test strip of the present invention
(1) detect the processing of sample: get disease pig pathological tissues, 1:1～5 add physiological saline and shred with scissors, and leachate is sample to be checked, and sick pig whole blood or serum are sample to be checked after adding the dilution of physiological saline 1:1～5.
(2) detect operation: test strip sample end of the present invention is inserted in sample liquid to be checked, and insertion depth is no more than mark line 9, after approximately 30 seconds, takes out test strips, horizontal positioned approximately 1～5 minute, simultaneously observations.
(3) result is judged: if only demonstrate a brownish red control line C on test strip cellulose membrane, represent that testing result is negative, illustrate in test sample not containing ApxIV antibody; If there is control line C in the cellulose membrane in test strip, detect trace place and occur detection line T, represent that testing result is positive, in sample to be checked, contain ApxIV antibody; If do not have control line C to show on cellulose membrane, show that test strips lost efficacy.
7. the expression and purification of Actinobacillus pleuropneumoniae ApxlV toxin
(1) design of primers: the complete genome sequence of ApxlV gene in the app gene of announcing according to GenBank, designs and synthesizes Auele Specific Primer, and add restriction enzyme site in upstream and downstream primer respectively.The cultivation of bacterial strain and the extraction of genomic DNA adopt conventional method to carry out.
(2) amplification of PCR product and qualification: add 5ul 10 × buffer, 4 ul 2.5 mmol/L dNTP, the each 2ul of upstream and downstream primer, template DNA 2ul in PCR reaction tube, add sterilizing H
20 to 50ul.95 DEG C of 5 min of PCR reaction conditions, 94 DEG C of sex change 30s, 56 DEG C of 30 s, 72 DEG C of extension 1.5 min, 30 circulations, last 72 DEG C are extended 10 min again, use kit purified pcr product.
(3) clone of genes of interest specific fragment: 18 DEG C of 5 h under the effect of T4DNA ligase by PCR purified product and PMD18-T, getting 10ul connects in product transformed competence colibacillus cell E.coli DH5 α, coat on the LB flat board of ammonia benzyl resistance, be coated with X-gal and IPTG simultaneously and carry out blue hickie screening, 37 DEG C of overnight incubation, choose white colony and shake bacterium, extract plasmid in a small amount, after enzyme is cut qualification, check order.
(4) structure of expression vector and expression: by positive colony plasmid and expression vector pET.28a (+) Hind III and NcoI double digestion, 37 DEG C of effect 4 h, reclaim kit purifying through glue.Then 16 DEG C connect 10 h, and in transformation receptor bacterium E.coliBL21 mono-DL3, obtain single bacterium colony, extract plasmid DNA, after double digestion qualification, positive colony is inoculated in to LB fluid nutrient medium (containing 50U/mL kanamycins), 0.5 mol/L lactose 10ul abduction delivering.
(5) purifying of expressing protein: with lysozyme and deoxycholate cracking bacterium, DNA enzyme bacterium for degrading discharges DNA, centrifuging and taking precipitation.To precipitate with 2% NP-40 washing 1 time, then use 8 mol/L urea cracking, centrifuging and taking supernatant, carries out preliminary purification through His-Bind resin.In the His-Bind of 500ul resin, add the bacterial lysate of 1ml, room temperature effect 30 min, constantly shake, is fully combined Ni-NTA with expressing protein during this time.With after urea washing 2 times, then use imidazoles solution (250mmol/L) wash-out 2 times.Collect eluent, through 12% SDS-PAGE electrophoresis detection purification result.
Embodiment mono-: the test strip of actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody
Referring to Fig. 1 and Fig. 2, in figure, 1 is supporting layer, make by hard plastic strip of foil, 2 is the sample adsorbing fiber layer of test lead, make with glass wool, 3 is gold mark fibres layer, absorption has the glass wool of the ApxIV of nanometer grade gold particle marker, prepare its gold mark thing glass wool 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, to number 2, 3, 4, 5 each layers stick on hard plastic strip of foil 1 from left end test lead to the right side, intersection crosses one another overlapping each other.On cellulose nitrate rete 4,6 is the detection trace T with the many of anti-pig IgG or the printing of SPA solution, 7 contrast trace C for printing with the monoclonal antibody of anti-ApxIV or how anti-solution, detect trace and contrast trace is orthoscopic or oblique line formula, the array configuration that two kinds of trace bands are arranged formation be "
||", "
//", "
?" in any.8-1 covers test lead sample adsorbing fiber layer 2 and golden labeling antibody fibrage 3 white diaphragm above; on the corresponding diaphragm 8-1 of 2 and 3 intersections position, be partial to sample adsorbing fiber layer 2 one side 0.5cm place and be printed on mark line 9; 9 right-hand member 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 bis-: the test strip of actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody, basic identical with embodiment mono-, difference is:
The 3 use absorption of gold labeling antibody fibrage have the glass wool of the SPA of gold grain mark to make, and prepare its gold mark SPA glass wool according to the preparation method described in above-mentioned embodiment 3; On cellulose nitrate rete 4,6 detection trace T for printing with ApxIV expressing protein solution, 7 print contrast trace C for the antibody-solutions with anti-SPA or anti-pig IgG, the array configuration that two kinds of trace bands are arranged formation be "
||", "
//", "
?" in any.Other comprises that detection sample preparation, method of operating and result judgement etc. are all identical with the method for operating in embodiment 6, no longer repeats.
Claims (8)
1. one kind is detected the test strip of actinobacillus pleuropneumoniae endotoxin (ApxIV) antibody, this test strips contains supporting layer and adsorbed layer, supporting layer is the lamella not absorbing water, adsorbed layer is attached on supporting layer, adsorbed layer is followed successively by the absorbent material layer of sample fiber layer, gold mark ApxIV or SPA fibrage, cellulose rete and handle end from test lead, on cellulose rete, be prepared with and detect trace and contrast trace, it is characterized in that gold mark ApxIV or the absorption of SPA fibrage have the expressing protein Apx of nanometer grade gold particle marker
or SPA, polyclonal antibody or the staphylococcal protein A (SPA) of sheep or the anti-pig IgG of rabbit for detection trace, or expressing protein Apx
print; The anti-Apx of contrast trace
monoclonal antibody or polyclonal antibody print, or with anti-Staphylococcus aureus A albumen (SPA) or the how anti-preparation of anti-pig IgG.
2. test strips according to claim 1, is characterized in that golden labeling antibody fibrage absorption has the expressing protein Apx of nanometer grade gold particle marker
, detect polyclonal antibody or the staphylococcal protein A (SPA) of trace sheep or the anti-pig IgG of rabbit and print, the anti-Apx of contrast trace
monoclonal antibody or polyclonal antibody print, or golden labeling antibody fibrage absorption has the SPA of nanometer grade gold particle marker, detects trace expressing protein Apx
preparation, the many anti-or how anti-preparations of anti-pig IgG of anti-Staphylococcus aureus A albumen (SPA) for contrast trace.
3. test strips according to claim 1 and 2, is characterized in that detecting the polyclonal antibody of trace sheep or the anti-pig IgG of rabbit or staphylococcal protein A (SPA) and prints i.e. polyclonal antibody or staphylococcal protein A solution with anti-pig IgG and prepare; Detect trace expressing protein Apx
printing is uses expressing protein Apx
solution preparation.
4. test strips according to claim 1, is characterized in that supporting layer the hard plastic slip or the cardboard bar that do not absorb water make; Test lead sample adsorbing fiber layer is made with glass wool; Gold mark Apx
or SPA fibrage is marked Apx with glass wool and gold
or SPA makes.
5. test strips according to claim 1, is characterized in that cellulose rete nitrocellulose filter or pure cellulose film or carboxylation cellulose membrane or polyvinylidene fluoride PVDF cellulose membrane make.
6. test strips according to claim 1, is characterized in that absorbent material layer thieving paper makes.
7. test strips according to claim 1, is characterized in that detecting trace and contrast trace is orthoscopic or oblique line formula, on cellulose rete, contain three and detect traces and one contrast trace, the spread pattern that detects trace and contrast trace be "
||", "
//", "
?" in any.
8. test strips according to claim 1; it is characterized in that containing layer protective layer above test strips adsorbed layer; protective seam is attached on adsorbed layer; on test lead sample adsorbing fiber layer, golden labeling antibody fibrage and absorbent material layer, be coated with diaphragm; on the test lead sample adsorbing fiber layer diaphragm corresponding with golden labeling antibody fibrage intersection, be printed with sample mark line, this mark line deflection test lead sample adsorbing fiber layer about 0.5cm of one side place.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310083890.2A CN103941015A (en) | 2013-03-15 | 2013-03-15 | Rapid detection test paper strip for porcine actinobacillus pleuropneumoniae endotoxin antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310083890.2A CN103941015A (en) | 2013-03-15 | 2013-03-15 | Rapid detection test paper strip for porcine actinobacillus pleuropneumoniae endotoxin antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103941015A true CN103941015A (en) | 2014-07-23 |
Family
ID=51188768
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310083890.2A Pending CN103941015A (en) | 2013-03-15 | 2013-03-15 | Rapid detection test paper strip for porcine actinobacillus pleuropneumoniae endotoxin antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103941015A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050130247A1 (en) * | 2001-11-14 | 2005-06-16 | Kiamars Hajizadeh | Rapid prion-detection device, system and test kit |
| CN1645148A (en) * | 2005-01-24 | 2005-07-27 | 江苏省农业科学院 | Reagent box for inspecting infection of swine pleuropneumonia actinomyces |
| CN101113980A (en) * | 2007-06-28 | 2008-01-30 | 华中农业大学 | Colloidal gold chemiluminescence immune analysis method for detecting pig pleuropneumonia antibody |
| CN101344524A (en) * | 2008-08-28 | 2009-01-14 | 河南省农业科学院 | Test paper strip for detecting Porcine Parvovirus and porcine encephalitis B virus antibody |
| WO2012129444A2 (en) * | 2011-03-23 | 2012-09-27 | Anp Technologies, Inc. | Rapid tests for the detection of inhibitors of enzymes and human exposure to the same |
| CN202512121U (en) * | 2012-02-27 | 2012-10-31 | 上海凯创生物技术有限公司 | Micro-albuminuria (MAU) colloidal gold quantitative detection kit |
-
2013
- 2013-03-15 CN CN201310083890.2A patent/CN103941015A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050130247A1 (en) * | 2001-11-14 | 2005-06-16 | Kiamars Hajizadeh | Rapid prion-detection device, system and test kit |
| CN1645148A (en) * | 2005-01-24 | 2005-07-27 | 江苏省农业科学院 | Reagent box for inspecting infection of swine pleuropneumonia actinomyces |
| CN101113980A (en) * | 2007-06-28 | 2008-01-30 | 华中农业大学 | Colloidal gold chemiluminescence immune analysis method for detecting pig pleuropneumonia antibody |
| CN101344524A (en) * | 2008-08-28 | 2009-01-14 | 河南省农业科学院 | Test paper strip for detecting Porcine Parvovirus and porcine encephalitis B virus antibody |
| WO2012129444A2 (en) * | 2011-03-23 | 2012-09-27 | Anp Technologies, Inc. | Rapid tests for the detection of inhibitors of enzymes and human exposure to the same |
| CN202512121U (en) * | 2012-02-27 | 2012-10-31 | 上海凯创生物技术有限公司 | Micro-albuminuria (MAU) colloidal gold quantitative detection kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104007261B (en) | Fowl three kinds of breathing problem three quick detection kit and application | |
| CN104459144B (en) | A kind of PRV velogen strain and vaccine strain differentiate Test paper | |
| CN104407137A (en) | Test paper for identifying and detecting virulent strain and low virulent strain of hog cholera virus | |
| CN106066398B (en) | A kind of indirect ELISA detection method of A types clostridium perfringens toxoid antibody | |
| CN101519447A (en) | Anti-rabbit hemorrhagic disease virus VP60 albumen monoclonal antibody | |
| CN105319359A (en) | Streptococcus pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof | |
| CN105158463A (en) | Colloidal gold test paper bar for antibody detection of sheep Brucella | |
| CN104991069B (en) | Acidovorax avenae subsp immune colloidal gold detection test paper strip and monoclonal antibody thereof and hybridoma cell strain | |
| CN104459142A (en) | Test paper for distinguishing virulent strain from attenuated strain of newcastle disease virus | |
| CN102477097A (en) | Preparation method of monoclonal antibody to chloramphenicol | |
| CN101424689B (en) | Bluetongue virus detection test paper | |
| CN103941013B (en) | Actinobacillus pleuropneumoniae endotoxin Rapid detection test strip | |
| CN101000347B (en) | Immunological chromatographic test paper for testing francisella tularensis and its preparation method | |
| CN105777900A (en) | Fruit-blotch monoclonal antibody and hybridoma cell strain | |
| CN102033128A (en) | Edwardsiella tarda rapid detection test paper as well as rapid detection method and application | |
| CN103941015A (en) | Rapid detection test paper strip for porcine actinobacillus pleuropneumoniae endotoxin antibody | |
| CN101550186A (en) | Sheep recombination prion protein and monoclonal antibody thereof and application | |
| CN104777302A (en) | Immunochromatographic assay test strip for bacterial fruit blotch of melons | |
| CN103235129B (en) | Marek's disease virus and J subgroup avian leucosis virus fast joint inspection test strips | |
| CN103941014B (en) | Swine enzootic pneumonia, porcine contagious pleuropneumonia and swine plague three test strip | |
| CN204495834U (en) | Detect the test strips of chicken trachitis virus | |
| CN103217527B (en) | Marek's disease virus and avian reticuloendotheliosis virus fast joint inspection test strips | |
| CN103235128B (en) | Avian reticuloendotheliosis virus and J subgroup avian leucosis virus fast joint inspection test strips | |
| CN102146138B (en) | Monoclonal antibody of chloramphenicol and application thereof | |
| CN101676724A (en) | Immunity-chropadography test paper for detecting Vibrio Cholerae and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140723 |