CN102062776A - Babesia caballi disease immunoblotting detection method and preparation of kit - Google Patents
Babesia caballi disease immunoblotting detection method and preparation of kit Download PDFInfo
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Abstract
The invention discloses a babesia caballi disease immunoblotting detection method and a kit. The babesia caballi disease immunoblotting detection method is established on the basis of obtaining a diagnostic antigen and positive control serum through a goat anti horse second antibody compound marked by horse radish peroxidase and a chemiluminescence substrate. The method has the advantages of less reagent dosage, low cost, high specificity, high sensitivity, quicker operation, no toxicity and harm, low required condition, easy judgment of a test result, long-term storage and stronger specificity than ELISA (Enzyme-Linked Immuno Sorbent Assay). By detecting by using the method, false positive and false negative results are not detected.
Description
Technical field
The present invention relates to this parasitosis immune blotting detection method of a kind of dull BABEI and kit preparation method, be particularly related to a kind of use western blot test and detect this parasitosis of dull BABEI (Babesia caballi) method of serum antibody and the preparation method of diagnostic kit thereof, belong to equus blood protozoosis detection range.
Background technology
An inferior horse this parasitosis of BABEI (Babesia caballi) is to parasitize caused blood protozoosis in the red blood cell of equus by this worm of dull BABEI, and clinical manifestation be symptoms such as high heat, anaemia, jaundice, oedema.The morbus acutissimus example is more rare, and sick horse is dead or dying during to discovery.This disease is widely distributed in the world, and in Europe, France, Italy and Russian south and most area of east until north latitude 58 degree areas, all have the popular report of this parasitosis of dull BABEI.France's southern dull BABEI this parasitosis serosurvey prevalence rate is 11.3%, in South America, all there is the report of dull this parasitosis of BABEI in Latin America, Africa, Asia.Brazil this worm of dull BABEI is popular in 59.4% to 65.5%, and also there is the popular report of this parasitosis of dull BABEI the Trinidad.Nicaragua's this worm positive rate of an inferior horse BABEI is 41%, and South Africa this parasitosis serosurvey positive rate of an inferior horse BABEI is up to 61.9%, and Mongolian this parasitosis of dull BABEI is widely distributed, and the positive animal of serosurvey reaches 40.1%.This parasitosis of China an inferior horse BABEI mainly is popular in ground such as northeast, east Inner Mongolia and Qinghai, has 14 provinces and regions all once to have this disease to distribute or endemoepidemic report in China.Gansu, Heilungkiang, Jilin, Xinjiang also are the popular areas of this parasitosis of dull BABEI.
After this parasitosis of the dull BABEI of the anti-mistake of horses, premunition is sustainable to reach 4 years.The horses in epidemic-stricken area are owing to often suffer biting of tick, and therefore repeated infection is not generally fallen ill or only showed light symptoms and anti-mistake, but enter the Singapore and Malaysia in epidemic-stricken area and newborn young coltfoal owing to do not have this immunity, morbidity easily by the other places.Do not having epidemic situation but the area of tick class activity is being arranged, external horses are being wanted strict carry out the quarantine of dull this parasitosis of BABEI, preventing to be with the worm horse to enter.
Because this disease is worldwide widely distributed, be serious transmissible disease, be difficult to eradicate, therefore various countries all classify this disease one of as horse disease of important monitoring, therefore sick to international trade generation material impact, OIE (OIE) classifies this disease as international trade is produced material impact disease, and China Ministry of Agriculture classifies them as two class zoonosis.In international trade, in the bilateral quarantine agreement of most countries signature, classify this disease as method inspection project.
Owing to behind this disease infection animal, often do not show as tangible clinical symptoms, therefore be difficult to diagnose from symptom.This type of disease mainly is that the chamber method of inspection is made a definite diagnosis by experiment.Cause of disease is identified mainly by blood smear and PCR, the laboratory inspection method often adopts the serology detection method, mainly contain IFA, complement fixation test (CFT), enzyme linked immunosorbent assay (OIE (OIE). terrestrial animal diagnosis and vaccine handbook [M], 2008:1-2.).The blood smear method has only animal to be in acute infection period to be only feasible diagnostic method, though PCR has set up at present but still needed development and perfect, and PCR is because of being limited by the extraction of cause of disease nucleic acid, and the susceptibility of its test and specificity are still waiting raising.IFA susceptibility is not strong, and is subjected to operator's subjective factor to influence the result easily to judge.Complement fixation test (CFT) now is the method for inspection that many countries are recommended, but this method can be subjected to the influence of anticomplement factor and experimental result that can not judgement sample, though enzyme linked immunosorbent assay is easy and simple to handle, susceptibility is stronger, but cross reaction easily takes place, false positive results occurs.Inquiring into the new detection method of research is the only way of scientific technological advance, also is the requirement of disease detection technical development.
Western blot is according to the specificity of the antigen-antibody method in conjunction with certain albumen in the detection of complex sample.This method is a kind of new immune biochemical technology that grows up on gel electrophoresis and solid-phase immunoassay technical foundation.Because Western blotting has the high specific and the susceptibility of high resolution and the solid-phase immunoassay of SDS-PAGE, has now become a kind of routine techniques of analysis of protein, but has been used for identifying certain albumen more, the research of using it for the eqpidemic disease context of detection is also few.
The domestic research that detects animal epidemic with Western blot seldom, not about the research report of this parasitosis immune blotting detection method of dull BABEI.
Summary of the invention
The inventor has carried out a large amount of experimental works, has set up the preparation method of this parasitosis Western blotting diagnostic kit of a kind of dull BABEI finally.The present invention gathers healthy this worm of naggy red blood cell in vitro culture an inferior horse BABEI, through enlarged culture, the culture that obtains is made the Western blotting diagnostic antigen, and obtain this worm antibody positive control serum of dull BABEI through the laboratory infection horses.Simultaneously, on the basis that obtains diagnostic antigen and positive control serum, set up this parasitosis immune blotting detection method of dull BABEI.
The object of the invention provides this parasitosis Western blotting diagnostic kit of a kind of dull BABEI and preparation method thereof, and the object of the invention is achieved through the following technical solutions:
(1) the preparation polypide is cultivated and uses red blood cell:
The healthy horse blood of aseptic collection uses the washing of VYM ' s damping fluid, suspension to obtain after the centrifugal treating;
Centrifugal condition: 4 ℃-8 ℃, low speed, 30 ± 5 min;
(2) set up and enlarge this polypide of dull BABEI and cultivate outward
Aseptic collection infects the anticoagulation of this parasitosis horse of dull BABEI, through centrifugal treating, use 3x VYM ' s damping fluid washing, suspend obtain to dye the dried female insect cell after, adopt HL-1 tissue culture medium to cultivate, when dye divide when the worm rate reaches 1.5%-2% be commissioned to train foster; Reach 2% or enlarged culture when above whenever dying the worm rate afterwards;
Culture environment is: 93%N
2, 2.0%O
2, 5%CO
2A cultivation cycle is 24 hours;
(3) this parasitosis western blot hybridization diagnostic antigen of the dull BABEI of preparation
When the worm rate of dying of step (2) culture surpasses 3%, collect culture,, be dissolved to limpid with PI buffer+1%NP40 at last through centrifugal, washing, add LDSsample buffer(4x again), ample Reducing Agent(10x), abundant mixing.Boil, cooling rapidly gets this parasitosis Western blotting diagnostic antigen of dull BABEI.
(4) the preparation positive control serum is logical:
With 25mL this dried female insect cell culture of dull BABEI with equivalent 10% conventional allos horse serum phosphate buffer mixing, in the naggy body that intravenous injection to spleen does not excise, the tick larva of not feeding with about 10000 ticks (about 0.5 gram) after 3 days is contained in the cloth bag, is placed on the naggy.Take the tick that becomes full pupa after 13 days away, tick was supported 6 days, tick was put into experiment with small-sized horses last 8 day in the 6th day, gathered serum after fortnight, and complement fixation test (CFT) can detect this worm antibody of dull BABEI, promptly obtains positive control serum.
Get this parasitosis Western blotting diagnostic antigen of (1) dull BABEI, (2) the goat-anti horse bond of horseradish peroxidase (HRP) mark, (3) positive control serum, negative control sera, (4) substrate (peroxide solution+Luminol enhancer solution), (5) molecular weight of albumen standard label (Marker), (6) 4-12 % Bis-Tris glue, (7) nitrocellulose membrane, (8) MOPS SDS Running Buffer (20x), (9) NuPAGE Antioxidant, (10) transfer printing damping fluid, (11) phosphate buffer (pH7.3), (12) confining liquid, (13) washing lotion, (14) sensitive film, (15) operation instructions are put in the papery box, obtain this parasitosis Western blotting detection kit of dull BABEI.
The advantage and the characteristics of the technical program are:
Antigen preparation only need surpass 3 % with the worm rate of dying of polypide and promptly can be used for the Western blotting diagnosis and use antigen, and the antigen preparation cycle is shorter, has shortened the cycle of production kit greatly.
Another object of the present invention provides this parasitosis immune blotting detection method of a kind of dull BABEI, and this method is that goat-anti horse two resistive connection compounds, the chemical luminous substrate with horseradish peroxidase-labeled set up on the basis that obtains diagnostic antigen and positive control serum.
The object of the invention is achieved through the following technical solutions:
(1) gets 4-12 % Bis-Tris offset plates, washes clean;
(2) be solvent with SDS Running Buffer and NuPAGE Antioxidant, adopt electrophoresis to separate antigen protein: the offset plate that obtains containing antigen protein;
(3) the transfer printing damping fluid with refrigeration is transferred to tunica fibrosa with antigen protein;
(4) serum and antigen are hatched:
There is the film of antigen protein to place confining liquid on shaking table, to hatch transfer printing; Afterwards film is taken out and place again with the blood serum sample of confining liquid by 1:100～1:500 dilution, vibration sense work;
(5) enzyme conjugates is hatched:
With the goat-anti horse two resistive connection compounds of film transposition in the horseradish peroxidase-labeled of pressing 1:5000～1:10000 dilution with confining liquid, the vibration sense is done;
(6) substrate is hatched: in substrate, the lucifuge sense is done with the film dislocation; Must contain substrate and hatch the film of thing;
(7) exposure, develop:
Blot the water on the film, film is wrapped, put in the dark folder, under the red light, get sensitive film and be placed on the film, buckle dark folder, exposure with preservative film; Under red light, take out film, put into developing machine and develop; Sensitive film must develop;
(8) result judges:
When positive serum a band occurs to impinging upon between 25kDa and 37kDa, the position is when the 33kDa place, and positive control is set up; When the no band of negative serum contrast occurred, negative control was set up; At this moment can carry out the judgement of test sample:, when the band position is identical with positive control, be judged to be positive when a band between 25kDa and 37kDa, occurring; When no band occurs between 25kDa and 37kDa, be judged to be negative sample.
Described substrate is peroxide solution+Luminol enhancer solution.
In order to obtain better effect, the preferred 1:1000 of the working concentration of antigen protein.
The advantage and the characteristics of the technical program are:
1, reagent dosage is few, cost is low
Two used anti-working concentrations of the present invention are 1:5000～1:10000, and two consumptions that resist are few, and cost has been saved in the consumption of having saved reagent.
2, operation is faster
The inventive method sensitivity, fast, and nontoxic, specificity is high.
3, low, the test findings of the required condition of test is easily judged and long preservation.
The inventive method required time is shorter, and whole test is finished in 4 h.Whole test can be finished at normal temperatures.And be that the reaction band is exposed on the film, but the result easily judge and long preservation, do not have the problem of fading.
4, the inventive method not only can detect this parasitosis serum antibody of dull BABEI, more can the result of other serological methods such as IFA, CFT, ELISA test positive be proved conclusively, and specificity is stronger than ELISA.
5, detect herds of horses serum with detection method of the present invention, testing result is not all found false positive and false-negative result.
Description of drawings
Figure: the feminine gender and the positive control of this parasitosis immune blotting detection method of dull BABEI
M: protein molecular weight standard; 1.1: this worm positive control serum of dull BABEI;
1.2: this parasitosis positive control serum of dull BABEI; 2.1: this parasitosis negative control sera of dull BABEI;
2.2: this parasitosis negative control sera of dull BABEI.
Embodiment
In order to understand the present invention, further specify the present invention with embodiment below, but do not limit the present invention.
This parasitosis Western blotting diagnostic kit of the dull BABEI of embodiment 1 preparation
Reagent preparation
1,1x VYM ' s damping fluid:
CaCl
2.2H
2O 0.016 g
KCl 0.400 g
KH
2PO
4 1.415 g
MgSO
4.7H
2O 0.154 g
Na
2HPO
4 0.077 g
NaCl 7.077 g
Glucose 20.500 g
ddH
2O 1 L
After the stirring and dissolving, add Adenine 0.0423 g, Guanosine 0.0708 g, adjust pH is to 7.0-7.2, vacuum filtration (0.22 μ m), and 4 ℃ of preservations are standby.
2, HL-1 tissue culture medium:
HL-1 nutrient culture media 400 mL
Horse serum 100 mL
Hepes 0.238 g
2 mmol L-glutamine 500μL
Antibiotic-antimycotic(100x) 1 mL
Magnetic agitation 5 min transfer pH to 7.2, vacuum filtration (0.22 μ m), and 4 ℃ of preservations are standby
3、 PI buffer：
Tris 3.03 g
Deionized water 500mL
Magnetic stirrer 1h transfers pH 8.0, adds
EDTA（SIGMA） 0.93g
IODAOACETAMIDE(C
2H
4INO)(SIGMA) 0.46g
Magnetic agitation is spent the night, and adds
TLCK（C
14H
22C
l2N
2O
3S） 18.45 mg
Preparation diagnosis antigen
(1) the preparation polypide is cultivated and uses red blood cell:
The healthy horse blood of aseptic collection 600 mL is to the sterilized vacuum triangular flask that beaded glass is housed, and the rotary triangle bottle is until white coagulum occurring.In super-clean bench, horse blood is poured in the centrifuge tube, 4 ℃ of 800 centrifugal 30 min of xg inhales and abandons plasma layer and leukocytic cream.It is inferior to use VYM ' s damping fluid to give a baby a bath on the third day after its birth, and as far as possible plasma layer and leukocytic cream suction is abandoned at every turn.VYM ' the s damping fluid that adds 2 times of volumes afterwards is with pipettor Red Blood Cells Suspension mud gently, 4 ℃ of 800 centrifugal 30 min of xg.Suction abandon add 2 times of volumes again behind plasma layer and the leukocytic cream VYM ' s damping fluid to red blood cell mud, suspend gently with pipettor, polypide is cultivated and to use red blood cell, put in the polypropylene tube, 4 ℃ of preservations, standby.
(2) setting up this polypide of dull BABEI cultivates outward:
Gather the anticoagulation that infects this parasitosis horse of dull BABEI, 4 ℃ of 800 centrifugal 30 min of xg abandons plasma layer and leukocytic cream with the pipettor suction.It is inferior to use 3x VYM ' s damping fluid to give a baby a bath on the third day after its birth, and inhales as far as possible at every turn and abandons supernatant and leukocytic cream, and 4 ℃ of 800 centrifugal 30 min of xg must dye the dried female insect cell afterwards.
1 mLHL-1 tissue culture medium is added in the 24 porocyte tissue culturing plates, draws 50 μ L and dye the dried female insect cell to cell hole, draw the gained polypide again and cultivate with red blood cell 150 μ L.The cell tissue culture plate put in 37 ℃ of incubators cultivate, culture environment is: 93%N
2, 2.0%O
2, 5%CO
2
Change nutrient solution every day 1 time, method is: do not stir the cellular layer suction and abandon about 1mL nutrient solution, add the new HL-1 nutrient solution of 1mL again.Plate is put continuation cultivation in the aforementioned incubator again.
When dye the worm rate when reaching 1.5%-2%, divide be commissioned to train foster.Method is: plate is taken out, inhale and abandon the 1mL nutrient solution, add the new HL-1 nutrient solution of 1mL again, with pipettor suspension culture gently, win for culture.In new cell hole, add 600 μ L HL-1 nutrient solutions, and get 600 μ L first generation cultures to new cell hole, gently mixing.Other gets 600 μ L HL-1 nutrient solutions and is added in the old cell hole.In new, old cell hole, respectively add 100 μ L polypides cultivation red blood cell.Plate is put aforementioned incubator to be continued to be cultured to and dyes the worm rate and surpass 2%.
(3) this polypide of dull BABEI enlarges cultivation outward:
At first polypide is cultivated and be extended to 4 cell holes.Method is: in the histocyte culture plate, add 600 μ L HL-1 tissue culture mediums in new cell hole, get 100 μ L polypides and cultivate with red blood cell to new cell hole; From old cell hole, inhale and abandon the old nutrient solution of about 1.2 mL, add the new nutrient solution of about 1.2 mL to old cell hole and draw 600 μ L cultures to new cell hole.Put in the aforementioned incubator and cultivated 24 hours.The preparation blood film dyes the worm rate and detects.
When dye the worm rate when surpassing 2% in triangular flask enlarged culture, method is: the triangular flask A that gets sterilization, after wherein adding 10 mL HL-1 tissue culture mediums, the culture of 1 mL polypide cultivation, put in the aforementioned incubator and cultivated 24 hours with red blood cell and 3 cell holes; Afterwards, triangular flask A is taken out, therefrom inhale and abandon the old nutrient solution of 7 mL-10 mL, supply with new HL-1 tissue culture medium and inhale the amount of abandoning, continue to put in the aforementioned incubator and cultivated 24 hours.
When dying the worm rate when surpassing 2%, enlarged culture once more, method is: other gets 2 triangular flasks, cultivates and uses red blood cell to wherein respectively adding new nutrient solution of 6 mL and 1 mL polypide; Suction is abandoned the nutrient solution among the triangular flask A and is added the new nutrient solution of equivalent, behind the mixing, draws respectively in 7 mL to 2 the new triangular flasks gently, they is put in the aforementioned incubator cultivated 24 hours.According to this method, when dying worm rate enlarged culture when surpassing 2%, from 2 triangular flasks to 4 again to 8 triangular flasks.
When the worm rate of dying in 8 triangular flasks surpasses 3%, dye dried female insect cell enlarged culture and finish.
Collect wherein in culture to the 2 50 mL centrifuge tube in 7 triangular flasks 4 ℃ of centrifugal 30 min of 1500 rpm; Supernatant is abandoned in suction, obtains to infect the red blood cell that dull this worm of BABEI is arranged.Put in the 1.5mL centrifuge tube 4 ℃ of centrifugal 2 min of 3000 rpm again; Supernatant is abandoned in suction, gets 750 μ L cell mud to the new centrifuge tube, adds 750 μ L distilled waters, the vortex mixing gently that vibrates, centrifugal 3 min of desk centrifuge 14000 rpm.Supernatant is abandoned in suction, adds 1.0 mL phosphate buffers (pH 7.3), uses pipettor pressure-vaccum mixing, centrifugal 3 min of 14000 rpm more gently; Supernatant is abandoned in suction, adds 1.0 mL distilled waters, still uses pipettor pressure-vaccum mixing gently, again with centrifugal 5 min of 14000 rpm; Supernatant is abandoned in suction.Add phosphate buffer, mixing, centrifugal 10 min of 14000 rpm repeat 1 time.Supernatant is abandoned in suction, adds 200 μ L PI buffer+1%NP40, is dissolved to limpid again.
Get in L to 1 new centrifuge tube of dissolved matter 200 μ, add 50 μ L LDS sample buffer(4x), 25 μ Lsample Reducing Agent(10x)
,Abundant mixing.Boiled 10 minutes, and put into ice chest rapidly and cool off, promptly get to diagnose and use antigen ,-20 ℃ of preservations, standby.
Preparation diagnosis positive control serum
Obtain positive control serum through band worm tick Boophilus microplus laboratory infection naggy:
With 25mL this dried female insect cell culture of dull BABEI with equivalent 10% conventional allos horse serum phosphate buffer mixing, in the naggy body that intravenous injection to spleen does not excise, the tick larva of not feeding with about 10000 ticks (about 0.5 gram) after 3 days is contained in the cloth bag, is placed on the naggy.Take the tick that becomes full pupa after 13 days away, tick was supported 6 days, tick was put into experiment with small-sized horses last 8 day in the 6th day, gathered serum after fortnight, when complement fixation test (CFT) detects this worm antibody of dull BABEI, promptly obtain this parasitosis western blot hybridization diagnosis positive control serum of dull BABEI;-20 ℃ of preservations, standby.
Get the goat-anti horse bond of (1) encapsulation diagnosis with antigen, (2) horseradish peroxidase (HRP) mark; (3) encapsulation positive control serum, negative control sera, (4) substrate (peroxide solution+Luminol enhancer solution), (5) molecular weight of albumen standard label (Marker), (6) 4-12 % Bis-Tris glue, (7) nitrocellulose membrane, (8) MOPS SDS Running Buffer (20x), (9) NuPAGE Antioxidant, (10) transfer printing damping fluid, (11) phosphate buffer (pH7.3), (12) confining liquid, (13) washing lotion, (14) sensitive film, (15) operation instructions are put in the papery box, stored refrigerated.
Embodiment 2: this parasitosis Western blotting of dull BABEI detects
The reagent preparation:
1,10x electrophoretic buffer:
Tris 121.1 g
Glycocoll 570 g
Distilled water 4 L
2, transfer printing damping fluid:
10x electrophoretic buffer 100 mL
Methyl alcohol 200 mL
Distilled water 700 mL
3,Confining liquid
:The phosphate buffer that contains 0.2 % Tween-20,10 % skimmed milk powers
4, washing lotion
:The phosphate buffer that contains 0.2 % Tween-20
Get NuPAGE 4%～12% Bis-Tris offset plate, wash the glue hole three times with 1 x SDS Running Buffer; Offset plate just forward-facing is placed in the Mini-Cell vertical electrophoresis instrument core frame locking.In electrophoresis tank, add 200 mL, 1 x SDS Running Buffer and 500 μ L NuPAGE Antioxidant, mix.Add 600 mL, 1 x SDS Running Buffer in the following groove.Add 7 μ L～10 μ L molecular weight of albumen standard labels (Marker) and embodiment 1 gained antigen samples in the glue hole, the working concentration of antigen protein is 1:1000, and 200V 350mA ran glue 1 hour, obtains containing the offset plate of antigen protein.
Afterwards antigen protein is transferred to nitrocellulose membrane:
Earlier glue, nitrocellulose membrane, transfer printing folder, filter paper, fiber mat are soaked with the transfer printing damping fluid, on the black flour of transfer printing folder, put fiber mat, filter paper, blob of viscose, tunica fibrosa, filter paper, fiber mat successively then, press bubble, fastening transfer printing folder.Transfer printing is installed in the transfer groove, in groove, pours into and be prepended to the transfer printing damping fluid that refrigerator and cooled is hidden earlier, groove is put on the magnetic stirring apparatus 150 V, 350 mA transfer printings 1 hour.
The film that transfer printing is had an antigen protein phosphate buffer (PBS, pH7.3) in flushing, be placed on then in the container that contains confining liquid, put and hatch 1 hour on the shaking table.The film taking-up is cut into some, and face down is placed on to be equipped with confining liquid and dilutes by 1:500 in the container of good blood serum sample, and the vibration sense is done 30 minutes on oscillator.Finish the back and wash 3 times each 5 minutes with washing lotion.
Outwell washing lotion, add then with in the goat-anti horse two resistive connection compounds of confining liquid by the horseradish peroxidase-labeled of 1:10000 dilution, the vibration sense is done 30 minutes.Wash 3 times each 5 minutes with washing lotion.
Outwell washing lotion, add peroxide solution+Luminol enhancer solution substrate, the lucifuge sense is done 5 minutes.
Blot water on the film with thieving paper, with preservative film film is wrapped, film is faced up to set to be placed in the mid-darkroom of dark folder, under the red light, gets 1 sensitive film, is placed on the film, buckles dark folder, exposure 30s-60 second.
Under red light, take out film, put into developing machine and develop.Sensitive film must develop.
Application examples
Decision method as a result
Get embodiment 2 gained development sensitive films, show that positive serum a band occurs to impinging upon between 25kDa and 37kDa, the position is about the 33kDa place, and is identical with positive control, is judged to be positive.
Claims (6)
1. this parasitosis Western blotting diagnostic kit of dull BABEI is characterized in that comprising:
(1) this parasitosis Western blotting diagnostic antigen of dull BABEI
(2) the goat-anti horse bond of horseradish peroxidase (HRP) mark
(3) positive control serum, negative control sera
(4) substrate
(5) molecular weight of albumen standard label
(6) 4-12 % Bis-Tris glue
(7) nitrocellulose membrane
（8）MOPS SDS Running Buffer
（9）NuPAGE Antioxidant
(10) transfer printing damping fluid
(11) phosphate buffer
(12) confining liquid
(13) washing lotion
(14) sensitive film
(15) operation instructions
Described substrate is peroxide solution+Luminol enhancer solution)
According to this parasitosis Western blotting diagnostic kit of the described a kind of dull BABEI of claim 1, it is characterized in that this parasitosis Western blotting diagnostic antigen of dull BABEI in the kit prepares by following method:
(1) the preparation polypide is cultivated and uses red blood cell:
The healthy horse blood of aseptic collection uses the washing of VYM ' s damping fluid, suspension to obtain after the centrifugal treating;
Centrifugal condition: 4 ℃-8 ℃, low speed, 30 ± 5 min;
(2) set up and enlarge this polypide of dull BABEI and cultivate outward
Aseptic collection infects the anticoagulation of this parasitosis horse of dull BABEI, through centrifugal treating, use 3x VYM ' s damping fluid washing, suspend obtain to dye the dried female insect cell after, adopt HL-1 tissue culture medium to cultivate, when dye divide when the worm rate reaches 1.5%-2% be commissioned to train foster; Reach 2% or enlarged culture when above whenever dying the worm rate afterwards;
Culture environment is: 93%N
2, 2.0%O
2, 5%CO
2A cultivation cycle is 24 hours;
(3) this parasitosis western blot hybridization diagnostic antigen of the dull BABEI of preparation
When the worm rate of dying of step (2) culture surpasses 3%, collect culture,, be dissolved to limpid with PI buffer+1%NP40 at last through centrifugal, washing, add LDSsample buffer(4x again), ample Reducing Agent(10x), abundant mixing; Boil, cooling promptly gets this parasitosis Western blotting diagnostic antigen of dull BABEI rapidly.
2. according to this parasitosis Western blotting diagnostic kit of the described a kind of dull BABEI of claim 1, it is characterized in that substrate is peroxide solution+Luminol enhancer solution.
3. according to this parasitosis Western blotting diagnostic kit of the described a kind of dull BABEI of claim 1, it is characterized in that the positive control serum in the kit prepares by following method:
With 25mL this dried female insect cell culture of dull BABEI with equivalent 10% conventional allos horse serum phosphate buffer mixing, in the naggy body that intravenous injection to spleen does not excise, the tick larva of not feeding with about 10000 ticks (about 0.5 gram) after 3 days is contained in the cloth bag, is placed on the naggy.
4.13 take the tick that has become full pupa after it away, tick was supported 6 days, tick was put into experiment with small-sized horses last 8 day in the 6th day, gathered serum after fortnight, complement fixation test (CFT) can detect this worm antibody of dull BABEI, promptly obtains positive control serum.
5. this parasitosis serum antibody immune blotting detection method of dull BABEI is the serum antibody immune blotting detection method that is based upon on claim 1 basis, it is characterized in that comprising step:
(1) gets 4～12 % Bis-Tris offset plates, washes clean;
(2) be solvent with SDS Running Buffer and NuPAGE Antioxidant, adopt electrophoresis to separate antigen protein: the offset plate that obtains containing antigen protein;
(3) the transfer printing damping fluid with refrigeration is transferred to tunica fibrosa with antigen protein;
(4) serum and antigen are hatched:
There is the film of antigen protein to place confining liquid on shaking table, to hatch transfer printing; Afterwards film is taken out and place again with the blood serum sample of confining liquid by 1:100～1:500 dilution, vibration sense work;
(5) enzyme conjugates is hatched:
With the goat-anti horse two resistive connection compounds of film transposition in the horseradish peroxidase-labeled of pressing 1:5000～1:10000 dilution with confining liquid, the vibration sense is done;
(6) substrate is hatched: in substrate, the lucifuge sense is done with the film dislocation; Must contain substrate and hatch the film of thing;
(7) exposure, develop:
Blot the water on the film, film is wrapped, put in the dark folder, under the red light, get sensitive film and be placed on the film, buckle dark folder, exposure with preservative film; Under red light, take out film, put into developing machine and develop; Sensitive film must develop;
(8) result judges:
When positive serum a band occurs to impinging upon between 25kDa and 37kDa, the position is when the 33kDa place, and positive control is set up; When the no band of negative serum contrast occurred, negative control was set up; At this moment can carry out the judgement of test sample:, when the band position is identical with positive control, be judged to be positive when a band between 25kDa and 37kDa, occurring; When no band occurs between 25kDa and 37kDa, be judged to be negative sample.
6. according to this parasitosis serum antibody immune blotting detection method of the described a kind of dull BABEI of claim 5, the working concentration that it is characterized in that antigen protein is 1:1000
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010105913319A CN102062776A (en) | 2010-12-16 | 2010-12-16 | Babesia caballi disease immunoblotting detection method and preparation of kit |
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| CN2010105913319A CN102062776A (en) | 2010-12-16 | 2010-12-16 | Babesia caballi disease immunoblotting detection method and preparation of kit |
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| CN105067817A (en) * | 2015-07-08 | 2015-11-18 | 上海清流生物医药科技有限公司 | Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips |
| CN105866220A (en) * | 2016-04-18 | 2016-08-17 | 苏州新赛美生物科技有限公司 | Efficient protein-to-membrane transferring buffer solution and preparation method thereof |
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| 《江西医学院学报》 20061231 王朝东等 Abeta-结合性酒精脱氢酶蛋白在缺氧性细胞中的表达及其对细胞缺氧性损害的保护作用 全文 1-6 第46卷, 第6期 * |
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Cited By (3)
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| CN105067817A (en) * | 2015-07-08 | 2015-11-18 | 上海清流生物医药科技有限公司 | Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips |
| CN105067817B (en) * | 2015-07-08 | 2017-05-10 | 上海清流生物医药科技有限公司 | Methods and devices for acquiring signals and tracking cells by adopting light sensitive chips |
| CN105866220A (en) * | 2016-04-18 | 2016-08-17 | 苏州新赛美生物科技有限公司 | Efficient protein-to-membrane transferring buffer solution and preparation method thereof |
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Application publication date: 20110518 |