CN104849470A - Enterohemorrhagic E.coli O157:H7 latex agglutination detection kit and application thereof - Google Patents
Enterohemorrhagic E.coli O157:H7 latex agglutination detection kit and application thereof Download PDFInfo
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Abstract
The invention relates to an enterohemorrhagic E.coli O157:H7 latex agglutination detection kit and an application of the detection kit in detection of enterohemorrhagic E.coli O157:H7. The kit comprises: a) a latex detection reagent; b) a sample treating fluid; c) a positive control sample; and d) a negative control sample. The latex detection reagent is a latex reagent which can be subjected to a specific reaction with enterohemorrhagic E.coli O157:H7, and is obtained from Intimin monoclonal antibody sensitized carboxylated polystyrene latex. The kit provided by the invention has the following advantages: 1, the kit is simple to operate, is convenient and fast, has low detection cost and is suitable for rapid on-site detection; 2, coupling rate of antibody and sensitivity and stability of the latex detection reagent are improved, and requirements of product research and mass production are met; and 3, the kit can be used to directly detect enterohemorrhagic E.coli O157:H7 in a sample and has characteristics of strong specificity, high sensitivity and short detection time.
Description
Technical field
The present invention relates to a kind of enterorrhagia Bacillus coil 0157: H7 latex agglutination assay kit and at enterorrhagia Bacillus coil 0157: the application during H7 detects, belong to bird immunity and to learn a skill field.
Background technology
Enterorrhagia Bacillus coil 0157: H7 is a kind of important foodborne bacterial pathogens, is one of main inducing of food security and the Sudden sensorineural hearing loss that the world today is on the increase.This bacterium energy infected poultry and other animal, the food morbidity of people mainly through polluting.Nineteen eighty-two, first O157:H7 finds in the U.S., after this distributes all over the world or local popular, has become global public health and food-safety problem.Therefore development fast, responsive, special detection method and reagent guarantees to drink water and food security, prevention Escherichia coli O 157: H7 infect the important means occurred.At present, the detection method reported both at home and abroad has multiplex PCR, genetic chip, bacteriophage typing, pulsed-field gel electrophoresis analysis, biology sensor etc., these methods have his own strong points in context of detection, but large multipair instrument and equipment requires higher, and therefore usable range has significant limitation.Be separated culture identification and be still the goldstandard detecting O157:H7, but it needs a couple of days just can obtain result usually, can not meet the requirement of quick diagnosis.At Escherichia coli O 157: in the immunological detection method of H7, antibody used mostly is polyclonal antibody, the polyclonal antibody of anti-O157:H7 also exists serious cross reaction with the antibody of other enteric bacteria anti-, common purifying can not meet accurate serologic test demand, and with the antigen detection method set up based on monoclonal antibody and the domestic and international rare report of reagent.Latex agglutination test take polystyrene latex particulate as carrier, is adsorbed onto on carrier by antigen or antibody, in order to detect the method for corresponding antibody or antigen.The method is easy and simple to handle, convenient and swift, cheap, does not need specific apparatus, and naked eyes can judged result, is applicable to field quick detection, is widely used in clinical detection.Mode mainly physisorphtion and the chemical crosslink technique of conventional sensitization latex, physisorphtion stability and susceptibility lower, be difficult to the needs of applicable product development.
Summary of the invention
The object of the invention is to the defect overcoming prior art, set up one and detect enterorrhagia Bacillus coil 0157 fast: H7 latex agglutination assay kit, this kit selects anti-enterorrhagia Bacillus coil 0157: H7Intimin protein monoclonal antibody sensitization carboxylated polystyrene latex reagent, and it is a kind of for detecting enterorrhagia Bacillus coil 0157 for core assembles to detect reagent with this latex: the kit of H7, this kit can detect enterorrhagia Bacillus coil 0157 fast: H7, to the epidemiology survey of this disease and prevention and control significant.
The present invention solves the problems of the technologies described above the technical scheme taked to be: a kind of enterorrhagia Bacillus coil 0157: H7 latex agglutination assay kit, it is characterized in that this kit comprises: a) latex detects reagent, b) sample treatment liquid, c) positive control sample, d) negative control sample, it is energy and enterorrhagia Bacillus coil 0157 that described latex detects reagent: the latex reagent of H7 specific reaction, adopts anti-Intimin protein monoclonal antibody sensitization carboxylated polystyrene latex gained.
By such scheme, the preparation method of described anti-Intimin protein monoclonal antibody is: screen from 5 strain of hybridoma building strain 1 strain antibody tire height, high specificity hybridoma expand and cultivate, simultaneously in BALB/c mouse body, induce mouse ascites manufacture order clonal antibody; Get the BALB/c mouse 20 in 8 week age, only, the injection of 7d pneumoretroperitoneum expands the hybridoma 5 × 10 cultivated to the incomplete freund adjuvant 0.5mL/ of lumbar injection
6~ 5 × 10
7/ only, breeding observing, until mouse web portion projection and by only extracting mouse ascites when being slow in action, the centrifugal 8min of 2000r/min, discard fat and collect upper strata clarification ascites, measure with indirect ELISA method every the mouse ascites collected and tire, will tire and be not less than 1:(1000 × 2
11) ascites mixing, mixed ascites is finally tired after measured as 1:(1000 × 2
12), hypotype is accredited as lgG2b.
The concrete preparation method of described anti-Intimin protein monoclonal antibody sensitization carboxylated polystyrene latex: (1) is got the carboxylated polystyrene latex 200ul shaken up and put into EP pipe; (2) wash 3 times, 12000rpm, 15min with pH9.6,0.1M carbonate buffer solution, centrifugally abandon supernatant; (3) use pH4.5 again, the phosphate buffer of 0.01M washes 3 times, 12000rpm, 15min, centrifugal, abandons supernatant; (4) the NHS 1000uL then adding EDC 1000uL, 20mM of 100mM is resuspended, mixing, and make both final concentrations be respectively 50mol/L and 10mol/L, in shaking table, 26 DEG C slowly jolt 4.5h; (5) wash 3 times, 12000rpin, 20min with pH8.0,0.01M borate buffer solution, centrifugally abandon supernatant; (6) suspend with pH8.0,0.01M borate buffer solution 1000ul, suspension latex concentration is 2%, 4 DEG C and saves backup; (7) the anti-enterorrhagia Bacillus coil 0157 that 1:4 doubly dilutes dropwise slowly is added: H7Intimin protein monoclonal antibody 1000ul, the protein content 2.78mg/mL of described protein monoclonal antibody, it is 1 hour that loading time controls, in 26 DEG C of shaking tables, slowly jolt 5h; (8) add 0.1M monoethanolamine 100ul, in 26 DEG C of shaking tables, slowly jolt 15min cessation reaction, 12000rpm, 20min, centrifugally abandon supernatant; (9) precipitation pH8.0,0.01M borate buffer solution washes 3 times, 12000rpin, 20min, centrifugally abandons supernatant; (10) precipitation pH8.0,0.01M borate buffer solution 2000ul suspends, and is the good latex of sensitization and detects reagent, 4 DEG C of preservations.
By such scheme, the component of described sample treatment liquid and content by weight/volume is: take tryptone 20.0g, No. 3 cholate 1.12g, lactose 5.0g, ADKP 4.0g, anhydrous potassium dihydrogenphosphate 1.5g, NaCl 5.0g, is dissolved in 1000ml ddH by mentioned component
2o, adjust pH is 6.9 ± 1,121 DEG C of autoclaving 15min, and the ovobiocin solution 20mg/L taking out filtration sterilization adds, and makes ultimate density be 20ug/L.
By such scheme, described positive control sample is the enterorrhagia Bacillus coil 0157 of deactivation: H7 bacteria suspension, and described negative control sample is not containing the autoclaved TSB nutrient solution of Escherichia coli O 157: H7.
The present invention selects chemical crosslink technique, the carboxylated polystyrene latex with carboxylic group (-COOH) is utilized to be carrier, with bifunctional reagent water-soluble carbodiimide (EDAC) for medium, by chemical reaction, antibody molecule is coupled to latex surface, its stability and susceptibility improve a lot.Intimin albumen (namely closely plain) is Escherichia coli O 157: the outer membrane protein of the eae gene code on H7LEE pathogenicity island; its mediating bacterial and epithelial close adhesion; it is most important adhesion factor; specific antibody sticking of bacterium capable of blocking of tight element and make it lose pathogenicity, there is stronger immunogenicity and immune protective.For this reason, select anti-enterorrhagia Bacillus coil 0157: H7Intimin protein monoclonal antibody sensitization carboxylated polystyrene latex detects reagent as latex of the present invention, and simple and convenient assembly is kit fast.
Enterorrhagia Bacillus coil 0157 of the present invention: the application of H7 latex agglutination assay kit, is characterized in that comprising following concrete steps: the measuring samples 37 DEG C of cultivation 4-6 hour in sample treatment liquid 1) will collected; 2) on clean slide or glass plate, 10ul positive control sample, 10ul negative control sample and 10ul measuring samples is dripped respectively, and then heliotropism control sample, negative control sample, the latex dripped in measuring samples described in 10ul detect reagent, abundant mixing, slow shake, after 1 minute under blue background observations; 3) result judges: control test occurs following result in 1 minute, and test can be set up: positive control sample and latex detect reagent effect and occur +++ above latex agglutination, and negative control sample and latex detect reagent effect not aggegation; Measuring samples occurred in 1 minute ++ above latex agglutination is judged to the positive, presents original even emulsus and is judged to be feminine gender.
Detection kit of the present invention can directly to the enterorrhagia Bacillus coil 0157 in sample: H7 detects, if containing enterohemorrhagic enterorrhagia Bacillus coil 0157: H7 in sample, then latex detection reagent occurred in 1 minute ++ above latex agglutination, can be judged to positive findings; If not containing enterorrhagia Bacillus coil 0157: H7 in sample, then latex detection reagent drop in 1 minute still presents original even emulsus, can be judged to negative findings.
The present invention compared with prior art, has the following advantages:
1, detection kit of the present invention is without the need to specialized operations technical ability and any special instruments and equipment, simple, convenient quick, testing cost is low, is applicable to on-the-spot quick detection;
The shortcomings such as when 2, detection kit of the present invention overcomes latex sensitizing antibody, Conjugate ratio is low, antibody easily comes off, improve susceptibility and stability that the Conjugate ratio of antibody and latex detect reagent, are applicable to the requirement of research and development of products and large-scale production;
3, detection kit of the present invention can directly to enterorrhagia Bacillus coil 0157 in sample: H7 detects, and has high specificity, the feature that highly sensitive, detection time is short.
Accompanying drawing explanation
Fig. 1 is latex agglutination result process decision chart.
Embodiment
Below in conjunction with specific embodiment, illustrate the present invention further.Should be appreciated that these examples are only not used in restriction the scope of protection of present invention for illustration of the present invention.
The anti-enterorrhagia Bacillus coil 0157 of embodiment 1: the preparation of H7intimin protein monoclonal antibody sensitization latex reagent
The preparation method of anti-Intimin protein monoclonal antibody is: screen from 5 strain of hybridoma building strain 1 strain antibody tire height, high specificity hybridoma expand and cultivate, simultaneously in BALB/c mouse body, induce mouse ascites manufacture order clonal antibody; Get the BALB/c mouse 20 in 8 week age, only, the injection of 7d pneumoretroperitoneum expands the hybridoma 5 × 10 cultivated to the incomplete freund adjuvant 0.5mL/ of lumbar injection
6~ 5 × 10
7/ only, breeding observing, until mouse web portion projection and by only extracting mouse ascites when being slow in action, the centrifugal 8min of 2000r/min, discard fat and collect upper strata clarification ascites, measure with indirect ELISA method every the mouse ascites collected and tire, will tire and be not less than 1:(1000 × 2
11) ascites mixing, mixed ascites is finally tired after measured as 1:(1000 × 2
12), hypotype is accredited as lgG2b;
Anti-enterorrhagia Bacillus coil 0157: the preparation of H7intimin protein monoclonal antibody monoclonal antibody sensitization latex reagent: 1. get the carboxylated polystyrene latex 200ul shaken up and put into 4mlEP pipe; 2. use pH9.6,0.1M carbonate buffer solution washes 3 times, 12000rpm, 15min, centrifugally abandons supernatant; 3. use pH4.5 again, the phosphate buffer of 0.01M washes 3 times, 12000rpm, 15min, centrifugal, abandons supernatant; 4. the EDC then adding 100mM is resuspended, and the NHS1000uL of 20mM is resuspended, mixing, and EDC and NHS makes both final concentrations reach 50mol/L and 10mol/L respectively, and in shaking table, 26 DEG C slowly jolt 4.5h; 5. coupling terminate after sensitization latex pH8.0,0.01M borate buffer solution washes 3 times, 12000rpin, 20min, centrifugally abandons supernatant; 6. use pH8.0,0.01M borate buffer solution suspends, and suspension latex concentration is 2%, 4 DEG C and saves backup; 7. the anti-enterorrhagia Bacillus coil 0157 that 1:4 doubly dilutes dropwise slowly is added: H7intimin protein monoclonal antibody 1000ul, the protein content 2.78mg/mL of described protein monoclonal antibody, it is 1 hour that loading time controls, in 26 DEG C of shaking tables, slowly jolt 5h; 8. add terminator 0.1M monoethanolamine and in 26 DEG C of shaking tables, slowly jolt 15min cessation reaction (being add 50uL ethanolamine solutions in the 1mL antibody latex of 1% by concentration), 12000rpm, 20min; 9. use 5. same method process sensitization carboxylated latex, resuspended, 4 DEG C save backup.
Anti-enterorrhagia Bacillus coil 0157: concrete preparation method's condition of H7intimin protein monoclonal antibody sensitization latex reagent is groped as follows:
1, the preparation of 2% carboxylated latex: 1. get the carboxylated latex that 200uL shakes up and put into 4mlEP pipe; 2. use pH9.6,0.1M carbonate buffer solution washes 3 times, 12000rpm, 15min, centrifugally abandons supernatant; 3. use pH4.5 again, the phosphate buffer of 0.01M washes 3 times, 12000rpm, 15min, centrifugal, abandons supernatant; 4. the EDC1000uL then adding 100mM is resuspended, the NHS1000uL mixing of 20mM, and EDC and NHS makes both final concentrations reach 50mol/L and 10mol/L respectively, and in shaking table, 26 DEG C slowly jolt 4.5h; 5. coupling terminate after sensitization latex pH8.0,0.01M borate buffer solution washes 3 times, 12000rpin, 20min, centrifugally abandons supernatant; 6. use pH8.0,0.01M borate buffer solution 1000ul suspends, and suspension latex concentration is 2%, 4 DEG C and saves backup.
2, the selection of optimum monoclonal antibody addition during sensitization: get above-mentioned 2% carboxylated latex prepared of 100uL, add the dilution monoclonal antibody of difference (doubling dilution 1:1,1:2,1:4,1:8,1:16) of 100uLBBS dilution, slowly 5h is jolted in 26 DEG C of shaking tables, add 10uL0.1M monoethanolamine terminator 15min cessation reaction in 26 DEG C of shaking tables, 12000rpm, 20min, get supernatant to measure, precipitation pH8.0,0.01M borate buffer solution washes 3 times, then use 200ul pH8.0,0.01M borate buffer solution is resuspended.With the sensitization latex prepared, the dilution positive control sample of difference is detected, carry out the detection from coagulation phenomena simultaneously.The supernatant reclaimed measures protein content through UV-120 ultraviolet spectrophotometer.Adding protein content before sensitization is X1mg/mL, and after sensitization, supernatant protein content is X2mg/mL, calculates the protein content X=Xl-X2 (test findings is as table 1, table 2) of coupling.Test findings shows: during monoclonal antibody dilution 1:4 times, Conjugate ratio is the highest, and susceptibility is the highest, and the latex not self-solidifying after sensitization.
Form 1 adds the relation of protein content and coupling protein amount
Form 2 adds protein content and sensitivity relationship
3, the selection of best sensitization time: get the above-mentioned 2% carboxylated latex liquid prepared of 400ul, add the monoclonal antibody that 400ul 1:4 doubly dilutes, sensitization 1h, 2h, 3h, 4h, 5h, 6h and 7h respectively, 100ul is got in 1.5mlEP pipe every 1 hour, add 5uL0.1M monoethanolamine terminator cessation reaction, measure protein content before and after sensitization, calculate coupling protein amount and Conjugate ratio (test findings is as table 3, table 4).As seen from table, when coupling time is 5h, Conjugate ratio is the highest, and susceptibility is the highest, and the latex not self-solidifying after sensitization.
The relation of form 3 coupling time and Conjugate ratio
The relation of form 4 coupling time and susceptibility
4, different sensitization paradigm is on the impact of Conjugate ratio
Adopt best sensitization temperature, best sensitization time, best sensitizing antibody amount, study the impact of different sensitization paradigm on Conjugate ratio, what selection coupling efficiency was the highest is best sensitization paradigm (test findings is as table 5).It is the highest that result shows to adopt " the slow jolting of shaking table, monoclonal antibody dropwise slowly add, control whole loading time is 1 hour " this mode Conjugate ratio.
The different sensitization paradigm of form 5 is on the impact of Conjugate ratio
5, self-solidifying inspection: replace measuring samples to detect with the PBS, sterile saline, sterile purified water etc. of equivalent, observe the self-solidifying (test findings is as table 6) of sensitization latex.The results are shown in lower form:
Table 6 self-solidifying is tested
Indicate: ++++be 100% latex agglutination, latex forms large particle aggregation in drop edge, and liquid presents completely transparent state (as Figure 1A); +++ be the latex agglutination of 75%, have obvious particle to be formed, liquid slightly muddy (as Figure 1B); ++ be the latex agglutination of 50%, have obviously but the formation of thinner particle, flowing fluid ratio more muddy (as Fig. 1 C); + there is a little aggegation, liquid is muddy state (as Fig. 1 D);-not aggegation, drop presents original even emulsus (as Fig. 1 E).Result is to occur ++ above latex agglutination is positive.
Embodiment 2 enterorrhagia Bacillus coil 0157: the using method of H7 latex agglutination assay kit
1, the process of sample: the measuring samples collected is placed in sample treatment liquid 37 DEG C and cultivates 4-6 hour.
2, the detection of test sample: 1) drip 10ul positive control sample, 10ul negative control sample and 10ul measuring samples respectively on clean slide or glass plate.And then heliotropism control sample, negative control sample, the latex dripped in measuring samples described in 10ul detect reagent, stir, to shake after 1 minute observations under blue background.2) result judges: control test occurred following result in 1 minute, and test can be set up: positive control sample and latex detect reagent effect and occur +++ above latex agglutination, and negative control sample and latex detect reagent not aggegation; Measuring samples occurred in 1 minute ++ above latex agglutination is judged to the positive, presents original even emulsus and is judged to be feminine gender.
Kit can directly to the enterorrhagia Bacillus coil 0157 in sample: H7 detects, if containing enterorrhagia Bacillus coil 0157: H7 in sample, then latex detects reagent and occurred in 1 minute ++ and above latex agglutination, can be judged to positive findings; If not containing enterorrhagia Bacillus coil 0157: H7 in sample, then latex detection reagent drop in 1 minute still presents original even emulsus, can be judged to negative findings.
Embodiment 3 enterorrhagia Bacillus coil 0157: the Preliminary Applications of H7 latex agglutination assay kit
1, sensitivity tests: the enterohemorrhagic Escherichia coli list bacterium colony on picking sorbierite Mai Kangkai plate after 37 DEG C of cultivation 18-24h, 18-24h is cultivated in 5mLTSB fluid nutrient medium, with colony counting method, bacterium is counted, and bacterium liquid is carried out different gradient dilution, with spectrophotometer bacterial detection bacterium liquid OD
600value, then carries out detecting (test findings is as table 7) with the latex reagent that sensitization is good.Test findings: bacterium liquid is diluted to 1:256 doubly (bacterium amount 2.3 × 10
7cFU/ml), time, still can occur obvious visible agglutinating reaction, test findings shows that susceptibility is better.
Form 7 sensitivity tests
2, specific test: latex reagent good for sensitization is carried out latex agglutination detection with cause of diseases such as salmonella, other serotypes of Escherichia coli, avian pasteurella multocida, ewcastle diseases respectively, its specificity is studied (test findings is as table 8).Test findings: sensitization latex reagent and Escherichia coli O 157: H7 reacts for positive, and react with the cause of disease such as avian pasteurella multocida, salmonella and be feminine gender, test findings shows that specificity is better.
Form 8 specific test
| Antigen | State of aggregation | Antigen | State of aggregation |
| Avian pasteurella multocida | - | Escherichia coli O78 | - |
| Salmonella | - | Escherichia coli O1 | - |
| Positive control | ++++ | Negative control | - |
| Escherichia coli O 157: H7 | ++++ | Ewcastle disease | - |
3, replica test
3.1, interior replica test is criticized: prepare 3 batches of enterorrhagia Bacillus coil 0157s: H7 latex agglutination antigen detection kit (kit lot number is respectively 1401,1402,1403), 10 every batch, in every batch of kit, randomly draw 3 kits detect positive control sample, negative control sample, enterorrhagia Bacillus coil 0157: H7 and salmonella, avian pasteurella multocida, observe its susceptibility, specificity and stability, and detect latex whether self-solidifying (test findings is as table 9).Result shows that its susceptibility, specificity and stability do not change, and illustrates that batch interior repeatability is good.
Replica test in form 9 batches
3.2, criticize between replica test: be chosen at 3 batches of enterorrhagia Bacillus coil 0157s prepared by different time: H7 latex agglutination antigen detection kit (kit lot number is respectively 1401,1402,1403), detect with a positive control sample, negative control sample, enterorrhagia Bacillus coil 0157: H7 and salmonella, avian pasteurella multocida, observe its susceptibility, specificity and stability, and detect latex whether self-solidifying (test findings is as table 10).Result shows that its susceptibility, specificity and stability do not change, and illustrates that between criticizing, repeatability is good.
Replica test between form 10 batches
4, coincidence rate test: the contaminated samples of manual simulation is detected.Select the ddH of 50 parts of sterilizings
2o, therefrom Stochastic choice 30 parts manually adds enterorrhagia Bacillus coil 0157: H7, adopts the latex agglutination antigen detection kit of the present invention's assembling and PCR method to detect (test findings is as table 11) 50 above-mentioned increment product respectively.The latex agglutination antigen detection kit that result shows the present invention's assembling detects positive 29 parts, and positive rate is 96.67%; Detect negative sample 21 parts, negative recall rate is 100%; Compare with PCR testing result, total coincidence rate is 98.00%.
Form 11 coincidence rate test findings
Embodiment 4 enterorrhagia Bacillus coil 0157: the assembling of H7 latex agglutination assay kit
1, enterorrhagia Bacillus coil 0157: H7 latex agglutination antigen detection kit, comprises
2, the preparation of related solution
1) preparation of sample treatment liquid: take tryptone 20.0g, No. 3 cholate 1.12g, lactose 5.0g, ADKP 4.0g, anhydrous potassium dihydrogenphosphate 1.5g, NaCl 5.0g, ddH
2water-soluble for mentioned component rear adjust pH is 6.9 ± 1,121 DEG C of autoclaving 15min by O1000ml, and take out the ovobiocin solution 20mg/L filtering sterilizing and add, make ultimate density be 20ug/L, packing after mixing, 4 DEG C save backup.
2) preparation of positive control sample: by enterorrhagia Bacillus coil 0157: H7 bacterial strain is 37 DEG C of cultivation 18-24 hour in autoclaved TSB, adding formalin makes its final concentration be 0.2%, 37 DEG C of slow jolting 72h in shaking table, be dispensed in 1.5mlEP pipe ,-20 DEG C save backup.
3) preparation of negative control sample: not containing enterorrhagia Bacillus coil 0157: the autoclaved TSB nutrient solution of H7 bacterial strain, be dispensed in 1.5mlEP pipe ,-20 DEG C save backup.
Claims (6)
1. an enterorrhagia Bacillus coil 0157: H7 latex agglutination assay kit, it is characterized in that this kit comprises: a) latex detects reagent, b) sample treatment liquid, c) positive control sample, d) negative control sample, it is energy and enterorrhagia Bacillus coil 0157 that described latex detects reagent: the latex reagent of H7 specific reaction, adopts anti-Intimin protein monoclonal antibody sensitization carboxylated polystyrene latex gained.
2. by enterorrhagia Bacillus coil 0157 according to claim 1: H7 latex agglutination assay kit, it is characterized in that the preparation method of described anti-Intimin protein monoclonal antibody is: screen from 5 strain of hybridoma building strain 1 strain antibody tire height, high specificity hybridoma expand and cultivate, simultaneously in BALB/c mouse body, induce mouse ascites manufacture order clonal antibody; Get the BALB/c mouse 20 in 8 week age, only, the injection of 7d pneumoretroperitoneum expands the hybridoma 5 × 10 cultivated to the incomplete freund adjuvant 0.5mL/ of lumbar injection
6~ 5 × 10
7/ only, breeding observing, until mouse web portion projection and by only extracting mouse ascites when being slow in action, the centrifugal 8min of 2000r/min, discard fat and collect upper strata clarification ascites, measure with indirect ELISA method every the mouse ascites collected and tire, will tire and be not less than 1:(1000 × 2
11) ascites mixing, mixed ascites is finally tired after measured as 1:(1000 × 2
12), hypotype is accredited as lgG2b.
3. by enterorrhagia Bacillus coil 0157 according to claim 1: H7 latex agglutination assay kit, is characterized in that the concrete preparation method of described anti-Intimin protein monoclonal antibody sensitization carboxylated polystyrene latex: (1) is got the carboxylated polystyrene latex 200ul shaken up and put into EP pipe; (2) wash 3 times, 12000rpm, 15min with pH9.6,0.1M carbonate buffer solution, centrifugally abandon supernatant; (3) use pH4.5 again, the phosphate buffer of 0.01M washes 3 times, 12000rpm, 15min, centrifugal, abandons supernatant; (4) the NHS 1000uL then adding EDC 1000uL, 20mM of 100mM is resuspended, mixing, and make both final concentrations be respectively 50mol/L and 10mol/L, in shaking table, 26 DEG C slowly jolt 4.5h; (5) wash 3 times, 12000rpin, 20min with pH8.0,0.01M borate buffer solution, centrifugally abandon supernatant; (6) suspend with pH8.0,0.01M borate buffer solution 1000ul, suspension latex concentration is 2%, 4 DEG C and saves backup; (7) the anti-enterorrhagia Bacillus coil 0157 that 1:4 doubly dilutes dropwise slowly is added: H7intimin protein monoclonal antibody 1000ul, the protein content 2.78mg/mL of described protein monoclonal antibody, it is 1 hour that loading time controls, in 26 DEG C of shaking tables, slowly jolt 5h; (8) add 0.1M monoethanolamine 100ul, in 26 DEG C of shaking tables, slowly jolt 15min cessation reaction, 12000rpm, 20min, centrifugally abandon supernatant; (9) precipitation pH8.0,0.01M borate buffer solution washes 3 times, 12000rpin, 20min, centrifugally abandons supernatant; (10) precipitation pH8.0,0.01M borate buffer solution 2000ul suspends, and is the good latex of sensitization and detects reagent, 4 DEG C of preservations.
4. by enterorrhagia Bacillus coil 0157 according to claim 1: H7 latex agglutination assay kit, it is characterized in that the component of described sample treatment liquid and content by weight/volume is: take tryptone 20.0g, No. 3 cholate 1.12g, lactose 5.0g, ADKP 4.0g, anhydrous potassium dihydrogenphosphate 1.5g, NaCl 5.0g, is dissolved in 1000ml ddH by mentioned component
2o, adjust pH is 6.9 ± 1,121 DEG C of autoclaving 15min, and the ovobiocin solution 20mg/L taking out filtration sterilization adds, and makes ultimate density be 20ug/L.
5. by enterorrhagia Bacillus coil 0157 according to claim 1: H7 latex agglutination assay kit, it is characterized in that described positive control sample is the enterorrhagia Bacillus coil 0157 of deactivation: H7 bacteria suspension, described negative control sample is not containing the autoclaved TSB nutrient solution of Escherichia coli O 157: H7.
6. enterorrhagia Bacillus coil 0157 according to claim 1: the application of H7 latex agglutination assay kit, is characterized in that comprising following concrete steps: the measuring samples 37 DEG C of cultivation 4-6 hour in sample treatment liquid 1) will collected; 2) on clean slide or glass plate, 10ul positive control sample, 10ul negative control sample and 10ul measuring samples is dripped respectively, and then heliotropism control sample, negative control sample, the latex dripped in measuring samples described in 10ul detect reagent, abundant mixing, slow shake, after 1 minute under blue background observations; 3) result judges: control test occurs following result in 1 minute, and test can be set up: positive control sample and latex detect reagent effect and occur +++ above latex agglutination, and negative control sample and latex detect reagent effect not aggegation; Measuring samples occurred in 1 minute ++ above latex agglutination is judged to the positive, presents original even emulsus and is judged to be feminine gender.
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Cited By (2)
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| CN106706904A (en) * | 2016-11-25 | 2017-05-24 | 湖北省农业科学院畜牧兽医研究所 | Pullorum disease antibody latex agglutination negative selection detection kit as well as preparation method and application thereof |
| CN108254559A (en) * | 2018-01-03 | 2018-07-06 | 北京市理化分析测试中心 | The method of quick detection microorganism |
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| CN106706904A (en) * | 2016-11-25 | 2017-05-24 | 湖北省农业科学院畜牧兽医研究所 | Pullorum disease antibody latex agglutination negative selection detection kit as well as preparation method and application thereof |
| CN106706904B (en) * | 2016-11-25 | 2018-06-29 | 湖北省农业科学院畜牧兽医研究所 | White diarrhea antibody latex agglutination bears selective mechanisms kit and preparation method and application |
| CN108254559A (en) * | 2018-01-03 | 2018-07-06 | 北京市理化分析测试中心 | The method of quick detection microorganism |
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| CN104849470B (en) | 2016-08-24 |
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