CN103529204B - Detect the latex agglutination kit of milk cow Streptococcusagalactiae antibody - Google Patents
Detect the latex agglutination kit of milk cow Streptococcusagalactiae antibody Download PDFInfo
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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Abstract
The invention discloses a kind of latex agglutination kit detecting milk cow Streptococcusagalactiae antibody, prepare method and the application thereof of kit.This kit by latex agglutination particle, positive control serum, negative control sera, sample diluting liquid, microslide and calibrated pipet composition; Adopt this kit to detect milk cow and whether suffer from mammitis, method is fast easy, high specificity, highly sensitive, does not need instrument and equipment, is suitable for basic unit animal doctor and detects.
Description
Technical field
The present invention relates to animal epidemic detection technique field, specifically, the present invention relates to a kind of kit and the application of kit in Streptococcusagalactiae antibody test that detect milk cow Streptococcusagalactiae antibody.
Technical background
For a long time, mastitis for milk cows is affect milk cow production development and the No.1 disease of dairy produce food security always, this disease is easy to get, easily recurrence, refractory, difficult elimination.In the existing milk cows of China, the clinical mammitis incidence of disease reaches 9.7% ~ 55.6%, and recessive mastitis positive rate reaches 61.03% ~ 79.62%(Feng Wan Yu etc., the application of biopreparate in mastitis for milk cows control, animal and veterinary magazine, 2010,4:31-33).Cause in the pathogen of mastitis for milk cows, Streptococcusagalactiae proportion is larger.The investigation result display of domestic scholars, 20% ~ 40%(Li Hong that China accounts for total incidence because of mastitis for milk cows that Streptococcusagalactiae causes wins, mastitis for milk cows pathogen Faunal distribution and occurrence regularity research, and animal medicine is in progress, 2005,26(6): 146-149).Therefore, the propagation and the infection that control Streptococcusagalactiae are one of important contents of mastitis for milk cows prevention and control.
Streptococcusagalactiae is gram-positive cocci, without pod membrane, and atrichia, diameter 0.5 ~ 1.0 μm is the former bacterium of most commonly encountered diseases that is acute, chronic mastitis (Lu Chengping, the veterinary microbiology (third edition) that cause ox and sheep, Beijing: Chinese agriculture publishing house, 2001:208 ~ 209).In order to prevention and corntrol mastitis for milk cows, domestic Duo Jia unit develops the mastitis for milk cows vaccine (Li Hongsheng etc. containing Streptococcusagalactiae composition in succession, the development of mastitis for milk cows multiple vaccines and application, Chinese agronomy circular (monograph in 2005): 151 ~ 156; Yang Dingxing etc., milk cow streptococcal mastitis inactivated vaccine and preparation method thereof: China, CN201010547036 [P] .2010-12-28.), the prevention and control for mastitis for milk cows provide effective weapon.Although domestic existing mastitis for milk cows vaccine, for the evaluation of immune effect of vaccine, particularly antibody surveillance after immunity, still lacks simple and rapid detection technique.This cause immunity whether qualified, cannot find out fast the need of situations such as booster immunizations, bring inconvenience to the prevention and control of mastitis for milk cows.
At present, the existing pathogeny detection technology for milk cow Streptococcusagalactiae, as biochemical identification, PCR and emulsion process (Hu Ruiping etc., the progress that Streptococcusagalactiae detects and mastadenitis of cow is prevented and treated, China's veterinary drug magazine, 2008,42(6): 54-57), but for the antibody detection method of this bacterium, then only report has ELISA detection method (Zhang Jie etc., the foundation of indirect ELISA detection mastitis for milk cows vaccine protected effect method, Chinese milk cow, 2009,11:38-41; Lang Jingmin etc., the prokaryotic expression of Streptococcusagalactiae sip proteantigen Predominance Area, ox source and the foundation of indirect ELISA detection method thereof, Chinese Preventive Veterinary Medicine report, 2011,33(1): 37-40).It is consuming time longer that ELISA method detects antibody, only testing process just cost more than 2 hours, and need by equipment such as microplate reader, and therefore the method is subject to a definite limitation on Clinical practice.Latex agglutination has huge advantage due to rapid, simple and need not be special technical ability and instrument and equipment, is applicable to basic unit and promotes.
Research shows that there is much conservative property albumen on Streptococcusagalactiae surface, has good immunogenicity (Shen Dingshu etc., the progress of Streptococcusagalactiae surface protein, Chinese microecology magazine, 2007,19(5): 472 ~ 473).The milk cow Streptococcusagalactiae antibody detection method of current foundation is all using the albumen of bacterial protein or Bacillus coli expression as antigen.Bacterial protein complicated component, specific aim is not strong, easily causes the detection method specificity set up to decline; The antigen cost that purifying is expressed from Escherichia coli is high, and antigen active is difficult to ensure.
Summary of the invention
The object of the present invention is to provide a kind of latex agglutination kit detecting milk cow Streptococcusagalactiae antibody, this kit can when by the Streptococcusagalactiae antibody detected fast when any instrument and equipment in cow serum, simple and practical.
Another object of the present invention is that the latex agglutination kit of Streptococcusagalactiae antibody is in the application detected in milk cow Streptococcusagalactiae antibody and the application of latex agglutination kit in the antibody test of mastitis for milk cows vaccine immunity.
Another object of the present invention is to provide the method for the latex agglutination kit detection mastitis for milk cows of Streptococcusagalactiae antibody.
Another object of the present invention is to provide Streptococcusagalactiae cvcc1886 bacterial strain and is detecting the application in mastitis for milk cows.
The invention discloses a kind of latex agglutination kit detecting milk cow Streptococcusagalactiae antibody, this kit comprises a) latex agglutination particle, b) positive control serum, c) negative control sera, d) sample diluting liquid, e) microslide and calibrated pipet, it is characterized in that: a) latex agglutination particle is the granules of polystyrene of Streptococcusagalactiae surface antigen sensitization, b) positive control serum is the milk cow product serum of Streptococcusagalactiae surface antigen immunity, and c) negative control sera is that non-immune milk cow produces serum; Streptococcusagalactiae is wherein cvcc1886 bacterial strain.
The invention discloses the preparation method of Streptococcusagalactiae surface antigen, Streptococcusagalactiae surface antigen is prepared through the following steps:
(1) Streptococcusagalactiae CVCC1886 strain is inoculated in the TSB nutrient culture media containing 5% hyclone, 37 DEG C of constant-temperature shaking culture;
(2) collected by centrifugation thalline, with by 10mMTris, 1mMEDTA, the TE damping fluid that pH value 8.0 forms is resuspended;
(3) ultrasonic disruption, 5000 revs/min of centrifugal 15min, supernatant is ultracentrifugation again, collecting precipitation, resuspended with the TE damping fluid containing 1.5%triton-X100, and room temperature is placed 30min and made it to dissolve;
(4) add isopyknic saturated ammonium sulfate solution, at 4 DEG C, precipitate 2 hours;
(5) collected by centrifugation protein precipitation, resuspended with TE damping fluid, load in bag filter and dialyse 3 days, change dislysate 3 times therebetween;
(6) collect the protein solution after dialysis, measure Streptococcusagalactiae surface antigen concentration, and be concentrated to 2mg/mL protein concentration, obtain Streptococcusagalactiae surface antigen.
The invention also discloses the granules of polystyrene preparation method of Streptococcusagalactiae surface antigen sensitization, the granules of polystyrene of Streptococcusagalactiae surface antigen sensitization is prepared through the following steps:
(1) Streptococcusagalactiae surface protein BBS is diluted to protein content is 600 μ g/mL, dropwise joins in pretreated 2% control latex of equal-volume, and dropping limit, limit is shaken, and makes it fully mix;
(2) be placed in 25 DEG C of constant temperature ovens and leave standstill 3 hours;
(3) dropwise adding 10% bovine serum albumin (BSA) to final concentration is 1%, after fully mixing, then is placed in the standing 30min of 37 DEG C of constant temperature ovens;
(4) 6000 revs/min of centrifugal 15min, abandon supernatant, the resuspended granules of polystyrene obtaining Streptococcusagalactiae surface antigen sensitization of precipitation BBS.
The latex agglutination kit that the invention discloses Streptococcusagalactiae antibody is detecting the application in milk cow Streptococcusagalactiae antibody.
The latex agglutination kit that the invention discloses Streptococcusagalactiae antibody detects the method for mastitis for milk cows, and the method comprises the following steps:
(1) gather milk cow venous blood, room temperature puts 1 hour, separation of serum;
(2) drip 10 microlitre blood serum sample to be checked, add isopyknic latex agglutination reagent, stir all with toothpick
Even, shake observations after 30 seconds, positive serum and negative serum are set in contrast simultaneously;
(3), when there is 50% and above latex agglutination phenomenon, be namely judged to positive findings, otherwise be judged to feminine gender.
The invention discloses Streptococcusagalactiae cvcc1886 bacterial strain and detect the application in mastitis for milk cows.
Streptococcusagalactiae type strain CVCC1886 strain in the present invention is published in the catalogue of China national veterinary microorganism DSMZ, and be in open state, scientific worker can ask for this DSMZ.
In the present invention, latex agglutination reagent is also latex agglutination particle.
Latex agglutination reagent in kit disclosed by the invention, follows these steps to preparation:
A, extraction Streptococcusagalactiae surface protein antigen, method is as follows:
(1) by Streptococcusagalactiae type strain, (CVCC1886 strain, is inoculated in the TSB nutrient culture media containing 5% hyclone purchased from National Veterinary Culture Collection, 37 DEG C of constant-temperature shaking culture about 6 hours.
(2) collected by centrifugation thalline, resuspended with TE damping fluid (10mMTris, 1mMEDTA, pH value 8.0).
(3) ultrasonic disruption, 5000 revs/min of centrifugal 15min, collect supernatant.
(4) ultracentrifugation, collecting precipitation, resuspended with the TE damping fluid containing 1.5%triton-X100, room temperature is placed 30min and is made it to dissolve.
(5) add isopyknic saturated ammonium sulfate solution, at 4 DEG C, precipitate 2 hours.
(6) collected by centrifugation protein precipitation, resuspended with TE damping fluid, load in bag filter and dialyse 3 days, change dislysate 3 times therebetween.
(7) collect the protein solution after dialysis, measure protein concentration with ultraviolet spectrophotometer, and be concentrated to 2mg/mL protein concentration.
(8) antigen is divided in 1.5ml centrifuge tube, put-20 DEG C frozen for subsequent use.
B, prepare Latex Particles suspension, method is as follows:
(1) by the BBS(borate buffer of the control latex pH value 8.2 of 10%) be diluted to 2%, and add the trypsase that final concentration is 1%, fully mix.
(2) put into 50 DEG C of thermostat water baths and leave standstill 12 hours, 5 ~ 6 times should be shaken therebetween.
(3) take out, 6000 revs/min of centrifugal 15min, precipitate and wash 2 times with BBS, and finally resuspended with BBS is that 2%, 4 DEG C of storages are for subsequent use to concentration.
C, antigen sensibilization latex particle, method is as follows:
(1) the Streptococcusagalactiae surface protein BBS of preparation being diluted to protein content is 600 μ g/mL, dropwise joins in isopyknic above-mentioned Latex Particles suspension, and dropping limit, limit is shaken, and makes it fully mix.
(2) potpourri is placed in 25 DEG C of constant temperature ovens and leaves standstill 3 hours.
(3) dropwise adding 10% bovine serum albumin (BSA) to final concentration is 1%, after fully mixing, then is placed in the standing 30min of 37 DEG C of constant temperature ovens.
(4) take out, 6000 revs/min of centrifugal 15min, abandon supernatant, and precipitation BBS is resuspended to centrifugal front volume.
(5) last room temperature places 1 hour, is latex agglutination reagent, is stored in 4 DEG C.
Above-mentioned latex agglutination kit, detecting the application in milk cow Streptococcusagalactiae antibody, the steps include:
(1) gather cow blood, room temperature puts 1 hour, centrifuging serum;
(2) drip 10 microlitre blood serum sample to be checked, add isopyknic latex agglutination reagent, stir with toothpick, shake observations after 30 seconds.Establish positive control serum and negative control sera simultaneously.
(3), when there is 50% and above latex agglutination phenomenon, be namely judged to positive findings, otherwise be judged to feminine gender.
Milk cow Streptococcusagalactiae antibody assay kit provided by the invention can carry out qualitative detection to the Streptococcusagalactiae antibody in blood serum sample.If containing Streptococcusagalactiae antibody in sample, then there is obvious agglutination phenomenon in 2 minutes, can the positive be judged to; If not containing Streptococcusagalactiae antibody in sample, then there will not be obvious aggegation in 2 minutes, be namely judged to feminine gender.This kit also can judge the antibody titer of serum by detecting the blood serum sample after diluting.
The present invention compared with prior art, has the following advantages:
1. do not need ELIAS secondary antibody, IgG and IgM antibody can be detected simultaneously, specificity, highly sensitive.
2. detection time is short, within 2 minutes, can go out result.
3. testing process is without any need for instrument and equipment, simple, be suitable for basic unit animal doctor personnel and the use of milk cattle cultivating family, and testing cost is low.
4) Streptococcusagalactiae cvcc1886 strain antigens high specificity, by extracting Streptococcusagalactiae surface protein as antigen, using polystyrene latex as carrier, establishing the latex agglutination methods of detection milk cow Streptococcusagalactiae antibody and preparing kit.This kit method is quick, easy, within 2 ~ 3 minutes, can obtain a result, and high specificity is highly sensitive, and does not need by instrument and equipment, is suitable for veterinary clinic application.
Accompanying drawing explanation
Fig. 1 is the Dot-blotting detection figure of Streptococcusagalactiae surface antigen and full bacterium antigen active
A represents that the Dot-blotting of Streptococcusagalactiae surface antigen detects, and tires and can reach 1:320
B represents that the Dot-blotting of the full bacterium antigen of Streptococcusagalactiae detects, and tires as there being 1:80
Fig. 2 is the result figure that kit detects positive control sample and negative control sample
A represents experiment positive findings
B represents experiment negative findings
Embodiment
The present invention is set forth further below in conjunction with specific embodiment.Should be appreciated that these embodiments are only for illustration of the present invention, and can not limit the scope of the invention.
The extraction of embodiment 1 Streptococcusagalactiae surface antigen
(1) Streptococcusagalactiae cvcc1886 is inoculated in the pancreas peptone soybean broth nutrient culture media (TSB) containing 5% hyclone, 37 DEG C of constant-temperature shaking culture about 6 hours.
(2) 10000 revs/min of centrifugal 5min collected by centrifugation thalline, resuspended with the TE damping fluid (10mMTris, 1mMEDTA, pH value 8.0) of precooling.
(3) ultrasonic disruption, 5000 revs/min of centrifugal 15min, collect supernatant.
(4) supernatant 18000 revs/min of ultracentrifugation 90min at 4 DEG C, collecting precipitation, resuspended with a small amount of TE damping fluid containing 1.5%triton-X100, room temperature is placed 30min and is made it to dissolve.
(5) add isopyknic saturated ammonium sulfate solution, at 4 DEG C, precipitate 2 hours.
At (6) 4 DEG C, 12000 revs/min of centrifugal 30min collect protein precipitation, resuspended with TE damping fluid, load in bag filter and dialyse 3 days, change dislysate 3 times therebetween.
(7) collect the protein solution after dialysis, measure protein concentration.With the absorbance value (OD) of ultraviolet spectrophotometer working sample when 280nm and 260nm wavelength, by formula 1.45 × OD
280nm-0.74 × OD
260nmcalculate protein concentration, and be concentrated to 2mg/mL protein concentration.
(8) proteantigen is divided in 1.5ml centrifuge tube, put-20 DEG C frozen for subsequent use.
The Dotblotting of embodiment 2 surface antigen activity detects
By Zhu's Dotblotting(material loyalty etc., dot blotting detects the clinical research of anti-Jo-1 antibody. Hainan medical science, 2010,21(21): activity 3-5.) detecting surface antigen.By surface antigen (concentration the is 600 μ g/ml) doubling dilution extracted, get different dilution antigen 3 μ l point sample on nitrocellulose filter, to put in 37 DEG C of incubators dry 30 minutes.Be immersed in confining liquid (PBST containing 1%BSA), in 37 DEG C of constant temperature ovens, close 30 minutes, put in PBST and wash 2 times.Add the ox Streptococcusagalactiae positive control serum of 1:20 dilution, place 30 minutes for 37 DEG C, PBST washs 2 times.Add goat-anti ox IgG-HRP, place 30 minutes for 37 DEG C.Concussion washing 3 times, each 3 minutes.Drip substrate solution (DAB-H
2o
2) colour developing.To occur that obvious brown spot is judged to the positive.The Streptococcusagalactiae ultrasonic disruption antigen (bacterial protein) of same concentrations is set in contrast.Result shows that the surface antigen extracted is tired and reaches 1:320, and display dot is obvious; The ultrasound wave antigen valence of same concentrations can only reach 1:80, develops the color smudgy (the results are shown in Figure 2) during 1:160 dilution.
Embodiment 3 is positive, the preparation of negative control sera
The Streptococcusagalactiae surface antigen of extraction is concentrated to 2mg/mL, with the Freund's adjuvant emulsification of equivalent.The mode immunity milk cow of musculi colli injection, per injection 5ml, interval after 15 days again immunity once, altogether immunity 3 times.First immunisation adopts Freund's complete adjuvant emulsification, and later booster immunization adopts incomplete Freund's adjuvant emulsification.Serum antibody titer reaches 1: after requirement, jugular vein blood collection, centrifuging serum.Blood sampling also detects antibody titer with this latex agglutination methods.When antibody titer reaches 1:64, gather jugular vein blood with vacuum test tube, centrifuging serum, adds penicillin and streptomysin by 1000U/mL, and 0.22 μm of filter membrane is degerming, as positive control.
Selecting immunity to cross streptococcus agalactiae vaccine and antibody test is negative healthy cow, jugular vein blood collection separation of serum, adds penicillin and streptomysin by 1000U/mL, degerming with 0.22 μm of membrane filtration, as negative control.
The foundation of embodiment 4 Streptococcusagalactiae antibody latex agglutination detection method
(1) preparation of Latex Particles suspension
By the BBS(borate buffer of the polystyrene latex of 10% by pH value 8.2, namely 0.05mol/L borax soln 350ml converts 0.2mol/L BAS 650ml) be diluted to 2%, and add the trypsase that final concentration is 1%, fully mix.Put into 50 DEG C of thermostat water baths and leave standstill 12 hours, 5 ~ 6 times should be shaken therebetween.Take out, 6000 revs/min of centrifugal 15min, precipitate and wash 2 times with BBS, and finally resuspended with BBS is 2% to concentration, and it is for subsequent use to be placed in 4 DEG C of storages.
(2) determination of best sensitization concentration
Adopt the best sensitization concentration of square formation titrimetry determination antigen.In the latex suspension of 1ml2%, add the Streptococcusagalactiae surface antigen of equal-volume variable concentrations, be incremented to 1200 μ g/ml from 200 μ g/ml, 400 μ g/ml, 600 μ g/ml, in 25 DEG C of thermostat water baths place 2 hours, then add final concentration be 1% bovine serum albumin(BSA) close.Positive control serum is detected respectively, using can with the antigen concentration of the positive control serum generation agglutinating reaction of most highly diluted multiple as the best sensitization concentration of antigen with the latex after sensitization.Result shows, antigen concentration at 600 more than μ g/ml time, sensitization effect is best, detects positive control serum and can reach most high-titer.Concrete outcome is in table 1.
The determination of the best sensitization concentration of table 1
Note: when there is 50% and above latex agglutination phenomenon, be namely judged to positive findings, otherwise be judged to feminine gender.
(3) selection of best sensitization time
On the basis determining best sensitization concentration, change the time acted in 25 DEG C of thermostat water baths, tiring of positive control serum determines the best sensitization time after testing.Find through test, latex and antigenic action 3 are constantly little, reach best sensitization effect, and tiring of detection positive control serum is the highest.Concrete outcome is in table 2.
The determination of table 2 best sensitization time
(4) selection of the suitableeest sealer
Determining on best sensitization concentration and sensitization time basis, use respectively the oralbumin (OVA) of 1%, the bovine serum albumin(BSA) (BSA) of 1%, the human serum albumins (HSA) of 1% and 0.5% casein (CS) as sealer, detect the sealing effect of latex after sensitization.Find through test, BSA and HAS sealing effect is best.Consider that BSA easily obtains, and low price, select BSA as sealer.Concrete outcome is in table 3.
The selection of the best sealer of table 3
(5) selection of best sensitization temperature
On the basis determining above best sensitization condition, carry out sensitization respectively at 10 DEG C, 25 DEG C, 37 DEG C, 56 DEG C, tiring of positive control serum determines the best sensitization time after testing.Find through test, latex sensitization effect under 25 DEG C and 37 DEG C of conditions is best.Because lower temperature sensitization is favourable to protein stability, select 25 DEG C of the most best sensitization temperature.Concrete outcome is in table 4.
The selection of the best sensitization temperature of table 4
(6) latex agglutination reagent stability test
By latex agglutination reagent respectively in 37 DEG C and 4 DEG C of environment, measure the stability of latex agglutination reagent.Wherein place 3 days at 37 DEG C, place 6 months at 4 DEG C.Taking-up shakes up, and observes with or without from coagulation phenomena, and detects negative, positive control serum, contrast place before and after the change of serum titer.Result shows, place in 37 DEG C of environment at 3 days or 4 DEG C and place 6 months, latex reagent does not occur from coagulation phenomena, and its Detection results is not affected, and stability is higher.Concrete outcome is in table 5.
The stability test of table 5 latex agglutination reagent
(7) foundation of antibody detection method
A, the BBS of the control latex pH value 8.2 of 10% is diluted to 2%, and adds the trypsase that final concentration is 1%, fully mix.
B, put into 50 DEG C of thermostat water baths leave standstill 12 hours, 5 ~ 6 times should be shaken therebetween.
C, by Streptococcusagalactiae surface protein antigen BBS(pH value 8.2) to be diluted to protein content be 600 μ g/mL, dropwise joins in the pretreated control latex of equal-volume 2%, dropping limit, limit is shaken, and makes it fully mix.
D, potpourri are placed in 25 DEG C of constant temperature ovens and leave standstill 3 hours.
E, dropwise to add 10% bovine serum albumin (BSA) to final concentration be 1%, after fully mixing, then be placed in 37 DEG C of constant temperature ovens and leave standstill 30min.
F, taking-up, 6000 revs/min of centrifugal 15min, abandon supernatant, and precipitation BBS is resuspended to centrifugal front volume.
G, last room temperature place 1 hour, are latex agglutination reagent, be stored in 4 DEG C for subsequent use.
H, collection milk cow venous blood, room temperature places 1 hour, centrifuging serum.
I, get 10 μ l serum to be checked, positive control serum and negative control sera respectively and drip on microslide, respectively add isopyknic latex agglutination reagent, stir and evenly mix and shake 30 seconds, observing response result under black background.
J, result judge: when detecting sample appearance 50% and above latex agglutination phenomenon, be namely judged to positive findings, otherwise be judged to feminine gender.The precondition that test is set up is: detect positive control serum 100% aggegation, detects negative control sera without agglutination phenomenon.
Embodiment 5 specific test
Staphylococcus aureus (abbreviation S. aureus L-forms) immune cattle serum, Escherichia coli positive serum, brucellosis abortus positive serum, ox aftosa positive serum, NBCS and feminine gender, positive control serum is detected by the method, result shows except positive control serum test positive, all the other serum are feminine gender, show that this detection method has good specificity.The results are shown in Table 6.
The specificity test of table 6 latex agglutination
Remarks: " ++++" representing whole latex agglutination, particle gathers in drop edge, and liquid is completely transparent; " +++ " represents most of latex agglutination, and particle is obvious, and liquid is slightly muddy; " ++ " represents about 50% latex agglutination, but particle is comparatively thin, and liquid is more muddy; "+" indicates a little aggegation, opaque; “ – " represent that drop is original even emulsus.To occur that " ++ " and above aggegation person are judged to the positive.
Embodiment 6 sensitivity tests
By the serum doubling dilution of 3 parts of Streptococcusagalactiae immune cattles, detect respectively with latex agglutination, result shows tiring of 3 parts of serum all can reach 1:64, illustrates that the method has comparatively hypersensitivity.The results are shown in Table 7.
The sensitivity tests of table 7 latex agglutination
The assembling of embodiment 7 kit
Prepare latex agglutination reagent, positive control serum, negative control sera according to the above-mentioned method set up, preparation BBS damping fluid is as serum dilution.The bottle packing of all liq reagent, covers bottle cap.Reagent constituents comprises: (1) latex agglutination reagent, (2) positive control serum and negative control sera (3) serum dilution (4) microslide (5) 10ul calibrated pipet.
The inspection of embodiment 8 latex agglutination antibody assay kit
The yin and yang attribute control serum of kit does steriling test, should be aseptic; The Sensitivity and Specificity inspection of kit is undertaken by the method in embodiment 5 and embodiment 6; Kit is placed 6 months at 4 DEG C, detects tiring of a yin and yang attribute serum every 1 month, the stability of checking kit.Result shows: the yin and yang attribute control serum asepsis growth after filtration sterilization; Detect blood serum sample after kit assembling, Sensitivity and Specificity is good; In the storage life of 6 months, detect tiring of yin and yang attribute serum and do not change, illustrate that kit has good stability, specifically in table 8.
The stability test of table 8 kit
Embodiment 9 latex agglutination kit method compares with ELISA detection method
Detect 20 parts of streptococcus agalactiae vaccine immune cattle serum and 20 parts of not immune cow's serums, the coincidence rate of contrast two kinds of methods by this detection method and ELISA detection method (Zhang Jie, 2009) simultaneously.Found that, two kinds of method coincidence rates reach 95%.Latex agglutination kit detects 20 parts of immune cattle serum and is the positive, and detect 20 parts of not immune cow's serums and be feminine gender, testing result conforms to completely with the immune background of serum; Indirect elisa method detects 20 parts of immune cattle serum and is the positive, detects 20 parts of not immune cow's serums and occurs 2 parts of positives, and all the other are negative (see table 9).This illustrates that this latex agglutination kit method has the susceptibility suitable with ELISA detection method, has higher specificity simultaneously.
The Pass Test of table 9 latex agglutination kit and ELISA
Embodiment 10: the application of latex agglutination antibody assay kit in veterinary clinic detects
The application process of 1 latex agglutination antibody assay kit
(a) sampling and detection
Gather milk cow tail vein to be checked, room temperature puts 1 hour, separation of serum; Drawing 10 μ l blood serum sample to be checked with calibrated pipet drips on microslide, adds isopyknic latex agglutination reagent, stirs with toothpick, shakes and under black background, observes testing result after 30 seconds.Establish positive serum controls and negative serum control in test, the disposal route of control serum is identical with sample.
If need the antibody titer measuring serum to tire, by blood serum sample serum dilution doubling dilution, different dilution serum can be got and detects according to the method described above.Serum titer is occur the most high dilution of the serum of positive reaction result.
B () result judges
The precondition that test is set up is: detect positive control serum aggegation degree for " ++++" (100% aggegation), detect negative control sera without agglutination phenomenon.When detecting sample appearance " ++ " (50% aggegation) and above latex agglutination phenomenon, be namely judged to positive findings, otherwise be judged to feminine gender.
2 application latex agglutination antibody assay kits detect clinical sample
(a) clinical sample
Year August in June, 2011 to 2011, ground gathered milk cow blood sample 34 parts from Wuhan, Hubei, Huang gang etc., and wherein 22 parts (numbering 1 ~ 22), from mammitis vaccine immunity ox, all the other 12 parts (numberings 23 ~ 34) are from non-immune cattle.After blood natural coagulation, collect the serum of separating out.
(b) sample detection
According to above-mentioned detection method, blood serum sample and isopyknic latex agglutination reagent are mixed, shake and within 30 seconds, make serum and latex particle react completely, observe and detect aggegation degree, record testing result.Establish yin and yang attribute serum control simultaneously.
(c) testing result
After testing, the testing result of 22 parts of immune cattle serum is the positive, illustrates that vaccine immunity serves effect; Have 11 parts in 12 parts of not immune cow's serums for negative, 1 part is positive.Learn after investigation, the non-immune cattle of this head of test positive has mammitis medical history, may obtain antibody because infecting.Concrete testing result is in table 10.
Table 10 clinical serum testing result
Note: " ++ " and above aggegation person are judged to the positive.
Above-mentioned test can illustrate, latex agglutination antibody assay kit method accuracy is higher, and detection time is short, only needs within 2 minutes, can complete sample detection.ELISA detection method, because there are the steps such as incubation, washing and colour developing, needs just can complete detection in more than 2 hours usually.In addition, this kit method does not need the special reagent such as ELIAS secondary antibody, nitrite ion, and also do not need by any instrument and equipment in testing process, testing cost is low, and simple, is particularly suitable for basic unit animal doctor personnel and the use of milk cattle cultivating family.
Claims (1)
1. one kind is detected the latex agglutination kit of milk cow Streptococcusagalactiae antibody, this kit comprises a) positive control serum, b) negative control sera, c) latex agglutination particle, d) sample diluting liquid, e) microslide and calibrated pipet, is characterized in that: a) positive control serum is the milk cow product serum of Streptococcusagalactiae surface antigen immunity
B) negative control sera is that non-immune milk cow produces serum,
C) latex agglutination particle is the granules of polystyrene of Streptococcusagalactiae surface antigen sensitization, and wherein the granules of polystyrene of Streptococcusagalactiae surface antigen sensitization is prepared through the following steps:
1) Streptococcusagalactiae cvcc1886 is inoculated in the TSB nutrient culture media containing 5% hyclone, 37 DEG C of constant-temperature shaking culture;
2) collected by centrifugation thalline, with by 10mMTris, 1mMEDTA, the TE damping fluid that pH value 8.0 forms is resuspended;
3) ultrasonic disruption, 5000 revs/min of centrifugal 15min, supernatant is ultracentrifugation again, collecting precipitation, resuspended with the TE damping fluid containing 1.5%triton-X100, and room temperature is placed 30min and made it to dissolve;
4) add isopyknic saturated ammonium sulfate solution, at 4 DEG C, precipitate 2 hours;
5) collected by centrifugation protein precipitation, resuspended with TE damping fluid, load in bag filter and dialyse 3 days, change dislysate 3 times therebetween;
6) collect the protein solution after dialysis, measure Streptococcusagalactiae surface antigen concentration, and be concentrated to 2mg/mL protein concentration, obtain Streptococcusagalactiae surface antigen;
7) diluting Streptococcusagalactiae surface protein to protein content with BBS is 600 μ g/mL, dropwise joins in pretreated 2% control latex of equal-volume, and dropping limit, limit is shaken, and makes it fully mix;
8) be placed in 25 DEG C of constant temperature ovens and leave standstill 3 hours;
9) dropwise adding 10% bovine serum albumin (BSA) to final concentration is 1%, after fully mixing, then is placed in the standing 30min of 37 DEG C of constant temperature ovens;
10) 6000 revs/min of centrifugal 15min, abandon supernatant, the resuspended granules of polystyrene obtaining Streptococcusagalactiae surface antigen sensitization of precipitation BBS.
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| CN103901204B (en) * | 2014-04-11 | 2016-01-27 | 深圳市儿童医院 | A kind of immunoturbidimetry kit and detection method thereof detecting Streptococcusagalactiae |
| CN107085103A (en) * | 2017-05-10 | 2017-08-22 | 中国农业大学 | A kind of antibodies toward salmonella latex agglutination detection method |
| CN107356752B (en) * | 2017-07-10 | 2020-07-17 | 湖北省农业科学院畜牧兽医研究所 | Riemerella anatipestifer latex agglutination antibody detection kit, detection method and storage method thereof |
| CN108896758A (en) * | 2018-05-09 | 2018-11-27 | 中国农业科学院兰州兽医研究所 | A kind of kit based on molten junket big coccus plate agglutination test antigen composition, preparation method and applications |
| CN112730845A (en) * | 2020-12-16 | 2021-04-30 | 广东海大畜牧兽医研究院有限公司 | Detection kit for Aeromonas schubertii latex agglutination antibody, and preparation method and application thereof |
| CN112730828A (en) * | 2020-12-16 | 2021-04-30 | 广东海大畜牧兽医研究院有限公司 | Nocardia fulvidraco latex agglutination antibody detection kit and preparation method and application thereof |
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