CN105866436B - A kind of Hua Nashi staphylococcuses indirect hemagglutination detection kit and its application - Google Patents
A kind of Hua Nashi staphylococcuses indirect hemagglutination detection kit and its application Download PDFInfo
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- CN105866436B CN105866436B CN201610382154.0A CN201610382154A CN105866436B CN 105866436 B CN105866436 B CN 105866436B CN 201610382154 A CN201610382154 A CN 201610382154A CN 105866436 B CN105866436 B CN 105866436B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
Abstract
The present invention provides a kind of Hua Nashi staphylococcuses indirect hemagglutination detection kit, it is characterised in that：The kit includes Hua Nashi staphylococcus indirect hemagglutination antigens, and the Hua Nashi staphylococcuses indirect hemagglutination antigen is made up of Hua Nashi staphylococcus CCTCC M 2016048 whole bacterial protein and the relation sheep red blood cell (SRBC) of hydroformylation.The kit of the present invention is simple, convenient quick, and without special installation and instrument, whole process was completed in 2~3 hours, was adapted to the investigation that in basic unit's popularization and application, can be widely applied to Hua Nashi staphylococosis epidemiology.Testing result is reproducible, stable, reliably, can accurately detect the staphylococcic antibody of Hua Nashi.
Description
Technical field
The invention belongs to the detection reagent technical field for microorganism, and in particular to a kind of Hua Nashi staphylococcuses are indirect
Hemagglutination detection kit and its application.
Background technology
Hua Nashi staphylococcuses belong to Gram-positive, negative catalase coccus, are the masters in humans and animals enteron aisle
Want flora.Since the 1990s, Hua Nashi staphylococcuses have been increasingly becoming the important conditionity pathogenic bacteria of humans and animals.
At present, Hua Nashi staphy lococcus infections rely primarily on laboratory bacterial separation, PCR molecular biology identifications, then lack in basic unit
Fast and effectively Hua Nashi staphylococcuses serum diagnostic method.The shortage of serological diagnostic method, it may make it that basic unit is to China
Na Shi staphy lococcus infections cause mistaken diagnosis and failed to pinpoint a disease in diagnosis, so as to cause unnecessary loss to Animal husbandry production.
The content of the invention
In order to solve problems of the prior art, the invention provides a kind of inspection of Hua Nashi staphylococcuses indirect hemagglutination
Test agent box and its application.
The application is using ultrasonic cell disintegration method extraction Hua Nashi staphylococcuses somatic antigen as detection antigen, sensitization aldehyde
Change relation sheep red blood cell (SRBC), exempt from rabbit anteserum with Hua Nashi staphylococcus height, Hua Nashi staphylococcus positive serums enter in the ranks
Connect blood coagulation tests.As a result show that this method specificity is good, high sensitivity, can be that the serology of Hua Nashi staphylococosises quickly be examined
Disconnected and epidemiology survey provides foundation.
The application provides a kind of Hua Nashi staphylococcuses indirect hemagglutination detection kit, and the kit includes Hua Nashi Portugals
Grape coccus indirect hemagglutination antigen, the Hua Nashi staphylococcuses indirect hemagglutination antigen is by Hua Nashi staphylococcus CCTCC M
2016048 whole bacterial protein and hydroformylation-relation sheep red blood cell (SRBC) composition.
Preferably, the concentration of the Hua Nashi staphylococcuses whole bacterial protein is 40 μ g/ml-200 μ g/ml.
Preferably, whole bacterial protein and the hydroformylation-relation sheep of the Hua Nashi staphylococcuses CCTCC M 2016048
The volume ratio of red blood cell is 1:1.
Preferably, the kit also includes standard positive serum.
Preferably, the kit also includes standard female serum.
Preferably, the kit also includes dilution.
Preferably, the preparation method of the Hua Nashi staphylococcuses indirect hemagglutination antigen is：
（1）The preparation of Hua Nashi staphylococcus somatic antigens
Culture is enlarged to Hua Nashi staphylococcuses, after thalline is harvested by centrifugation, washs, is resuspended, with super in ice bath
The broken 10min of sound wave interval, centrifuging and taking supernatant, obtains Hua Nashi staphylococcus somatic antigen suspension；
（2）The fixation of red blood cell and hydroformylation-relation
It is red to sheep thin according to a conventional method by the ram blood being stored in Alsever's liquid after 4 DEG C stand 3-7 days
Born of the same parents are washed, glutaraldehyde is fixed and tannic acid processing, solution used are the PBS that the concentration of pH 7.2 is 0.15M, are prepared
Sheep red blood cell (SRBC) be 0.15M with pH7.2 concentration PBS be suspended into 5% concentration, obtain the red blood cell after hydroformylation-relation and hang
Liquid；
（3）The preparation of Hua Nashi staphylococcus indirect hemagglutination antigens
With step（1）Obtained Hua Nashi staphylococcus somatic antigen sensitization steps（2）Obtained red blood cell, obtains Warner
Family name's staphylococcus indirect hemagglutination antigen, the final concentration of 40 μ g/ml-200 μ g/ml of Hua Nashi staphylococcus indirect hemagglutination antigens.
The present invention also provides application of the mentioned reagent box in Hua Nashi staphylococcuses are detected, and the application is non-disease
Diagnosis and the application of therapeutic purposes.
Preferably, n is detected simultaneously（n≤9）The specific method of individual determined antigen is：
（1）Be separately added into serum to be checked in the hole of titer plate 1-n row each column the 1st, (n+1)th, n+2, n+3 be separately added into same body
Long-pending standard positive serum, standard female serum and blank control, by 1-n+2 arrange from the 1st hole proceed by 4 times of doubling dilutions to
Last hole；
（2）Hua Nashi staphylococcus indirect hemagglutination antigens are added per hole；
（3）Titer plate is shaken, fully mixes, puts 37 DEG C of insulating box 1.5-2h；
（4）According to erythrocyte agglutination deciding degree serum titer to be checked, to there is the maximum dilution of 50% erythrocyte agglutination times
Antibody titer of the number as this part of serum.
The superiority of the present invention includes following aspect：（1）Antigen is Hua Nashi staphylococcus somatic antigens in the present invention, tool
There is very strong specificity, can truly detect Hua Nashi Staphylococcal antibodies；（2）It is red with protein sensitization hydroformylation-relation sheep
Cell, the shortcomings that fresh sheep erythrocytes holding time is short, sensitization potency is low is avoided, be easy to preserve for a long time, hemagglutinative titer
It is high.（3）Simple, convenient quick, without special installation and instrument, whole process was completed in 2~3 hours, is adapted to push away in basic unit
Wide application, can be widely applied to the investigation of Hua Nashi staphylococosis epidemiology.（4）As a result reproducible, stabilization,
Reliably, the staphylococcic antibody of Hua Nashi can accurately be detected.（5）The present invention is current domestic unique detection Hua Nashi Portugals
The diagnostic reagent of grape coccus antibody, and cheap, safety and stability, non-toxic, non-environmental-pollution.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used, is city unless otherwise specified in following embodiments
Sell.
The preparation of the Hua Nashi staphylococcus indirect hemagg lutination diagnostic reagent boxes of embodiment 1
The component of Hua Nashi staphylococcus indirect hemagg lutination diagnostic reagent boxes is as follows：
（1）Hua Nashi staphylococcus indirect hemagglutination antigen 1 bottles, 10ml/ bottles；
（2）1 bottle of standard positive serum, 1ml/ bottles；
（3）1 bottle of standard female serum, 1ml/ bottles；
（4）2 bottles of dilution, 10ml/ bottles.
The storage life of the kit is 1 year.
The preparation of 1 Hua Nashi staphylococcus indirect hemagglutination antigens
1.1 culture mediums are prepared
Prepare THB fluid nutrient mediums 2000ml：By powdered beef 4.0g, tryptone 40g, glucose 40g, sodium chloride
6.0g, Na2HPO4 5.8g, KH2PO4 0.6g, Yeast extracts 6.0g add deionized water, are settled to 2000mL, fully
Stirring and dissolving, use anhydrous Na2CO3PH value is adjusted to 7.4.Autoclave sterilization, after culture medium cools, it is put into 4 DEG C and saves backup.
It is prepared by 1.2 Hua Nashi staphylococcuses somatic antigens
By Hua Nashi staphylococcusesStaphylococcus warneriQH (velogen strain, Qinghai is isolated from by this laboratory
Certain sheep field pathological material of disease) it is preserved in China typical culture collection center, preservation address：Wuhan City, Hubei Province Wuchang District Bayi Road
No. 299 Wuhan Universitys, deposit number are CCTCC M 2016048, and preservation date is：On January 18th, 2016.Single bacterium colony connects respectively
Kind is positioned in 37 DEG C of incubators in 20ml THB fluid nutrient mediums, and after culture 10h or so, bacterium is estimated according to Maxwell opacity tube
Liquid concentration is about 4-5 × 108During cfu, the bacterium solution for then drawing the 20ml is inoculated into new 0.5LTHB fluid nutrient mediums, constant temperature
Bacterium solution, 8000 r/min centrifugation 30min harvest thalline, sediment 0.15mol/L pH7.4 PBS are harvested after 37 DEG C of culture 16h
Suspend, after as above washing 3 times, then by the PBS of 50 times of thalline volume amount plus 0.15mol/L pH7.4 be diluted to 125 mL, be made into
Concentrated antigen.10min is crushed with ultrasonic wave interval in ice bath, then 4000 r/min centrifugations 20min, Aspirate supernatant, then
10000 r/min centrifuge 20min, and the supernatant concentrate after centrifugation is antigen stock solution, adds final concentration 0.1g/L thimerosal ,-
20 DEG C save backup.
The fixation of 1.3 red blood cells and hydroformylation-relation
By the ram blood preserved with the Alsever's liquid of equivalent at 4 DEG C after 3-7 days stable, centrifuge washing, with
3000rpm is centrifuged 10 minutes, abandons supernatant.The red blood cell of deposition is with the PBS that the pH of 10 times of volumes is that 7.2 concentration are 0.15M
By the washing of above-mentioned centrifugal condition three times, 95 milliliters of the PBS that the concentration of pH 7.2 is 0.15M is added in every 5 milliliters of red blood cells, made
It is 5% red cell suspension into volumetric concentration.Put and stirred on magnetic stirrer, every 100 milliliter of 5% red cell suspension adds 2.5%
20 milliliters of glutaraldehyde, continue stirring 1 hour after reordering at room temperature, centrifuged 3 minutes with 3000rpm, abandon supernatant.PH is used again
The PBS for being 0.15M for 7.2 concentration, after the pelleted by centrifugation washing three times of 3 minutes is centrifuged with 3000rpm, add what is newly prepared
2.5% 100 milliliters of tannic acid, it is sufficiently mixed in rearmounted 37 DEG C of water baths 10 minutes, supernatant is abandoned in centrifugation, then with the concentration of pH 7.2
For 0.15M PBS centrifuge washings three times after, then with pH be that PBS that 7.2 concentration are 0.15M is made into 5% hydroformylation-relation red thin
Born of the same parents' suspension, it is standby to put preservation at 4 DEG C.
The preparation of 1.4 Hua Nashi staphylococcus indirect hemagglutination antigens（That is Hua Nashi staphylococcuses somatic antigen sensitization aldehyde
The preparation of change-relation sheep red blood cell (SRBC)）
Hua Nashi staphylococcus somatic antigens are diluted to 200 μ g/ml, take 1 parts by volume Hua Nashi staphylococcus thalline to resist
Original is well mixed with the hydroformylation of 1 parts by volume 5%-relation sheep red blood cell (SRBC)（Sensitization concentration is 10 μ g/ml）, make in 37 DEG C of water-baths
With 30 min, it is stirred continuously therebetween；Then it is PBS that 7.2 concentration are 0.15 M with 3000rpm centrifuge washing red blood cells with pH
Three times, every time 5 minutes.It is again that the PBS that 0.15mol/L, pH are 7.2 is washed with the concentration containing the 1% healthy rabbit anteserum (NRS) of inactivation
Wash three times, sediment is with added with 0.1%（Volumetric concentration）Sodium azide (NaN3 ) 2%（Volumetric concentration）It is red that NRS is made into 1 % sensitization
Cell suspension, as indirect hemagglutination diagnostic antigen.It is standby to put preservation at 4 DEG C.
The preparation of 2 standard positive serums
The preparation of incomplete Freund's adjuvant：Lanolin and saxol are pressed 1:4 (V/V) are well mixed, 103.4kPa
(121 DEG C) autoclaving 30min, it is standby to put 4 DEG C of refrigerators.Antigen and incomplete Freund's adjuvant mixed in equal amounts, emulsification during use.
Freund's complete adjuvant is mycobacteria (the final concentration 3mg/ that nontoxic inactivation is added in incomplete Freund's adjuvant
mL).Antigen is mixed with the Freund's complete adjuvant of equivalent, emulsified during use.
Used from the kg of body weight 1.5 or so healthy rabbits as when being immunized.By two kinds of serotype Hua Nashi staphylococcuses
Bacterial strain（QH1 and QH2 bacterial strains, separated and preserved by Lanzhou veterinary institute.）Continuous three generations is carried out after being recovered respectively on THB flat boards
Rejuvenation, and even spread does expansion culture, 37 DEG C of incubated 16h, then adds the formalin of volumetric concentration 10% and cultivates
In good bacterium solution, make bacterium solution final concentration of 0.2%, with adding with shaking, be sufficiently mixed it, put 37 DEG C of inactivation 20h, shaken during culture
Shake 4 ~ 5 times, inactivate fully.First immunisation examines qualified antigen to be mixed with isometric Freund's complete adjuvant with above-mentioned inactivation
Fully emulsified, at every rabbit women's headgear lymph node and dorsal sc multiple spot is inoculated with, inoculum concentration 1mL；After 7d, second it is immune with
Isometric incomplete Freund's adjuvant emulsification antigen, dosage of inoculation 1.5mL, multiple spot are inoculated in every rabbit shoulder muscle and the back of the body
Portion is subcutaneous；After 10d, take the antigen containing isometric incomplete Freund's adjuvant prepared that 1.5mL is subcutaneously injected in every rabbit back,
It is immune to carry out third time, is taken a blood sample after 14d, carries out auricular vein and takes serum.After blood sampling, subcutaneously directly noted in every rabbit back
The Hua Nashi staphylococcus bacterium solution 1mL of fresh cultured are penetrated, it are carried out to attack poison, to improve antibody titer.After last time is immune,
The serum collected after 14d is as rabbit hyper-immune serum.
The preparation of 3 standard female serum
Choose and turn out to be the negative healthy goat of Hua Nashi Staphylococcal antibodies through serology, taken a blood sample with the method for routine,
Sterile separation serum.
The preparation of 4 dilutions
Weigh NaCl 4.25 g, KH2PO4 2.858 g、Na2HPO4·12H2The g of O 19.339, be dissolved in 1000 mL go from
In sub- water, dissolving is sufficiently stirred, it is 7.2 ~ 7.4 to adjust pH, and quantitative separating is 10ml/ bottles.
The determination of the optimal sensitization concentration of 5 kits
It is with 0.15mol/L pH7.2 PBS doubling dilutions in 1.5ml Eppendorf pipes by somatic antigen stock solution
1:2、1:4、1:8、1:16、1:32、1：64 totally 6 dilution gradients, the relation sheep red blood cell (SRBC) sensitization of hydroformylation with 5%, is surveyed respectively
Determine Hua Nashi staphylococcus rabbit hyper-immune serums, determine that the optimal sensitization of antigen is dense according to the definition of potency height and aggegation image
Degree.Concrete outcome is referring to table 1.
The measure of the antigen optimum concentration of table 1
By 1:2,1:4,1:8,1:16,1:32,1:The 64 concentrated antigen difference sensitized erythrocytes diluted in proportion, with 1:2,
1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28The positive serum of doubling dilution does indirect blood agglutination test.As a result show
Show, with bacterial strain QH1 and QH2, concentrated antigen is less than or equal to 1:During 4 dilution factor sensitization, HA-HI test no longer significantly improves, so really
QH1 and QH2 concentrated antigens are determined with 1:4 are diluted to standard sensitization concentration.The 5% sensitized erythrocyte blood clotting prepared with this density antigen
Potency is high, through being repeatedly measured, effect stability and image clearly.It the results are shown in Table 1.
Through experiment, the optimal sensitization concentration of Hua Nashi staphylococcus indirect hemagglutination antigens is 40 μ in kit of the invention
g/ml-200μg/ml。
Embodiment 2
Determined antigen is detected using the kit of the present invention, specific method is as follows：
First in the upper dropwise addition dilution 75 μ L per hole of the row 12 of 96 holes " ∨ " type polystyrene titer plates 8 row；Then it is every in 1-9 row
Arrange the 1st hole and be separately added into the tested μ L of serum 25,1 row of every part of tested serum, the 10th, 11, which arrange the 1st hole, is separately added into standard positive
Each 25 μ L of serum, standard female serum, the 12nd is classified as blank control.1-11 is arranged after the 1st hole fully mixes with the volley of rifle fire and draws 25 μ
L adds the 2nd hole, and serial dilution to the 8th hole, 25 μ L are discarded after mixing from the 8th hole successively；It is last that 1% sensitized erythrocyte is added dropwise per hole
Suspension（I.e. concentration is 100 μ g/ml Hua Nashi staphylococcus indirect hemagglutination diagnostic antigens）25 μ L, sample-adding, which finishes, titrates ∨ types
Plate, which is placed on micro oscillator, shakes 1-2min, covers glass plate, puts 37 DEG C of insulating box 1.5-2h, result of determination.
Criterion：Red blood cell whole aggegation（++++）；75% erythrocyte agglutination（+++）；50% red blood cell coagulates
Collection（++）；25% erythrocyte agglutination（+）；Without aggegation（-）.The hole of positive serum controls 1~8 should present " ++++~+
+ " aggegation；"-" should be presented in negative serum and blank control.On the premise of control wells are qualified, each hole of serum to be checked is observed, with
Antibody titer of the maximum dilution multiple of " ++ " aggegation as this part of serum is presented.Potency >=1: 8（++）The positive is judged to, is imitated
Valency≤1: 4（++）It is judged to feminine gender.Potency, which falls between, to be judged to suspicious, need to be redeterminated, is still judged to the positive to be suspicious.
The specific test of the Hua Nashi staphylococcus indirect hemagglutination detection kits of the present invention of embodiment 3
With the present invention Hua Nashi staphylococcus indirect hemagglutination detection kits, to healthy rabbit anteserum, Hua Nashi grape balls
Bacterium, streptococcus positive serum, staphylococcus positive serum, enterococcus faecalis positive serum, VREF positive serum are detected,
As a result in addition to Hua Nashi staphylococcuses, remaining is feminine gender（Referring to table 2）, illustrate that the antigen has high specificity.
The Hua Nashi staphylococcus indirect hemagg lutination diagnostic reagent specific test results of table 2
The repeated experiment of the Hua Nashi staphylococcus indirect hemagglutination detection kits of the present invention of embodiment 4
Repeated experiment
First, replica test in criticizing
3 different operators, take 5 kinds of Hua Nashi staphylococcuses sun of certain a batch of kit of the invention while detection
Property, negative serum, observe indirect hemagglutination potency situation of change.
2nd, repeat to test between criticizing
The kit of the invention of 3 parts of different batches is taken, while detects the staphylococcic positive serums of 5 kinds of Hua Nashi and the moon
Property serum, observe indirect hemagglutination potency situation of change.
3rd, field sample detection
114 parts of field blood serum samples of the ground censorship such as Gansu, Qinghai are detected with the kit of the present invention, to understand
Popularity degree of the disease in NORTHWEST CHINA area.
Repeated experiment result
1st, replica test in criticizing：3 bit manipulation persons jointly with a batch of kit of the invention to 5 kinds of Hua Nashi Portugals
Grape coccus positive serum, negative serum detect once every January, total to detect 3 times, the complete phase of testing result of each indirect hemagglutination
Together, variation within batch coefficient is：0.
2nd, replica test between criticizing：Using 3 batches of kits (lot number 201409,201410,201411) of the invention to 5 kinds
Hua Nashi staphylococcuses positive serum and negative serum repeat detection 3 times with IHA, and each result is consistent (P substantially>0.05).Say
Bright kit of the invention is stable.
3rd, the detection of field sample
Blood serum sample amounts to 126 parts, and the positive rate in Gansu is 25%, and the positive rate in Qinghai is 38.9%, and average positive rate is
30.95%.Testing result is shown in Table 3.
The kit field sample detection result of the present invention of table 3
The sensitivity experiments of the Hua Nashi staphylococcus indirect hemagglutination detection kits of the present invention of embodiment 5
Detected after Hua Nashi staphylococcus positive serums are diluted in proportion, take 5 parts of immune Hua Nashi staphylococcuses
Positive serum is carried out while detected with agar agglutination test with the kit of the present invention, compares sensitiveness.To 5 experiment immunization pigs
With 5 tribal chief's work infected pigs in immunity inoculation or infection 0d, 7d, 14d, 21d, 30d, the blood serum sample of 45d collections, it is anti-to detect it
The growth and decline change of body and antibody generation time are to determine the sensitiveness of the IHA laboratory diagnostic methods and be used as method of early diagnosis
Feasibility.
QH1 and QH2 positive serums are diluted in proportion, detected with the indirect hemagglutination diagnostic antigen, 1:256（1:28）
The testing result of dilution is still the positive.The positive serum of 5 parts of immune rabbits is taken, indirect hemagglutination test is used respectively after diluting in proportion
（IHA）Test and detect with AGP test aggegation, the diffusion aggegation experiment potency of serum is 1:128；IHA potency is 1:256, specifically
As a result referring to table 4.
The testing result of the sensitivity tests of table 4
Finally it should be noted that：The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To be modified to the technical scheme described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic.
Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., it should be included in the present invention's
Within protection domain.
Claims (7)
- A kind of 1. Hua Nashi staphylococcuses indirect hemagglutination detection kit, it is characterised in that：The kit includes Hua Nashi Portugals Grape coccus indirect hemagglutination antigen, the Hua Nashi staphylococcuses indirect hemagglutination antigen is by Hua Nashi staphylococcus CCTCC M 2016048 whole bacterial protein and hydroformylation-relation sheep red blood cell (SRBC) composition；The concentration of the Hua Nashi staphylococcuses whole bacterial protein For 40 μ g/ml-200 μ g/ml；The whole bacterial protein and hydroformylation-relation silk floss of the Hua Nashi staphylococcuses CCTCC M 2016048 The volume ratio of sheep red blood cell is 1:1.
- 2. kit according to claim 1, it is characterised in that：The kit also includes standard positive serum.
- 3. kit according to claim 1, it is characterised in that：The kit also includes standard female serum.
- 4. kit according to claim 1, it is characterised in that：The kit also includes dilution.
- 5. according to any described kits of claim 1-4, it is characterised in that：The Hua Nashi staphylococcuses indirect hemagglutination resists Former preparation method is：（1）The preparation of Hua Nashi staphylococcus somatic antigensCulture is enlarged to Hua Nashi staphylococcuses, after thalline is harvested by centrifugation, washs, is resuspended, with ultrasonic wave in ice bath The broken 10min of interval, centrifuging and taking supernatant, obtains Hua Nashi staphylococcus somatic antigen suspension；（2）The fixation of red blood cell and hydroformylation-relationBy the ram blood being stored in Alsever's liquid after 4 DEG C stand 3-7 days, sheep red blood cell (SRBC) is entered according to a conventional method Row washing, glutaraldehyde fix and tannic acid processing, solution used be the concentration of pH 7.2 be 0.15M PBS, the silk floss prepared The PBS that sheep red blood cell is 0.15M with pH7.2 concentration is suspended into 5% concentration, obtains the red cell suspension after hydroformylation-relation；（3）The preparation of Hua Nashi staphylococcus indirect hemagglutination antigensWith step（1）Obtained Hua Nashi staphylococcus somatic antigen sensitization steps（2）Obtained red blood cell, obtain Hua Nashi Portugals Grape coccus indirect hemagglutination antigen, the final concentration of 40 μ g/ml-200 μ g/ml of Hua Nashi staphylococcus indirect hemagglutination antigens.
- 6. application of any described kits of claim 1-4 in Hua Nashi staphylococcuses are detected, the application is non-disease The diagnosis of disease and the application of therapeutic purposes.
- 7. application according to claim 6, it is characterised in that：While the specific method for detecting n test serum is：（1）It is separately added into serum to be checked in the hole of titer plate 1-n row each column the 1st, (n+1)th, n+2, n+3 arrange the 1st hole and be separately added into together Standard positive serum, standard female serum and the blank control of sample volume, 1-n+2 are arranged to proceed by 4 times of multiple proportions from the 1st hole dilute Release to last hole；Wherein, n≤9；（2）Hua Nashi staphylococcus indirect hemagglutination antigens are added per hole；（3）Titer plate is shaken, fully mixes, puts 37 DEG C of insulating box 1.5-2h；（4）According to erythrocyte agglutination deciding degree serum titer to be checked, made with there is the maximum dilution multiple of 50% erythrocyte agglutination For the antibody titer of this part of serum.
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| CN108646018A (en) * | 2018-05-09 | 2018-10-12 | 中国农业科学院兰州兽医研究所 | A kind of Hai Shi enterococcus slide agglutination Serum Antibody Detection kit, preparation method and applications |
| CN109682977A (en) * | 2018-06-21 | 2019-04-26 | 中国农业科学院兰州兽医研究所 | A kind of Hai Shi enterococcus indirect hemagglutination antibody assay kit and preparation method thereof |
| CN108957000A (en) * | 2018-06-21 | 2018-12-07 | 中国农业科学院兰州兽医研究所 | A kind of staphylococcus saprophyticus indirect hemagglutination antibody assay kit and preparation method thereof |
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| CN104007269B (en) * | 2014-05-21 | 2015-10-21 | 中国农业科学院兰州兽医研究所 | A kind of riemerella anatipestifer indirect hemagglutination antibody assay kit and application thereof |
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