CN107664699A - Detect indirect ELISA reagent kit, the preparation method and applications of many animals erysipelothrix ruhsiopathiae antibody - Google Patents
Detect indirect ELISA reagent kit, the preparation method and applications of many animals erysipelothrix ruhsiopathiae antibody Download PDFInfo
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Abstract
The present invention is erysipelothrix ruhsiopathiae (bacterial strain number：YN15075, bacterial strain preserving number：CGMCC No.14468) and its antibody indirect ELISA detection kit, preparation method and application.Kit asks B, negative control sera, the staphylococcal protein A (HRP SPA) of horseradish peroxidase-labeled, skimmed milk power, 100 × tmb substrate liquid, 10 × colour developing dilution, 3% (v/v) H by ELISA Plate, serum-dilution plate, envelope antigen, coating buffer solution, positive control serum A, positive control blood2O2Solution, terminate liquid, 25 × lavation buffer solution assemble.The kit can detect pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae IgG antibody, its result criterion：On the premise of the validity of guarantee test, pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae antibody positive standard are respectively that (S represents the OD of tested serum for S/P≤0.30, S/P≤0.28, S/P≤0.26 and S/P≤0.33450nmValue, P represent the OD of 2 hole positive control serums450nmThe average value of value), otherwise it is determined as feminine gender, kit can be used for erysipelothrix ruhsiopathiae Serum Antibody Detection and epidemiology survey.
Description
Technical field
Patent of the present invention is related to a kind of erysipelothrix ruhsiopathiae, and (Erysipelothrix rhusiopathiae, are abbreviated as：
E.rhusiopathiae, bacterial strain number are：YN15075, bacterial strain preserving number are：CGMCC No.14468) and its antibody indirect
ELISA detection kit, preparation method and application.Between particularly a kind of detection pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae antibody
Connect ELISA kit.
Background technology
Erysipelothrix ruhsiopathiae (Erysipelothrix rhusiopathiae) is also known as bacillus rhusiopathiae suis or erysipelothrix porci,
A variety of mammals and birds, fish and the people such as energy infected pigs, ox, sheep and rabbit, it is a kind of larger Amphixenosis of harm
Opportunistic pathogen.Erysipelothrix ruhsiopathiae infection typically results in a large amount of acute deaths of animal and the erysipeloid of people, and the disease is in worldwide distribution.
In recent years, erysipelothrix ruhsiopathiae the incidence of infection and the death rate dramatically increase, and the new hair situation of old disease are presented, it is considered to be Yi Zhongfu
Hair property disease, therefore, research and development one kind can be with quick detection many animals erythema for serious threat animal health and mankind's public health
The prevention and control tool that the kit of Erysipelothrix antibody is infected erysipelothrix ruhsiopathiae are of great significance.
At present, the method for being used to detect erysipelothrix ruhsiopathiae antibody both at home and abroad mainly has agglutination test (Agglutination
Test, AT), Dot-ELISA (Dot-PAA-ELISA) and the ELISA antibody test sides based on erysipelothrix ruhsiopathiae SpaA albumen
Method.But because agglutination test has sensitiveness low and cannot be used for the detection of a large amount of samples, make its application by a definite limitation；It is existing
Some ELISA methods are also due to it can only detect erysipelothrix rhusiopathiae antibody, it is impossible to detect many animals erysipelothrix ruhsiopathiae
Antibody, its application is set also to be subject to certain restrictions；Urgently research and develop a kind of many animals erysipelothrix ruhsiopathiae antibody of can detecting
Sensitive, quick, high-flux detection method.
The content of the invention
Present invention aims to overcome that above-mentioned existing erysipelothrix ruhsiopathiae agglutination test antibody detection method sensitiveness it is low and
The defects of being unsuitable for high flux sample detection and existing ELISA kit can not detect many animals erysipelothrix ruhsiopathiae antibody
The defects of, there is provided a kind of indirect ELISA reagent kit that can detect pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae antibody, this method
Also there is the advantages of sensitive, quick, suitable for high flux sample detection.
The technical proposal for solving the technical problem of the invention is：One kind detection many animals erysipelothrix ruhsiopathiae antibody
Indirect ELISA reagent kit by ELISA Plate, serum-dilution plate, envelope antigen, coating buffer solution, positive control serum A and the positive
Serum control B, negative control sera, the staphylococcal protein A (HRP-SPA) of horseradish peroxidase-labeled, skimmed milk power,
100 × tmb substrate liquid, 10 × colour developing dilution, 3% (v/v) H2O2Solution, terminate liquid and 25 × cleaning solution assemble.
Specific preparation method is as follows：
(1), envelope antigen is the erysipelothrix ruhsiopathiae isolated in Yunnan province YN15075 good by screening antigenicity, is inoculated with
Nutrient agar panel, 37 DEG C of culture 48h, is eluted thalline with sterile saline, adjusts its concentration as 2 × 108CFU/
ML, ice-bath ultrasonic 100 times, ultrasound 10 seconds, are spaced 10 seconds every time, and ultrasonic power is 100 watts, by ultrasonic degradation protein solution
5000r/min centrifuges 10min, and it is ELISA envelope antigens to take supernatant, and it is 0.2mg/mL to determine its protein concentration with BCA methods, is added
Enter final concentration of 0.01% (m/v) thiomersal preservative, -20 DEG C save backup；
(2) it is by by NaHCO, to be coated with buffer solution (pH9.6 0.05M carbonate buffer solutions)3 0.16g、NaHCO3
0.29g is dissolved in 100mL ultra-pure waters and prepares gained, and 4 DEG C save backup；
(3), positive control serum A is by the way that the good erysipelothrix ruhsiopathiae YN15075 separation strains of antigenicity are inoculated with into battalion
Agar plate is supported, 37 DEG C of culture 48h, thalline is eluted with sterile saline, adjusts its concentration as 2 × 1010CFU/mL,
Add 37 DEG C of inactivation 6h of final concentration of 0.05% (v/v) formalin and prepare erysipelothrix ruhsiopathiae immunizing antigen；Take 2mL immune anti-
Former musculi colli injects the sodium selenite of the 1 first non-vaccine inoculation of 70 age in days, immune once at interval of 7d, each immunization method phase
Together, it is immunized 4 times altogether, 10d vena cava anteriors are taken a blood sample after the 4th is immune, and centrifugation prepares positive control serum A, and 4 DEG C of preservations are standby
With；
(4), positive control serum B takes erysipelothrix ruhsiopathiae immunizing antigen 2mL necks in above-mentioned steps (3) to be subcutaneously injected 1
The only healthy goat of 10 monthly ages non-vaccine inoculation, it is immunized once at interval of 7d, each immunization method is identical, immune 4 times altogether, and the 4th
Secondary immune rear 10d jugular vein blood collections, centrifugation prepare positive control serum B, and 4 DEG C save backup；
(5), negative control sera is by the healthy goat from 1 10 monthly ages non-vaccine inoculation, isolated rearing 10d
Afterwards, jugular vein blood collection, centrifugation prepare negative control sera, and 4 DEG C save backup；
(6), HRP-SPA freeze-dried powders are purchased from KPL companies of the U.S., with 50% (v/v) sterile glycerol dissolved dilution to 0.1mg/
ML, final concentration of 0.01% (m/v) thiomersal preservative is added, -20 DEG C save backup；
(7), skimmed milk power is purchased from U.S. company BD, and room temperature preservation is standby；
(8), 100 × tmb substrate liquid (1%TMB) is by the way that 0.1g TMB (3,3', 5,5'- tetramethyl benzidine) is molten
Solution prepares gained in 10mL dimethyl sulfoxide (DMSO)s, and 4 DEG C save backup, and wherein TMB and dimethyl sulfoxide (DMSO) are purchased from Sigma Co., USA；
(9), 10 × colour developing dilution (1mol/L sodium acetates/citric acid) is surpassed by the way that 21g citric acids are dissolved in into 100mL
Pure water obtains citric acid solution；8.2g sodium acetates are dissolved in 100mL ultra-pure waters and obtain sodium acetate solution, then take above-mentioned 3.5mL
Citric acid solution, which is added in 100mL sodium acetate solutions, prepares gained 10 × colour developing dilution, and 4 DEG C save backup；
(10), 3%H2O2Solution is purchased from Sigma Co., USA, and room temperature preservation is standby；
(11), terminate liquid (2mol/L H2SO4) it is by the way that the 22mL concentrated sulfuric acids are slowly added into 178mL ultra-pure waters along bottle wall
Obtained by middle preparation, 4 DEG C save backup, and wherein the concentrated sulfuric acid is purchased from Chongqing Chuan Dong Chemical Co., Ltd.s；
(12), 25 × lavation buffer solution is by by disodium hydrogen phosphate (Na2HPO4·12H2O) 7.3g, potassium dihydrogen phosphate
(KH2PO4) 0.5g, potassium chloride (KCl) 0.5g, sodium chloride (NaCl) 20g, polysorbas20 (Tween-20) 1.3mL be dissolved in 100mL
Ultra-pure water prepares gained, and room temperature preservation is standby；
(13), ELISA Plate is purchased from NUNC companies of Denmark, and room temperature preservation is standby；
(14), serum-dilution plate：It is standby purchased from clean special (JET) the biofiltration Co., Ltd in Guangzhou, room temperature preservation；
(15), the term of validity of this kit is 12 months.
The use operating procedure of indirect ELISA reagent kit of present invention detection many animals erysipelothrix ruhsiopathiae antibody is：
(1) antigen coat ELISA Plate：Envelope antigen coating buffer solution 1/400 is diluted, added in each hole of ELISA Plate
The μ L of envelope antigen 100 of dilution are stated, 4 DEG C of coatings are overnight；
Before next-step operation is carried out, following 3 kinds of reagents need to be configured：1. 1 × lavation buffer solution must be prepared：It will be fitted in kit
1/25 times of 25 × lavation buffer solution progress of amount, which is diluted in ultra-pure water, to be produced；2. the preparation of confining liquid：Will be suitable in kit
The skimmed milk power of amount is diluted in above-mentioned 1 × lavation buffer solution, its concentration is produced for 4% (m/v)；3. serum/secondary antibody dilution
The preparation of liquid：The appropriate skimmed milk power in kit will be taken to be diluted in above-mentioned 1 × lavation buffer solution, make its concentration be
0.4% (m/v) is produced；
(2) board-washing：Each hole of ELISA Plate adds 400 μ L lavation buffer solutions, stands 1min, inhales the cleaning solution abandoned in hole, each hole
In add 400 μ L lavation buffer solutions, stand 1min, inhale the cleaning solution abandoned in hole, repeat above-mentioned washing process 5 times；
(3) close：Each hole of ELISA Plate adds 100 μ L confining liquids, and 1h is closed in 37 DEG C of wet box；
(4) board-washing：Each hole of enzyme mark is washed 5 times by plate washing method in above-mentioned (2)；
Before next-step operation is carried out, serum need to be subjected to 1/30 times of pre-dilution with serum-dilution plate, such as：Take 3 μ L above-mentioned
Serum is added to mix in 87 μ L serum dilutions and produced；
(5) primary antibody is incubated：90 μ L serum dilutions first are added in each hole of ELISA Plate, by 1/30 times in above-mentioned serum-dilution plate
10 μ L are into each hole of above-mentioned ELISA Plate for the corresponding transfer of prediluted serum to be checked, positive control serum, negative control sera, 37 DEG C
1h is incubated in wet box；
(6) board-washing：Each hole of ELISA Plate is washed 5 times by plate washing method in above-mentioned (2)；
Before next-step operation is carried out, HRP-SPA secondary antibodies need to be subjected to pre-dilution：If it is used for pig and rabbit anteserum sample detection
HRP-SPA is then subjected to 1/60000 dilution；If being used for ox and sheep blood serum sample detection, need HRP-SPA carrying out 1/2000
Dilution；
(7) secondary antibody is incubated：The above-mentioned prediluted HRP-SPA secondary antibodies of 100 μ L are added in each ELISA Plate hole, in 37 DEG C of wet box
It is incubated 1h；
(8) board-washing：Each hole of enzyme mark is washed 5 times by plate washing method in above-mentioned (2)；
Before next-step operation is carried out, 1 × nitrite ion need to be prepared, it is comprised the following steps that：Take in kit appropriate 10 ×
Colour developing dilution, which is added to mix in the ultra-pure water of 9 times of volumes, produces 1 × colorbuffer；Again in every 10mL above-mentioned 1 × colour developing
100 μ 100 × tmb substrates of L liquid and 35 μ L 3% (v/v) H are added in buffer solution2O2Solution, which mixes, produces 1 × nitrite ion；
(9) develop the color：100 μ 1 × nitrite ions of L, color development at room temperature 10min are added in each hole of ELISA Plate；
(10) color development stopping is reacted：Each hole of ELISA Plate adds 100 μ L terminate liquids；
(11) OD is read with ELIASA450nmIt is worth and calculates S/P values (the tested serum OD of S expressions450nmValue, P represent that the 2 hole positives are right
According to serum OD450nmThe average value of value)；
(12) result judgement：(the positive control serum OD on the premise of the validity of guarantee test450nmThe Ping Jun Zhi of value≤
0.8, and negative control sera OD450nmThis experiment side of Ping Jun Zhi≤0.08 of value is effective；Otherwise it is invalid, it should reform and once examine
Survey), judge pig, ox, the standard of sheep and rabbit erysipelothrix ruhsiopathiae antibody positive be respectively S/P≤0.30, S/P≤0.28, S/P≤
0.26 and S/P≤0.33, otherwise it is determined as feminine gender, the kit can be used for pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae serum antibody
Detection and epidemiology survey；
In clinical serum sample detection, when there is erysipelothrix ruhsiopathiae specific antibody in blood serum sample to be checked, specifically
Property antibody and ELISA Plate in envelope antigen specifically bind, non-specific antibody is then washed buffer solution and washed away；Add energy
With the HRP-SPA of a variety of mammal IgG specific bindings, the HRP-SPA not combined with specific antibody is then washed slow
Fliud flushing washes away, and specific antibody HRP-SPA more at most in connection is more, and addition nitrite ion colour developing is deeper, i.e., specific
Antibody titer (concentration) is higher, and its colour developing is also deeper；Therefore, the ELISA method of the invention can be not only used for Erysipelothrix silk
Bacterium blood asks the qualitative detection of antibody, and can pass through the depth or OD of colour developing450nmThe size of value is entered to erysipelothrix ruhsiopathiae antibody
Row half-quantitative detection.The present invention from a large amount of erysipelothrix ruhsiopathiae bacterial strains by screening the good erysipelothrix ruhsiopathiae cloud of antigenicity
Southern separation strains YN15075, by cultivating, washing, ultrasound preparation erysipelothrix ruhsiopathiae envelope antigen；Pass through mass propgation, inactivation
Erysipelothrix ruhsiopathiae YN15075 thalline as immunizing antigen, sodium selenite and the goat of not inoculated vaccine are immunized respectively, point
Positive control serum A and positive control serum B are not prepared；Pass through the healthy goat from non-vaccine inoculation, jugular vein blood collection system
Standby negative control sera；It can detect by using foundation can be reached as secondary antibody with the HRP-SPA that a variety of mammal IgG are combined
Pig, ox, the purpose of sheep and the general indirect ELISA reagent kit of rabbit erysipelothrix ruhsiopathiae antibody.
The envelope antigen activity that the present invention is prepared from the bacterial strain is higher, greatly reduces the concentration of envelope antigen and (reduces
To 0.5 μ g/mL), the dosage for the envelope antigen not only saved, and the specificity of the indirect ELISA is improved, acquirement well has
Beneficial effect.And the ultrasonic degradation preparation process of envelope antigen has novelty, it is also beneficial to save the dosage of envelope antigen and carries
High indirect ELISA specificity, adding thiomersal preservative is beneficial to extend the antigen term of validity.
The indirect ELISA reagent kit of the present invention cannot be only used for determining for pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae serum antibody
Property detection, and available for serum antibody half-quantitative detection.
In the indirect ELISA method that the present invention establishes, existed using thalline ultrasonic degradation albumen as envelope antigen, its advantage
It is simple in envelope antigen preparation process, thalline ultrasonic degradation need to only be produced, and envelope antigen stablizes easy preservation, freezing its
It is multiple to eliminate expression, purifying of the recombinant protein needed for recombinant protein as envelope antigen etc. up to more than 12 months for the term of validity
Miscellaneous process.
Kit of the present invention is to assemble a variety of consumptive materials (ELISA Plate and serum-dilution plate) and various reagents, especially
Envelope antigen in kit is solution dosage, can freezen protective, extend the term of validity of kit, it is unique；Kit
In skimmed milk power sealer for solid dosage forms it is unique；There are two kinds of positive control serum (positive control blood in kit
Clear A and positive control serum B), help to reduce different animals OD450nmBackground value, increase positive and developed the color with negative sample
The difference of the depth, help to visually observe the depth of colour developing, naked eyes judgement is carried out to testing result；The operating procedure of the kit
With characteristics such as novelty, simplicity.
The present invention is using the multiple direct immunization pig of erysipelothrix ruhsiopathiae YN15075 thalline of Formalin inactivation and goat point
Positive control serum A and positive control serum B are not prepared, eliminates the complex process for adding expensive immunologic adjuvant, its mistake
Journey has novelty.It is that make use of pig and goat quick to erysipelothrix ruhsiopathiae to prepare positive control serum from pig and goat in addition
The antigenic good characteristic of the characteristic and erysipelothrix ruhsiopathiae YN15075 of sense.
The present invention is applied to detection many animals erysipelothrix ruhsiopathiae serum antibody from HRP-SPA as secondary antibody
ELISA method, it is that can reach indirect ELISA examination with the characteristic of a variety of mammal IgG antibody specific bonds using SPA
Agent box can detect the purpose of pig, ox, sheep and rabbit many animals serum antibody, add the usage range of the kit.
The method that the present invention judges ELISA testing results using S/P values as critical value, greatly reduces laboratory temperature
Error caused by the factors such as change, operating personnel, operating process, considerably increase the invention ELISA kit stability and
Repeatability.
The present invention has the advantages that sensitive, quick, high flux detects, be economical, easy, available for erysipelothrix ruhsiopathiae serum
Antibody test and epidemiology survey.
Brief description of the drawings
Illustrated embodiment below in conjunction with the accompanying drawings, the concrete application of the kit is further illustrated, but present invention protection model
Enclose not limited to this embodiment.
1~No. 24 blood to be checked of Fig. 1 present invention asks the ELISA testing results of sample antibody.
Wherein, ELISA Plate A1~H1 holes are respectively 1~No. 8 Swine serum antibody ELISA testing result to be checked；A2~D2 holes point
Wei not 9~No. 12 rabbit anteserum antibody ELISA testing results to be checked；A3~H3 holes are respectively 13~No. 20 cow's serum antibody to be checked
ELISA testing results；A4~D4 holes are respectively the ELISA testing results of 21~No. 24 sheep blood serum antibody to be checked.
Embodiment
Embodiment 1：Detect the preparation method of the indirect ELISA reagent kit of many animals erysipelothrix ruhsiopathiae antibody
Kit is by ELISA Plate, serum-dilution plate, envelope antigen, coating buffer solution, positive control serum A, positive control
Serum B, negative control sera, HRP-SPA, skimmed milk power, 100 × tmb substrate liquid, 10 × colour developing dilution, 3% (v/v) H2O2
Solution, terminate liquid, 25 × cleaning solution assemble, and the specific method of various components is as follows：
(1), the preparation of envelope antigen：The good erysipelothrix ruhsiopathiae isolated in Yunnan province YN15075 of screening antigenicity, inoculation
Nutrient agar panel, 37 DEG C of culture 48h, is eluted thalline with sterile saline, adjusts its concentration as 2 × 108CFU/
ML, ice-bath ultrasonic 100 times, ultrasound 10 seconds, are spaced 10 seconds every time, and ultrasonic power is 100 watts, by ultrasonic degradation protein solution
5000r/min centrifuges 10min, and it is ELISA envelope antigens to take supernatant, and it is 0.2mg/mL to determine its protein concentration with BCA methods, is added
Enter final concentration of 0.01% (m/v) thiomersal preservative, the μ L of envelope antigen 100 are housed in each kit, -20 DEG C of preservations are standby
With；
(2), it is coated with the preparation of buffer solution：By NaHCO30.16g, NaHCO3 0.29g are dissolved in the preparation of 100mL ultra-pure waters
Gained, equipped with coating buffer solution 100mL in each kit, 4 DEG C save backup；
(3), positive control serum A preparation：The good erysipelothrix ruhsiopathiae YN15075 separation strains of antigenicity are inoculated with battalion
Agar plate is supported, 37 DEG C of culture 48h, thalline is eluted with sterile saline, adjusts its concentration as 2 × 1010CFU/mL,
Add 37 DEG C of inactivation 6h of final concentration of 0.05% (v/v) formalin and prepare erysipelothrix ruhsiopathiae immunizing antigen；Take 2mL immune anti-
Former musculi colli injects the sodium selenite of the 1 first non-vaccine inoculation of 70 age in days, immune once at interval of 7d, each immunization method phase
Together, it is immunized 4 times altogether, 10d carries out vena cava anterior blood sampling after the 4th is immune, and 5000r/min centrifugation 10min, it is the positive to take supernatant
Control serum A, final concentration of 0.01% (m/v) thiomersal preservative is added, positive control serum A is housed in each kit
200 μ L, -20 DEG C save backup；
(4), positive control serum B preparation：Take the erysipelothrix ruhsiopathiae immunizing antigen 2mL musculi collis in above-mentioned (3)
The healthy goat of 1 10 monthly ages non-vaccine inoculation is injected, is immunized once at interval of 7d, each immunization method is identical, and 4 are immunized altogether
Secondary, 10d carries out jugular vein blood collection after the 4th is immune, and 5000r/min centrifugation 10min, it is positive control serum B to take supernatant, is added
Enter final concentration of 0.01% (m/v) thiomersal preservative, equipped with the μ L of positive control serum B 200 in each kit, -20 DEG C
Save backup；
(5), the preparation of negative control sera：From the healthy goat of 1 10 monthly ages non-vaccine inoculation, isolated rearing
10d carries out jugular vein blood collection, and 5000r/min centrifugation 10min, it is negative control sera to take supernatant, is added final concentration of
0.01% (m/v) thiomersal preservative, the μ L of negative control sera 200 are housed in each kit, -20 DEG C save backup；
(6), the preparation of HRP-SPA secondary antibodies：The SPA freeze-dried powders of HRP marks are purchased from KPL companies of the U.S., are gone out with 50% (v/v)
Bacterium glycerine dissolved dilution adds final concentration of 0.01% (m/v) thiomersal preservative, filled in each kit to 0.1mg/mL
There are the μ L of HRP-SPA 100, -20 DEG C save backup；
(7), skimmed milk power：Purchased from U.S. company BD, skimmed milk power 5g is housed in each kit, room temperature preservation is standby；
(8), the preparation of 100 × tmb substrate liquid：0.1g TMB (3,3', 5,5'- tetramethyl benzidine) are dissolved in 10mL
Dimethyl sulfoxide (DMSO) prepares gained, 100 × tmb substrate liquid 1mL is housed in each kit, 4 DEG C save backup, wherein TMB and two
Methyl sulfoxide is purchased from Sigma Co., USA；
(9), the preparation of 10 × colour developing dilution：It is to obtain citric acid by the way that 21g citric acids are dissolved in into 100mL ultra-pure waters
Solution；8.2g sodium acetates are dissolved in 100mL ultra-pure waters and obtain sodium acetate solution；Above-mentioned 3.5mL citric acid solutions are taken to add again
Mixed into 100mL sodium acetate solutions and produce 10 × colour developing dilution, 10 × colour developing dilution 10mL is housed in each kit,
4 DEG C save backup；
(10), 3%H2O2The preparation of solution：3% (v/v) H2O2Purchased from Sigma Co., USA, it is equipped with each kit
3% (v/v) H2O2Solution 0.5mL, room temperature preservation are standby；
(11), the preparation of terminate liquid：The 22mL concentrated sulfuric acids are slowly added into 178mL ultra-pure waters along bottle wall and prepare gained,
Liquor 100mL is terminated equipped with sulfuric acid in each kit, room temperature preservation is standby, and wherein the concentrated sulfuric acid has purchased from Chongqing Chuan Dong chemical industry
Limit company；
(12), the preparation of 25 × lavation buffer solution：By disodium hydrogen phosphate (Na2HPO4·12H2O) 7.3g, potassium dihydrogen phosphate
(KH2PO4) 0.5g, potassium chloride (KCl) 0.5g, sodium chloride (NaCl) 20g, polysorbas20 (Tween-20) 1.3mL be dissolved in 100mL
Ultra-pure water prepares gained, 25 × lavation buffer solution 120mL is housed in each kit, room temperature preservation is standby；
(13), ELISA Plate：Purchased from NUNC companies of Denmark, 5 pieces of ELISA Plates are housed in each kit, room temperature preservation is standby；
(14), serum-dilution plate：Purchased from clean special (JET) the biofiltration Co., Ltd in Guangzhou, 5 pieces are equipped with each kit
Serum-dilution plate.
Embodiment 2：Using indirect ELISA reagent kit detection pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae antibody method
The indirect ELISA reagent kit cannot be only used for the qualitative inspection of pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae serum antibody
Survey, and available for the half-quantitative detection of serum antibody.Have sensitive, quick, high flux detection, economy and simplicity etc. excellent simultaneously
Point, available for erysipelothrix ruhsiopathiae Serum Antibody Detection and epidemiology survey.Treated with 1~No. 8 Swine serum to be checked, 9~No. 12
Examine the antibody ELISA detection of totally 24 parts of blood serum samples of rabbit anteserum, 13~No. 20 cow's serums to be checked, 21~No. 24 sheep blood serums to be checked
Comprise the following steps that：
(1), antigen coat ELISA Plate：Take 12.5 μ L envelope antigens to be added in 5mL coating buffer solutions and mix, ELISA Plate
The μ L of envelope antigen 100 of above-mentioned dilution are added in 1st~4 each hole of row, 4 DEG C of coatings are overnight；
Before next-step operation is carried out, following 3 kinds of reagents need to be configured：1. 1 × lavation buffer solution must be prepared：Take in kit
25 × lavation buffer solution 8mL is added to mix in 192mL ultra-pure waters and produced；2. the preparation of confining liquid：Take the degreasing in kit
Milk powder 0.2g is added to mix in the above-mentioned 1 × lavation buffer solutions of 5mL and produced；3. the preparation of serum/secondary antibody dilution：Take kit
In skimmed milk power 0.1g be added to mix in the above-mentioned 1 × lavation buffer solutions of 25mL and produce；
(2), board-washing：ELISA Plate the 1st~4, which arranges, adds 400 μ 1 × lavation buffer solutions of L in each hole, stand 1min, and suction is abandoned each
Cleaning solution in hole, 400 μ 1 × lavation buffer solutions of L are added in each hole, stand 1min, inhale the cleaning solution abandoned in each hole, repeated
Above-mentioned washing process 5 times；
(3), close：ELISA Plate the 1st~4 is arranged in each hole and adds 100 μ L confining liquids, and 1h is closed in 37 DEG C of wet box.
(4), board-washing：ELISA Plate the 1st~4 is arranged into each hole by plate washing method in above-mentioned (2) to wash 5 times；
Before next-step operation is carried out, tested serum, positive control serum and negative control sera need to be carried out to 1/30 times in advance
Dilution, its specific method are as follows：1. pig blood asks the dilution of sample：It is dilute that 87 μ L serum are added in serum-dilution plate the 1st~4 arranges each hole
Liquid is released, then 3 μ L, 1~No. 8 Swine serum to be checked is separately added into A1~H1 holes；2. rabbit blood asks the dilution of sample：In A2~D2 holes point
3 μ L, 9~No. 12 rabbit anteserums to be checked are not added, and each add in 3 μ L positive control serums A, G2~H2 holes respectively adds in E2~F2 holes
3 μ L negative control seras；3. ox blood asks the dilution of sample：3 μ L, 13~No. 20 cow's serums to be checked are sequentially added in A3~H3 holes；
4. sheep blood asks the dilution of sample：3 μ L, 21~No. 24 sheep blood serums to be checked are separately added into A4~D4 holes, 3 are respectively added in E4~F4 holes
3 μ L negative control seras are respectively added in μ L positive control serums B, G4~H4 holes, above-mentioned serum-dilution plate is placed on oscillator
Concussion 2min can be used to test in next step；
(5), primary antibody is incubated：Add 90 μ L serum dilutions in ELISA Plate the 1st~4 arranges each hole, then by serum-dilution plate
Pre-dilution serum in A1~H4 holes respectively takes 10 μ L to be correspondingly added in ELISA Plate A1~H4 holes, and ELISA Plate is placed on oscillator
2min is shaken, 1h is incubated in 37 DEG C of wet box；
(6), board-washing：ELISA Plate the 1st~4 is arranged into each hole by plate washing method in above-mentioned (2) to wash 5 times；
Before next-step operation is carried out, HRP-SPA secondary antibodies need to be subjected to pre-dilution：The row of ELISA Plate the 1st~2 are used in 16 holes totally
Swine serum and rabbit anteserum antibody are detected, the HRP-SPA of the dilutions of 4mL 1/60000 need to be prepared, it is comprised the following steps that：First take 2 μ L
HRP-SPA is added in 58 μ L 50% (v/v) sterile glycerols and mixed；The HRP-SPA of above-mentioned 1/30 dilutions of 2 μ L is taken to be added to again
The HRP-SPA of 1/60000 dilution is produced in 4mL antibody diluents.Totally 16 holes are used to detect cow's serum the row of ELISA Plate the 3rd~4
With sheep blood serum antibody, the HRP-SPA of the dilutions of 4mL 1/2000 need to be prepared, it is comprised the following steps that：2 μ L HRP-SPA are taken to be added to
The HRP-SPA of 1/2000 dilution is produced in 4mL antibody diluents；
(7), secondary antibody is incubated：ELISA Plate the 1st~2 arranges in each hole and adds above-mentioned 1/60000 prediluted HRP-SPA of 100 μ L
Secondary antibody, for pig and rabbit anteserum antibody test；It is prediluted that 100 μ L above-mentioned 1/2000 are added in 3rd~4 each hole of row ELISA Plate
HRP-SPA secondary antibodies, for ox and sheep blood serum antibody test, 1h is incubated in 37 DEG C of wet box；
(8), board-washing：ELISA Plate the 1st~4 is arranged into each hole by plate washing method in above-mentioned (2) to wash 5 times.
Before next-step operation is carried out, 1 × nitrite ion need to be prepared, it is comprised the following steps that：Take 1mL 10 × colour developing dilution
It is added in 9mL ultra-pure waters and mixes, adds 100 μ 100 × tmb substrates of L liquid and 35 μ L 3% (v/v) H2O2Solution mixes
Obtain 1 × nitrite ion；
(9), develop the color：ELISA Plate the 1st~4 arranges in each hole and adds the 100 above-mentioned 1 × nitrite ions of μ L, color development at room temperature 10min；
(10), color development stopping is reacted：ELISA Plate the 1st~4 arranges in each hole and adds 100 μ L terminate liquids；
(11), OD is read with ELIASA450nmValue：It the results are shown in Table 1；
The pig of table 1, ox, the OD values of sheep and rabbit anteserum sample
(12) S/P values, are calculated：ELISA Plate the 1st~2 arranges each hole and is respectively used to 1~No. 8 Swine serum of detection and 9~No. 12 rabbits
Serum antibody, its 2 common hole positive control serum A OD450The average value of nm values (E2~F2 holes) is 1.107, more than 0.8,
Its common negative control sera OD450The average value of nm values (G1~H1) is 0.048, less than 0.08, this detection pig and rabbit blood
The ELISA experiments of clear antibody are effective；The OD of 1~No. 8 Swine serum450Nm values (A1~H1 holes) divided by 1.107 produce 1~No. 8 pig blood
Clear S/P values are respectively 1.010,0.766,0.743,0.041,0.911,0.995,0.045 and 0.041.9~No. 12 rabbit anteserums
OD450Nm values (A2~D2 holes) divided by the 1.107 S/P values for producing 9~No. 12 rabbit anteserums are respectively 0.858,0.138,0.041
With 0.044.Each hole of the row of ELISA Plate the 3rd~4 is respectively used to 13~No. 20 cow's serums of detection and 21~No. 24 sheep blood ask antibody, and it is common
The OD of 2 same hole positive control serum B450The average value of nm values (E4~F4 holes) is 1.106, more than 0.8, its common feminine gender
Control serum OD450The average value of nm values (G4~H4) is 0.051, less than 0.08, this detection ox and sheep blood serum antibody
ELISA experiments are effective；13~No. 24 blood are asked into the OD values (A3~D4) of sample divided by 1.106 produce what 13~No. 24 blood to be checked were asked
S/P values are respectively 0.454,0.359,0.041,0.040 etc. (table 2).
The pig of table 2, ox, the S/P values of sheep and rabbit anteserum sample
(13), result judgement：Pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae antibody positive standard are respectively S/P≤0.30, S/P
≤ 0.28, S/P≤0.26 and S/P≤0.33, standard is to 8 parts of Swine serums, 4 parts of rabbit anteserums, 8 parts of cow's serums and 4 parts of sheep blood accordingly
Final proof product erysipelothrix ruhsiopathiae antibody is judged.Such as：The S/P values of No. 1 pig anteserum sample (A1 holes) are 1.010, more than pig
Seropositivity critical value 0.30, it is determined as erysipelothrix rhusiopathiae antibody positive；The S/P values of No. 4 pig anteserum samples (D1 holes) are
0.041, less than Swine serum antibody positive critical value 0.30, it is determined as erysipelothrix rhusiopathiae negative antibody；No. 9 rabbit anteserum samples
S/P values (A2 holes) be 0.859, more than rabbit anteserum antibody positive critical value 0.33, be determined as rabbit erysipelothrix ruhsiopathiae antibody sun
Property；The S/P values (B2 holes) of No. 10 rabbit anteserum samples are 0.138, less than rabbit anteserum antibody positive critical value 0.33, are determined as that rabbit is red
Spot Erysipelothrix negative antibody；The S/P values (A3 holes) of No. 13 cow's serums are 0.454, more than cow's serum antibody positive critical value
0.28, it is determined as ox erysipelothrix ruhsiopathiae antibody positive；The S/P values (D4 holes) of No. 24 sheep blood serums are 0.643, are resisted more than sheep blood serum
Body positive critical value 0.26, it is determined as sheep erysipelothrix ruhsiopathiae antibody positive；By this decision method to other 18 parts of serum erythema
Erysipelothrix antibody is judged, produces 24 parts of blood serum sample erysipelothrix ruhsiopathiae antibody test results (table 3).
The pig of table 3, ox, sheep and rabbit anteserum sample erysipelothrix ruhsiopathiae antibody test result
Claims (4)
1. a kind of indirect ELISA reagent kit for detecting many animals erysipelothrix ruhsiopathiae antibody, it is characterised in that indirect ELISA tries
Agent box is by ELISA Plate, serum-dilution plate, envelope antigen, coating buffer solution, positive control serum A, positive control serum B, feminine gender
Control serum, the staphylococcal protein A (HRP-SPA) of horseradish peroxidase-labeled, skimmed milk power, 100 × tmb substrate liquid,
10 × colour developing dilution, 3% (v/v) H2O2 solution, terminate liquid and 25 × cleaning solution assemble.
A kind of 2. preparation method for the indirect ELISA reagent kit for detecting many animals erysipelothrix ruhsiopathiae antibody, it is characterised in that
Preparation process is：
(1), envelope antigen is the erysipelothrix ruhsiopathiae isolated in Yunnan province YN15075 good by screening antigenicity, is inoculated with nutrition
Agar plate, 37 DEG C of culture 48h, is eluted thalline with sterile saline, it is 2 × 108CFU/mL to adjust its concentration, ice
Bath ultrasound 100 times, ultrasound 10 seconds, are spaced 10 seconds every time, and ultrasonic power is 100 watts, by ultrasonic degradation protein solution
5000r/min centrifuges 10min, and it is ELISA envelope antigens to take supernatant, and it is determined with BCA (bicinchoninic acid) method
Protein concentration is 0.2mg/mL, adds final concentration of 0.01% (m/v) thiomersal preservative, -20 DEG C save backup；
(2) it is by by 0.16g NaHCO3,0.29g NaHCO3, to be coated with buffer solution (pH9.6 0.05M carbonate buffer solutions)
It is dissolved in 100mL ultra-pure waters and prepares gained, 4 DEG C saves backup；
(3), positive control serum A is by the way that the good erysipelothrix ruhsiopathiae YN15075 separation strains of antigenicity are inoculated with into nutrition fine jade
Fat flat board, 37 DEG C of culture 48h, is eluted thalline with sterile saline, it is 2 × 1010CFU/mL to adjust its concentration, is added
Enter 37 DEG C of inactivation 6h of final concentration of 0.05% (v/v) formalin and prepare erysipelothrix ruhsiopathiae immunizing antigen；Take 2mL immunizing antigens
Musculi colli injects the sodium selenite of the 1 first non-vaccine inoculation of 70 age in days, is immunized once at interval of 7d, and each immunization method is identical,
It is immunized 4 times altogether, 10d vena cava anteriors are taken a blood sample after the 4th is immune, and centrifugation prepares positive control serum A, and 4 DEG C save backup；
(4), positive control serum B takes erysipelothrix ruhsiopathiae immunizing antigen 2mL necks in above-mentioned (3) that 1 10 monthly age is subcutaneously injected
The healthy goat of non-vaccine inoculation, it is immunized once at interval of 7d, each immunization method is identical, is immunized 4 times altogether, after the 4th is immune
10d jugular vein blood collections, centrifugation prepare positive control serum B, and 4 DEG C save backup；
(5), negative control sera be by the healthy goat from 1 10 monthly ages non-vaccine inoculation, after isolated rearing 10d, neck
Venous blood collection, centrifugation prepare negative control sera, and 4 DEG C save backup；
(5), HRP-SPA freeze-dried powders are purchased from KPL companies of the U.S., with 50% (v/v) sterile glycerol dissolved dilution to 0.1mg/mL, add
Enter final concentration of 0.01% (m/v) thiomersal preservative, -20 DEG C save backup；
(7), skimmed milk power is purchased from U.S. company BD, and room temperature preservation is standby；
(8), 100 × tmb substrate liquid (1%TMB) is by the way that 0.1g TMB (3,3', 5,5'- tetramethyl benzidine) are dissolved in
10mL dimethyl sulfoxide (DMSO)s (abbreviation DMSO) prepare gained, and 4 DEG C save backup；
(9), 10 × colour developing dilution (1mol/L sodium acetates/citric acid) is by the way that 21g citric acids are dissolved in into 100mL ultra-pure waters
Citric acid solution is obtained, 8.2g sodium acetates are dissolved in into 100mL ultra-pure waters obtains sodium acetate solution, then takes above-mentioned 3.5mL lemons
Acid solution, which is added in 100mL sodium acetate solutions, prepares gained, and 4 DEG C save backup；
(10), 3%H2O2 solution is purchased from Sigma Co., USA, and room temperature preservation is standby；
(11), terminate liquid (2mol/L H2SO4) is by the way that the 22mL concentrated sulfuric acids are slowly added into 178mL ultra-pure waters along bottle wall
Gained is prepared, 4 DEG C save backup, and wherein the concentrated sulfuric acid is purchased from Chongqing Chuan Dong Chemical Co., Ltd.s；
(12), 25 × lavation buffer solution is by by disodium hydrogen phosphate (Na2HPO412H2O) 7.3g, potassium dihydrogen phosphate
(KH2PO4) 0.5g, potassium chloride (KCl) 0.5g, sodium chloride (NaCl) 20g, polysorbas20 (Tween-20) 1.3mL are dissolved in 100mL
Ultra-pure water prepares gained, and room temperature preservation is standby；(13), ELISA Plate is purchased from NUNC companies of Denmark, and room temperature preservation is standby；(14), blood
It is thin to release plate：It is standby purchased from clean special (JET) the biofiltration Co., Ltd in Guangzhou, room temperature preservation.
A kind of 3. application method for the indirect ELISA reagent kit for detecting many animals erysipelothrix ruhsiopathiae antibody, it is characterised in that
Concrete operation step is：
(1), antigen coat ELISA Plate：Envelope antigen coating buffer solution 1/400 is diluted, added in each hole of ELISA Plate above-mentioned dilute
The μ L of envelope antigen 100 released, 4 DEG C of coatings are overnight；Before next-step operation is carried out, following 3 kinds of reagents need to be configured：1. 1 × washing is slow
Fliud flushing must be prepared：1/25 times of 25 × lavation buffer solution progress appropriate in kit is diluted in ultra-pure water and produced；2. close
The preparation of liquid：Appropriate skimmed milk power in kit is diluted in above-mentioned 1 × lavation buffer solution, it is 4% (m/ to make its concentration
V) produce；3. the preparation of serum/secondary antibody dilution：The appropriate skimmed milk power in kit will be taken to be diluted in above-mentioned 1 × washing
In buffer solution, its concentration is set to be produced for 0.4% (m/v)；
(2), board-washing：Each hole of ELISA Plate adds 400 μ L lavation buffer solutions, stands 1min, inhales the cleaning solution abandoned in hole, each Kong Zhongzai
400 μ L lavation buffer solutions are added, stand 1min, inhale the cleaning solution abandoned in hole, the above-mentioned washing process of repetition 5 times；
(3), close：Each ELISA Plate hole adds 100 μ L confining liquids, and 1h is closed in 37 DEG C of wet box；
(4), board-washing：Each hole of enzyme mark is washed 5 times by plate washing method in above-mentioned (2)；Before next-step operation is carried out, serum need to be used dilute
Release plate and serum is subjected to 1/30 times of pre-dilution, such as：The 3 above-mentioned serum of μ L are taken to be added to mix in 87 μ L serum dilutions and produce；
(5), primary antibody is incubated：First in each ELISA Plate hole per 90 μ L serum dilutions of middle addition, by 1/ in above-mentioned serum-dilution plate
30 times of prediluted serum to be checked, positive control serum, 10 μ L of the corresponding transfer of negative control sera are into above-mentioned ELISA Plate hole, and 37
1h is incubated in DEG C wet box；
(6), board-washing：Each hole of ELISA Plate is washed 5 times by plate washing method in above-mentioned (2)；, need to be by HRP- before next-step operation is carried out
SPA secondary antibodies carry out pre-dilution：Need HRP-SPA carrying out 1/60000 dilution if for pig and rabbit anteserum sample detection；Such as it is used for
Ox and sheep blood serum sample detection, then need HRP-SPA carrying out 1/2000 dilution；
(7), secondary antibody is incubated：The above-mentioned prediluted HRP-SPA secondary antibodies of 100 μ L are added in each hole of ELISA Plate, are incubated in 37 DEG C of wet box
1h；
(8), board-washing：Each hole of enzyme mark is washed 5 times by plate washing method in above-mentioned steps (2)；
Before next-step operation is carried out, 1 × nitrite ion need to be prepared, it is comprised the following steps that：Take 10 × colour developing appropriate in kit
Dilution, which is added to mix in the ultra-pure water of 9 times of volumes, produces 1 × colorbuffer；Again in every 10mL above-mentioned 1 × colour developing buffering
100 μ 100 × tmb substrates of L liquid are added in liquid and 35 μ L 3% (v/v) H2O2 solution mix and produce 1 × nitrite ion；
(9), develop the color：100 μ 1 × nitrite ions of L, color development at room temperature 10min are added in each hole of ELISA Plate；
(10), color development stopping is reacted：Each hole of ELISA Plate adds 100 μ L terminate liquids；
(11) OD450nm values, are read with ELIASA and calculate S/P values, wherein S represents tested serum OD450nm values, and P represents 2 holes sun
The average value of property control serum OD450nm values；
(13), result judgement：On the premise of the validity of guarantee test, four kinds of pig, ox, sheep and rabbit animal Erysipelothrix are judged
The positive standard of silk bacteria antibody is respectively S/P≤0.30, S/P≤0.28, S/P≤0.26 and S/P≤0.33, is otherwise determined as the moon
Property, the kit can be used for pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae Serum Antibody Detection and epidemiology survey；Before its is described
Put be positive control serum OD450nm values Ping Jun Zhi≤0.8, and Ping Jun Zhi of negative control sera OD450nm values≤
0.08 this experiment side is effective；Otherwise it is invalid, one-time detection should be reformed.
A kind of 4. application for the indirect ELISA reagent kit for detecting many animals erysipelothrix ruhsiopathiae antibody, it is characterised in that the examination
Agent box is used to detect pig, ox, sheep and rabbit totally four kinds of animal erysipelothrix ruhsiopathiae IgG antibodies, and the result of the kit judges
Standard：On the premise of the validity of guarantee test, pig, ox, sheep and rabbit erysipelothrix ruhsiopathiae antibody positive standard are respectively S/P
≤ 0.30, S/P≤0.28, S/P≤0.26 and S/P≤0.33, wherein S represent the OD450nm values of tested serum, and P represents 2 holes sun
The average value of the OD450nm values of property control serum, is otherwise determined as feminine gender, the kit can be used not only for erysipelothrix ruhsiopathiae blood
Clear antibody qualitative detection, and the half-quantitative detection of serum antibody can be used for and applied to the detection of erysipelothrix ruhsiopathiae immune antiboidy and
Epidemiology survey；It is Ping Jun Zhi≤0.8 of positive control serum OD450nm values under the premise of it is described, and negative control blood
This experiment side of Ping Jun Zhi≤0.08 of clear OD450nm values is effective；Otherwise it is invalid, one-time detection should be reformed.
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