CN107219364A - Detect indirect ELISA reagent kit and its detection method and its application of A type haemophilus paragallinarum antibody - Google Patents
Detect indirect ELISA reagent kit and its detection method and its application of A type haemophilus paragallinarum antibody Download PDFInfo
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Abstract
The invention discloses a kind of indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody and its detection method and its application, it is related to technical field of biological.The ELISA kit of the present invention includes：Coated elisa plate, cleaning solution, serum dilution, substrate nitrite ion, rabbit-anti chicken ELIAS secondary antibody, terminate liquid, A type Hpg standard positive serums and A type Hpg standard female serum；Wherein, the coated elisa plate is to be used as envelope antigen using A types haemophilus paragallinarum restructuring hemagglutinin.The ELISA kit of the present invention is used for the antibody for detecting A type haemophilus paragallinarums, has the advantages that efficient, sensitive specificity and repeatability are good, and the kit is easy to operate, quick and with low cost, it is adaptable to clinical practice and popularization.
Description
Technical field
The present invention relates to technical field of biological, more particularly to a kind of detection A type haemophilus paragallinarum antibody is indirect
ELISA kit and its detection method and its application.
Background technology
Haemophilus paragallinarum (Haemophilus paragallinarum, Hpg) is the cause of disease of infectious coryza of chicken, the disease
Clinical symptoms are the acute upper respiratory infection of chicken, nasal cavity and nasal sinus inflammation, eye conjunctiva inflammation, are shed tears, and diseased chicken egg production declines,
It is slow-growing.Infectious coryza of chicken takes place frequently in each area in the world, in endemicity, although the sick fatal rate is relatively
It is low, but haemophilus paragallinarum can cause laying rate to reduce in chicken group's long-term existence, the production performance of reduction chicken, Growing Chicken growth
It is slow, and the ability that diseased chicken resists Other diseases is reduced, easily secondary other infectious diseases in the case where resistance is weaker
And it is dead, huge economic loss is caused to raiser, the development of aquaculture is seriously hindered.
Research data at present on haemophilus paragallinarum molecule aetology is also seldom, and relevant report concentrates on diagnosis and treatment.It is right
It is unsuitable big in evaluation method such as clinical diagnosis, hemagglutination test etc. of the diagnostic method and immune effect of vaccine of infectious coryza of chicken
The sample detection of scope, for infectious coryza of chicken epidemic monitoring and effective assessment of vaccine be required to set up it is more sensitive,
Efficiently detection method.Hemagglutinin (HA) is important composition in Hpg antigens, is also the basis of Hpg partings, with preferably anti-
Former reactivity, can react with Hpg positive serum, epidemiology detection and immune effect for infectious coryza of chicken
Monitoring be with a wide range of applications.
The content of the invention
In view of this, the embodiments of the invention provide a kind of indirect ELISA reagent of detection A type haemophilus paragallinarum antibody
Box and its detection method and its application, main purpose are to provide a kind of easy, quick and accurate detection A types haemophilus paragallinarum and resisted
The method of body.
To reach above-mentioned purpose, invention broadly provides following technical scheme：
On the one hand, the embodiments of the invention provide a kind of indirect ELISA reagent of detection A type haemophilus paragallinarum antibody
Box, the kit includes：Coated elisa plate, cleaning solution, serum dilution, substrate nitrite ion, rabbit-anti chicken ELIAS secondary antibody, termination
Liquid, A type Hpg standard positive serums and A type Hpg standard female serum；Wherein, the coated elisa plate is bloodthirsty with the secondary chicken of A types
Bacillus restructuring hemagglutinin is used as envelope antigen.
Preferably, the nucleotides sequence list of the A types haemophilus paragallinarum restructuring hemagglutinin is
SEQ.ID.No.1。
As preferred, the preparation method of the A types haemophilus paragallinarum restructuring hemagglutinin includes：
Synthetic primer is designed, the genome using A type haemophilus paragallinarum Hpg-8 bacterial strains is obtained as template by PCR amplifications
The amplified production of hemagglutinin, the amplified production connects its expression vector and while carries out double digestion with construction recombination plasmid；Will
Positive recombinant plasmid conversion e. coli bl21 (DE3) competent cell selected, obtains positive plasmid monoclonal bacterium, the sun
Property grain monoclonal bacterium carries out protein expression under IPTG inductions and obtains induced product, and the induced product is purified successively and multiple
Property after obtain recombinate hemagglutinin；Wherein, the synthetic primer is：
Sense primer：5'-CGCGGATCCATGAAAAAAACTGCAATCG-3',
Anti-sense primer：5'-CCGCTCGAGCTCGTTACCTTTAACTGAGAT-3'.
Preferably, the cleaning solution is the phosphate buffer containing 0.05% Tween-20, pH is 7.4.
As preferred, the serum dilution is the phosphate buffer of the skimmed milk power containing 50g/L；The substrate nitrite ion
For the tetramethyl biphenyl amine aqueous solution containing hydrogen peroxide.
As preferred, the terminate liquid is 2M sulfuric acid solutions.
As preferred, the coating concentration of the envelope antigen is 5.0 μ g/mL；The coated elisa plate is through 50g/L defatted milks
Powder PBST is closed, and is fitted into hermetic bag and is placed in 4 DEG C of preservations.
As preferred, the extension rate of the rabbit-anti chicken ELIAS secondary antibody is 1:2500.
On the other hand, the embodiments of the invention provide the indirect ELISA reagent of above-mentioned detection A types haemophilus paragallinarum antibody
The detection method of box, the detection method comprises the following steps：
With serum dilution with volume ratio 1：200 dilution proportion serum to be checked obtains dilute serum；
Dilute serum described in 100 μ is added into the every hole of 96 hole elisa Plates, holes A type Hpg standard males are respectively added to ELISA Plate
Property serum and A types Hpg standard females serum as control, 1h are incubated in 37 DEG C, liquid in reacting hole is discarded, then added to every hole
300 μ L cleaning solutions are simultaneously washed 3 times, 5 minutes every time, are abandoned cleaning solution, are patted dry for the last time；
100 μ L are added with volume ratio 1 into the every hole of 96 hole elisa Plates:Rabbit-anti chicken ELIAS secondary antibody after 2500 dilutions, in 37
DEG C be incubated 1h, discard liquid in reacting hole, added into every hole 300 μ L cleaning solutions and wash 3 times, 5 minutes every time, abandon washing
Liquid, is patted dry for the last time；
100 μ L substrate nitrite ions are added into the every hole of 96 hole elisa Plates, lucifuge is acted on 10 minutes at room temperature；
100 μ L terminate liquids are added to 96 hole elisa Plates per hole to react with color development stopping；
Each hole absorption photometric (OD) value is determined under 450nm wavelength with ELIASA；
Judged according to OD values, work as OD450During value >=0.34, judge that A type Hpg antibody levels, as the positive, work as OD450Value ＜ 0.34
When, judge A type Hpg antibody levels as feminine gender.
Another aspect, the embodiments of the invention provide the indirect ELISA reagent of above-mentioned detection A types haemophilus paragallinarum antibody
Application of the box in detection A type haemophilus paragallinarum antibody.
Compared with prior art, the beneficial effects of the invention are as follows：
The indirect ELISA reagent kit of the present invention, is made using A types haemophilus paragallinarum (Hpg) restructuring hemagglutinin (HA) albumen
For envelope antigen, the albumen is that A types Hpg recombinates HA gene amplification products, using pET-28a-HA as the expression product of expression vector,
Easily prepared and purifying, its immunoreactivity is good；As envelope antigen after restructuring HA protein purifications, its concentration is easy to determine
And control, be conducive to a large amount of productions and cost control of the kit.
The kit of the present invention is used for the antibody for detecting A type haemophilus paragallinarums, with efficient, sensitive specificity and repetition
Property good advantage, the kit is easy to operate, quick, with low cost, is suitable for being promoted in clinical practice, is A
The quick detection of type haemophilus paragallinarum provides reliable technological means.
Brief description of the drawings
Fig. 1 is the pET-28a-HA/B2L expression bacterium positive monoclonal identification nucleic acid agaroses that the embodiment of the present invention 1 is provided
(in figure, M is DM2000plus DNA Marker to gel electrophoresis figure, and 1 is the analysis of pET-28a-HA double digestions, and 2 be that HA genes expand
Increase production thing)；
Fig. 2 is the induced expression and expression-form for the A type haemophilus paragallinarum HA recombinant proteins that the embodiment of the present invention 1 is provided
Analysis and purifying figure (in figure, M is wide spectrum albumen Marker, and 1 is not induce pET-28a-HA, 2-4 induce respectively 4h, 5h and
6h pET-28a-HA, 5 be the HA recombinant proteins of purifying)；
(in figure, M is pre-dyed albumen Marker to the Western blot qualification figures that Fig. 3 embodiment of the present invention 1 is provided, and 1 is A
Type haemophilus paragallinarum positive serum, 2 be SPF chicken serums).
Embodiment
For further illustrate the present invention to reach the technological means and effect that predetermined goal of the invention is taken, below with compared with
Good embodiment, to embodiment, technical scheme, feature and its effect according to the present patent application, is described in detail as after.Under
State it is bright in multiple embodiments in special characteristic, structure or feature can be combined by any suitable form.
Embodiment 1
The structure of 1.A type haemophilus paragallinarums HA (HA refers to hemagglutinin) protein expression vector：
The genomic DNA of haemophilus paragallinarum Hpg-8 bacterial strains (being purchased from China Veterinary Drugs Supervisory Inst.) is extracted, synthetic primer is designed
HAF:5'-CGCGGATCCATGAAAAAAACTGCAATCG-3'(underscores are BamHI digestions point), HAR:5'-
CCGCTCGAGCTCGTTACCTTTAACTGAGAT-3'(underscores are XhoI restriction enzyme sites), amplification obtains HA gene magnifications production
Thing, amplified production length is 1032bp, and its nucleotide sequence refers to the SEQ.ID.No.1, (amplification of glue reclaim HA genes purpose fragment
Product), cloning vector pEASY-Tl is connected, 16 DEG C of connections are stayed overnight, connection product conversion Trans5 α clone's competent cells, and
The LB flat boards of kalamycin resistance (100mg/ml) are applied to, in 37 DEG C of overnight incubations, picking monoclonal, in kalamycin resistance
LB culture mediums in after shaken cultivation 10h, extract plasmid, digestion identification；
Choose and identify correct pEASY-T1-HA plasmids, pET-28a empty plasmids, respectively with BamHI and XhoI double digestions,
Digestion products enter row agarose gel electrophoresis, glue reclaim pET-28a carrier ribbons and HA gene purpose bands, by recovery product 16
DEG C connection is stayed overnight；Connection product conversion Trans5 α clone's competent cells, routinize selection positive colony, extracts thalline plasmid
Digestion is identified, is accredited as positive plasmid and is sent company to be sequenced；Correct recombinant plasmid will be sequenced and is named as pET-28a-HA, such as Fig. 1
It is shown.
2. the induced expression of recombinant plasmid
Correct recombinant plasmid pET-28a-HA will be built and be transferred to competent escherichia coli cell BL21, routinize selection sun
Property clone, extract the identification of thalline plasmid enzyme restriction, positive plasmid is named as pET-28a-HA/BL21, is forwarded to by volume fraction 1%
The fresh LB culture mediums containing kalamycin resistance of 5mL, shaken cultivation to exponential phase (3.5h or so) adds 0.1%IPTG
Induced, continue Fiber differentiation 6h, take 1ml bacterium solutions, 4 DEG C, 12000r/min centrifugation 5min collection precipitations add 100 μ L
PBS (7.4) phosphate buffer, PH is) is resuspended after thalline, 25 μ L SDS-PAGE sample-loading buffers of addition, boiling water boiling 10min,
12000r/min centrifuges 10min, carries out SDS-PAGE electrophoretic analysis, electrophoresis result is with reference to protein molecular quality standard, it is seen that lure
It is 37ku to lead expression and obtain albumen size, as shown in Fig. 2 being consistent with predicting the outcome, it was demonstrated that the recombinant plasmid is in Escherichia coli
Induced expression is obtained；
1ml is taken to induce 6h bacterium solution, 8000r/min centrifugations 5min collects bacterial sediment, adds 100 μ L PBS and be resuspended, surpass
Raw cracking, 4 DEG C, 12000r/min centrifugation 10min centrifugation supernatants and inclusion body, is distinguished with SDS-PAGE sample-loading buffers
Supernatant and precipitation are handled, SDS-PAGE electrophoretic analysis is carried out, is expressed in precipitation by the visible restructuring HA albumen of electrophoresis result.
The Western blot experiments of 3.A types haemophilus paragallinarum restructuring HA albumen
6h bacterium solutions after induction are taken to carry out PAGE-SDS electrophoresis, it is not dyed by the PAGE-SDS running gels after electrophoresis, directly
Connect and be transferred to albumen on PADF films with 150mA with transfer device, transfer 1.5h, it is careful to take out PADF films after transferring film is finished,
Rinsed in TBST, be placed in room temperature closing 1h (slow to shake) in confining liquid (TBST for containing 2.5% skimmed milk power)；Abandon closing
PADF films after liquid, incubation are washed 3 times with TBST, each 10min, and PADF films are put into confining liquid properly than the primary antibody of row dilution
In (chicken A type Hpg positive serums), 4 DEG C are incubated overnight (slow shake), abandon primary antibody, and the PADF films TBST after incubation washes 3 times, often
Secondary 10min, is put into PADF films the secondary antibody (the anti-chicken lgG secondary antibodies of enzyme mark) diluted with confining liquid, steady to shake, and is incubated at room temperature 1h.
Secondary antibody is abandoned, is washed with PADF films with TBST 3 times, each 10min, is developed the color according to eECL Western Blot kit specifications,
Darkroom tabletting exposure.
4. recombinate the purification renaturation of HA albumen
PET-28a-HA/BL21 is added into 5mL LB culture mediums, activation culture is stayed overnight, take 5mL bacterium solutions to add 500mL LB
In culture medium, culture to logarithmic phase is added after 0.1%IPTG (IPTG refers to isopropylthiogalactoside, a kind of derivant),
Fiber differentiation 6h, collects bacterium solution 5000rpm centrifugation 10min, abandons supernatant, add 200mLPBS (phosphate buffer, Ph=
7.4) it is resuspended, 5000rpm centrifuges 10min again, abandons supernatant；Thalline is resuspended in the bacterial lysate for adding 50mL, excusing from death is broken,
12000rpm centrifuges 10min, collects precipitation；It is resuspended and is precipitated with inclusion body cleaning solution, 12000rpm centrifugation 10min abandons supernatant, altogether
Wash 3 times；The solubilization of inclusion bodies liquid of the 5mL urea containing 8mol/L is added, room-temperature dissolution 30min, 12000rpm centrifugation 10min takes
Supernatant is filtered with 0.45 μm of filter, is added to ProteinIso Ni-NTA and is purified, is washed with 180mmol/L imidazoles
Wash, collect 1mL cleaning solutions, eluted with 300mmol/L eluent, 1mL eluent is collected every time, is even collected 10 times,
Examined through SDS-PAGE, it is seen that purification effect is good, the albumen is easily prepared and purifies；
A certain amount of albumen is fitted into the bag filter that trapped molecular weight is 15ku, with the urea (6,4,2 of various concentrations
And 1mol/L) dialyzate A, dialyzate B (20mmol/L Tris-HCI, 0.6mmol/L L-arginines, 10% glycerine,
2mmol/L EDTA, 0.5mol/L urea, pH 8.0) and dialyzate C (20mmol/L Tris-HCI, 25mmol/L NaC1,
0.2mol/L urea) in carry out the gradient renaturation of albumen, totally six stages, each stage 12h, in 4 DEG C of slow magnetic agitations；
Dialysis terminates, and collects recombinant protein, and it is 1mg/mL to determine protein concentration with trace of albumin concentration measuring apparatus, it is seen that the albumen is easy
In purification renaturation.
5. the preparation of sample diluting liquid, cleaning solution, terminate liquid
Coating buffer is 0.05M carbonate buffer solutions：Na2CO31.59g, NaHCO32.93g, plus distilled water are settled to
1000mL, PH are 9.6；
Sample diluting liquid is the cleaning solution of the skimmed milk power containing 50g/L；
Cleaning solution is the phosphate buffer that the 0.01M pH containing 0.05%Tween-20 are 7.4, and its formula is：
Nacl：8.5g, NaH2PO42H2O：0.356g, NaH2PO412H2O：2.772g, after being mixed, then is used
Distilled water dissolves and is settled to 1000mL, then addition 0.5mL Tween-20s obtain above-mentioned cleaning solution thereto；
Terminate liquid is sulfuric acid solution containing 2M：21.8mL diluting concentrated sulfuric acids are settled to 200mL and obtain above-mentioned 2M sulfuric acid solutions.
6. the determination of the most suitable coating concentration of antigen (restructuring HA albumen) and the optimal dilution of serum
The most suitable coating concentration of antigen is determined using orthogonal test, serum optimal dilution and ELIAS secondary antibody best effort are dense
Degree；Restructuring HA albumen after purification renaturation is diluted to 2.5 μ g/mL, 5.0 μ g/mL and 7.5 μ g/mL tri- with coating buffer successively
Dilution factor, is added drop-wise in 96 hole polystyrene micro-reaction plates with the amount in 100 μ L/ holes, constitutes square formation, and 4 DEG C of coatings are stayed overnight；Discard
Antigen liquid, is fully washed 3 times, 300 μ L/ holes, 5min/ times with cleaning solution, is closed with 50g/L skimmed milks, 200 μ L/ holes, 37 DEG C of works
Use 1h；Confining liquid is discarded, is washed in aforementioned manners；A type Hpg standard positive serums, negative serum are once done 1:50、1:
100、1:200、1:After 400 times of dilutions, it is added drop-wise to the amount in 100 μ L/ holes in 96 hole polystyrene micro-reaction plates, 37 DEG C of effects
1h；Serum is abandoned, is washed in aforementioned manners, 1 is added:The ELIAS secondary antibody of 2500 times of dilutions, 96 holes are added drop-wise to the amount in 100 μ L/ holes
In polystyrene micro-reaction plate, 1h is acted in 37 DEG C；ELIAS secondary antibody is discarded, is washed in aforementioned manners, substrate nitrite ion is added,
100 μ L/ holes, 37 DEG C of lucifuges colour developing 10min, ultimately join terminate liquid terminating reaction；With ELIASA OD is determined under 450nm wavelength
Value；Compare the OD of positive and negative serum450Value, that is, select positive OD450Value (P values) is about 1.0, negative sample OD450It is worth (N
Value) 0.1 or so, and P/N values it is maximum when be optimal antigen coat concentration and serum and secondary antibody dilution factor.
Orthogonal experiments show that serum is with 1 when the dilution factor of antigen is 5.0 μ g/mL every holes:200 times of dilutions, enzyme mark two
Resist with 1:During 2500 times of dilutions, positive OD450Value (P values) is about 1.0, negative sample OD450It is worth (N values) 0.1 or so, and
And P/N values are 10.93 maximum, it is thus determined that the optimal coating concentration of restructuring HA albumen is 5.0 μ g/mL, serum optimum dilution degree
For 1:200, ELIAS secondary antibody optimum dilution degree is 1:2500, table 1 below is indirect ELISA orthogonal experiments.
7. the optimization of action time
Based on antigen, serum and ELIAS secondary antibody the best use of concentration, optimize (37 DEG C of the optimal off-period of antigen
1h, 1.5h and 2h), the seroreaction time (37 DEG C of 0.5h, 1h, 1.5h), the optimal incubation time of ELIAS secondary antibody (37 DEG C of 0.5h, 1h,
1.5h) and the condition such as optimal developing time (37 DEG C, 10min, 20min, 30min), positive OD is taken450Value (P values) is about
1.0, negative sample OD450It is worth (N values) 0.1 or so, and is optimal action time during P/N values maximum, it is final to determine to resist
Former optimal off-period is 1h, and the seroreaction time is 1h, and the optimal incubation time of ELIAS secondary antibody is 1h, and optimal developing time is
10min。
8. the determination of yin and yang attribute standard cut-off value
It is negative blood serum sample to take 25 parts of A type haemophilus paragallinarums antibody, is carried out according to the condition of determination indirect
ELISA, OD values are determined with ELIASA under 450nm wavelength, calculate OD450Average valueAnd standard variance (s), To eliminate during each indirect ELISA detection, the influence of experimental enviroment and operation, when detecting every time
Add standard positive serum and standard female serum is used as control, positive serum OD450It is approximately equal to 1.0, otherwise result is invalid；Through
Calculate, 25 parts of negative serum OD450The average value of valueStandard deviation s ≈ 0.05, therefore indirect ELISA yin and yang attribute is critical
ValueTherefore, as sample OD450During value >=0.34, A type haemophilus paragallinarum antibody levels are judged as the positive, when
OD450During value ＜ 0.34, A type haemophilus paragallinarum negative antibodies are judged.
9. the determination (determination of the detection method of kit) of indirect ELISA operation sequence
Every identified optimum operation condition, determines that indirect ELISA operation sequence is more than：Taking-up has been coated with simultaneously
The elisa plate bar closed, 1 is done with serum dilution by serum to be checked：200 times of dilutions, add each hole of ELISA Plate, per the μ of hole 100
L, respectively adds a hole, A types haemophilus paragallinarum is negative, positive serum respectively adds holes, 37 DEG C of effect 1h；Liquid in hole is discarded, per hole
Washed 3 times with 300 μ L cleaning solutions, each 5min, after having washed for the last time, patted on blotting paper, abandon liquid in most hole；Often
Hole adds 100 μ L ELIAS secondary antibodies, 37 DEG C of effect 1h；Liquid in hole is discarded, 3 times are washed with 300 μ L cleaning solutions per hole, every time
5min, after having washed for the last time, pats on blotting paper, abandons liquid in most hole；100 μ LTMB (3,3', 5,5'- are added per hole
Tetramethyl benzidine) after substrate nitrite ion, room temperature lucifuge colour developing 10min adds 100 μ L terminate liquids；With ELIASA in 450nm ripples
It is long to read each hole OD values, result of determination.
The indirect ELISA orthogonal experiments of table 1.
Embodiment 2
The specific test of ELISA kit：
By the indirect ELISA operating method set up, detect that the known secondary chicken of A types is bloodthirsty respectively using ELISA kit
Bacillus, newcastle disease, bird flu, salmonella, staphylococcus aureus, pasteurella multocida and Escherichia coli positive serum
Sample；Each hole OD values are determined under 450nm wavelength with ELIASA, the specificity of the indirect ELISA detection method is determined；
Testing result shows, the only OD of A types haemophilus paragallinarum positive serum450>0.34, other each point of serum OD450
Respectively less than 0.34, illustrate that indirect ELISA testing result specificity is good.
Embodiment 3
ELISA kit is with batch interior repetition experiment：
HA albumen is recombinated using the A types haemophilus paragallinarum prepared with batch induction purifying, according to optimal coating concentration coating
ELISA Plate, with built indirect ELISA operating method, detection A type haemophilus paragallinarum antibody levels different positive serum and A
Each 3 parts of type haemophilus paragallinarum negative serum, detects 6 parts of randomly selected serum in different time, is existed with ELIASA respectively
OD values are determined under 450nm wavelength；According to the OD in every hole450Value, calculates the average value of each part serumStandard deviation S, the coefficient of variation
CV；Testing result shows, 6 parts of serum OD450The coefficient of variation of value illustrates bloodthirsty with the secondary chicken of batch A types between 1.5%-3.7%
Bacillus restructuring HA Protein Detections result repeatability is good.
Embodiment 4
Repeated experiment between ELISA kit batch：
HA albumen is recombinated using the A types haemophilus paragallinarum after different batches purification renaturation, it is dense according to optimal antigen coat
Coated elisa plate is spent, with the indirect ELISA operating method set up, detection is with 1 part of A type haemophilus paragallinarum positive serum and 1
Part A type haemophilus paragallinarum negative serums, 4 repetitions are set per hole, OD values are determined under 450nm wavelength with ELIASA；According to
OD per hole450Value, calculates the average value of each part serumStandard deviation S, coefficient of variation CV；Testing result shows, 2 parts of serum
OD450The coefficient of variation of value illustrates that synantigen detection repeatability is not good between 6.5%-8%.
The indirect ELISA reagent kit of the present invention, is made using A types haemophilus paragallinarum (Hpg) restructuring hemagglutinin (HA) albumen
For envelope antigen, the albumen is that A types Hpg recombinates HA gene amplification products, the expression product of pET-28a-HA expression vectors, it is easy to
Prepare and purify, its immunoreactivity is good；As envelope antigen after restructuring HA protein purifications, its concentration is easy to determine and controlled
System, is conducive to a large amount of productions and cost control of the kit.
The kit of the present invention is used for the antibody for detecting A type haemophilus paragallinarums, with efficient, sensitive specificity and repetition
Property good advantage, the kit is easy to operate, quick, with low cost, is suitable for being promoted in clinical practice, is A
The quick detection of type haemophilus paragallinarum provides reliable technological means.
Not most part in the embodiment of the present invention, those skilled in the art can select from the prior art.
Disclosed above is only the embodiment of the present invention, but protection scope of the present invention is not limited thereto, and is appointed
What those familiar with the art the invention discloses technical scope in, change or replacement can be readily occurred in, all should
It is included within the scope of the present invention.Therefore, protection scope of the present invention should using above-mentioned scope of the claims as
It is accurate.
SEQUENCE LISTING
<110>Yangling Polytechnic college, Xingping City crowd and modern ecological Farming Ltd.
<120>Detect indirect ELISA reagent kit and its detection method and its application of A type haemophilus paragallinarum antibody
<160> 1
<210> 1
<211> 1032
<212> DNA
<213>A type haemophilus paragallinarums
<400> 1
atgaaaaaaa ctgcaatcgc attagtaatc gctggtttaa ctgccgcgtc agtagcacaa 60
gctgcaccac aagcaaatac tttctatgct ggtgcaaaag cgggctgggc atctttccac 120
gatggtttaa accaatttga aaactcacaa aatgcatatg gtacattgcg taattctgta 180
acttatgggg tgtttggtgg ttatcaaatt actgataatt ttgctgttga gctaggttat 240
gatgactttg gacgtgcgaa acttcgtcaa gacggtgaaa ctgttggaaa acatacaaat 300
cacggggctc atttaagctt aaaagcaagt tatccagtgc ttgaaggatt agatgtttat 360
gctcgcgttg gagctgcgtt aattcgttct gattataaac caactaaaag agcagcccct 420
aatgagacgc acgaacatag cttaaaagtt tctccagtgt ttgctggtgg cttagagtat 480
aacttaccat cattaccaga acttgcatta cgtgttgaat atcaatgggt gaataaagta 540
gggcgttggg aaaaagatgg tagccgtgta gattatacac caagcatcgg ttctgtaact 600
gctggtttat cttaccgttt tggccaaagt gcaccagttg ttgaacctaa ggttgttgca 660
aaaacatttg cattaaattc agacgttact ttcgcatttg gtaaagcaaa tttacgtcca 720
gaagcacaaa atgtattaga cggtatttat ggtgaaatcg cacagttaaa atcagtacaa 780
gtagatcttg ctggttatac tgaccgtatt ggtagcgaag cagccaactt aaaattatca 840
caacgtcgtg ctgacactgt ggctaactac ttagtttcta aaggtgttgc tcaagaagtg 900
atttcttcaa caggttatgg tgaagcgaac ccagtaactg gtgcgaaatg tgatgcggtt 960
aaaggtcgca aagcattaat cgcttgttta gcagacgatc gtcgtgtaga aatctcagtt 1020
aaaggtaacg ag 1032
Claims (10)
1. a kind of indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody, it is characterised in that the kit includes：
Coated elisa plate, cleaning solution, serum dilution, substrate nitrite ion, rabbit-anti chicken ELIAS secondary antibody, terminate liquid, A type Hpg standard positives
Serum and A type Hpg standard female serum；Wherein, the coated elisa plate is to recombinate hemagglutinin with A types haemophilus paragallinarum
It is used as envelope antigen.
2. the indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody according to claim 1, it is characterised in that
The nucleotides sequence list of the A types haemophilus paragallinarum restructuring hemagglutinin is SEQ.ID.No.1.
3. the indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody according to claim 1, it is characterised in that
The preparation method of the A types haemophilus paragallinarum restructuring hemagglutinin includes：
Synthetic primer is designed, the genome using A type haemophilus paragallinarum Hpg-8 bacterial strains obtains blood clotting as template by PCR amplifications
The amplified production of element, the amplified production connects its expression vector and while carries out double digestion with construction recombination plasmid；It will select
Positive recombinant plasmid conversion e. coli bl21 (DE3) competent cell, obtain positive plasmid monoclonal bacterium, the positive matter
Grain monoclonal bacterium carries out protein expression under IPTG inductions and obtains induced product, the induced product is purified successively and renaturation after
Obtain recombinating hemagglutinin；Wherein, the synthetic primer is：
Sense primer：5'-CGCGGATCCATGAAAAAAACTGCAATCG-3',
Anti-sense primer：5'-CCGCTCGAGCTCGTTACCTTTAACTGAGAT-3'.
4. the indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody according to claim 1, it is characterised in that
The cleaning solution is the phosphate buffer containing 0.05% Tween-20, and pH is 7.4.
5. the indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody according to claim 1, it is characterised in that
The serum dilution is the phosphate buffer of the skimmed milk power containing 50g/L；The substrate nitrite ion is to contain hydrogen peroxide
Tetramethyl biphenyl amine aqueous solution.
6. the indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody according to claim 1, it is characterised in that
The terminate liquid is 2M sulfuric acid solutions.
7. the indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody according to claim 1, it is characterised in that
The coating concentration of the envelope antigen is 5.0 μ g/mL；The coated elisa plate is closed through 50g/L skimmed milk powers PBST, is loaded close
4 DEG C of preservations are placed in envelope.
8. the indirect ELISA reagent kit of detection A type haemophilus paragallinarum antibody according to claim 1, it is characterised in that
The extension rate of the rabbit-anti chicken ELIAS secondary antibody is 1:2500.
9. the detection side of the indirect ELISA reagent kit of the detection A type haemophilus paragallinarum antibody described in claim any one of 1-8
Method, it is characterised in that the detection method comprises the following steps：
With serum dilution with volume ratio 1：200 dilution proportion serum to be checked obtains dilute serum；
Dilute serum described in 100 μ is added into the every hole of 96 hole elisa Plates, holes A type Hpg standard positive blood is respectively added to ELISA Plate
Cleer and peaceful A types Hpg standard females serum is incubated 1h as control in 37 DEG C, discards liquid in reacting hole, then add 300 μ to every hole
L cleaning solutions are simultaneously washed 3 times, 5 minutes every time, are abandoned cleaning solution, are patted dry for the last time；
100 μ L are added with volume ratio 1 into the every hole of 96 hole elisa Plates:Rabbit-anti chicken ELIAS secondary antibody after 2500 dilutions, is incubated in 37 DEG C
1h is educated, liquid in reacting hole is discarded, 300 μ L cleaning solutions are added into every hole and are washed 3 times, 5 minutes every time, cleaning solution are abandoned, most
Once pat dry afterwards；
100 μ L substrate nitrite ions are added into the every hole of 96 hole elisa Plates, lucifuge is acted on 10 minutes at room temperature；
100 μ L terminate liquids are added to 96 hole elisa Plates per hole to react with color development stopping；
Each hole absorption photometric (OD) value is determined under 450nm wavelength with ELIASA；
Judged according to OD values, work as OD450During value >=0.34, judge that A type Hpg antibody levels, as the positive, work as OD450During value ＜ 0.34,
Judge A type Hpg antibody levels as feminine gender.
10. the indirect ELISA reagent kit of the detection A type haemophilus paragallinarum antibody described in claim any one of 1-8 is in detection A
Application in type haemophilus paragallinarum antibody.
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| CN108956985A (en) * | 2018-05-22 | 2018-12-07 | 山东农业大学 | A kind of indirect ELISA testing kit detecting novel goose astrovirus antibody and application |
| CN109055253A (en) * | 2018-06-15 | 2018-12-21 | 北京市农林科学院 | A kind of blood clotting of pair chicken poultry bacillus inhibits the preparation method of antigen and the application of antigen |
| CN109142727A (en) * | 2018-10-10 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of the visualization quick detection kit and its application of O-shaped antibodies against foot-and-mouth disease virus |
| CN110376388A (en) * | 2019-08-16 | 2019-10-25 | 南京农业大学 | A kind of haemophilus parasuis antibody detection method and its kit |
| CN110904256A (en) * | 2019-12-19 | 2020-03-24 | 内蒙古自治区农牧业科学院 | Primer group for simultaneously detecting salmonella, pasteurella multocida, staphylococcus aureus and avibacterium paragallinarum and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108956985A (en) * | 2018-05-22 | 2018-12-07 | 山东农业大学 | A kind of indirect ELISA testing kit detecting novel goose astrovirus antibody and application |
| CN109055253A (en) * | 2018-06-15 | 2018-12-21 | 北京市农林科学院 | A kind of blood clotting of pair chicken poultry bacillus inhibits the preparation method of antigen and the application of antigen |
| CN109055253B (en) * | 2018-06-15 | 2020-09-29 | 北京市农林科学院 | Preparation method of hemagglutination inhibition antigen of avibacterium paragallinarum and application of antigen |
| CN109142727A (en) * | 2018-10-10 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of the visualization quick detection kit and its application of O-shaped antibodies against foot-and-mouth disease virus |
| CN110376388A (en) * | 2019-08-16 | 2019-10-25 | 南京农业大学 | A kind of haemophilus parasuis antibody detection method and its kit |
| CN110376388B (en) * | 2019-08-16 | 2021-10-19 | 南京农业大学 | Haemophilus parasuis antibody detection method and kit thereof |
| CN110904256A (en) * | 2019-12-19 | 2020-03-24 | 内蒙古自治区农牧业科学院 | Primer group for simultaneously detecting salmonella, pasteurella multocida, staphylococcus aureus and avibacterium paragallinarum and application |
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Application publication date: 20170929 |