CN104502588A - ELISA kit for detecting canine distemper virus IgG antibody and preparation method of ELISA kit - Google Patents
ELISA kit for detecting canine distemper virus IgG antibody and preparation method of ELISA kit Download PDFInfo
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- CN104502588A CN104502588A CN201410758118.0A CN201410758118A CN104502588A CN 104502588 A CN104502588 A CN 104502588A CN 201410758118 A CN201410758118 A CN 201410758118A CN 104502588 A CN104502588 A CN 104502588A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/13—Canine distemper virus
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Abstract
The invention discloses an ELISA kit for detecting a canine distemper virus IgG antibody and a preparation method of the ELISA kit, belonging to the technical field of biology. According to the kit, a canine distemper virus F protein is used as a coating antigen, and a canine distemper virus-like IgG antibody in canine serum is detected according to the indirect ELISA principle. A coating antigen in a 96-well plate in the kit is an F protein which has favorable antigenicity. The ELISA kit disclosed by the invention comprises a coated 96-well plate, a positive control, a negative control, a rabbit-anti-dog IgG polyclonal antibody labeled by horseradish peroxidase, a concentrated scrubbing solution, a serum diluent, a TMB substrate and a stop solution. The kit disclosed by the invention can be used for screening a large number of samples, and a main reagent in the kit is provided as a working solution, so that the kit is convenient to use.
Description
Technical field
The present invention is the detection kit detecting CDV IgG antibody, belongs to biological technical field.Specifically, the present invention relates to ELISA kit detecting antibody and preparation method thereof in canine distemper communicable disease.
Background technology
CDV (canine distemper virus, CDV) is paramyxovirus section, and Morbillivirus is a kind of single strand RNA virus.CDV infected dogs is the most ancient a kind of, virus that clinical meaning is maximum.The initial infection of CDV starts from virus attack upper respiratory tract epithelial cell, virus very fast diffusion to regional nodes's tissue, tonsil and bronchial lymph nodes.
Infect CDV reveal any symptoms varied, relevant with age of virus virulence, environmental baseline, host and immune state, laboratory often adopts ELISA method to measure Serum Antibody titre to evaluate protectiveness.F protein is one of structural proteins of CDV, has good antigenicity.
Summary of the invention
The invention discloses kit based on indirect ELISA method detecting CDV IgG antibody and preparation method thereof, can be used for a large amount of examinations of the hot IgG antibody of pest.
Present invention also offers that a kind of F genophore builds, the technique of the Expression and purification of albumen.
Present invention also offers preparation technology and the horseradish peroxidase-labeled method of a kind of rabbit anti-dog IgG polyclonal antibody.
The object of the invention is based on the recombinant protein of prokaryotic expression, develop a kind of indirect ELISA reagent kit detecting CDV IgG antibody by Enzyme-multiplied immune technique.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: a kind of indirect ELISA reagent kit for detecting CDV IgG antibody, and described kit comprises by the rabbit of horseradish peroxidase-labeled anti-dog IgG polyclonal antibody.
Also comprise elisa plate bar, serum dilution and concentrated cleaning solution, substrate nitrite ion, stop buffer, monoclonal antibody linked with peroxidase, positive control, negative control.
The above-mentioned indirect ELISA reagent kit for detecting CDV IgG antibody comprises:
Described elisa plate bar: each kit is equipped with 1 piece of ELISA microwell plate, every block elisa plate bar is the ELISA microwell plate of the envelope antigen of energy realizing self disassembling, and envelope antigen is the F protein of prokaryotic expression, and specification is 8 hole × 12;
Described serum dilution and concentrated cleaning solution: serum dilution is instant, cleansing solution is 25 times and concentrates, and uses front dilution;
Described substrate nitrite ion: instant TMB nitrite ion, 10mL;
The H of described stop buffer: 2mol/L
2sO
4solution, 10mL;
Described enzyme mark polyclonal antibody: the rabbit anti-dog IgG polyclonal antibody of described horseradish peroxidase-labeled, uses front 100 times of dilutions;
Positive control is: through the dog serum dilution after vaccine immunity of high-temperature inactivation.
Negative control is: through the not ill of high-temperature inactivation and without the dog serum dilution of vaccine immunity.
Described indirect ELISA reagent kit is detecting the application in CDV IgG antibody.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail:
Technical scheme of the present invention is as follows:
One, antigen preparation
1. the expression of CDV F protein
According to distemper virus F gene order in GenBank, the design and synthesis 1 pair of primer, adopt PCR method from DNA, amplify F genetic fragment, be cloned in expression vector pET28b, construct recombinant plasmid pET-F, expressive host bacterium BL21 (DE3) is transformed into, through IPTG abduction delivering after sequence verification.
2. the purifying of CDV F protein
After induction terminates, thalline after collected by centrifugation induction, washs resuspended thalline with PBS, and ultrasonic degradation (ultrasonic 1s, interval 1s, altogether 10min), 12000rpm is centrifugal, collects upper cleer and peaceful precipitation respectively, carries out SDS-PAGE analysis.Use nickel ion affinity chromatograph post during protein purification, after purifying, identify through SDS-PAGE, western blotting, get single band, and possess good antigenicity.Protein content after use BCA method mensuration purifying, after purifying, the OD value of F protein is 2.25, after comparing with standard items, and its concentration is 1700 μ g/ml.
Two, the preparation of the anti-dog IgG of horseradish peroxidase-labeled rabbit
1. the preparation of purifying dog IgG
Learn from else's experience the dog that is up to the standards as blood sampling with dog, and before blood sampling, body condition is better, and body temperature, appetite, stool and urine are normal, without other diseases.After sterilization, jugular vein blood collection, separation of serum, IgG purification scheme is sad-ammonium sulfate precipitation method.
Get 1 part of pretreated serum and add 2 parts of 0.06mol/L pH5.0 acetate buffer solutions, adjust pH to 4.8 with 1mol/LHCl; Reset and add the sad ratio of 11ul in every milliliter of diluted blood, it is sad dropwise to add under stirring at room temperature, adds in 30 minutes, and 4 DEG C leave standstill 2 hours, take out the centrifugal 30min of 12000r/min, abandon precipitation; Supernatant filters (125um) through nylon mesh, adds the 0.01mol/L PBS of 1/10 volume, adjusts pH to 7.2 with 1mol/L NaOH; At 4 DEG C, add saturated ammonium sulfate to 45% saturation degree, mix 30min gently, leave standstill 1 hour; Centrifugal 30 minutes of 12000r/min, abandons supernatant; Precipitation is dissolved in appropriate PBS, and dialyse to the PBS of 50-100 times of volume, 4 DEG C are spent the night; Take out the centrifugal 30min of 12000r/min, removing infusible precipitate, packing, frozen for subsequent use.
2. the horseradish peroxidase-labeled of rabbit anti-dog IgG polyclonal antibody
The purifying dog IgG of above-mentioned preparation is as immunogene, and immune adult healthy rabbit, after Culling heart blood, purifying obtains rabbit anti-dog IgG polyclonal antibody.
The preparation scheme of the enzyme marker of the polyclonal antibody after purifying is Over-voltage protection, and concrete steps are as follows:
Taking 5mg HRP is dissolved in 1ml distilled water; Add the 0.1MNaIO4 solution that 0.2ml newly joins wherein, room temperature lucifuge stirs 20 minutes; Dialyse in the sodium-acetate buffer of 1mM pH4.4,4 DEG C are spent the night; Add the carbonate buffer solution of 20 μ l 0.2M pH9.5 wherein again, the pH of above hydroformylation thing is made to be elevated to 9.0, then the polyclonal antibody sterling 5mg (10mg/ml) being dissolved in 0.01M carbonate buffer solution is added immediately, room temperature lucifuge gentle agitation 2 hours; Add the 4mg/ml NaBH4 that 0.1ml newly joins, mixing, place 2 hours for 4 DEG C; Products therefrom is to 4 DEG C of dialysed overnight in 0.1M pH7.4 PBS.
After having dialysed, in stirring, dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour.The centrifugal 30min of 3000r/min, abandons supernatant.Sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of a small amount of 0.15M pH 7.4.Loaded in bag filter by above-mentioned solution, dialyse to the PBS damping fluid of 0.1M pH7.4, take out ammonium ion, the centrifugal 30min of 10000r/min removes precipitation, and supernatant is enzyme conjugates, after packing, frozen.
Three, the foundation of kit
1. the Cleaning Principle of kit of the present invention
Adopt indirect elisa method, hot for the pest of restructuring albumen F is coated in microwell plate, then with 1%BSA, ELISA Plate is closed, add sample to be tested and standard positive control, negative control.Canine distemper IgG antibody in sample or standard items can with ELISA Plate in wrap the F antigen-reactive of quilt, after adding enzyme mark rabbit anti-dog IgG polyclonal antibody, enzyme labelled antibody and previous step are reacted the F antigen-antibody complex terminated and are combined.Subsequently, add horseradish peroxidase substrate TMB to develop the color, stop buffer cessation reaction, by microplate reader under 450nm wavelength, measure each hole absorbance, the size (depth of the rear color of color development stopping reaction) of OD value is directly proportional to the content of canine distemper IgG antibody in sample to be tested.
2. the composition of kit of the present invention
A) the best preparation method of ELISA Plate
Be buffered liquid with the carbonate buffer solution of pH9.6 0.05M as bag, after F protein dilution of recombinating after the purifying of above-mentioned preparation, add in microwell plate by 100ul/ hole, ensure that the F protein content in every hole is 0.2ug.4 DEG C of bags are spent the night, and next day, discard coating buffer, add the confining liquid of 1%BSA by 200ul/ hole, and 37 DEG C leave standstill 2 hours, and washing dries.Load packaging bag after drying at room temperature, add drying agent, vacuum is preserved.
B) configuration of working reagent
Cleansing solution (pH 7.4,0.15M PBS): KH
2pO
40.2g, Na
2hPO
4-12H
2o 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 (0.05%) 0.5ml, adding distil water is to 1000ml.Be condensed into 25 times as storing liquid.
Serum dilution: bovine serum albumin(BSA) 0.1g, adds lavation buffer solution to 100ml
Substrate buffer solution (acid of pH 5.0 phosphate citrate): 0.2M Na
2hPO
425.7ml, 0.1M citric acid 24.3ml, adding distil water 50ml
TMB (tetramethyl benzidine) uses liquid: TMB (10mg/5ml absolute ethyl alcohol) 0.5ml, substrate buffer solution 10ml, 0.75%H
2o
232ul
Stop buffer (2M H
2sO
4): distilled water 178.3ml, dropwise adds the concentrated sulphuric acid (98%) 21.7ml
C) establishment of the indirect ELISA reagent kit of canine distemper IgG antibody is detected
Set up the ELISA kit detecting CDV IgG antibody, comprise following component:
96 hole ELISA Plate of F recombinant protein bag quilt
The rabbit anti-dog IgG polyclonal antibody of horseradish peroxidase-labeled
Standard positive control
Standard negative control
Concentrated cleaning solution
Serum dilution
Tmb substrate
Stop buffer
Product description
Four, the detection of canine distemper IgG antibody in sample
1. detect with the kit of above-mentioned preparation
1) test serum, positive control and negative control antibody diluent are diluted in proportion, every hole 100 μ l adds ELISA Plate, hatches 1 hour for 37 DEG C.Cleansing solution cleaning 3-5 time.
2) every hole adds the rear enzyme mark rabbit anti-dog IgG polyclonal antibody 100 μ l of dilution, hatches 30min for 37 DEG C, cleansing solution cleaning 3-5 time.
3) every hole adds tmb substrate liquid 100 μ l, room temperature lucifuge reaction 10-15 minute.
4) every hole adds 100 μ l 2M H
2sO
4cessation reaction, microplate reader detects 450nm absorbance.
2. Analysis of test results
1), when the difference detecting the OD value of positives control serum (PC) and the OD value of negative control sera (NC) must be greater than 0.4, detect and be considered to effective.
PC-NC≥0.4
Cut Off＝NC+0.05
Wherein NC=negative control sera OD value
PC=positive control serum OD value
2) use multiple hole when detecting, final utilization OD value is the mean value of two.
When the OD value of serum sample is lower than Cut Off value, this sample is that CDV IgG antibody is negative.
When the OD value of serum sample higher than Cut Off value is, this sample is that CDV IgG antibody is positive.
Above one embodiment of the present of invention have been described in detail, but described content being only preferred embodiment of the present invention, can not being considered to for limiting practical range of the present invention.All equalizations done according to the present patent application scope change and improve, and all should still belong within patent covering scope of the present invention.
Claims (6)
1. detect the ELISA kit of CDV IgG antibody, it is characterized in that: described ELISA detection kit with restructuring the hot F protein of pest for envelope antigen, sample and standard positive control, negative control is added after closing, with enzyme mark rabbit anti-dog IgG polyclonal antibody for labelled antibody, by this enzymic-labelled antibody, set up indirect elisa method and detect CDV IgG type antibodies, then add horseradish peroxidase substrate TMB and develop the color, stop buffer cessation reaction, measures each hole absorbance.
2. detect the ELISA kit of CDV IgG antibody according to claim 1, it is characterized in that: described kit comprises elisa plate bar, serum dilution and concentrated cleaning solution, substrate nitrite ion, stop buffer, monoclonal antibody linked with peroxidase, positive control, negative control.
3. the ELISA kit of detection CDV IgG antibody according to claim 2, is characterized in that:
Described elisa plate bar: each kit is equipped with 1 piece of 96 hole ELISA microwell plate, every block elisa plate bar be can realizing self disassembling wrap by the ELISA microwell plate of F protein, specification is 8 hole × 12;
Described serum dilution is the bovine serum albumin solution of instant;
Described cleansing solution is 25 times of concentrated phosphate buffers, uses front dilution;
Described substrate nitrite ion: instant TMB nitrite ion, 10mL;
The H of described stop buffer: 2mol/L
2sO
4solution, 10mL;
Described monoclonal antibody linked with peroxidase: the rabbit anti-dog IgG polyclonal antibody of described horseradish peroxidase-labeled, uses front 100 times of dilutions;
Described positive control is: through the dog serum dilution after vaccine immunity of high-temperature inactivation.
Described negative control is: through the not ill of high-temperature inactivation and without the dog serum dilution of vaccine immunity.
4. prepare the ELISA kit of detection CDV IgG antibody according to claim 1, it is characterized in that: the method for the rabbit anti-dog IgG polyclonal antibody of described horseradish peroxidase-labeled, adopt caprylic acid-ammonium purifying dog serum IgG, prepare the dog IgG of purifying, and with this IgG purification immunizing rabbit, prepare rabbit anti-dog IgG polyclonal antibody; Over-voltage protection is adopted to carry out horseradish peroxidase-labeled to above-mentioned polyclonal antibody.
5. prepare the ELISA kit of detection CDV IgG antibody according to claim 1, it is characterized in that: described distemper virus F protein be adopt gene recombination technology, the mode of external evoked expression, purifying protein obtains.
6. the ELISA kit of detection CDV IgG antibody according to claim 1 is detecting the application in CDV IgG type antibodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410758118.0A CN104502588A (en) | 2014-12-12 | 2014-12-12 | ELISA kit for detecting canine distemper virus IgG antibody and preparation method of ELISA kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410758118.0A CN104502588A (en) | 2014-12-12 | 2014-12-12 | ELISA kit for detecting canine distemper virus IgG antibody and preparation method of ELISA kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110568204A (en) * | 2019-06-10 | 2019-12-13 | 青岛海润检测股份有限公司 | Chemiluminescence kit for one-step quantitative detection of canine distemper virus-canine parvovirus antibody |
| CN112876561A (en) * | 2021-01-14 | 2021-06-01 | 中国农业科学院特产研究所 | Antibody pair for detecting canine distemper virus and application thereof |
-
2014
- 2014-12-12 CN CN201410758118.0A patent/CN104502588A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110568204A (en) * | 2019-06-10 | 2019-12-13 | 青岛海润检测股份有限公司 | Chemiluminescence kit for one-step quantitative detection of canine distemper virus-canine parvovirus antibody |
| CN110568204B (en) * | 2019-06-10 | 2022-04-01 | 青岛海润检测股份有限公司 | Chemiluminescence kit for one-step quantitative detection of canine distemper virus-canine parvovirus antibody |
| CN112876561A (en) * | 2021-01-14 | 2021-06-01 | 中国农业科学院特产研究所 | Antibody pair for detecting canine distemper virus and application thereof |
| CN112876561B (en) * | 2021-01-14 | 2021-11-16 | 中国农业科学院特产研究所 | Antibody pair for detecting canine distemper virus and application thereof |
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