CN102323428B - Riemerella anatipestifer antibody indirect ELISA method detection kit and application thereof - Google Patents
Riemerella anatipestifer antibody indirect ELISA method detection kit and application thereof Download PDFInfo
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- CN102323428B CN102323428B CN201110235293.8A CN201110235293A CN102323428B CN 102323428 B CN102323428 B CN 102323428B CN 201110235293 A CN201110235293 A CN 201110235293A CN 102323428 B CN102323428 B CN 102323428B
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Abstract
The invention relates to a riemerella anatipestifer antibody indirect ELISA method detection kit and an application thereof; the kit comprises: a) an antibody detection plate, b) enzyme conjugate working fluid, c) a positive control, d) a negative control, e) a sample diluent, f) 10X concentrated washing liquid, g) a substrate coloured solution A, h) a substrate coloured solution B, and i) a stopping solution; the beneficial and positive effects of the invention are that: the kit is simple in operation; the requirements for apparatuses needed are not high; the kit can be operated by everyone, can meet the requirements with different levels such as epidemic disease monitoring, hygienic epidemic prevention, intensive culture, and individual culture, is suitable for large-scope popularization and application, and has wide market prospects.
Description
Technical field
The present invention relates to fast diagnosis method and the utensil of a kind of bacterial antibodies of duck, more specifically a kind of Riemerella anatipestifer antibody indirect ELISA method detection kit and application thereof.
Background technology
Riemerella anatipestifer disease (Riemerellla anatipestifer infection, RAI) is a kind of social disease of a duck, turkey and multiple other birds.This disease is world wide distribution at present, and the country having produced all intensive foster ducks finds.This disease is mainly encroached on 1～8 week age (especially 2～8 week age) duckling, young goose and poult.This disease is often acute or chronic septicemia process, and mainly take nervous symptoms and fibrinous pericarditis, the gentle capsulitis of perihepatitis is feature.This sick incidence of disease can reach more than 9%, and fatal rate can be up to more than 75%, and the sick duck of resistance to mistake usually grows up to inferior duck or stiff duck, and feed conversion rate reduces, growth retardation.In addition by the caused salpingitis of this disease, be also to affect one of most important reason that the adult rear laying rate of duck is low.
The serotype of riemerella anatipestifer is complicated, generally acknowledge and have 21 kinds of serotypes at present, in China, serotype development becomes increasingly complex, chemoprophylaxis is that China controls one of this sick major measure, but at present a lot of research reports show riemerella anatipestifer and the most common drug on producing are produced to drug resistance, meanwhile, use in a large number medicine also to food security, to cause great threat, immunity inoculation becomes one of safe and effective measure of controlling this disease by replacing gradually chemoprophylaxis.
If carry out the popularity of Riemerella anatipestifer disease and infect the investigation of scope, be necessary to set up a kind of serology quick, responsive, special, that be convenient to large-scale application and do not need to slaughter animal and detect diagnostic method.
Therefore develop for riemerella anatipestifer antibody test special, responsive, that bio-safety degree is high, be suitable for the utensil that diagnosis that basic unit uses detects, to duck immune level monitor in real time and set up science, flexibly riemerella anatipestifer immune programme for children and to the prevention of epidemic disease with control significant.
Through relevant report, show that the outer membrane protein ompA gene of riemerella anatipestifer is present in all reference strain.In different serotype, ompA gene demonstrates less heterologous.OmpA is a conservative and strong antigenic determinant, therefore, not only can be used as one of candidate's composition of subunit vaccine, but also have good serology, detects value.
Summary of the invention
The invention provides a kind of indirect ELISA testing kit for detection of riemerella anatipestifer antibody, by pET-28a expression vector establishment can express the carrier of riemerella anatipestifer outer membrane protein gene ompA, and the recombinant protein of expressing is set up and detected corresponding antibodies, can be used for the detection of riemerella anatipestifer antibody.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of riemerella anatipestifer antibody indirect ELISA detection kit, it is characterized in that this kit includes: a) antibody test plate, b) enzyme conjugates working fluid, c) positive control, d) negative control, e) sample diluting liquid, f) 10 * concentrated cleaning solution, g) substrate nitrite ion A, h) substrate nitrite ion B and i) stop buffer, wherein: described antibody test plate is the removable 96 hole polystyrene ELISA Plate of coated riemerella anatipestifer outer membrane protein, enzyme conjugates working fluid is the anti-duck IgY of horseradish peroxidase-labeled rabbit polyclonal antibody, positive control is that riemerella anatipestifer reference culture immunity duckling is prepared hyper-immune serum, negative control is the negative serum through the duckling of the not immune riemerella anatipestifer of screening acquisition.
Press such scheme, the preparation method of described riemerella anatipestifer outer membrane protein is: the gene order of utilizing round pcr amplification riemerella anatipestifer outer membrane protein, utilize the prokaryotic expression plasmid of genetic engineering recombinant technique structure riemerella anatipestifer outer membrane protein, called after pET-28a-OmpA; Plasmid pET-28a-OmpA is proceeded in Host Strains Escherichia coli and becomes genetic engineering bacterium, called after BLpET-28a-OmpA, BLpET-28a-OmpA is cultivated in LB nutrient culture media, abduction delivering also shows that through SDS-PAGE electrophoretic analysis recombination outer membrane protein is present in thalline with insoluble inclusion body form, and recombination outer membrane protein is carried out to renaturation after purifying by affinity chromatography.
Press such scheme, the preparation method of described antibody test plate is: by the recombination outer membrane protein after purification renaturation, with antigen coated liquid dilution, be the final concentration of 1.5 μ g/mL, in each polystyrene micropore of removable 96 hole ELISA Plate, add 100 μ L, coated 2h under 37 ℃ of conditions, with cleansing solution, wash 5min * 4 time, dry, every hole adds confining liquid 100 μ L, 37 ℃ of reaction 3h, with PBST damping fluid washing 5min * 4 time, dry, add again 20%(w/v) sucrose phosphate buffer room temperature protection 3 hours, put hothouse dry after, pack into containing in the packaging bag of drying agent and save backup.
Press such scheme, described antigen coated liquid is PBS damping fluid, and described cleansing solution is PBST damping fluid, and described confining liquid is 1%(w/v) the PBST damping fluid of bovine serum albumin(BSA).
Press such scheme, the described anti-duck IgY of horseradish peroxidase-labeled rabbit polyclonal antibody dilutes with phosphate buffer.
Press such scheme, described sample diluting liquid is for by 0.1%(w/v) add the PBST damping fluid of bovine serum albumin(BSA); 10 * concentrated cleaning solution is for containing 0.5%(v/v) the 0.1M phosphate buffer of Tween-20; The tetramethyl biphenyl amine aqueous solution that substrate nitrite ion A is 0.2mg/ml, substrate nitrite ion B is for containing 0.05%(v/v) citric acid-phosphate buffer of hydrogen peroxide urea; Stop buffer is 2M sulfuric acid solution.
The application process of described riemerella anatipestifer antibody indirect ELISA detection kit, is characterized in that including following steps:
1) 10 times of 10 * concentrated cleaning solution dilutions are to cleansing solution;
2) by the dilution in by volume 1: 100 of sample diluting liquid for serum to be checked, by 100 μ l/ holes, join in antibody test plate, establish negative control, positive control, and establish and only add 100 μ l sample diluting liquids as blank, hatch 20-45 minute for 37 ℃, dry;
3) every hole adds the cleansing solution 200 μ l of step 1), washs 4 times, and every minor tick 1 minute, pats dry;
4) every hole adds the enzyme conjugates working fluid of 100 μ l, and blank does not add, and hatches 20-45 minute for 37 ℃, dries;
5) every hole adds the cleansing solution 200 μ l of step 1), washs 4 times, and every minor tick 1 minute, pats dry;
6) every hole adds 50 μ l substrate nitrite ion A and 50 μ l substrate nitrite ion B successively, and 37 ℃ of lucifuges are hatched 5-15 minute;
7) every hole adds 50 μ l stop buffers, reads every hole absorbance value by microplate reader under 630nm wavelength.
Riemerella anatipestifer antibody indirect ELISA detection kit, its criterion that detects sample is: under best operating condition, 30 parts that the collect duck serum without riemerella anatipestifer antibody are carried out to indirect ELISA mensuration, trying to achieve OD mean value (Average) X is 0.099, standard deviation (Standard Difference) SD is 0.022, and to set yin and yang attribute critical value be that X+3SD is 0.166, be greater than this value and be decided to be the positive, be less than negative.
The good effect that the present invention is useful is: kit is simple to operate, required instrument is less demanding, everybody can operate, can better meet the needs of different levels, as epidemic disease monitoring, health and epidemic prevention, intensive culture arrive individual cultivation etc., be easy to apply on a large scale, there are wide market outlook, there is following advantages:
1) high specificity, susceptibility is high, and security is good.Riemerella anatipestifer antibody indirect ELISA detection kit be take the recombination outer membrane protein of gene engineering expression and is prepared from as basis; Relevant report shows that the outer membrane protein gene of riemerella anatipestifer is present in the reference strain of all riemerella anatipestifers.Recombination outer membrane protein security is good, containing irrelevant foreign protein, can with riemerella anatipestifer positive serum specific bond, not with other epidemic disease positive serum generation cross reaction of duck.There is good antigenicity, therefore there is very high specificity and susceptibility;
2) easy and simple to handle quick.While using riemerella anatipestifer antibody indirect ELISA detection kit to detect riemerella anatipestifer antibody, without separately joining other reagent, sample, without aseptic process, can be judged testing result by kit explanation in 1-2 hour;
3) result is judged image, accurate, reliable.Riemerella anatipestifer antibody indirect ELISA detection kit can detect hole and occur that blueness is positive according to the depth preliminary judgement testing result of colour developing, and colourless negative, result is judged image.Adopt again the machine-readable number of microplate reader, can more accurate testing result, reduce subjectivity, accurately and reliably;
4) low, the small investment of cost.This kit can mass detection, settles at one go, with low cost, small investment, instant effect.
Accompanying drawing explanation
Fig. 1 is the pcr amplification product of riemerella anatipestifer outer membrane protein ompA gene;
Wherein: swimming lane M is DL2000 DNA Marker; Swimming lane 1 is the PCR product of riemerella anatipestifer outer membrane protein ompA;
Fig. 2 recombinant plasmid pET28a (+)-ompA double digestion is identified;
Wherein: swimming lane M1 is DL2000 DNA Marker; Swimming lane M2 is DL15000 DNA Marker; Swimming lane 1 is that recombinant plasmid pET28a (+)-ompA double digestion is identified;
SDS-PAGE after Fig. 3 recombinant protein ompA expression and purifying analyzes;
Wherein: swimming lane M is high molecular weight protein Marker; Swimming lane 1 is the bacterium liquid precipitate albumen of ultrasonication; Swimming lane 2 is the destination protein ompA after affinitive layer purification;
Fig. 4 recombinate ompA for albumen western blot test (SDS-PAGE) analyze;
Wherein: swimming lane M is high molecular weight protein Marker; The recombination outer membrane protein ompA that swimming lane 1 detects for riemerella anatipestifer standard positive serum.
Embodiment
Below in conjunction with embodiment, the present invention is described in more detail, but this explanation can not be construed as limiting the invention.
A kind of riemerella anatipestifer antibody indirect ELISA detection kit, this kit includes: a) antibody test plate, b) enzyme conjugates working fluid, c) positive control, d) negative control, e) sample diluting liquid, f) 10 * concentrated cleaning solution, g) substrate nitrite ion A, h) substrate nitrite ion B and i) stop buffer, wherein: described antibody test plate is the removable 96 hole polystyrene ELISA Plate of coated riemerella anatipestifer outer membrane protein (ompA), enzyme conjugates working fluid is the anti-duck IgY of horseradish peroxidase-labeled rabbit polyclonal antibody, positive control is that riemerella anatipestifer reference culture immunity duckling is prepared hyper-immune serum, negative control is the negative serum through the duckling of the not immune riemerella anatipestifer of screening acquisition.
The preparation method of described riemerella anatipestifer outer membrane protein is: the gene order of utilizing round pcr amplification riemerella anatipestifer outer membrane protein, utilize the prokaryotic expression plasmid of genetic engineering recombinant technique structure riemerella anatipestifer outer membrane protein, called after pET-28a-OmpA; Plasmid pET-28a-OmpA is proceeded in Host Strains Escherichia coli and becomes genetic engineering bacterium, called after BLpET-28a-OmpA, BLpET-28a-OmpA is cultivated in LB nutrient culture media, abduction delivering also shows that through SDS-PAGE electrophoretic analysis recombination outer membrane protein is present in thalline with insoluble inclusion body form, and recombination outer membrane protein is carried out to renaturation after purifying by affinity chromatography.
The preparation method of described antibody test plate is: by the recombination outer membrane protein after purification renaturation, with antigen coated liquid dilution, be the final concentration of 1.5 μ g/mL; in each polystyrene micropore of removable 96 hole ELISA Plate, add 100 μ L; coated 2h under 37 ℃ of conditions; with cleansing solution, wash 5min * 4 time; dry; every hole adds confining liquid 100 μ L; 37 ℃ of reaction 3h; with PBST damping fluid washing 5min * 4 time; dry; add again 20%(w/v) sucrose phosphate buffer room temperature protection 3 hours, put hothouse dry after, pack in the packaging bag containing drying agent and save backup.
Described antigen coated liquid is PBS damping fluid, and described cleansing solution is PBST damping fluid, and described confining liquid is 1%(w/v) the PBST damping fluid of bovine serum albumin(BSA) (BSA).
The described anti-duck IgY of horseradish peroxidase-labeled rabbit polyclonal antibody is pressed 1:5000 dilution proportion with phosphate buffer.
Described sample diluting liquid is for by 0.1%(w/v) add the PBST damping fluid of bovine serum albumin(BSA); 10 * concentrated cleaning solution is for containing 0.5%(v/v) the 0.1M phosphate buffer of Tween-20; The tetramethyl biphenyl amine aqueous solution that substrate nitrite ion A is 0.2mg/ml, substrate nitrite ion B is for containing 0.05%(v/v) citric acid-phosphate buffer of hydrogen peroxide urea; Stop buffer is 2M sulfuric acid solution.
The application process of described riemerella anatipestifer antibody indirect ELISA detection kit, is characterized in that including following steps:
1) 10 times of 10 * concentrated cleaning solution dilutions are to cleansing solution;
2) by the dilution in by volume 1: 100 of sample diluting liquid for serum to be checked, by 100 μ l/ holes, join in antibody test plate, establish negative control, positive control, and establish and only add 100 μ l sample diluting liquids as blank, hatch 20-45 minute for 37 ℃, dry;
3) every hole adds the cleansing solution 200 μ l of step 1), washs 4 times, and every minor tick 1 minute, pats dry;
4) every hole adds the enzyme conjugates working fluid of 100 μ l, and blank does not add, and hatches 20-45 minute for 37 ℃, dries;
5) every hole adds the cleansing solution 200 μ l of step 1), washs 4 times, and every minor tick 1 minute, pats dry;
6) every hole adds 50 μ l substrate nitrite ion A and 50 μ l substrate nitrite ion B successively, and 37 ℃ of lucifuges are hatched 5-15 minute;
7) every hole adds 50 μ l stop buffers, reads every hole absorbance value by microplate reader under 630nm wavelength.
Invention main points of the present invention are: utilize pET-28a Prokaryotic Expression Systems to build the prokaryotic expression carrier of ompA gene, and the recombinant protein after expressing is carried out to purifying, set up on this basis ELISA method assembling diagnostic kit, and tentatively apply to clinical.
Concrete preparation method of the present invention is as follows:
One, clone, expression and the purifying of riemerella anatipestifer outer membrane protein ompA gene
(1) structure of riemerella anatipestifer outer membrane protein ompA gene magnification and expression vector
According to the ompA gene order of delivering in NCBI Genbank, a pair of expression primer of designed, designed (OmpAF upstream primer 5 '-CCATGGATCCATGGACAAGGAGTTTATG-3 '; OmpAR downstream primer 5 '-CGCCTCGAGTTATTTTCTTTTCTTTTTTAC-3 ').Take riemerella anatipestifer reference culture as template, adopt round pcr to amplify the genes of interest of ompA, size is about 1200bp, as shown in Figure 1.By BamHI/XhoI double digestion object fragment, obtain ompA gene, be cloned into pET-28a carrier, with BamHI/XhoI enzyme, cut evaluation, positive colony called after pET-28a-OmpA, as shown in Figure 2, and serves the order-checking of marine growth Engineering Co., Ltd, sees SEQ1.
(2) abduction delivering and the analysis of riemerella anatipestifer outer membrane protein ompA gene
Plasmid pET-28a-OmpA is proceeded in Host Strains Escherichia coli and becomes genetic engineering bacterium, called after BLpET-28a-OmpA, the single bacterium colony of picking is 37 ℃ of cultivations in LB fluid nutrient medium, until A600 value, reach at 0.4 ~ 0.6 o'clock, add Isopropylthio-β-D-galactosi de (IPTG) to final concentration be 1.0mmol/L, carry out abduction delivering.Collect the thalline of induction rear 1h, 2h, 3h, 4h expression, on ice bath, carry out ultrasonication, 4 ℃ of centrifugal 10min of 15000 * g, get cleer and peaceful precipitation and carry out SDS-PAGE electrophoresis, result has as seen obvious recombination outer membrane protein to express in precipitation, and recombination outer membrane protein is present in thalline with insoluble inclusion body form.
(3) purifying of recombination outer membrane protein, renaturation and analysis
According to the instructions of nickel affinity chromatography post purification kit, carry out the purifying of albumen by affinity chromatography, then make its renaturation by the method for dialysis, the destination protein band of the visible 42KDa of result, shows that affinity chromatography can obtain purer destination protein.Western blot analyzes and shows that the recombination outer membrane protein after renaturation has good immunologic competence.This albumen can be used for the detection of riemerella anatipestifer positive serum.
Two, take riemerella anatipestifer outer membrane protein prepares the antibody test plate of ELISA as envelope antigen, detects the antibody horizontal of riemerella anatipestifer in duck serum, and the various conditions of impact test are selected.
1. the selection of antigen coated concentration and serum optimum dilution degree
Adopt square formation titration, horizontally-arranged is made antigen doubling dilution (12
μg/mL, 6
μg/mL, 3
μg/mL, 1.5
μg/mL, 0.75
μg/mL, 0.375
μg/mL, 0.188
μg/mL, 0.099
μg/mL), tandem is also carried out positive serum and negative serum respectively doubling dilution (1:50,1:100,1:200,1:400,1:800, done 12 doubling dilutions) after Escherichia coli lysate is processed, and each dilutability repeats 1 time, gets its mean value.As a result, when antigen concentration is 1.5 μ g/mL, when serum to be checked dilutes with 1:100, P/N value (positive OD value and negative OD are worth ratio) maximum is non-specific lower.Therefore the serum-concentration of the antigen coated amount of every hole 150ng and 1:100 dilution is optimum response concentration.
2. antigen coated condition determines
By the antigen coated ELISA Plate of optimum concentration, coated condition is respectively: 3h under 37 ℃ of conditions, 4 ℃ are spent the night, under 37 ℃ of conditions 2h again 4 ℃ spend the night, 37 ℃ of 3h again 4 ℃ spend the night.With 0.5% BSA solution sealing, add the positive serum of 1:100 dilution to carry out ELISA mensuration, 4 ℃ of synthesis result are spent the night coated best.
3. best sealing condition determines
Add respectively 0.5%BSA, 1%BSA, 0.5% skimmed milk power and 1% skimmed milk power, in 37 ℃ of incubators, act on 30Min, 60Min, 90Min, 120Min.Add the positive serum of 1:100 dilution to carry out ELISA mensuration, result 1%BSA sealing 60Min is best.
4. serum optimum reacting time determines
Add after serum, in 37 ℃ of incubators, act on 30Min, 60Min, 90Min, 120Min respectively.With 0.5% BSA solution sealing, add the positive serum of 1:100 dilution to carry out ELISA mensuration, result serum effect to be checked 30Min is best.
5. two anti-optimum reacting times determines
After adding two of 1:5000 dilution to resist, in 37 ℃ of incubators, acting on 30Min, 60Min, 90Min, 120Min respectively.With adding the positive serum of 1:100 dilution to carry out ELISA mensuration, two anti-effect 60Min are best as a result.
6. substrate optimum reacting time determines
Add after substrate solution, act on 5Min, 10Min, 12Min, 15Min under lucifuge condition, result shows that under lucifuge condition, 5Min is best.
7. indirect ELISA critical value determines
Under best operating condition, 30 parts that collect 100 times of laggard row indirect ELISAs of dilution of duck serum without riemerella anatipestifer antibody are measured, trying to achieve OD mean value (Average) X is 0.099, standard deviation (Standard Difference) SD is 0.022, and to set yin and yang attribute critical value be that X+3SD is 0.166, be greater than this value and be decided to be the positive, be less than negative.
Three, the preparation of riemerella anatipestifer antibody indirect ELISA kit
1. the preparation of antibody test plate
By antigen coated liquid (PBS for the outer membrane protein after purification renaturation, 0.05mol/L, the carbonate buffer solution of pH9.6) dilution is the final concentration of 1.5 μ g/mL, in each polystyrene micropore of removable 96 hole ELISA Plate, add 100 μ L, coated 2h under 37 ℃ of conditions, with cleansing solution (PBST, PBS damping fluid containing 0.05%Tween-20) washing 5min * 4 time, dry, every hole adds the PBST damping fluid 100 μ L of confining liquid 1% bovine serum albumin(BSA) (BSA), 37 ℃ of effect 3h, PBST damping fluid washing 5min * 4 time, dry, add again 20% sucrose phosphate buffer room temperature protection 3 hours, put hothouse dry after, pack into containing in the packaging bag of drying agent and preserve.
2. enzyme is in conjunction with the preparation of working fluid
The phosphate buffer of pH7.0 (PBS) is pressed the anti-duck IgY of rabbit of 1:5000 dilution proportion HRP mark.
3. the preparation of sample cleansing solution, dilution, stop buffer
10 * concentrated cleaning solution is the 0.1M phosphate buffer (KH containing 0.5% Tween-20
2pO
42g, Na
2hPO
412H
2o 29g, NaCI 80g, is settled to 1000ml, pH7.4, then add 5ml Tween-20); Sample diluting liquid measures 0.1g BSA for containing 1 * sample cleansing solution of 0.1%BSA joins in 1 * sample cleansing solution (PBST) of 100ml; Stop buffer is 2M sulfuric acid solution, measures the 111.2ml concentrated sulphuric acid (18M) dilution and is settled to 1000ml.
4. the preparation of positive control and negative control
Prepare according to a conventional method positive serum and negative serum.Select healthy sheldrake 10 plumages of 7 ages in days, with three immunity of the oil-emulsion inactivated vaccinating agent of 1 type riemerella anatipestifer, each immune interval 14d.Collection serum is standby.
It is standby after screening that negative serum is directly adopted non-immune 15 age in days duckling serum.
Negative serum, positive serum are used to 1: 800 times of dilution (positive serum OD630nm >=1.00 of sample diluting liquid, negative serum≤0.10) by 1000U/ml, add penicillin and streptomysin, aseptic filtration, as negative control and the positive control of riemerella anatipestifer outer membrane protein antibody indirect ELISA detection kit.
5. the preparation of substrate nitrite ion
Take 200mg tetramethyl benzidine (TMB), after dissolving with 100ml absolute ethyl alcohol or DMSO, with distilled water, be settled to 1000ml, join substrate nitrite ion A; Take 21g Citric Acid Mono, 28.2g ADSP (Na
2hPO
4), 6.4ml0.75% hydrogen peroxide urea, distilled water is settled to 1000ml, and adjust pH, to 4.5-5.0, is prepared substrate nitrite ion B.
6. kit detects running program
Its trace routine is:
1) 10 times of 10 * concentrated cleaning solution dilutions are to cleansing solution;
2) serum to be checked is done to dilution in 1: 100 with sample diluting liquid, 100 μ l/ holes are added in antibody test plate, establish blank (only adding 100 μ l sample diluting liquids), negative control (duck standard female serum), positive control (riemerella anatipestifer standard positive serum) simultaneously, hatch 20-45 minute for 37 ℃, dry;
3) every hole adds cleansing solution 200 μ l, washs 4 times, and every minor tick 1 minute, pats dry;
4) every hole adds 100 μ l enzyme conjugates working fluids (blank does not add), hatches 20-45 minute for 37 ℃, dries; 5) every hole adds cleansing solution 200 μ l, washs 4 times, and every minor tick 1 minute, pats dry;
6) add successively 50 μ l substrate nitrite ion A and 50 μ l substrate nitrite ion B, mix, 37 ℃ of lucifuges are hatched 5-15 minute;
7) every hole adds 50 μ l stop buffers, reads every hole absorbance value (OD630nm value) by microplate reader under 630nm wavelength.
7. result criterion yin and yang attribute critical value is 0.166, is greater than this value and is decided to be the positive, is less than negative.
Four) evaluation of Riemerella anatipestifer antibody indirect ELISA method detection kit
1. the specificity of indirect ELISA method detection kit and replica test
Utilize the indirect ELISA method detection kit that riemerella anatipestifer outer membrane protein ompA sets up to detect the positive serums such as duck source Escherichia coli, duck pasteurella multocida, duck streptococcus, reovirus-originated duck, DHV and duck plague virus infected duck serum, result OD value is all less than 0.166, negative, show the method and above-mentioned virus and bacterium no cross reaction; To tiring, three parts of high, medium and low serum are measured, calculate between the plate of this test, the coefficient of variation (CV in plate, the standard deviation of individual parameter and the ratio of average), result is respectively in 5% and 9%, show that the method has good repeatability, this detection kit has good clinical value.
2. the blocking test of indirect ELISA method detection kit
By each 4 parts of positive serums and negative serum respectively with the concentration riemerella anatipestifer outer membrane protein solution that is 1.5mg/ml in 1:10(V/V) ratio mix, 37 ℃ of effect 1h, 10000rpm is centrifugal, and 20min gets supernatant.After the positive and negative serum 1:100 dilution of processing with this, measure by the indirect ELISA method detection kit of having set up with the positive and negative serum that does not add antigen processing, relatively before and after blocking-up, the OD630nm value of serum changes.Result shows to be significantly less than by the OD value of the positive serum of recombination outer membrane protein processing the positive serum OD value that does not add antigen processing, and significant difference (P < 0.05), and by the negative serum OD value that recombination outer membrane protein is processed, be also significantly less than the OD value of the negative serum that does not add antigen, illustrate that this albumen can well block the antibody in serum.
3. the replica test of indirect ELISA method detection kit
(1) criticize interior repetition
By the antigen coated ELISA Plate with a collection of preparation, get 4 parts of serum to be checked, 1 part known of positive serum, 1 part of negative serum, every part of serum repeats 3 holes.The results are shown in Table 2, through SSPS statistical analysis software, analysis shows, in batch, replica test result, without significant difference, illustrates that the repeatability of this ELISA detection method is good.Operation steps is the same.
Reproducible results in table 1 batch
(2) repeat between criticizing
By the antigen coated ELISA Plate of different batches, get 4 parts of serum to be checked, 1 part of known 1 part of positive serum, negative serum detects, and every part of serum detects 3 holes, duplicate detection 3 times.Relatively OD630nm value and S/N, calculate each sample standard deviation and the coefficient of variation.The results are shown in Table 3, through SSPS statistical analysis software, analysis shows, between batch, the revision test coefficient of variation is less than 8.8%, shows that the indirect ELISA method of setting up has good repeatability, can detect the antibody horizontal of riemerella anatipestifer fast and accurately.
Reproducible results between table 2 batch
Sequence table
<110> Inst. of Veterinary Science, Hubei Academy of Agricultural Science
<120> Riemerella anatipestifer antibody indirect ELISA method detection kit and application thereof
<160> 1
<210> 1
<211> 1266
<212> DNA
<213> riemerella anatipestifer
<400> 1
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggacaa ggagtttatg 120
ttgatgactg gtcttggtct tcagcttaaa tttgctggtc ttctttttgg aaacgaagat 180
gcgtggtttg acccttatgt aagagttgga gccaactatt tgagacacga ctatacaggt 240
cttacgttcc ctgtgactga tagctacaat gatgtaactt acgcggggta tagcgaaaat 300
aaaccataca ctcaaggaag agcggatcat tttgctttat caacaggttt aggtacaaac 360
atttggttaa ctaagaactt tggtcttggt atccaagggg attatgtttc tactccagta 420
gataagtctg gattggctaa cttttggcaa gcgtcagctt cattgaactt tagatttggt 480
aacagagata aggataagga tggagtgtta gataaagacg atttatgtcc agaaatacca 540
ggtttacctg aattccaagg ttgtccagat acagatggcg atggagttcc agataaagat 600
gataactgtc cagaagttgc aggaccagtt gaaaacaacg gttgtccttg gccagataca 660
gacaaagatg gtgtattgga taaagacgat gcttgtgttg atgtagctgg acctgctgaa 720
aacaatggtt gtccttggcc agatacagat aatgatggag tattagataa agatgataag 780
tgtcctaatg ttccaggtct tccagaatac aaaggttgtc ctaagcctca ggaagcgtat 840
gcagttgaag caacaggagc attaaagggt atattcttca actttaataa agcatctatc 900
agacctgaat ctaatactaa gttagatcaa gctgctgaag tgattaagtc ttctaacgga 960
ggtactttct tagtggtagg tcatacggat gttaagggta atgctaacta caacttgaaa 1020
ctttctagag aaagagctgc atctgtagta gctgctttag aagctagagg agttaatcca 1080
tctcagttaa aatctaaagg ggttggttct gctgaagcta cagtaccagc gtctgcttct 1140
aacgaagaga gaatgaaaga cagaaaagtg gttgtagaag caatcagcgg atctgcttgg 1200
gaagctcttc aaaagtctga tcttccagta gtgaagaaaa aagtagtaaa aaagaaaaga 1260
aaataa
1266
Claims (4)
1. a riemerella anatipestifer antibody indirect ELISA detection kit, it is characterized in that this kit includes: a) antibody test plate, b) enzyme conjugates working fluid, c) positive control, d) negative control, e) sample diluting liquid, f) 10 * concentrated cleaning solution, g) substrate nitrite ion A, h) substrate nitrite ion B and i) stop buffer, wherein: described antibody test plate is the removable 96 hole polystyrene ELISA Plate of coated riemerella anatipestifer outer membrane protein, enzyme conjugates working fluid is the anti-duck IgY of horseradish peroxidase-labeled rabbit polyclonal antibody, positive control is that riemerella anatipestifer reference culture immunity duckling is prepared hyper-immune serum, negative control is the negative serum through the duckling of the not immune riemerella anatipestifer of screening acquisition, the preparation method of described riemerella anatipestifer outer membrane protein is: the gene order of utilizing round pcr amplification riemerella anatipestifer outer membrane protein, utilize genetic engineering recombinant technique to build the prokaryotic expression plasmid of riemerella anatipestifer outer membrane protein, called after pET-28a-OmpA, plasmid pET-28a-OmpA is proceeded in Host Strains Escherichia coli and becomes genetic engineering bacterium, called after BLpET-28a-OmpA, BLpET-28a-OmpA is cultivated in LB nutrient culture media, abduction delivering also shows that through SDS-PAGE electrophoretic analysis recombination outer membrane protein is present in thalline with insoluble inclusion body form, recombination outer membrane protein is carried out to renaturation after purifying by affinity chromatography, the preparation method of described antibody test plate is: by the recombination outer membrane protein after purification renaturation, with antigen coated liquid dilution, be the final concentration of 1.5 μ g/mL, in each polystyrene micropore of removable 96 hole ELISA Plate, add 100 μ L, coated 2h under 37 ℃ of conditions, with cleansing solution, wash 5min * 4 time, dry, every hole adds confining liquid 100 μ L, 37 ℃ of reaction 3h, with PBST damping fluid washing 5min * 4 time, dry, add again 20%(w/v) sucrose phosphate buffer room temperature protection 3 hours, put hothouse dry after, pack into containing in the packaging bag of drying agent and save backup.
2. by riemerella anatipestifer antibody indirect ELISA detection kit claimed in claim 1, it is characterized in that: described antigen coated liquid is PBS damping fluid, described cleansing solution is PBST damping fluid, and described confining liquid is 1%(w/v) the PBST damping fluid of bovine serum albumin(BSA).
3. riemerella anatipestifer antibody indirect ELISA detection kit according to claim 1, is characterized in that: the described anti-duck IgY of horseradish peroxidase-labeled rabbit polyclonal antibody dilutes with phosphate buffer.
4. riemerella anatipestifer antibody indirect ELISA detection kit according to claim 1, is characterized in that: described sample diluting liquid is for by 0.1%(w/v) add the PBST damping fluid of bovine serum albumin(BSA); 10 * concentrated cleaning solution is for containing 0.5%(v/v) the 0.1M phosphate buffer of Tween-20; The tetramethyl biphenyl amine aqueous solution that substrate nitrite ion A is 0.2mg/ml, substrate nitrite ion B is for containing 0.05%(v/v) citric acid-phosphate buffer of hydrogen peroxide urea; Stop buffer is 2M sulfuric acid solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102675469A (en) * | 2012-03-08 | 2012-09-19 | 福建省农业科学院畜牧兽医研究所 | Novel duck reovirus recombinant sigma B protein antigen, preparation method and application |
| CN103558377B (en) * | 2013-11-06 | 2016-04-13 | 湖北省农业科学院畜牧兽医研究所 | A kind of enterorrhagia Bacillus coil 0157: H7ELISA antibody assay kit and using method thereof |
| CN103926414B (en) * | 2014-04-30 | 2015-08-26 | 四川农业大学 | Riemerella anatipestifer surface antigen D15 brachymemma recombinant protein sensitization latex reagent and its preparation method and application |
| CN103923193B (en) * | 2014-04-30 | 2015-09-23 | 四川农业大学 | Riemerella anatipestifer surface antigen D15 brachymemma recombinant protein and its preparation method and application |
| CN104931493B (en) * | 2015-06-16 | 2018-07-10 | 四川农业大学 | Kit and its application based on riemerella anatipestifer OmpH intercept recombinant proteins |
| CN104987369B (en) * | 2015-06-16 | 2020-09-15 | 四川农业大学 | Riemerella anatipestifer OmpH recombinant protein and ELISA kit thereof |
| CN105181963B (en) * | 2015-07-30 | 2017-08-01 | 西北农林科技大学 | A kind of preparation method of the ELISA detection kit of the pseudo- mycobacterium tuberculosis antibody of goat |
| CN105675862B (en) * | 2016-01-20 | 2017-09-05 | 西南大学 | A kind of enzyme linked immunological quarantine method of Riemerellosis Anatipestifer infected duck |
| CN107356752B (en) * | 2017-07-10 | 2020-07-17 | 湖北省农业科学院畜牧兽医研究所 | Riemerella anatipestifer latex agglutination antibody detection kit, detection method and storage method thereof |
| CN108753811A (en) * | 2018-06-29 | 2018-11-06 | 山东省农业科学院家禽研究所 | A kind of recombinant plasmid, structure, expression and application for riemerella anatipestifer novel subunit vaccine |
| CN110041426B (en) * | 2018-12-31 | 2022-11-25 | 湖北省农业科学院畜牧兽医研究所 | Riemerella anatipestifer positive serum and preparation method and application thereof |
| CN112180087B (en) * | 2020-10-23 | 2022-06-14 | 温氏食品集团股份有限公司 | ELISA method for detecting riemerella anatipestifer antibody, kit and application thereof |
| CN114636818B (en) * | 2022-05-19 | 2022-07-29 | 天津德祥生物技术有限公司 | Coating liquid, erythrocyte membrane coating liquid containing coating liquid and application of erythrocyte membrane coating liquid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101839917A (en) * | 2010-05-18 | 2010-09-22 | 河南科技大学 | Enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody, test method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2332246A1 (en) * | 1998-05-15 | 1999-11-25 | Marshall K. Brinton | Compositions and method for immunizing poultry |
| CN102405058B (en) * | 2009-04-15 | 2015-11-25 | 法国诗华大药厂 | For restructuring avian herpetoviruses carrier and the vaccine of immunity inoculation aquatic bird species |
-
2011
- 2011-08-17 CN CN201110235293.8A patent/CN102323428B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101839917A (en) * | 2010-05-18 | 2010-09-22 | 河南科技大学 | Enzyme-linked immunosorbent assay (ELISA) kit for duck hepatitis virus type-I serum antibody, test method and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| 1型鸭疫里氏杆菌OmpA蛋白间接ELISA方法的建立;孙？ 等;《中国预防兽医学报》;20110815;第33卷(第8期);635-639 * |
| 1型鸭疫里默氏杆菌OmpA蛋白间接ELISA方法的建立及应用;孙？;《中国优秀硕士学位论文全文数据库》;20101015;第2010年卷(第10期);18-44 * |
| 周祖涛.鸭疫里氏杆菌免疫诊断方法及在感染鸭肝脏差异表达基因的研究.《中国博士学位论文全文数据库》.2010,第2010年卷(第7期),34-62. |
| 孙？ 等.1型鸭疫里氏杆菌OmpA蛋白间接ELISA方法的建立.《中国预防兽医学报》.2011,第33卷(第8期),635-639. |
| 孙？.1型鸭疫里默氏杆菌OmpA蛋白间接ELISA方法的建立及应用.《中国优秀硕士学位论文全文数据库》.2010,第2010年卷(第10期),18-44. |
| 霍翠梅.鸭疫里默氏杆菌检测方法的建立.《中国优秀硕士学位论文全文数据库》.2008,第2008年卷(第1期),34-65. |
| 鸭疫里氏杆菌免疫诊断方法及在感染鸭肝脏差异表达基因的研究;周祖涛;《中国博士学位论文全文数据库》;20100715;第2010年卷(第7期);34-62 * |
| 鸭疫里默氏杆菌检测方法的建立;霍翠梅;《中国优秀硕士学位论文全文数据库》;20080115;第2008年卷(第1期);34-65 * |
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