CN106124772A - A kind of ELISA detection kit based on OMP18 detection campylobacter jejuni and application thereof - Google Patents
A kind of ELISA detection kit based on OMP18 detection campylobacter jejuni and application thereof Download PDFInfo
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- CN106124772A CN106124772A CN201610427396.7A CN201610427396A CN106124772A CN 106124772 A CN106124772 A CN 106124772A CN 201610427396 A CN201610427396 A CN 201610427396A CN 106124772 A CN106124772 A CN 106124772A
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Abstract
The invention provides a kind of ELISA detection kit based on OMP18 detection campylobacter jejuni, including following components: be coated with detection plate, cleaning mixture, the rabbit anti-igg solution of two antienzyme labellings, stop buffer, sample diluting liquid, the substrate nitrite ion of the B cell antigen epitope polypeptide of OMP18.The present invention is with the B cell antigen epitope polypeptide of OMP18 as envelope antigen, according to the immunoreation principle of indirect method, by the B cell antigen epitope polypeptide of detection OMP18, campylobacter jejuni is carried out detection and analyzes.By immunoreactivity and the immunologic opsonin of the B cell antigen epitope polypeptide of detection OMP18, result shows that the B cell antigen epitope polypeptide of three OMP18 is respectively provided with higher immunoreactivity and immunologic opsonin, as envelope antigen, the serum of Cj infection can be carried out ELISA detection.
Description
Technical field
The invention belongs to biological medicine and technical field of medical examination, relate to immunochemistry detection technique, be specifically related to one
Campylobacter jejuni immunological detecting kit, is more particularly to the campylobacter jejuni of a kind of B cell antigen epi-position based on OMP18
ELISA detection kit and preparation method thereof
Background technology
Campylobacter jejuni (Campylobacter jejuni) is common Amphixenosis pathogen, is widely present in
In the animal bodies such as bird, fowl, Canis familiaris L., cat, be also a kind of borne Parasitic Encephalopathy cause of disease bacterium, can by pollute poultry meat and metabolism product and without
The water disinfected and cause infecting, such as the Carnis Gallus domesticus not boiled, the halfway milk of pasteurize, egg products, green ham etc..Separately
Outward, campylobacter jejuni exists in the reproductive tract and intestinal of the poultry such as cattle, sheep, Canis familiaris L. in a large number, therefore can be dirty by childbirth or Excreta
Dye food and drinking-water.
Campylobacter jejuni can cause the multiple disease of the mankind, and such as, acute enteritis, Guillain-Barre syndrome are it is considered to be people
One of Main Pathogenic Bacteria of bacterioid diarrhoea, is the bacillary enteric pathogenic bacteria of Zoonosis important in global range, its sense
Dye rate is the most in rising trend.Jejunum campylobacter bacteria pathogenic is strong, and crowd is the most susceptible, and less than 5 years old child sends out
Sick rate is the highest, and fly also plays important instrumentality, also can be through being infected by contact with, and the puerpera simultaneously infected can be transmitted to when childbirth
Fetus.
Campylobacter jejuni all can be caused damage by hot and cold, sunshine in food processing, storage and transportation and oxygen etc.
Evil, but the often quantity of the campylobacter jejuni in food is few and active relatively low, and conventional method is difficult to detection and obtains.It addition, jejunum is curved
Aspergillosis In vitro culture difficulty, growth conditions requires harshness, and conventional culture methods has necessarily with qualification for detecting fast and accurately
Difficulty.Big and the to be detected thing quantity of In vitro culture difficulty brings challenges to less the most correct detection.In recent years, nucleic acid molecules is miscellaneous
Friendship technology and PCR equimolecular biological detection method have made great progress, and have the most successively carried out some for jejunum
The gene test of Campylobacter spp, for target gene mainly include 16S rDNA, 3S rDNA, hipO, flagellin gene (flaA
Or flaB) etc., the detection method of PCR-based amplification technique is that nucleic acid level detection campylobacter jejuni provides and effectively detects hands
Section, but owing to using PCR method to need the quantity of pathogen is required, extract the DNA fragmentation of pathogenic bacterium, operator is had one
Determining the requirement of specialty background, from preparing to obtaining, the testing result time is longer simultaneously, it is impossible to meet testing requirement fast and accurately.
Summary of the invention
In view of this, object of the present invention is to provide a kind of B cell antigen epitope polypeptide based on OMP18 detection sky
The enzyme linked immunological kit of intestinal Campylobacter spp, makes described test kit meet the demand that campylobacter jejuni is used for quickly detecting report.
The invention provides the enzyme linked immunological of a kind of B cell antigen epitope polypeptide based on OMP18 detection campylobacter jejuni
Test kit, including following components:
(1) the detection plate of the B cell antigen epitope polypeptide of OMP18 it is coated with；
(2) cleaning mixture；
The rabbit anti-igg solution of (3) two antienzyme labellings；
(4) stop buffer；
(5) sample diluting liquid；
(6) substrate nitrite ion.
Preferably, described detection plate is vacuum sealed package.
Preferably, described stop buffer is sulfuric acid solution or the sodium hydroxide solution of 2～3mol/L of 1～2mol/L.
Preferably, described sample diluting liquid be pH value 7.4～8.0, molar concentration 0.1～0.25mol/L, containing 45～
The phosphate buffer of 50% methanol.
Preferably, the concentration that is coated of the B cell antigen epitope polypeptide antigen of described OMP18 is 4～9 μ g/ml.
Preferably, the enzyme that described enzyme labelling rabbit anti-igg is used is HRP, and described substrate nitrite ion is TMB.
Preferably, described cleaning mixture be comprise 0.05～0.1%Tween 20 PBS solution.
Preferably, the Staphylococal Protein A epitope amino acid sequence of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18 is
STKSTSVSGDSSVDSNRGSGGSDGWDID。
The B epitope peptide sequence of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18 is
EGNCDEWGTDEY。
Preferably, the C epitope peptide sequence of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18 is
SYGETNPVCTEKTKACDAQNRR。
The invention provides the application in detection campylobacter jejuni of the test kit described in technique scheme, its feature exists
In, comprise the following steps:
A, testing sample is carried out pre-treatment；
B, described step a is processed after testing sample add the B cell antigen epitope polypeptide being coated with OMP18
In detection plate hole, mixing, hatch, wash with cleaning mixture；
C, described detection plate hole add two anti-solution, mixing, hatch, wash with cleaning mixture；
D, addition substrate nitrite ion, hatch, and adds stop buffer mixing, measures OD value.
The invention provides the enzyme linked immunological of a kind of B cell antigen epitope polypeptide based on OMP18 detection campylobacter jejuni
Test kit, including detection plate, cleaning mixture, the rabbit anti-igg of two antienzyme labellings of the B cell antigen epitope polypeptide being coated with OMP18
Solution, stop buffer, sample diluting liquid and substrate nitrite ion.The present invention is anti-for being coated with the B cell antigen epitope polypeptide of OMP18
Former, according to the immunoreation principle of indirect method, B cell antigen epitope polypeptide based on OMP18 detects campylobacter jejuni.This
The test kit of bright offer is easy to carry and preserves；Easy to operate, method is stable, result is accurate, for clinical diagnosis and treatment detection
Use provide and greatly facilitate, detection simultaneously quickly, meets the needs of quickly detection clinically.
Accompanying drawing explanation
Fig. 1 is the B cell antigen epitope polypeptide immunoreactivity testing result block diagram of 3 OMP18
Fig. 2 is the B cell antigen epitope polypeptide immunologic opsonin testing result block diagram of 3 OMP18
Detailed description of the invention
The invention provides the enzyme linked immunological of a kind of B cell antigen epitope polypeptide based on OMP18 detection campylobacter jejuni
Test kit, including following components: (1) is coated with the detection plate of the B cell antigen epitope polypeptide of OMP18；(2) cleaning mixture；(3) two
The rabbit anti-igg solution of antienzyme labelling；(4) stop buffer；(5) sample diluting liquid；(6) substrate nitrite ion.
The enzyme linked immunological kit that the present invention provides, by anti-as being coated using the B cell antigen epitope polypeptide of OMP18
Former, detect whether campylobacter jejuni exists.It is special that test kit of the present invention has good repeatability, specificity, sensitivity
Point, can apply to the laboratory discriminatory analysis to campylobacter jejuni, detects prevention for campylobacter jejuni and has certain meaning
Justice.
The enzyme linked immunological kit that the present invention provides includes the detection plate being coated with the B cell antigen epitope polypeptide of OMP18.
The structure of described detection plate is not particularly limited by the present invention, and detection plate well known to those skilled in the art all can use,
Detection plate well known to those skilled in the art is coated the B cell antigen epitope polypeptide of OMP18.Heretofore described detection
Plate, uses vacuum sealed package.
In the present invention, described detection plate is coated with the B cell antigen epitope polypeptide of OMP18.Described campylobacter jejuni
The aminoacid sequence of the Staphylococal Protein A epi-position of the B cell antigen epitope polypeptide of OMP18 is
STKSTSVSGDSSVDSNRGSGGSDGWDID；The B antigen table of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18
The aminoacid sequence of position peptide is EGNCDEWGTDEY；The C antigen of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18
The aminoacid sequence of epitope peptide is SYGETNPVCTEKTKACDAQNRR.
In the present invention, the concentration that is coated of the B cell antigen epitope polypeptide antigen of described OMP18 is preferably 4～9 μ g/
Ml, more preferably 5～8 μ g/ml.The present invention does not has special limit to the source of the B cell antigen epitope polypeptide of described OMP18
System, uses the B cell antigen epitope polypeptide acquisition methods of OMP18 well-known to those skilled in the art, and the application implements
Example uses Suspension array technique to obtain the B cell antigen epitope polypeptide of OMP18.
In the present invention, the preparation method of the B cell antigen epitope polypeptide of described OMP18 is the most preferred: use solid phase many
Peptide symthesis technology, with N-α-Fmoc protection aminoacid as raw material, Fmoc-AA-Wang resin is carrier, after HBTU method coupling
Obtain polypeptide crude product, after being identified by crude product polypeptide analysis, use preparative reversed-phase liquid chromatography (RP-HPLC) method to be purified,
And with HPLC and MS Analysis and Identification.
The enzyme linked immunological kit that the present invention provides includes cleaning mixture.In the present invention, described cleaning mixture preferably comprises
The PBS solution of 0.05～0.1%Tween 20.The source of described cleaning mixture is not particularly limited by the present invention, uses this area
PBS solution known to technical staff, in the embodiment of the present application, PBS solution used is prepared voluntarily.
The enzyme linked immunological kit that the present invention provides includes stop buffer.Described stop buffer is the sulfuric acid solution of 1～2mol/L
Or the sodium hydroxide solution of 2～3mol/L.The source of described stop buffer is not particularly limited by the present invention, uses art technology
Stop buffer known to personnel, in the embodiment of the present application, stop buffer used is that oneself configuration obtains.
The enzyme linked immunological kit that the present invention provides includes sample diluting liquid.Described sample diluting liquid be pH value 7.4～
8.0, molar concentration 0.1～0.25mol/L, containing 45～50% phosphate buffer of methanol.The present invention is dilute to described sample
The source releasing liquid is not particularly limited, and uses sample diluting liquid well-known to those skilled in the art, the embodiment of the present application
Middle specimen in use diluent is that oneself preparation obtains.
The enzyme linked immunological kit that the present invention provides includes two antienzyme labelling rabbit anti-igg.Described two antienzyme labelling rabbit anti-igg
The enzyme used is HRP.The source of described enzyme labelling rabbit anti-igg is not particularly limited by the present invention, uses people in the art
Enzyme labelling rabbit anti-igg known to Yuan, in the embodiment of the present application, enzyme labelling rabbit anti-igg used is purchased from the biological section of pool, Hangzhou weighing apparatus
Skill company limited.
The test kit that the present invention provides includes substrate nitrite ion.In the present invention, described substrate nitrite ion is preferably TMB
(3,3,5,5-tetramethyl benzidine).
The invention provides the application in detection campylobacter jejuni of the test kit described in technique scheme, its feature exists
In, comprise the following steps:
A, testing sample is carried out pre-treatment；
B, described step a is processed after testing sample add the B cell antigen epitope polypeptide being coated with OMP18
In detection plate hole, mixing, carry out hatching for the first time, carry out washing for the first time with cleaning mixture；
C, described detection plate hole add two anti-solution, mixing, carry out second time and hatch, and carry out second time with cleaning mixture and wash
Wash；
D, addition substrate nitrite ion, carry out third time and hatch, and adds stop buffer mixing, measures OD value.
In the present invention, described pre-treatment is for be diluted testing sample.The method of described dilution is not had by the present invention
Particular restriction, uses the technical scheme of known dilution as well known to those skilled in the art.The dilution factor of described sample is excellent
Elect 1:10～100 as.
After described pre-treatment, the B cell that the sample addition through described pre-treatment has been coated with OMP18 is resisted by the present invention
In the detection plate hole of former epitope polypeptide, mixing, carry out hatching for the first time.In the present invention, the environment temperature that described first time hatches
Degree is preferably 35～38 DEG C, more preferably 37 DEG C；The time that described first time hatches preferably 20～35min, more elect 30min as.
After having hatched, the present invention uses cleaning mixture to carry out washing for the first time to detection plate.In the present invention, described first
The number of times of secondary washing is preferably 4～5 times.
After cleaning mixture carries out washing for the first time, present invention detection plate after described washing adds two anti-solution, described
The addition of two anti-solution and sample introduction are 1:1～2 by volume, and the mass concentration of described two anti-solution is 2mg/ml,
Mixing, carries out second time and hatches.In the present invention, the environment that described second time is hatched is preferably 37 DEG C, and incubation time is preferably
30min。
The present invention carries out second time to detection plate after second time has been hatched and washs.In the present invention, described second time
The number of times of washing is preferably 4～5 times.
Detection plate after washing adds substrate nitrite ion, carries out third time and hatches.In the present invention, described substrate colour developing
The consumption of liquid and sample introduction are 1:1 by volume.In the present invention, described three times hatch condition and technique scheme
Described first time incubation conditions is identical.
After described third time is hatched, the present invention adds stop buffer mixing in described detection plate, and detection plate is measured OD value.
In the present invention, the consumption of described stop buffer is 50 μ l；Described mensuration OD value is measured under 450nm wavelength.
In order to verify whether the B cell antigen epitope polypeptide of OMP18 is applicable to detect campylobacter jejuni as envelope antigen
Experiment, the immunoreactivity of the B cell antigen epitope polypeptide of OMP18 is measured, comprises the following steps: by the present invention
1) use is coated the B cell antigen epitope polypeptide of liquid dilution antigen OMP18, and the antigen after described dilution is joined inspection
In drafting board, overnight, negative control is carried out with BSA as envelope antigen；
2) by described step 1) the detection plate being coated with antigen that obtains washs, empty dry after, to being coated with antigen
Adding confining liquid in detection plate, close overnight, detection plate is washed by next day, seals；
3) to described step 2) gained detection plate add the anti-campylobacter jejuni of an anti-rabbit full bacterium IgG, hatch, then outwell rabbit
Anti-campylobacter jejuni full bacterium IgG, adds cleaning mixture washing, adds the anti-solution of goat anti-rabbit igg two of the enzyme labelling after dilution, hatches,
Outwell described two anti-solution washings, add substrate colour developing, detect OD value；
4) according to negative control sample OD value+3SD as positive criterion.
The present invention B cell antigen epitope polypeptide being coated liquid dilution antigen OMP18.In the present invention, described antigen
Dilution factor is preferably 1:1000～64000, more elects 1: 1000,1: 4000,1: 16000,1: 64000 as.
In the present invention, the B cell antigen of negative control uses the concentration of BSA, volume, dilution factor and described OMP18
The concentration dilution degree volume of epitope polypeptide is identical.
After obtaining being coated with the detection plate of described antigen, under the conditions of 4 DEG C, add confining liquid 200～300 μ l, be preferably
300 μ l, the time of described closing is 12～14h.
Described confining liquid be mass concentration be the calf serum solution of 5%.The source of described calf serum is not had by the present invention
There is particular restriction, use calf serum well-known to those skilled in the art, calf blood used in the embodiment of the present application
Clear source is commercially available commodity.
After closing, the present invention adds cleaning mixture in described detection plate and carries out washing for the first time.In the present invention, described first
Secondary washing is at room temperature carried out；Washing times is preferably 3～6 times, more elects 4～5 times as；Cleaning mixture addition is 50 μ l.
Described cleaning mixture is the PBS solution comprising 0.05%Tween 20.The source of described cleaning mixture is not had by the present invention
Particular restriction, uses PBS solution well-known to those skilled in the art, and in the embodiment of the present application, PBS solution used is
Oneself configuration obtains.
After completing to wash for the first time, the present invention adds an anti-campylobacter jejuni of anti-rabbit in the detection plate after described closing complete
Bacterium IgG.In the present invention, described one anti-dilution factor is preferably 1:3000.
The temperature hatched after described addition one is anti-is preferably 35～38 DEG C, more preferably 37 DEG C, described in time of hatching preferred
20～35min.More preferably 30min.
Add one anti-hatch after, the present invention adds cleaning mixture to detection plate and carries out second time and wash.In the present invention, second
Secondary washing is identical with washing methods for the first time.
After second time washing, the present invention adds the anti-solution of goat anti-rabbit igg two of the enzyme labelling after dilution in detection plate.?
In the present invention, the dilution factor of described two anti-solution is preferably 1:8000～12000, more preferably 1:10000；Described two anti-solution
Addition be 50 μ l.
Add two anti-after incubation conditions be that temperature is preferably 35～38 DEG C, more preferably 37 DEG C, described in time of hatching excellent
Select 20～35min, more elect 30min as；Stand in described incubation time, it is to avoid shake.
Add two anti-hatch after, the present invention adds cleaning mixture in detection plate and carries out third time and wash.In the present invention,
Three washings are identical with washing methods for the first time.
After third time washing, the present invention adds substrate colour developing in detection plate.In the present invention, addition substrate is TMB, right
The described amount of addition substrate and the concentration of substrate do not have particular/special requirement, and the concentration of substrate being well known to those skilled in the art is i.e.
Can.
Described substrate is TMB.The source of described TMB is not particularly limited by the present invention, uses those skilled in the art institute
Known to TMB, TMB source used in the embodiment of the present application is commercially available commodity.
After adding substrate, detection plate is carried out the detection of OD value.In the present invention, detection plate is carried out the detection of OD value to be used
Microplate reader kind and model be not particularly limited, the microplate reader being well known to those skilled in the art.Described detection
Wavelength is 450nm.
In order to verify whether the B cell antigen epitope polypeptide of OMP18 is applicable to detect campylobacter jejuni as envelope antigen
Experiment, also the immunologic opsonin of the B cell antigen epitope polypeptide of OMP18 is measured, specifically includes the following step:
A) epitope peptide is coated in detection plate, is coated overnight, use BSA solution as negative control；
B) described step a) gained envelope antigen cleaning mixture is washed, then close overnight with confining liquid, to detection plate
Wash, then seal；
C) described step b) gained detection plate is added an anti-campylobacter jejuni full bacterium rabbit anti-igg solution of dilution, hatches,
Then outwell a described anti-campylobacter jejuni full bacterium rabbit anti-igg solution, add cleaning mixture washing, the enzyme after detection plate adds dilution
The anti-solution of rabbit anti-igg two of labelling, hatches, and outwells described two anti-solution, washing, adds substrate colour developing to detection plate, detects OD
Value；
D) according to negative control sample OD value+3SD as positive criterion.
The present invention B cell antigen epitope polypeptide being coated liquid dilution antigen OMP18.In the present invention, described antigen
Dilution factor is preferably 1: 64000.
In the present invention, the B cell antigen of negative control uses the concentration of BSA, volume, dilution factor and described OMP18
The concentration dilution degree volume of epitope polypeptide is identical.
After obtaining being coated with the detection plate of described antigen, under the conditions of 4 DEG C, add confining liquid 200～300 μ l, be preferably
300 μ l, the time of described closing is 12～14h.
Described confining liquid be mass concentration be the calf serum solution of 5%.The source of described calf serum is not had by the present invention
There is particular restriction, use calf serum well-known to those skilled in the art, calf blood used in the embodiment of the present application
Clear source is commercially available commodity.
After closing, the present invention adds cleaning mixture in described detection plate and carries out washing for the first time.In the present invention, described first
Secondary washing is at room temperature carried out；Washing times is preferably 3～6 times, more elects 4～5 times as.
Described cleaning mixture is the PBS solution comprising 0.05%Tween 20.The source of described cleaning mixture is not had by the present invention
Particular restriction, uses PBS solution well-known to those skilled in the art, and in the embodiment of the present application, PBS solution used is
Oneself configuration obtains.
After completing to wash for the first time, the present invention adds an anti-campylobacter jejuni of anti-rabbit in the detection plate after described closing complete
Bacterium IgG.In the present invention, described one anti-dilution factor is preferably 1:3000.
The temperature hatched after described addition one is anti-is preferably 35-38 DEG C, more preferably 37 DEG C, described in time preferably 20 of hatching
～35min.More preferably 30min.
A described anti-solution is respectively Cj infection rabbit anteserum, Healthy Rabbits serum, infection due to Escherichia coli Sanguis Leporis seu oryctolagi
Clearly, Shigella dysenteriae infected rabbits serum and infection by Salmonella typhi rabbit anteserum.The present invention is the most special to described one source resisted
Limit, use well-known to those skilled in the art described one to resist, described one anti-coming used in the embodiment of the present application
Source is commercially available commodity.
Add one anti-hatch after, the present invention adds cleaning mixture to detection plate and carries out second time and wash.In the present invention, second
Secondary washing is identical with washing methods for the first time.
After second time washing, the present invention adds the anti-solution of goat anti-rabbit igg two of the enzyme labelling after dilution in detection plate.?
In the present invention, the dilution factor of described two anti-solution is preferably 1:8000～12000, more preferably 1:10000.
Add two anti-after incubation conditions be that temperature is preferably 35～38 DEG C, more preferably 37 DEG C, described in time of hatching excellent
Select 20～35min, more elect 30min as,
Add two anti-hatch after, the present invention adds cleaning mixture in detection plate and carries out third time and wash.In the present invention,
Three washings are identical with washing methods for the first time.
After third time washing, the present invention adds substrate colour developing in detection plate.In the present invention, addition substrate is TMB, right
The described amount of addition substrate and the concentration of substrate do not have particular/special requirement, and the concentration of substrate being well known to those skilled in the art is i.e.
Can.
Described substrate is TMB.The source of described TMB is not particularly limited by the present invention, uses those skilled in the art institute
Known to TMB, TMB source used in the embodiment of the present application is commercially available commodity.
After adding substrate, detection plate is carried out the detection of OD value.In the present invention, detection plate is carried out the detection of OD value to be used
Microplate reader kind and model be not particularly limited, the microplate reader being well known to those skilled in the art.Described detection
Wavelength is 450nm.
Result display OMP18 three epitope polypeptides of B cell antigen at dilution factor 1: 1000,1: 4000,1: 16000,1
: when 64000, immunoreactivity all has and substantially increases, and wherein dilution factor increases the most obvious when 1: 1000；Meanwhile, three
The B cell antigen epitope polypeptide of OMP18 has preferable immunologic opsonin, and therefore, the B cell antigen epi-position of three OMP18 is many
Peptide can carry out ELISA detection as envelope antigen to the serum of Cj infection.
The enzyme linked immunological kit provided the present invention below in conjunction with embodiment is described in detail, but can not be it
Be interpreted as limiting the scope of the present invention.
Embodiment 1
Use TMHMM Server V2.0, analysis of biological information software and the tool analysis such as DNA Star, NCBI/Blast
The structure of campylobacter jejuni major outer membrane protein OMP18 and characterization of molecules, territory predicted protein functional structure shows to deposit in OMP18
At OMP A domain and closely related with the adhesive capacity of campylobacter jejuni.Secondary structure analysis result shows, at OMP18 egg
Having 3 Linear B Cell Epitopes in Bai, it has good antigenicity and hydrophilic, uses Suspension array technique, specifically uses solid phase
Peptide synthesis technology, with N-α-Fmoc protection aminoacid as raw material, Fmoc-AA-Wang resin is carrier, HBTU method coupling
Rear acquisition polypeptide crude product, after being identified by crude product polypeptide analysis, uses preparative reversed-phase liquid chromatography (RP-HPLC) method to carry out pure
Change, and with HPLC and MS Analysis and Identification, filter out the B cell Linear antigenic table of 3 campylobacter jejuni major outer membrane protein OMP18
Position polypeptide.
Find through order-checking, the Staphylococal Protein A epitope amino acid sequence of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18
It is classified as STKSTSVSGDSSVDSNRGSGGSDGWDID；The B of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18 resists
Former Epitope peptide sequences is EGNCDEWGTDEY；The C epitope of the B cell antigen epitope polypeptide of described campylobacter jejuni OMP18
Peptide sequence is SYGETNPVCTEKTKACDAQNRR.
Embodiment 2
The B cell antigen epitope polypeptide immunoreactivity assay method of OMP18, comprises the following steps:
1) three kinds of antigen epitope polypeptides that embodiment 1 obtains are respectively Staphylococal Protein A, B antigen, C antigen, carry out every kind of antigen
According to 1:1000, the ratio of 1:4000,1:16000,1:64000 is diluted, with OMP18Pro as positive control；
2) adding the 50 above-mentioned dilution antigens of μ L in the 96 each holes of hole ELISA Plate, 4 DEG C are coated overnight, each epitope peptide
Sample standard deviation repeats three holes, and equivalent BSA is the negative control of envelope antigen；
3) the secondary daily PBS containing 0.05%Tween 20 washs 4 times, then closes overnight with 5% calf serum 4 DEG C；
4) the anti-campylobacter jejuni of the rabbit anti-solution of full bacterium IgG mono-of the 50ul that the ELISA Plate after closing dilutes with 1:3000,
Hatch 30min for 37 DEG C；
5) hatch rear ELISA Plate and discarded a unreacted anti-solution, wash 5 with the PBS containing 0.05%Tween 20
Secondary；
6) goat-anti of the 2mg/ml HRP labelling of the 50ul mass concentration of the ELISA Plate addition 1:10000 dilution after washing
The anti-solution of rabbit igg two, hatches 30min for 37 DEG C；
7) ELISA Plate after having hatched discards unreacted two anti-solution, washs 4 times, adds substrate MB, 450nm wavelength
Lower mensuration OD value.
8) using the negative control sample OD value+3SD standard deviation of OD value (SD refer to) as positive criterion.
Experimental result is as follows, and table 1 is that the B cell antigen epitope polypeptide immunity of 3 OMP18 of different gradient concentration is anti-
Answering property ELISA detects OD value.
The B cell antigen epitope polypeptide immunoreactivity ELISA detection OD value of 3 OMP18 of the different gradient concentration of table 1
Embodiment 3
The B cell antigen epitope polypeptide immunologic opsonin assay method of OMP18, comprises the following steps:
A) in 96 hole ELISA Plate, add the B cell antigen epi-position of the 50 above-mentioned three kinds of OMP18 of μ l of 1:64000 dilution proportion
Polypeptide solution, 4 DEG C are coated in detection plate overnight, and each epitope peptide sample standard deviation repeats three holes, uses BSA solution as feminine gender
Comparison；
B) described step a) gained is coated with the ELISA Plate PBS washing containing 0.05%Tween20 5 times of antigen, so
Close overnight with 5% calf serum 4 DEG C afterwards；
C) described step b) gained detection plate will be separately added into the Cj infection rabbit anteserum of dilution, healthy Sanguis Leporis seu oryctolagi
Clearly, infection due to Escherichia coli rabbit anteserum, Shigella dysenteriae infected rabbits serum and each 50ul of infection by Salmonella typhi rabbit anteserum, 37 DEG C
Hatch 30min；
D) ELISA Plate after hatching outwells described anti-liquid, washs 5 times with the PBS containing 0.05%Tween 20；
E) add the dilution of 1:10000 dilution to detection plate after, the rabbit of the HRP labelling of the 2mg/ml of 50ul mass concentration resists
The anti-solution of IgG bis-, hatches 30min for 37 DEG C,
F) ELISA Plate after hatching outwells the two anti-solution that unreacted completes, and washs 5 with the PBS containing 0.05%Tween 20
Secondary, add substrate TMB colour developing to detection plate, under 450nm wavelength, measure OD value；
G) according to negative control sample OD value+3SD as positive criterion.
Experimental result is as shown in table 2 below, and table 2 is the B cell antigen epitope polypeptide immunologic opsonin ELISA inspection of 3 OMP18
Survey OD value.
The B cell antigen epitope polypeptide immunologic opsonin ELISA detection OD value of 23 OMP18 of table
As seen from the above embodiment, result display OMP18 three epitope polypeptides of B cell antigen at dilution factor 1:
1000,1: 4000,1: 16000,1: 64000 time immunoreactivity all have and substantially increase, wherein dilution factor increases when 1: 1000
The most obvious；Meanwhile, the B cell antigen epitope polypeptide of three OMP18 has preferable immunologic opsonin, therefore, three
The B cell antigen epitope polypeptide of OMP18 can carry out ELISA detection as envelope antigen to the serum of Cj infection.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. an enzyme linked immunological kit for B cell antigen epitope polypeptide based on OMP18 detection campylobacter jejuni, its feature exists
In, including following components:
(1) the detection plate of the B cell antigen epitope polypeptide of OMP18, described detection plate vacuum sealed package it are coated with；
(2) cleaning mixture；
The rabbit anti-igg solution of (3) two antienzyme labellings；
(4) stop buffer；
(5) sample diluting liquid；
(6) substrate nitrite ion.
Test kit the most according to claim 1, it is characterised in that described stop buffer is the sulfuric acid solution or 2 of 1～2mol/L
～the sodium hydroxide solution of 3mol/L.
Test kit the most according to claim 1, it is characterised in that described sample diluting liquid be pH value 7.4～8.0, mole
Concentration 0.1～0.25mol/L, containing volumetric concentration 45～the phosphate buffer of 50% methanol.
Test kit the most according to claim 1, it is characterised in that the bag of the B cell antigen epitope polypeptide antigen of described OMP18
It is 4～9 μ g/ml by concentration.
Test kit the most according to claim 1, it is characterised in that the enzyme used in the rabbit anti-igg solution of described enzyme labelling
For HRP, described substrate nitrite ion is TMB.
Test kit the most according to claim 1, it is characterised in that described cleaning mixture is for comprising mass concentration 0.05～0.1%
The PBS solution of Tween 20.
7. according to the test kit described in Claims 1 to 4 any one, it is characterised in that the B cell antigen table of described OMP18
The Staphylococal Protein A epitope amino acid sequence of position polypeptide is STKSTSVSGDSSVDSNRGSGGSDGWDID.
8. according to the test kit described in Claims 1 to 4 any one, it is characterised in that the B cell antigen table of described OMP18
The B epitope peptide sequence of position polypeptide is EGNCDEWGTDEY.
9. according to the test kit described in Claims 1 to 5 any one, it is characterised in that the B of described campylobacter jejuni OMP18
The C epitope peptide sequence of cell antigen epitope polypeptide is SYGETNPVCTEKTKACDAQNRR.
10. the application in detection campylobacter jejuni of the test kit described in claim 1～9 any one, it is characterised in that bag
Include following steps:
A, testing sample is carried out pre-treatment；
B, described step a is processed after testing sample add the detection of B cell antigen epitope polypeptide being coated with OMP18
In plate hole, mixing, hatch, wash with cleaning mixture；
C, described detection plate hole add two anti-solution, mixing, hatch, wash with cleaning mixture；
D, addition substrate nitrite ion, hatch, and adds stop buffer mixing, measures OD value.
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Application publication date: 20161116 Assignee: Shanghai Huiyun Biological Technology Co.,Ltd. Assignor: Jinhua College of Vocation and Technology Contract record no.: X2019980000956 Denomination of invention: ELISA (enzyme linked immunosorbent assay) detection kit for detecting campylobacter jejuni on basis of OMP18 (outer membrane protein 18) and application of detection kit Granted publication date: 20180504 License type: Exclusive License Record date: 20191210 |
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