CN109884291A - Campylobacter spp polypeptide, antibody capture device and kit - Google Patents

Abstract

Campylobacter spp polypeptide, antibody capture device and kit.The present invention discloses core sequence, the Campylobacter spp polypeptide containing core sequence, antibody capture device, kit and the detection method of Campylobacter spp polypeptide shown in a kind of SEQ ID:1, belongs to immunological technique field.The technical scheme is that clonal expression contains the Campylobacter spp polypeptide of SEQ ID:1 core sequence, the antibody in the polypeptide capture sample containing core sequence, has the function that auxiliary diagnosis Crohn disease by solid phase carrier and thereon.

Description

Campylobacter spp polypeptide, antibody capture device and kit

Technical field

Antibody capture device and kit the present invention relates to a kind of core sequence of Campylobacter spp polypeptide, comprising polypeptide, belong to Technical field of immunological detection.

Background technique

Inflammatory bowel disease (inflammatory bowel disease, IBD) is a kind of autoimmune enteropathy, including is burst Ulcer colitis (ulcerative colitis, UC) and Crohn disease (Crohn ' s disease, CD), exist during the nearly last ten years China's disease incidence is in gradually to increase trend.The complicated clinical manifestation multiplicity of IBD, not only has symptom of digestive tract, can also there is parenteral table It is existing.Since symptom is the non-specific enteritis performance such as abdominal pain, diarrhea, hematochezia, CD and UC and IBD and tuberculous enteritis etc. its He is difficult to discriminate between diagnosis at enteron aisle chronic disease.Serological index helps to identify CD and UC and IBD and non-IBD.

The pathogenesis of IBD is complicated, and the mechanism of one of them is to make intestinal mucosa since the factors such as heredity, environment influence Barrier dysfunction, immune system is immunoreacted antigenic product existing for normal bowel, shows as can be detected in vivo pair The antibody of enteric microorganism antigen and metabolin.It, can auxiliary diagnosis autoimmunity enteropathy packet by the detection to these antibody Include IBD.At present explicitly with the effect of IBD auxiliary diagnosis antimicrobial antibody include anti-CBirl antibody, anti-Fla-X resist The bacterial flagellums antibody such as body, anti-4aFla-2 antibody and anti-Pseudomonas fluorescens I2 antibody and anti-Escherichia coli outer membrane duct Albumen OmpC antibody etc..Due to the easy variability and polymorphism of microbial gene, new microbial antigen is continuously emerged, wherein Some immunological regulations for participating in intestinal mucosa.The IBD related antigen of Campylobacter spp polypeptide and existing report that the present invention is announced is more Peptide significant difference, but analyzed by sequence, it is certain core sequences that discovery generates related with Campylobacter spp polypeptide autoantibody, is contained There is the Campylobacter spp polypeptide of the core sequence to verify through the clinical samples of clinical definite, there is the energy for identifying Healthy People and Crohn disease Power.

Summary of the invention

The first aspect of the present invention announces Campylobacter spp polypeptide and core sequence shown in SEQ ID:1, connects core sequence Between amino acid sequence can there are many variation.

The polypeptide includes three core sequences, as shown in SEQ ID:2 and SEQ ID:3.

The second aspect of the present invention provides a kind of preparation method of antibody capture device, and the Campylobacter spp polypeptide is straight It connects or is indirectly fixed on solid phase carrier, for capturing the counter-bending bacteria antibody in sample.

Solid phase carrier includes microwell plate, fluorescent microsphere, latex beads, resin microsphere, magnetic microsphere, film, microporous barrier, nitre Acid cellulose film etc..

In one preference, solid phase carrier is microwell plate, is magnetic microsphere in another preference, in the 3rd preference Fluorescent microsphere and nitrocellulose membrane have been selected simultaneously, are colloidal gold in the 4th preference.

The method that polypeptide is fixed on solid phase carrier is direct coated or is coated with indirectly by carrier protein, it is preferable that carrier Albumen is BSA or biotin-streptavidin.

In one preference, the polypeptide and BSA are coupled, are then coated on solid phase carrier again.

In another preference, first polypeptide and biotin reaction, then biotinylated polypeptide and Streptavidin Coupling, forms the compound of polypeptide and streptavidin, then compound is coated on solid phase carrier.

The third aspect of the present invention discloses application of the antibody capture device above-mentioned in Crohn disease, of the present invention Application be to be included the following steps: to the measuring method of bacteria antibody counter-bending in sample based on immunological response

(1) sample to be detected acts on antibody capture device, makes counter-bending bacteria antibody and antibody capture device in sample On polypeptide combine；(2) pass through the content of bending resistance aspergillus polypeptide antibody in marker measurement sample.

Campylobacter spp antigen or epitope conjugate can be detected in sample by current any immunological detection method Counter-bending bacteria antibody, such as ELISA, board-like chemiluminescence, chemiluminescence, immunochromatography, immunity percolation etc. are corresponding Marker is respectively horseradish peroxidase, alkaline phosphatase, acridinium ester, rare earth element and colloidal gold etc..

Embodiment 4 provides the board-like kit of enzymatic and detection method of two kinds of counter-bending bacteria antibodies, Campylobacter spp polypeptide coating On microwell plate, antigen-antibody complex is formed with the antibody response in sample, anti-human igg and IgA enzyme are marked, it is used Enzyme is horseradish peroxidase and alkaline phosphatase, and sample used is excrement and urine.

Embodiment 5 provides the chemical luminescence reagent kit and detection method of a kind of acridinium ester label, and the Campylobacter spp polypeptide is raw By being coated on magnetic microsphere with streptavidin after object element, antigen-antibody is formed with the antibody response to be checked in plasma sample Compound carries out quantitative detection to antigen antibody complex with the anti-human antibody of acridinium ester label after cleaning.Detection method tool Have the advantages that high sensitivity, detection time be short, full-automatic detection.

Embodiment 6 provides a kind of fluorescence immune chromatography kit of bending resistance aspergillus polypeptide antibody and method, the method are Campylobacter spp polypeptide is coated with directly or by biotin-streptavidin onto the fluorescent microsphere containing rare earth element, while polypeptide It is coated on detection line T line, forms the counter-bending bacteria antibody content in dual-antigen sandwich method detection serum sample.The detection side Method has many advantages, such as that quick, detection time is short, high sensitivity.

Embodiment 7 provides the colloid gold reagent and method of a kind of bending resistance aspergillus polypeptide antibody, and the method is Campylobacter spp Polypeptide colloid gold particle or color latex microballoon label, are sprayed in sample pad, while polypeptide is coated on detection line T line, Form bending resistance aspergillus polypeptide antibody in dual-antigen sandwich method detection whole blood sample.The detection method can visual results, have Fast, the advantages that instrument cost is small.

Compared with prior art, Campylobacter spp polypeptide and antibody capture device provided by the invention, when being used for Crohn disease, tool There are high sensitivity, performance stabilization, simple operation and other advantages.

Detailed description of the invention

Fig. 1 chemiluminescence quantitative measurement standard curve

The specificity and sensitivity of Fig. 2 chemiluminescence quantitative detection

Fig. 3 fluorescence immune chromatography reagent standard curve

Specific embodiment

Biotin used in the present invention, goat anti-human igg, goat-anti people IgA are purchased from Sigma.Avidin is purchased from Pan Gu's gene.Magnetic Property microballoon and the pre-coated magnetic microsphere for having Streptavidin be purchased from Nanjing enlightening Gneuss.Microwell plate is purchased from Suzhou castor.Latex Microballoon, color latex microballoon, resin microsphere are purchased from Tianjin Sai Er groups.Rare earth element fluorescent microsphere and time-resolved fluorescence reader Purchased from Shenzhen micrometering.Other reagents are purchased from the raw work of traditional Chinese medicines reagent and Shanghai.

Combined with specific embodiments below and attached drawing, the present invention is further explained.

The preparation of 1 Campylobacter spp polypeptide of embodiment

1, SEQ ID:2 and the preparation of SEQ ID:3 polypeptide

SEQ ID:2 and SEQ ID:3 whole genome sequence is synthesized by Jin Weizhi.

1) reagent

Cloning vector pCR2.1 T-vector, expression plasmid pET28a (+), transfection Escherichia coli host strain DH5 α, BL21 (DE3), archaeal dna polymerase rTaq, T4DNA ligase, archaeal dna polymerase rTaq, LA Taq and restriction enzyme BamH I, Hind III and EcoR I, BamH I, DL2000DNA Marker, T4DNA ligase, low molecular weight standard protein, DNA glue return Receive kit, IPTG etc.

2) instrument

Common shaking table SCS-24；Water isolation type constant temperature electric heating incubator；Biophotometer spectrophotometer, desk-top freezing Centrifuge Centrifuge5810R, desk centrifuge MiniSpin；High speed freezing centrifuge；Protein electrophorese instrument and gel Imaging system；PCR instrument；Ultrasound cracking instrument；Constant-temperature metal bath；HIS protein purification column etc..

2. experimental method

1) vector construction:

Design primer, PCR amplification goes out SEQ ID:2 and SEQ ID:3 segment, plastic recovery kit recycling from template DNA It is connected to pCR2.1 cloning vector after segment and carries out sequencing identification.It will identify that correct sequence is cloned into expression vector pET28a In (+), restriction enzyme site is EcoR I, BamH I, while having 6 × HIS label on carrier, is convenient for subsequent protein purification.

2) it expresses

Obtained plasmid is converted to e. coli bl21 (DE3), resistance screening positive expression bacterial strain is passed through.It will filter out The positive bacterium solution come is inoculated in the LB culture medium added with Kan resistance in 1: 1000 ratio, and each two pipe of bacterium inoculation a, pipe is used for Induction, another pipe is used for non-induced control, while to be inoculated with a pipe empty plasmid bacterium and compare.37 DEG C are incubated overnight to OD600 value When about 0.4-0.6, IPTG to final concentration of 1mmol/L is added in induction pipe and empty control plasmid pipe, and non-induced control is not added, most Two high bacterium of selection ability to express eventually, the expression for a large amount of albumen.And according to conventional SDS-PAGE method to expression Product is identified.

3) it purifies

By the product of expression HIS protein purification column purification albumen, and dialysis desalting, SEQ ID:2 and SEQ after purification ID:3 cryo-conservation is spare.

Embodiment 2 prepares bending resistance aspergillus polypeptide antibody

1. animal immune

Mouse is immunized with SEQ ID:2 and SEQ ID:3 respectively, immunizing dose is 50 μ g/.After generating positive serum, add Strong immune primary rear progress cell fusion.

Such as need to prepare polyclonal antibody, can same method immune goat or rabbit, take serum obtain polyclonal antibody.

2. cell fusion

PEG1500 pre-temperature draws 1 × 10⁷A SP2/0 myeloma cell's suspension and 5 × 10⁷It is a it is immune after mouse spleen B Lymphocyte suspension (cell number 1: 5) is merged with the 50%PEG1500 solution of pre-temperature, and supernatant is abandoned in centrifugation, and 10ml HAT is added (SIGMA) cell is resuspended in culture medium, is seeded to and has been covered with 96 porocyte culture plates of trophocyte and is cultivated.

3. screening and clone

After fused cell culture 10-14 days, with SEQ ID:2 or SEQ ID:3 coating microwell plate screened, positive hole into Then row limiting dilution is further cultured for 10~14 days, 3~4 times repeatedly, obtains anti-SEQ ID:2 monoclonal antibody cell strain and anti-SEQ ID:3 monoclonal antibody cell strain.

4. prepared by ascites

Ascites is collected after mouse ascites accumulation in monoclonal cell Mice Inoculated abdominal cavity.By ascites under the conditions of 4 DEG C, 10000 revs/min are centrifuged 10 minutes, remove lipid material.Supernatant is drawn after centrifugation, and is filtered with 0.45 μm of film. Protein G is purified.Monoclonal antibody concentration mensuration after purification, dispense, freeze it is spare.

The preparation of the counter-bending bacteria antibody capture device of embodiment 3

1, SEQ ID:2 and SEQ ID:3 is coated with microwell plate

Concentration with phosphate buffered saline SEQ ID:2 and SEQ ID:3 is about 5ug/mL, every hole 100uL coating 96 Hole microwell plate, 4 DEG C overnight, discard coating buffer, and board-washing is primary, with Block buffer every hole 200uL containing 1% calf serum, 37 DEG C be incubated for 2 hours, pat dry, be dried for standby.

2, SEQ ID:2 and SEQ ID:3 is coated with magnetic microsphere

Appropriate amino magnetic microsphere is taken, is cleaned and is resuspended with PBS buffer solution, 5% glutaraldehyde solution is added, is stirred at room temperature anti- It answers 1 hour；It is cleaned and is resuspended with PBS buffer solution, after ultrasonic disperse, SEQ ID:2 or SEQ is added by 100 μ g/mg magnetic microspheres It is reacted at room temperature 6 hours after ID:3；It is cleaned and is resuspended with PBS buffer solution, after 1%BSA closing being added 30 minutes, it is sweet to add 1% Propylhomoserin is resuspended after being cleaned after closing 30 minutes with PBS buffer solution to get SEQ ID:2 magnetic microsphere and SEQ ID:3 magnetic microsphere.

3, SEQ ID:2 and SEQ ID:3 biotinylation

Biotin, dicyclohexylcarbodiimide (DCC) and n-hydroxysuccinimide (NHS) is taken to be dissolved in DMF, room temperature magnetic Power is stirred to react 3 hours, and supernatant is collected by centrifugation；Supernatant is added dropwise in SEQ ID:2 or SEQ ID:3 solution, 2-8 It DEG C is stirred to react 6 hours；Reaction solution taking-up is dialysed with phosphate buffer, obtains biotinylation SEQ ID:2 and biotinylation SEQ ID:3.

4, SEQ ID:2 and SEQ ID:3 coating fluorescent microsphere and detection line T line

1) fluorescent microsphere, color latex microballoon, resin microsphere label

Take fluorescent microsphere, color latex microballoon or resin microsphere 200 μ l (210nm) (1% stoste), 13000rpm, 4 DEG C from Heart 10min abandons supernatant, and 400ul deionized water ultrasound 10S is added and mixes；Centrifugation is added MES buffer (50mM, PH6.0) 400 μ l, ultrasound mix；EDC solution is added in centrifugation, reacts at room temperature 15 minutes；Centrifugation, be added labelled antibody (SEQ ID:2 and SEQ ID:3 and rabbit-anti chicken IgY) 100ug, room temperature shaker middling speed reaction 2 hours；Confining liquid is added in centrifugation, and ice water ultrasound mixes, Room temperature shaker middling speed is reacted 1 hour；400 μ l borate buffer solutions (20mM PH8.0) are added in centrifugation, and ice water ultrasound mixes, and 4 It DEG C saves stand-by.

2) detection line T line is coated with

With phosphate buffer SEQ ID:2 and SEQ ID:3 polypeptide is diluted to 1mg/ml respectively, draws liquid by 1 μ l/cm Amount is drawn film instrument with metal spraying and is uniformly drawn to preparation T line on NC film；The NC film pulled is placed in 37 DEG C of drying boxes, it is dry 16h。

5, SEQ ID:2 and SEQ ID:3 marks colloidal gold

Colloidal gold 10ml is taken, appropriate 0.1M K is added₂CO₃Adjust pH.Appropriate formula SEQ ID:2 or SEQ are added after mixing ID:3 continues to stir 30min；Be added 10%BSA to its final concentration of 1%, continue stir 30min；4 DEG C of 10000rpm centrifugations 20min collects precipitating, with colloidal gold dilution (0.2M BB, 1%BSA, 3% trehalose, 0.03%procline300) constant volume To 1ml to get SEQ ID:2 and SEQ ID:3 colloidal gold composite.

4 Campylobacter spp polypeptide of embodiment is used to prepare the board-like detection kit of enzymatic

1, SEQ ID:2 and SEQ ID:3 polypeptide are coated with microwell plate

(1) SEQ ID:2 and SEQ ID:3 buffer are diluted to 5 μ g/ml, in coating to microwell plate, 100 μ l of every hole, and 4 DEG C It is incubated for 16h or 37 DEG C of incubation 2h.

(2) with PBST washing 3 times, drying；

(3) it is closed with the protein solution containing 1% bovine serum albumin(BSA), 200uL confining liquid is added in every hole, and 37 DEG C anti- 2h is answered, hole inner sealing liquid is discarded, is dried；

(4) coating plate is placed in 37 DEG C of baking oven 4h, that is, completes coating, is sealed with aluminium foil bag, it is standby to deposit in -20 DEG C of preservations With.

2, anti-polypeptide IgG antibody detection

(1) it is added to after diluting 10 healthy human faecal mass and 10 cd patient fecal samples with Sample dilution In the microwell plate being coated with, react at room temperature 1h, board-washing 4 times；

(2) it is added the goat anti-human igg of horseradish peroxidase-labeled, 37 DEG C of reaction 0.5h, board-washing 4 times；

(3) plus horseradish peroxidase substrate develops the color；

(4) it terminates, reading.

3, anti-polypeptide IgA antibody detection

(1) coating is added to after diluting 10 Healthy People urines and 10 cd patient urines with Sample dilution In good microwell plate, 1h is reacted at room temperature, board-washing 4 times；

(2) it is added the goat anti-human igg of alkali phosphatase enzyme mark, 37 DEG C of reaction 0.5h, board-washing 4 times；

(3) plus alkaline phosphatase substrate develops the color；

(4) it terminates, reading.

4, data are analyzed

The mean OD value of 10 Healthy People samples is calculated, 2.5 times of OD value is higher than as negative and positive numerical value is distinguished This value is positive (+), is negative (-) less than or equal to this value, counts the inspection of 10 patient's fecal samples and urine specimen As a result measured data prompts, the antibody positive rate of anti-SEQ ID:2 and SEQ ID:3 polypeptide is distinguished in the excrement of cd patient For 20% and 30% (being shown in Table 1)；Positive rate in urine specimen is respectively 10% and 20%.

Testing result of the 1 Campylobacter spp polypeptide antibody of table in fecal sample

Embodiment 5 is used to prepare acridinium ester label chemiluminescence detection kit

It can be there are two types of different implementation methods:

The first: (1) pressing step 2 method of embodiment 3, SEQ ID:2 and SEQ ID:3 direct coated in magnetic microsphere On, this microspheres solution is R1；(2) R2 is goat anti-human igg/IgA antibody of acridinium ester label.When detection sample to be detected with R1 reaction, Magnetic Isolation removes sample impurity later, cleans 3 times, reacts with R2, is formed sandwich containing Ag-Ab-antiantibody Complex, compound carry out acridinium ester quantitative fluorescence analysis.

Second: (1) pressing step 3 method of embodiment 3, SEQ ID:2 and SEQ ID:3 biotinylation, this solution is R1；(2) R2 is the magnetic microsphere of pre-coated SA；(2) R3 is goat anti-human igg/IgA antibody of acridinium ester label.Detect Shi Keyong One-step method or two step method, the former is for sample sheet, R1 and R2 simultaneous reactions；The latter is first SA magnetic microsphere and biotinylated R1 It is added after reaction, Magnetic Isolation and washing for sample sheet.Two methods are equally effective.One-step method or two step method are formed containing anti- Antigen-antibody-antiantibody sandwich complex, compound carry out acridinium ester quantitative fluorescence analysis.

1) standard curve making

The goat anti-human igg of acridinium ester label or IgA antibody are diluted 5 concentration, respectively: the goat-anti people of acridinium ester label IgG and IgA antibody respectively dilute 5 concentration, respectively 0ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 400ng/ml.With Above-mentioned the first and second detection method make standard curve, and wherein the anti-human igg of second method and IgA standard curve are shown in Fig. 1, R²Value is respectively 0.9978 and 0.9973.

2) pattern detection

100 human normal plasmas or serum and 100 clone grace patients are detected with above-mentioned the first and second method Blood plasma or serum, the sensitivity of two kinds of detection methods with second be it is excellent, wherein the ROC curve of second method detection is shown in Fig. 2, But the IgG of bending resistance aspergillus polypeptide is significantly better than the diagnosis efficiency of IgA antibody, and area is only under the ROC curve of SEQ ID:2 and 3 0.615 and 0.683 (data are not shown).As shown in table 2, the area under the curve of SEQ ID:2 and SEQ ID:3 anti-human igg is distinguished For 0.693 and 0.753, when illustrating for diagnosing Crohn disease, SEQ ID:3 is better than SEQ ID:2.

The IgG area under the curve of 2 anti-SEQ ID:2 and SEQ ID:3 of table

6 Campylobacter spp polypeptide of embodiment is used to prepare rare earth element fluorescence immune chromatography detection kit

1, the preparation of fluorescence immune chromatography kit

(1) label of fluorescent microsphere and detection line T line coating

The method of SEQ ID:2 and SEQ ID:3 polypeptide marker fluorescent microsphere is shown in 3 step 4 of embodiment.

(2) preparation of sample pad and bonding pad

Glass fibre element film with containing surfactant buffer (formula: 100mM pH 7.4PB, wherein containing 2% NaCl, 2%BSA, 0.5% casein, 0.1% Tween-20,0.5%S9 and 5% sucrose) impregnate carry out in advance close after, 37 DEG C It is dried overnight, sample pad is prepared；

The sample pad prepared is taken, draws SEQ ID:2 and SEQ the ID:3 polypeptide that film instrument will be marked with fluorescent microsphere with metal spraying It is sprayed onto the wide sample pad for 1cm of pretreatment with rabbit-anti chicken IgY antibody according to the amount of 5 μ l/cm, 37 DEG C of dry 5h are prepared into To bonding pad.

(3) coating of nitrocellulose filter (NC film)

With phosphate buffer SEQ ID:2 and SEQ ID:3 polypeptide is diluted to 1mg/ml respectively, is used to prepare T line；It will Chicken IgY antibody is diluted to 0.5mg/ml, is used to prepare C line；Liquid measure is drawn by 1 μ l/cm, drawing film instrument with metal spraying will be above two molten Liquid is uniformly drawn to preparation T line and C line on NC film；The NC film pulled is placed in 37 DEG C of drying boxes, dry 16h.

(4) it assembles

The bonding pad that step 2) is obtained is laminated on the one end for the nitrocellulose filter that step 3) obtains, and water absorption pad is consolidated Surely it is laminated on the other end of nitrocellulose filter, the sample pad for finally obtaining step 2) is laminated on the bonding pad other end, and use is micro- The automatic cutting machine of computer is cut by the width of every 5.5mm, and is fitted into chromatography strip shell to get finished product.

2, standard curve

The anti-SEQ ID:2 monoclonal antibody and anti-SEQ ID:3 monoclonal antibody prepared with embodiment 2 dilutes 5 concentration respectively, respectively: The 50 above calibration objects of μ l are added drop-wise on well by 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 200ng/ml respectively, It is detected after 15 minutes with fluorescence detector, fluorescence can be collected on detection line T and nature controlling line location of C.With sample concentration and T/C Value carries out quadratic polynomial fitting, draws standard curve, the R of SEQ ID:2 and 3²Respectively 0.9904 and 0.9970, see Fig. 3. According to the detection to 200 Healthy Human Serum samples, the anti-SEQ ID:2 of 90% Healthy People and SEQ ID:3 antibody are judged as yin The cut-off value of property is 30ng/ml and 32ng/ml respectively.

3. sample detection

The blood sample to be checked for taking 50 μ l, is added drop-wise on well, is detected after 15 minutes with fluorescence detector, if detection line goes out Existing band, illustrates the polypeptide antibody of aspergillus containing bending resistance in sample, and content can the acquisition of establishing criteria curve calculation formula.20 samples Testing result to be shown in Table the detection specificity of 3, SEQ ID:2 and SEQ ID:3 be respectively 70% and 80%, sensitivity is respectively 40% and 50%.

3 fluorescence immune chromatography test result of table

7 Campylobacter spp polypeptide of embodiment is used to prepare gold calibration property detection kit

1, SEQ ID:2 and SEQ ID:3 gold marked reagent blocking are standby

1) preparation of colloidal gold

In round-bottomed flask be added 0.01% chlorauric acid solution of 100ml, be placed in and be heated to boiling on electric jacket, at once plus Enter 2ml1% citric acid three sodium solution, continues to stir 15min, be saved backup for 4 DEG C after natural cooling.

2) colloid gold label

See 3 step 5 of embodiment.

3) preparation of sample pad and gold-labelled pad

Glass fibre element film with treatment fluid liquid (formula: 100mM pH 7.4PB, wherein contain 2%BSA, 0.5% tween- 20,0.5%S9 and 5% sucrose) impregnate carry out in advance close after, 37 DEG C are dried overnight, and sample pad is prepared；

Take the sample pad prepared, with metal spraying draw film instrument by be marked with SEQ ID:2 and SEQ ID:3 gold mark compound and The gold mark compound of rabbit-anti chicken IgY antibody label is sprayed onto the wide sample pad for 1cm of pretreatment according to the amount of 5 μ l/cm, and 37 DEG C Dry 5h, is prepared gold-labelled pad.

4) coating of nitrocellulose filter (NC film)

SEQ ID:2 and SEQ ID:3 polypeptide is diluted to 1mg/ml with phosphate buffer, is used to prepare T line；By chicken IgY antibody is diluted to 0.5mg/ml, is used to prepare C line；Liquid measure is drawn by 1 μ l/cm, draws film instrument for above two solution with metal spraying It is uniform to draw to preparation T line and C line on NC film；The NC film pulled is placed in 37 DEG C of drying boxes, dry 16h.

5) it assembles

The auxiliary materials such as above-mentioned gold-labelled pad, sample pad, the NC film being coated with and water absorption pad are assembled into gold-labeled kit.

2, kit detects

(1) detection method

Sampling originally to take 100 μ l after Sample dilution (BB, 0.5%s9,1%BSA) dilution, is directly added into sample in chromatography strip Product window；After 15min, Visual observations.

(2) result judgement

Negative findings (-): only there is nature controlling line, no detection line；

Positive findings (+): nature controlling line occurs simultaneously with detection line；

Null result: nature controlling line does not occur, and shows operating mistake or kit failure.

(3) it detects

The standard items and 20 whole blood samples for taking 100 μ l embodiments 5 to prepare are separately added into sample window in chromatography strip； After 15min, Visual observations (table 4), SEQ ID:2 and SEQ ID:3 is used to judge that the specificity of Crohn disease to be respectively 70% and 80%, sensitivity is respectively 30% and 40%.

4 gold marked reagent test result of table

Claims (7)

1. Campylobacter spp polypeptide shown in a kind of SEQ ID:1, which is characterized in that include 3 core sequences and catenation sequence xxx.

2. Campylobacter spp polypeptide described in claim 1, which is characterized in that catenation sequence xxx can make a variation, it is preferable that be SEQ ID: 2 or SEQ ID:3 polypeptide.

3. a kind of antibody capture device, which is characterized in that bending described in solid phase carrier and thereon coated claim 1~2 Bacterium polypeptide.

4. solid phase carrier as claimed in claim 3, which is characterized in that including but not limited to fluorescent microsphere, latex beads, resin are micro- Ball, magnetic microsphere, colloid gold particle, microwell plate, film, microporous barrier, nitrocellulose filter.

5. a kind of Crow grace detection kit and method, which is characterized in that including the polypeptide or power described in claim 1~2 Benefit require 3~4 described in antibody capture device, method comprising steps of

(1) sample is acted on into antibody capture device；

(2) pass through the content of bending resistance aspergillus polypeptide antibody in marker measurement sample.

6. sample described in claim 5, which is characterized in that serum, blood plasma, whole blood, urine or excrement.

7. marker described in claim 5, which is characterized in that include but is not limited to horseradish peroxidase, alkaline phosphatase, Acridinium ester, rare earth element and colloidal gold.