CN109879940A - Flagellum G polypeptide, antibody capture device and kit - Google Patents

Abstract

Flagellum G 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 flagellum G polypeptide shown in a kind of SEQ ID:1, belongs to immunological technique field.The technical scheme is that clonal expression contains the flagellum G 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

Flagellum G polypeptide, antibody capture device and kit

Technical field

The present invention relates to a kind of core sequences of flagellum G polypeptide, and it includes the antibody capture devices and kit of polypeptide, belong to In immunological technique field.

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 flagellum G polypeptide that the present invention is announced is related to the flagellin of existing report anti- Former polypeptide significant difference, but analyzed by sequence, it is certain core sequences that discovery generates related with flagellum G polypeptide autoantibody Column, the flagellum G polypeptide containing the core sequence are verified through the clinical samples of clinical definite, have and identify Healthy People and Crow grace The ability of disease.

Summary of the invention

The first aspect of the present invention, announce SEQ ID:1 shown in flagellum G polypeptide and core sequence, connection core sequence it Between amino acid sequence can there are many variation.

The polypeptide includes 2~3 core sequences, and wherein SEQ ID:2 and SEQ ID:3 contains three core sequences simultaneously, SEQ ID:4 contains two core sequences.

The second aspect of the present invention provides a kind of preparation method of antibody capture device, and the flagellum G polypeptide is direct Or be indirectly fixed on solid phase carrier, for capturing the anti-flagellum G 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, the third aspect of the present invention, the open reagent being made of antibody capture device above-mentioned Box and the application in diagnosis of Crohn disease, application of the present invention are based on immunological response, to enterobacteriaceae anti-in sample The measuring method of antibody includes the following steps: that (1) sample to be detected acts on antibody capture device, makes the anti-flagellum in sample G antibody is in conjunction with the polypeptide on antibody capture device；(2) contained by anti-flagellum G polypeptide antibody in marker measurement sample Amount.

Flagellum G antigen or epitope conjugate can be detected in sample by current any immunological detection method Anti- flagellum G antibody, such as ELISA, board-like chemiluminescence, chemiluminescence, immunochromatography, immunity percolation etc., corresponding mark Remember that object 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 anti-flagellum G antibody, and flagellum G polypeptide is coated on On microwell plate, antigen-antibody complex is formed with the antibody response in sample, anti-human igg and IgA enzyme are marked, enzyme used For horseradish peroxidase and alkaline phosphatase, sample used is excrement and urine.

Embodiment 5 provides the chemical luminescence reagent kit and detection method of a kind of acridinium ester label, the flagellum G polypeptide biology By being coated on magnetic microsphere with streptavidin after elementization, 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 the fluorescence immune chromatography kit and method of a kind of anti-flagellum G polypeptide antibody, and the method is handle Flagellum G polypeptide is coated with directly or by biotin-streptavidin onto the fluorescent microsphere containing rare earth element, while polypeptide packet By to the anti-flagellum G antibody content on detection line T line, formed in dual-antigen sandwich method detection serum sample.Detection method tool Have the advantages that quick, detection time is short, high sensitivity.

Embodiment 7 provides the colloidal gold chromatographic kit and method of a kind of anti-flagellum G polypeptide antibody, and the method is whip Hair G polypeptide colloid gold particle or color latex microballoon label, are sprayed in sample pad, while polypeptide is coated on detection line T line On, form anti-flagellum G polypeptide antibody in dual-antigen sandwich method detection whole blood sample.The detection method can visual results, tool Have the advantages that quick, instrument cost is small.

Compared with prior art, flagellum G 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 and Abcam.Avidin is purchased from Pan Gu Gene.Magnetic microsphere and the pre-coated magnetic microsphere for having Streptavidin are purchased from Nanjing enlightening Gneuss.Microwell plate is purchased from Su Zhouhai Leopard cat.Latex beads, color latex microballoon, resin microsphere are purchased from Tianjin Sai Er groups.Rare earth element fluorescent microsphere and time resolution are glimmering Optical reader is 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 flagellum G polypeptide of embodiment

1, SEQ ID:2, SEQ ID:3 and the preparation of SEQ ID:4 polypeptide

SEQ ID:2, SEQ ID:3 and SEQ ID:4 whole genome sequence are 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, SEQ ID:3 and SEQ ID:4 segment, glue recycling examination from template DNA PCR2.1 cloning vector, which is connected to, after agent box recycling segment carries out sequencing identification.It will identify that correct sequence is cloned into expression vector In pET28a (+), restriction enzyme site is EcoR I, BamH I, while having 6 × HIS label on carrier, pure convenient for subsequent albumen Change.

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, one is effective In 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 When value is 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 Add, two high bacterium of final choice ability to express, the expression for a large amount of albumen.And according to conventional SDS-PAGE method Expression product is identified.

3) it purifies

By the product of expression HIS protein purification column purification albumen, and dialysis desalting, SEQ ID:2, SEQ after purification ID:3 and SEQ ID:4 cryo-conservation is spare.

Embodiment 2 prepares anti-flagellum G polypeptide antibody

1. animal immune

Mouse is immunized with SEQ ID:2, SEQ ID:3 and SEQ ID:4 respectively, immunizing dose is 50 μ g/.It generates positive After serum, booster immunization once carries out cell fusion afterwards.

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, sieved with SEQ ID:2, SEQ ID:3 or SEQ ID:4 coating microwell plate Choosing, positive hole carry out limiting dilution, are then further cultured for 10~14 days, 3~4 times repeatedly, it is thin to obtain anti-SEQ ID:2 monoclonal antibody Born of the same parents' strain, anti-SEQ ID:3 monoclonal antibody cell strain and anti-SEQ ID:4 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 anti-flagellum G antibody capture device of embodiment 3

1, SEQ ID:2, SEQ ID:3 and SEQ ID:4 are coated with microwell plate

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

2, SEQ ID:2, SEQ ID:3 and SEQ ID:4 are coated with magnetic microsphere

Magnetic microsphere and the pre-coated magnetic microsphere for having Streptavidin are purchased from Nanjing enlightening Gneuss.

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, SEQ is added by 100 μ g/mg magnetic microspheres It is reacted at room temperature 6 hours after ID:3 or SEQ ID:4；It is cleaned and is resuspended with PBS buffer solution, after 1%BSA closing being added 30 minutes, It adds after 1% glycine is closed 30 minutes after being cleaned with PBS buffer solution and is resuspended to get SEQ ID:2 magnetic microsphere, SEQ ID: 3 magnetic microspheres and SEQ ID:4 magnetic microsphere.

3, SEQ ID:2, SEQ ID:3 and SEQ ID:4 biotinylation

Biotin, dicyclohexylcarbodiimide (DCC) and n-hydroxysuccinimide (NHS) is taken to be dissolved in DMF, room temperature Magnetic agitation is reacted 3 hours, and supernatant is collected by centrifugation；Supernatant is added dropwise to SEQ ID:2, SEQ ID:3 or SEQ ID: In 4 solution, 2-8 DEG C is stirred to react 6 hours；Reaction solution taking-up is dialysed with phosphate buffer, obtains biotinylation SEQ ID: 2, biotinylation SEQ ID:3 and biotinylation SEQ ID:4.

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

1) fluorescent microsphere, color latex microballoon and 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；Labelled antibody (ID:2~4 SEQ are added in centrifugation With rabbit-anti chicken IgY) 100ug, room temperature shaker middling speed is reacted 2 hours；Confining liquid is added in centrifugation, and ice water ultrasound mixes, and room temperature is shaken Bed middling speed is reacted 1 hour；400 μ l borate buffer solutions (20mM PH8.0) are added in centrifugation, and ice water ultrasound mixes, 4 DEG C of preservations For use.

2) detection line T line is coated with

With phosphate buffer the polypeptide of SEQ ID:2~4 is diluted to 1mg/ml respectively, draws liquid measure by 1 μ l/cm, use metal spraying Film instrument is drawn uniformly to draw to preparation T line on NC film；The NC film pulled is placed in 37 DEG C of drying boxes, dry 16h.

5, SEQ ID:2, SEQ ID:3 and SEQ ID:4 mark colloidal gold

Colloidal gold 10ml is taken, appropriate 0.1M K is added₂CO₃Adjust pH.Appropriate formula SEQ ID:2, SEQ ID are added after mixing: 3 or SEQ ID:4 continues to stir 30min；Be added 10%BSA to its final concentration of 1%, continue stir 30min；10000rpm 4 DEG C of centrifugation 20min, collect precipitating, with colloidal gold dilution (0.2M BB, 1%BSA, 3% trehalose, 0.03% Procline300 it is settled to 1ml) to get SEQ ID:2, SEQ ID:3 and SEQ ID:4 colloidal gold composite.

4 flagellum G polypeptide of embodiment is used to prepare the board-like detection kit of enzymatic

1, the polypeptide of SEQ ID:2~4 is coated with microwell plate

(1) buffer of SEQ ID:2~4 is diluted to 5 μ g/ml, is coated with onto microwell plate, every hole 100 μ l, 4 DEG C of incubation 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 high as the numerical value for distinguishing feminine gender and the positive It is positive (+) in this value, is negative (-) less than or equal to this value, counts 10 patient's fecal samples and urine specimen As a result detection data prompts, the antibody positive rate of the anti-polypeptide of SEQ ID:2~4 is respectively in the excrement of cd patient 20%, 20% and 30% (being shown in Table 1)；Positive rate in urine specimen is respectively 10%, 10% and 20%.

Testing result of the 1 flagellum G 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, the direct coated of SEQ ID:2~4 on magnetic microsphere, this is micro- Ball solution is R1；(2) R2 is goat anti-human igg/IgA antibody of acridinium ester label.Sample to be detected is reacted with R1 when detection, Magnetic Isolation removes sample impurity later, cleans 3 times, reacts with R2, is formed and contains Ag-Ab-antiantibody sandwich complex, Compound carries out acridinium ester quantitative fluorescence analysis.

Second: (1) pressing step 3 method of embodiment 3, the biotinylation of SEQ ID:2~4, 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 first reacts SA magnetic microsphere with biotinylated R1, It is added after Magnetic Isolation and washing for sample sheet.Two methods are equally effective.One-step method or two step method form anti-containing antigen- Body-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. Standard curve is made with above-mentioned the first and second detection method, wherein the anti-human igg of second method and IgA standard curve See Fig. 5, R²Value is respectively 0.9953 and 0.9989.

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 figure 6, the area under ROC curve is it is found that the diagnosis efficiency of the IgG antibody of flagellum G polypeptide is better than IgA.As shown in table 2, SEQ ID: 2, the area under the curve of SEQ ID:3 and SEQ ID:4 anti-human igg is respectively 0.720,0.733 and 0.703, is prompted for diagnosing When Crohn disease, the detection sensitivity and specificity of SEQ ID:3 is best, followed by SEQ ID:2 and SEQ ID:4.SEQ ID: The area under the curve of 2~4 anti-human IgA is respectively 0.706,0.717 and 0.665 (data are not shown).

The IgG area under the curve of anti-ID:2~4 SEQ of table 2

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

Antibody standard substance used in this experiment is the anti-SEQ ID:2 monoclonal antibody of 2 method of embodiment preparation, anti-SEQ ID:3 monoclonal antibody And anti-SEQ ID:4 monoclonal antibody.

1, the preparation of fluorescence immune chromatography kit

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

The method of the polypeptide marker fluorescent microsphere of SEQ ID:2~4 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 the polypeptide of SEQ ID:2~4 and rabbit-anti that film instrument will be marked with fluorescent microsphere with metal spraying Chicken IgY antibody is sprayed onto the wide sample pad for 1cm of pretreatment according to the amount of 5 μ l/cm, and combination is prepared in 37 DEG C of dry 5h Pad.

(3) coating of nitrocellulose filter (NC film)

With phosphate buffer the polypeptide of SEQ ID:2~4 is diluted to 1mg/ml respectively, is used to prepare T line；Chicken IgY is resisted Body is diluted to 0.5mg/ml, is used to prepare C line；Liquid measure is drawn by 1 μ l/cm, it is uniform by above two solution to draw film instrument with metal spraying It draws 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

Anti- SEQ ID:2 monoclonal antibody, anti-SEQ ID:3 monoclonal antibody and the anti-SEQ ID:4 monoclonal antibody difference prepared with embodiment 2 is dilute 5 concentration are released, respectively: 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, by the 50 above calibration objects of μ l point It is not added drop-wise on well, is detected after 15 minutes with fluorescence detector, can be collected on detection line T and nature controlling line location of C glimmering Light.Quadratic polynomial fitting is carried out with sample concentration and T/C value, draws standard curve, the R2 difference of the polypeptide of SEQ ID:2~4 For 0.9973,0.997 and 0.9931 (see Fig. 7).According to the detection to 200 Healthy Human Serum samples, 90% Healthy People is anti- The cut-off value that the antibody of SEQ ID:2~4 is judged as negative is 30ng/ml, 35ng/ml and 30ng/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 that, containing anti-flagellum G polypeptide antibody in sample, content can the acquisition of establishing criteria curve calculation formula.20 samples Testing result to be shown in Table the detection specificity of ID:2~4 3, SEQ be respectively 80%, 90% and 80%, sensitivity is respectively 40%, 50% and 30%.

3 fluorescence immune chromatography test result of table

7 flagellum G polypeptide of embodiment is used to prepare gold calibration property detection kit

1, the gold marked reagent blocking of SEQ ID:2~4 is 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；

The sample pad prepared is taken, draws the gold that film instrument will be marked with SEQ ID:2, SEQ ID:3 and SEQ ID:4 with metal spraying The gold mark compound of mark compound and rabbit-anti chicken IgY antibody label is sprayed onto the wide sample for 1cm of pretreatment according to the amount of 5 μ l/cm On product pad, gold-labelled pad is prepared in 37 DEG C of dry 5h.

4) coating of nitrocellulose filter (NC film)

SEQ ID:2, SEQ ID:3 and SEQ ID:4 polypeptide are diluted to 1mg/ml with phosphate buffer, are used to prepare T Line；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-mentioned two with metal spraying Kind solution 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.

5) 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 marked reagent by assembling Box.

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), ID:2~4 SEQ are for judging that the specificity of Crohn disease is 80%, sensitivity Respectively 40%, 40% and 30%.

4 gold marked reagent test result of table

Claims (7)

1. flagellum G polypeptide shown in a kind of SEQ ID:1, which is characterized in that include 2~3 core sequences and catenation sequence xxx.

2. flagellum G 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, SEQ ID:3 or SEQ ID:4 polypeptide.

3. a kind of antibody capture device, which is characterized in that flagellum G described in solid phase carrier and thereon coated claim 1~2 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 anti-flagellum G 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, colloidal gold and colored latex.