CN109884291B - Campylobacter polypeptide, antibody capture device and kit - Google Patents

Abstract

The invention discloses a polypeptide shown in SEQ ID:1, campylobacter polypeptide containing the core sequence, an antibody capture device, a kit and a detection method, and belongs to the technical field of immunology. The technical scheme of the invention is that the expression of the polypeptide containing SEQ ID:1 core sequence, and capturing an antibody in a sample by a solid phase carrier and the polypeptide containing the core sequence on the solid phase carrier to achieve the effect of assisting in diagnosing Crohn's disease.

Description

Campylobacter polypeptide, antibody capture device and kit

Technical Field

The invention relates to a core sequence of campylobacter polypeptide, an antibody trap containing the polypeptide and a kit, belonging to the technical field of immunological detection.

Background

Inflammatory Bowel Disease (IBD) is an autoimmune bowel disease including Ulcerative Colitis (UC) and Crohn's Disease (CD), and the incidence of the disease has been increasing in our country for over 10 years. IBD has complex and diverse clinical manifestations, including digestive tract symptoms and extra-intestinal manifestations. Due to the symptoms of nonspecific enteritis such as abdominal pain, diarrhea, hematochezia, and the like, the differential diagnosis of other chronic intestinal diseases such as CD, UC, IBD, tuberculous enteritis, and the like is difficult. Serological indicators help to identify CD from UC, as well as IBD from non-IBD.

IBD has complex pathogenesis, one of which is the barrier dysfunction of intestinal mucosa and immune response of immune system to antigen products existing in normal intestinal tract, which is manifested by in vivo detection of antibodies to intestinal microbial antigens and metabolites due to genetic and environmental influences. Detection of these antibodies can aid in the diagnosis of autoimmune bowel diseases, including IBD. The antimicrobial antibodies with IBD auxiliary diagnosis effect which have been clarified at present include bacterial flagella antibodies such as anti-CBirl antibody, anti-Fla-X antibody and anti-4 aFla-2 antibody, as well as anti-Pseudomonas fluorescens I2 antibody and anti-Escherichia coli outer membrane porin OmpC antibody. Due to the variable nature and polymorphism of microbial genes, new microbial antigens continue to emerge, some of which are involved in the immunomodulation of the intestinal mucosa. The campylobacter polypeptide disclosed by the invention has a remarkable difference with IBD-related antigen polypeptides reported in the prior art, but sequence analysis shows that certain core sequences are related to the production of campylobacter polypeptide autoantibodies, and the campylobacter polypeptide containing the core sequences has the capability of identifying healthy people and Crohn's disease through the verification of clinically confirmed patient samples.

Disclosure of Invention

In the first aspect of the invention, the campylobacter polypeptide shown in SEQ ID:1 and the core sequence are disclosed, and the amino acid sequence connecting the core sequences can have various variations.

The polypeptide comprises three core sequences shown as SEQ ID:2 and SEQ ID: 3.

A campylobacter polypeptide having the amino acid sequence set forth in SEQ ID:2 or SEQ ID: 3.

In a second aspect of the present invention, a method for preparing an antibody capture device is provided, wherein the campylobacter polypeptide is directly or indirectly immobilized on a solid phase carrier for capturing an anti-campylobacter antibody in a sample.

The solid phase carrier comprises a microporous plate, fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, a film, a microporous film, a nitrocellulose film and the like.

In a preferred embodiment, the solid phase carrier is a microporous plate, in another preferred embodiment, the solid phase carrier is a magnetic microsphere, in the 3 rd preferred embodiment, a fluorescent microsphere and a nitrocellulose membrane are simultaneously selected, and in the 4 th preferred embodiment, the solid phase carrier is colloidal gold.

The method for immobilizing the polypeptide on the solid phase carrier is directly coating or indirectly coating through a carrier protein, preferably, the carrier protein is BSA or biotin-streptavidin.

In a preferred embodiment, the polypeptide is coupled to BSA and then coated on a solid support.

In another preferred embodiment, the polypeptide is reacted with biotin, followed by coupling of the biotinylated polypeptide to streptavidin to form a complex of the polypeptide and streptavidin, and coating the complex on a solid support.

In a third aspect of the present invention, there is disclosed an application of the antibody capture device described above to crohn's disease, the application of the present invention is a method for measuring an anti-campylobacter antibody in a sample based on an immunological reaction, comprising the steps of:

(1) acting a sample to be detected on an antibody capture device to enable an anti-campylobacter antibody in the sample to be combined with the polypeptide on the antibody capture device; (2) and determining the content of the anti-campylobacter polypeptide antibody in the sample by the marker.

The campylobacter antigen or epitope conjugate can be used for detecting an anti-campylobacter antibody in a sample by any current immunological detection method, such as ELISA, plate-type chemiluminescence, immunochromatography, immunodiafiltration, and the like, and the corresponding markers are horseradish peroxidase, alkaline phosphatase, acridinium ester, rare earth elements, colloidal gold, and the like.

Example 4 provides two enzymatic plate kits and methods for detecting campylobacter antibodies, in which campylobacter polypeptides are coated on a microplate and reacted with antibodies in a sample to form an antigen-antibody complex, and anti-human IgG and IgA enzymes are labeled, the enzymes used are horseradish peroxidase and alkaline phosphatase, and the samples used are feces and urine.

Embodiment 5 provides an acridinium ester labeled chemiluminescence kit and a detection method, after biotinylation, campylobacter polypeptide is coated on a magnetic microsphere through streptavidin, the magnetic microsphere reacts with an antibody to be detected in a plasma sample to form an antigen-antibody complex, and after washing, the antigen-antibody complex is quantitatively detected by an acridinium ester labeled anti-human antibody. The detection method has the advantages of high sensitivity, short detection time, full-automatic detection and the like.

Embodiment 6 provides a fluorescence immunochromatographic kit and a method for detecting an anti-campylobacter polypeptide antibody, wherein the method comprises the steps of coating campylobacter polypeptide on a fluorescent microsphere containing rare earth elements directly or through biotin-streptavidin, and coating the polypeptide on a detection line T line to form a double-antigen sandwich method for detecting the content of the anti-campylobacter antibody in a serum sample. The detection method has the advantages of high speed, short detection time, high sensitivity and the like.

Embodiment 7 provides a colloidal gold reagent for detecting campylobacter polypeptide antibodies and a method thereof, wherein the method comprises the steps of marking campylobacter polypeptides by using colloidal gold particles or colored latex microspheres, spraying the colloidal gold particles or the colored latex microspheres on a sample pad, and meanwhile, coating the polypeptides on a detection line T line to form a double-antigen sandwich method for detecting the campylobacter polypeptide antibodies in a whole blood sample. The detection method can be used for observing results by naked eyes, and has the advantages of rapidness, low instrument cost and the like.

Compared with the prior art, the campylobacter polypeptide and the antibody capturing device provided by the invention have the advantages of high sensitivity, stable performance, simplicity in operation and the like when used for treating Crohn's disease.

Drawings

FIG. 1 standard curve for quantitative measurement of chemiluminescence

FIG. 2 specificity and sensitivity of chemiluminescence quantitative detection

FIG. 3 standard curve of fluorescence immunochromatography reagent

Detailed Description

Biotin, goat anti-human IgG and goat anti-human IgA used in the present invention were purchased from Sigma. Avidin was purchased from the orthodiscal gene. Magnetic microspheres and streptavidin pre-coated magnetic microspheres were purchased from Nanjing Diegos. Microplates were purchased from suzhou beaver. Latex microspheres, colored latex microspheres, resin microspheres were purchased from the Tianjincel group. The rare earth element fluorescent microspheres and the time-resolved fluorescence reader are purchased from Shenzhen micrometering. Other reagents were purchased from national medicine reagents and Shanghai Producers.

The invention will be further elucidated with reference to the specific embodiments and the accompanying drawings.

EXAMPLE 1 preparation of Campylobacter Polypeptides

1. Preparation of the polypeptides of SEQ ID:2 and SEQ ID:3

The entire genome sequences of SEQ ID:2 and SEQ ID:3 were synthesized by Kingchi.

1) Reagent

Cloning vector pCR2.1T-vector, expression plasmid pET28a (+), transfection Escherichia coli host bacteria DH5 alpha, BL21(DE3), DNA polymerase rTaq, T4DNA ligase, DNA polymerase rTaq, LA Taq and restriction endonucleases BamH I, Hind III, EcoR I, BamH I, DL2000DNA Marker, T4DNA ligase, low molecular weight standard protein, DNA glue recovery kit, IPTG and the like

2) Instrument for measuring the position of a moving object

Common shaking table SCS-24; a water-proof constant-temperature electric heating incubator; biophotometer spectrophotometer, benchtop refrigerated Centrifuge centrifue 5810R, benchtop Centrifuge MiniSpin; a high speed refrigerated centrifuge; protein electrophoresis apparatus and gel imaging system; a PCR instrument; an ultrasonic cracker; constant temperature metal bath; HIS protein purification column, etc.

2. Experimental methods

1) Vector construction:

designing a primer, amplifying the fragments of SEQ ID:2 and SEQ ID:3 from the template DNA by PCR, recovering the fragments by using a gel recovery kit, and connecting the fragments to a pCR2.1 cloning vector for sequencing identification. The correctly identified sequence is cloned into an expression vector pET28a (+), the enzyme cutting sites are EcoR I and BamH I, and meanwhile, the vector is provided with a 6 XHIS label, which is convenient for the subsequent protein purification.

2) Expression of

The resulting plasmid was transformed into E.coli BL21(DE3), and positive expression strains were selected by resistance selection. Inoculating the screened positive bacteria liquid into LB culture medium with Kan resistance in the ratio of 1 to 1000, inoculating each bacteria into two tubes, one tube for induction and the other tube for non-induction control, and inoculating one tube of empty plasmid bacteria as control. When the culture is carried out at 37 ℃ overnight until the OD600 value is about 0.4-0.6, IPTG is added into the inducing tube and the empty plasmid control tube until the final concentration is 1mmol/L, and the non-inducing control is not added, and finally two bacteria with high expression capacity are selected for the expression of a large amount of protein. And the expression product was identified according to a conventional SDS-PAGE method.

3) Purification of

Purifying the expressed product with HIS protein purification column, dialyzing to desalt, and preserving the purified SEQ ID:2 and SEQ ID:3 at low temperature for later use.

EXAMPLE 2 preparation of anti-Campylobacter polypeptide antibodies

1. Animal immunization

Mice were immunized with SEQ ID:2 and SEQ ID:3, respectively, at an immunization dose of 50. mu.g/mouse. After positive serum was generated, cell fusion was performed after one booster immunization.

If polyclonal antibody is required to be prepared, goat or rabbit can be immunized in the same way, and serum is taken to obtain the polyclonal antibody.

2. Cell fusion

PEG1500 Pre-warming, suction 1X 10⁷SP2/0 myeloma cell suspension and 5X 10⁷The spleen B lymphocyte suspension (cell number 1:5) of each immunized mouse is fused with a pre-warmed 50% PEG1500 solution, centrifuged, the supernatant is discarded, 10ml HAT (SIGMA) culture medium is added to resuspend the cells, and the cells are inoculated to a 96-well cell culture plate which is paved with trophoblasts for culture.

3. Screening and cloning

And (3) after culturing the fusion cells for 10-14 days, coating a microporous plate with SEQ ID:2 or SEQ ID:3 for screening, performing limited dilution on positive holes, culturing for 10-14 days, and repeating the steps for 3-4 times to obtain the anti-SEQ ID:2 monoclonal antibody cell strain and the anti-SEQ ID:3 monoclonal antibody cell strain.

4. Preparation of ascites

Inoculating the monoclonal cells into the abdominal cavity of the mouse, and collecting ascites after the ascites of the mouse accumulates. The ascites was centrifuged at 10000rpm for 10 minutes at 4 ℃ to remove lipid. After centrifugation, the supernatant was aspirated and filtered through a 0.45 μm membrane. protein G was purified. And (5) measuring the concentration of the purified monoclonal antibody, subpackaging and freezing for later use.

EXAMPLE 3 anti-Campylobacter antibody Capture device preparation

1. Coated microporous plate of SEQ ID:2 and SEQ ID:3

Preparing the concentration of SEQ ID:2 and SEQ ID:3 by phosphate buffer solution to be about 5ug/mL, coating a 96-well micropore plate by 100uL of each well, standing overnight at 4 ℃, removing a coating solution, washing the plate once, incubating for 2 hours at 37 ℃ by 200uL of a blocking buffer solution containing 1% calf serum in each well, patting dry, and drying for later use.

2. SEQ ID 2 and SEQ ID 3 coated magnetic microspheres

Taking a proper amount of amino magnetic microspheres, washing with PBS buffer solution and suspending, adding 5% glutaraldehyde solution, and stirring at room temperature for reaction for 1 hour; washing with PBS buffer solution and resuspending, after ultrasonic dispersion, adding 100 mug/mg of magnetic microspheres into SEQ ID:2 or SEQ ID:3, and reacting for 6 hours at room temperature; washing and resuspending by PBS buffer solution, adding 1% BSA for blocking for 30 minutes, adding 1% glycine for blocking for 30 minutes, washing by PBS buffer solution, and resuspending to obtain the magnetic microspheres of SEQ ID:2 and SEQ ID: 3.

3. Biotinylation of SEQ ID:2 and SEQ ID:3

Dissolving biotin, Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) in DMF, reacting for 3 hours at room temperature under magnetic stirring, centrifuging and collecting supernatant; dropwise adding the supernatant into the solution of SEQ ID:2 or SEQ ID:3, and stirring and reacting for 6 hours at the temperature of 2-8 ℃; the reaction solution was removed and dialyzed against phosphate buffer to obtain biotinylated SEQ ID:2 and biotinylated SEQ ID: 3.

4. Fluorescent microsphere coated with SEQ ID:2 and SEQ ID:3 and detection line T line

1) Fluorescent microsphere, color latex microsphere and resin microsphere mark

Taking 200 mul (210 nm) of fluorescent microspheres, color latex microspheres or resin microspheres (1 percent stock solution), centrifuging for 10min at 13000rpm and 4 ℃, discarding supernatant, adding 400ul deionized water, and ultrasonically mixing for 10S; centrifuging, adding 400 μ l MES buffer (50mM, pH6.0), and ultrasonically mixing; centrifuging, adding EDC solution, and reacting for 15 minutes at room temperature; centrifuging, adding 100ug of labeled antibody (SEQ ID:2, SEQ ID:3 and rabbit anti-chicken IgY), and reacting at medium speed in a shaker at room temperature for 2 hours; centrifuging, adding the confining liquid, ultrasonically mixing the confining liquid and the ice water uniformly, and reacting for 1 hour in a shaking table at room temperature at medium speed; centrifuging, adding 400 μ l borate buffer (20 mM pH 8.0), mixing with ice water under ultrasound, and storing at 4 deg.C.

2) Detection line T quilt

Respectively diluting the polypeptides of SEQ ID:2 and SEQ ID:3 to 1mg/ml by using a phosphate buffer solution, uniformly scratching an NC membrane by using a gold spraying film scratching instrument according to the scratching amount of 1 mu l/cm to prepare a T line; the scribed NC film was placed in a 37 ℃ drying oven and dried for 16 h.

5. Colloidal gold labeled with SEQ ID:2 and SEQ ID:3

Taking 10ml of colloidal gold, adding a proper amount of 0.1M K₂CO₃The pH is adjusted. Mixing, adding appropriate amount of SEQ ID:2 or SEQ ID:3, and stirring for 30 min; adding 10% BSA to a final concentration of 1%, and stirring for 30 min; centrifuging at 10000rpm at 4 deg.C for 20min, collecting precipitate, and diluting with colloidal gold diluent (0.2M BB, 1% BSA, 3% trehalose, 0.03% procline 300) to 1ml to obtain colloidal gold complexes of SEQ ID:2 and SEQ ID: 3.

Example 4 use of Campylobacter Polypeptides for the preparation of enzymatic plate-type assay kits

1. Polypeptide coated microporous plate of SEQ ID:2 and SEQ ID:3

(1) The buffer solutions of SEQ ID:2 and SEQ ID:3 were diluted to 5. mu.g/ml, coated on a microplate, 100. mu.l per well, incubated at 4 ℃ for 16h or at 37 ℃ for 2 h.

(2) Washing with PBST for 3 times, and spin-drying;

(3) blocking with protein solution containing 1% bovine serum albumin, adding 200uL of blocking solution into each well, and performing reverse reaction at 37 deg.C

And (4) taking out for 2 hours, discarding sealing liquid in the holes, and spin-drying;

(4) and (3) putting the coated plate in a 37 ℃ oven for 4h to finish coating, sealing the coated plate by using an aluminum foil bag, and storing the sealed coated plate at-20 ℃ for later use.

2. Anti-polypeptide IgG antibody detection

(1) Diluting 10 cases of excrement of healthy people and 10 cases of excrement samples of patients with Crohn's disease by using a sample diluent, adding the diluted sample into a coated microporous plate, reacting for 1h at room temperature, and washing the plate for 4 times;

(2) adding horse radish peroxidase labeled goat anti-human IgG, reacting at 37 ℃ for 0.5h, and washing the plate for 4 times;

(3) adding a horseradish peroxidase substrate for color development;

(4) and (4) terminating and reading.

3. Anti-polypeptide IgA antibody detection

(1) Diluting 10 cases of urine of healthy people and 10 cases of urine of Crohn's disease patients with a sample diluent, adding the diluted urine into a coated microporous plate, reacting for 1h at room temperature, and washing the plate for 4 times;

(2) adding alkaline phosphatase-labeled goat anti-human IgG, reacting at 37 ℃ for 0.5h, and washing the plate for 4 times;

(3) adding alkaline phosphatase substrate for color development;

(4) and (4) terminating and reading.

4. Data analysis

Calculating the average OD value of 10 healthy human samples, taking the OD value of 2.5 times as a value for distinguishing negative from positive, taking positive (+) above the value and taking negative (-) below or equal to the value, and counting the detection data of the feces samples and urine samples of 10 patients, wherein the results indicate that the positive rates of the antibodies against the polypeptides of SEQ ID:2 and SEQ ID:3 in the feces of the Crohn patients are 20% and 30% respectively (see Table 1); the positive rates in urine samples were 10% and 20%, respectively.

TABLE 1 detection results of Campylobacter polypeptide antibodies in fecal samples

Example 5 preparation of acridinium ester-labeled chemiluminescent assay kit

There may be two different implementations:

the first method comprises the following steps: (1) according to the method of step 2 in example 3, the magnetic microspheres coated with SEQ ID:2 and SEQ ID:3 were prepared by coating them with R1 as a solution, and (2) R2 was an acridinium ester-labeled goat anti-human IgG/IgA antibody. During detection, a sample to be detected reacts with R1, then sample impurities are removed through magnetic separation, the sample is washed for 3 times and reacts with R2 to form a sandwich complex containing the antigen-antibody-anti-antibody, and the complex is subjected to acridinium ester fluorescence quantitative analysis.

And the second method comprises the following steps: (1) biotinylation of SEQ ID:2 and SEQ ID:3 was performed according to the method of step 3 of example 3, and the solution was R1, (2) R2 was magnetic microspheres pre-coated with SA; (2) r3 is an acridinium ester labeled goat anti-human IgG/IgA antibody. During detection, a one-step method or a two-step method can be adopted, wherein the one-step method or the two-step method simultaneously reacts a substitute detection sample, R1 and R2; in the latter, SA magnetic microspheres are reacted with biotinylated R1, magnetically separated and washed, and then added to a substitute sample. Both methods are equally effective. Forming a sandwich complex containing the antigen-antibody-anti-antibody by a one-step method or a two-step method, and carrying out acridinium ester fluorescence quantitative analysis on the complex.

1) Standard Curve preparation

Acridinium ester-labeled goat anti-human IgG or IgA antibodies were diluted to 5 concentrations, respectively: acridinium ester-labeled goat anti-human IgG and IgA antibodies were diluted 5 times at concentrations of 0ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, and 400ng/ml, respectively. Making the target by the first and second detection methodsQuasi-curve, where the second method is shown in FIG. 1 for anti-human IgG and IgA standard curves, R²The values are 0.9978 and 0.9973, respectively.

2) Sample detection

The sensitivity of the first and second methods described above for 100 healthy human plasma or serum and 100 cloned enzyne patient plasma or serum was optimized for the second method, where the second method detected a ROC curve as shown in figure 2, but IgG anti-campylobacter polypeptides were significantly better than the diagnostic efficiency of IgA antibodies, and the areas under the ROC curves of SEQ ID nos. 2 and 3 were only 0.615 and 0.683 (data not shown). As shown in Table 2, the areas under the curves of the anti-human IgG of SEQ ID:2 and SEQ ID:3 were 0.693 and 0.753, respectively, indicating that SEQ ID:3 is superior to SEQ ID:2 when used to diagnose Crohn's disease.

TABLE 2 IgG area under the curve for anti-SEQ ID:2 and SEQ ID:3

Example 6 application of Campylobacter polypeptide to preparation of rare earth element fluorescence immunochromatography detection kit

1. Preparation of fluorescence immunochromatography kit

(1) Labeling and detection line T-shaped coating of fluorescent microspheres

The method for labeling fluorescent microspheres with the polypeptides of SEQ ID:2 and SEQ ID:3 is shown in step 4 of example 3.

(2) Preparation of sample pad and conjugate pad

The glass cellulose membrane was pre-blocked by soaking in a buffer solution containing a surfactant (formulation: 100mM pH 7.4 PB containing 2% NaCl, 2% BSA, 0.5% casein, 0.1% Tween-20, 0.5% S9 and 5% sucrose), and then dried overnight at 37 ℃ to prepare a sample pad;

and (3) spraying the polypeptides SEQ ID:2 and SEQ ID:3 marked with fluorescent microspheres and the rabbit anti-chicken IgY antibody on the sample pad which is pre-treated and has the width of 1cm by using a gold spraying and membrane scratching instrument according to the amount of 5 mu l/cm, and drying for 5h at 37 ℃ to prepare the combined pad.

(3) Coating of nitrocellulose membranes (NC membranes)

Respectively diluting the polypeptides of SEQ ID:2 and SEQ ID:3 to 1mg/ml by using a phosphate buffer solution for preparing a T line; diluting the chicken IgY antibody to 0.5mg/ml for preparing a C line; uniformly scratching the two solutions onto an NC film by a gold spraying film scratching instrument according to the scratching amount of 1 mul/cm to prepare a T line and a C line; the scribed NC film was placed in a 37 ℃ drying oven and dried for 16 h.

(4) Assembly

Laminating the bonding pad obtained in the step 2) on one end of the nitrocellulose membrane obtained in the step 3), fixing and laminating the water absorption pad on the other end of the nitrocellulose membrane, finally laminating the sample pad obtained in the step 2) on the other end of the bonding pad, cutting each sample pad by a microcomputer automatic cutting machine according to the width of 5.5mm, and filling the sample pad into a chromatography strip shell to obtain a finished product.

2. Standard curve

The anti-SEQ ID:2 monoclonal antibody and the anti-SEQ ID:3 monoclonal antibody prepared in example 2 were diluted by 5 concentrations, respectively: 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml and 200ng/ml, respectively dripping more than 50 μ l of calibrator on the sample adding hole, detecting by a fluorescence detector after 15 minutes, and collecting fluorescence at the positions of a detection line T and a quality control line C. Second order polynomial fitting of sample concentration to T/C values to draw a standard curve, R of SEQ ID:2 and 3²0.9904 and 0.9970, respectively, see fig. 3. According to the detection of 200 cases of healthy human serum samples, cut-off values of 90% of healthy human antibodies negative to SEQ ID:2 and SEQ ID:3 were 30ng/ml and 32ng/ml, respectively.

3. Sample detection

And (3) dripping 50 mu l of blood sample to be detected on the sample adding hole, detecting by using a fluorescence detector after 15 minutes, and if a strip appears in a detection line, indicating that the sample contains an anti-campylobacter polypeptide antibody, wherein the content of the anti-campylobacter polypeptide antibody can be obtained according to a standard curve calculation formula. The results of the detection of 20 samples are shown in Table 3, and the detection specificity of SEQ ID:2 and SEQ ID:3 are 70% and 80%, respectively, and the sensitivity is 40% and 50%, respectively.

TABLE 3 fluorescence immunochromatography test results

Example 7 application of Campylobacter polypeptide to preparation of gold-labeled qualitative detection kit

1. Preparation of gold-labeled reagent cards of SEQ ID:2 and SEQ ID:3

1) Preparation of colloidal gold

Adding 100ml of 0.01% chloroauric acid solution into a round-bottom flask, placing on an electric heating jacket, heating to boil, immediately adding 2ml of 1% trisodium citrate solution, continuing stirring for 15min, naturally cooling, and storing at 4 ℃ for later use.

2) Colloidal gold labeling

See example 3, step 5.

3) Preparation of sample pad and gold-labeled pad

The glass cellulose membrane was pre-blocked by soaking in a treatment solution (formulation: 100mM pH 7.4 PB containing 2% BSA, 0.5% Tween-20, 0.5% S9 and 5% sucrose), and then dried overnight at 37 ℃ to prepare a sample pad;

and (3) spraying the gold-labeled compound marked with SEQ ID:2 and SEQ ID:3 and the gold-labeled compound marked with the rabbit anti-chicken IgY antibody on the sample pad which is pre-treated and has the width of 1cm according to the amount of 5 mu l/cm by using a gold spraying and membrane scratching instrument, and drying for 5 hours at 37 ℃ to prepare the gold-labeled pad.

4) Coating of nitrocellulose membranes (NC membranes)

Diluting the polypeptides of SEQ ID:2 and SEQ ID:3 to 1mg/ml with phosphate buffer solution for preparing T line; diluting the chicken IgY antibody to 0.5mg/ml for preparing a C line; uniformly scratching the two solutions onto an NC film by a gold spraying film scratching instrument according to the scratching amount of 1 mul/cm to prepare a T line and a C line; the scribed NC film was placed in a 37 ℃ drying oven and dried for 16 h.

5) Assembly

And (3) assembling the gold label pad, the sample pad, the coated NC membrane, the water absorption pad and other auxiliary materials into the gold label kit.

2. Kit detection

(1) Detection method

Diluting the sample with sample diluent (BB, 0.5% s9, 1% BSA), taking 100. mu.l, and directly adding into the sample window of the chromatographic strip; after 15min, the results were visually observed.

(2) Determination of results

Negative result (-): only a quality control line appears, and no detection line exists;

positive result (+): the quality control line and the detection line appear at the same time;

invalid result: the absence of a quality control line indicates an operational error or a kit failure.

(3) Detection of

100 mul of the standard substance prepared in the example 5 and 20 examples of the whole blood samples are respectively added into a sample window in the chromatographic strip; after 15min, the results were visually observed (Table 4), with specificity of 70% and 80% for the determination of Crohn's disease for SEQ ID:2 and 40% for sensitivity, respectively.

TABLE 4 gold-labeled reagent test results

Claims (9)

1. A Campylobacter polypeptide having an amino acid sequence as set forth in SEQ ID:2 or SEQ ID: 3.

2. An antibody capture device comprising a solid support and the campylobacter polypeptide of claim 1 coated thereon.

3. The antibody capture device of claim 2, wherein the solid support is selected from fluorescent microspheres, latex microspheres, resin microspheres, magnetic microspheres, colloidal gold particles, microwell plates, or membranes.

4. The antibody capture device of claim 3, wherein the solid support is selected from the group consisting of microporous membranes.

5. The antibody capture device of claim 4, wherein the solid support is selected from nitrocellulose membranes.

6. A Crohn's assay kit comprising the Campylobacter polypeptide of claim 1 or the antibody capture device of any one of claims 2 to 5.

7. The Crohn detection kit of claim 6, wherein the kit further comprises a marker for determining the amount of anti-Campylobacter polypeptide antibody in the sample.

8. The Crohn's test kit of claim 7, wherein the sample is selected from serum, plasma, whole blood, urine, or feces.

9. The Crohn detection kit of claim 7, wherein the label is selected from horseradish peroxidase, alkaline phosphatase, acridinium esters, rare earth elements, or colloidal gold.

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