CN109879948A - Flagellum polypeptide, antibody capture device and kit - Google Patents
Flagellum polypeptide, antibody capture device and kit Download PDFInfo
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Abstract
Flagellum polypeptide, antibody capture device and kit.The present invention discloses core sequence, the flagellum polypeptide containing core sequence, antibody capture device, kit and the detection method of flagellum polypeptide shown in a kind of SEQ ID:1, belongs to immunological technique field.The technical scheme is that clonal expression contains the flagellum polypeptide of SEQ ID:1 core sequence, the antibody in the polypeptide capture sample containing core sequence, has the function that auxiliary diagnosis autoimmune enteropathy by solid phase carrier and thereon.
Description
Technical field
The present invention relates to a kind of core sequences of flagellum polypeptide, and it includes the antibody capture devices and kit of polypeptide, belong to
In 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 phase of enteric microorganism flagellum polypeptide and existing report that the present invention is announced
Antigen polypeptide significant difference is closed, but is analyzed by sequence, it is certain cores that discovery generates related with flagellum polypeptide autoantibody
Sequence, the flagellum polypeptide containing the core sequence are verified through the clinical samples of clinical definite, are had and are identified Healthy People and exempt from self
The ability of epidemic disease enteropathy.
Summary of the invention
The first aspect of the present invention, announce SEQ ID:1 shown in flagellum polypeptide and core sequence, connection core sequence it
Between amino acid sequence can there are many variation.
The polypeptide includes three core sequences, is SEQ ID:2, shown in SEQ ID:3, SEQ ID:4 and SEQ ID:5.
The second aspect of the present invention provides a kind of preparation method of antibody capture device, and the flagellum polypeptide is direct
Or be indirectly fixed on solid phase carrier, for capturing the anti-flagellar 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 (or color latex or resin 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 the autoimmune of flagellum more polypeptide above-mentioned, antibody capture device composition
Enteropathy detection kit and measuring method, kit of the present invention and method are to be resisted based on immunological response in sample
The measuring method of flagellum polypeptide antibody, includes the following steps:
(1) sample to be detected acts on antibody capture device, makes anti-flagellum polypeptide antibody and antibody capture device in sample
Polypeptide on part combines;(2) pass through the content of anti-flagellum more polypeptide antibody in marker measurement sample.
Flagellar antigen or epitope conjugate can be detected in sample by current any immunological detection method
Anti- flagellar 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 of two kinds of anti-flagellar antibodies and detection method, flagellum polypeptide are coated on micro-
On orifice plate, antigen-antibody complex is formed with the antibody response in sample, anti-human igg and IgA enzyme are marked, enzyme used is
Horseradish peroxidase and alkaline phosphatase, sample used are excrement and urine.
Embodiment 5 provides the chemical luminescence reagent kit and detection method of a kind of acridinium ester label, the flagellum 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 detection method of a kind of anti-flagellum polypeptide antibody, the method
It is directly or by biotin-streptavidin coating flagellum polypeptide on the fluorescent microsphere containing rare earth element, while polypeptide
It is coated on detection line T line, forms the anti-flagellar antibody content in dual-antigen sandwich method detection serum sample.The detection method
Have many advantages, such as that quick, detection time is short, high sensitivity.
Embodiment 7 provides the colloidal gold chromatographic kit and method of a kind of anti-flagellum polypeptide antibody, and the method is whip
Hair 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 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 polypeptide and antibody capture device provided by the invention are used for autoimmune enteropathy
When, there is 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 are read
Device 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 polypeptide of embodiment
1, SEQ ID:2, SEQ ID:3, the preparation of SEQ ID:4 and SEQ ID:5 polypeptide
SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ ID:5 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, SEQ ID:4 and SEQ ID:5 piece from template DNA
Section, pCR2.1 cloning vector, which is connected to, after plastic recovery kit recycling segment carries out sequencing identification.It will identify correct sequence gram
Grand into expression vector pET28a (+), 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, 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, SEQ ID:4 and SEQ ID:5 cryo-conservation are spare.
Embodiment 2 prepares anti-flagellum polypeptide antibody
1. animal immune
Mouse is immunized with SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ ID:5 respectively, immunizing dose is 50 μ g/.
After generating positive 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, micropore is coated with SEQ ID:2, SEQ ID:3, SEQ ID:4 or SEQ ID:5
Plate is screened, and positive hole carries out limiting dilution, is then further cultured for 10~14 days, 3~4 times repeatedly, is obtained anti-SEQ
ID:2 monoclonal antibody cell strain, anti-SEQ ID:3 monoclonal antibody cell strain, SEQ ID:4 monoclonal antibody cell strain and SEQ ID:5 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-flagellar antibody capture device of embodiment 3
1, SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ ID:5 are coated with microwell plate
Concentration with phosphate buffered saline SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ ID:5 is about 5ug/
ML, every hole 100uL are coated with 96 hole microwell plates, and 4 DEG C overnight, discard coating buffer, and board-washing is primary, with the closing for containing 1% calf serum
The every hole 200uL of buffer, 37 DEG C are incubated for 2 hours, pat dry, are dried for standby.
2, SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ ID:5 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, SEQ ID:4 or SEQ ID:5;It is cleaned and is resuspended with PBS buffer solution, 1%BSA closing is added
After 30 minutes, it is micro- to get SEQ ID:2 magnetism that resuspension after cleaning with PBS buffer solution is added after 1% glycine is closed 30 minutes
Ball, SEQ ID:3 magnetic microsphere and SEQ ID:4 magnetic microsphere.
3, SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ ID:5 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, SEQ ID:4
Or in SEQ ID:5 solution, 2-8 DEG C is stirred to react 6 hours: reaction solution taking-up being dialysed with phosphate buffer, obtains biology
Elementization SEQ ID:2, biotinylation SEQ ID:3, biotinylation SEQ ID:4 and biotinylation SEQ ID:5.
4, SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ ID:5 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:2~5 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:2~5 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, SEQ ID:4 and SEQ ID:5 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, SEQ ID:4 or SEQ ID:5 continues to stir 30min;Be added 10%BSA to its final concentration of 1%, continue stir 30min;
4 DEG C of centrifugation 20min of 10000rpm, 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, SEQ ID:4 and SEQ ID:5 colloidal gold composite.
4 flagellum polypeptide of embodiment is used to prepare the board-like detection reagent of enzymatic
1, the polypeptide of SEQ ID:2~5 is coated with microwell plate
(1) buffer of SEQ ID:2~5 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) after 10 healthy human faecal mass and 10 autoimmune enteropathy patient's fecal samples being diluted with Sample dilution
Be added 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) it is added after diluting 10 Healthy People urines and 10 autoimmune enteropathy Urine in Patients with Sample dilution
Into 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 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:2~5 point in the excrement of autoimmune enteropathy patient
It Wei not 20%, 20%, 30% and 30% (being shown in Table 1);Positive rate in urine specimen is respectively 10%, 10%, 20% and 20%
(data are not shown).
Testing result of the 1 flagellum polypeptide antibody of table in fecal sample
Embodiment 5 is used to prepare acridinium ester label chemiluminescence detection reagent
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:2~5 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:2~5, 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. 1, R2Value is respectively 0.9937 and 0.9988.
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
2, it can determine whether from ROC curve, the IgG diagnosis effect of anti-flagellum polypeptide is better than IgA.As shown in table 2, SEQ ID:2, SEQ
The area under the curve of ID:3, SEQ ID:4 and SEQ ID:5 anti-human igg is respectively 0.700,0.724,0.720 and 0.736, explanation
When for diagnosing autoimmune enteropathy, the detection sensitivity and specificity of SEQ ID:5 is best, followed by SEQ ID:3, SEQ
ID:4 and SEQ ID:2.
The IgG area under the curve of anti-ID:2~5 SEQ of table 2
6 flagellum polypeptide of embodiment is used to prepare rare earth element fluorescence immune chromatography detection kit
Antibody standard substance used in this experiment be the anti-SEQ ID:2 monoclonal antibody of 2 method of embodiment preparation, anti-SEQ ID:3 monoclonal antibody,
Anti- SEQ ID:4 monoclonal antibody and anti-SEQ ID:5 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:2~5 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 pH7.4PB, 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:2~5 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:2~5 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, anti-SEQ ID:4 monoclonal antibody and the anti-SEQ prepared with embodiment 2
ID:5 monoclonal antibody dilutes 5 concentration respectively, respectively: 0ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, by 50 μ
1 or more calibration object is added drop-wise on well respectively, is detected after 15 minutes with fluorescence detector, can be in detection line T and nature controlling line C
Fluorescence is collected on position.Quadratic polynomial fitting is carried out with sample concentration and T/C value, draws standard curve, ID2~5 SEQ
R2 be respectively 0.9924,0.9923,0.9979 and 0.9931 (see Fig. 3).According to the inspection to 200 Healthy Human Serum samples
It surveys, the cut-off value that the anti-antibody of SEQ ID:2~5 of 90% Healthy People is judged as negative is 30ng/ml.
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 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:2~5 3, SEQ be respectively 80%, 80%, 80% and 90%, sensitivity difference
It is 40%, 50%, 40% and 50%.
3 fluorescence immune chromatography test result of table
7 flagellum polypeptide of embodiment is used to prepare gold calibration property detection kit
1, the gold marked reagent blocking of SEQ ID:2~5 is standby
1) preparation of colloidal gold
100ml0.01% chlorauric acid solution is added in round-bottomed flask, is placed in and is heated to boiling on electric jacket, be added immediately
2ml1% citric acid three sodium solution continues to stir 15min, save 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 pH7.4PB, 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, SEQ ID:2, SEQ ID:3, SEQ ID:4 and SEQ will be marked with by drawing film instrument with metal spraying
ID:5 gold mark compound and rabbit-anti chicken IgY antibody label gold mark compound according to the amount of 5 μ l/cm be sprayed onto pretreatment it is wide
For in the sample pad of 1cm, 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, SEQ ID:4 and SEQ ID:5 polypeptide are diluted to 1mg/ml with phosphate buffer,
It is 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 with metal spraying
Instrument uniformly draws above two solution 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), ID:2~5 SEQ are used to judge that the specificity of autoimmune enteropathy to be respectively
80%, 80%, 90% and 80%, sensitivity is respectively 30%, 40%, 40% and 50%.
4 gold marked reagent test result of table
Claims (7)
1. flagellum polypeptide shown in a kind of SEQ ID:1, which is characterized in that include 3 core sequences and catenation sequence xxx.
2. flagellum polypeptide described in claim 1, which is characterized in that catenation sequence xxx can make a variation, it is preferable that for SEQ ID:2,
SEQ ID:3, SEQ ID:4 or SEQ ID:5 polypeptide.
3. a kind of antibody capture device, which is characterized in that flagellum 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 autoimmune enteropathy detection kit and method, which is characterized in that including more described in claim 1~2
Antibody capture device described in peptide or claim 3~4, method comprising steps of
(1) sample is acted on into antibody capture device;
(2) pass through the content of anti-flagellum 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 color latex.
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