CN108333368A - The kit and preparation method of calprotectin in a kind of detection human faecal mass - Google Patents
The kit and preparation method of calprotectin in a kind of detection human faecal mass Download PDFInfo
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- CN108333368A CN108333368A CN201810123018.9A CN201810123018A CN108333368A CN 108333368 A CN108333368 A CN 108333368A CN 201810123018 A CN201810123018 A CN 201810123018A CN 108333368 A CN108333368 A CN 108333368A
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- calprotectin
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
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- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims description 2
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- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims 2
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- 230000002550 fecal effect Effects 0.000 description 12
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4727—Calcium binding proteins, e.g. calmodulin
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention relates to biotechnologies, and in particular to a kind of kit and preparation method detecting calprotectin in human faecal mass.The kit substitutes traditional sealer using specific microballoon conjugate closed reagent, being capable of accurate, quantitative, quick, the sensitive calprotectin detected in human faecal mass.
Description
Technical field
The present invention relates to a kind of kit of calprotectin in biotechnology more particularly to detection human faecal mass and preparations
Method.
Background technology
Calprotectin be 14kD by two molecular weight heavy chain and a molecular weight be 8kD light chain to be covalently keyed
Calcium-binding protein heterotrimer composition, be neutrophil leucocyte and protein important in the macrophage matter of activation, permitted
Content increases under more inflammatory conditions.As a kind of intestinal inflammation marker, it can clearly distinguish organic diseases associated with inflammation IBD
With functional disease IBS, it can assess inflammatory bowel disease mucous membrane as a kind of activity index of evaluation inflammatory bowel disease, evaluate medicine
Object therapeutic effect is cured and predictive disease recurrence.Conventional endoscopic inspection and biopsy histology analysis are invasive inspection, indium
For radioactive element, this affects to patient health mark leucocyte, and test operation is more complex；Pass through detection
Calcium is defended content and is distinguished to it, is avoided that cost of use is expensive and invasive colonoscopy.
Currently, the method for detection calprotectin content mainly has enzyme-linked immunization and colloidal gold chromatography.Wherein, using enzyme
The German Buhlmann Laboratories AG calprotectin kits of linked immunosorbent assay, the kit can accurately test excrement
The content of middle calprotectin, but ELISA needs board-washing, is incubated, and enzyme and substrate is stringent to temperature environment, cumbersome to show
Field detecting, testing time are long.Xiamen uses for calprotectin detection kit prepared by positive bio tech ltd colloid
Golden method tests calprotectin content, since colloid gold particle with labeled albumen is combined by electrostatic interaction, in conjunction with unstable
It is easily interfered by environment, sensitivity is low, no standard measure detection.Moreover, these existing detection kits defend the calcium in excrement
Before albumen is detected, it usually needs extracting solution dissolves excrement, concussion 10 minutes or more, and concussion extraction time is longer.
Invention content
In view of this, the present invention provides a kind of kit and preparation method detecting calprotectin in human faecal mass.The present invention
Substituting kit made from traditional sealer using specific microballoon conjugate closed reagent can be accurate, quantitative, quick, clever
The content of calprotectin in quick detection human faecal mass so that entire detection process only needs 11min.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The kit, including box body itself of calprotectin and the Test paper in kit in a kind of detection human faecal mass
Item；The test strip includes bottom plate and the sample pad being successively set on bottom plate, bonding pad 1, bonding pad 2, reaction film
And blotting paper；
The bonding pad 1 is coated with the second of the first anti-calprotectin antibodies-fluorescent microsphere conjugate and biotin labeling
Anti-calprotectin antibody；
The bonding pad 2 is coated with Avidin；
It is set gradually by chromatography direction on the reaction film and detects T lines and Quality Control C lines, T line positions institute on the reaction film
State detection T lines coating biotin-bovine serum albumin(BSA) conjugate or biotin-chicken ovalbumin conjugate, the Quality Control C lines
Upper coating sheep anti-mouse igg antibody；
The anti-calprotectin antibodies fluorescent microsphere conjugate is prepared by the following method：
The fluorescent microsphere of activation and the first anti-calprotectin antibodies are coupled, blocking agent is then used, obtain described the
One anti-calprotectin antibodies fluorescent microsphere conjugate, it is to include 1~3wt% gelatin, 2 that the sealer, which is comprising the sealer,
~4wt%NH225~50mM MES buffer solutions of-PEG and 0.5~1wt% trehaloses.
Wherein, the first anti-calprotectin antibodies and the second anti-calprotectin antibody are mouse source antibody, can resist also may be used more
To be monoclonal antibody, two kinds of antibody can be combined with calprotectin, and identify that the epitope of calprotectin is different.
The testing principle of kit of the present invention is：
When in fecal sample to be measured there are when calprotectin, as chromatography acts on, when sample to be tested passes through bonding pad 1, with
The secondary antibody of the first anti-calprotectin antibodies-fluorescent microsphere conjugate and biotin labeling on bonding pad combines, and forms folder
Heart compound：Second anticalcium of the first anti-calprotectin antibodies-fluorescent microsphere conjugate-calprotectin-biotin labeling is defended
Protein antibodies；As chromatography acts on, when by bonding pad 2, the biotin in the sandwich complex and the Avidin on bonding pad 2
In conjunction with the second anticalcium for forming the first anti-calprotectin antibodies-fluorescent microsphere conjugate-calprotectin-biotin labeling is defended
Protein antibodies-Avidin compound, the compound of the combination Avidin detect T line packets when by reaction film, by reaction film
The bi-BSA conjugates (or bi-OVA) of quilt capture.Excessive first anti-calprotectin antibodies-fluorescent microsphere conjugate passes through matter
It is captured by coated sheep anti-mouse igg antibody on Quality Control C lines when controlling line C, detects T lines and Quality Control C lines through laser excitation, generate fluorescence
Signal is read by instrument and obtains signal reaction value.The signal reaction value that detection is obtained substitutes into preset standard curve, meter
Calculate the concentration for obtaining calprotectin in sample to be tested.
The present invention also provides the non-detections for medical diagnosis on disease and therapeutic purposes based on kit provided by the invention
Method includes the following steps：
Calprotectin standard items are taken, the standard items sample of a certain concentration gradient is made, standard items sample is taken to be added drop-wise to this hair
Detection in bright kit have a try paper slip sample pad on, be placed at room temperature for 10min.Test strip is inserted into special fluorescent quantitation
Detector reads the content of calprotectin, with the fluorescence intensity level (A of detection line T linesT) with the fluorescence intensity level of nature controlling line C line
(AC) ratio (AT/AC) it is ordinate, using calprotectin concentration of standard solution as abscissa, draw standard curve；
In the sample pad for taking the test strip that sample to be tested is added drop-wise in kit of the present invention, it is placed at room temperature for 10min, it will
The fluorescence intensity that test strip is inserted into special fluorescent quantitative detector reading test strips sample to be tested is obtained according to standard curve
Obtain calprotectin content in sample to be tested.
Preferably, in detection method of the present invention, the calprotectin standard items sample used when standard curve is established
A concentration of 2000 μ g/g, 1600 μ g/g, 1200 μ g/g, 600 μ g/g, 300 μ g/g, 100 μ g/g, the 30 μ g/ of calprotectin in this
g、10μg/g、0μg/g。
Preferably, when drawing standard curve, the test strips that the test strip that uses produces for same batch, each
Concentration determination 6 times.
The fluorescence intensity level of sample to be tested, which is substituted into above-mentioned standard curve, can calculate calprotectin in acquisition sample to be tested
Concentration.
In the present invention, it is coated with Avidin on bonding pad 2, its role is to by biotinylated antibody and biotin-cow's serum
Albumin conjugate (or biotin-chicken serum albumin conjugate) combines.Specifically, it is obtained when sample to be tested passes through bonding pad 1
Obtain sandwich complex, i.e. the second of the first anti-calprotectin antibodies-fluorescent microsphere conjugate-calprotectin-biotin labeling
Anti-calprotectin antibodies will be in the sandwich complex and reaction film detection T lines then with the Avidin on bonding pad 2 for " bridge "
Biotin-bovine serum albumin(BSA) conjugate (or biotin-chicken ovalbumin conjugate) connection, reach multilayer amplification effect
Fruit.
In specific embodiment provided by the invention, the sealer is to include 2wt% gelatin, 2wt%NH2- PEG and
The 50mM MES buffer solutions of 0.5wt% trehaloses.
Preferably, the bonding pad 1 and the bonding pad 2 are glass fibre element film；The reaction film is cellulose nitrate
Plain film.
Preferably, the bottom plate is viscosity PVC bottom plates.
Preferably, kit of the present invention, further includes excrement Extraction buffer, the excrement Extraction buffer is packet
Containing 2.5~4.0M urea, 0.025~0.05M CaCl2, 0.1~0.5wt%SDS, 10~20mM EDTA, 0.25M~0.5M
0.25~0.5M Tris-HCl bufferings that the pH of citric acid, 0.02~0.05wt% Sodium azides and 1.25-5wt%BSA is 8.0
Liquid.
In specific embodiment provided by the invention, the excrement Extraction buffer is to include 2.5M urea, 0.05M
CaCl2, 0.25wt%SDS, 20mM EDTA, 0.5M citric acids, 0.05% Sodium azide and 2wt%BSA pH be 8.0 0.5M
Tris-HCl buffer solutions.
The present invention the study found that using in kit of the present invention extracting solution and fecal sample mixing to be measured after surveyed
The recovery rate of examination, calprotectin is 93.4~100.2%, what effect was far superior to extracted and then tested using physiological saline
As a result.Show that kit of the present invention can fast and efficiently extract calprotectin from fecal sample to be measured, and then realizes fast
Speed, the purpose for accurately detecting calprotectin content in human faecal mass.
The present invention also provides the preparation methods of the kit, including：
The preparation of sample pad：Sample pad is impregnated with the Tris-HCl buffer solutions of 0.25~0.5M of the trehalose containing 5-10%
5-10min, 37 DEG C of dry 2h, sealed storage are spare；
The preparation of reaction film：It will make T lines in BSA-bi coatings to nitrocellulose filter, dilution sheep anti-mouse igg coating arrived
Make C lines on nitrocellulose filter, 37 DEG C of dry 16-22h, sealed storage is spare；
The preparation of bonding pad 1：The first anti-calprotectin antibodies-fluorescent microsphere conjugate and biotin labeling are prepared respectively
Second anti-calprotectin antibodies, are then sprayed on glass fibre membrane, 37 DEG C of dry 2h sealed storages, spare；
The preparation of conjugate pad 2：Avidin is sprayed on glass fibre element film, 37 DEG C of dry 2h sealed storages are spare；
The assembling of test strip：Sample pad, conjugate pad 1 will be overlapped on sticky PVC bottom plates successively, conjugate pads 2 nitre
Acid cellulose film, cushion pad, blotting paper are simultaneously cut into one fixed width, and the test strip is made；
The excrement Extraction buffer is fitted into reagent bottle, is placed in box body, obtains together with test strip obtained
To the kit.
In some embodiments, in the preparation of the bonding pad 1, the first anti-calprotectin antibodies-fluorescent microsphere is even
Connection object is prepared by the following method：
The fluorescent microsphere that grain size is 200-300nm is chosen, the MES buffer solutions for being 6.0-6.5 with pH wash fluorescent microsphere, add
Enter NHS and EDC, the reaction of room temperature mixing, the fluorescent microsphere activated；
The first anti-calprotectin antibodies are added in the microballoon of above-mentioned activation, then the reaction of room temperature mixing is sealed with sealer
It closes, obtains the first anti-calprotectin antibodies-fluorescent microsphere conjugate；The sealer be comprising 1~3wt% gelatin, 2~
4wt%NH20.25~0.5M MES buffer solutions of-PEG and 0.5~1wt% trehaloses.
In some embodiments, in the preparation of the bonding pad 1, the second anti-calprotectin antibodies of the biotin labeling
ByS μ lfo-NHS-LC-Biotin biotinylation kits (being purchased from thermo companies) defend the second anticalcium of mouse source
Protein antibodies biotinylation is prepared.
Kit of the present invention using specific microballoon conjugate closed reagent (comprising 1~3wt% gelatin, 2~
4wt%NH20.25~0.5M MES buffer solutions of-PEG and 0.5~1wt% trehaloses) substitute the examination obtained of traditional sealer
Agent box, calprotectin that can be in accurate, quantitative, quick, sensitive detection human faecal mass, and entire detection process only needs 11min.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is the test strip in kit of the present invention；
Fig. 2 is the detection principle diagram using the test strip in kit of the present invention；
Fig. 3 is that kit of the present invention contains calprotectin with Buhlmann Laboratories AG companies of Switzerland kits
Measure the correlation curve figure being detected.
Specific implementation mode
The invention discloses the kit and preparation method of calprotectin in a kind of human faecal mass, those skilled in the art can be with
Present disclosure is used for reference, technological parameter realization is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications are to ability
It is it will be apparent that they are considered as being included in the present invention for field technique personnel.The method and application of the present invention has been led to
Preferred embodiment is crossed to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to this paper institutes
The methods and applications stated are modified or suitably change and combine, to realize and apply the technology of the present invention.
To the explanation of the disclosed embodiments, enable those skilled in the art to implement or use the present invention.To this
A variety of modifications of a little embodiments will be apparent to those skilled in the art, as defined herein general
Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, the present invention will not
It can be intended to be limited to the embodiments shown herein, and be to fit to consistent with the principles and novel features disclosed in this article
Widest range.
With reference to embodiment, the present invention is further explained：
The preparation of 1 kit of the present invention of embodiment
Use thermo companies EZ-S μ lfo-NHS-LC-Biotin biotinylation kit bovine serum albumin(BSA)s
(BSA) biotinylation, dialysis purification obtain Bi-BSA conjugates keep in it is spare.
1. the preparation of sample pad：Sample pad is impregnated with the 0.25~0.5M Tris-HCl buffer solutions of the trehalose containing 5-10%
5-10min, 37 degree of dry 2h sealed storages.
2. the preparation of reaction film：It will make T lines in biotin-bovine serum albumin(BSA) conjugate coating to nitrocellulose filter,
By the sheep anti-mouse igg antibody coating after dilution to making C lines on nitrocellulose filter, 37 DEG C of dry 16-22h, sealed storage is standby
With.
3. the preparation of bonding pad 1
The preparation of 3.1 anti-calprotectin antibodies biotinylation conjugates
Use thermo companiesS μ lfo-NHS-LC-Biotin biotinylation kits are by another plant of mouse source
Anti-calprotectin antibodies biotinylation.
The preparation of 3.2 first anti-calprotectin antibodies-fluorescent microsphere conjugate
The 50mM MES for being 7.5 with pH by the first anti-calprotectin antibodies (being purchased from Zhuhai bio tech ltd Bo Mei)
Buffer solution dialyse three times it is spare.
The fluorescent microsphere that grain size is 200nm is chosen, is washed twice fluorescent microsphere with the pH MES buffer solutions for being 6.0,
10000r/min is centrifuged, and is resuspended with the MES buffer solutions of pH6.0, then presses every 100 μ l microballoons and 0.2-0.4 μ gNHS and 0.2- is added
NHS and EDC is added in 0.4 μ gEDC, and room temperature mixing reacts 30-40min, the fluorescent microsphere activated；
By the fluorescent microsphere 10000r/min centrifugation of above-mentioned activation, remove supernatant, with the 50mM MES buffer solutions of pH=7.0 from
The heart washs 2 times, and the first anticalcium that the addition of the first anti-calprotectin antibodies of 0.1-0.2mg has been dialysed, which is added, by every 100 μ l microballoons defends
Protein antibodies, room temperature mixing react 1-2h；Then sealer is added to be closed, it is micro- to obtain the anti-calprotectin antibodies fluorescence
Ball conjugate；Wherein, the sealer is to include 2wt% gelatin, 2wt%NH2The 50mM MES of-PEG and 0.5wt% trehaloses
Buffer solution；
The 10mmol/L boron for being 8.5 by above-mentioned the first anti-calprotectin antibodies-fluorescent microsphere conjugate pH prepared
Acid solution 10000r/min is washed twice, is added and is preserved liquid preservation, and the preservation liquid is 1%-5%BSA, 1%-2% trehaloses
The 10mmol/L boric acid solutions of pH=8.5.
3.3. the preparation of the second anti-calprotectin antibodies of biotin labeling
Use EZ-S μ lfo-NHS-LC-Biotin biotinylation kits (be purchased from thermo companies) are by mouse source
The second anti-calprotectin antibodies of biotin labeling are made in second anti-calprotectin antibodies biotinylation.
3.4 by anti-calprotectin antibodies fluorescent microsphere conjugate and anti-calprotectin antibodies biotinylation conjugate and with suitable
When concentration is sprayed on glass, 37 degree of dry 2h sealed storages are spare.
4. the preparation of conjugate pad 2
Avidin is sprayed on 5.2 μ g/ml on glass fibre element film, 37 DEG C of dry 2h, sealed storage is spare.
5. the preparation of reaction film
With coating buffer (PBS buffer solution of the trehalose containing 5%-10%) by biotin-bovine serum albumin(BSA) conjugate with 1-
Make T lines in the concentration coating to NC films of 2mg/ml, dilutes and make C lines on sheep anti-mouse igg antibody to 1-2mg/ml coatings to NC films, 37
DEG C dry 16-22h, sealed storage are spare.
6. the assembling of test strips
Overlap the above-mentioned sample pad prepared, conjugate pad 1, conjugate pad 2, reaction film successively on sticky PVC bottom plates
(nitrocellulose filter), blotting paper are cut into the test strips of 4mm width, are loaded, and the test strip are made, as shown in Figure 1.
7. the preparation of excrement Extraction buffer
It prepares comprising 2.5 urea, 0.05M CaCl2, 0.25wt%SDS, 20mM EDTA, 0.5M citric acids, 0.05wt%
The 0.5M Tris-HCl buffer solutions that the pH of Sodium azide and 2wt%BSA are 8.0.
Prepared excrement Extraction buffer is fitted into reagent bottle, box is placed in together with the above-mentioned test strip made
In vivo, the kit is obtained.
The preparation of 2 kit of the present invention of embodiment
In the kit in the preparation of test strip step 3 bonding pad 1 preparation, the sealer used is bright for 2wt%
Glue (w/w), 2wt%NH2The 0.5M MES buffer solutions of-PEG and 0.5wt% trehaloses, other steps are same as Example 1.
The preparation of 3 kit of the present invention of embodiment
The formula of excrement Extraction buffer is in the kit：4.0M urea, 0.025M CaCl2, 0.5wt%SDS,
10mM EDTA, 0.5M citric acids, 0.05 Sodium azide and 5wt%BSA pH be 8.0 0.25M Tris-HCl buffer solutions, other
Step is same as Example 1.
4 kit of the present invention of embodiment and the correlation of Buhlmann Laboratories AG companies of Switzerland kit are surveyed
Examination
50mg fecal samples to be measured are put into test tube with sampler, 49 times of Extraction buffer of sample net weight is added
Mixing in (the excrement Extraction buffer in 1 kit of embodiment), as sample to be tested solution.Sample to be tested is diluted to concentration
The test strip in 1 kit of embodiment and Switzerland Buhlmann is respectively adopted in different 1~samples of sample 20
The fecal calprotectin of Laboratories AG companies defends protein detection kit (enzyme-linked immunization) and is carried out at the same time test.
The test method of kit of the present invention：
Calprotectin standard items are taken to prepare a concentration of 2000 μ g/g, 1600 μ g/g, 1200 μ g/g, the 600 μ g/ of calprotectin
G, the standard items sample of 300 μ g/g, 100 μ g/g, 30 μ g/g, 10 μ g/g, 0 μ g/g.Above-mentioned standard product sample is taken to be added drop-wise to respectively
Detection in kit of the present invention have a try paper slip sample pad on, be placed at room temperature for 10min, each concentration of specimens tests 6 times, then
Test strip is inserted into the content that special fluorescent quantitative detector reads calprotectin, with the fluorescence intensity level of detection line T lines
(AT) with the fluorescence intensity level (A of nature controlling line C lineC) ratio (AT/AC) it is ordinate, be with calprotectin concentration of standard solution
Abscissa draws standard curve；
Take above-mentioned sample to be tested solution (1~sample of sample 20), the Test paper being added drop-wise to respectively in kit of the present invention
In the sample pad of item, it is placed at room temperature for 10min, each concentration of specimens tests 6 times, test strip is then inserted into special fluorescence and is determined
The fluorescence intensity that detector reads test strips sample to be tested is measured, according to standard curve, obtains calprotectin content in sample to be tested.
Meanwhile defending protein detection kit (enzyme-linked immunization) according to the fecal calprotectin of Buhlmann Laboratories AG companies of Switzerland
Method sample 1~20 is detected, testing result is as shown in table 1.
The correlation of 1 kit of the present invention of table and Buhlmann Laboratories AG companies of Switzerland kits
Using the test result of the test strip in the kit of embodiment 1 as abscissa, with Switzerland Buhlmann
It is that ordinate establishes correlation curve that the fecal calprotectin of Laboratories AG companies, which defends protein detection kit test result, is seen
Fig. 3.
The results show that in the range of calprotectin concentration is 8-2100 μ g/g, the inspection in 1 kit of the embodiment of the present invention
Test paper slip and the fecal calprotectin of Buhlmann Laboratories AG companies of Switzerland defend protein detection kit (enzyme linked immunological
Method) it compares, test result has good consistency, correlation coefficient r2=0.9687, show the survey of test strip of the present invention
Test result is accurate and reliable, can be effectively as the foundation of clinical examination.
5 kit precision of the present invention of embodiment is tested
3 parts of excrement for choosing high, medium and low value calprotectin content, through Buhlmann Laboratories AG fecal calprotectins
It is respectively 1500.2 μ g/g, 603.0 μ g/g, 42.8 μ g/g to defend protein detection kit and measure the content of calprotectin.Using this
The kit of inventive embodiments 1 is detected, every part of sample replication 10 times in homogeneous experiment, calculate separately average value,
Standard deviation calculates coefficient of variation CV (%) in experiment；It measures 1 time daily, METHOD FOR CONTINUOUS DETERMINATION 10 days, calculates test bay CV (%) value.
Calculation formula is：CV (%)=standard deviation/average value × 100%, the results are shown in Table 2.
2 precision test result of table
Note：Refer to being tested repeatedly identical test repeatedly in primary experiment in experiment；Refer to identical test between experiment not
With retest in the time.
The results show that kit of the present invention, which meets, is immunized the requirement that class measures the usual precision ＜ of project 10%, precision
Well.
The accuracy of 6 kit of the present invention of embodiment is tested
Two parts of excrement samples of protein detection kit measurement are defended in selection with Buhlmann Laboratories AG fecal calprotectins
This, calprotectin concentration is respectively 200 μ g/g and 1200 μ g/g, and the supernatant after extraction is respectively with 1：4、1：1 and 9：1 ratio is mixed
The sample solution of 3 parts of various concentrations is synthesized, the theoretical value of calprotectin concentration is respectively 1000,700 and 300 μ g/g.Using this
The kit of inventive embodiments 1 is detected, and calculates the consistency of detected value and theoretical value, the i.e. rate of recovery.Calculation formula is:
The rate of recovery=test value/theoretical value × 100%
Rate of recovery result of calculation is shown in Table 3.
3 rate of recovery testing result of table
| Test | Low serum (μ l) | High serum (μ L) | Theoretical value (μ g/g) | Test value (μ g/g) | The rate of recovery (μ g/g) |
| 1 | 2 | 8 | 1000 | 1020 | 102 |
| 2 | 5 | 5 | 700 | 690.2 | 98.6 |
| 3 | 9 | 1 | 300 | 273.3 | 91 |
The results show that the rate of recovery of kit of the present invention, between 98.6%-102%, accuracy is high.
7 Extraction buffer of the present invention of embodiment is compared with the effect of traditional extraction liquid
The embodiment is by the extraction element of excrement Extraction buffer and Buhlmann companies in kit of the present inventionIn the extraction effect of extract (0.9%NaCl solution) be compared.
The 10 of the determination of protein detection kit are defended with the fecal calprotectin of Buhlmann Laboratories AG companies of Switzerland
Part fecal sample, the theoretical concentration of wherein calprotectin are：25.8μg/g、61.2μg/g、200.5μg/g、312.3μg/g、
520.7 μ g/g, 580.6 μ g/g, 832.1 μ g/g, 1212.5 μ g/g, 1424.6 μ g/g, 1920.5 μ g/g, weigh 50mg excrement by
1：50 (g/ml) are separately added into above two extracting solution.Then it is tested, is tied with the test strip in 1 kit of embodiment
Fruit is shown in Table 4.
The extracting solution of 4 kit of the present invention of table is compared with the test result of traditional extraction liquid
The results show that using being tested in the extracting solution in kit of the present invention and 1min after excrement mixing, calcium defends egg
White recovery rate is 93.4~100.2%, and effect is far superior to the extraction element of Buhlmann companiesIn Cap
The test result of 15min is tested and shaken in extract 1min at once.
8 inventive closure agent of embodiment is compared with the effect of conventional sealer
Use the phosphate buffer that the pH containing 10%BSA is 7.2 0.02M as sealer, other steps with embodiment 1,
Contrast agents box 1 is prepared.
Test, test sample Buhlmann are carried out at the same time using the kit and contrast agents box 1 of the embodiment of the present invention 1
15 clinical negative samples that Laboratories AG kits determine.Test result be shown in Table 5 table, 5 inventive closure agent with often
The testing result of rule sealer compares
Note："-" indicates negative, and " ± " indicates that weak sun, "+" indicate positive.
The results show that all using the test result of the clinical negative sample of kit pair 15 of the embodiment of the present invention 1
Feminine gender uses the pH containing 10%BSA to have 7 samples as the contrast agents box 1 of sealer for the phosphate buffer of 7.2 0.02M
Product are false positive, false positive rate 42%.
The kit of Example 2~3 carries out the testing experiment of embodiment 4~8, as a result same as Example 1 or close,
Without significant difference (p>0.05).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (8)
1. the kit of calprotectin in a kind of detection human faecal mass, which is characterized in that including box body itself and in kit
Test strip；The test strip includes bottom plate and the sample pad being successively set on bottom plate, bonding pad 1, combines
Pad 2, reaction film and blotting paper；
The bonding pad 1 is coated with the second anticalcium of the first anti-calprotectin antibodies-fluorescent microsphere conjugate and biotin labeling
Defend protein antibodies；
The bonding pad 2 is coated with Avidin；
It is set gradually by chromatography direction on the reaction film and detects T lines and Quality Control C lines, examined described in T line positions on the reaction film
T lines coating biotin-bovine serum albumin(BSA) conjugate or biotin-chicken ovalbumin conjugate are surveyed, is wrapped on the Quality Control C lines
By sheep anti-mouse igg antibody；
The first anti-calprotectin antibodies fluorescent microsphere conjugate is prepared by the following method：
The fluorescent microsphere of activation and the first anti-calprotectin antibodies are coupled, blocking agent is then used, it is anti-to obtain described first
Anti-calprotectin antibody fluorescent microsphere conjugate, the sealer are to include 1~3wt% gelatin, 2~4wt%NH2- PEG and 0.5
25~50mM MES buffer solutions of~1wt% trehaloses.
2. kit according to claim 1, which is characterized in that the sealer is to include 2wt% gelatin, 2wt%NH2-
The 50mM MES buffer solutions of PEG and 0.5wt% trehaloses.
3. kit according to claim 1, which is characterized in that the bonding pad 1 and the bonding pad 2 are glass fibre
Plain film.
4. kit according to claim 1, which is characterized in that the reaction film is nitrocellulose filter.
5. according to Claims 1 to 4 any one of them kit, which is characterized in that further include excrement Extraction buffer, it is described
Excrement Extraction buffer is to include 2.5~4.0M urea, 0.025~0.05M CaCl2, 0.1~0.5wt%SDS, 10~20mM
The pH of EDTA, 0.25M~0.5M citric acids, 0.02~0.05wt% Sodium azides and 1.25-5wt%BSA be 8.0 0.25~
0.5M Tris-HCl buffer solutions.
6. kit according to claim 5, which is characterized in that the excrement Extraction buffer be comprising 2.5M urea,
0.05M CaCl2, 0.25wt%SDS, 20mM EDTA, 0.5M citric acids, 0.05% Sodium azide and 2wt%BSA pH be 8.0
0.5M Tris-HCl buffer solutions.
7. the preparation method of the kit described in claim 5 or 6 any one, which is characterized in that including：
The preparation of sample pad：Sample pad 5- is impregnated with the Tris-HCl buffer solutions of 0.25~0.5M of the trehalose containing 5-10%
10min, 37 DEG C of dry 2h, sealed storage are spare；
The preparation of reaction film：It will make T lines in BSA-bi coatings to nitrocellulose filter, by dilution sheep anti-mouse igg coating to nitric acid
Make C lines on cellulose membrane, 37 DEG C of dry 16-22h, sealed storage is spare；
The preparation of bonding pad 1：The second of the first anti-calprotectin antibodies-fluorescent microsphere conjugate and biotin labeling is prepared respectively
Then anti-calprotectin antibodies are sprayed on glass fibre membrane, 37 DEG C of dry 2h sealed storages, spare；
The preparation of conjugate pad 2：Avidin is sprayed on glass fibre element film, 37 DEG C of dry 2h sealed storages are spare；
The assembling of test strip：Sample pad will be overlapped on sticky PVC bottom plates successively, conjugate pad 1, it is fine that conjugate pads 2 nitric acid
The plain film of dimension, cushion pad, blotting paper are simultaneously cut into one fixed width, the test strip are made；
The excrement Extraction buffer is fitted into reagent bottle, is placed in box body together with test strip obtained, institute is obtained
State kit.
8. preparation method according to claim 7, which is characterized in that in the preparation of the bonding pad 1, first anticalcium defends egg
White antibody-fluorescent microballoon conjugate is prepared by the following method：
The fluorescent microsphere that grain size is 200-300nm is chosen, the MES buffer solutions for being 6.0-6.5 with pH wash fluorescent microsphere, are added
NHS and EDC, the reaction of room temperature mixing, the fluorescent microsphere activated；
The first anti-calprotectin antibodies are added in the microballoon of above-mentioned activation, then the reaction of room temperature mixing is used blocking agent, obtained
Obtain the first anti-calprotectin antibodies-fluorescent microsphere conjugate；The sealer is to include 1~3wt% gelatin, 2~4wt%
NH225~50mM MES buffer solutions of-PEG and 0.5~1wt% trehaloses.
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| CN201810123018.9A CN108333368A (en) | 2018-02-07 | 2018-02-07 | The kit and preparation method of calprotectin in a kind of detection human faecal mass |
| PCT/CN2018/093606 WO2019153630A1 (en) | 2018-02-07 | 2018-06-29 | Kit for detecting calprotectin in human feces and preparation method |
| NZ758859A NZ758880B2 (en) | 2017-07-01 | 2018-06-29 | Refrigerator |
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| NZ758859A (en) | 2021-10-29 |
| WO2019153630A1 (en) | 2019-08-15 |
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