CN104569411A - Immunochromatography kit for fluorescently and quantitatively detecting MBL (mannose-binding lectin) - Google Patents
Immunochromatography kit for fluorescently and quantitatively detecting MBL (mannose-binding lectin) Download PDFInfo
- Publication number
- CN104569411A CN104569411A CN201510005651.4A CN201510005651A CN104569411A CN 104569411 A CN104569411 A CN 104569411A CN 201510005651 A CN201510005651 A CN 201510005651A CN 104569411 A CN104569411 A CN 104569411A
- Authority
- CN
- China
- Prior art keywords
- mbl
- fluorescent
- fluorescence
- monoclonal antibody
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to an immunochromatography kit for fluorescently and quantitatively detecting MBL (mannose-binding lectin). The immunochromatography kit comprises a fluorescence test strip and sample diluent, wherein the fluorescence test strip comprises a bottom plate, a sample pad, a fluorescence binding pad, an enveloped membrane and absorbent paper are sequentially bonded on the upper surface of the bottom plate from one end to the other end, the inner end of the sample pad is lap-jointed with one end of the fluorescence binding pad, the other end of the fluorescence binding pad is lap-jointed with one end of the enveloped membrane, and the other end of the enveloped membrane is lap-jointed with the absorbent paper, wherein the enveloped membrane comprises a detection zone and a quality control zone, the detection zone is enveloped with anti-MBL monoclonal antibody, and the quality control zone is enveloped with goat anti-rabbit IgG; and the fluorescence binding pad contains fluorescently-labeled MBL monoclonal antibody and fluorescently-labeled goat anti-rabbit IgG. The immunochromatography kit for fluorescently and quantitatively detecting MBL has strong specificity and high sensitivity, the detection result acquisition time is short, the operating mode is convenient and simple, and the detection result is accurate and reliable.
Description
Technical field
The present invention relates to a kind of fluorescent quantitation and detect immune chromatography reagent kit of MBL and preparation method thereof.
Background technology
Sweet dew (gathering) sugar is in conjunction with lectin, and English name: mannan-binding lectin, is abbreviated as MBL.Sweet dew (gathering) sugar is a kind of change of serum C type agglutinin that liver produces in conjunction with lectin, the acute phase protein that can be combined with mannose, the pathogen of band mannose can be nursed one's health, activating complement system, participates in innate immune response, activating complement, adjustment inflammation, promotes opsonophagocytosis and remove apoptotic cell.Be one of immune important component, in immune defense, play multiple action.When the shortage of adaptive immune system still immature (infancy) or suppressed (after such as organ transplant, or during cancer chemotherapy) MBL usually may cause the increase of susceptibility infection.MBL is not enough and autoimmune disease has important relevant, as systemic loupus erythematosus, and rheumatoid arthritis, the immunocompromised host disease etc. after the cystic fibrosis that prognosis is poor and tumor chemoradiotherapy.MBL constitutes the first line of defence of body congenital immunity, early stage at cause pathogeny imcrobe infection, and before specific antibody is formed, MBL has just passed through its " Carbohydrate recognition domain " in conjunction with pathogenic microorganism.Therefore, detect MBL (MBL) content in human serum or blood plasma, monitor and assess the basic value of MBL serum-concentration, and treatment and intervene after MBL level, for integrating clinical Clinics and Practices, the individuality of early detection height risk, accurate instruction also adjusts clinical treatment in time, realizes the aspects such as the individualized treatment of disease significant.
About the detection means to MBL, the detection method existed at present both at home and abroad comprises that MBL protein concentration detects, MBL biological function measures and mbl gene type detection etc., but only has that to carry out Concentration Testing to MBL albumen be unique directly detection means.
The detection of MBL biological function: can indirectly reflect MBL content.At present, the method detecting MBL biological function mainly detects the opsonic action of MBL to phagocyte phagocytosis and the activation to complement system, i.e. the MBL activated pathway of complement.Opsonophagocytosis experimental implementation is very loaded down with trivial details, and can only carry out half-quantitative detection, at present to a great extent by complement activation test replace.The most frequently used hemolytic experiment of complement activation effect of MBL detects, and also can detect the deposition conditions of intermediate product on solid phase carrier in MBL complement activation pathway by ELISA method, indirectly to reflect the functional activity of MBL.But, because the activation of complement also has classical pathway and alternative route, therefore complement activation experiment must first fully suppress the classical pathway of complement activation and alternative pathway before detection, so operation is also more loaded down with trivial details, also more to the influence factor of result precision.
The detection of mbl gene type: the level indirectly can estimating MBL.At present, the method that major part detects mbl gene type all builds on the basis of PCR (polymerase chain reaction), and the new method derived by PCR constantly occurs.Earlier trials chamber interior adopt restriction fragment length polymorphism method, amplification refractory mutation system and Heteroduplex analysis is very complicated, effort, expense is relatively costly, poor accuracy.The nineties in 20th century, the biochip technology automaticity of Later development was high, and analysis time shortens greatly, and precision of analysis also improves a lot, but cost is relatively high, is only limited to laboratory study, was difficult to clinically to promote on a large scale.The limitation of genotype research is that it can not prove the relation between MBL concentration and disease.
The detection of Serum MBL concentrations: uniquely a kind of can the method for direct-detection MBL concentration.What MBL proteantigen detection method was commonly used at present has mannan to catch determination method, Antibody-capture ELISA and enzyme connection agglutinin immmunosorbent assay, but does not all become kit.MBL in the world for clinical diagnosis detects BioPorto company of reagent Jin You Denmark kit developed, principle is as seizure antibody by the monoclonal antibody for MBL CRD (mannan-binding lectin carbohydrate recognition domain), the identical antibody of biotin labeling is as detection antibody, HRP-SA (Streptavidin of horseradish peroxidase-labeled) is as amplifying Color Appearance System, the method detection required time is longer 4 hours, operation is more loaded down with trivial details, and sensitivity can detect 0.5ng/ml.But need to adopt robotization import instrument and kit to detect, the costliness that price is suitable and cannot sample be realized and detect fast in enormous quantities, make to apply being restricted.And the domestic MBL detection kit that there is no independent development.
Therefore, at present clinically in the urgent need to a kind of Method and kit for that can carry out fast MBL level in patients serum, accurately detect.Fluorescent microsphere mark chromatography detection technique in recent years continues after colloidal gold-labeled method, and the one of carrying out on the near patient bed side that fluorochrome label technically grows up detects analytical technology fast, is called for short POCT.POCT is the detection technique of a class great potential, and it is fast and convenient, and efficiency is high, and cost is low, has the advantages such as round of visits is short, sample consumption is few.Meanwhile, its stable reagent and be convenient to preserve and carry, be widely used in clinical, even oneself detect.Data processing and self-reacting device similar, have the software analysis process of design compilation in advance, improve POCT detect accuracy and reliability.The fluorescent microsphere of different-grain diameter is combined with the biomacromolecule material containing amino, carboxyl, hydroxyl, sulfydryl, when protein (antigen/antibody) antigen/antibody be coated on nitrocellulose filter of fluorescent microsphere mark is caught, fluorescent microsphere is assembled in corresponding region, under the exciting light of a wavelength range irradiates, send the visible ray of particular color.By detecting the fluorescence of laser excitation on lath, quantitatively detect single or multiple mark on the detection lath in units of pg/ml.Detection system is made up of Fluorescent reader and check-out console usually.The multiplex chromatography of check-out console, analyze thing and form immune complex in the process of movement, become certain ratio by the difference of the fluorescence signal value in surveyed area, Quality Control region with the variable concentrations analyzing thing, obtain calibration curve, the concentration analyzing thing can be detected in unknown sample.Can reach and measure suitable susceptibility and specificity with Routine Test Lab euzymelinked immunosorbent assay (ELISA).
Summary of the invention
The object of this invention is to provide a kind of high specificity, highly sensitive, the time obtaining testing result is short, and mode of operation is easy, and testing result accurately and reliably fluorescent quantitation detects the immune chromatography reagent kit of MBL.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, comprise fluorescent test paper strip and Sample dilution, fluorescent test paper strip comprises base plate, the upper surface of base plate is pasted with sample pad, fluorescence pad, coated film and thieving paper from one end successively to the other end, the inner end of sample pad is connected with one end overlap joint of fluorescence pad, the other end of fluorescence pad overlaps with one end of described coated film and is connected, the other end of coated film overlaps with described thieving paper and is connected, and described coated film comprises detection zone and quality control region; Described detection zone is coated with monoclonal antibody against MBL, and described quality control region bag is by goat anti-rabbit igg; Containing fluorescently-labeled MBL monoclonal antibody and fluorescently-labeled rabbit igg in described fluorescence pad.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, the monoclonal antibody against MBL of wherein said detection zone bag quilt is produced by the hybridoma cell strain of the monoclonal antibody of anti-MBL by being used as bag, being used as bag is CGMCC No.10091 by the preserving number of the hybridoma cell strain of the monoclonal antibody of anti-MBL, produced by the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking containing fluorescently-labeled MBL monoclonal antibody in described fluorescence pad, be CGMCC No.10092 with the preserving number of the hybridoma cell strain of the monoclonal antibody of anti-MBL of marking, being used as bag is had different from epi-position by the hybridoma cell strain of the monoclonal antibody of anti-MBL and the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, and wherein said quality control region bag is 0.2-4mg/ml by the coating buffer concentration of goat anti-rabbit igg, and consumption is 30 μ l/30cm; The coating buffer concentration of the MBL monoclonal antibody of described detection zone is 0.2-4mg/ml, and consumption is 30 μ l/30cm.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, and wherein said quality control region bag is 0.5-1.5mg/ml by the coating buffer concentration of goat anti-rabbit igg, and the coating buffer concentration of the MBL monoclonal antibody of described detection zone is 1-2mg/ml.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, wherein said fluorescence pad comprises glass fibre, fluorescence pad is sprayed on glass fibre after being mixed by fluorescently-labeled MBL monoclonal antibody and fluorescently-labeled rabbit igg and makes through freeze-drying, the concentration of the fluorescently-labeled MBL monoclonal antibody on glass fibre in label is 2-5 μ g/ml, and the concentration of fluorescently-labeled rabbit igg is 2-5 μ g/ml.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, and wherein said Sample dilution comprises sodium hydrogen phosphate concentration 20mmol, sodium chloride concentration 150mmol, bovine serum albumin(BSA) concentration 1%-2%, Tween-20 concentration 0.1%, and all the other are water.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, fluorescently-labeledly on wherein said fluorescence pad excites electromagnetic wavelength to be 310-550nm, and emission wavelength is the electromagnetic wave of 340-620nm.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, and wherein said fluorescence pad adopts following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
After the fluorescein of 18-22mg or 480-520mg fluorescent microsphere are mixed with 50mgMBL monoclonal antibody, slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide while stirring, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides content in its whole reaction system is made to be 0.1-0.3mg, the content of N-hydroxy-succinamide is 0.1-0.2mg, spends the night 4 DEG C of lucifuge reactions; With Sephadex G25 gel column, antibody fluorescence label is carried out purifying, remove impurity, redissolve with fluorescence protective agent; Save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
After the fluorescein of 18-22mg or 480-520mg fluorescent microsphere are mixed with 50mg rabbit igg, slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide while stirring, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides content in its whole reaction system is made to be 0.1-0.3mg, the content of N-hydroxy-succinamide is 0.1-0.2mg, spends the night 4 DEG C of lucifuge reactions; With Sephadex G25 gel column, antibody fluorescence label is carried out purifying, remove impurity, redissolve with fluorescence protective agent; Save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, and wherein said fluorescence pad also can adopt following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
By the fluorescein of 18-22mg or 480-520mg Fluorescent microsphere marker and 65mgMBL monoclonal antibody, be placed in 4 DEG C, regulation system pH6.8-9.0, stir and slowly add 0.2-1% glutaraldehyde; Reaction 2-5 hour, carries out purifying with Sephadex G25 gel column by antibody fluorescence label, removes impurity, redissolves with fluorescence protective agent; Save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
After the fluorescein of 18-22mg or 480-520mg fluorescent latex label are mixed with rabbit igg, be placed in 4 DEG C, regulation system pH6.8-9.0, stir and slowly add 0.2-1% glutaraldehyde; Reaction 2-5 hour, carries out purifying with Sephadex G25 gel column by antibody fluorescence label, removes impurity, redissolves with fluorescence protective agent; Save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, and wherein said fluorescence combines to pay somebody's debt and expect repayment later and following methods can be adopted to make:
Prepare the MBL antibody of fluorescent particle mark:
Dissolve 45mg MBL monoclonal antibody with pH8.0-pH9.6 carbonate buffer solution, dissolve with pH8.0-pH9.6 carbonate buffer solution or suspension 18-22mg fluorescein or 480-520mg fluorescent microsphere; While stirring the fluorescein of above-mentioned dissolving or fluorescent microsphere are added in antibody-solutions gradually, after adding, continue lucifuge and stir 10-14 hour, in conjunction with after, load in bag filter, by above-mentioned carbonic acid buffer saline dialysed overnight, redissolve with fluorescence protective agent, save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
Dissolve 45mg rabbit igg with pH8.0-pH9.6 carbonate buffer solution, dissolve with pH8.0-pH9.6 carbonate buffer solution or suspension 18-22mg fluorescein or 480-520mg fluorescent microsphere; While stirring the fluorescein of above-mentioned dissolving or fluorescent microsphere are added in antibody-solutions gradually, after adding, continue lucifuge and stir 10-14 hour, in conjunction with after, load in bag filter, by above-mentioned carbonic acid buffer saline dialysed overnight, redissolve with fluorescence protective agent, save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad.
The hybridoma cell strain being used as the monoclonal antibody of people's MBL of bag quilt of the present invention, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on November 25th, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCC No.10091, and the Classification And Nomenclature of suggestion is: produce and be used as bag by the hybridoma cell strain of the monoclonal antibody of anti-MBL; The hybridoma cell strain of the monoclonal antibody with anti-MBL of marking of the present invention, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on November 25th, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCC No.10092, and the Classification And Nomenclature of suggestion is: the hybridoma cell strain producing the monoclonal antibody with anti-MBL of marking.
The Cleaning Principle that fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL is double antibody sandwich method, fluorescein or fluorescent microsphere and MBL monoclonal antibody covalent bond.To detect after sample (serum or blood plasma) dilutes 100 times with Sample dilution adds in well, and its fluorescence labeling MBL monoclonal antibody can be combined by the MBL in blood, forms compound; Upwards on chromatography and coated film, the MBL monoclonal antibody of detection zone T forms antibody-antigen-antibody compound.When adding in the sample pad of test strips containing MBL sample drop, upwards the fluorescence labeling MBL monoclonal antibody of chromatography on fluorescence labeling pad is combined, mixed liquor continues to move forward along coated film, in sample, contained MBL amount is more, the fluorescent-labeled antibody that can be combined with MBL antibody is more, thus makes to record fluorescence and detect value and raise.Result judges: a. is shows green fluorescence trace when detection line T and nature controlling line C is under 450nm exciting light irradiates while, represents that testing result is positive; B. when detection line T not shows green fluorescence trace under 450nm exciting light irradiates, only have nature controlling line C shows green fluorescence trace simultaneously under excitation light, represent that testing result be feminine gender; C. nature controlling line C under excitation light not shows green fluorescence trace time, this test strips can not be used.When scanning fluorescence signal intensity by fluorescence detector, MBL content in sample can be detected.
MBL fluorescence immune chromatography test paper bar and GC/MS, HPLC isochromatic spectrum instrument and enzyme of the immune chromatography reagent kit of fluorescent quantitation detection MBL of the present invention are exempted from method and are detected compared with MBL, there is simple operations one step complete, be applicable to varying number pattern detection, fast, result, sensitivity more advantages of higher within about 15 minutes, can be had.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, employing fluorescence labeling liquid frozen dried uniform spreading is coated in the method on import glass fibre, compared with other drying processes, the preci-sion and accuracy of its batch production reaches best effects, better compared to fluorescent marker fluid preservation stability, easy and simple to handle.Fluorescent quantitative detector can reach and only within 10 seconds, just can carry out sensitive quantitative measurement to MBL, contained MBL amount in faster more accurate working sample; MBL chromatograph test strip has good accuracy, the recovery is 90%-110%, quantitative deviation is little, quantitative deviation within 20%, high sensitivity, the sample size of needs is few, be only 100ul, operate very easy, testing result clock like precision is reliable, has outstanding substantive distinguishing features and significant progress.
Below in conjunction with accompanying drawing, the immune chromatography reagent kit that fluorescent quantitation of the present invention detects MBL is described in further detail.
Accompanying drawing explanation
Fig. 1 is the stereographic map that fluorescent quantitation of the present invention detects the structural representation of fluorescent test paper strip in MBL chromatography kit;
Fig. 2 is Fig. 1 vertical view;
Fig. 3 is that fluorescent quantitation of the present invention detects in MBL chromatography kit for placing the schematic diagram of ELISA test strip card.
Embodiment
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, comprise fluorescent test paper strip and Sample dilution, as depicted in figs. 1 and 2, fluorescent test paper strip comprises base plate 7, the upper surface of base plate 7 is pasted with sample pad 1, fluorescence pad 2, coated film 3 and thieving paper 6 from one end successively to the other end, the inner end of sample pad 1 is connected with one end overlap joint of fluorescence pad 2, the other end of fluorescence pad 2 overlaps with one end of described coated film 3 and is connected, the other end of coated film 3 overlaps with described thieving paper 6 and is connected, and described coated film 3 comprises detection zone 4 and quality control region 5; Described detection zone 4 is coated with monoclonal antibody against MBL, and described quality control region 5 is wrapped by goat anti-rabbit igg; Containing fluorescently-labeled MBL monoclonal antibody and fluorescently-labeled rabbit igg in described fluorescence pad 2.
Number of patent application is 201310390393.7, denomination of invention is the Chinese invention patent application of " monoclonal antibody of anti-MBL and kit ", disclose a kind of hybridoma cell strain, it comprises as bag by the hybridoma cell strain of the monoclonal antibody of anti-MBL, its preserving number is CGMCC No.7592, also comprise the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking, its preserving number is CGMCC No.7591, being used as bag is had different from epi-position by the hybridoma cell strain of the monoclonal antibody of anti-MBL and the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking.
The monoclonal antibody of above-mentioned anti-MBL, comprise the monoclonal antibody being used as bag and being produced by the hybridoma cell strain of the monoclonal antibody of anti-MBL, also comprise the monoclonal antibody produced with the hybridoma cell strain of the monoclonal antibody of anti-MBL of marking.
Above-mentioned mannose binding lectin immue quantitative detection reagent box, comprise the monoclonal antibody being used as bag and being produced by the hybridoma cell strain of the monoclonal antibody of anti-MBL, also comprise the monoclonal antibody produced with the hybridoma cell strain of the monoclonal antibody of anti-MBL of marking.
The monoclonal antibody against MBL of quilt is wrapped in the fluorescent quantitation of the present invention described detection zone 4 detected in the immune chromatography reagent kit of MBL, can utilize that number of patent application is 201310390393.7, denomination of invention be that " monoclonal antibody of anti-MBL and kit " disclosed preserving number is CGMCC No.7592, is used as bag by the monoclonal antibody of the hybridoma cell strain of the monoclonal antibody of anti-MBL generation.
Fluorescent quantitation of the present invention to detect in the described fluorescence pad 2 in the immune chromatography reagent kit of MBL containing fluorescently-labeled MBL monoclonal antibody, can utilize that number of patent application is 201310390393.7, denomination of invention be the monoclonal antibody that disclosed in " monoclonal antibody of anti-MBL and kit ", preserving number is CGMCC No.7591, produces with the hybridoma cell strain of the monoclonal antibody of anti-MBL of marking.
The immune chromatography reagent kit of the fluorescent quantitation detection MBL of the monoclonal antibody that the hybridoma cell strain that the monoclonal antibody using preserving number to produce for CGMCC No.7592 hybridoma cell strain and preserving number are CGMCC No.7591 produces, testing requirement of the present invention can be met completely, but its sensitivity can only reach the level of 1.0ng/ml usually, sensitivity is not as the hybridoma cell strain of the present invention again preservation.
As improvement of the present invention, the monoclonal antibody against MBL that quilt is wrapped in above-mentioned detection zone 4 is produced by the hybridoma cell strain of the monoclonal antibody of anti-MBL by being used as bag, being used as bag is CGMCC No.10091 by the preserving number of the hybridoma cell strain of the monoclonal antibody of anti-MBL, produced by the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking containing fluorescently-labeled MBL monoclonal antibody in described fluorescence pad 2, be CGMCC No.10092 with the preserving number of the hybridoma cell strain of the monoclonal antibody of anti-MBL of marking, being used as bag is had different from epi-position by the hybridoma cell strain of the monoclonal antibody of anti-MBL and the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking.Sandwich ratio juris is utilized to detect sample.
The immune chromatography reagent kit of the fluorescent quantitation detection MBL of the monoclonal antibody that the hybridoma cell strain that the monoclonal antibody using the new preserving number of the invention described above to produce for CGMCC No.10091 hybridoma cell strain and new preserving number are CGMCC No.10092 produces, its sensitivity can reach 0.2ng/ml.
As a further improvement on the present invention, it is 0.2-4mg/ml that above-mentioned quality control region 5 is wrapped by the coating buffer concentration of goat anti-rabbit igg, and consumption is 30 μ l/30cm; The coating buffer concentration of the MBL monoclonal antibody of described detection zone 4 is 0.2-4mg/ml, and consumption is 30 μ l/30cm.
As a further improvement on the present invention, it is 0.5-1.5mg/ml that above-mentioned quality control region 5 is wrapped by the coating buffer concentration optimization of goat anti-rabbit igg, and the coating buffer concentration optimization of the MBL monoclonal antibody of described detection zone 4 is 1-2mg/ml.
As a further improvement on the present invention, above-mentioned fluorescence pad 2 comprises glass fibre, fluorescence pad 2 is sprayed on glass fibre after being mixed by fluorescently-labeled MBL monoclonal antibody and fluorescently-labeled rabbit igg and makes through freeze-drying, the concentration of the fluorescently-labeled MBL monoclonal antibody on glass fibre in label is 2-5 μ g/ml, and the concentration of fluorescently-labeled rabbit igg is 2-5 μ g/ml.
Above-mentioned Sample dilution comprises sodium hydrogen phosphate concentration 20mmol, sodium chloride concentration 150mmol, bovine serum albumin(BSA) concentration 1%-2%, Tween-20 concentration 0.1%, and all the other are water.
Fluorescently-labeledly on above-mentioned fluorescence pad 2 excite electromagnetic wavelength to be 310-550nm, emission wavelength is the electromagnetic wave of 340-620nm.
Above-mentioned fluorescence pad 2 can adopt following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
After the fluorescein of 18-22mg or 480-520mg fluorescent microsphere are mixed with 50mgMBL monoclonal antibody, slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide while stirring, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides content in its whole reaction system is made to be 0.1-0.3mg, the content of N-hydroxy-succinamide is 0.1-0.2mg, spends the night 4 DEG C of lucifuge reactions; With Sephadex G25 gel column, antibody fluorescence label is carried out purifying, remove impurity, redissolve with fluorescence protective agent; Save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
After the fluorescein of 18-22mg or 480-520mg fluorescent microsphere are mixed with 50mg rabbit igg, slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide while stirring, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides content in its whole reaction system is made to be 0.1-0.3mg, the content of N-hydroxy-succinamide is 0.1-0.2mg, spends the night 4 DEG C of lucifuge reactions; With Sephadex G25 gel column, antibody fluorescence label is carried out purifying, remove impurity, redissolve with fluorescence protective agent; Save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad 2.
Above-mentioned fluorescence pad 2 also can adopt following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
By the fluorescein of 18-22mg or 480-520mg Fluorescent microsphere marker and 65mgMBL monoclonal antibody, be placed in 4 DEG C, regulation system pH6.8-9.0, stir and slowly add 0.2-1% glutaraldehyde; Reaction 2-5 hour, carries out purifying with Sephadex G25 gel column by antibody fluorescence label, removes impurity, redissolves with fluorescence protective agent; Save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
After the fluorescein of 18-22mg or 480-520mg Fluorescent microsphere marker are mixed with rabbit igg, be placed in 4 DEG C, regulation system pH6.8-9.0, stir and slowly add 0.2-1% glutaraldehyde; Reaction 2-5 hour, carries out purifying with Sephadex G25 gel column by antibody fluorescence label, removes impurity, redissolves with fluorescence protective agent; Save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad 2.
Above-mentioned fluorescence pad 2 can also adopt following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
Dissolve 45mgMBL monoclonal antibody with pH8.0-pH9.6 carbonate buffer solution, dissolve with pH8.0-pH9.6 carbonate buffer solution or suspension 18-22mg fluorescein or 480-520mg fluorescent microsphere; While stirring the fluorescein of above-mentioned dissolving or fluorescent microsphere are added in antibody-solutions gradually, after adding, continue lucifuge and stir 10-14 hour, in conjunction with after, load in bag filter, by above-mentioned carbonic acid buffer saline dialysed overnight, redissolve with fluorescence protective agent, save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
Dissolve 45mg rabbit igg with pH8.0-pH9.6 carbonate buffer solution, dissolve with pH8.0-pH9.6 carbonate buffer solution or suspension 18-22mg fluorescein or 480-520mg fluorescent microsphere; While stirring the fluorescein of above-mentioned dissolving or fluorescent microsphere are added in antibody-solutions gradually, after adding, continue lucifuge and stir 10-14 hour, in conjunction with after, load in bag filter, by above-mentioned carbonic acid buffer saline dialysed overnight, redissolve with fluorescence protective agent, save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad 2.
Fluorescence labeling pad 2 of the present invention is that the label of fluorescein and albumen or the label of fluorescent microsphere and albumen are sprayed on glass fiber material and make, and wherein used fluorescein can be wherein one or more such as fluorescein isothiocynate, RB 200, TRITC, phycoerythrin, many dinoflagellates chlorophyll protein, lanthanide chelate, Fluoresceincarboxylic acid, cumarin.
Embodiment one
In this embodiment, fluorescent quantitation detects MBL immune chromatography reagent kit, includes the preparation of fluorescent test paper strip making and Sample dilution.
In this embodiment, T line place, the detection zone 0.5mg/ml monoclonal antibody against MBL coating buffer of coated film, use amount is 30ul/30cm.The anti-rabbit IgG being 1mg/ml at the quality control region C line place working concentration of coated film carries out bag quilt, and use amount is all 30ul/30cm.For the rabbit igg of combined with fluorescent mark, for the validity of test strip.
In this embodiment, fluorescence labeling liquid excites by 310nm, and emission wavelength is 340nm.In this embodiment, the preparation of fluorescence labeling liquid adopts the preparation method (A) of carboxylic Fluorescent microsphere marker (the present embodiment use fluorescein be umbelliferone microballoon), and step is as follows:
After the umbelliferone microballoon of 500mg is mixed with 50mgMBL antibody or 50mg rabbit igg respectively, slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS) while stirring, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides (EDC) and N-hydroxy-succinamide (NHS), EDC adds 0.2mg, NHS adds 0.1mg, the content of EDC in its whole reaction system is made to be 0.2mg, NHS content is 0.1mg, and 2-8 DEG C of lucifuge reaction is spent the night at low temperatures.With Sephadex G25 gel column, antibody fluorescence label is carried out purifying, remove impurity; Redissolve with fluorescence protective agent.
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated with on the glass fibers by 500ul/30cm uniform spreading, freeze-drying saves backup.
The sample pad cut, fluorescence pad, coated film and thieving paper are pasted onto on base plate with mutually overlapping successively, cut into the wide film bar of uniform 3--4mm with cutting cutter, be assembled in the fluorescence detection test strip namely becoming finished product in the plastic clip shell (test card) fastened by plastics upper casing and plastics lower casing.Coordinate by sodium hydrogen phosphate concentration 20mmol, sodium chloride concentration 150mmol, bovine serum albumin(BSA) concentration 1%-2%, Tween-20 concentration 0.1%, all the other sample diluting liquids be mixed with for water can detect clinical blood sample.
Embodiment two
In this embodiment, fluorescent quantitation detects MBL immune chromatography reagent kit, comprises making and the Sample dilution preparation of fluorescent test paper strip.
In this embodiment, T line place, the detection zone 1mg/ml monoclonal antibody against MBL coating buffer of coated film, use amount is 30ul/30cm.The anti-rabbit IgG being 1mg/ml at the quality control region C line place working concentration of coated film carries out bag quilt, and use amount is all 30ul/30cm, for the rabbit igg of combined with fluorescent mark, for the validity of test strip.
In this embodiment, after fluorescence labeling liquid excites by 550nm, emission wavelength is 620nm.In this embodiment, the preparation of fluorescence labeling liquid adopts the preparation method containing amino Fluorescent microsphere marker (the present embodiment use fluorescein be TRITC microballoon), and step is as follows:
After 500mg fluorescent latex label is mixed with 65mgMBL antibody or 65mg rabbit igg respectively, be placed in 4 DEG C of environment, regulation system pH7.0-8.5, stir and slowly add 0.4-0.6% glutaraldehyde; Reaction 2-5 hour, carries out purifying with Sephadex G25 gel column by antibody fluorescence label, removes impurity, redissolves with fluorescence protective agent.
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, so that two kinds of antibody concentration are all 5ug/ml respectively, are coated with on the glass fibers by 500ul/30cm uniform spreading, and freeze-drying is preserved.
The sample pad cut, fluorescence pad, coated film and thieving paper are pasted onto on base plate with mutually overlapping successively, cut into the wide film bar of uniform 3--4mm with cutting cutter, be assembled in the fluorescence detection test strip namely becoming finished product in the plastic clip shell (test card) fastened by plastics upper casing and plastics lower casing.Coordinate by sodium hydrogen phosphate concentration 20mmol, sodium chloride concentration 150mmol, bovine serum albumin(BSA) concentration 1%-2%, Tween-20 concentration 0.1%, all the other sample diluting liquids be mixed with for water can detect clinical blood sample.
Embodiment three
In this embodiment, fluorescent quantitation detects MBL immune chromatography reagent kit, comprises making and the Sample dilution preparation of fluorescent test paper strip.
In this embodiment, T line place, the detection zone 1.5mg/ml monoclonal antibody against MBL coating buffer of coated film, use amount is 30ul/30cm.The anti-rabbit IgG being 2mg/ml at the quality control region C line place working concentration of coated film carries out bag quilt, and use amount is all 30ul/30cm, for the rabbit igg of combined with fluorescent mark, for the validity of test strip.
In this embodiment, fluorescence labeling liquid excites by 490nm, and emission wavelength is 530nm.In this embodiment, the preparation of fluorescence labeling liquid adopts the preparation method of the fluorescein of sulfur-bearing carbon acylamino (the present embodiment use fluorescein be fluorescein isothiocynate), and step is as follows:
Dissolve 45mgMBL antibody or 45mg rabbit igg respectively with pH8.0-pH9.6 carbonic acid buffer, dissolve or suspension 20mg fluorescein isothiocynate with pH8.0-PH9.6 carbonic acid buffer; While stirring above-mentioned fluorescein isothiocynate is added in globulin solution gradually, after adding, continues lucifuge and stir 12 hours, in conjunction with after, load in bag filter, by above-mentioned carbonic acid buffer saline dialysed overnight at low temperatures, redissolve with fluorescence protective agent.
By the MBL antibody of the above-mentioned fluorescent-substance markers prepared and the mixing of fluorescent latex particles mark rabbit igg, so that two kinds of antibody concentration are all 3ug/ml respectively, and be coated with on the glass fibers by 500ul/30cm uniform spreading, freeze-drying saves backup.
MBL immunochromatographytest test kit of the present invention, in instantiation, semi-manufacture are assembled by following operation: overlapped in turn be pasted onto on base plate by sample pad, fluorescence pad, coated film, thieving paper, form test strips, can again with 7 card shells of the prior art, as shown in Figure 3, be fixed into test card, described card shell scribbles can for the product information coding of luminoscope scanning recognition.With the ID chip of written information.The quantitative computing formula that ID chip of the prior art adopts is semilog straight line equation or other calculation equations of detected signal value and calculating concentration, automatically can carry out result judgement.The Sample dilution installed is divided to become kit with other assembling fittings.
Fluorescent quantitation of the present invention detects the immune chromatography reagent kit of MBL, in use, as shown in Figure 3, fluorescent test paper strip can be assembled in the plastic clip shell 7 (test card) fastened by plastics upper casing and plastics lower casing, plastics upper casing is provided with two perforates, well 9 and display window 8, and well 9 faces the sample pad 1 of fluorescent test paper strip, result display window 8 faces toward detection zone and the quality control region of coated film 3, and this fluorescent test paper strip can take out from this plastic clip shell 7.
Be used for testing the fluorescent quantitation spectral detection system (Immunofluorescence test instrument) of immuno-chromatographic test paper strip, mainly comprise fluorescence light source system, detection system and automatic software analysis and Control system.
In one embodiment of the present of invention, detect sample to need by following operation: the sample (serum or blood plasma) drawing a certain amount of such as 10ul mixes with 990ul Sample dilution, 100ul is drawn after mixing, add toward horizontal positioned test card well, do not bring bubble into, start chromatography reaction.React 15 minutes, by fluorescence detector read test result.
Quantitative MBL immunochromatographytest test kit described in embodiments of the invention 1-3 compares with the measurement result of Immunofluorescence test instrument to 200 routine samples (serum/plasma) and shows: the accuracy measuring MBL within the scope of 0.2-100ng/ml is high: quantitation curves linear coefficient is all greater than 0.99, containing 0.5 in sample with its accuracy of MBL of 1ng/ml concentration is 80%-120%, and kit detects and is limited to 0.2ng/ml.The relatively general colloidal gold method (qualitative) of employing and the testing result of Enzyme-Linked Immunospot detection kit have better sensitivity and specificity.Table specific as follows.
Be in the process of the hybridoma cell strain of CGMCC No.10092 to produce the new preserving number of the present invention be CGMCC No.10091 hybridoma cell strain and new preserving number, inventor with MBL (MBL) as immunogene, the animals such as immune mouse, rat, rabbit, cavy, goat or sheep, from by the splenocyte of MBL immune animal or lymphocyte and myeloma cell fusion, obtain above-mentioned 2 kinds of hybridoma cell strains of the present invention.
Above-mentioned splenocyte or lymphocytic separation, can adopt known method to carry out, and to the screening of hybridoma, known method can be adopted to carry out.
The hybridoma cell strain of the new preserving number of the present invention to be CGMCC No.10091 hybridoma cell strain and new preserving number be CGMCC No.10092, obtain in accordance with the following methods: by mouse, by the immunogene containing MBL, at three soles and subcutaneous routine immunization, immunity is repeated after 3 to 4 weeks, tail vein booster immunization once after 2 weeks, gets its splenocyte and SP2/0 myeloma cell is conventionally merged after 3 to 4 days, and hybridoma is with limiting dilution assay cloning; Pick out high titre hybridoma.
Monoclonal antibody of the present invention produces by hybridoma of the present invention.The method producing monoclonal antibody of the present invention from hybridoma of the present invention is this area routine or known method, such as: can obtain from the tissue culture medium of cultivating hybridoma of the present invention, also can breed by being inoculated in Mice Body, being separated and obtaining from the ascites of collecting or serum.Described cultivation hybridoma of the present invention can carry out according to this area routine or known method.Described inoculation, to collect ascites or serum, from ascites or serum, be separated the method for monoclonal antibody of the present invention can be the conventional or known method in this area.
Embodiment
The foundation of preserving number to be CGMCC No.10091 hybridoma cell strain and preserving number the be hybridoma cell strain of CGMCC No.10092
Immunity: the female BAl BIc/c mouse in 8-12 age in week, with the amount of every 20ugMBL (in Health China human plasma purifying), at three soles and subcutaneous routine immunization, 3-4 repeats immunity after week, and within 2 weeks, afterwards tail vein booster immunization is once.
Merge: from the mouse extracting spleen cell of last booster immunization after 3 days, mix with myeloma cell's (Nat'l Pharmaceutical & Biological Products Control Institute's Cytology Lab provides) ratio with 5: 1, merge with 50%PEG liquid.37 DEG C of 5%CO in HAT nutrient solution
2cultivate.
The detection of hybridoma and selectivity are cultivated: cultivate to measure with HAT nutrient solution half afterwards for 3-4 days and change liquid, until Growth of Cells detects to during 1/3-1/2 hole, envelope antigen MBL, get hybridoma supematant (primary antibodie) jointly to hatch with antigen, unconjugated antibody is removed in washing, and enzyme-added mark sheep anti mouse two resists, the chromogenic reaction positive (OD value, i.e. absorbance, is greater than 2 with negative control ratio) be clone to be selected.Change HT nutrient solution after first time clone, change DMEM nutrient solution after second time clone and cultivate.
Hybridoma is with limiting dilution assay cloning: washed down hybridoma nutrient solution to be cloned in 96 orifice plates, and count and be diluted to 20 cell/ml, every hole 0.1ml inoculates 96 orifice plates, contains 0.5 cell through 3 two-fold dilutions and every hole, 37 DEG C of 5%CO
2cultivate.Have the hole of Growth of Cells to add nutrient solution after 3-5 days to continue to cultivate, during naked eyes visible cell clone, carry out antibody test, now clone is 100% positive, can build strain.
Pick out 2 plant height titre hybridomas altogether.
The preparation of monoclonal antibody and purifying
Inoculation: get male BALB/c mouse in 8 week age, the aseptic wax oil 0.5ml/ of lumbar injection only, inoculates 2*10 after 5 days respectively
7/ ml is by embodiment 1 gained hybridoma.Collect after there is more ascites, can repeatedly collect.
Caprylic acid precipitation method purification monoclonal antibody: dilute the ascites collected with the acetate buffer solution (glacial acetic acid 1.8ml+500ml distilled water) of 4 times of volumes, adjusts ph4.5 with 0.1N NaOH.Under stirring, the sample after every ml dilution slowly adds 25ul caprylic acid.Place after 30 minutes, centrifugal 30 minutes of room temperature 10000rpm.Filter supernatant, remove thin slag.Add 10 times of concentrated PBS (9 increment product+1 part of 10*PBS), adjust ph7.5 with 5N NaOH.Supernatant is cooled to 4 DEG C, 45% saturated ammonium sulphate (0.277g/ml), stirs 30 minutes, 4 DEG C, centrifugal 15 minutes of 5000rpm.With a small amount of PBS dissolution precipitation, by the PBS dialysed overnight of 50-100 times of volume, 4 DEG C.After dialysis, sample adds 0.1% thimerosal, and 4 DEG C of preservations, namely obtain monoclonal antibody B3 and B10 respectively.
Protein A chromatography method monoclonal antibody: HiTrap rProtein A FF (Amersham Bioscinces) fills binding buffer liquid (the 1.5M glycocoll with more than 5 times column volumes after post, 3M NaCl, ph8.9) column equilibration is washed, monoclonal antibody sample syringe loading after sad precipitation is extracted, the binding buffer liquid of 10 times of column volumes washes post, flow velocity 1ml/min, eluent (the 0.1M citrate buffer of 5 times of column volumes, pH5) post is washed, protein peak is collected in monitoring, obtain antibody purification, SDS-PAGE identifies purity.
Mark HRP (horseradish peroxidase): antibody purification 8-10mg above-mentioned steps obtained loads bag filter, by carbonate buffer solution 4 DEG C of dialysed overnight of 0.01M pH9.5.Get HRP4mg, be dissolved in the pure water of 1.0ml, slowly add 0.1M NaIO4, addition is 0.1ml, and under room temperature, lucifuge stirs 40 minutes, loads bag filter, by acetate buffer 4 DEG C of dialysed overnight of 0.001M pH4.5.The sodium carbonate 0.05ml adding 0.1M in HRP (horseradish peroxidase) adjusts pH to 9.5, and then mix with antibody, lucifuge stirs 2 hours.Add the NaBH of 4mg/ml
40.1ml, 4 DEG C leave standstill 3 hours, 4 DEG C of PBS dialysed overnight.Add isopyknic saturated ammonium sulfate, stir 40 minutes, 4 DEG C leave standstill 3 hours, and centrifugal 30 minutes of 3000rpm, abandons supernatant.Gained precipitation repeats aforementioned operation one time with the ammonium sulfate of 50% again.Gained precipitation is dissolved in 1ml PBS, and 4 DEG C of PBS dialyse 48 hours, obtain monoclonal antibody linked with peroxidase HRP-B3 and HRP-B10.
The mensuration that enzyme labelled antibody is tired: wrap the reaction plate being purified antigen, enzyme labelled antibody is done serial dilution, 37 DEG C of reactions colour developing in 1 hour, judge that enzyme labelled antibody is tired, its result is: HRP-B3 is 1: 8000, HRP-B10 is 1: 2000.
Sds polyacrylamide gel electrophoresis: prepare 5.5% separation gel and 5% concentrated glue, Casting of gels, another preparation electrophoresis rushes liquid (25mmol Tris-Cl, 250mmol glycocoll, 0.1%SDS).Sample is suspended from SDS-PAGE sample loading buffer, boils 5min, loading after the centrifugal 1min of 12000rpm, loading volume is 20ul/ hole.During electrophoresis, when sample is positioned at concentrated glue, voltage is set to 70V, and after sample enters separation gel, voltage is set to 100V, stops electrophoresis when band bromjophenol blue arrives bottom separation gel.Soak was dyeed 2-3 as a child in coomassie brilliant blue R_250 dye liquor, with the destainer decolouring containing 20% ethanol and 10% glacial acetic acid to the colourless rear observation of background.
According to molecular weight standard, calculate the molecular weight of two strain monoclonal antibodies, drawn the degree of monoclonal antibody by gel imaging analysis, result shows that the purity of monoclonal antibody of the present invention can be used for preparing external diagnosis reagent.
Claims (10)
1. fluorescent quantitation detects the immune chromatography reagent kit of MBL, it is characterized in that: comprise fluorescent test paper strip and Sample dilution, fluorescent test paper strip comprises base plate (7), the upper surface of base plate (7) is pasted with sample pad (1) from one end successively to the other end, fluorescence pad (2), coated film (3) and thieving paper (6), the inner end of sample pad (1) is connected with one end overlap joint of fluorescence pad (2), the other end of fluorescence pad (2) overlaps with one end of described coated film (3) and is connected, the other end of coated film (3) overlaps with described thieving paper (6) and is connected, described coated film (3) comprises detection zone (4) and quality control region (5), described detection zone (4) is coated with monoclonal antibody against MBL, and described quality control region (5) bag is by goat anti-rabbit igg, containing fluorescently-labeled MBL monoclonal antibody and fluorescently-labeled rabbit igg in described fluorescence pad (2).
2. fluorescent quantitation according to claim 1 detects the immune chromatography reagent kit of MBL, it is characterized in that: the monoclonal antibody against MBL of described detection zone (4) bag quilt is produced by the hybridoma cell strain of the monoclonal antibody of anti-MBL by being used as bag, being used as bag is CGMCC No.10091 by the preserving number of the hybridoma cell strain of the monoclonal antibody of anti-MBL, produced by the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking containing fluorescently-labeled MBL monoclonal antibody in described fluorescence pad (2), be CGMCC No.10092 with the preserving number of the hybridoma cell strain of the monoclonal antibody of anti-MBL of marking, being used as bag is had different from epi-position by the hybridoma cell strain of the monoclonal antibody of anti-MBL and the hybridoma cell strain of the monoclonal antibody with anti-MBL of marking.
3. fluorescent quantitation according to claim 2 detects the immune chromatography reagent kit of MBL, it is characterized in that: described quality control region (5) bag is 0.2-4mg/ml by the coating buffer concentration of goat anti-rabbit igg, and consumption is 30 μ l/30cm; The coating buffer concentration of the MBL monoclonal antibody of described detection zone (4) is 0.2-4mg/ml, and consumption is 30 μ l/30cm.
4. fluorescent quantitation according to claim 3 detects the immune chromatography reagent kit of MBL, it is characterized in that: described quality control region (5) bag is 0.5-1.5mg/ml by the coating buffer concentration of goat anti-rabbit igg, and the coating buffer concentration of the MBL monoclonal antibody of described detection zone (4) is 1-2mg/ml.
5. the immune chromatography reagent kit of MBL is detected according to the fluorescent quantitation in Claims 1-4 described in any one, it is characterized in that: described fluorescence pad (2) material is glass fibre, fluorescence pad (2) is sprayed on glass fibre after being mixed by fluorescently-labeled MBL monoclonal antibody and fluorescently-labeled rabbit igg and makes through freeze-drying, the concentration of the fluorescently-labeled MBL monoclonal antibody on glass fibre in label is 2-5 μ g/ml, and the concentration of fluorescently-labeled rabbit igg is 2-5 μ g/ml.
6. fluorescent quantitation according to claim 5 detects the immune chromatography reagent kit of MBL, it is characterized in that: described Sample dilution comprises sodium hydrogen phosphate concentration 20mmol, sodium chloride concentration 150mmol, bovine serum albumin(BSA) concentration 1%-2%, Tween-20 concentration 0.1%, all the other are water.
7. fluorescent quantitation according to claim 6 detects the immune chromatography reagent kit of MBL, it is characterized in that: described fluorescence pad (2) is above fluorescently-labeled excites electromagnetic wavelength to be 310-550nm, and emission wavelength is the electromagnetic wave of 340-620nm.
8. fluorescent quantitation according to claim 7 detects the immune chromatography reagent kit of MBL, it is characterized in that: described fluorescence pad (2) adopts following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
After the fluorescein of 18-22mg or 480-520mg fluorescent microsphere are mixed with 50mgMBL monoclonal antibody, slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide while stirring, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides content in its whole reaction system is made to be 0.1-0.3mg, the content of N-hydroxy-succinamide is 0.1-0.2mg, spends the night 4 DEG C of lucifuge reactions; With Sephadex G25 gel column, antibody fluorescence label is carried out purifying, remove impurity, redissolve with fluorescence protective agent; Save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
After the fluorescein of 18-22mg or 480-520mg fluorescent microsphere are mixed with 50mg rabbit igg, slowly add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides and N-hydroxy-succinamide while stirring, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides content in its whole reaction system is made to be 0.1-0.3mg, the content of N-hydroxy-succinamide is 0.1-0.2mg, spends the night 4 DEG C of lucifuge reactions; With Sephadex G25 gel column, antibody fluorescence label is carried out purifying, remove impurity, redissolve with fluorescence protective agent; Save backup through freeze-drying;
By the above-mentioned fluorescently-labeled MBL antibody for preparing and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad (2).
9. fluorescent quantitation according to claim 7 detects the immune chromatography reagent kit of MBL, it is characterized in that: described fluorescence pad (2) also can adopt following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
By the fluorescein of 18-22mg or 480-520mg Fluorescent microsphere marker and 65mgMBL monoclonal antibody, be placed in 4 DEG C, regulation system pH6.8-9.0, stir and slowly add 0.2-1% glutaraldehyde; Reaction 2-5 hour, carries out purifying with Sephadex G25 gel column by antibody fluorescence label, removes impurity, redissolves with fluorescence protective agent; Save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
After the fluorescein of 18-22mg or 480-520mg Fluorescent microsphere marker are mixed with rabbit igg, be placed in 4 DEG C, regulation system pH6.8-9.0, stir and slowly add 0.2-1% glutaraldehyde; Reaction 2-5 hour, carries out purifying with Sephadex G25 gel column by antibody fluorescence label, removes impurity, redissolves with fluorescence protective agent; Save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad (2).
10. fluorescent quantitation according to claim 7 detects the immune chromatography reagent kit of MBL, it is characterized in that: described fluorescence pad (2) also can adopt following methods to make:
Prepare the MBL antibody of fluorescent particle mark:
Dissolve 45mg MBL monoclonal antibody with pH8.0-pH9.6 carbonate buffer solution, dissolve with pH8.0-pH9.6 carbonate buffer solution or suspension 18-22mg fluorescein or 480-520mg fluorescent microsphere; While stirring the fluorescein of above-mentioned dissolving or fluorescent microsphere are added in antibody-solutions gradually, after adding, continue lucifuge and stir 10-14 hour, in conjunction with after, load in bag filter, by above-mentioned carbonic acid buffer saline dialysed overnight, redissolve with fluorescence protective agent, save backup through freeze-drying;
Prepare fluorescent particle mark rabbit igg:
Dissolve 45mg rabbit igg with pH8.0-pH9.6 carbonate buffer solution, dissolve with pH8.0-pH9.6 carbonate buffer solution or suspension 18-22mg fluorescein or 480-520mg fluorescent microsphere; While stirring the fluorescein of above-mentioned dissolving or fluorescent microsphere are added in globulin solution gradually, after adding, continue lucifuge and stir 10-14 hour, in conjunction with after, load in bag filter, by above-mentioned carbonic acid buffer saline dialysed overnight, redissolve with fluorescence protective agent, save backup through freeze-drying;
By the MBL antibody of the above-mentioned fluorescent particle mark prepared and the mixing of fluorescent particle mark rabbit igg, to make two kinds of antibody concentration respectively all for 2ug/ml, be coated on the glass fibre of bar shaped by 500ul/30cm uniform spreading, then the glass fibre of bar shaped is blocked make described fluorescence pad (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510005651.4A CN104569411A (en) | 2015-01-07 | 2015-01-07 | Immunochromatography kit for fluorescently and quantitatively detecting MBL (mannose-binding lectin) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510005651.4A CN104569411A (en) | 2015-01-07 | 2015-01-07 | Immunochromatography kit for fluorescently and quantitatively detecting MBL (mannose-binding lectin) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104569411A true CN104569411A (en) | 2015-04-29 |
Family
ID=53085964
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510005651.4A Pending CN104569411A (en) | 2015-01-07 | 2015-01-07 | Immunochromatography kit for fluorescently and quantitatively detecting MBL (mannose-binding lectin) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104569411A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105259352A (en) * | 2015-10-19 | 2016-01-20 | 山西隆克尔生物制药有限公司 | Fluorescence immunoassay strip based on coumarin dendrimer as well as preparation method and application of fluorescence immunoassay strip |
| CN105548535A (en) * | 2016-01-21 | 2016-05-04 | 成都微瑞生物科技有限公司 | Classical swine fever antibody detection card and preparation method thereof |
| CN105606798A (en) * | 2016-04-08 | 2016-05-25 | 四川新健康成生物股份有限公司 | Sample diluent for immunofluorescence chromatography detection |
| CN107656045A (en) * | 2017-11-01 | 2018-02-02 | 上海凯创生物技术有限公司 | Procalcitonin near-infrared fluorescent detection reagent card, kit and application thereof |
| CN108732344A (en) * | 2018-04-12 | 2018-11-02 | 江苏维尔生物科技有限公司 | A kind of test paper for quickly detecting taxol and preparation method thereof, kit |
| CN109085349A (en) * | 2018-08-22 | 2018-12-25 | 宁波奥丞生物科技有限公司 | A kind of immune chromatography reagent kit of multinomial joint-detection |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102023211A (en) * | 2010-11-19 | 2011-04-20 | 广州万孚生物技术有限公司 | Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof |
| CN102323406A (en) * | 2011-10-21 | 2012-01-18 | 广州万孚生物技术有限公司 | Kit for carrying out fluorescence quantitative on ractopamine through and preparation method of fluorescence labeling liquid |
| CN103614341A (en) * | 2013-09-02 | 2014-03-05 | 佛山市天海医药科技有限公司 | Anti-mannan-binding lectin monoclonal antibodies and kit |
-
2015
- 2015-01-07 CN CN201510005651.4A patent/CN104569411A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102023211A (en) * | 2010-11-19 | 2011-04-20 | 广州万孚生物技术有限公司 | Immunochromatographic test strip for full quantitative detection of C-reactive protein and preparation method thereof |
| CN102323406A (en) * | 2011-10-21 | 2012-01-18 | 广州万孚生物技术有限公司 | Kit for carrying out fluorescence quantitative on ractopamine through and preparation method of fluorescence labeling liquid |
| CN103614341A (en) * | 2013-09-02 | 2014-03-05 | 佛山市天海医药科技有限公司 | Anti-mannan-binding lectin monoclonal antibodies and kit |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105259352A (en) * | 2015-10-19 | 2016-01-20 | 山西隆克尔生物制药有限公司 | Fluorescence immunoassay strip based on coumarin dendrimer as well as preparation method and application of fluorescence immunoassay strip |
| CN105548535A (en) * | 2016-01-21 | 2016-05-04 | 成都微瑞生物科技有限公司 | Classical swine fever antibody detection card and preparation method thereof |
| CN105606798A (en) * | 2016-04-08 | 2016-05-25 | 四川新健康成生物股份有限公司 | Sample diluent for immunofluorescence chromatography detection |
| CN107656045A (en) * | 2017-11-01 | 2018-02-02 | 上海凯创生物技术有限公司 | Procalcitonin near-infrared fluorescent detection reagent card, kit and application thereof |
| CN108732344A (en) * | 2018-04-12 | 2018-11-02 | 江苏维尔生物科技有限公司 | A kind of test paper for quickly detecting taxol and preparation method thereof, kit |
| CN108732344B (en) * | 2018-04-12 | 2021-06-11 | 江苏维尔生物科技有限公司 | Test paper for rapidly detecting paclitaxel, preparation method thereof and kit |
| CN109085349A (en) * | 2018-08-22 | 2018-12-25 | 宁波奥丞生物科技有限公司 | A kind of immune chromatography reagent kit of multinomial joint-detection |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104569411A (en) | Immunochromatography kit for fluorescently and quantitatively detecting MBL (mannose-binding lectin) | |
| CN101988924B (en) | Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method | |
| Birchler et al. | Infrared photodetection for the in vivo localisation of phage-derived antibodies directed against angiogenic markers | |
| CN101226194B (en) | Malachite green vestigial ELISA detection kit and usage method thereof | |
| CN103439514B (en) | Pesticide and veterinary drug multi-residue detection method based on microarray detection chip | |
| CH621873A5 (en) | ||
| CN106124757A (en) | Signal during lateral flow and related immune measure amplifies | |
| CN102680676A (en) | Myeloperoxidase (MPO) determination kit (by using latex enhanced turbidimetric immunoassay) | |
| CN109406778A (en) | The time-resolved fluorescence quantitative test paper item and its preparation method and application of ralstonia solanacearum in a kind of detection tobacco leaf | |
| CN104280549B (en) | Epstein-Barr virus VCA-IgA antibody test reagent and preparation method thereof | |
| CN101241134A (en) | ELISA kit for detecting ractopamine residue and method of use thereof | |
| CN110488006A (en) | A kind of immunochromatographydetection detection card and preparation method thereof of 3 sample albumen 1 of quick detection chitinase | |
| CN107764992A (en) | The orientation coupling method of a kind of microballoon and antibody and application | |
| CN106226522A (en) | The detection method of a kind of GP73, detectable and detection kit | |
| CN109265404A (en) | A kind of preparation method and application of carbendazim haptens and antigen | |
| Zhou et al. | Ultrasensitive paper sensor for simultaneous detection of alpha-amanitin and beta-amanitin by the production of monoclonal antibodies | |
| CN108761075A (en) | A kind of deoxynivalenol quantifies rapid detection card and its detection method | |
| CN105388286B (en) | A kind of test strips for detecting iprodione and its preparation method and application | |
| JPH02259570A (en) | Assay of bone-derived alkali phosphatase | |
| JP2001519894A (en) | Early detection method for lysosomal storage disease | |
| CN100476439C (en) | ELISA kit for detecting quinolones in animal derived food | |
| CN105334323A (en) | Method and test strip for detecting zilpaterol, and application of test strip | |
| CN109180519A (en) | A kind of olaquindox metabolite antigen, antibody and enzyme-linked immunologic detecting kit and detection method | |
| CN107505459A (en) | Quantitatively detect people H FABP time-resolved fluoroimmunoassay chromatograph test strip, kit and preparation method thereof | |
| CN1979169A (en) | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20160629 Address after: 201206 No. 1888, Jinqiao Road, building 7, building 503, Shanghai, Pudong New Area Applicant after: Yang Yang Address before: Three road 528231 Guangdong city of Foshan province Dali Town, Nanhai District No. 181 Hong Ling China Academy of biomedical science and technology industrial center of the South China Sea block B room 207 Applicant before: FOSHAN TIANHAI MEDICAL TECHNOLOGY CO., LTD. |
|
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150429 |
|
| RJ01 | Rejection of invention patent application after publication |