CN106771239A - Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method - Google Patents
Serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one to determine kit and preparation method Download PDFInfo
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- CN106771239A CN106771239A CN201611165670.4A CN201611165670A CN106771239A CN 106771239 A CN106771239 A CN 106771239A CN 201611165670 A CN201611165670 A CN 201611165670A CN 106771239 A CN106771239 A CN 106771239A
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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
- G01N33/68—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
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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
- G01N33/53—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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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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
- G01N33/53—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
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- 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
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- 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
- G01N2333/4737—C-reactive protein
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- 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
- G01N2333/575—Hormones
- G01N2333/585—Calcitonins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The present invention is that a kind of serum amyloid A protein/Procalcitonin/C reactive proteins are three-in-one determines agent box, is provided with test card, it is characterised in that the test card is sequentially provided with from the bottom to top：Rare earth Eu is adsorbed with PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad3+Antiserum amyloid A, anti-Procalcitonin, three kinds of monoclonal antibody microballoon coupled complexes of anti-C reactive proteins, a diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide that fluorescent microsphere is marked respectively3+, it is stable under ground state, the fluorescence of wavelength 615nm is launched under the excitation source effect of 337nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, and the cell strain of monoclonal antibody for epitopes such as 26 different serum amyloid A proteins, Procalcitonin, C reactive proteins is derived from respectively.Have the advantages that easy to operate, reaction is quick, sensitivity is high, high specificity.
Description
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically one kind can be quick and precisely
While to the infection of the serum amyloid A protein in serum, blood plasma and whole blood sample, Procalcitonin, C reactive protein etc. three
Inflammation index of correlation carries out the three-in-one measure kit of serum amyloid A protein/Procalcitonin/C reactive protein of quantitative analysis
And preparation method.
Background technology：
Serum amyloid A protein (SAA) belongs to human body acute phase protein, and low-level is present in blood, when human body is thin
Bacterium or virus etc. infection after, be inflamed or activity lesion, tissue damage after quickly raise.It is acute in inflammation or infection
Phase can raise rapidly, and decline rapidly in the convalescence of disease.Research display, it is athero- in bacterium, fungi, virus infection, artery
SAA is raised in can detect serum in the diseases such as hardening, angiocardiopathy, acute transplantation rejection reaction.
Procalcitonin (procalcitonin, PCT) be it is a kind of contain 116 amino acid without hormonal activity glycoprotein.When tight
Its level in blood plasma is raised when weight bacterium, fungi, parasitic infection and pyemia and MOFE.PCT is not
Only it is the specific index of serious bacterial inflammation and fungal infection, and is also the pyemia multi viscera relevant with course inflammatory activity
The reliability index of exhaustion, be it is a kind of for bacterium infection early diagnosis, antidiastole, treatment monitoring and Index for diagnosis with wound
The diagnosis index of new meaning.
C reactive protein (C-reaction protein, CRP), is infected or during tissue damage in blood plasma in body
The protein that one class steeply rises, is mainly synthesized by liver cell.Albumen can be combined with pneumococcal cell walls C- polysaccharide, therefore
Referred to as CRP.CRP has various biological function, participates in various own physiologicals and pathophysiological process, and CRP can be with activating complement
With strengthen phagocyte phagocytosis and rise opsonic action so that remove invasion body pathogenic microorganism and damage, necrosis, apoptosis
Histocyte, important protective effect is played during the innate immunity of body.Additionally, CRP is that an acute phase is anti-
Albumen is answered, under normal circumstances atomic (the Neonatal CRP concentration of content<CRP is dense in 2mg/L, children and normal adult human serum
Degree<10mg/L), quickly, drastically raised with its blood concentration when infecting in acute injury.Common CRP is raised and is seen infection, inflammation
Disease, heart infarction, operation and wound etc., are controlled when infection, CRP declines rapidly when wound disappears.Current CRP is scorching as general
The index of disease reaction, is clinically the most frequently used acute-phase response index, diagnosis in clinical many diseases, treatment and pre-
Middle extensive use afterwards.
SAA/PCT/CRP joint-detections have certain clinical value for inflammation comprehensive descision.
Immuno analytical method is detected such as using the immune response between trace antigen and corresponding antibody with high specificity
It is living in the organisms such as hormone, medicine, protein, polypeptide, enzyme, tumor associated antigen, micro-element, virus, bacterium and metallic element
Property material.Immuno analytical method includes labelling immunoassay, non-marked immunoassay and instrument immunoassay.This kit is utilized
The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be belonging to one kind of labelling immunoassay.
Fluoroimmunoassay (FIA) and radiommunoassay (RIA) since the advent of the world, experienced the development of decades, but
It is that people increasingly feel FIA because of naturally local too high, interference detection results；RIA uses isotope marks, has pole to human body
Big harm is simultaneously made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme is unstable in itself, pushes away
Wide application is restricted.The beginning of the eighties, people begin one's study and replace fluorescent material and isotope-labelled protein with rare earth element
Or antibody, TIME RESOLVED TECHNIQUE is incorporated into field of biological detection, establish new ultramicron time-resolved fluoroimmunoassay point
Analysis technology (Time resolved Fluoroimmunoassay, abbreviation TrFIA).The technology uses multidisciplinary advanced technology, collection
The characteristics of having tied other immunoassays, in fields such as immunology, molecular biology, cytology and medical science, obtains significant progress
And extensive use.
TrFIA make use of trivalent rare earth ion and chelate with unique fluorescent characteristic be tracer replace fluorescent material,
Enzyme, isotope, chemiluminescent substance, labelled antibody, antigen, hormone, polypeptide, protein, nucleic acid probe and biological cell treat anti-
Answer system (such as antigen-antibody reaction, nucleic acid probe hybridization, biotin-labeled pentylamine reaction and the killing of target cell pairing effect cell
Effect etc.) occur after, with TrFIA detectors determine product in fluorescence intensity.According to product fluorescence intensity and relatively glimmering
The ratio of luminous intensity, judges the concentration of analyte in reaction system, so as to reach quantitative analysis.In common fluoremetry,
Due to containing various fluorescent components in test sample, background fluorescence (causes from the colloidal solid and solvent molecule in sample
The non-specific fluorescence that scattering light and Proteins in Serum and other compounds send) intensity is big, disturb strong, as fluorescence point
The bottleneck that analysis method is promoted on a large scale.Why TrFIA can turn into after the new sensitive detection method of the latter of EIA, RIA,
Depend primarily on the wavelength resolution and time-delay technique that are used in the unique fluorescence feature of lanthanide series, detection and dissociation-
Enhancing technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, has in 17, be usually used in TrFIA mainly have europium (Eu),
Samarium (Sm), terbium (Tb), dysprosium (Dy).Lanthanide series has unique fluorescence radiation feature, compared with common fluorescent, lanthanide ion chela
Compound fluorescence decay time is long, is the 10 of conventional fluorescent3-106Times.If the fluorescence decay time of lanthanide ion chelate is in 60-
900 μ s, conventional Eu3+Fluorescence decay time is 714 μ s, and the fluorescence decay time of fluorogen only has in common fluorescent immunoassay
1-100 μ s, the fluorescence decay time of some protein is only 1-10 μ s in sample, therefore utilizes TIME RESOLVED TECHNIQUE, postpones one
Measured after fixing time, just can obtain Eu3+Specific fluorescence signal.Simultaneously because decay time is long, Eu3+Label is in measurement
Between in can be excited repeatedly, ground state is transitted to by excitation state quickly after exciting every time, just there is fluorescence to send, then again can be weighed
Newly excite, so it is per second have 1000 times excite so that the relative specific activity of TrFIA fluorescent markers is very high.Lanthanide series is glimmering
The maximum of light spectrum is characterized in that the Stokes displacements between exciting light and launching light are larger, Eu3+Excitation wavelength is 337nm, transmitting
Wavelength is 615nm, and Stokes displacements are up to 278nm；While Eu3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is very
Sharply, can adjust instrument to be determined in extremely narrow wave-length coverage, thus almost completely eliminate the interference of background fluorescence, after
And pass through time delay and wavelength resolution, and strong specificity fluorescent and background fluorescence are distinguished into open (therefore referred to as time resolution), make interference
Reach almost nil.
In view of the application of above labeling method and detection technique, this kit has good detection specific, higher
The fluorescent marker of sensitivity, the simplicity of operation and stabilization ensure that the accuracy of detection.
The content of the invention：
The present invention is for shortcoming and defect present in prior art, it is proposed that a kind of utilization fluorescence immune chromatography it is sensitive
Property, combined with fluorescent immunochromatographiassays assays instrument realize sensitivity it is high, fast and simple, can simultaneously accurate quantitative analysis detection serum amyloid
Sample albumin A, Procalcitonin, the three-in-one measure reagent of serum amyloid A protein/Procalcitonin/C reactive protein of DDi
Box and preparation method.
The present invention can be reached by following measures：
A kind of three-in-one measure kit of serum amyloid A protein/Procalcitonin/C reactive protein, is provided with test card, its
It is characterised by that the test card is sequentially provided with from the bottom to top：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads,
Antiserum amyloid A, anti-Procalcitonin, anti-C- reactions that rare-earth fluorescent microballoon is marked respectively are wherein adsorbed with pad
Three kinds of monoclonal antibodies of albumen-microballoon coupled complex, a diameter of 100-250nm of the rare-earth fluorescent microballoon, rare-earth fluorescent
Microballoon containing one or more in rare earth lanthanide, under the excitation source effect of 300-400nm launch by the stabilization under ground state
Go out the fluorescence that wave-length coverage is 550-650nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, is originated respectively
It is thin in the monoclonal antibody for epitopes such as 2-6 different serum amyloid A protein, Procalcitonin, C reactive proteins
Born of the same parents' strain.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 120-150nm；The rare-earth fluorescent microballoon
Preferably comprise one or more rare earth lanthanides；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 on pad
The monoclonal cell cell line of individual different epitopes.
Pad of the present invention is obtained using following steps：Glass fibre membrane is soaked in 150mM Tris-HCL treatment
In liquid (X-100 containing 1.0%Triton, 2.5%BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 small
When, it is standby.Glass fibre membrane is placed on Bio-DotXYZ3050 three-dimensional specking platforms, is connect with Bio-Jet Quanti300 are non-
Antiserum amyloid A, anti-Procalcitonin and anti-C reactive protein that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon
Three kinds of coupled complexes of antibody are sprayed onto on glass fibre membrane after mixing, and 37 DEG C of drying are obtained after 1 hour.
Serum amyloid A protein/Procalcitonin/the C- of the rare-earth fluorescent microballoon mark in the present invention on pad is anti-
Three kinds of monoclonal antibodies are obtained using following steps in answering the three-in-one measure agent box of albumen：
Step 1：The acquisition of cell strain of monoclonal antibody：Respectively egg is reacted with serum amyloid A protein, Procalcitonin, C-
Bai Chunpin distinguishes immune mouse, and the monoclonal antibody of specific high-affinity is prepared using the method for preparing monoclonal antibody of standard
Cell line, the monoclonal antibody cell line to being obtained carries out pairing screening, is preferably gone out for trying according to pairing result and affinity data
The monoclonal antibody cell line of agent box；
Step 2：The preparation of monoclonal antibody：Antiserum amyloid egg is prepared and purified using the ascites production technology of standard
Three kinds of mouse resource monoclonal antibodies such as white A antibody, anti-Procalcitonin antibody and anti-C reactive protein antibody, are stored in -20 after packing
It is DEG C standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate of pH 9.5 delays
Fliud flushing, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：Antiserum amyloid A antibody, anti-Procalcitonin antibody and the anti-C- that rare-earth fluorescent microballoon is marked are anti-
Answer three kinds of preparations of coupled complex such as protein antibodies：Three kinds of antibody-microspheres coupled complexes are individually coupled, and operation is such as
Under：Choose from 2 antiserum amyloid A monoclonal antibodies of the monoclonal cell cell line of different epitopes, press
According to mass ratio 1:1 by 2mg serum amyloid A proteins monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, then
Mix with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C anti-
Answer 4 hours；Isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is added, 4 DEG C were closed
Night；Then use the buffer solution of 50mM Tris-HCL, pH7.4 to be washed 3 times using centrifugal process, be resuspended in the 50mM Tris- of 100 μ l
In HCL buffer solutions (contain 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.Same operation difference
Anti- Procalcitonin antibody-microspheres coupled complex and anti-C reactive protein antibody-microspheres coupled complex are prepared, 4 DEG C of lucifuges are protected
Deposit standby.
The nitrocellulose filter for being coated with detection line and nature controlling line of the present invention is obtained by following steps：
Step 1：Using with antiserum amyloid A used on pad, anti-Procalcitonin and anti-C reactive protein
Deng the different cell line of three kinds of cell strain of monoclonal antibody, antiserum starch is prepared and purified using the ascites production technology of standard
Three kinds of mouse resource monoclonal antibodies such as sample protein A antibody, anti-Procalcitonin antibody and anti-C reactive protein antibody, obtain anti-with mark
Body pairing monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Step 2：Above-mentioned three kinds of mouse resource monoclonal antibodies and goat anti-mouse igg antibody are adjusted dense with coating dilution respectively
1-3mg/ml is spent, film liquid amount is 1-3 μ l/cm, and they are sprayed on into cellulose nitrate as detection line is parallel with nature controlling line
It is coated with plain film, at intervals of 3mm between three detections line and with nature controlling line, is subsequently placed in baking oven, 37 DEG C of drying 2 is small
When.
Sample pad of the present invention is obtained by following steps：Glass fibre membrane is soaked in and contains 1.0%Triton X-
100,2.5%BSA, 0.15M Tris buffer solutions, in the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, are subsequently placed in baking oven
In, 37 DEG C dry 2 hours.
Serum amyloid A protein, Procalcitonin, the C- reactions realized present invention also offers a kind of kit as described above
The three-in-one assay method of albumen, it is characterised in that comprise the following steps：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, kept flat；
Step 2：Card Reader：IC-card is placed on dry type fluorescence immunity analyzer labeling position, relevant information is read, it is described glimmering
Light immunochromatographiassays assays instrument is a kind of Systems for optical inspection, and detection range is respectively：SAA：5.00-200mg/L、PCT:0.1-
40ng/mL、CRP：0.5-200mg/L；
Step 3：Sample-adding：10 μ L serum, plasma sample or 15 μ L whole blood samples are taken to be added in a pipe buffer solution, it is fully mixed
It is even, it is vertical to be added dropwise at 100 μ L mixed liquors to test card sample-adding, it is careful not to suck bubble during sampling；
Step 4：Detection, can be detected, automatic test using automatic test or immediately test both of which：By test card
Insert on the carrier of dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis detection to test card automatically；
Immediately test：After test card room temperature places 15min, insert on the carrier of dry type fluorescence immunity analyzer, click on test immediately.
The present invention provides a kind of using rare earth Eu3+Serum prepared by the fluorescence immune chromatography technology of carboxyl latex microballoon mark
Amyloid A/Procalcitonin/C reactive protein is three-in-one to determine agent box, while it is adapted to serum, blood plasma and whole blood sample, and
It is adapted to clinically single part detection, relative to qualitative colloid gold reagent, serum amyloid A protein that can be in quantitative determination sample,
The content of Procalcitonin and C reactive protein, it is quick, clever with easy to operate, reaction with more specific Clinical significance of MG
Sensitivity is high, high specificity, be adapted to Site Detection and it is economical and practical the advantages of.
Brief description of the drawings：
Accompanying drawing 1 is the structural representation of test card in the present invention.
Accompanying drawing 2 to accompanying drawing 10 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
Specific embodiment：
The present invention is further illustrated with reference to the accompanying drawings and examples：
As shown in Figure 1, present invention firstly provides a kind of serum amyloid A protein/Procalcitonin/C reactive protein three
Unification determines agent box, and test card is provided with box, and the test card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, pad
3rd, nitrocellulose filter 4 and adsorptive pads 5, are wherein adsorbed with the difference antiserum starch of rare-earth fluorescent microballoon mark on pad 3
Three kinds of monoclonal antibodies such as sample albumin A, anti-Procalcitonin and anti-C reactive protein, the rare-earth fluorescent microballoon it is a diameter of
150nm, rare-earth fluorescent microballoon includes rare earth lanthanide Eu3+, it is stable under ground state, issued in the excitation source effect of 337nm
Project the fluorescence of wavelength 615nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, is derived from respectively for 2-
The cell strain of monoclonal antibody of the epitopes such as 6 different serum amyloid A proteins, Procalcitonin, C reactive proteins.
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 150nm；The rare-earth fluorescent microballoon preferably comprises dilute
Native lanthanide series europium (Eu3+)；The antibody of rare-earth fluorescent microballoon mark is preferably derived from for 2 different epitopes on pad
Monoclonal cell cell line.
Embodiment 1：
The three-in-one each part for determining test card in agent box of serum amyloid A protein/Procalcitonin/C reactive protein
Can be obtained by following measures：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15M Tris buffer solutions,
In the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, be subsequently placed in baking oven, 37 DEG C dry 2 hours.2nd, fluorescent microsphere is adsorbed
The preparation of the pad 3 of labelled antibody：
Glass fibre membrane is soaked in (X-100 containing 1.0%Triton, 2.5% in 150mM Tris-HCL treatment fluids
BSA, pH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby.Glass fibre membrane is placed on Bio-
On DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare-earth fluorescent microballoon with Bio-Jet Quanti300 noncontacts
The serum amyloid A protein of mark, three kinds of coupled complexes of Procalcitonin and C reactive protein are sprayed onto glass fibre membrane after mixing
On, 37 DEG C drying 1 hour after be obtained.
The aldehyde radical of rare-earth fluorescent Nano microsphere：5mg rare-earth fluorescent Nano microspheres are taken, with 20mM, the carbonate of pH9.5 delays
Fliud flushing, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbon of 100 μ l
In phthalate buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Rare earth Eu3+Antiserum amyloid A antibody, anti-Procalcitonin antibody and anti-C- reaction eggs that fluorescent microsphere is marked
Three kinds of preparations of coupled complex such as Bai Kangti：Three kinds of antibody-microspheres coupled complexes are individually coupled, and operate as follows：Choosing
Fetch from 2 antiserum amyloid A monoclonal antibodies of the monoclonal cell cell line of different epitopes, according to quality
Than 1:1 by 2mg serum amyloid A proteins monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, then with it is above-mentioned
The rare-earth fluorescent microballoon mixing of aldehyde radical, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C of reactions 4 are small
When；Isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is added, 4 DEG C of closings are overnight；So
The buffer solution of 50mM Tris-HCL, pH7.4 is used to be washed 3 times using centrifugal process afterwards, the 50mM Tris-HCL for being resuspended in 100 μ l delay
In fliud flushing (contain 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.Same operation prepares anti-respectively
Procalcitonin antibody-microspheres coupled complex and anti-C reactive protein antibody-microspheres coupled complex, 4 DEG C keep in dark place it is standby.
3rd, it is coated with the preparation of the nitrocellulose filter 4 of detection line and nature controlling line：
Using with three kinds of monoclonals such as serum amyloid A protein used on pad, Procalcitonin and C reactive protein
The different cell line of antibody cell strain, prepared using the ascites production technology of standard and purified antiserum amyloid A antibody,
Three kinds of mouse resource monoclonal antibodies such as anti-Procalcitonin antibody and anti-C reactive protein antibody, obtain the Dan Ke matched with labelled antibody
Grand antibody, be stored in after packing -20 DEG C it is standby；
Above-mentioned three kinds of mouse resource monoclonal antibodies and goat anti-mouse igg antibody are adjusted into concentration with coating dilution respectively to arrive
1.5mg/ml, film liquid amount is 1.5 μ l/cm, and they are sprayed on into nitrocellulose filter as detection line is parallel with nature controlling line
On be coated with, at intervals of 3mm between three detections line and with nature controlling line, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
The assembling of test card：Paste treated sample pad 2 successively in PVC board 1, be adsorbed with rare-earth fluorescence labeling
The pad 3 of antibody, the nitrocellulose filter 4 and adsorptive pads 5 that are coated with detection line and nature controlling line, obtain test paper big after assembling
Plate, it is wide to cut into 4mm as requested, loads in plastic clip and form test card test paper.
The equipment and raw material selected in above steps preferably following raw material：
The species specificity of serum amyloid A protein, Procalcitonin, C reactive protein etc. three matches somebody with somebody antagonist；Serum amyloid sample egg
The three special quality control product such as white A, Procalcitonin, C reactive protein：Landau laboratory diagnosis Co., Ltd of Britain；Rare-earth fluorescent microballoon：On
Hai Zhenzhun bio tech ltd；Nitrocellulose (NC) film：Millipore Products；Bovine serum albumin(BSA) (BSA),
Polyethylene glycol PEG20000, caseinhydrolysate：Sigma products, other common agents are AR.
Embodiment 2：Accuracy test
From above-mentioned test card and fluorescence immune chromatography analyzer (model：NEO-007), fluorescence immunity analyzer parameter
Setting：After test card technological parameter is set on fluorescence immunity analyzer, take the above-mentioned test card for assembling, respectively with 5,
20th, 40,60,80, the 100, SAA of 200mg/L, 0.1,1,5,10,20,30, the PCT of 40ng/mL, 0.5,5,20,50,100,
150th, the CRP calibration objects of 200mg/L, are measured with test card, obtain the fluorescence intensity level of each calibration object, and result is input to
In the parameter of analyzer, the setting of the parameter of analyzer is completed.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 300 parts of latex enhancing immune turbidimetry definite value samples
Sheet, wherein 100 parts of serum sample, 100 parts of plasma sample, 100 parts of whole blood sample, serum amyloid A protein/Procalcitonin/C-
Reactive protein content distribution interval is respectively：SAA：5.00-200mg/L、PCT:0.1-40ng/mL、CRP：0.5-200mg/L.
Detection method：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, kept flat；
Step 2：Card Reader：IC-card is placed on dry type fluorescence immunity analyzer labeling position, relevant information is read；
Step 3：Sample-adding：10 μ L serum, plasma sample or 15 μ L whole blood samples are taken to be added in a pipe buffer solution, it is fully mixed
It is even, it is vertical to be added dropwise at 100 μ L mixed liquors to test card sample-adding, it is careful not to suck bubble during sampling；
Step 4：Detection, can be detected, automatic test using automatic test or immediately test both of which：By test card
Insert on the carrier of dry type fluorescence immunity analyzer, by feeler switch, instrument will be scanned analysis detection to test card automatically；
Immediately test：After test card room temperature places 15min, insert on the carrier of dry type fluorescence immunity analyzer, click on test immediately.
Test result analysis：After the completion of prepared by clinical sample detection reagent, all clinical samples are carried out by detection method
Detection, and analyze testing result.
Result of the test：
As shown in accompanying drawing 2 to accompanying drawing 10, the detected value with experimental system as Y-axis, the test value with contradistinction system as X-axis,
Scatter diagram is drawn, and carries out correlation analysis.Clinical sample detection is to 300 parts of clinical definite value pattern detections, sample mean deviation
Value is less than 10%, and maximum deviation is less than 20%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection examination for preparing
Agent box is functional, is suitable for clinical detection, meets the differentiation needs of the different detection occasions of different clients.
The present invention provides serum shallow lake prepared by a kind of fluorescence immune chromatography technology by the use of rare earth element as mark substance
Powder sample albumin A/Procalcitonin/C reactive protein is three-in-one to determine agent box, is adapted to serum, blood plasma and whole blood sample, and be adapted to face
Single part detection on bed, relative to qualitative colloid gold reagent, serum amyloid A protein, drop in energy simultaneous quantitative detection sample
Calcium element is former, three kinds of contents of material of C reactive protein, fast with easy to operate, reaction with more specific Clinical significance of MG
Speed, sensitivity high, high specificity, be adapted to Site Detection and it is economical and practical the advantages of.
Claims (7)
1. a kind of serum amyloid A protein/Procalcitonin/C reactive protein is three-in-one determines agent box, is provided with test card, its feature
It is that the test card is sequentially provided with from the bottom to top：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein
Rare earth Eu is adsorbed with pad3+Antiserum amyloid A, anti-Procalcitonin, anti-C- reactions that fluorescent microsphere is marked respectively
Three kinds of monoclonal antibodies of albumen-microballoon coupled complex, a diameter of 100-250nm of the rare-earth fluorescent microballoon, rare-earth fluorescent
Microballoon Eu containing rare earth lanthanide3+, it is stable under ground state, launch the glimmering of wavelength 615nm under the excitation source effect of 337nm
Light；The monoclonal antibody is the monoclonal antibody for mixing after purification, is derived from respectively for 2-6 different serum amyloid
The cell strain of monoclonal antibody of the epitopes such as sample albumin A, Procalcitonin, C reactive protein.
2. a kind of three-in-one measure agent of serum amyloid A protein/Procalcitonin/C reactive protein according to claim 1
Box, it is characterised in that the diameter of the rare-earth fluorescent microballoon of the pad is 120-150nm；The rare-earth fluorescent microballoon doped with
Rare earth lanthanide Eu3+, three kinds of antibody of rare-earth fluorescent microballoon mark are derived from for 2 different epitopes on pad
Monoclonal cell cell line.
3. a kind of three-in-one measure agent of serum amyloid A protein/Procalcitonin/C reactive protein according to claim 2
Box, it is characterised in that rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+；.
4. a kind of three-in-one measure agent of serum amyloid A protein/Procalcitonin/C reactive protein according to claim 1
Box, it is characterised in that the pad is obtained using following steps：Glass fibre membrane is soaked in 150mM Tris-HCL treatment
In liquid (X-100 containing 1.0%Triton, 2.5%BSA, pH7.4), 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 small
When, it is standby, glass fibre membrane is placed on Bio-DotXYZ3050 three-dimensional specking platforms, connect with Bio-Jet Quanti300 are non-
Serum amyloid A protein, three kinds of idols of Procalcitonin and C reactive protein that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon
Connection compound mix after be sprayed onto on glass fibre membrane, 37 DEG C drying 1 hour after be obtained.
5. a kind of three-in-one measure agent of serum amyloid A protein/Procalcitonin/C reactive protein according to claim 4
Box, it is characterised in that the serum amyloid A protein/Procalcitonin of the rare-earth fluorescent microballoon mark on pad/C- reactions
The three-in-one three kinds of monoclonal antibodies determined in agent box of albumen are obtained using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：It is pure with serum amyloid A protein, Procalcitonin, C reactive protein respectively
Product distinguish immune mouse, and the Monoclonal Antibody Cell of specific high-affinity is prepared using the method for preparing monoclonal antibody of standard
Strain, the monoclonal antibody cell line to being obtained carries out pairing screening, is preferably gone out for kit according to pairing result and affinity data
Monoclonal antibody cell line；
Step 2：The preparation of monoclonal antibody：Antiserum amyloid A is prepared and purified using the ascites production technology of standard
Three kinds of mouse resource monoclonal antibodies such as antibody, anti-Procalcitonin antibody and anti-C reactive protein antibody, be stored in after packing -20 DEG C it is standby
With；
Step 3：Rare earth Eu3+The aldehyde radical of fluorescent microsphere：5mg rare-earth fluorescent microballoons are taken, with 20mM, the carbonate buffer of pH9.5
Liquid, is washed 3 times using centrifugal process, and centrifugal speed is 12000rpm, and the time is 5 minutes, is finally resuspended in the above-mentioned carbonic acid of 100 μ l
In salt buffer, the glucan of 500 μ l aldehyde radicals is added, mixed, dark reaction 4 hours, are washed using same centrifugal process at room temperature
In washing and being resuspended to the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：Rare earth Eu3+Antiserum amyloid A antibody, anti-Procalcitonin antibody and anti-C- reactions that fluorescent microsphere is marked
Three kinds of preparations of coupled complex such as protein antibodies：Three kinds of antibody-microspheres coupled complexes are individually coupled, and operate as follows：
Choose from 2 antiserum amyloid A monoclonal antibodies of the monoclonal cell cell line of different epitopes, according to matter
Amount compares 1:1 by 2mg serum amyloid A proteins monoclonal antibody with above-mentioned carbonate buffer solution in 4 DEG C of dialysed overnights, then with it is upper
The rare-earth fluorescent microballoon mixing of aldehyde radical is stated, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM, 4 DEG C of reactions 4 are added
Hour；Isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is added, 4 DEG C of closings are overnight；
Then use the buffer solution of 50mM Tris-HCL, pH7.4 to be washed 3 times using centrifugal process, be resuspended in the 50mM Tris-HCL of 100 μ l
In buffer solution (contain 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.Same operation is prepared respectively
Anti- Procalcitonin antibody-microspheres coupled complex and anti-C reactive protein antibody-microspheres coupled complex, 4 DEG C keep in dark place it is standby
With.
6. a kind of three-in-one measure agent of serum amyloid A protein/Procalcitonin/C reactive protein according to claim 1
Box, it is characterised in that the nitrocellulose filter for being coated with detection line and nature controlling line is obtained by following steps：
Step 1：Using with three kinds of Dan Ke such as serum amyloid A protein used on pad, Procalcitonin and C reactive protein
The different cell line of grand antibody cell strain, is prepared and purified using the ascites production technology of standard antiserum amyloid A and resist
Three kinds of mouse resource monoclonal antibodies such as body, anti-Procalcitonin antibody and anti-C reactive protein antibody, obtain and labelled antibody pairing
Monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Step 2：Above-mentioned three kinds of mouse resource monoclonal antibodies and goat anti-mouse igg antibody are adjusted into concentration with coating dilution respectively to arrive
1.5mg/ml, film liquid amount is 1.5 μ l/cm, and they are sprayed on into nitrocellulose filter as detection line is parallel with nature controlling line
On be coated with, detection line and nature controlling line are subsequently placed in baking oven at intervals of 3mm, 37 DEG C dry 2 hours.
7. a kind of three-in-one measure agent of serum amyloid A protein/Procalcitonin/C reactive protein according to claim 1
Box, it is characterised in that the sample pad is obtained by following steps：Glass fibre membrane is soaked in and contains 1.0%Triton X-
100,2.5%BSA, 0.15M Tris buffer solutions, in the treatment fluid of pH7.5,4 hours are soaked in 4 DEG C, are subsequently placed in baking oven
In, 37 DEG C dry 2 hours.
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| CN109633163A (en) * | 2018-11-28 | 2019-04-16 | 浙江聚康生物工程有限公司 | The two-in-one detection kit of Procalcitonin/c reactive protein |
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