CN106872716A - Serum amyloid A protein and two-in-one measure kit and the preparation method of Procalcitonin - Google Patents
Serum amyloid A protein and two-in-one measure kit and the preparation method of Procalcitonin Download PDFInfo
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- CN106872716A CN106872716A CN201710158011.6A CN201710158011A CN106872716A CN 106872716 A CN106872716 A CN 106872716A CN 201710158011 A CN201710158011 A CN 201710158011A CN 106872716 A CN106872716 A CN 106872716A
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- procalcitonin
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- monoclonal antibody
- antibody
- serum amyloid
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- 102000054727 Serum Amyloid A Human genes 0.000 title claims abstract description 57
- 101710190759 Serum amyloid A protein Proteins 0.000 title claims abstract description 40
- 108010048233 Procalcitonin Proteins 0.000 title claims abstract description 38
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 title claims abstract description 38
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N2400/02—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates involving antibodies to sugar part of glycoproteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
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- G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
- G01N2400/14—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar alpha-D-Glucans, i.e. having alpha 1,n (n=3,4,6) linkages between saccharide units, e.g. pullulan
- G01N2400/16—Starch, amylose, amylopectin
Abstract
The present invention is a kind of serum amyloid A protein and two-in-one measure kit and the preparation method of Procalcitonin, 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, two kinds of monoclonal antibody microballoon coupled complexes of anti-Procalcitonin, 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, derive from respectively for 26 different serum amyloid A proteins, the cell strain of monoclonal antibody of Procalcitonin epitope, 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 the serum amyloid A protein in serum, blood plasma and whole blood sample, two infection inflammation indexs of correlation of Procalcitonin are entered
The serum amyloid A protein and Procalcitonin of row quantitative analysis are two-in-one to determine kit 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.
SAA/PCT 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, the sensitivity serum amyloid A protein and Procalcitonin two high, fast and simple that combined with fluorescent immunochromatographiassays assays instrument is realized
Unification determines kit and preparation method.
The present invention can be reached by following measures：
A kind of serum amyloid A protein and the two-in-one measure kit of Procalcitonin, are provided with test card, it is characterised in that institute
Test card is stated to be sequentially provided with from the bottom to top：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad
On be adsorbed with antiserum amyloid A, two kinds of monoclonal antibody-microballoons of anti-Procalcitonin that rare-earth fluorescent microballoon is marked respectively
Coupled complex, a diameter of 100-250nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon is containing in rare earth lanthanide
Plant or several, the stabilization under ground state, it is 550-650nm's to launch wave-length coverage under the excitation source effect of 300-400nm
Fluorescence；The monoclonal antibody is the monoclonal antibody for mixing after purification, derives from being formed sediment for 2-6 different serum respectively
Powder sample albumin A, the cell strain of monoclonal antibody of Procalcitonin epitope.
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, connect with Bio-Jet Quanti300 are non-
Antiserum amyloid A, two kinds of coupled complexes of anti-Procalcitonin that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon
Be sprayed onto on glass fibre membrane after mixing, 37 DEG C drying 1 hour after be obtained.
The serum amyloid A protein and Procalcitonin two of the rare-earth fluorescent microballoon mark in the present invention on pad are closed
Two kinds of monoclonal antibodies are obtained using following steps in one measure agent box：
Step 1：The acquisition of cell strain of monoclonal antibody：It is immunized respectively with serum amyloid A protein, Procalcitonin respectively small
Mouse, the cell strain of monoclonal antibody of specific high-affinity is prepared using the method for preparing monoclonal antibody of standard, to being obtained
Monoclonal antibody cell line carry out pairing screening, according to pairing result and affinity data preferably go out for kit monoclonal antibody cell
Strain；
Step 2：The preparation of monoclonal antibody：Antiserum amyloid egg is prepared and purified using the ascites production technology of standard
White A antibody, two kinds of mouse resource monoclonal antibodies of anti-Procalcitonin antibody, be stored in after packing -20 DEG C it is 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：The antiserum amyloid A antibody of rare-earth fluorescent microballoon mark, the two kinds of couplings of anti-Procalcitonin antibody
The preparation of compound：Two kinds of antibody-microspheres coupled complexes are individually coupled, and operate as follows：Choose and come from 2 not synantigens
The antiserum amyloid A monoclonal antibody of the monoclonal cell cell line of epitope, according to mass ratio 1:1 forms sediment 2mg serum
The above-mentioned carbonate buffer solution of powder sample albumin A monoclonal antibody in 4 DEG C of dialysed overnights, then with the rare-earth fluorescent of above-mentioned aldehyde radical
Microballoon mixes, and 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C are reacted 4 hours；Add isometric
Confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 50mM Tris-HCL are used,
The buffer solution of pH7.4 is washed 3 times using centrifugal process, (containing 1.2% in being resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l
NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby, same operation prepare respectively anti-Procalcitonin antibody-
Microballoon coupled complex, 4 DEG C keep in dark place it is 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, two kinds of monoclonal antibodies of anti-Procalcitonin
The different cell line of cell line, prepares and purifies antiserum amyloid A antibody, anti-drop using the ascites production technology of standard
Calcium element two kinds of mouse resource monoclonal antibodies of original antibody, obtain the monoclonal antibody matched with labelled antibody, and -20 DEG C are stored in after packing
It is standby；
Step 2：Above two mouse resource monoclonal antibody 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 two detection lines 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；
It is two-in-one present invention also offers a kind of serum amyloid A protein of kit realization as described above, Procalcitonin
Assay method, 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；
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 is two-in-one to determine agent box, while being adapted to serum, blood plasma and whole blood sample, and is adapted to clinically single
Person-portion is detected, relative to qualitative colloid gold reagent, serum amyloid A protein that can be in quantitative determination sample, Procalcitonin contain
Amount, with more specific Clinical significance of MG, with easy to operate, reaction is quick, sensitivity high, high specificity, is adapted to scene
The advantages of detecting and be economical and practical.
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 7 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 serum amyloid A protein and the two-in-one measure reagent of Procalcitonin
Box, is provided with test card in box, the test card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, pad 3, cellulose nitrate
The difference antiserum amyloid A of rare-earth fluorescent microballoon mark is adsorbed with plain film 4 and adsorptive pads 5, wherein pad 3, is resisted
Two kinds of monoclonal antibodies of Procalcitonin, a diameter of 150nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon includes rare earth group of the lanthanides
Element Eu3+, 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, is derived from respectively for 2-6 different serum amyloid A protein, drop calcium
The cell strain of monoclonal antibody of plain Proantigen epitope.
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：
Serum amyloid A protein/Procalcitonin it is two-in-one determine agent box in test card each part can by with
Lower measure is obtained：
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, two kinds of coupled complexes of Procalcitonin are sprayed onto on glass fibre membrane after mixing, 37 DEG C of drying 1
It is obtained after hour.
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+The antiserum amyloid A antibody of fluorescent microsphere mark, the two kinds of couplings of anti-Procalcitonin antibody are compound
The preparation of thing：Two kinds of antibody-microspheres coupled complexes are individually coupled, and operate as follows：Choose from 2 different epitopes
Monoclonal cell cell line antiserum amyloid A monoclonal antibody, according to mass ratio 1:1 by 2mg serum amyloid samples
The above-mentioned carbonate buffer solution of albumin A monoclonal antibody in 4 DEG C of dialysed overnights, then with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical
Mixing, 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C are reacted 4 hours；Add isometric closing
Liquid (50mM Tris-HCL, pH7.4, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 50mM Tris-HCL are used,
The buffer solution of pH7.4 is washed 3 times using centrifugal process, (containing 1.2% in being resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l
NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby；Same operation prepare respectively anti-Procalcitonin antibody-
Microballoon 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 serum amyloid A protein used on pad, two kinds of cell strain of monoclonal antibody differences of Procalcitonin
Cell line, antiserum amyloid A antibody, anti-Procalcitonin antibody are prepared and purified using the ascites production technology of standard
Two kinds of mouse resource monoclonal antibodies, obtain with labelled antibody match monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Above two mouse resource monoclonal antibody 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 two detection lines 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：
Serum amyloid A protein, the species specificity of Procalcitonin two match somebody with somebody antagonist；Serum amyloid A protein, Procalcitonin two
Special quality control product：Landau laboratory diagnosis Co., Ltd of Britain；Rare-earth fluorescent microballoon：Shanghai Zhen Zhun bio tech ltd；Nitric acid
Cellulose (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 calibration objects of 40ng/mL, carried out with test card
Determine, obtain the fluorescence intensity level of each calibration object, result is input in the parameter of analyzer, the parameter for completing analyzer sets
It is fixed.
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 content
Distributed area is respectively：SAA：5.00-200mg/L、PCT:0.1-40ng/mL.
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 7, the detected value with experimental system as Y-axis, as X-axis paint by the test value with contradistinction system
Scatter diagram processed, and carry out correlation analysis.Clinical sample detection is to 300 parts of clinical definite value pattern detections, sample mean deviation
Less than 10%, maximum deviation is less than 20%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection reagent for preparing
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 is two-in-one to determine agent box, is adapted to serum, blood plasma and whole blood sample, and be adapted to clinically single part inspection
Survey, relative to qualitative colloid gold reagent, serum amyloid A protein, two kinds of things of Procalcitonin in energy simultaneous quantitative detection sample
The content of matter, with more specific Clinical significance of MG, with easy to operate, reaction is quick, sensitivity high, high specificity, suitable
Close Site Detection and it is economical and practical the advantages of.
Claims (7)
1. a kind of serum amyloid A protein and the two-in-one measure kit of Procalcitonin, are provided with test card, it is characterised in that described
Test card is sequentially provided with from the bottom to top：On PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad
It is adsorbed with rare earth Eu3+Antiserum amyloid A, two kinds of monoclonal antibodies of anti-Procalcitonin-micro- that fluorescent microsphere is marked respectively
Ball coupled complex, a diameter of 100-250nm of the rare-earth fluorescent microballoon, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+,
The stabilization under ground state, launches the fluorescence of wavelength 615nm under the excitation source effect of 337nm；The monoclonal antibody is pure
The monoclonal antibody mixed after change, derives from for 2-6 different serum amyloid A protein, Procalcitonin antigen respectively
The cell strain of monoclonal antibody of epitope.
2. a kind of serum amyloid A protein according to claim 1 and the two-in-one measure kit of Procalcitonin, its feature
The diameter for being the rare-earth fluorescent microballoon of the pad is 120-150nm；The rare-earth fluorescent microballoon is doped with rare earth group of the lanthanides
Element Eu3+, two kinds of antibody of rare-earth fluorescent microballoon mark are derived from for 2 monoclonals of different epitopes on pad
Cell strain.
3. a kind of serum amyloid A protein according to claim 1 and the two-in-one measure kit of Procalcitonin, its feature
It is rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+；.
4. a kind of serum amyloid A protein according to claim 1 and the two-in-one measure kit of Procalcitonin, its feature
It is that the pad is obtained using following steps：Glass fibre membrane is soaked in 150mMTris-HCL treatment fluids and (is contained
1.0%Triton X-100,2.5%BSA, pH7.4), 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 hours, standby
With, glass fibre membrane is placed on Bio-DotXYZ3050 three-dimensional specking platforms, declined with Bio-Jet Quanti300 noncontacts
It is sprayed onto after serum amyloid A protein, two kinds of coupled complexes mixings of Procalcitonin that quantitation nozzle marks rare-earth fluorescent microballoon
On glass fibre membrane, 37 DEG C drying 1 hour after be obtained.
5. a kind of serum amyloid A protein according to claim 1 and the two-in-one measure kit of Procalcitonin, its feature
Be on pad the rare-earth fluorescent microballoon mark serum amyloid A protein/Procalcitonin it is two-in-one determine agent box in
Two kinds of monoclonal antibodies using following steps be obtained：
Step 1：The acquisition of cell strain of monoclonal antibody：It is immunized respectively with serum amyloid A protein, Procalcitonin sterling respectively small
Mouse, the cell strain of monoclonal antibody of specific high-affinity is prepared using the method for preparing monoclonal antibody of standard, to being obtained
Monoclonal antibody cell line carry out pairing screening, according to pairing result and affinity data preferably go out for kit monoclonal antibody cell
Strain；
Step 2：The preparation of monoclonal antibody：Antiserum amyloid A is prepared and purified using the ascites production technology of standard
Antibody, two kinds of mouse resource monoclonal antibodies of anti-Procalcitonin antibody, be stored in after packing -20 DEG C it is standby；
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+The antiserum amyloid A antibody of fluorescent microsphere mark, the two kinds of couplings of anti-Procalcitonin antibody are multiple
The preparation of compound：Two kinds of antibody-microspheres coupled complexes are individually coupled, and operate as follows：Choose and come from 2 not synantigen tables
The antiserum amyloid A monoclonal antibody of the monoclonal cell cell line of position, according to mass ratio 1:1 by 2mg serum amyloids
, in 4 DEG C of dialysed overnights, then the rare-earth fluorescent with above-mentioned aldehyde radical is micro- for the above-mentioned carbonate buffer solution of sample albumin A monoclonal antibody
Ball mixes, and 4 DEG C of reactions are overnight；Then, sodium borohydride to final concentration 5mM is added, 4 DEG C are reacted 4 hours；Add isometric envelope
Liquid (50mMTris-HCL, pH7.4, containing 2%BSA, 5% sucrose) is closed, 4 DEG C of closings are overnight；Then 50mM Tris-HCL are used,
The buffer solution of pH7.4 is washed 3 times using centrifugal process, (containing 1.2% in being resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l
NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C keep in dark place it is standby.Same operation prepare respectively anti-Procalcitonin antibody-
Microballoon coupled complex, 4 DEG C keep in dark place it is standby.
6. a kind of serum amyloid A protein according to claim 1 and the two-in-one measure kit of Procalcitonin, its feature
The nitrocellulose filter that detection line and nature controlling line are coated with described in being is obtained by following steps：
Step 1：Using with serum amyloid A protein used on pad, two kinds of cell strain of monoclonal antibody of Procalcitonin not
Same cell line, is prepared and purified using the ascites production technology of standard antiserum amyloid A antibody, anti-Procalcitonin and resist
Two kinds of mouse resource monoclonal antibodies of body, obtain with labelled antibody match monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Step 2：Above two mouse resource monoclonal antibody 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 serum amyloid A protein according to claim 1 and the two-in-one measure kit of Procalcitonin, its feature
It is 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, 37 DEG C of drying
2 hours.
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| CN107167597A (en) * | 2017-07-18 | 2017-09-15 | 深圳市惠安生物科技有限公司 | Quantitatively detection SAA, CRP, PCT immunofluorescence chromatographs kit and preparation method thereof |
| CN109085358A (en) * | 2018-07-02 | 2018-12-25 | 威海纽普生物技术有限公司 | Serum amyloid A protein assay kit and production method |
| CN109541235A (en) * | 2018-08-19 | 2019-03-29 | 重庆早柒天生物科技股份有限公司 | Heparin-binding protein and Procalcitonin two joint detection reagent box and preparation method thereof |
| 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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| CN104714033A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | Procalcitonin detection kit and detection method |
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| CN109541235A (en) * | 2018-08-19 | 2019-03-29 | 重庆早柒天生物科技股份有限公司 | Heparin-binding protein and Procalcitonin two joint detection reagent box and preparation method thereof |
| 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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Application publication date: 20170620 |