CN106706931A - Progesterone measurement kit and preparation method - Google Patents
Progesterone measurement kit and preparation method Download PDFInfo
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- CN106706931A CN106706931A CN201611161994.0A CN201611161994A CN106706931A CN 106706931 A CN106706931 A CN 106706931A CN 201611161994 A CN201611161994 A CN 201611161994A CN 106706931 A CN106706931 A CN 106706931A
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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- G01—MEASURING; TESTING
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- 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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Abstract
The invention relates to the technical field of fluorescence immunochromatography in medical immunology and particularly relates to a progesterone measurement kit and a preparation method. The progesterone measurement kit is provided with a test paper card and is characterized in that the test paper card is provided with a PVC plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad sequentially from bottom to top, wherein a progesterone monoclonal antibody marked by rare earth Eu<3+> fluorescent microspheres is adsorbed on the combination pad; the diameter of the rare earth fluorescent microspheres is 150nm; the rare earth fluorescent microspheres contain rare earth lanthanide Eu<3+>, are stable in a ground state and emit fluorescence with the wavelength of 615nm under the effect of a 337nm excitation light source; the monoclonal antibody is purified and then mixed, comes from monoclonal antibody cell lines for 2-6 different progesterone epitopes; and the kit has the advantages of easiness in operation, quick reaction, high sensitivity, strong specificity and the like.
Description
Technical field：
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, including protein-crosslinking technology, film layer analysis skill
Art, labelling immunoassay technology etc..Specifically one kind can fast and accurately to pregnant in the samples such as serum, blood plasma and whole blood
The progesterone that ketone carries out quantitative analysis determines kit and preparation method.
Background technology：
Progesterone (Progesterone, PROG) is a kind of steroid hormones for containing 21 carbon, is steroid hormone building-up process
In mesostate.It is transformed by isomerase through pregnenolone by cholesterol.Ovum in unrecognizing factor, ovary
Thecacells produces progesterone, and gestation, progesterone is then mainly secreted by placenta syneytiotrophoblast layer, therefore progesterone level master in blood
It is related to placental weight and hemoperfusion amount.Pregnant early stage rises relatively slowly, is speeded after 13 weeks, and peak is reached when mature.It is pregnant in blood
Ketone is mainly combined with cortico steroid-binding globulin (CBG) and albumin, and most progesterone are mating type in blood, seldom
Number is sequestered.Progesterone is main in liver degraded, pregnanediol is reduced into, after pregnanediol is combined with sulfuric acid or glucuronic acid again
Discharged through kidney.Progesterone is to the regulation menstrual cycle and maintains gestation with important physiological action, can promote the endometrial secretion phase
Conversion, for fertilization egg implantation prepare；Lax uterus muscle, reduces to prostaglandin and the reactivity of oxytocins；Suppress defeated ovum
Pipe Rythmic contractions characteristic.Progesterone also has rush mammary gland alveolus hyperplasia, increases energetic supersession, raises basal body temperature, promotes what water sodium was excluded
Effect.Progesterone there is positive-negative feedback to act on hypothalamus-pituitary system, the synthesis and secretion of controllable pituitary gonadotropic hormone.
Preovulatory low dose of progesterone collaboration E3 induces preovulatory LH peaks to be occurred, and heavy dose of progesterone is to hypothalamus-pituitary system after ovulation
Negative feedback is then presented.
Women progesterone in blood is nearly all as produced by corpus luteum or placenta；Male's progesterone level is very low, mainly by kidney
Upper gland cortex is produced.Progesterone is an important hormone, and it not only plays a significant role in the regulation of menstrual cycle, is being maintained
It is also essential in gestation.Progesterone grows body of gland in Uterine mucosa in later stage menstrual cycle, and metryperemia, inner membrance increases
Thickness, for the fertilization implantation of ovum is got ready；Then it is allowed to produce placenta after the fertilization implantation of ovum, and reduces the excitability of gravid uterus, suppression
Its activity is made, fetal well-being is grown.Progesterone in the serum concentration after ovulation is raised rapidly, because of the miscarriage that may occur and different
The means of normal gestation.Progesterone is determined for determining ovulation, progestogen therapy monitoring and the evaluation of early pregnancy situation, is judging yellow
Body function state and aspect have especially important meaning, are research ovarian physiology and the indispensable means of pathologic, physiologic.
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 that combined with fluorescent immunochromatographiassays assays instrument is realized is high, fast and simple, can determine kit with the progesterone of accurate quantitative analysis
And preparation method.
The present invention can be reached by following measures：
A kind of progesterone determines kit, 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+Fluorescent microsphere mark
Antiprogestin monoclonal antibody-microballoon the coupled complex of note, a diameter of 100-250nm of the rare-earth fluorescent microballoon, rare earth is glimmering
Containing one or more in rare earth lanthanide, the stabilization under ground state is issued light microballoon in the excitation source effect of 300-400nm
It is the fluorescence of 550-650nm to project wave-length coverage；The monoclonal antibody is the monoclonal antibody for mixing after purification, from pin
The cell strain of monoclonal antibody of the progesterone epitope different to 2-6.
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-
The antiprogestin monoclonal antibody coupled complex that the micro- quantitation nozzle of touch marks rare-earth fluorescent microballoon is sprayed onto glass fibre membrane, 37
DEG C drying 1 hour after be obtained.
The antiprogestin monoclonal antibody of the rare-earth fluorescent microballoon mark in the present invention on pad uses following steps
It is obtained：
Step 1：The acquisition of cell strain of monoclonal antibody：With progesterone sterling immune mouse, using the monoclonal antibody of standard
Preparation method prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line to being obtained carries out pairing sieve
Choosing, the monoclonal antibody cell line for kit is preferably gone out according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Antiprogestin monoclonal is prepared and purified using the ascites production technology of standard to resist
Body, 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 preparation of the antiprogestin monoclonal antibody of rare-earth fluorescent microballoon mark：Choose and come from 2 not synantigen tables
The monoclonal antibody of the monoclonal cell cell line of position, according to mass ratio 1:1 by the above-mentioned carbonate of 2mg progesterone monoclonal antibodies
Then buffer solution mixes in 4 DEG C of dialysed overnights with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, and 4 DEG C of reactions are overnight；Then, add
To final concentration 5mM, 4 DEG C are reacted 4 hours sodium borohydride；(50mM Tris-HCL, pH7.4, contain to add isometric confining liquid
2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then the buffer solution of 50mM Tris-HCL, pH7.4 is used using centrifugal process washing 3
Time, it is resuspended in (containing 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C in the 50mM Tris-HCL buffer solutions of 100 μ l
Keep in dark place 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 the cell line different from antiprogestin cell strain of monoclonal antibody used on pad, using standard
Ascites production technology is prepared and purifies antiprogestin monoclonal antibody, be stored in -20 DEG C it is standby；
Step 2：Above-mentioned mouse source antiprogestin monoclonal antibody and goat anti-mouse igg antibody are adjusted with coating dilution respectively
To 1-3mg/ml, film liquid amount is 1-3 μ l/cm to concentration, and they are sprayed on into nitric acid fibre as detection line is parallel with nature controlling line
It is coated with the plain film of dimension, detection line and nature controlling line are subsequently placed in baking oven at intervals of 3-7mm, 37 DEG C dry 2 hours.
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.
Present invention also offers the progesterone preparation method that a kind of kit as described above is realized, it is characterised in that including following
Step：
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 the measurement range to progesterone is 0.30-50ng/mL；
Step 3：Sample-adding：Serum/plasma：100 μ L serum/plasmas samples are taken vertically to drop at test card sample-adding；Whole blood：
150 μ L whole blood samples are taken vertically to drop at 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 10min, insert on the carrier of dry type fluorescence immunity analyzer, click on test immediately.
The present invention provides progesterone survey prepared by a kind of fluorescence immune chromatography technology of utilization rare earth carboxyl latex microballoon mark
Determine kit, while being adapted to serum, blood plasma and whole blood sample, and be adapted to clinically single part detection, relative to the qualitative glue of progesterone
Body gold reagent, the progesterone content in energy quantitative determination sample, with more specific Clinical significance of MG, with easy to operate, anti-
The advantages of answering quick, sensitivity high, high specificity, be adapted to Site Detection 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 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 3 is the accuracy analysis result schematic diagram of embodiment 2 in the present invention.
Accompanying drawing 4 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, kit is determined present invention firstly provides a kind of progesterone, test card, the examination is provided with box
Paper card is sequentially provided with from the bottom to top：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads 5, wherein pad
On be adsorbed with rare earth Eu3+Antiprogestin monoclonal antibody-microballoon the coupled complex of fluorescent microsphere mark, the rare-earth fluorescent microballoon
A diameter of 150nm, rare-earth fluorescent microballoon Eu containing rare earth lanthanide3+, the stabilization under ground state, in the excitation source work of 337nm
Launch the fluorescence of wavelength 615nm under；The monoclonal antibody is the monoclonal antibody for mixing after purification, from for 2-
6 cell strain of monoclonal antibody of different progesterone epitopes.
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：
Each part that progesterone determines test card in kit 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, and glass fibre membrane is placed on into Bio-
On DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare earth Eu with Bio-Jet Quanti300 noncontacts3+Fluorescence is micro-
The antiprogestin monoclonal antibody coupled complex of ball mark is sprayed onto glass fibre membrane, and 37 DEG C of drying are obtained after 1 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 preparation of the antiprogestin monoclonal antibody of fluorescent microsphere mark：Choose from 2 different epitopes
The monoclonal antibody of monoclonal cell cell line, according to mass ratio 1:1 delays 2mg antiprogestins monoclonal antibody with above-mentioned carbonate
Then fliud flushing mixes in 4 DEG C of dialysed overnights with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical, and 4 DEG C of reactions are overnight；Then, boron is added
To final concentration 5mM, 4 DEG C are reacted 4 hours sodium hydride；Add isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%
BSA, 5% sucrose), 4 DEG C of closings are overnight；Then the buffer solution of 50mM Tris-HCL, pH7.4 is used to be washed 3 times using centrifugal process,
It is resuspended in the 50mM Tris-HCL buffer solutions of 100 μ l (containing 1.2%NaCL, 0.5%BSA, 0.1%Tween 20), 4 DEG C are kept away
Light is saved backup.
3rd, it is coated with the preparation of the nitrocellulose filter 4 of detection line and nature controlling line：
Using the cell line different from antiprogestin cell strain of monoclonal antibody used on pad, given birth to using the ascites of standard
Production. art is prepared and purifies antiprogestin monoclonal antibody, be stored in -20 DEG C it is standby；
Above-mentioned mouse source antiprogestin monoclonal antibody and goat anti-mouse igg antibody are adjusted into concentration with coating dilution respectively to arrive
1mg/ml, film liquid amount is 1 μ l/cm, and using them, as detection line, parallel with nature controlling line to be sprayed on nitrocellulose filter enterprising
Row coating, detection line and nature controlling line are subsequently placed in baking oven at intervals of 4mm, and 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：
Progesterone specific pairs antibody；Progesterone quality-control product：Landau laboratory diagnosis Co., Ltd of Britain；Rare-earth fluorescent microballoon：
Shanghai Zhen Zhun 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),
The setting of fluorescence immunity analyzer parameter：After test card technological parameter is set on fluorescence immunity analyzer, take
The above-mentioned test card for assembling, respectively with 0.3,3,6,10,20,40, the progesterone calibration object of 50ng/mL, surveyed with test card
It is fixed, the fluorescence intensity level of each calibration object is obtained, 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, progesterone content distributed area is 0.3-50ng/
Between 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：Serum/plasma：Take 100 μ L serum/plasmas samples vertically to drop at test card sample-adding, whole blood：
Take 150 μ L whole blood samples vertically to drop at test card sample-adding, be 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 10min, 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 detected by detection method, and analyze inspection
Survey result.
Result of the test：
As shown in Figure 2, as Y-axis, the test value with contradistinction system draws scatterplot to the detected value with experimental system as X-axis
Figure, and carry out correlation analysis.Clinical sample detection is less than to 300 parts of clinical definite value pattern detections, sample mean deviation
10%, maximum deviation is less than 20%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection kit for preparing
Can be good, it is suitable for clinical detection, meet the differentiation needs of the different detection occasions of different clients.
The present invention provides progesterone fast quantification immunochromatography detection prepared by a kind of utilization rare-earth fluorescent immunochromatography technique
Kit, while being adapted to serum/plasma and whole blood sample, and is adapted to clinically single part detection, relative to the qualitative colloid of progesterone
Gold reagent, the progesterone content in energy quantitative determination sample, with more specific Clinical significance of MG, with easy to operate, reaction
Quickly, sensitivity is high, high specificity, be adapted to Site Detection and it is economical and practical the advantages of.
Claims (6)
1. a kind of progesterone determines kit, is provided with test card, it is characterised in that the test card is sequentially provided with from the bottom to top：PVC
Rare earth Eu is adsorbed with plate, sample pad, pad, nitrocellulose filter and adsorptive pads, wherein pad3+Fluorescent microsphere mark
Antiprogestin monoclonal antibody-microballoon coupled complex, a diameter of 100-250nm of the rare-earth fluorescent microballoon, rare-earth fluorescent is micro-
Ball containing one or more in rare earth lanthanide, under the excitation source effect of 300-400nm launch by the stabilization under ground state
Wave-length coverage is the fluorescence of 550-650nm；The monoclonal antibody is the monoclonal antibody for mixing after purification, from for 2-
6 cell strain of monoclonal antibody of different progesterone epitopes.
2. a kind of progesterone according to claim 1 determines kit, it is characterised in that the rare-earth fluorescent of the pad is micro-
The diameter of ball is 120-150nm；The antibody sources of rare-earth fluorescent microballoon mark are in for 2 different epitopes on pad
Monoclonal cell cell line.
3. a kind of progesterone according to claim 1 determines kit, it is characterised in that the pad uses following steps
It is obtained：During glass fibre membrane is soaked in into 150mM Tris-HCL treatment fluids (containing 1.0%TritonX-100,2.5%BSA,
PH7.4), 4 DEG C are soaked 2 hours, then take out 37 DEG C of oven for drying 4 hours, standby, and glass fibre membrane is placed on into Bio-
On DotXYZ3050 three-dimensional specking platforms, quantitation nozzle is declined by rare earth Eu with Bio-Jet Quanti300 noncontacts3+Fluorescence is micro-
The antiprogestin monoclonal antibody coupled complex of ball mark is sprayed onto glass fibre membrane, and 37 DEG C of drying are obtained after 1 hour.
4. a kind of progesterone according to claim 1 determines kit, it is characterised in that the rare-earth fluorescent on pad
The progesterone monoclonal antibody of microballoon mark is obtained using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：With progesterone sterling immune mouse, prepared using the monoclonal antibody of standard
Method prepares the cell strain of monoclonal antibody of specific high-affinity, and the monoclonal antibody cell line to being obtained carries out pairing screening, root
Preferably go out the monoclonal antibody cell line for kit according to pairing result and affinity data；
Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify progesterone monoclonal antibody, point
Be stored in after dress -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 buffer solution of pH9.5,
Washed 3 times using centrifugal process, centrifugal speed is 12000rpm, the time is 5 minutes, is finally resuspended in the above-mentioned carbonate of 100 μ l
In buffer solution, the glucan of 500 μ l aldehyde radicals is added, mixed, at room temperature dark reaction 4 hours, washed using same centrifugal process
Be resuspended in the above-mentioned carbonate buffer solution of 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The preparation of the progesterone monoclonal antibody of rare-earth fluorescent microballoon mark：Choose from 2 lists of different epitopes
The monoclonal antibody of clone cell cell line, according to mass ratio 1:1 by the above-mentioned carbonate buffer solution of 2mg progesterone monoclonal antibodies
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, hydroboration is added
To final concentration 5mM, 4 DEG C are reacted 4 hours sodium；Add isometric confining liquid (50mM Tris-HCL, pH7.4, containing 2%BSA,
5% sucrose), 4 DEG C of closings are overnight；Then the buffer solution of 50mM Tris-HCL, pH7.4 is used to be washed 3 times using centrifugal process, it is resuspended
(contain 1.2%NaCL, 0.5%BSA, 0.1%Tween 20) in the 50mM Tris-HCL buffer solutions of 100 μ l, 4 DEG C of lucifuges are protected
Deposit standby.
5. a kind of progesterone (PROG) according to claim 1 determines kit (fluorescence immune chromatography method), 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 the cell line different from progesterone monoclonal antibody cell line used on pad, given birth to using the ascites of standard
Production. art is prepared and purifies progesterone monoclonal antibody, be stored in -20 DEG C it is standby；
Step 2：Above-mentioned mouse source antiprogestin monoclonal antibody and goat anti-mouse igg antibody are adjusted into concentration with coating dilution respectively
To 1mg/ml, film liquid amount is 1 μ l/cm, and they are sprayed on nitrocellulose filter as detection line is parallel with nature controlling line
It is coated with, detection line and nature controlling line are subsequently placed in baking oven at intervals of 4mm, 37 DEG C dry 2 hours.
6. a kind of progesterone according to claim 1 determines kit, it is characterised in that the sample pad passes through following steps
It is obtained：Glass fibre membrane is soaked in containing 1.0%Triton X-100,2.5%BSA, 0.15MTris buffer solution, pH7.5's
In treatment fluid, 4 hours are soaked in 4 DEG C, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
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|---|---|---|---|
| CN201611161994.0A CN106706931A (en) | 2016-12-15 | 2016-12-15 | Progesterone measurement kit and preparation method |
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| CN201611161994.0A CN106706931A (en) | 2016-12-15 | 2016-12-15 | Progesterone measurement kit and preparation method |
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| CN201611161994.0A Pending CN106706931A (en) | 2016-12-15 | 2016-12-15 | Progesterone measurement kit and preparation method |
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| CN109813889A (en) * | 2017-11-18 | 2019-05-28 | 镇江亿特生物科技发展有限公司 | The time-resolved fluoroimmunoassay for detecting progesterone chromatographs quantitative testing test paper item |
| CN111141917A (en) * | 2019-12-19 | 2020-05-12 | 云奥生物科技(常州)有限公司 | Combined chromatography detection card of progesterone and β -hCG, preparation method and detection method thereof |
| CN115975028A (en) * | 2022-11-02 | 2023-04-18 | 厦门康基生物科技有限公司 | Progesterone antibody, preparation method and application thereof |
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Application publication date: 20170524 |