CN107703315A - Folic acid detection kit and preparation method thereof - Google Patents
Folic acid detection kit and preparation method thereof Download PDFInfo
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- CN107703315A CN107703315A CN201710852510.5A CN201710852510A CN107703315A CN 107703315 A CN107703315 A CN 107703315A CN 201710852510 A CN201710852510 A CN 201710852510A CN 107703315 A CN107703315 A CN 107703315A
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- G—PHYSICS
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- 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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
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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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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- 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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Abstract
The present invention relates to fluorescence immune chromatography technical field in Medical Immunology, specifically a kind of folic acid detection kit and preparation method, provided with test card, it is characterised in that the test card is provided with by lower from being above sequentially provided with：PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, the folic acid monoclonal antibody of rare-earth fluorescent microballoon mark is wherein adsorbed with pad, a diameter of 60 120nm of the rare-earth fluorescent microballoon, the rare earth doped lanthanide series of rare-earth fluorescent microballoon, it is stable under ground state, launch fluorescence of the wave-length coverage in 540 600nm under 340 380nm excitation source effect；The monoclonal antibody is the monoclonal antibody mixed after purification, from the cell strain of monoclonal antibody for 26 different folic acid epitopes, has the advantages that easy to operate, rapid reaction, high sensitivity, 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
Folic acid detection kit and preparation method thereof that quantitative analysis is carried out to folic acid.
Background technology：
Folic acid is by petrin pyridine, the compound of the composition such as p-aminobenzoic acid and glutamic acid.It is present in the folic acid of nature
There are two kinds of forms of dihydrofoilic acid and tetrahydrofolic acid.And only have tetrahydrofolic acid that just there is physiological function in human body.Tetrahydrofolic acid is
The carrier of one carbon unit, and one carbon unit refers to the group containing a carbon atom during human metabolism, such as
Methyl, methylol etc..Exactly these active one carbon unit tetrahydrofolic acids, take part in the generation and metabolism of many compounds.Leaf
Acid is most active group in various enzymes, and it almost take part in the lived biochemical metabolism process of institute：1. the division life of pair cell
Long and nucleic acid, the synthesis of amino acid, protein play an important role；2. pair huge a red gemmule property anaemia and sprue have treatment
Effect；3. antitumor action；4. the nerve cell of couple infant has facilitation with brain cell development；5. other are acted on, can conduct
The auxiliary therapeutical agent of schizophrenic patients, available for atrophic gastritis, suppress the conversion of branch organ squamous and preventing and treating because of height
Coronary atherosclerosis, myocardial damage, miocardial infarction etc. caused by plasma homocysteine.
The harm of folic acid deficiency：Cause megaloblastic anemia mass formed by blood stasis；Cutaneous lesions：Psoriasis, sore rash shape dermatitis, brandy nose,
Eczema, exfoliative dermatitis etc.；The obstacle of obstetrics：Folic acid deficiency is the Etiological that NTD occurs in pregnant woman's body.In addition,
It can also result in the birth prevalence increase of the organs such as eye, lip, palate, intestines and stomach, sustainer, kidney, skeleton.It is it is moreover found that pregnant
Woman lacks folic acid and placental abruption, spontaneous abortion, pre-eclampsia, development of fetus is bad and low birth weight waits until that disease has
Close；DPN and phrenoblabia：The shortage of folic acid can show mental disease syndrome, and people has phenomena such as depressed.
The excessive harm of folic acid supplement：Intake folic acid, which crosses multipotency, causes senile dementia occur：It is nearest U.S. Ruo Shi universities
What Marta doctor Mao Lisi of medical center was had found by studying, this conclusion allows Europe about whether being added in food
The discussion of folic acid further heats up；The probability large dose oral administration folic acid that pregnant woman's excessive use folic acid increase suffers from breast cancer can reduce blood
The content of vitamin B12 in clear；A large amount of folic acid may cause human gene to change：Newcastle from Australian Olympic bar
Researcher mark of university strangles Cork and the assistant common vetch thatches of Leeds, England university exist《Science of heredity is commented on naturally》On write articles
Point out, the gene composition of population may be changed at leisure by strengthening the way of folic acid in food, so as to cause offspring couple
Weaken in the resistance of some fatal diseases；Folic acid supplement excessively makes one hypomnesia during age.
Immuno analytical method is using the immune response between trace antigen and corresponding antibody with high specificity, to detect such as
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 utilizes
The carboxyl latex microballoon labelling immunoassay technology containing rare earth element be to belong 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
It is big to endanger and made troubles to experiment.EIA enzyme immunoassay (EIA) is larger by other influences factor also because enzyme itself is unstable, pushes away
Wide application is restricted.At the beginning of the eighties, people begin one's study replaces fluorescent material and isotope-labelled protein with rare earth element
Or antibody, TIME RESOLVED TECHNIQUE is incorporated into field of biological detection, establishes new ultramicron time-resolved fluoroimmunoassay point
Analysis technology (Timeresolved 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, obtain significant progress
And extensive use.
TrFIA make use of trivalent rare earth ion and chelate with unique fluorescent characteristic for 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 reaction product in fluorescence intensity.According to product fluorescence intensity and relatively glimmering
The ratio of luminous intensity, the concentration of analyte in reaction system is judged, so as to reach quantitative analysis.In common fluoremetry,
Due to containing a variety of fluorescent components in test sample, background fluorescence is (caused by the colloidal solid and solvent molecule in sample
The non-specific fluorescence that scattering light and Proteins in Serum and other compounds are sent) intensity is big, it is strong to disturb, turn into 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 EIA, RIA latter,
Depend primarily in the unique fluorescence feature of lanthanide series, detection the wavelength resolution used and time-delay technique and dissociation-
Enhancing technology.
Lanthanide series (lanthanide, Ln) belongs to rare earth element, in sharing 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 grown, and 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, delay one
Measured after fixing time, Eu can be obtained3+Specific fluorescence signal.Simultaneously because decay time is grown, 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 has 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 exciting light and the Stokes displacements launched between light are larger, Eu3+Excitation wavelength is 337nm, transmitting
Wavelength is 615nm, and Stokes displacements are up to 278nm；Eu simultaneously3+The fluorescence light belt being excited is extremely narrow, and the emission peak of fluorescence is very
Sharply, instrument adjustment can be made to be determined in extremely narrow wave-length coverage, thus almost completely eliminate the interference of background fluorescence, after
And passage time delay and wavelength resolution, 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
Sensitivity, the simplicity of operation and stable fluorescent marker 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 to utilize the sensitive of fluorescence immune chromatography
Property, the high sensitivity, fast and simple realized with reference to fluorescence immune chromatography analyzer can be with the folic acid detection kit of accurate quantitative analysis
And preparation method.
The present invention can be reached by following measures：
A kind of folic acid detection kit, provided with test card, it is characterised in that the test card is sequentially provided with from the bottom to top：
PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, rare-earth fluorescent microballoon mark is wherein adsorbed with pad
Folic acid monoclonal antibody, a diameter of 60-120nm of the rare-earth fluorescent microballoon, the rare earth doped lanthanide series of rare-earth fluorescent microballoon,
It is stable under ground state, launch fluorescence of the wave-length coverage in 540-600nm under 340-380nm excitation source effect；It is described
Monoclonal antibody is the monoclonal antibody mixed after purification, from the monoclonal for 2-6 different folic acid epitopes
Antibody cell strain.
The diameter of the rare-earth fluorescent microballoon of pad of the present invention is preferably 90-110nm；The rare-earth fluorescent microballoon is excellent
Choosing is doped with rare earth lanthanide, for any one or a few of the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd)
Mixture；The preferably rare earth doped complex compound of rare-earth fluorescent microballoon；The antibody that rare-earth fluorescent microballoon marks on pad is excellent
Choosing derives from the monoclonal cell cell line for 3 different epitopes.
Pad of the present invention is made using following steps：Glass fibre membrane is soaked in 200mMTris-HCL processing
In liquid (X-100 containing 1.5%Triton, 1.5%BSA, pH7.5), 4 DEG C are soaked 4 hours, and it is small to then take out 37 DEG C of oven for drying 4
When, it is standby, it is non-contact with Bio-Jet Quanti300 by glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platforms
The folic acid monoclonal antibody that rare-earth fluorescent microballoon marks is sprayed onto glass fibre membrane by the quantitation nozzle that declines, after 37 DEG C dry 2 hours
It is made.
The folic acid monoclonal antibody of rare-earth fluorescent microballoon mark in the present invention on pad uses following steps system
：
Step 1：The acquisition of cell strain of monoclonal antibody：With reference to folic acid amino acid sequence, the strong site people of selection antigenicity
Work synthesizes the peptide sequence of 20 amino acid or so, is linked on KLH, is prepared using the method for preparing monoclonal antibody of standard special
The cell strain of monoclonal antibody of different in nature high-affinity, by monoclonal antibody corresponding to the cell line obtained carry out pairing experiment and
Affinity determination experiment, capture antibody and detection antibody are determined according to experimental result；
Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify the folic acid for detection
Monoclonal antibody, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：3mg rare-earth fluorescent microballoons are taken, with 50mM, pH 9.5 carbonate delays
Fliud flushing, washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbon
In phthalate buffer, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In the above-mentioned carbonate buffer solution for washing and being resuspended to 100 μ l, be placed in 4 DEG C it is standby；
Step 4：The preparation of the folic acid monoclonal antibody of rare-earth fluorescent microballoon mark：The folic acid Dan Ke that 2mg is used to detect
Grand antibody, in 4 DEG C of dialysed overnights, is then mixed, 4 DEG C with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical
Reaction is overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid
(100mM Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL are used,
PH7.5 buffer solution is washed 3 times using centrifugal process, is resuspended in 100 μ l 100mM Tris-HCL buffer solutions and (is contained 1.2%
NaCL, 0.5%BSA, 0.2%Tween 20), 4 DEG C be kept in dark place it is standby.
It is of the present invention to be coated with detection line and the nitrocellulose filter of nature controlling line is made by following steps：
Step 1：According to foregoing pairing experiment and affinity determination experiment, select for monoclonal corresponding to the antibody of capture
Antibody cell strain, prepared according to the ascites production technology of standard and purify the folic acid monoclonal antibody for capture, be stored in -20
It is DEG C standby；
Step 2：Folic acid monoclonal antibody and goat anti-mouse igg antibody are adjusted into concentration to 1- with coating dilution respectively
5mg/ml, film liquid amount are 1-2 μ l/cm, are sprayed on using them as detection line is parallel with nature controlling line on nitrocellulose filter
It is coated with, detection line and nature controlling line are subsequently placed in baking oven, 37 DEG C dry 2 hours at intervals of 3-7mm.
Sample pad of the present invention is made by following steps：Glass fibre membrane is soaked in containing 2.0%TritonX-
100,2%BSA, 0.1M Tris buffer solutions, in pH7.5 treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in baking oven, 37
DEG C drying 2 hours.
Present invention also offers a kind of folic acid preparation method that kit is realized as described above, it is characterised in that including following
Step：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Accurate to draw 25 μ l serum samples, sample is drawn 40 μ l samples, is added in sample aperture when being whole blood, then
100 μ l Sample dilutions are added in the buffering fluid apertures of bottom immediately, Sample dilution is using physiological saline or PBS, 15-30 points
In clock result is quantitatively judged with fluorescence immune chromatography analyzer；
Step 3：After the relevant parameter for setting fluorescence immune chromatography analyzer, test card is put into storehouse and detected,
Instrument would indicate that the quantified results of sample concentration, and the fluorescence immune chromatography analyzer is a kind of Systems for optical inspection,
Detection range to folic acid is 0-20ng/ml.
The present invention is provided a kind of folic acid fast quantification immunochromatography prepared using rare-earth fluorescent immunochromatography technique and detected
Kit, while it is adapted to serum and whole blood sample, and it is adapted to clinically single part detection, tried relative to the qualitative collaurum of folic acid
Agent, can quantify detection sample in folate content, there is more specific Clinical significance of MG, with easy to operate, rapid reaction,
High sensitivity, high specificity, be adapted to Site Detection and it is economical and practical the advantages that.
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.
Subordinate list 3 is the Precision Analyze result data of embodiment 3 in the present invention.
Reference：PVC board 1, sample pad 2, pad 3, nitrocellulose filter 4, adsorptive pads 5.
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 folic acid detection kit, box is interior to be provided with test card, the examination
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
The folic acid monoclonal antibody of rare-earth fluorescent microballoon mark is adsorbed with 3, a diameter of 60-120nm of the rare-earth fluorescent microballoon is dilute
The native rare earth doped lanthanide series of fluorescent microsphere, it is stable under ground state, launch wavelength under 340-380nm excitation source effect
Fluorescence of the scope in 540-600nm；The monoclonal antibody is the monoclonal antibody mixed after purification, from for 2-6
The cell strain of monoclonal antibody of different folic acid epitopes；
The diameter of the rare-earth fluorescent microballoon of the pad 3 is preferably 90-110nm；The rare-earth fluorescent microballoon is preferably mixed
It is miscellaneous to have rare earth lanthanide, it is any one or a few mixed of the lanthanide series such as europium (Eu), samarium (Sm), erbium (Er), neodymium (Nd)
Compound；The preferably rare earth doped complex compound of rare-earth fluorescent microballoon；The antibody that rare-earth fluorescent microballoon marks on pad preferably comes
Come from the monoclonal cell cell line for 3 different epitopes.
Embodiment 1：
Each part of test card can be made by following measures in folic acid detection kit：
1st, the preparation of sample pad 2：
Glass fibre membrane is soaked in containing 2.0%Triton X-100,2%BSA, 0.1M Tris buffer solutions, pH7.5
Treatment fluid in, in 4 DEG C soak 4 hours, be subsequently placed in baking oven, 37 DEG C dry 2 hours.
2nd, the preparation of the pad 3 of fluorescent microsphere labelled antibody is adsorbed：
Glass fibre membrane is soaked in 200mM Tris-HCL treatment fluids (X-100 containing 1.5%Triton, 1.5%
BSA, pH7.5), 4 DEG C are soaked 4 hours, then take out 37 DEG C of oven for drying 4 hours, standby.By glass fibre membrane in Bio-
On DotXYZ3050 three-dimensional specking platforms, with the non-contact quantitation nozzles that decline of Bio-Jet Quanti300 by rare-earth fluorescent microballoon
The folic acid monoclonal antibody of mark is sprayed onto glass fibre membrane, and 37 DEG C dry 2 hours, standby；
The aldehyde radical of rare-earth fluorescent nanoparticle：3mg rare-earth fluorescent nanoparticles are taken, with 50mM, pH9.5 carbonate delays
Fliud flushing, washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbon
In phthalate buffer, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, using same centrifugal process
In the above-mentioned carbonate buffer solution for washing and being resuspended to 100 μ l, be placed in 4 DEG C it is standby；
Rare-earth fluorescent nanoparticle marks the preparation of folic acid monoclonal antibody：The 2mg folic acid monoclonals for being used to detect are resisted
Body, in 4 DEG C of dialysed overnights, is then mixed, 4 DEG C of reactions with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical
Overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid (100mM
Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL, pH7.5 buffering are used
Liquid is washed 3 times using centrifugal process, be resuspended in 100 μ l 100mM Tris-HCL buffer solutions (contain 1.2%NaCL, 0.5%BSA,
0.2%Tween 20), 4 DEG C be kept 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：
According to foregoing pairing experiment and affinity determination experiment, select thin for monoclonal antibody corresponding to the antibody of capture
Born of the same parents' strain, prepared according to the ascites production technology of standard and purify the folic acid monoclonal antibody for capture, be stored in -20 DEG C it is standby
With；
Folic acid monoclonal antibody and goat anti-mouse igg antibody are adjusted into concentration to 1-5mg/ml with coating dilution respectively,
Film liquid amount is 1-2 μ l/cm, is wrapped them as be sprayed on nitrocellulose filter on parallel with nature controlling line of detection line
Quilt, detection line and nature controlling line are subsequently placed in baking oven, 37 DEG C dry 2 hours at intervals of 3-7mm；
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 for being coated with detection line and nature controlling line, it is big to obtain test paper after assembling
Plate, it is wide to cut into 4mm as requested, and test paper is loaded in plastic clip and forms test card.
The preferably following raw material of equipment and raw material selected in above steps：
Folic acid specific pairs antibody；Folic acid 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), fluorescence immunity analyzer parameter
Setting：After test card technological parameter is set on fluorescence immunity analyzer, the above-mentioned test card assembled is taken, is used respectively
0.2nd, 0.5,1,2,5,20ng/ml folic acid calibration object, is measured with test card, obtains the fluorescence intensity level of each calibration object,
Result is input in the parameter of analyzer, completes the setting of the parameter of analyzer.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 200 parts of Roche Electrochemiluminescence immunoassay definite values
Sample, wherein 100 parts of serum sample, 100 parts of whole blood sample, folate content distributed area is between 0-20ng/mL.
Preparation method：
Step 1：Detection reagent and sample are balanced to room temperature, test card is taken out, keeps flat；
Step 2：Accurate to draw 25 μ l serum samples, sample is drawn 50 μ l samples, is added in sample aperture when being whole blood, then
100 μ L Sample dilutions (physiological saline or PBS) are added in the buffering fluid apertures of bottom immediately, 15-30 minutes, interior fluorescence was exempted from
Epidemic disease chromatographic analysis instrument quantitatively judges result；
Step 3：Set test card to be put into storehouse after instrument relevant parameter and detected, instrument would indicate that sample is dense
The quantified results of degree.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, all clinical samples are detected by preparation method, and analyze inspection
Survey result.
Result of the test：
As shown in Figure 2, using the detected value of experimental system as Y-axis, using the test value of contradistinction system as X-axis, scatterplot is drawn
Figure, and carry out correlation analysis.Clinical sample detection is respectively less than to 200 parts of clinical definite value pattern detections, sample mean deviation
10%, maximum deviation is less than 25%, R2>0.98, consistency coefficient>0.90.Testing result shows the detection kit prepared
Can be good, it is suitable for clinical detection, meets the differentiation needs of the different detection occasions of different clients.
Embodiment 3：Precision test
Using the test card and measuring system of embodiment 2, the test card and fluorescence immune chromatography analyzer of the present invention are entered
Row Precision Experiment.
Predominantly detect material：Clinical sample is obtained by relevant hospital, totally 2 parts of Chemiluminescence immunoassay definite value serum samples,
Wherein low value definite value sample clinical measures are 0.24ng/ml, and high level definite value sample clinical measures are 1.78ng/ml.
Preparation method：
Using the test card and measuring system of embodiment 2, replication is respectively carried out 20 times to 2 parts of definite value samples.
Test result analysis：
After the completion of prepared by clinical sample detection reagent, clinical sample is detected by preparation method, and analyzes detection knot
Fruit.
Result of the test：
As shown in table 1, the low value definite value sample and clinical measures that are 0.24ng/ml to clinical measures are 1.78ng/
Average value, standard deviation and CV, the reality of the acquired results display present invention are calculated after ml high level definite value sample replication 20 times
Check system test low value definite value sample CV is 8.13%, and test high level definite value sample CV is 5.61%.Testing result shows to prepare
Detection kit it is functional, be suitable for clinical detection, meet the differentiation needs of the different detection occasion of different clients.
The present invention is provided a kind of folic acid fast quantification immunochromatography prepared using rare-earth fluorescent immunochromatography technique and detected
Kit, while it is adapted to serum and whole blood sample, and it is adapted to clinically single part detection, tried relative to the qualitative collaurum of folic acid
Agent, can quantify detection sample in folate content, there is more specific Clinical significance of MG, with easy to operate, rapid reaction,
High sensitivity, high specificity, be adapted to Site Detection and it is economical and practical the advantages that.
Claims (7)
1. a kind of folic acid detection kit, provided with test card, it is characterised in that the test card is provided with by lower from being above sequentially provided with：
PVC board, sample pad, pad, nitrocellulose filter and adsorptive pads, rare-earth fluorescent microballoon mark is wherein adsorbed with pad
Folic acid monoclonal antibody, a diameter of 60-120nm of the rare-earth fluorescent microballoon, the rare earth doped lanthanide series of rare-earth fluorescent microballoon,
It is stable under ground state, launch fluorescence of the wave-length coverage in 540-600nm under 340-380nm excitation source effect；It is described
Monoclonal antibody is the monoclonal antibody mixed after purification, from the monoclonal for 2-6 different folic acid epitopes
Antibody cell strain.
2. a kind of folic acid detection kit according to claim 1, it is characterised in that the rare-earth fluorescent of the pad is micro-
The diameter of ball is preferably 90-110nm；The rare-earth fluorescent microballoon is doped with rare earth lanthanide.
3. a kind of folic acid detection kit according to claim 2, it is characterised in that the rare-earth fluorescent microballoon doping is dilute
Native complex compound；The antibody sources that rare-earth fluorescent microballoon marks on pad are in the monoclonal cell for 3 different epitopes
Cell line.
4. a kind of folic acid quality detection kit according to claim 1, it is characterised in that the pad is using following step
It is rapid to be made：Glass fibre membrane is soaked in 200mM Tris-HCL treatment fluids, 4 DEG C are soaked 4 hours, then take out 37 DEG C of baking ovens
Drying 4 hours, it is standby, by glass fibre membrane on Bio-DotXYZ3050 three-dimensional specking platforms, with Bio-Jet Quanti300
The folic acid monoclonal antibody that rare-earth fluorescent microballoon marks is sprayed onto glass fibre membrane, 37 DEG C of drying 2 by the non-contact quantitation nozzle that declines
It is made after hour.
5. a kind of folic acid quality detection kit according to claim 4, it is characterised in that the rare earth on pad is glimmering
The folic acid monoclonal antibody of light microballoon mark is made using following steps：
Step 1：The acquisition of cell strain of monoclonal antibody：With reference to folic acid amino acid sequence, the strong site of selection antigenicity is manually closed
Into the peptide sequence of 20 amino acid or so, it is linked on KLH, specificity is prepared using the method for preparing monoclonal antibody of standard
The cell strain of monoclonal antibody of high-affinity, by monoclonal antibody corresponding to the cell line obtained carry out pairing experiment and it is affine
Power determination experiment, capture antibody and detection antibody are determined according to experimental result；
Step 2：The preparation of monoclonal antibody：Prepared using the ascites production technology of standard and purify the folic acid Dan Ke for detection
Grand antibody, be stored in after packing -20 DEG C it is standby；
Step 3：The aldehyde radical of rare-earth fluorescent microballoon：3mg rare-earth fluorescent microballoons are taken, with 50mM, pH9.5 carbonate buffer solution,
Washed 3 times, centrifugal speed 12000rpm using centrifugal process, the time is 5 minutes, is finally resuspended in 100 μ l above-mentioned carbonate
In buffer solution, the glucan of 300 μ l aldehyde radicals is added, is mixed, at room temperature dark reaction 4 hours, is washed using same centrifugal process
Be resuspended in 100 μ l above-mentioned carbonate buffer solution, be placed in 4 DEG C it is standby；
Step 4：The preparation of the folic acid monoclonal antibody of rare-earth fluorescent microballoon mark：The 2mg folic acid monoclonals for being used to detect are resisted
Body, in 4 DEG C of dialysed overnights, is then mixed, 4 DEG C of reactions with above-mentioned carbonate buffer solution with the rare-earth fluorescent microballoon of above-mentioned aldehyde radical
Overnight；Then, sodium borohydride is added to final concentration 10mM, and 4 DEG C are reacted 4 hours；Add isometric confining liquid (100mM
Tris-HCL, pH7.5, containing 2%BSA, 5% sucrose), 4 DEG C of closings are overnight；Then 100mM Tris-HCL, pH7.5 buffering are used
Liquid using centrifugal process wash 3 times, be resuspended in 100 μ l 100mM Tris-HCL buffer solutions, 4 DEG C be kept in dark place it is standby.
6. a kind of folic acid quality detection kit according to claim 1, it is characterised in that described to be coated with detection line and matter
The nitrocellulose filter of control line is made by following steps：
Step 1：According to foregoing pairing experiment and affinity determination experiment, select for monoclonal antibody corresponding to the antibody of capture
Cell line, prepared according to the ascites production technology of standard and purify the folic acid monoclonal antibody for capture, be stored in -20 DEG C it is standby
With；
Step 2：Folic acid monoclonal antibody and goat anti-mouse igg antibody are adjusted into concentration to 1-5mg/ with coating dilution respectively
Ml, film liquid amount are 1-2 μ l/cm, are carried out them as be sprayed on nitrocellulose filter on parallel with nature controlling line of detection line
Coating, detection line and nature controlling line are subsequently placed in baking oven, 37 DEG C dry 2 hours at intervals of 3-7mm.
7. a kind of folic acid quality detection kit according to claim 1, it is characterised in that the sample pad passes through following step
It is rapid to be made：Glass fibre membrane is soaked in containing 2.0%Triton X-100,2%BSA, 0.1M Tris buffer solutions, pH7.5's
In treatment fluid, 4 hours are soaked in 4 DEG C, are subsequently placed in baking oven, 37 DEG C dry 2 hours.
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| CN201710852510.5A CN107703315A (en) | 2017-09-19 | 2017-09-19 | Folic acid detection kit and preparation method thereof |
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| CN108761103A (en) * | 2018-06-05 | 2018-11-06 | 宁波奥丞生物科技有限公司 | A kind of immunofluorescence technique kit of detection folic acid |
| CN113325171A (en) * | 2021-08-02 | 2021-08-31 | 天津康博尔生物基因技术有限公司 | Kit for detecting human body erythrocyte folic acid content, detection method and application |
| CN113933502A (en) * | 2021-10-19 | 2022-01-14 | 青岛汉唐生物科技有限公司 | Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography |
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| CN104714025A (en) * | 2014-11-28 | 2015-06-17 | 威海纽普生物技术有限公司 | NT-proBNP detection kit and detection method |
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| CN108761103A (en) * | 2018-06-05 | 2018-11-06 | 宁波奥丞生物科技有限公司 | A kind of immunofluorescence technique kit of detection folic acid |
| CN113325171A (en) * | 2021-08-02 | 2021-08-31 | 天津康博尔生物基因技术有限公司 | Kit for detecting human body erythrocyte folic acid content, detection method and application |
| CN113933502A (en) * | 2021-10-19 | 2022-01-14 | 青岛汉唐生物科技有限公司 | Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography |
| CN113933502B (en) * | 2021-10-19 | 2024-02-02 | 青岛汉唐生物科技有限公司 | Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography |
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