CN104749385A - Immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and preparation method of immunochromatography reagent strip - Google Patents
Immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and preparation method of immunochromatography reagent strip Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and discloses an immunochromatography reagent strip for quantitative detection of 25-hydroxyvitamin D and a preparation method of the immunochromatography reagent strip. The immunochromatography reagent strip for quantitative detection of the 25-hydroxyvitamin D comprises a sample adding region, a conjugate release region, a reaction region and a water absorption region, which are sequentially arranged; the conjugate release region is a glass cellulose membrane enveloped with colloidal gold or a quantum dot or an up-conversion luminescent material UCP-marked mouse-anti-human 25-hydroxyvitamin D monoclonal antibody; the reaction region is a test region and a control region; the test region is enveloped with 25-hydroxyvitamin D-coupling protein; and the control region is enveloped with a nitrocellulose membrane of a goat anti-mouse IgG antibody. The immunochromatography reagent strip is capable of accurately, quantitatively, rapidly and sensitively detecting the 25-hydroxyvitamin D in human whole blood, serum and plasma; the required instrument is different from a traditional large expensive instrument for a clinical laboratory; and only one corresponding miniature immunity analyzer is required, therefore, real-time detection is realized.
Description
Technical field
The present invention relates to biological technical field, be specifically related to immunochromatography reagent bar quantitatively detecting 25-hydroxy-vitamin D and preparation method thereof.
Background technology
Vitamin D is steroid derivatives, belongs to liposoluble vitamin.Vitamin D in human body only has fraction to derive from food, and 95% of vitamin D required in body derives from sunlight middle-ultraviolet lamp and irradiates skin generation.Vitamin D enters liver through blood circulation, 25-hydroxy-vitamin D is converted under the effect of 25-hydroxylase, then in Renal proximal tubular epithelial cell mitochondria α hydroxylase system effect under change 1 into, 25-dihydroxyvitamin D, it is the maximum biological agent form of vitamin D, can promote the synthesis of enteron aisle calbindin.And 25-hydroxyvitamin D is the main circulation form in metabolism, can reflects the vitamin D storage level of body and be associated with the clinical symptoms that vitamin D lacks, therefore 25-hydroxy-vitamin D is the most reliable clinical indices weighing level in vitamin D body.
The main work of vitamin D regulates Ca,P metabolism, by promoting calcium and the absorption of phosphorus in enteron aisle, and the mode of reabsorption calcium phosphorus, improve the calcium phosphorus level in blood, and by the method for calcification, with parathyroid hormone and calcitonin and other hormone actings in conjunction, sclerotin is made to become hard.Except having traditional bone effect, also there is non-bone effects such as affecting autoimmune disease, cardiovascular disease, diabetes, tumour.Research finds, vitamin D lacks the expression that directly can affect human gene, and these genes are relevant with numerous disease such as rheumatic arthritis and diabetes.Vitamin D shortage can cause the osteomalacia of juvenile's rickets and adult.Research also shows that vitamin D deficiency may make the onset risk of some cancer, angiocardiopathy, autoimmune disease and infectious diseases raise.
Clinically conventional sense project and health check-up project are become to the quantitative detection of vitamin D.Set up fast a kind of, easy, sensitive, the method detecting vitamin D level is accurately and reliably particularly important.The assay method of current 25-hydroxyvitamin D mainly contains euzymelinked immunosorbent assay (ELISA), radioimmunology, liquid chromatography-mass spectroscopic assays and chemoluminescence method.Euzymelinked immunosorbent assay (ELISA) automaticity is not high, and is affected by human factors larger.Radioimmunology also exists problem of environmental pollution; Liquid chromatography mass complicated operation, the used time is longer; And though chemoluminescence method sensitivity is high, testing cost is higher, needs specified chemical light-emitting appearance, and these reasons cause its range of application less.
Summary of the invention
The present invention is for avoiding the weak point existing for above-mentioned prior art, immunochromatography reagent bar of a kind of quantitative detection 25-hydroxy-vitamin D and preparation method thereof is provided, there is the advantages such as sampling is easy, easy and simple to handle, susceptibility is high, high specificity, good stability, be adapted at the unit such as basic hospital, clinic and MEC to use, reduction vitamin D testing cost, universal body vitamin D are detected, to reduce clinical blindness medication significant.
In order to realize foregoing invention object, the invention provides following technical scheme:
An immuno-chromatographic test paper strip for quantitative detection 25-hydroxy-vitamin D, comprise set gradually sample application zone, bond release district, reaction zone and suction zones; Described bond release district be bag by the plain film of glass fibre of the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody of collaurum (or quantum dot or up-conversion luminescent material UCP); Described reaction zone is test section and control zone, and test section bag is by 25(OH)VD-coupling protein, and control zone bag is by the nitrocellulose filter of sheep anti-mouse igg antibody.
Described collaurum, quantum dot, up-conversion luminescent material are all as the label in immunochromatography technique, indirectly read the concentration of determinand.
Collaurum has developed into the most frequently used label arrived in immunochromatography technique.But it mainly carries out qualitative or half-quantitative detection, limit its range of application.
The principle of the quantitative immunochromatographic technology of collaurum: colloid gold particle is at a particular wavelength, relevant to the amount of colloid gold particle with scattering to the absorption of light, detects colloid gold particle to the absorption of light and scattering by photoelectric sensor, thus obtains the amount of determined antigen.
Quantum dot is a kind of semiconductor nano, its exciting light spectrum width, in continuous distribution, and emission spectrum is narrow, good and the Color tunable of monochromaticity, luminous intensity is 20 ~ 50 times of traditional organic fluorescence element, and has lasting photochemical stability, and the optical property of its uniqueness makes it obtain extensive concern as a kind of novel fluorescence probe.Quantum dot has higher sensitivity as label than collaurum.
Quantum dot shortcoming: preparation condition harshness, reactions steps more complicated, reagent cost is high, toxicity is larger.
Up-converting phosphor technology is the immunoassay technology that immunochromatography principle combines with photoelectricity transformation principle, using up-conversion luminescence particle as tracer, after infrared ray excited, upload light-emitting particles launch visible ray, realize energy upper turn, by the conversion to optical signalling, can quantitatively detect determinand fast.
The advantage of up-conversion luminescent material: effectively can reduce the decline that photo ionization causes host material; Do not need strict phase matching, not high to the stability requirement of excitation wavelength; Output wavelength has certain property coordinated.
The shortcoming of up-conversion luminescent material: the reaction time is longer; Standard curve range relative narrower; Luminescence efficiency is lower, less stable.
The bond release district of the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody being coated with collaurum (or quantum dot or up-conversion luminescent material UCP) connects together with the test section being coated with 25-hydroxy-vitamin D-coupling protein by the immuno-chromatographic test paper strip of quantitative detection 25-hydroxy-vitamin D of the present invention, utilize collaurum (or quantum dot or up-conversion luminescent material UCP) immunochromatographic method competitive binding monoclonal antibody principle, namely detected person's serum 25-hydroxy vitamin be coated on the principle of the 25-hydroxy-vitamin D-coupling protein competitive binding 25-hydroxy-vitamin D monoclonal antibody on nitrocellulose filter, by detecting the shade (or the fluorescence intensity of the quantum dot be excited or the luminous intensity of UCP particle that is excited) of test section, 25-hydroxy-vitamin D in quantitative detection human serum.
When there is not 25-hydroxy-vitamin D in serum, the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody of collaurum (or quantum dot or up-conversion luminescent material UCP) moves to test section along bond release district due to capillarity, the 25-hydroxy-vitamin D be coated on nitrocellulose filter with this test section is combined, thus occurs an aubergine band (or produce strong fluorescence signal or produce strong utilizing emitted light after exciting after exciting) in test section, when there is 25-hydroxy-vitamin D in serum, the 25-hydroxy-vitamin D of mouse-anti people 25-hydroxy-vitamin D monoclonal antibody first in serum of collaurum (or quantum dot or up-conversion luminescent material UCP) is combined and forms compound, then test section is moved to by capillarity along bond release district, but do not have or only have the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody of a small amount of collaurum (or quantum dot or up-conversion luminescent material UCP) to be combined with the 25-hydroxy-vitamin D-coupling protein of test section, test section line color disappears or shoal (or can only produce weak fluorescence signal or can only produce weak utilizing emitted light after exciting after exciting), under certain condition, concentration in the depth of test section line color (or the intensity of fluorescence or radiative intensity) and sample is negative correlation, adopt corresponding immunochromatography instrument can 25-hydroxy-vitamin D concentration in rational judgment human serum.
And control zone bag is by sheep anti-mouse igg antibody, the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody of collaurum (or quantum dot or up-conversion luminescent material UCP) is combined with the sheep anti-mouse igg antibody of bag quilt, thus an aubergine band (or produce strong fluorescence signal or produce strong utilizing emitted light after exciting after exciting) is there is in control zone, test strips and testing result can be judged whether effectively, also as the inner quality standard measuring reagent.If control zone does not produce aubergine band (or do not produce strong fluorescence signal or do not produce strong utilizing emitted light after exciting after exciting), then illustrate that test strips lost efficacy.
Further, 25-hydroxy-vitamin D test strip of the present invention also comprises PVC backer board, and what described sample application zone, bond release district, reaction zone, suction zones overlapped successively mutually is pasted onto on PVC backer board.
Because the concentration of 25-hydroxy-vitamin D is finally decided by the shade (or the fluorescence intensity that produces after exciting of quantum dot or up-conversion luminescent material UCP produce after exciting emitted luminescence intensity) of collaurum, therefore 25-hydroxy-vitamin D monoclonal antibody plays the key player connecting collaurum (or quantum dot or up-conversion luminescent material UCP) and these two kinds of materials of 25-hydroxy-vitamin D, can combine with collaurum (or quantum dot or up-conversion luminescent material UCP), can be combined with 25-hydroxy-vitamin D again, wherein 25-hydroxy-vitamin D monoclonal antibody is combined with 25-hydroxy-vitamin D is a spontaneous process, and the combination of 25-hydroxy-vitamin D monoclonal antibody and collaurum (or quantum dot or up-conversion luminescent material UCP) needs artificial promotion.An important step of producing is exactly collaurum (or quantum dot or up-conversion luminescent material UCP) 25-hydroxy-vitamin D monoclonal antibody.
Wherein, as preferably, the preparation method of the 25-hydroxy-vitamin D monoclonal antibody of collaurum of the present invention (or quantum dot or up-conversion luminescent material UCP) is to 1mL pH7.4,2 μm of ol collaurums (or quantum dot or up-conversion luminescent material UCP) and 1mg mouse-anti people 25-hydroxy-vitamin D monoclonal antibody is added in the phosphate buffer of 50mM, gentle vibration 2 hours under room temperature, then the centrifugal 30min of 12000r/min, gets precipitation and get final product.
The preparation method of described 25(OH)VD-coupling protein comprises the following steps:
10mg vitamin D is dissolved in 100 μ L organic solvents, then under agitation to 10mL, concentration is the vitamin D after dropwise adding dissolving in the coupling protein solution of 5mg/mL, dropwise add again concentration of volume percent be 50% glutaraldehyde water solution to the concentration of volume percent of glutaraldehyde in solution be 1% ~ 3%, stirring reaction 16h ~ 18h at moving to 4 DEG C after stirring 5min ~ 20min, reacted reactant liquor PBS buffer solution is dialysed 8h at 4 DEG C, liquid is changed 2 ~ 3 times in dialysis procedure, supernatant is got after dialysate is centrifugal, finally by supernatant 0.45 μm of membrane filtration, obtain the conjugate of vitamin D and coupling protein, i.e. 25(OH)VD-coupling protein,-20 DEG C save backup.
Wherein, described organic solvent is the one in methenyl choloride, methyl alcohol, ethanol, normal hexane, acetonitrile, dimethyl sulfoxide (DMSO), Isosorbide-5-Nitrae-dioxane.
Wherein, described coupling protein is the one in bovine serum albumin(BSA), ovalbumin (OVA) or hemocyanin (KLH).
Wherein, described mouse-anti people 25-hydroxy-vitamin D monoclonal antibody, 25-hydroxy-vitamin D and sheep anti-mouse igg antibody well known to a person skilled in the art, can be obtained by commercial sources or be prepared by method disclosed in prior art.
Collaurum (or quantum dot or up-conversion luminescent material UCP) as signal-obtaining is the emphasis during test strips quantitatively detects.The size of collaurum (or quantum dot or up-conversion luminescent material UCP), uniformity, stability all can affect the last reading of product.As preferably, in 25-hydroxy-vitamin D test strip of the present invention, described collaurum particle diameter is 20 ~ 40nm, and absorbing wavelength is 530nm(or quantum point grain diameter is 5nm, and excitation wavelength is 365nm, and emission wavelength is 605nm; Or upper forwarding luminescent material UCP particle diameter is 5nm).
Another object of the present invention is achieved through the following technical solutions:
The invention provides the preparation method of the immuno-chromatographic test paper strip of described quantitative detection 25-hydroxy-vitamin D, comprise the following steps:
Step 1: the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody that collaurum (or quantum dot or up-conversion luminescent material UCP) marks, by 1ml solution paving 20cm
2ratio, be sprayed on glass fibre element film with spray film instrument and obtain bond release district;
Step 2: the solution with 0.01M pH 7.4 phosphate buffer, 25-hydroxy-vitamin D being configured to 0.8 ~ 1.6mg/ml, nitrocellulose filter wraps tested district with spray film instrument with the amount of 1 ~ 1.5 ul/cm; Another solution sheep anti-mouse igg antibody being configured to 1 ~ 3mg/ml, with spraying film instrument with the amount bag of 1 ~ 1.5 ul/cm by control zone; Nitrocellulose filter after line is dry 8-12 hour at 36-38 DEG C, makes bag by the detection zone of 25-hydroxy-vitamin D and bag by the nitrocellulose filter of the control zone of sheep anti-mouse igg;
Step 3: be less than the condition of 30% in relative humidity under, get PVC backer board, the nitrocellulose filter wrapping quilt is pasted onto the middle part of plastic base, pastes bond release district in the side, detection zone of nitrocellulose filter, in bond release district, opposite side pastes sample application zone; In the stickup suction zones, side, control zone of nitrocellulose filter; Adjacent interface laminates 1 ~ 2mm mutually, and the large plate pasted is cut into the wide test strip of 3 ~ 5mm.
the immuno-chromatographic test paper strip beneficial effect compared to existing technology of quantitative detection 25-hydroxy-vitamin D of the present invention is:
Combine the immunochromatography instrument with the built-in typical curve of 25-hydroxy-vitamin D when the present invention uses, realize the Quantitative detection to 25-hydroxy-vitamin D concentration, be applicable to serum, blood plasma, whole blood, be applicable to micro-example.There is the advantages such as sampling is easy, easy and simple to handle, susceptibility is high, high specificity, good stability, be adapted at the unit such as basic hospital, clinic and MEC to use, reduction vitamin D testing cost, universal body vitamin D are detected, to reduce clinical blindness medication significant.
Accompanying drawing explanation
Fig. 1 shows the schematic diagram of the immuno-chromatographic test paper strip of quantitative detection 25-hydroxy-vitamin D of the present invention; Wherein 1: sample application zone, 2: bond release district, 3: reaction zone, 4: suction zones; 5:PVC backer board; T: detection zone; C: control zone;
Fig. 2 is Roche chemoluminescence method 25-hydroxy-vitamin D measurement result schematic diagram;
Fig. 3 is the front view (Kato stretches out) of described intelligent immunity quantitative analysis instrument;
Fig. 4 is the front view (Kato withdrawal) of described intelligent immunity quantitative analysis instrument;
Fig. 5 is the lateral side view of described intelligent immunity quantitative analysis instrument;
Fig. 6 is the upward view of described intelligent immunity quantitative analysis instrument;
Fig. 7 is the cut-away view of described intelligent immunity quantitative analysis instrument.
Embodiment
The invention discloses immuno-chromatographic test paper strip of a kind of quantitative detection 25-hydroxy-vitamin D and preparation method thereof, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In immuno-chromatographic test paper strip of a kind of quantitative detection 25-hydroxy-vitamin D provided by the invention and preparation method thereof, biomaterial used and reagent all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
embodiment 1
A kind of 25(OH)VD quantitative testing test paper bar, comprise set gradually sample application zone 1, bond release district 2, reaction zone 3 and suction zones 4; Described bond release district wraps by the glass fibre of mouse-anti people 25-hydroxy-vitamin D monoclonal antibody element film, and described mouse-anti people 25-hydroxy-vitamin D monoclonal antibody is by collaurum, quantum dot or up-conversion luminescent material UCP bag quilt; Described reaction zone is divided into test section T and control zone C, and described test section is coated with 25(OH)VD-coupling protein, and described control zone is the nitrocellulose filter being coated with sheep anti-mouse igg antibody.
Further, described quantitative testing test paper bar also comprises PVC backer board (5), and what described sample application zone, bond release district, reaction zone, suction zones overlapped successively mutually is pasted onto on PVC backer board.
Further, the preparation method of the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody of collaurum or quantum dot or up-conversion luminescent material UCP is: in phosphate buffer, add collaurum or quantum dot or up-conversion luminescent material UCP and mouse-anti people 25-hydroxy-vitamin D monoclonal antibody, gentle vibration 2 hours under room temperature, then centrifugal, get precipitation and namely obtain described mouse-anti people 25-hydroxy-vitamin D monoclonal antibody.
Further, the preparation method of described collaurum is: chlorauric acid solution is heated to boiling, adds citric acid three sodium solution rapidly, stir, the collaurum that the colloid gold particle that to be reduced into by gold chloride by diameter be 20nm ~ 40nm is formed.
Further, described quantum point grain diameter is 5nm, and excitation wavelength is 365nm, and emission wavelength is 605nm; The particle diameter of described up-conversion luminescent material and UCP is 5nm.
Further, described coupling protein is the one in bovine serum albumin(BSA), ovalbumin or hemocyanin.
Further, the preparation method of described 25(OH)VD-coupling protein comprises the following steps: be dissolved in by 10mg vitamin D in 100 μ L organic solvents, then under agitation to 10mL, concentration is the vitamin D after dropwise adding dissolving in the coupling protein solution of 5mg/mL, dropwise add again concentration of volume percent be 50% glutaraldehyde water solution to the concentration of volume percent of glutaraldehyde in solution be 1% ~ 3%, stirring reaction 16h ~ 18h at moving to 4 DEG C after stirring 5min ~ 20min, reacted reactant liquor PBS buffer solution is dialysed 8h at 4 DEG C, liquid is changed 2 ~ 3 times in dialysis procedure, supernatant is got after dialysate is centrifugal, finally by supernatant 0.45 μm of membrane filtration, obtain the conjugate of vitamin D and coupling protein, i.e. 25(OH)VD-coupling protein,-20 DEG C save backup.
Wherein, described organic solvent is the one in methenyl choloride, methyl alcohol, ethanol, normal hexane, acetonitrile, dimethyl sulfoxide (DMSO), Isosorbide-5-Nitrae-dioxane
The preparation method of described 25-hydroxy-vitamin D quantitative testing test paper bar, described preparation method comprises the following steps:
Step 1: will the collaurum of mouse-anti people 25-hydroxy-vitamin D monoclonal antibody or quantum dot or up-conversion luminescent material UCP be marked with, be sprayed on glass fibre element film with spray film instrument, be placed in temperature 20-30 DEG C, dry under relative humidity 30-35%, obtain bond release district;
Step 2: with 10mM, pH 7.4 PB damping fluid 25-hydroxy-vitamin D-coupling protein is configured to the solution of 1mg/ml, nitrocellulose filter wraps tested district with spray film instrument with the amount of 1 μ l/cm; Another solution sheep anti-mouse igg antibody being configured to 1mg/ml, with spraying film instrument with the amount bag of 1 μ l/cm by control zone; Bag, by the nitrocellulose filter behind test section and control zone at 38 DEG C dry 8 hours, is made bag by the detection zone of 25-hydroxy-vitamin D-coupling protein and bag by the control zone of sheep anti-mouse igg, is namely obtained reaction zone;
Step 3: being namely pasted onto on PVC backer board of mutually being overlapped in sample application zone, bond release district, reaction zone and suction zones obtain described 25-hydroxy-vitamin D quantitative testing test paper bar.
The using method of described 25-hydroxy-vitamin D quantitative testing test paper bar is, described 25-hydroxy-vitamin D quantitative testing test paper bar is placed in intelligent immunity quantitative analysis instrument, described intelligent immunity quantitative analysis instrument is made to be in holding state, described 25-hydroxy-vitamin D quantitative testing test paper bar is placed in intelligent immunity quantitative analysis instrument, testing sample is added to reagent strip sample application zone, at intelligent immunity quantitative analysis instrument interface hit testing, the self-clocking of intelligent immunity quantitative analysis instrument is carried out after 15 minutes detecting and showing test results.
embodiment 2: the preparation in bond release district
To 1mL pH7.4,2 μm of ol collaurums (or quantum dot or up-conversion luminescent material UCP) and 1mg mouse-anti people 25-hydroxy-vitamin D monoclonal antibody is added in the phosphate buffer of 50mM, gentle vibration 2 hours under room temperature, then the centrifugal 30min of 12000r/min, gets precipitation and get final product.
The mouse-anti people 25-hydroxy-vitamin D monoclonal antibody that collaurum (or quantum dot or up-conversion luminescent material UCP) marks, by 1ml solution paving 20cm
2ratio, be sprayed on glass fibre element film with spray film instrument, be placed in temperature 20-30 DEG C, dry 5 hours of relative humidity 30-35%, obtain bond release district.
embodiment 3:the preparation of reaction zone
With 0.01M pH 7.4 phosphate buffer, 25-hydroxy-vitamin D-coupling protein is configured to the solution of 0.8 ~ 1.6mg/ml, nitrocellulose filter wraps tested district with spray film instrument with the amount of 1 ~ 1.5 μ l/cm; Another solution sheep anti-mouse igg antibody being configured to 1 ~ 3mg/ml, with spraying film instrument with the amount bag of 1 ~ 1.5 μ l/cm by control zone; Nitrocellulose filter after line is dry 8-12 hour at 36-38 DEG C, makes bag by the detection zone of 25-hydroxy-vitamin D-coupling protein and bag by the nitrocellulose filter of the control zone of sheep anti-mouse igg.
embodiment 4:the preparation of test strips
Be less than the condition of 30% in relative humidity under, get PVC backer board, the nitrocellulose filter wrapping quilt is pasted onto the middle part of plastic base, paste bond release district in the T side, detection zone of nitrocellulose filter, in bond release district, opposite side pastes sample application zone; In the stickup suction zones, side, control zone of nitrocellulose filter; Adjacent interface laminates 1 ~ 2mm mutually, and the large plate pasted is cut into the wide test strip of 3 ~ 5mm.
comparative example
Quantitative detecting method based on test strips provided by the invention compares with Roche chemoluminescence method 25-hydroxy-vitamin D test strip correlativity.
Get each 100 μ L of 25-hydroxy-vitamin D standard items that concentration is 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL and 100ng/mL, add the sample application zone of test strips prepared by the embodiment of the present invention 4, room temperature places 15min.Test strips is inserted intelligent immunity quantitative analysis instrument and read 25-hydroxy-vitamin D content, each concentration replication 3 times.According to concentration and the corresponding reading of 25-hydroxy-vitamin D, drawing standard curve.
Get 100 parts of blood samples, by its decile, test strips prepared by a employing the present invention and special immune chromatographic detector measure, and another part adopts Roche chemoluminescence method 25-hydroxy-vitamin D to measure.Measurement result is shown in Fig. 2, and result shows no difference of science of statistics between fine r=0.9905, the P > of its correlativity 0.05, two kinds of methods.
Described intelligent immunity quantitative analysis instrument is Chinese utility model patent " a kind of portable intelligent immunity quantitative analysis instrument " (application number: 2014208245854, the applying date: on Dec 23rd, 2014, applicant: Pu Maide (Beijing) Science and Technology Ltd.) disclosed in.As shown in Figure 3, Figure 4, described quantitative analysis instrument comprises upper shell 100 and the lower house 200 with its fastening, preferably, upper shell 100 is designed to plane and horizontal by certain angle, meet the Visual Observations Observations custom of people, upper shell 100 and lower house 200 all adopt the material that magnetic and fluorescent effect do not occur to make, and such as: acrylonitrile-butadiene-styrene (ABS), are applicable on the panimmunity chromatographic detectors such as Colloidal Gold, immunomagnetic beads method and immunofluorescence technique.
As shown in Figure 3, Figure 4, upper shell 100 is provided with screen 101 and the paper delivery module for printing examining report on paper slip, screen 101 is positioned at the middle part of upper shell 100, parallel with the surface of upper shell 100, arrange at an angle with lower house 200 bottom surface, conveniently allow user directly check and analyze detection case report; Paper delivery module comprise can take off conjunction store paper groove 103 and the sawtooth 102 for severing paper slip, store the center section that paper groove 103 is positioned at upper casing 100 upper end, its underpart is provided with printing mechanism, for printing examining report, printed examining report is from storing the lower end of paper groove 103 out, sawtooth 102 is positioned at the side storing paper groove 103 paper delivery, for the examining report that severing is printed; Lower house 200 is provided with interface socket and the Kato 203 for holding test strips, and Kato 203 shifts out can change test strips, and interface socket is used for the connection of this quantitative analysis instrument and peripheral apparatus.
The position that the surface of upper shell 100 stores between paper groove 103 and screen 101 is provided with logo, and paper groove 103 left and right sides that stores of upper shell 100 is provided with groove 104, conveniently stores the unlatching of paper groove.
As shown in Figure 5, lower house 200 rear end is provided with interface socket and power switch 205, and front end is provided with Kato 203 and Kato opens the door 204, and specific in the present embodiment, the rear end of lower house 200 is provided with interface socket and power switch 205.Wherein, as shown in Figure 5, power switch 205 is positioned at the rear end of lower house 200, prevents user to be not intended to touch power switch in use and causes error cut-off machine.
As shown in Figure 5, interface socket comprises outlet 202 and one or more peripheral hardware socket 201, preferably, and the arrangement in yi word pattern of peripheral hardware socket 201 and outlet 202.Specific in the present embodiment, have the peripheral hardware socket 201 that 5 can connect different peripheral equipment, be respectively a PS2 interface, network interface, a Type B USB interface and a miniUSB interface, be isolated from each other between 5 peripheral hardware sockets 201 and outlet 202, person easy to use connects peripheral apparatus quickly and easily.
As shown in Figure 6, the bottom surface of lower house 200 is provided with a lithium battery groove 206, and specific in the present embodiment, lithium battery groove is used for erecting equipment lithium battery, equipment can be made to break away from dependence to power supply, realize removableization truly.
During use, after quantitative analysis instrument is switched on power by outlet 202, be connected with clinical assay devices by peripheral hardware socket 201, turn on the power switch 205, press key 105, can start to carry out detection and analyze; Detect after analyzing, screen 101 can directly demonstrate to detect analyzes situation report, can print on heat-sensitive paper, by stupefied 102 severings of sawtooth by detection analysis report by the printing mechanism stored under paper groove 103; Open and store paper groove 103 and can safeguard printing mechanism and clear up.When the complete Kato of ELISA test strip stretches out, test strips can be changed in Kato 203 position.
As shown in Figure 7, colloidal gold immune chromatography test is detected, operational outfit, preheating 10min.Sample will be detected as serum, blood plasma, whole blood or urine etc., and add in test strips well on request, after certain hour, put into the Kato 203 of equipment, touching test button, instrument detects automatically, and immediately prints testing result, or is sent by interface socket or pour out test result.
In sum, described quantitative analysis instrument comprises upper shell 100 and the lower house 200 with its fastening, upper shell 100 being provided with key, can checking the screen 101 of analysis situation and the paper delivery module for printing examining report on paper slip, paper delivery module comprises and stores paper groove 103 and the sawtooth 102 for severing paper slip, store the center section that paper groove 103 is positioned at upper shell 100 upper end, sawtooth 102 is positioned at and stores paper lid 103 paper delivery side, facilitate severing examining report, lower house 200 is provided with interface socket and opens the door 204 for the Kato 203 that holds test strips and Kato.
Claims (10)
1. a 25(OH)VD quantitative testing test paper bar, is characterized in that, comprise set gradually sample application zone (1), bond release district (2), reaction zone (3) and suction zones (4); Described bond release district wraps by the glass fibre of mouse-anti people 25-hydroxy-vitamin D monoclonal antibody element film, and described mouse-anti people 25-hydroxy-vitamin D monoclonal antibody is by collaurum, quantum dot or up-conversion luminescent material UCP bag quilt; Described reaction zone is divided into test section (T) and control zone (C), and described test section is coated with 25(OH)VD-coupling protein, and described control zone is the nitrocellulose filter being coated with sheep anti-mouse igg antibody.
2. 25(OH)VD quantitative testing test paper bar according to claim 1, is characterized in that, also comprises PVC backer board (5), and what described sample application zone, bond release district, reaction zone, suction zones overlapped successively mutually is pasted onto on PVC backer board.
3. 25(OH)VD quantitative testing test paper bar according to claim 1, it is characterized in that, the preparation method of the mouse-anti people 25-hydroxy-vitamin D monoclonal antibody of collaurum or quantum dot or up-conversion luminescent material UCP is: in phosphate buffer, add collaurum or quantum dot or up-conversion luminescent material UCP and mouse-anti people 25-hydroxy-vitamin D monoclonal antibody, gentle vibration 2 hours under room temperature, then centrifugal, get precipitation and namely obtain described mouse-anti people 25-hydroxy-vitamin D monoclonal antibody.
4. the 25-hydroxy-vitamin D test strip according to claim 1 or 3, it is characterized in that, the preparation method of described collaurum is: chlorauric acid solution is heated to boiling, add citric acid three sodium solution rapidly, stir, the collaurum that the colloid gold particle that to be reduced into by gold chloride by diameter be 20nm ~ 40nm is formed.
5. 25-hydroxy-vitamin D test strip according to claim 4, is characterized in that, described quantum point grain diameter is 5nm, and excitation wavelength is 365nm, and emission wavelength is 605nm; The particle diameter of described up-conversion luminescent material and UCP is 5nm.
6. the immuno-chromatographic test paper strip of quantitative detection 25-hydroxy-vitamin D according to claim 1, is characterized in that, described coupling protein is the one in bovine serum albumin(BSA), ovalbumin or hemocyanin.
7. the immuno-chromatographic test paper strip of quantitative detection 25-hydroxy-vitamin D according to claim 1, is characterized in that, the preparation method of described 25(OH)VD-coupling protein comprises the following steps:
10mg vitamin D is dissolved in 100 μ L organic solvents, then under agitation to 10mL, concentration is the vitamin D after dropwise adding dissolving in the coupling protein solution of 5mg/mL, dropwise add again concentration of volume percent be 50% glutaraldehyde water solution to the concentration of volume percent of glutaraldehyde in solution be 1% ~ 3%, stirring reaction 16h ~ 18h at moving to 4 DEG C after stirring 5min ~ 20min, reacted reactant liquor PBS buffer solution is dialysed 8h at 4 DEG C, liquid is changed 2 ~ 3 times in dialysis procedure, supernatant is got after dialysate is centrifugal, finally by supernatant 0.45 μm of membrane filtration, obtain the conjugate of vitamin D and coupling protein, i.e. 25(OH)VD-coupling protein,-20 DEG C save backup.
8. the immuno-chromatographic test paper strip of quantitative detection 25-hydroxy-vitamin D according to claim 7, is characterized in that, described organic solvent is the one in methenyl choloride, methyl alcohol, ethanol, normal hexane, acetonitrile, dimethyl sulfoxide (DMSO), Isosorbide-5-Nitrae-dioxane.
9. a preparation method for the 25-hydroxy-vitamin D quantitative testing test paper bar as described in claim 1-8 any one, it is characterized in that, described preparation method comprises the following steps:
Step 1: will the collaurum of mouse-anti people 25-hydroxy-vitamin D monoclonal antibody or quantum dot or up-conversion luminescent material UCP be marked with, be sprayed on glass fibre element film with spray film instrument, be placed in temperature 20-30 DEG C, dry under relative humidity 30-35%, obtain bond release district;
Step 2: with 10mM, pH 7.4 PB damping fluid 25-hydroxy-vitamin D-coupling protein is configured to the solution of 1mg/ml, nitrocellulose filter wraps tested district with spray film instrument with the amount of 1 μ l/cm; Another solution sheep anti-mouse igg antibody being configured to 1mg/ml, with spraying film instrument with the amount bag of 1 μ l/cm by control zone; Bag, by the nitrocellulose filter behind test section and control zone at 38 DEG C dry 8 hours, is made bag by the detection zone of 25-hydroxy-vitamin D-coupling protein and bag by the control zone of sheep anti-mouse igg, is namely obtained reaction zone;
Step 3: being namely pasted onto on PVC backer board of mutually being overlapped in sample application zone, bond release district, reaction zone and suction zones obtain described 25-hydroxy-vitamin D quantitative testing test paper bar.
10. the using method of the 25-hydroxy-vitamin D quantitative testing test paper bar as described in claim 1-8 any one, it is characterized in that, described 25-hydroxy-vitamin D quantitative testing test paper bar is placed in intelligent immunity quantitative analysis instrument, described intelligent immunity quantitative analysis instrument is made to be in holding state, described 25-hydroxy-vitamin D quantitative testing test paper bar is placed in intelligent immunity quantitative analysis instrument, testing sample is added to reagent strip sample application zone, at intelligent immunity quantitative analysis instrument interface hit testing, the self-clocking of intelligent immunity quantitative analysis instrument is carried out after 15 minutes detecting and showing test results.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105352958A (en) * | 2015-11-28 | 2016-02-24 | 宁波美康生物科技股份有限公司 | Detection reagent kit for overall 25-hydroxy-vitamin-D |
| CN106405072A (en) * | 2016-09-06 | 2017-02-15 | 北京华科泰生物技术有限公司 | Activated fluorescent latex microspheres used for 25-hydroxyvitamin D fluorescence immunochromatography |
| CN108918848A (en) * | 2018-05-22 | 2018-11-30 | 德康润生物科技（北京）有限公司 | Vitamin D releasing reagent and the preparation method and application thereof |
| CN108982882A (en) * | 2018-06-22 | 2018-12-11 | 卢氏实验室公司 | A kind of digital information vitamin D rapid detection system and method |
| CN110441537A (en) * | 2019-08-23 | 2019-11-12 | 北京丹大生物技术有限公司 | A method of measurement 25-hydroxy-vitamin D content |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014042979A1 (en) * | 2012-09-11 | 2014-03-20 | Cornell University | Apparatus and method for point-of-collection measurement of a biomolecular reaction |
| WO2014100715A2 (en) * | 2012-12-21 | 2014-06-26 | Ehrenkranz Joel R L | Supplements and monitoring systems for dosing of the supplements |
| CN104062440A (en) * | 2014-06-30 | 2014-09-24 | 韩雅君 | Test strip for rapidly detecting vitamin D |
-
2015
- 2015-02-06 CN CN201510063491.9A patent/CN104749385A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014042979A1 (en) * | 2012-09-11 | 2014-03-20 | Cornell University | Apparatus and method for point-of-collection measurement of a biomolecular reaction |
| WO2014100715A2 (en) * | 2012-12-21 | 2014-06-26 | Ehrenkranz Joel R L | Supplements and monitoring systems for dosing of the supplements |
| CN104062440A (en) * | 2014-06-30 | 2014-09-24 | 韩雅君 | Test strip for rapidly detecting vitamin D |
Cited By (15)
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| CN105352958B (en) * | 2015-11-28 | 2019-02-26 | 美康生物科技股份有限公司 | Total 25-hydroxy-vitamin D detection kit |
| CN105352958A (en) * | 2015-11-28 | 2016-02-24 | 宁波美康生物科技股份有限公司 | Detection reagent kit for overall 25-hydroxy-vitamin-D |
| CN106405072A (en) * | 2016-09-06 | 2017-02-15 | 北京华科泰生物技术有限公司 | Activated fluorescent latex microspheres used for 25-hydroxyvitamin D fluorescence immunochromatography |
| CN106405072B (en) * | 2016-09-06 | 2018-01-02 | 北京华科泰生物技术有限公司 | 25 hydroxy-vitamine D fluorescence immune chromatographies activation fluorescent latex microballoon |
| CN111936837A (en) * | 2017-02-08 | 2020-11-13 | Essenlix公司 | QMAX determination and uses |
| CN111936837B (en) * | 2017-02-08 | 2024-06-07 | 上海宜晟生物科技有限公司 | QMAX assay and use |
| CN108918848A (en) * | 2018-05-22 | 2018-11-30 | 德康润生物科技（北京）有限公司 | Vitamin D releasing reagent and the preparation method and application thereof |
| CN108982882A (en) * | 2018-06-22 | 2018-12-11 | 卢氏实验室公司 | A kind of digital information vitamin D rapid detection system and method |
| CN110441537A (en) * | 2019-08-23 | 2019-11-12 | 北京丹大生物技术有限公司 | A method of measurement 25-hydroxy-vitamin D content |
| CN110441537B (en) * | 2019-08-23 | 2022-09-13 | 北京丹大生物技术有限公司 | Method for measuring content of 25-hydroxy vitamin D |
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| CN113495159A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | System reaction liquid, 25-hydroxy vitamin D quantitative detection kit and use method thereof |
| CN114702572A (en) * | 2021-11-30 | 2022-07-05 | 南京岚煜生物科技有限公司 | Preparation method of 25-hydroxyvitamin D3 antigen and immunochromatography test strip |
| CN114702572B (en) * | 2021-11-30 | 2023-08-15 | 南京岚煜生物科技有限公司 | Preparation method of 25-hydroxy vitamin D3 antigen and immunochromatography test strip |
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