CN207148125U - Soluble ST2 test strip - Google Patents

Abstract

It the utility model is related to a kind of soluble ST2 test strip, including bottom plate, be sequentially distributed on bottom plate sample-adding portion, ST2 labelled antibodies pad, in capture soluble ST2 antibody test section, be coated with and can capture the Quality Control portion, chromatographic film and the blotting paper that mark soluble ST2 antibody materials, described sample-adding portion is at least sequentially overlapped by three layers of sample pad and formed.By being improved to chromatograph test strip sample-adding portion so that after sample-adding, its liquid significantly improves test strips to the releasability of the label on pad, and faster, the amount of release is more for speed.

Description

Soluble ST2 test strip

Technical field

It the utility model is related to vitro diagnostic techniques field, and in particular to technical field of biological, it is more particularly to a kind of Soluble ST2 test strip.

Background technology

With the increase of living-pattern preservation and aging population degree, the quantity day of China's Patients with Cardiovascular/Cerebrovascular Diseases Benefit increase；Heart failure (abbreviation heart failure) is due to that cardiac structure or dysfunction cause ventricular filling or penetrate what blood ability was damaged One group of complex clinical syndrome, its main clinical manifestation for expiratory dyspnea and it is weak, activity tolerance is limited, and fluid retention, Pulmonary venous pleonaemia and periphery oedema.In heart failure last stage for the serious of various heart diseases and eventually, the incidence of disease and case fatality rate are high, and it is deposited for 5 years Motility rate is close with malignant tumour, is one of current most important angiocardiopathy.

Clinically a variety of difficulties be present to heart failure management at present, such as：The detection of early screening is not popularized；After patient discharge It is difficult to monitor；The discharge guide do not fixed；The species of medication can not be adjusted and determine the dosage of medication；It can not assess and reenter The risk of institute；The high-risk patient for needing most treatment can not be differentiated.

Modern medicine study realizes the management to heart failure by various biomarkers；Common heart failure mark is main Including following 7 class：Myocardial injury markers, myocardium stretching mark, myocardial fibrosis and remodeling thing, marker of inflammation, Oxidative stress mark, Neurohumoral activation mark and renal function mark；It reflects different pathologic, physiologics respectively Process.As myocardial fibrosis and remodeling thing, Soluble growth stimulates the expression factor ((Growth Stim μ Lating Express Gene 2, sST2, or soluble ST2) application in patients with heart failure management more and more widely paid close attention to, 《U.S.'s Heart failure guide 2013》、《Chinese diagnosing patients with heart failure and treatment guidelines 2014》It is middle to propose that soluble ST2 reflecting myocardiums are fine Dimensionization, extraneous information can be provided in the prediction, prognosis and risk stratification of acute and chronic heart failure, be clinically most potential at present , heart failure Management flag thing of new generation.

In summary, soluble ST2 detections have important value to the clinical management of patients with heart failure.

The detection to soluble ST2 is substantially detected using the immunological method of double antibody sandwich method at present, including： Enzyme linked immunosorbent assay (patent：CN106556705A), colloidal gold chromatography (patent：CN103487586B), fluorescence immunoassay layer Analysis method (patent：CN204287196U), chemoluminescence method (patent：CN106199002A), immunoturbidimetry (patent： CN106018789A) etc..

Traditional immuno-chromatographic test paper strip is typically at least made up of sample pad, pad, chromatographic film, adsorptive pads；Due to layer The structure of test strips is analysed, causes differing greatly (batch internal difference is larger) for different test strips.And soluble ST2 in clinical practice in sentencing The critical value (＞ 35ng/mL) of disconnected heart failure with the reference value (8.9~41ng/mL) of normal population closely, and fall ill by heart failure Soluble ST2 concentration elevation amplitude is smaller (median about 45ng/mL) afterwards；It is therefore desirable to the standard of soluble ST2 detection kits True property is higher, especially accuracy preferably (coefficient of variation in batch is small).

The reason for causing Traditional immunochromatographic test strips variation within batch coefficient larger is more, e.g., the environment that test strips use Label release in factor, pad not in time and the reason such as insufficient, wherein, especially with the label release on pad not It is timely and it is insufficient be main cause.To find out its cause, it is because the structure of traditional chromatograph test strip, is to push down knot with sample pad Close pad, pad pushes down chromatographic film, blotting paper pushes down the structure of chromatographic film, and sample pad pushes down the part of pad, due to hair Spy is with the combination pad part for causing to be held down, and label thereon will not be released or rate of release is slower；Especially work as difference Clinical sample, its property (the mainly viscosity of the sample caused by the factors such as blood fat) differs greatly, and causes sample to be added dropwise After to sample pad swimming drawn by capillary action to pad, its releasing degree on the label on pad, speed influence pole Greatly；Above-mentioned reason, it is to cause soluble ST2 immuno-chromatographic assay technologies using one of the main reason for being limited with clinical diagnosis.

The content of the invention

In order to solve the above problems, the utility model provides a kind of soluble ST2 test strip, including bottom plate, bottom Be sequentially distributed on plate sample-adding portion, ST2 labelled antibodies pad, in capture soluble ST2 antibody test section, be coated with Quality Control portion, chromatographic film and the blotting paper for marking soluble ST2 antibody materials can be captured, described sample-adding portion is at least by three layers of sample Product pad, which is sequentially overlapped, to be formed.

More preferably, described sample-adding portion is formed by stacking by upper strata sample pad, intermediate layer print pad and bottom sample pad.

More preferably, described intermediate layer sample pad caves in form " recessed " shape structure.

More preferably, between described pad insertion upper strata sample pad and bottom sample pad, and it is connected with meso sample pad； Levels sample pad and pad form interlayer structure.

More preferably, described chromatographic film is by nitrocellulose, cellulose acetate film, nylon membrane, pvdf membrane or Fusion 5 Filter paper is made.

Specifically, cave in form the structure of " recessed " shape with the sample of three layers of sample pad superposition and intermediate layer, by pad Insertion is simultaneously connected with the sample pad in intermediate layer, forms interlayer structure；The sample pad of the test strips, pad, chromatographic film, adsorptive pads It is sequentially connected etc. structure；Wherein, by the ST2 labelled antibody speckings of detection on pad, the bag such as capture antibody (detection line) By in chromatographic film, the soluble ST2 test strips that production technology is relatively simple but detection performance significantly improves are formed.

Described Quality Control portion is coated with the material that can be combined with label, wherein described mark substance, including：Colloid Gold, fluorescein (microballoon) or latex beads etc..

During detection, sample to be measured is added in sample pad, interlayer structure is formed through single layered substrate swimming to detection line, it is right Soluble ST2 albumen in sample to be tested is detected.By being improved to chromatograph test strip sample application zone (sample pad) so that Test strips are after sample-adding, and its liquid (sample or buffer solution) significantly improves to the releasability of the label on pad, speed Faster, the amount of release is more.

The improved chromatograph test strip, after sample to be tested adds, three layers of sample pad is comprehensive to parcel from upper, middle and lower Chromatography swimming is carried out in interior pad, so as to accelerate the releasability of label on pad, including accelerates the speed of release Degree, and improve release rate.

Pass through the abundant release of label on pad so that the detection of soluble ST2 test strips of the invention It can significantly improve, being mainly manifested in accuracy (variation within batch coefficient, CV) significantly reduces, and accelerates the detection speed of test strips Degree.

The beneficial effects of the utility model mainly have：

(1) detection performance of test strips significantly improves, and especially the accuracy (variation within batch coefficient, CV) of testing result is aobvious Writing reduces；

(2) detection speed of test strips is improved.

Brief description of the drawings

The top view of Fig. 1 test strip structures of the present utility model

The side view of Fig. 2 test strip structures of the present utility model

The enlarged drawing in sample-adding portion in Fig. 3 test strip structures of the present utility model

Part reference：1- sample-addings portion, 11- upper stratas sample pad, 12- intermediate layers sample pad, 13- bottom sample pads, 2- Pad, 3- test sections, 4- Quality Controls portion；5- chromatographic films, 6- bottom plates, 7- blotting papers

Embodiment

In order to be more clearly understood that the technology contents of the utility model patent, described in detail especially exemplified by following examples. The preparation process of the test strips of the soluble ST2 detection methods of patent of the present invention is summarized as follows, it should be appreciated that these embodiments are only used In the explanation present invention rather than limitation the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, lead to Often according to normal condition, such as Sambrook et al., molecule clone technology Laboratory Manual (New York:Cold Spring Harbor Laboratory Press, 2005) condition described in, or according to the condition proposed by manufacturer.

The soluble ST2 test strips structure of embodiment 1

A kind of soluble ST2 test strip, including bottom plate 6, it is anti-that sample-adding portion 1, ST2 marks are sequentially distributed on bottom plate Body pad 2, in capture soluble ST2 antibody test section 3, be coated with and can capture the soluble ST2 antibody thing of mark Quality Control portion 4, chromatographic film 5 and the blotting paper 7 of matter, sample-adding portion 1 are at least sequentially overlapped by three layers of sample pad and formed.

Sample-adding portion 1 is sequentially overlapped and formed by upper strata sample pad 11, intermediate layer sample 12 and bottom sample pad 13；Wherein, in Interbed sample pad 12 is caved in form " recessed " shape structure, and pad 2 is embedded between upper strata sample pad 11 and bottom sample pad 13, and with Meso sample pad 12 connects；Upper and lower layer sample pad and pad 2 form interlayer structure.

The fluorescence labeling specific antibody of embodiment 2

Cleaning：Fluorescent microsphere is taken (to be purchased from Bangs Lab, article No. within 2014：11233) into centrifuge tube, 0.1M MES are added (pH 5.0) buffer solution mixes, and with 13000rpm, 15min, 4 DEG C of pelleted by centrifugation, abandons supernatant, delayed with 0.1M MES (pH 5.0) Fliud flushing is resuspended stand-by.Activate and clean：By activator EDC, NHS and fluorescent microsphere according to mass ratio ratio 2:1:2 amount is lived Change, concrete operations are as follows：

Weigh EDC, NHS and add in 0.1M MES (pH 5.0) buffer solution and dissolve, take cleaned in right amount to 4.3.3.1 rapidly In complete fluorescent microsphere, sealed with sealed membrane and be placed on room temperature on 200rpm shaking tables and shake up 30min, 13000rpm after taking-up, 30min, 4 DEG C are centrifuged off supernatant, and ultrasound mixing is resuspended with equivalent 0.1M MES (pH 6.5) buffer solution and cleans, is repeated once Operation is cleaned twice above.It is stand-by that supernatant is discarded after the completion of centrifugation.

Mark：Latex after activation is resuspended into 0.1M MES (pH 6.5) buffer solution, is separately added into specificity rapidly (mouse monoclonal antibody, Britain BBI Solutions, article No. are purchased from reference to soluble ST2 antibody within 2016：102321), mix, place Room temperature, shake up 4 hours on 200rpm shaking tables.

Closing：The good microballoon of above-mentioned mark is taken to be centrifuged off supernatant in 10000rpm, 30min, 4 DEG C, immediately into microballoon Equivalent confining liquid is added, ultrasound is placed room temperature after being resuspended, shaken up on 200rpm shaking tables 1 hour again.10000rpm after the completion of reaction, 20min, 4 DEG C of centrifugations, takes supernatant to be checked.

Detection content	Quality standard	Detection method
			Package amount	The μ g antibody of package amount >=100/mg microballoons	BCA determination of protein concentration methods

＆ is cleaned to be resuspended：Re-suspension liquid is added, ultrasound piping and druming is resuspended, with high speed freezing centrifuge 10000rpm, 20min, 4 DEG C Centrifugation, discards supernatant；Re-suspension liquid is added, ultrasound piping and druming is resuspended.

The microspheres solution being resuspended carries out mark, standby.

The specking of the pad of embodiment 3

The specking method for specifically binding soluble ST2 pads filter paper is as follows：Fluorescence labeling solution dilutes：With fluorescence mark Remember that the fluorescence labeling conjugate (coming from embodiment 1) of above-mentioned preparation is diluted 2 times by re-suspension liquid；Point film instrument is set, opens point film instrument Power supply, set specking program, specking amount is 8 μ L/cm；No. 1 pipeline is specking passage；Point film instrument initializes：No. 1 pipeline is put It is resuspended in fluorescence labeling in solution, selects initialization program, initializes 6 circulations；Specking：By Whatman Fusion5 filter paper Lain in by fixed position in point film instrument, press on control panel " GO " key and start specking, removed after having put, check the good knot of specking Close pad, the fluorescence labeling solubility ST2 specific antibodies band of specking uniformly, the continuous and whole straight line of insertion be qualified specking product, It is unqualified specking product to occur breakpoint in straight line；A piece of pad filter paper is often put, it is specking to press " GO " key on a control panel Once (a piece of)；Specking terminates, and the pad of specking is placed in room temperature and spontaneously dried 1 hour, specking vestige should be can't see on film.

The preparation (specific antibody) of the detection line of embodiment 4 and nature controlling line

The antibody for specifically binding soluble ST2 (mouse monoclonal antibody, is purchased from Britain BBI Solutions, article No. in 2016： 102322) taken out from refrigerator, place room temperature, it is standby.

Preparation method is as follows：The anti-soluble ST2 of mouse antibody 500ug is taken, is added in 5ml graduated centrifuge tubes, antibody dilution Liquid is to 1ml, Container Tag T flag.Sheep anti-mouse igg antibody is taken (to open Thailand, article No. purchased from Hangzhou within 2016：B103-GMI01)10μ L, it is added in 5ml graduated centrifuge tubes, antibody diluent to 1ml, Container Tag C marks.Point film instrument is set, opens the electricity of point film instrument Source, specking program is set, specking amount is 1 μ L/cm；No. 1 pipeline is detection line specking passage, and No. 2 pipelines are that nature controlling line specking leads to Road；Point film instrument initializes：No. 1 pipeline is placed in detection line solution, No. 2 pipelines are placed in nature controlling line solution, selection is initial Change program, initialize 6 circulations；Specking：Nitrocellulose filter (NC films) is lain in point film instrument by fixed position, by control " GO " key starts specking on panel, is removed after having put, and checks the good detection line of specking, detection line and nature controlling line be two uniformly, Continuous and insertion straight line is qualified specking product, and it is unqualified specking product to occur breakpoint in two straight lines；Often put it is a piece of, by once " GO " key on control panel for specking once (a piece of)；Specking terminates, and it is small that the NC films of specking are placed in into natural drying 1 in room temperature When, specking vestige should be can't see on film.

Embodiment 5 assembles

The protection sheet of wider portion on bottom plate is removed, along the lower edge of protection sheet above, the good NC films of specking (come from Embodiment 3), by nature controlling line up in a manner of be attached on bottom plate, stick absorbent filter above NC films；In the most lower of bottom plate Sample pad is sticked by side, then pad is attached between sample pad and NC films, and pad both ends are press respectively against NC films and sample On pad (coupling part is generally 1~3mm or so)；Hydrophilic pressure-sensitive glue (3M, article No. are coated in bottom sample pad：CA- 40H), the sample pad in intermediate layer is then sticked, intermediate layer sample pad is alignd with pad and connection of trying one's best；Finally in intermediate layer sample Hydrophilic pressure-sensitive glue is coated on product pad, then sticks upper strata sample pad；Upper strata sample pad covers pad about 1~3mm.

Kilocalorie is assembled into according to said procedure.

Embodiment 6 is cut

Cutting electromechanical source is connected, setting cuts film program, sets cutting width as 4mm；Kilocalorie (coming from embodiment 4) is kept flat Enter in cutting machine platform track, face-up, press on guidance panel " GO " key, start to cut；A piece of kilocalorie certified products are often put, are pressed On guidance panel " GO " key once, until cut all kilocalorie certified products；After the completion of cutting, test strips are sticked in into bottom side by side On plate, test strip is formed.

The test strips of embodiment 7 assemble

Above-mentioned test strips (coming from embodiment 5) are fitted into cartridge, form detection card.

Take aluminium foil bag and drier；Open heat sealing machine, preheating；Detection card to be packed, 1 bag of drier are loaded into aluminium foil bag In；According to aluminium foil bag of the length cut-out of regulation equipped with detection card and drier；Aluminium foil bag is sealed with heat sealing machine；It is labelled.

Soluble ST2 in the ELISA test strip clinical sample of embodiment 8

By above-mentioned detection card (coming from embodiment 6), verified with clinical sample.

Sample to be tested is added drop-wise in the sample aperture of detection card, is subsequently placed in fluorescence detector and carries out reading.

Testing result and goldstandard (double antibody sandwich ELISA, the U.S. Critical of soluble ST2 detections Diagnostics companies) it is as follows：

	Double antibody sandwich method is positive	Double antibody sandwich method is negative	It is total
				Detection card detection is positive	72	1	73
Detection card detection is negative	0	24	24
				It is total	72	25	97

It can be obtained according to upper table：

The detection sensitivity of the test strips is：72/ (72+0) × 100%=100%；

The detection specificity of the test strips is：24/ (24+1)=96%.

Above testing result shows that the accuracy rate that this test strip detects soluble ST2 is higher, disclosure satisfy that clinical answer It is required that.

The ELISA test strip solubility ST2 of embodiment 9 accuracy

Above-mentioned detection card (coming from embodiment 7), and sample application zone improved soluble ST2 detections card (component is not made into From embodiment 1~3, only kilocalorie process is assembled according to the conventional method, with sample pad, pad, chromatographic film and absorbent filter The mode being sequentially connected), verified with quality-control product.

Quality-control product of the soluble ST2 concentration for 35ng/mL is used, the detection card of embodiment 6 blocks with not doing improved detection It is each to detect 10 times；Sample to be tested is added drop-wise in the sample aperture of detection card, is subsequently placed in fluorescence detector and carries out reading；Detection As a result it is used to calculate variation within batch coefficient (CV), it is as a result as follows：

Above testing result shows that the accuracy that the present invention improves the soluble ST2 of test strip detection of sample application zone shows Write and improve, can preferably meet the requirement of clinical practice.

The ELISA test strip solubility ST2 of embodiment 10 time

Above-mentioned detection card (coming from embodiment 6), and sample application zone improved soluble ST2 detections card (component is not made into From embodiment 1~3, only kilocalorie process is assembled according to the conventional method, with sample pad, pad, chromatographic film and absorbent filter The mode being sequentially connected), verified with quality-control product.

Quality-control product of the soluble ST2 concentration for 35ng/mL is used, the detection card of embodiment 6 blocks with not doing improved detection It is each to detect 10 times；Sample to be tested is added drop-wise in the sample aperture of detection card, is subsequently placed in fluorescence detector and carries out observation timing, Untill the nature controlling line fluorescent value started with sample-adding to detection card does not change, the detection time (second, s) of detection card is designated as；And The average detected time of two kinds of cards is calculated, it is as a result as follows：

Testing result above shows that the speed that this test strip detects soluble ST2 significantly improves (detection time drop Low more than 50%) requirement of clinical practice can preferably, be met.

It should be noted that the foregoing is only preferred embodiment of the present utility model, this reality is not limited to With new scope, all made any modifications within the spirit and principles of the utility model, equivalent replacement and improvement Deng should be included within the scope of protection of the utility model.

Claims (5)

1. a kind of soluble ST2 test strip, including bottom plate, sample-adding portion, ST2 labelled antibody knots have been sequentially distributed on bottom plate Close pad, in capture soluble ST2 antibody test section, be coated with and can capture the matter for marking soluble ST2 antibody materials Control portion, chromatographic film and blotting paper, it is characterised in that described sample-adding portion is at least sequentially overlapped by three layers of sample pad and formed.

2. soluble ST2 according to claim 1 test strip, it is characterised in that described sample-adding portion is by upper strata Sample pad, intermediate layer print pad and bottom sample pad are formed by stacking.

3. soluble ST2 according to claim 2 test strip, it is characterised in that described intermediate layer sample pad Cave in form " recessed " shape structure.

4. soluble ST2 according to claim 3 test strip, it is characterised in that in described pad insertion Between layer print pad and bottom sample pad, and it is connected with meso sample pad.

5. the test strip of the soluble ST2 according to any one of claim 1-4, it is characterised in that described layer Analysis film is made up of nitrocellulose, cellulose acetate film, nylon membrane, pvdf membrane or the filter paper of Fusion 5.