CN104345150A - Glycated albumin detection immunochromatography test trip and preparation method thereof - Google Patents
Glycated albumin detection immunochromatography test trip and preparation method thereof Download PDFInfo
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Abstract
The invention provides a glycated albumin detection immunochromatography test trip and a preparation method thereof. The test trip comprises a liner, an analysis membrane disposed in the middle part of the liner, a water absorption pad arranged at one end of the analysis membrane upper part, a conjugate pad arranged at the other end of the analysis membrane upper part, and a sample pad disposed at one end of the conjugate pad upper part. The analysis membrane is provided with detection lines and a quality control line. The detection lines include a glycated albumin detection line and a hemoglobin detection line. The preparation method comprises: preparation of a phenylboronic acid marker, preparation of the sample pad, preparation of the conjugate pad, preparation of the detection line and quality control line on the analysis membrane, preparation of the water absorption pad and preparation of the glycated albumin detection immunochromatography test trip. The glycated albumin detection immunochromatography test trip provided by the invention can realize quantitative detection of the glycated albumin content in 3-5min only with a trace amount of a whole blood sample, greatly improves the screening speed, and has the advantages of high sensitivity, good specificity and simple structure.
Description
Technical field
The present invention relates to technical field of immunoassay, particularly relate to a kind of detection glycosylated albumin immuno-chromatographic test paper strip and preparation method thereof.
Background technology
Blood sugar monitoring is a very important link in diabetes daily diagnosis and treatment work, and good glycemic control effectively can delay generation and the development of the acute and chronic complication of diabetes.Blood sugar will be made in clinical position comprehensively up to standard for a long time, and the good control of short-term averaging blood sugar is extremely important.Glycated albumin (glycated albumin, GA) is one of index of blood sugar monitoring clinically, for evaluating diabetic's recent glycemic control situation and reflecting that the change of blood sugar level in a short time has higher clinical value.
In human body, glucose and protein generation nonenzymatic glycosylation react and namely form glycated protein.Glycosylated hemoglobin (HbA1c) is glucose and endoerythrocytic haemoglobin a kind of glycoprotein that non-enzymatic reaction is formed in 120 days of its life in blood, therefore the overall glycemic level measuring patient in first 2 ~ 3 months can be reflected, it is the goldstandard evaluating long-term blood glucose control in the world, because of haemoglobin long half time, its measured value is difficult to the recent blood sugar level of reflection patient; Fructosamine (GSP) is the product that non-enzymatic reaction occurs for haemocyanin (mainly albumin) and glucose, because the albuminous half life period is 17 ~ 19 days, therefore its value can reflect the average level measuring first 2 ~ 3 weeks blood sugar, it is the index being used for judging parameters of short term glycemic control at present clinically.But be glycosylated plasma protein matter total in reflection blood plasma because GSP measures, its value is subject to the impact of protein concentration in blood, cholerythrin, chyle and lower-molecular substance etc., especially transforms abnormal patient at Hypoproteinemia and albumin; Because this reaction also can occur material non-specific in serum, the nonenzymatic glycosylation reaction rate of different protein component is different in addition, therefore GSP detection method poor specificity.And GA detection is to the quantitative measurement that GA carries out on the basis of GSP, utilize serum glycated albumin and sero-abluminous number percent to represent the level of GA, thus eliminate the impact of serum albumin levels on testing result, therefore comparatively GSP is more accurate for GA.
As far back as the eighties in last century, Japanese scholars just have developed high pressure liquid-phase ion exchange (HPLC method) and carries out GA mensuration.HPLC method measures the aggregate level that GA accurately can detect patient's glycemic control in a short time, but at that time because it is of a high price, process sample size is little, is not suitable for routine clinical development and is not used widely.The enzyme process of the application liquid reagent in recent years developed by Japan detects GA(GA-L) be a kind of simple, fast, sensitive, the detection method of accurate quantitative analysis, develop liquid reagent on the basis that solid enzyme process (the glycated albumin assay method that the species specificity developed by the U.S. for 2002 is higher) measures glycated serum protein, decrease dissolution process, improve operability, add with glycated amino acid to remove the impact of endogenous glycated amino acid on testing result, and utilize the bromcresol purple higher to oxidisability albumin specificity to substitute original bromcresol green, decrease the impact of globulin on measurement result, therefore result is more accurate.GA-L detects has good dilution rectilinearity, in a few days repeatability and stability in the daytime, and has good consistance (r=0.879) with HPLC detection method.(the Yamaguchi M such as Yamaguchi in 2005, Kambe S, Eto T, et al.Biosens Bioelectron, 2005,21:426-432) report a kind of detection system applying the enzymatic assays GA of dry chemical reagent, this detector needs blood specimen amount little, can measure GA numerical value within 5min, compare with GA-L, its accuracy of detection shows good linear correlation (r=0.879).
GA detects average blood glucose levels and the change that accurately can reflect diabetic's short-term all sidedly, has important clinical value.The improvement of GA detection method, from the HPLC method that last century is applied at first, to the liquid reagent method in recent years developed, so that the dryness enzyme process recently reported, detection method is progressively tending towards easy, fast, accurate and practical.GA can be used as the Con trolling index of the recent blood sugar of diabetic and metabolic condition clinically, to early detection diabetes, instructs the treatment of diabetes to have greater significance.
Method in sum, all also exists many problems to a certain extent, urgently proposes a kind of new method and solves these problems.
Summary of the invention
An object of the present invention is to provide a kind of detection glycosylated albumin immuno-chromatographic test paper strip, it needs the whole blood sample of trace, the content quantitatively detecting glycosylated albumin can be realized in 3 ~ 5 minutes, substantially increase the speed of examination, have highly sensitive, specificity good and the simple advantage of structure.
Another object of the present invention is to provide a kind of preparation method detecting glycosylated albumin immuno-chromatographic test paper strip, and its preparation method is simple, is easy to large-scale production.
Technical scheme of the present invention is achieved in that
A kind of detection glycosylated albumin immuno-chromatographic test paper strip, comprise liner, be arranged at the analyzing film in the middle part of described liner, be arranged at the adsorptive pads of described analyzing film upper one end, be arranged at the bond pad of the described analyzing film top other end and be arranged at the sample pad of described bond pad upper one end, described analyzing film is provided with detection line and nature controlling line, described detection line comprises glycosylated albumin detection line and albumin detection line, described nature controlling line, described albumin detection line and described glycated protein detection line are arranged in order along described analyzing film upper one end to the other end.
Preferably, described adsorptive pads 1 ~ 2mm overlapping with described analyzing film, described bond pad 1 ~ 2mm overlapping with described analyzing film, described sample pad and the overlapping 1 ~ 2mm of described bond pad.
Detect a preparation method for glycosylated albumin immuno-chromatographic test paper strip, comprise the steps:
1) preparation of phenyl boric acid label: diluted by fluorescent material damping fluid, adds phenyl boric acid lysate, and 20 ~ 25 DEG C are reacted at least 1 hour, cross column separating purification with the gel column that specifications and models are G25, collect label, are phenyl boric acid label;
2) preparation of sample pad: with cellulose membrane as sample pad solid phase material, soak with the first phosphate buffer that substance withdrawl syndrome is 0.01 ~ 0.3mol/L, described first phosphate buffer pH value is 7.2 ~ 7.6, after immersion, be dried process, obtain sample pad;
3) preparation of bond pad: with glass fibre element film as bond pad solid phase material, with substance withdrawl syndrome be 0.01 ~ 0.1mol/L, pH value be 7.2 the second phosphate buffer dilute described phenyl boric acid label, make suspension, described suspension is sprayed on glass fibre element film, drying process is carried out to it, obtains bond pad;
4) preparation of detection line and nature controlling line on analyzing film: with nitrocellulose filter as solid phase carrier, be analyzing film, with the antibody that triphosphate damping fluid dilution detection line and nature controlling line use, adopt on the Membrane jetter detection line that is sprayed on described analyzing film respectively and nature controlling line position, analyzing film after spray film is carried out drying process, obtains the analyzing film with detection line and nature controlling line;
5) preparation of adsorptive pads: the filter paper selecting 1mm thick, as adsorptive pads solid phase material, is cut into the band of 25 × 300mm, obtains adsorptive pads;
6) preparation of glycosylated albumin immuno-chromatographic test paper strip is detected: first stick on liner centre position by described analyzing film, adsorptive pads and bond pad is adhered in described analyzing film upper end, sample pad is pasted again in described bond pad upper end, by described liner and be arranged at the described sample pad on described liner top, described bond pad, described analyzing film and described adsorptive pads and together cut into strip, be and detect glycosylated albumin immuno-chromatographic test paper strip.
Preferably, in described step 1, the first damping fluid is identical with the first phosphate-buffered fluid component in described step 2, it comprises: be calculated in mass percent, the polyglycol of 0.01% ~ 0.5%, the bovine serum albumin(BSA) of 1% ~ 5% and 0.01% ~ 0.05% first surface activating agent.
Preferably, in described step 3, phosphate buffer comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 1% ~ 5%, the polyglycol of 0.1 ~ 2%, the sucrose of 0.5 ~ 2% and the second surface activating agent of 0.01% ~ 0.1%.
Preferably, in described step 4, in triphosphate damping fluid, substance withdrawl syndrome is 0.05mol/L, pH value is 7.4 ~ 7.6, comprises: be calculated in mass percent, the methyl alcohol of 0.5 ~ 1% and the bovine serum albumin(BSA) of 0.8 ~ 1.5% in described triphosphate damping fluid.
Further, described first surface activating agent and second surface activating agent are the one in Tween20, Triton X-100 and tetronic1307.
Preferably, in described step 4, antibody is the one in anti-glycosylated albumin monoclonal antibody, antialbumin monoclonal antibody and sheep anti-mouse igg antibody.
Preferably, in described step 6 liner for be made up of pet material.
The beneficial effect that the present invention produces is: one aspect of the present invention proposes a kind of detection glycosylated albumin immuno-chromatographic test paper strip, it needs the whole blood sample of trace, the content quantitatively detecting glycosylated albumin can be realized in 3-5 minute, substantially increase the speed of examination, have highly sensitive, specificity good and the simple advantage of structure; Propose a kind of preparation method detecting glycosylated albumin immuno-chromatographic test paper strip on the other hand, its preparation method is simple, is easy to large-scale production.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is that the present invention's one detects phenyl boric acid label reaction principle schematic diagram in glycosylated albumin immuno-chromatographic test paper strip;
Fig. 2 is a kind of structural representation detecting glycosylated albumin immuno-chromatographic test paper strip of the present invention;
Fig. 3 is that the present invention's one detects glycosylated albumin immuno-chromatographic test paper strip test example 1 neutral line examination criteria working curve diagram;
Fig. 4 is that a kind of detection in glycosylated albumin immuno-chromatographic test paper strip test example 5 contrasts detection regression curve;
In figure: 1 sample pad; 2 bond pads; 3 analyzing films; 4 glycosylated albumin detection lines; 5 albumin detection lines; 6 nature controlling lines; 7 adsorptive pads; 8 liners.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The total technical scheme of the present invention is as follows:
As Figure 1-4: a kind of detection glycosylated albumin immuno-chromatographic test paper strip, comprise liner 8, be arranged at analyzing film 3 in the middle part of liner, be arranged at the adsorptive pads 7 of analyzing film upper one end, be arranged at the bond pad 2 of the analyzing film top other end and be arranged at the sample pad 1 of bond pad upper one end, analyzing film is provided with detection line and nature controlling line 6, described nature controlling line, described albumin detection line and described glycated protein detection line are arranged in order along described analyzing film upper one end to the other end.
Adsorptive pads 1 ~ 2mm overlapping with analyzing film, bond pad 1 ~ 2mm overlapping with analyzing film, sample pad 1 ~ 2mm overlapping with bond pad.Detection line comprises glycosylated albumin detection line 4 and albumin detection line 5.Glycosylated albumin detection line, albumin detection line, nature controlling line interval 5mm successively.
In the present invention, sample pad detects the position that glycosylated albumin immuno-chromatographic test paper strip in use drips testing sample.Be fixed with phenyl boric acid material marking fluorescent material and fluorescent material marks antibody isoreactivity molecular bond in bond pad, after adding testing sample, start Ag-Ab immune response occurs at this.Analyzing film is the core of chromatographic test paper, is fixed with glycosylated albumin detection line, albumin detection line and nature controlling line in its surface respectively; Detection line contains, with glycosylated albumin in testing sample, immunoreactive antibody occurs, and detection line contains, with albumin in testing sample, immunoreactive antibody occurs, and nature controlling line contains and the raw immunoreactive antibody of fluorescent material marks produce.Adsorptive pads, in whole testing process, provides liquid to flow through the power of whole test strips by syphonic effect.Overlapping region is had, to ensure the continuity that liquid flows in test strips between each several part.
The principle of the detection glycosylated albumin immuno-chromatographic test paper strip that the present invention proposes is: when detecting, sample drop is added in sample pad, sample enters bond pad by infiltration and syphonic effect, fluorescent material marks thing is wherein made to dissolve release, under the syphonic effect of adsorptive pads, liquid enters analyzing film, flows through glycosylated albumin detection line, albumin detection line and nature controlling line successively, and there is specific immune response, produce and there is tell-tale fluorescence signal.
Detect a preparation method for glycosylated albumin immuno-chromatographic test paper strip, comprise the steps:
1) preparation of phenyl boric acid label: diluted by fluorescent material damping fluid, adds phenyl boric acid lysate, and 20 ~ 25 DEG C are reacted at least 1 hour, cross column separating purification with the gel column that specifications and models are G25, collect label, are phenyl boric acid label;
2) preparation of sample pad: with cellulose membrane as sample pad solid phase material, soak with the first phosphate buffer that substance withdrawl syndrome is 0.01 ~ 0.3mol/L, the first phosphate buffer pH value is 7.2 ~ 7.6, after immersion, be dried process, obtain sample pad;
3) preparation of bond pad: with glass fibre element film as bond pad solid phase material, with substance withdrawl syndrome be 0.01 ~ 0.1mol/L, pH value be 7.2 the second phosphate buffer dilution phenyl boric acid label, make suspension, suspension is sprayed on glass fibre element film, drying process is carried out to it, obtains bond pad;
4) preparation of detection line and nature controlling line on analyzing film: with nitrocellulose filter as solid phase carrier, be analyzing film, with the antibody that the second damping fluid dilution detection line and nature controlling line use, adopt on the Membrane jetter detection line that is sprayed on analyzing film respectively and nature controlling line position, analyzing film after spray film is carried out drying process, obtains the analyzing film with detection line and nature controlling line;
5) preparation of adsorptive pads: the filter paper selecting 1mm thick, as adsorptive pads solid phase material, is cut into the band of 25 × 300mm, obtains adsorptive pads;
6) preparation of glycosylated albumin immuno-chromatographic test paper strip is detected: first sticked to by analyzing film on liner centre position, adsorptive pads and bond pad is adhered in described analyzing film upper end, sample pad is pasted again in described bond pad upper end, by described liner and be arranged at the described sample pad on described liner top, described bond pad, described analyzing film and described adsorptive pads and together cut into strip, be and detect glycosylated albumin immuno-chromatographic test paper strip.
In the present invention, fluorescent material can be carbazole, pyrazine, thiazole derivative or metal porphyrins, the present inventor is different through the best performance of overtesting determination metal porphyrins, preferable alloy porphyrin fluorescent material in the present invention, metalloporphyrin is platinum/palladium porphyrin, the excitation light spectral limit of metalloporphyrin is 390 ~ 420nm, and wavelength of transmitted light scope is 600 ~ 700nm.
In the present invention; damping fluid, the first phosphate buffer, the second phosphate buffer can be identical with its substance withdrawl syndrome of triphosphate damping fluid, pH value and component; can be different; it is all within protection scope of the present invention, for different component out cited by specific embodiment be the present inventor do experiment in some of them preferred embodiment.
The glycosylated albumin immuno-chromatographic test paper strip of phenyl boric acid labelling technique is detecting the application in biological sample, and detected object is the content detection of glycosylated albumin in whole blood sample.
In the present invention, for quantitative test item, by setting up determinand standard items and fluorescence signal intensity typical curve, realize quantitative detection.
When carrying out sample detection, be added in by sample drop in sample pad, sample enters bond pad by infiltration and syphonic effect, the bond that phosphor material is wherein marked dissolves again, and under the syphonic effect of adsorptive pads, discharge from bond pad and enter analyzing film, flow to adsorptive pads direction.In analyzing film in moving process, between fluorescent marker, target determinand, detection line, nature controlling line, specific immune response will be there is, and at detection line and nature controlling line generation, there is tell-tale light signal.
Embodiment 1
Detect a preparation method for glycosylated albumin immuno-chromatographic test paper strip, comprise the steps:
1) preparation of phenyl boric acid label:
By antialbumin monoclonal antibody and sheep anti-mouse igg antibody, be 0.1mol/L, pH with substance withdrawl syndrome be respectively that the sodium bicarbonate-carbonate solution dilution of 9.6 is to 1mg/ml, respectively get 5ml antibody-solutions, add 30mg fluorescent material metalloporphyrin lysate respectively, stir evenly, incubated at room 1 hour, every mixing in 15 minutes once.Finally cross column separating purification with the gel column that specifications and models are G25, collect the metalloporphyrin mark anti-albumin antibodies and sheep anti-mouse igg antibody that have marked, with substance withdrawl syndrome be 0.01mol/L, pH value be 7.2 first phosphate buffer dilution, wherein the first phosphate buffer comprise containing mass percent be 0.1% polyglycol, the bovine serum albumin(BSA) of 2.0%, the first surface activating agent of 0.05%, pack with reagent bottle, preserve under 2 ~ 8 DEG C of conditions.
2) preparation of sample pad:
Select cellulose membrane as the solid support material of sample pad, cut into the band of 5 × 300mm specification.By sample pad as in rectangular tank, with substance withdrawl syndrome be 0.05mol/L, pH value be 7.4 first phosphate buffer dilution soak 30min, wherein the first phosphate buffer comprise containing mass percent be 0.2% polyglycol, the bovine serum albumin(BSA) of 2.5%, the first surface activating agent of 0.03%.After immersion treatment, sample pad is taken out, is placed on clean network, the drying box inner drying putting into 60 DEG C after 80 minutes taking-up aluminium foil bag to vacuumize sealing for subsequent use.
3) preparation of bond pad:
Select glass fibre element film as the solid phase carrier of bond pad, cut into the band of 5 × 300mm specification.Phenyl boric acid material marking fluorescent material, fluorescence labeling anti-albumin antibodies and sheep anti-mouse igg antibody that 2 ~ 8 DEG C are saved backup, with substance withdrawl syndrome be 0.01mol/L, pH value be 7.2 second phosphate buffer dilution make suspension, wherein the second phosphate buffer comprise containing mass percent be 0.3% polyglycol, the bovine serum albumin(BSA) of 2.4%, the sucrose of 2% and 0.05% second surface activating agent.With the line of Membrane jetter spray film, film liquid measure is 10ul/mm, is then placed on clean network, the drying box inner drying putting into 37 DEG C after 60 minutes taking-up aluminium foil bag vacuumize sealing and preserve.
4) preparation of detection line and nature controlling line in analyzing film:
The preparation of glycosylated albumin detection line coating buffer: with 50ml substance withdrawl syndrome be 0.05mol/L, pH value is the phosphate buffer of 7.4, wherein, be calculated in mass percent, this phosphate buffer includes methyl alcohol 0.5%, trehalose 0.8%, bovine serum albumin(BSA) 1.5%, dilutes anti-glycosylated albumin antibody to final concentration 1.6mg/ml.
The preparation of albumin detection line coating buffer: with 50ml substance withdrawl syndrome be 0.05mol/L, pH value is the phosphate buffer of 7.6, wherein, be calculated in mass percent, this phosphate buffer includes methyl alcohol 1%, trehalose 0.6%, bovine serum albumin(BSA) 0.8%, and dilution anti-albumin antibodies is to final concentration 2mg/ml.
The preparation of nature controlling line coating buffer: with 50ml substance withdrawl syndrome be 0.05mol/L, pH value is the phosphate buffer of 7.4, wherein, be calculated in mass percent, this phosphate buffer includes methyl alcohol 1%, bovine serum albumin(BSA) 0.8%, and dilution mouse IgG antibody is to final concentration 0.5mg/ml.
Select nitrocellulose filter as solid phase carrier, be analyzing film, cut into the band of 25 × 300mm specification.Rule successively on the analyzing film that 25mm is wide with Membrane jetter, the line of the film of 10mm place spray from the bottom up, film liquid measure is 2ul/mm, as glycosylated albumin detection line detection line; Interval 5mm place again, the line of the film of 15mm place spray from the bottom up, film liquid measure is 2ul/mm, as albumin detection line detection line; With Membrane jetter on the analyzing film that 25mm is wide, the line of the film of 20mm place spray from the bottom up, film liquid measure is 1.5ul/mm, as nature controlling line.Glycosylated albumin detection line detection line, albumin detection line detection line and nature controlling line interval 5mm successively, line fine uniform, places 37 DEG C of drying box process 50 minutes by analyzing film, vacuumize pack sealing save backup after taking-up with aluminium foil bag.
5) preparation of adsorptive pads:
The filter paper selecting 1mm thick, as adsorptive pads solid phase material, is cut into the band of 25 × 300mm.Adsorptive pads saves backup at dry environment.
6) preparation detects the immuno-chromatographic test paper strip of glycosylated albumin:
First analyzing film is sticked on liner centre position that pet material makes, adsorptive pads and bond pad is adhered in analyzing film upper end, sample pad is pasted again in bond pad upper end, by liner and be arranged at the sample pad on liner top, bond pad, analyzing film and adsorptive pads and together cut into strip, be and detect glycosylated albumin immuno-chromatographic test paper strip.
First surface activating agent and second surface activating agent are the one in Tween20, Triton X-100 and tetronic1307, and preferably first surface activating agent and second surface activating agent are Tween20 in the present embodiment.
Embodiment 2
Identical with embodiment 1, difference is:
In step 1 and step 2, damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.05mol/L, and pH value is 7.4, it comprises: be calculated in mass percent, the polyglycol of 0.01%, the bovine serum albumin(BSA) of 1% and 0.01% first surface activating agent.
In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.5mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 1%, the polyglycol of 0.1%, the sucrose of 0.5% and the second surface activating agent of 0.01%.
First surface activating agent and second surface activating agent are Triton X-100.
Embodiment 3
Identical with embodiment, difference is:
In step 1 and step 2, damping fluid is identical with the first phosphate-buffered fluid component, and its substance withdrawl syndrome is 0.3mol/L, and pH value is 7.6, it comprises: be calculated in mass percent, the polyglycol of 0.5%, the bovine serum albumin(BSA) of 5% and 0.03% first surface activating agent.
In step 3, its substance withdrawl syndrome of the second phosphate buffer is 0.1mol/L, and pH value is 7.2, and it comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 5%, the polyglycol of 2%, the sucrose of 1% and the second surface activating agent of 0.1%.
First surface activating agent and second surface activating agent are tetronic1307.
Through test, its performance of glycosylated albumin immuno-chromatographic test paper strip that embodiment 1 is prepared is best, and it is highly sensitive, and the method for the performance of test glycosylated albumin immuno-chromatographic test paper strip is the method that those skilled in the art generally apply, and does not repeat at this.
Below the glycosylated albumin immuno-chromatographic test paper strip that embodiment 1 is prepared is set forth further by the performance of test example to the glycosylated albumin immuno-chromatographic test paper strip that the present invention proposes.
Test example 1
The glycosylated albumin immuno-chromatographic test paper strip that embodiment 1 is prepared is detected, its concrete detection method is: whole blood sample 20ul to be detected is added the pH value that 500ul contains hemolysin is 7.2, substance withdrawl syndrome is the phosphate buffer of 0.05mol/L, add in test card well again, after question response 3min, with the detection line in biological sensor interpretation detection window and nature controlling line, to obtain a result.
The drafting of standard working curve:
Getting GA is that the serum specimen of the artificial preparation of 100.0% is made into the serum specimen that GA is 100.0,80.0,60.0,40.0,20.0,10.0,0.0%, measure its GA respectively again, to be made into value for X measured value for Y drawing standard working curve, expression formula through statistical fit standard working curve lists regression equation: Y=1.97X-1.46, fitting coefficient square be R
2=0.999.The results are shown in accompanying drawing 3, Fig. 3 is linearity test standard working curve, and glycosylated albumin measurement range is 3.2% ~ 68.1%.
Test example 2
Replica test:
Replica test in batch: get same patients serum's sample, continuous duplicate detection 20 times, calculates CV value simultaneously.Replica test between batch: same patients serum's sample is distributed into 20 parts, is stored in refrigerator, get 1 part of detection every day, totally 20 days, calculate CV value.In batch, CV value is 3.5%.Between batch, (in the daytime) CV value is 4.2%.According to the U.S. clinical trial room standardization council (NCCLS) documentation requirements batch in, batch between imprecision level should be less than 5%, the method meets the requirements.
Embodiment 3
Recovery test: according to interior addition method, adds respectively by the reference material of 100 μ l variable concentrations in 1000 μ l patients serum samples, and the recovery test carrying out high, medium and low variable concentrations detects.The recovery test result of high, medium and low variable concentrations is as shown in table 1, and average recovery rate is 95.4%, substantially meets clinical trial requirement.
Table 1
Test example 4
Interference test:
By a fresh serum sample, be divided into 3 parts, every part of 1000 μ l, the 1st part of haemoglobin adding 196mg/dl, 2nd part adds the serum 100 μ l that total bilirubin is 29.8mg/dl, and the 3rd part adds the serum 100 μ l that chyle is 200mmol/L, measure GA value respectively after mixing.
Result shows, haemoglobin be below 196mg/dl haemolysis sample, the piarhemia of total bilirubin to be the jaundice sample of below 29.8mg/dL and chyle be below 200mmol/L measures GA without obvious interference to this method method.
Test example 5
Actual detection contrast test
The test strips of embodiment 1 is carried out to the mensuration of aspect of performance, get the fresh serum sample of 28 routine patients, every part is carried out double-blind study detection with Japanese Asahi Kasei Pharma Corp GA reagent and native system respectively.Result application correlation regression and paired t-test analytical approach are compared.Result shows, and two method correlativitys are good, no significant difference (n=28, Y=0.996X+0.846, r=0.993, t=0.0870, P>0.05).Regression curve is shown in accompanying drawing 4, and accompanying drawing 4 is contrast detection regression curve.In Fig. 4, solid dot represents saccharification standard (glycated standard), and hollow dots represents purification of albumin (purified albumin).
From above-mentioned detection, detection method has higher sensitivity, and realization batch in, batch between accurate quantification detection while there is good repeatability.
The detection glycosylated albumin immuno-chromatographic test paper strip that the present invention proposes, it needs the whole blood sample of trace, the content quantitatively detecting glycosylated albumin can be realized in 3 ~ 5 minutes, substantially increase the speed of examination, have highly sensitive, specificity good and the simple advantage of structure, and its preparation method is simple, is easy to large-scale production.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. one kind is detected glycosylated albumin immuno-chromatographic test paper strip, it is characterized in that: comprise liner, be arranged at the analyzing film in the middle part of described liner, be arranged at the adsorptive pads of described analyzing film upper one end, be arranged at the bond pad of the described analyzing film top other end and be arranged at the sample pad of described bond pad upper one end, described analyzing film is provided with detection line and nature controlling line, described detection line comprises glycosylated albumin detection line and albumin detection line, described nature controlling line, described albumin detection line and described glycated protein detection line are arranged in order along described analyzing film upper one end to the other end.
2. detection glycosylated albumin immuno-chromatographic test paper strip as claimed in claim 1, it is characterized in that: described adsorptive pads 1 ~ 2mm overlapping with described analyzing film, described bond pad 1 ~ 2mm overlapping with described analyzing film, described sample pad and the overlapping 1 ~ 2mm of described bond pad.
3. the preparation method detecting glycosylated albumin immuno-chromatographic test paper strip as claimed in claim 1, is characterized in that, comprise the steps:
1) preparation of phenyl boric acid label: diluted by fluorescent material damping fluid, adds phenyl boric acid lysate, and 20 ~ 25 DEG C are reacted at least 1 hour, cross column separating purification with the gel column that specifications and models are G25, collect label, are phenyl boric acid label;
2) preparation of sample pad: with cellulose membrane as sample pad solid phase material, soak with the first phosphate buffer that substance withdrawl syndrome is 0.01 ~ 0.3mol/L, described first phosphate buffer pH value is 7.2 ~ 7.6, after immersion, be dried process, obtain sample pad;
3) preparation of bond pad: with glass fibre element film as bond pad solid phase material, with substance withdrawl syndrome be 0.01 ~ 0.1mol/L, pH value be 7.2 the second phosphate buffer dilute described phenyl boric acid label, make suspension, described suspension is sprayed on glass fibre element film, drying process is carried out to it, obtains bond pad;
4) preparation of detection line and nature controlling line on analyzing film: with nitrocellulose filter as solid phase carrier, be analyzing film, with the antibody that triphosphate damping fluid dilution detection line and nature controlling line use, adopt on the Membrane jetter detection line that is sprayed on described analyzing film respectively and nature controlling line position, analyzing film after spray film is carried out drying process, obtains the analyzing film with detection line and nature controlling line;
5) preparation of adsorptive pads: the filter paper selecting 1mm thick, as adsorptive pads solid phase material, is cut into the band of 25 × 300mm, obtains adsorptive pads;
6) preparation of glycosylated albumin immuno-chromatographic test paper strip is detected: first stick on liner centre position by described analyzing film, adsorptive pads and bond pad is adhered in described analyzing film upper end, sample pad is pasted again in described bond pad upper end, by described liner and be arranged at the described sample pad on described liner top, described bond pad, described analyzing film and described adsorptive pads and together cut into strip, be and detect glycosylated albumin immuno-chromatographic test paper strip.
4. the preparation method detecting glycosylated albumin immuno-chromatographic test paper strip as claimed in claim 3, it is characterized in that: in described step 1, damping fluid is identical with the first phosphate-buffered fluid component in described step 2, it comprises: be calculated in mass percent, the polyglycol of 0.01% ~ 0.5%, the bovine serum albumin(BSA) of 1% ~ 5% and 0.01% ~ 0.05% first surface activating agent.
5. the preparation method detecting glycosylated albumin immuno-chromatographic test paper strip as claimed in claim 3, it is characterized in that: in described step 3, the second phosphate buffer comprises: be calculated in mass percent, the bovine serum albumin(BSA) of 1% ~ 5%, the polyglycol of 0.1 ~ 2%, the sucrose of 0.5 ~ 2% and the second surface activating agent of 0.01% ~ 0.1%.
6. the preparation method detecting glycosylated albumin immuno-chromatographic test paper strip as claimed in claim 3, it is characterized in that: in described step 4, in triphosphate damping fluid, substance withdrawl syndrome is 0.05mol/L, pH value is 7.4 ~ 7.6, comprise in described triphosphate damping fluid: be calculated in mass percent, the methyl alcohol of 0.5 ~ 1% and the bovine serum albumin(BSA) of 0.8 ~ 1.5%.
7. the preparation method of the detection glycosylated albumin immuno-chromatographic test paper strip as described in claim 4 or 5 or 6, is characterized in that: described first surface activating agent and second surface activating agent are the one in Tween20, TritonX-100 and tetronic1307.
8. the preparation method detecting glycosylated albumin immuno-chromatographic test paper strip as claimed in claim 3, is characterized in that: in described step 4, antibody is the one in anti-glycosylated albumin monoclonal antibody, antialbumin monoclonal antibody and sheep anti-mouse igg antibody.
9. the preparation method detecting glycosylated albumin immuno-chromatographic test paper strip as claimed in claim 3, is characterized in that: in described step 6, liner is for be made up of pet material.
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| CN109828112A (en) * | 2019-03-02 | 2019-05-31 | 浙江康特生物科技有限公司 | Glycosylated albumin antibody complex preparation method and application |
| CN113311171A (en) * | 2021-07-01 | 2021-08-27 | 南京无书化工有限公司 | Method for detecting glycated serum albumin by using boric acid affinity principle |
| CN115950868A (en) * | 2022-12-26 | 2023-04-11 | 深圳市雷诺华科技实业有限公司 | Testing device and testing method for determining glycated albumin by antibody-spot method |
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