CN102353787B - Colloidal gold test paper strip for semiquantitatively detecting concentration of GFAP (Glial Fibrillary Acidic Protein) in human serum - Google Patents
Colloidal gold test paper strip for semiquantitatively detecting concentration of GFAP (Glial Fibrillary Acidic Protein) in human serum Download PDFInfo
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- CN102353787B CN102353787B CN 201110295520 CN201110295520A CN102353787B CN 102353787 B CN102353787 B CN 102353787B CN 201110295520 CN201110295520 CN 201110295520 CN 201110295520 A CN201110295520 A CN 201110295520A CN 102353787 B CN102353787 B CN 102353787B
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Abstract
The invention discloses a colloidal gold test paper strip for semiquantitatively detecting the concentration of a GFAP (Glial Fibrillary Acidic Protein) in a human serum. The colloidal gold test paper strip is produced by adhering a detection diaphragm, a colloidal gold combined diaphragm and a water-absorbing paper layer to a PVC (Poly Vinyl Chloride) board, wherein the colloidal gold combined diaphragm is coated by a colloidal gold-labeled antibody for resisting the GFAP; a detection line and a quality control line are arranged on the detection diaphragm; a capture antibody for resisting the GFAP is fixedly arranged on the detection line; a rabbit anti-mouse IgG (Immunoglobulin G) is fixedly arranged on the quality control line; and a sampling part, the colloidal gold combined diaphragm, the detection line, the quality control line and the water-absorbing paper are sequentially arranged on the test paper strip from the bottom up. The test paper strip disclosed by the invention can be used for semiquantitatively detecting the GFAP, has the advantages of high specificity, high sensitiveness, high accuracy, quickness, simpleness and convenience in operation, low expense, no need of any apparatus, time-and-labor saving and the like as well as has favorable application prospect.
Description
Technical field
The present invention relates to a kind of colloidal gold strip, relate in particular to the colloidal gold strip of glial fibrillary acidic protein in a kind of half-quantitative detection human serum (glial fibrillary acidic protein, GFAP) concentration.
Background technology
Epidemiological investigation shows, three large reasons of present cranial vascular disease and heart disease, the death of malignant tumour formation human diseases.Compare with western developed country, the M ﹠ M of China's cerebrovascular disease is apparently higher than cardiovascular disease.It is estimated, the annual new patients with cerebral apoplexy in the whole nation is about 2,000,000 people; The patient who dies from cerebral apoplexy every year is 1,500,000 people approximately; Survival patient number 6,000,000～7,000,000; And 70%～80% survivor leaves over the handicaps such as paralysis, aphasia, brings heavy burden for society and family.Its midbrain hemorrhage is all high common disease, frequently-occurring diseases of a kind of fatal rate and disability rate, and the serious harm mankind are the elderly's health particularly, threatens patient's life, has a strong impact on quality of life of patients.
Glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) is that a kind of molecular weight is the acidic protein of 50～52KDa, belongs to cytoskeleton albumen, is the intracytoplasmic specific proteins of astrocyte, marker protein.GFAP is upset when inducing reaction at astroglia, and its expression changes, and abundant, unique expression is arranged in astrocyte.Research finds that GFAP and dementia, multiple sclerosis and cerebral apoplexy have obvious correlativity.Neuron and Deiter's cells suffer damage after the cerebral hemorrhage, and a large amount of plasmosin matter spills cell and enters intercellular fluid, and the GFAP of solubility enters cerebrospinal fluid by intercellular fluid, and the blood-brain barrier that passes destruction enters blood circulation.There are some researches show that serum GFAP level namely obviously raises in the patients with cerebral hemorrhage 6 hours of onset, and serum GFAP level and Normal group no significant difference in the ischemic cerebral stroke patients 6 hours, so think that GFAP is the biological markers of acute cerebral hemorrhage.In addition, also there are some researches show between the GFAP in the serum of patients with acute intracerebral hemorrhage and the cerebral hemorrhage volume close positive correlation is arranged, therefore can judge by serum of patients with intracerebral hemorrhage GFAP level patient's the state of an illness and prognosis.
At present, detection to GFAP in the clinical diagnosis mainly is the ELISA experimental technique, the method because of its specificity, highly sensitively be widely used at recent two decades, but the method detection method is loaded down with trivial details simultaneously, grow required proving time (1～2 hour), and often need to save bit by bit after some samples, just can detect, detection is incured loss through delay, and the collaurum rule has a lot of advantages, can compensate its defect: a) quick: all testing process only needs 5-10 minute, has greatly shortened detection time.B) easy: as not need other any instrument and equipment, operate also extremely simply, can carry out whenever and wherever possible.C) testing result is directly perceived.Testing result directly with the color demonstration, judge easily, do not need special instrument and equipment, only needs test strips by naked eyes, as long as sample is done very simple processing or do not needed to do pre-treatment.D) can single part of detection: can detect in batch sample, again can single part of detection, the patient can take the result at once, needn't wait for.E) good stability: gold marked reagent is stable, is not subjected to the impact of the external environments such as temperature, but long preservation.Yet result for retrieval shows, utilizes the document of GFAP concentration in the colloidal gold method detection human serum to yet there are no report.
Summary of the invention
For the deficiencies in the prior art, the problem to be solved in the present invention provides the colloidal gold strip that a kind of rapid semi-quantitative detects glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) concentration in the human serum.
The colloidal gold strip of GFAP concentration in the half-quantitative detection human serum of the present invention, to make in conjunction with diaphragm and absorbent paper layer at PVC plate adhesion detection diaphragm, collaurum, it is characterized in that: described collaurum is in conjunction with being coated with resisting-GFAP antibody of colloid gold label on the diaphragm, described detection diaphragm is provided with detection line and nature controlling line, wherein be fixed with anti--GFAP capture antibody on the detection line, be fixed with rabbit anti-mouse igg on the nature controlling line; Described test strips is followed successively by application of sample place, collaurum from bottom to top in conjunction with diaphragm, detection line, nature controlling line, thieving paper.
In the above-mentioned half-quantitative detection human serum in the colloidal gold strip of GFAP concentration, described colloid gold label anti--on GFAP antibody and the detection line coated anti--the GFAP capture antibody be for identifying the monoclonal antibody of different epi-positions.
In the colloidal gold strip of GFAP concentration, described collaurum gets by the glass fibre element is made of paper in conjunction with diaphragm in the above-mentioned half-quantitative detection human serum, and described detection diaphragm is made by nitrocellulose filter.
In the above-mentioned half-quantitative detection human serum in the colloidal gold strip of GFAP concentration, described detection line can be made as many and interval setting, preferred embodiment be 4 and interval setting, the package amount of fixing resisting-GFAP capture antibody has represented the GFAP concentration that can detect on the detection line, is followed successively by 16ng/ml, 8ng/ml, 4ng/ml, 2ng/ml from top to bottom.
The application of the colloidal gold strip of GFAP concentration in the preparation detection kit in the half-quantitative detection human serum of the present invention, the kit of GFAP concentration comprises following component in the wherein said half-quantitative detection human serum:
1) 10 single part of test card; Test card is made of test strips shell and test strips, test strips is made of collaurum diaphragm (being sprayed with the collaurum by anti--GFAP labeling of monoclonal antibody), detection diaphragm (detection line is coated with, and anti--GFAP catches monoclonal antibody, and nature controlling line is coated with rabbit anti-mouse igg antibody), thieving paper, lining form sheet; 2) instructions is a; 3) standard color comparison card is one.
Use the method for GFAP concentration in the mentioned reagent box half-quantitative detection human serum, step is as follows:
(1) test card is placed on the clean water flat surface, horizontal positioned also performs mark.
(2) draw 120 μ l samples with pipettor, vertically drip at the application of sample place.
(3) wait for that detection line place aubergine band occurs, carry out the sxemiquantitative interpretation with range estimation normative reference colorimetric card.
The principle that colloidal gold colloidal gold detection test paper strip of the present invention carries out GFAP concentration in the half-quantitative detection human serum is: resist-the GFAP monoclonal antibody with colloid gold label, this colloid gold label thing is coated on collaurum in conjunction with on the diaphragm; Be coated with a certain amount of resisting-GFAP at the detection line that detects diaphragm and catch monoclonal antibody.When serum to be checked be added to collaurum in conjunction with diaphragm after, GFAP in the serum on collaurum anti--GFAP antibody is combined, when detection line is arrived in this colloid gold label compound swimming, GFAP albumen on can tested survey line anti--GFAP catches monoclonal antibody and catches, colloidal gold aggregation is on detection line and develop the color; If the GFAP concentration in the test serum is low be not enough to fully in conjunction with on the detection line anti-GFAP capture antibody the time, the few or aggregated colloids gold of the collaurum quantity that detection line is assembled will develop the color shallow or do not develop the color.The colour developing band is more, and the band color is darker, illustrates that the GFAP protein concentration in the test serum is higher, therefore can be according to the content of how much judging GFAP in the test serum of band.When showing 5 band, the GFAP amount in the serum of the certain volume that drips that illustrates makes the binding site that detects antibody on the diaphragm saturated, at this moment, need to dilute serum, detect again, and the concentration of GFAP multiply by extension rate in the concentration of detection line representative and is measured concentration in the test serum, has namely realized test serum is carried out half-quantitative detection.That the present invention has realized is convenient, quick, economical, the purpose of GFAP concentration in the simple half-quantitative detection serum.
The present invention further preferably arranges many at the detection diaphragm and detects detection lines.Distance on the collaurum diaphragm from the near to the remote, detection line shows that the GFAP concentration of representative increases successively when positive.Can according to what and position of detection line colour developing, judge the concentration range of GFAP in the test serum.
The present invention is coated on the antibody on the collaurum diaphragm and the anti-GFAP capture antibody that is fixed on the detection line is monoclonal antibody, to reduce the interference of the cross reaction in the testing process as far as possible, to improve the accuracy of ELISA test strip.
The present invention further optimization arranges nature controlling line at the detection diaphragm, is coated with sheep anti-mouse igg on the described nature controlling line, as the foundation of paper for monitoring bar validity and reagent contamination.
Test strip of the present invention is attached to the film reaction system on the corresponding PVC lath of size, saves cost, and don't affects testing result.
To sum up, the present invention when GFAP detects in the serum, can be easy fast, accurately and effectively GFAP is carried out half-quantitative detection, have high specific, high sensitivity, operation is fast and convenient, expense is cheap, without any need for instrument, the characteristics such as time saving and energy saving.
Description of drawings
Fig. 1 colloidal gold strip film reaction of the present invention layer reaction system structural representation;
Wherein: 1: thieving paper; 2: nature controlling line; 3: detection line; 3a: detectable concentration is 16ng/ml; 3b: detectable concentration is 8ng/ml; 3c: detectable concentration is 4ng/ml; 3d: detectable concentration is 2ng/ml; 4: collaurum is in conjunction with diaphragm; 5: the application of sample place.
Fig. 2 a testing result of the present invention is understood schematic diagram---and expression GFAP concentration is less than 2ng/ml;
Fig. 2 b testing result of the present invention is understood schematic diagram---and expression GFAP concentration is 2ng/ml～4ng/ml;
Fig. 2 c testing result of the present invention is understood schematic diagram---and expression GFAP concentration is 4ng/ml～8ng/ml;
Fig. 2 d testing result of the present invention is understood schematic diagram---and expression GFAP concentration is 8ng/ml～16ng/ml;
Fig. 2 e testing result of the present invention is understood schematic diagram---and expression GFAP concentration detects after should diluting greater than 16ng/ml again.
Embodiment
With the PBS damping fluid will resist-GFAP catches monoclonal antibody and is diluted to four different concentration, from high to low respectively with some film instrument point on nitrocellulose filter, every line is put 20 μ l, live width 1～1.5mm, distance between centers of tracks 5mm.Its package amount representative can detect 2ng/ml, 4ng/ml, 8ng/ml, 16ng/ml.Same method, as nature controlling line, live width 1～1.5mm is apart from nearest detection line 8mm at the sheep anti-mouse igg of using 200 μ g/ml on the some film instrument point behind the detection line.Sealed 2 hours with the PBS damping fluid that contains 1%BSA, room temperature is dried in the shade, 4 ℃ of preservations.
The step 2 collaurum is in conjunction with the preparation of diaphragm
1) anti--GFAP antibody
Monoclonal antibody is available from Santa Cruz Biotechnology.
2) preparation of anti--GFAP antibody-colloidal gold composite
Adopting trisodium citrate reduction method to prepare diameter is the 20nm collaurum.With purified water 1% gold chloride is diluted to 0.01%, adds thermal agitation at magnetic stirring apparatus and boil, every 100ml 0.01% gold chloride adds 2ml 1% trisodium citrate, continues to boil that heating stops when taking on a red color to liquid, is cooled to room temperature, adds water to original volume.
Colloid gold label GFAP antibody.Transfer collaurum to pH9.0 at magnetic stirring apparatus with the sal tartari damping fluid of 0.1M, add anti--GFAP monoclonal antibody according to 1～3 μ g/ml collaurum, continue to stir 10min, add BSA as stabilizing agent, to final concentration be 1%, left standstill 1 hour.Under 4 ℃ of mentioned solutions, 3000r/min low-speed centrifugal 40min carefully draws supernatant, discards precipitation; Supernatant with 4 ℃ of high speed centrifugation 30min of 12000r/min, is abandoned supernatant again,, spends the night after fully stable to original volume with PBS damping fluid (including 1%BSA, the 0.04 sodium azide) dissolution precipitation of 0.01M, pH7.2.With 4 ℃ of high speed centrifugation 30min of 12000r/min, abandon supernatant again, store for future use with to original volume 1/5,4 ℃ of PBS damping fluid (including 1%BSA, the 0.04 sodium azide) dissolution precipitation of 0.01M, pH7.2.
3) collaurum-anti-GFAP antibody complex uses the sal tartari damping fluid of 0.1M pH9.0 to be diluted to setting concentration, is applied on the glass fibre element paper with spraying equipment, and then vacuum drying namely gets collaurum in conjunction with diaphragm.
4) assembling of test strips
From bottom to top adhere to detect successively diaphragm, collaurum in conjunction with diaphragm on the PVC plate, last layer is water accepting layer, is comprised of 3～5 metafiltration paper, and the outside seals with adhesive tape, becomes portion of the handle.
The above material that assembles is cut into wide little of 3mm, namely makes the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum of the present invention.
The application of the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum
(1) with GFAP concentration in the ELISA test strip human serum
Draw 120 μ l serum samples with pipettor, the vertical application of sample place that drips at colloidal gold strip.The beginning timing waits for that red stripes occurs, and compares with standard color comparison card in 10 minutes, surpasses 20 minutes, and the result is invalid.
(2) result explains
In this colloidal gold strip from top to bottom related four concentration gradient markings (being detection line) be: 16ng/ml, 8ng/ml, 4ng/ml, 2ng/ml.Testing result is explained as follows:
When a line of nature controlling line only occurring, illustrate that GFAP concentration is less than 2ng/ml.
When occurring containing the two-lines of nature controlling line, illustrate that GFAP concentration is at 2ng/ml～4ng/ml.
When occurring containing three lines of nature controlling line, illustrate that GFAP concentration is at 4ng/ml～8ng/ml.
When occurring containing four lines of nature controlling line, illustrate that GFAP concentration is at 8ng/ml～16ng/ml.
When occurring containing five lines of nature controlling line, GFAP concentration is described greater than 16ng/ml, measure again after should diluting.
Claims (1)
1. the preparation method of the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum, be adhere at the PVC plate detect diaphragm, collaurum is made in conjunction with diaphragm and absorbent paper layer, it is characterized in that: comprise following preparation process:
The preparation of step 1, detection diaphragm:
With the PBS damping fluid will resist-GFAP catches monoclonal antibody and is diluted to four different concentration, from high to low respectively with some film instrument point on nitrocellulose filter, every line is put 20 μ l, live width 1～1.5mm, distance between centers of tracks 5mm; Its package amount representative can detect 2ng/ml, 4ng/ml, 8ng/ml, 16ng/ml; Same method, as nature controlling line, live width 1～1.5mm is apart from nearest detection line 8mm at the sheep anti-mouse igg of using 200 μ g/ml on the some film instrument point behind the detection line; Sealed 2 hours with the PBS damping fluid that contains 1%BSA, room temperature is dried in the shade, 4 ℃ of preservations;
Step 2, collaurum are in conjunction with the preparation of diaphragm:
1) anti--GFAP antibody
Monoclonal antibody is available from Santa Cruz Biotechnology;
2) preparation of anti--GFAP antibody-colloidal gold composite
Adopting trisodium citrate reduction method to prepare diameter is the 20nm collaurum: with purified water 1% gold chloride is diluted to 0.01%, adding thermal agitation at magnetic stirring apparatus boils, every 100ml 0.01% gold chloride adds 2ml 1% trisodium citrate, continue to boil that heating stops when taking on a red color to liquid, be cooled to room temperature, add water to original volume;
Colloid gold label GFAP antibody: transfer collaurum to pH9.0 with the sal tartari damping fluid of 0.1M at magnetic stirring apparatus, add anti--GFAP monoclonal antibody according to 1～3 μ g/ml collaurum, continue to stir 10min, add BSA as stabilizing agent, to final concentration be 1%, left standstill 1 hour; Under 4 ℃ of mentioned solutions, 3000r/min low-speed centrifugal 40min carefully draws supernatant, discards precipitation; Supernatant with 4 ℃ of high speed centrifugation 30min of 12000r/min, is abandoned supernatant again, uses the PBS damping fluid dissolution precipitation of 0.01M, pH7.2 to original volume, spends the night after fully stablizing; With 4 ℃ of high speed centrifugation 30min of 12000r/min, abandon supernatant again, store for future use with to original volume 1/5,4 ℃ of the PBS damping fluid dissolution precipitation of 0.01M, pH7.2;
3) collaurum-anti-GFAP antibody complex uses the sal tartari damping fluid of 0.1MpH9.0 to be diluted to setting concentration, is applied on the glass fibre element paper with spraying equipment, and then vacuum drying namely gets collaurum in conjunction with diaphragm;
4) assembling of test strips
From bottom to top adhere to detect successively diaphragm, collaurum in conjunction with diaphragm on the PVC plate, last layer is water accepting layer, is comprised of 3～5 metafiltration paper, and the outside seals with adhesive tape, becomes portion of the handle;
The above material that assembles is cut into wide little of 3mm, namely makes the colloidal gold strip of GFAP concentration in the described half-quantitative detection human serum.
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| CN104142404B (en) * | 2013-05-10 | 2016-10-05 | 深圳市安群生物工程有限公司 | Fluorescence immune chromatography test paper of detection people's GFAP albumen and preparation method thereof |
| CN105021596B (en) * | 2014-04-18 | 2017-09-29 | 曾嵘斌 | Multilayer film dry chemical detection strip based on concentration gradient |
| CN105527438A (en) * | 2015-12-25 | 2016-04-27 | 广州甘蔗糖业研究所 | Colloidal gold test strip for semi-quantitative detection of alpha-glucan, and detection method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005029087A1 (en) * | 2003-09-24 | 2005-03-31 | Roche Diagnostics Gmbh | Use of gfap for identification of intracerebral hemorrhage |
| CN1963514A (en) * | 2005-11-10 | 2007-05-16 | 北京庄笛浩禾生物医学科技有限公司 | Test paper bar for testing colloidal gold of capsular antibody of Bacillus anthracis |
| CN101566634A (en) * | 2008-04-22 | 2009-10-28 | 上海一滴准生物科技有限公司 | Troponin I serum quick test kit (colloidal gold method) |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005029087A1 (en) * | 2003-09-24 | 2005-03-31 | Roche Diagnostics Gmbh | Use of gfap for identification of intracerebral hemorrhage |
| CN1963514A (en) * | 2005-11-10 | 2007-05-16 | 北京庄笛浩禾生物医学科技有限公司 | Test paper bar for testing colloidal gold of capsular antibody of Bacillus anthracis |
| CN101566634A (en) * | 2008-04-22 | 2009-10-28 | 上海一滴准生物科技有限公司 | Troponin I serum quick test kit (colloidal gold method) |
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