CN102353787A - 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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- CN102353787A CN102353787A CN 201110295520 CN201110295520A CN102353787A CN 102353787 A CN102353787 A CN 102353787A CN 201110295520 CN201110295520 CN 201110295520 CN 201110295520 A CN201110295520 A CN 201110295520A CN 102353787 A CN102353787 A CN 102353787A
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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 glial fibrillary acidic protein in a kind of half-quantitative detection human serum (glial fibrillary acidic protein, the GFAP) colloidal gold strip of concentration.
Background technology
Epidemiological investigation shows that cranial vascular disease and heart disease, malignant tumour constitute three dead big reasons of human diseases at present.Compare with western developed country, the M & M of China's cerebrovascular disease is apparently higher than cardiovascular disease.It is estimated that national annual New Development patients with cerebral apoplexy is about 2,000,000 people; Die from about 1,500,000 people of patient of cerebral apoplexy every year; Survival patient number 6,000,000～7,000,000; And 70%～80% survivor leaves over handicaps such as paralysis, aphasia, brings heavy burden for society and family.Its midbrain hemorrhage is a kind of fatal rate and disability rate all high common disease, frequently-occurring disease, and the serious harm mankind are the elderly's health particularly, threatens patient's life, has a strong impact on patient's quality of life.
(glial fibrillary acidic protein is that a kind of molecular weight is the acidic protein of 50～52KDa GFAP) to glial fibrillary acidic protein, belongs to the cell skeletin, 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.Discover 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 gets into intercellular fluid, and the GFAP of solubility gets into cerebrospinal fluid through intercellular fluid, and the blood-brain barrier that passes destruction gets into blood circulation.There are some researches show that serum GFAP level promptly obviously raises in the patients with cerebral hemorrhage morbidity 6 hours, and serum GFAP level and normal control 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 have research to show between GFAP among the acute cerebral hemorrhage patients serum and the cerebral hemorrhage volume close positive correlation is arranged, therefore can judge patient's the state of an illness and prognosis through patients with cerebral hemorrhage serum GFAP level.
At present; Detection to GFAP in the clinical diagnosis mainly is the ELISA experimental technique; This method because of its specificity, highly sensitively be widely used at recent two decades; But this method detection method is loaded down with trivial details simultaneously; Grow required proving time (1～2 hour); And often need 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 shortened detection time greatly.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 shows with color that directly naked eyes are judged easily, do not need special instrument and equipment, only need test strips, as long as sample is done very simple processing or need not be done 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 influence of external environments such as temperature, but long preservation.Yet result for retrieval shows that the document that utilizes colloidal gold method to detect GFAP concentration in the human serum does not also appear in the newspapers.
Summary of the invention
To the deficiency of prior art, the problem that the present invention will solve provides a kind of rapid semi-quantitative and detects glial fibrillary acidic protein (glial fibrillary acidic protein, the GFAP) colloidal gold strip of concentration in the human serum.
The colloidal gold strip of GFAP concentration in the half-quantitative detection human serum according to the invention; Be on the PVC plate, to adhere to detection diaphragm, collaurum combination diaphragm and absorbent paper layer to process; It is characterized in that: said collaurum combines to be coated with on the diaphragm the anti--GFAP antibody of colloid gold label; Said 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; Said test strips is followed successively by the application of sample place from bottom to top, collaurum combines diaphragm, detection line, nature controlling line, thieving paper.
In the colloidal gold strip of GFAP concentration, the anti--GFAP capture antibody that encapsulates on the anti--GFAP antibody of said colloid gold label and the detection line is for discerning the monoclonal antibody of different epi-positions in the above-mentioned half-quantitative detection human serum.
In the colloidal gold strip of GFAP concentration, said collaurum combines diaphragm by plain made of paper the getting of glass fibre in the above-mentioned half-quantitative detection human serum, and said detection diaphragm is made by nitrocellulose filter.
In the above-mentioned half-quantitative detection human serum in the colloidal gold strip of GFAP concentration; Said detection line can be made as many and setting at interval; Preferred embodiment be 4 and setting at interval; The package amount of fixing anti--GFAP capture antibody has been 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 according to the 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 up of test strips shell and test strips; Test strips is made up 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 following:
(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 according to the 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 combines on the diaphragm; On the detection line that detects diaphragm, encapsulate a certain amount of resisting-GFAP and catch monoclonal antibody.After serum to be checked is added to collaurum combination diaphragm; Anti--GFAP antibodies on GFAP in the serum and the collaurum; When detection line is arrived in this colloid gold label compound swimming; GFAP albumen anti--GFAP on can seized survey line catches monoclonal antibody and catches, and 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 combine fully on the detection line anti-GFAP capture antibody the time, the collaurum quantity that detection line is assembled is the aggregated colloids gold less or not, will develop the color shallow or does not develop the color.The colour developing band is many more, and the band color is dark more, illustrates that the GFAP protein concentration in the test serum is high more, therefore can be according to the content of how much judging GFAP in the test serum of band.When showing 5 bands; 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 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 promptly 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 is provided with many and detects detection lines on the detection diaphragm.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 judge the concentration range of GFAP in the test serum according to what and position of detection line colour developing.
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 that test strips detects.
The present invention further optimization is provided with nature controlling line on the detection diaphragm, be coated with sheep anti-mouse igg on the said 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, practices thrift cost, and don't influences 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 that high specific, high sensitivity, operation are fast and convenient, cost, without any need for instrument, 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 combines diaphragm; 5: the application of sample place.
Fig. 2 a testing result of the present invention is understood synoptic diagram---and expression GFAP concentration is less than 2ng/ml;
Fig. 2 b testing result of the present invention is understood synoptic diagram---and expression GFAP concentration is 2ng/ml～4ng/ml;
Fig. 2 c testing result of the present invention is understood synoptic diagram---and expression GFAP concentration is 4ng/ml～8ng/ml;
Fig. 2 d testing result of the present invention is understood synoptic diagram---and expression GFAP concentration is 8ng/ml～16ng/ml;
Fig. 2 e testing result of the present invention is understood synoptic 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 appearance 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 quadrat 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 appearance point behind the detection line.PBS damping fluid with containing 1%BSA sealed 2 hours, and room temperature is dried in the shade, 4 ℃ of preservations.
The step 2 collaurum combines 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%, heated and stirred is boiled on magnetic stirring apparatus, and every 100ml 0.01% gold chloride adds 2ml 1% trisodium citrate, continues to boil when liquid takes on a red color, to heat to stop, and is cooled to room temperature, adds water to original volume.
Colloid gold label GFAP antibody.On magnetic stirring apparatus, transfer collaurum to pH9.0, add anti--GFAP monoclonal antibody, continue to stir 10min, add BSA as stabilizing agent according to 1～3 μ g/ml collaurum with the sal tartari damping fluid of 0.1M, to final concentration be 1%, left standstill 1 hour.Under 4 ℃ of above-mentioned solution, 3000r/min low-speed centrifugal 40min carefully draws supernatant, discards deposition; 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 1/5,4 ℃ of PBS damping fluid (including 1%BSA, 0.04 sodium azide) dissolution precipitation to the original volume 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 plain paper of glass fibre with spraying equipment, and vacuum drying then promptly gets collaurum and combines diaphragm.
4) assembling of test strips
On the PVC plate, from bottom to top adhere to detect diaphragm successively, collaurum combines diaphragm, last layer is a water accepting layer, is made up of 3～5 metafiltration paper, seal with adhesive tape the outside, becomes portion of the handle.
The above material that assembles is cut into wide little of 3mm, promptly makes the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum according to the invention.
The application of the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum
(1) detects GFAP concentration in the human serum with test strips
Draw 120 μ l serum samples with pipettor, the vertical application of sample place that drips at colloidal gold strip.Pick up counting, wait for that red stripes occurs, with the standard color comparison card comparison, surpass 20 minutes in 10 minutes, the result is invalid.
(2) result explains
Four concentration gradient markings (being detection line) related from top to bottom in this colloidal gold strip are: 16ng/ml, 8ng/ml, 4ng/ml, 2ng/ml.Testing result is explained as follows:
When 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 (4)
1. the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum; Be on the PVC plate, to adhere to detection diaphragm, collaurum combination diaphragm and absorbent paper layer to process; It is characterized in that: said collaurum combines to be coated with on the diaphragm the anti--GFAP antibody of colloid gold label; Said 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; Said test strips is followed successively by the application of sample place from bottom to top, collaurum combines diaphragm, detection line, nature controlling line, thieving paper.
2. the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum as claimed in claim 1; It is characterized in that the anti--GFAP capture antibody that encapsulates on the anti--GFAP antibody of said colloid gold label and the detection line is for discerning the monoclonal antibody of different epi-positions.
3. the colloidal gold strip of GFAP concentration is characterized in that in the half-quantitative detection human serum as claimed in claim 1, and said collaurum combines diaphragm by plain made of paper the getting of glass fibre, and said detection diaphragm is made by nitrocellulose filter.
4. the colloidal gold strip of GFAP concentration in the half-quantitative detection human serum as claimed in claim 1; It is characterized in that; Said detection line is 4 and is provided with at interval; The concentration of the GFAP that the package amount representative of fixing anti--GFAP capture antibody can detect on the detection line is followed successively by 16ng/ml, 8ng/ml, 4ng/ml, 2ng/ml from top to bottom.
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| CN 201110295520 CN102353787B (en) | 2011-09-27 | 2011-09-27 | Colloidal gold test paper strip for semiquantitatively detecting concentration of GFAP (Glial Fibrillary Acidic Protein) in human serum |
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| CN 201110295520 CN102353787B (en) | 2011-09-27 | 2011-09-27 | Colloidal gold test paper strip for semiquantitatively detecting concentration of GFAP (Glial Fibrillary Acidic Protein) in human serum |
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| CN102353787A true CN102353787A (en) | 2012-02-15 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104142404A (en) * | 2013-05-10 | 2014-11-12 | 深圳市安群生物工程有限公司 | Fluorescent immunochromatographic test paper for detecting human GFAP protein, and preparation method thereof |
| CN105021596A (en) * | 2014-04-18 | 2015-11-04 | 曾嵘斌 | Concentration gradient based dry chemical test strip with multiple layers of films |
| 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 |
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| 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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2011
- 2011-09-27 CN CN 201110295520 patent/CN102353787B/en active Active
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) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104142404A (en) * | 2013-05-10 | 2014-11-12 | 深圳市安群生物工程有限公司 | Fluorescent immunochromatographic test paper for detecting human GFAP protein, and preparation method thereof |
| CN105021596A (en) * | 2014-04-18 | 2015-11-04 | 曾嵘斌 | Concentration gradient based dry chemical test strip with multiple layers of films |
| 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 |
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| CN102353787B (en) | 2013-03-06 |
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