CN1425775A - Tuberculosis antibody gold label test strip and its preparing method - Google Patents
Tuberculosis antibody gold label test strip and its preparing method Download PDFInfo
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- CN1425775A CN1425775A CN 01131834 CN01131834A CN1425775A CN 1425775 A CN1425775 A CN 1425775A CN 01131834 CN01131834 CN 01131834 CN 01131834 A CN01131834 A CN 01131834A CN 1425775 A CN1425775 A CN 1425775A
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Abstract
The gold label test strip includes lining sheet and protecting layer. There are adhered hand-held end water-absorbing layer and test end water-absorbing layer made of filter paper on two ends of the lining sheet; test layer of cellulose film between the two said water-absorbing layers; gold label antibody layer between the test end water-absorbing layer and the test layer; test line and control line of tuberculosis antigen formed by rabbit and sheep to resisting tubercle bacillus on the test layer; and tuberculosis antigen with gold label soaked in glass fiber or non-woven fabric on the label antibody layer. The test strip of the present invention has high specificity and high sensitivity.
Description
Technical field
The present invention relates to a kind of tuberculosis antibody gold label test strip and preparation method thereof.
Background technology
Various data show that about 1/3rd populations in the whole world have infected tubercule bacillus.8,000,000 new cases are arranged every year approximately, and year death toll surpasses 1,500,000.Tuberculosis becomes the emphasis controlled member that 2000-2001 WHO prevents and treats transmissible disease for this reason.On December 20th, 200, the Ministry of Health announced national tuberculosis epidemiological random sampling survey result the 4th time: China's tuberculosis state of an illness is quite serious, in some areas even have the tendency to spread.The whole nation has 1/3 above population to infect tubercule bacillus according to investigations, wherein has 10% people to fall ill; As not adopting an effective measure, in following 10 years, have 3,000 ten thousand people tuberculosis takes place.China's infectivity tuberculosis morbidity is 157.8 people/100,000 at present, it is estimated, existing infectivity lunger 2,000,000 people in the whole nation, the state of an illness of western 12 provinces is especially serious, at west area infectivity pulmonary tuberculosis morbidity rate up to 197 people/100,000, and be spreading trend, now existing infectivity pulmonary tuberculosis patient 650,000 people.
The emphasis of controlling tuberculosis is the positive tuberculosis patient of monitoring phlegm smear, and the morbidity patient is obtained medical treatment early.To the tubercle bacillus affection person monitor in early days and treatment prevent the morbidity.
The method of current diagnosis tuberculosis mainly contains: methods such as the inspection of phlegm smear tubercule bacillus, tuberculin test (PPD), tubercule bacillus polymerase chain reaction (PCR) and mycobacterium tuberculosis antibody detection.The specificity of phlegm smear method is up to 100% in aforesaid method, but susceptibility low be 22.2%.Tuberculin experiment (PPD test) susceptibility increases, but has poor specificity, be subject to the influence of BCG antibody, and the result judges that the time is oversize.Though there is poor specificity simultaneously in PCR susceptibility height; And the test apparatus apparatus expensive, to operator and environmental requirement height, personal errors is bigger.The susceptibility that mycobacterium tuberculosis antibody detects is up to 60-80%, but has the not high problem of specificity too.Therefore, in the diagnosis of tuberculosis, several method can cooperatively interact.
Compare in above-mentioned several method, the tuberculosis antibody detection method is easier, fast.Help the tuberculosis early diagnosis.The clinical tuberculosis antibody detection method of having used has enzyme linked immunosorbent assay ELISA at present, but ELISA method experimentation needs the 2-3 step, and the result judges needs 1-2 hour; And need dedicated experiments facility and professional operator.
Of the present invention open
Main purpose of the present invention is for a kind of tuberculosis antibody gold label test strip is provided, and this test strip has the specific specificity and the highly sensitive of height, and speed is fast, easy and simple to handle, need not specialized facilities.
Another object of the present invention is for a kind of preparation method of tuberculosis antibody gold label test strip is provided, and this method is simple, stability, good reproducibility.
Main purpose of the present invention can realize by following measure:
A kind of tuberculosis antibody gold label test strip comprises liner plate and protective layer; Be pasted with handheld terminal water accepting layer and the test lead water accepting layer of making by filter paper respectively at the two ends of liner plate; On the liner plate between handheld terminal water accepting layer and the test lead water accepting layer, be provided with the detection layers that cellulose membrane is made; Between test lead water accepting layer and detection layers, be provided with tuberculosis antigen gold labeling antibody layer; On detection layers, be provided with detection line and control line.
Another object of the present invention can realize by following measure:
A kind of preparation method of tuberculosis antibody gold label test strip comprises golden labeling antibody layer and detection layers and goes up the preparation of wrapping tested survey line and control line, and the assembling of test strip.
The preparation of described golden labeling antibody layer comprises: the preparation of i Radioactive colloidal gold, in concentration is that the 1.2-1.8%, the concentration that add its volume in the hydrochloro-auric acid of 0.01-0.05% is 1% trisodium citrate, boiled 10-20 minute, and be reduced into the Radioactive colloidal gold atom liquid of 15-50 nanometer; The preparation of ii tuberculosis antigen is got tubercule bacillus type strain TBRV strain culture and is dissolved in the physiological saline centrifugation and gets throw out, throw out is dissolved in centrifugation again behind the physiological saline again, and so repetitive scrubbing is removed the substratum composition for 4-8 time; Then with throw out ultrasonication 12-15 time, each 30 seconds, crushed material through 3.8-4.5 ten thousand g high speed centrifugations, is got supernatant liquor and crossed gel chromatography column, collect the active peak of tuberculosis antigen; Iii tuberculosis antigen colloid gold label adds the tuberculosis antigen that the above-mentioned ii of 2-10mg prepares in the 100ml colloidal gold solution; In above-mentioned colloidal gold solution, press 0.1-0.6g/100ml again and add animal serum albumin; In above-mentioned colloidal gold solution, press the saline solution of 6-12ml/100ml adding 10% then again; The last centrifugal precipitation of going, get supernatant centrifugal again throw out, throw out is dissolved in by 4-10ml/100ml 0.02M, PH7.4Tris-HCl contain the 0.1-0.6% animal serum albumin, 0.02% sodium azide gets the tuberculosis antigen colloidal gold solution; Iv is impregnated with glass fibre or non-woven fabrics with the tuberculosis antigen colloidal gold solution, and the immersion amount is with till immersing liquid and beginning outwards to flow out, then at 37 ℃ of dry down golden labeling antibody layers.
Described gel chromatography column is selected from a kind of among Sephardex G100, Sephardex G200, SepharclyS100, Sepharcly S200, the Sepharcly S300.
The preparation of described detection layers adds fixing agent in the tuberculosis antigen of preparation and rabbit, goat-anti mycobacterium tuberculosis antibody, Membrane jetter is sprayed on tuberculosis antigen and rabbit, goat-anti mycobacterium tuberculosis antibody and forms detection line and control line on the cellulose membrane then; Contain 10% calf serum sealing rinsing in 30 minutes, the dry detection layers that gets with 0.01MPH7.0PBS liquid again.
Described fixing agent is selected from a kind of in formaldehyde, the acetone.
The present invention has following advantage compared to existing technology:
Adopt single stage method when 1, gold test strip bar of the present invention detects tuberculosis antibody, specificity and highly sensitive with height, it detects fast, result's judgement was finished in 3-15 minute, need not dedicated experiments facility, test conditions and special operator, be fit to tuberculosis infection crowd's primary dcreening operation and epidemiology survey.
2, the preparation method of test strip of the present invention have simple for production, stability, good reproducibility.
The drawing explanation
Fig. 1 is a structural representation of the present invention
1-liner plate 2-protective layer 3-handheld terminal water accepting layer 4-detection layers
5-detection line 6-control line 7-gold mark antigen layer 8-test lead water accepting layer
Embodiments of the present invention
The present invention also will be described in further detail in conjunction with the embodiments:
Embodiment one:
With reference to Fig. 1, a kind of tuberculosis antibody gold label test strip comprises liner plate 1 and protective layer 2; Be respectively equipped with handheld terminal water accepting layer 3 and the test lead water accepting layer of making by multilayer filter paper 8 at the two ends of liner plate 1; On the liner plate 1 between handheld terminal water accepting layer 3 and the test lead water accepting layer 8, be provided with detection layers 4; Between test lead water accepting layer 8 and detection layers 4, be provided with golden labeling antibody layer 7; On detection layers 4, be provided with detection line 5 and control line 6.
A kind of preparation method of tuberculosis antibody gold label test strip comprises golden labeling antibody layer 7 and detection layers 4 and goes up the preparation of wrapping tested survey line 5 and control line 6, and the assembling of test strip.
The preparation of described golden labeling antibody layer 7 comprises: the preparation of i Radioactive colloidal gold is the trisodium citrate that adds its volume 1.6% in the hydrochloro-auric acid of 0.01-0.03% in concentration, boils 10-20 minute, is reduced into the Radioactive colloidal gold atom liquid of 15-50 nanometer; The preparation of ii tuberculosis antigen, get tubercule bacillus type strain TBRV strain culture 20ml (scrape get the tuberculosis bacterium colony from solid medium) and be dissolved in 20ml, 0.9% NaCl solution, through 3000 rev/mins separated in centrifugal 10 minutes throw out, again throw out is dissolved in 20ml, the 0.9%NaCl solution, through 3000 rev/mins of centrifugations, so repetitive scrubbing is removed the substratum composition 6 times again; At last throw out is dissolved in 20ml, the 0.9%NaCl solution ultrasonication 14 times, 30 seconds/time, crushed material through 40000G high speed centrifugation 1 hour, is got supernatant liquor and crossed Sepharcly S100 gel chromatography column, with 0.9%NaCl eluant solution, flow velocity 0.6ml/ branch, collect the active peak of tuberculosis antigen; Iii tuberculosis antigen colloid gold label adds the tuberculosis antigen that the above-mentioned ii of 6mg prepares in the 100ml colloidal gold solution; In above-mentioned colloidal gold solution, press 0.24g/100ml again and add bovine serum albumin; In above-mentioned colloidal gold solution, press the NaCl solution of 8ml/100ml adding 10% then again; After 2000 rev/mins went precipitation in centrifugal 10 minutes, get supernatant again through 10000 rev/mins centrifugal 1 hour throw out, with throw out by 8ml/100ml be dissolved in 0.02M, PH7.4Tris-HCl liquid gets the tuberculosis antigen colloidal gold solution; Wherein contain 0.25% bovine serum albumin, 0.02% sodium azide; Iv is impregnated with glass fibre or non-woven fabrics with the tuberculosis antigen colloidal gold solution, and the immersion amount is with till immersing liquid and beginning outwards to flow out, then at 37 ℃ of dry down golden labeling antibody layers 7.
The preparation of described detection layers 4 is to add 2% fixing agent in the tuberculosis antigen of 2mg/ml and the rabbit anti-mycobacterium tuberculosis antibody in the concentration of preparation, and Membrane jetter is sprayed on tuberculosis antigen and rabbit anti-mycobacterium tuberculosis antibody and forms detection line 5 and control line 6 on the cellulose membrane then; Contain 10% calf serum sealing rinsing in 30 minutes, the dry detection layers 4 that gets with 0.01MPH7.0PBS liquid again.Described fixing agent is selected from a kind of in formaldehyde, the acetone.
The last filter paper of pasting handheld terminal water accepting layer 3 and test lead water accepting layer 8 at the two ends of plastics lining board 1 respectively; and section is pasted the detection layers 4 that cellulose membrane is made therein; folder pastes the antigen layer 7 that contains the antigenic glass fibre of gold mark between test lead water accepting layer 8 and detection layers 4; its 4/5 part is clipped in test lead water accepting layer 8; 1/5 part is pressed on the detection layers 4, encloses the protective layer 2 of non-setting adhesive then on handheld terminal water accepting layer 3 and test lead water accepting layer 8.
Embodiment two:
Except that the cellulose membrane upper control line 6 of detection layers 4 is the goat-anti mycobacterium tuberculosis antibody, all the other conditions all with example together.
Claims (6)
1, a kind of tuberculosis antibody gold label test strip comprises liner plate (1) and protective layer (2); It is characterized in that being respectively equipped with handheld terminal water accepting layer (3) and test lead water accepting layer (8) at the two ends of liner plate (1); On the liner plate (1) between handheld terminal water accepting layer (3) and the test lead water accepting layer (8), be provided with detection layers (4); Between test lead water accepting layer (8) and detection layers (4), be provided with tuberculosis antigen gold labeling antibody layer (7); On detection layers (4), be provided with detection line (5) and control line (6).
2, the preparation method of the described tuberculosis antibody gold label test strip of a kind of claim 1 is characterized in that comprising golden labeling antibody layer (7) and detection layers (4) and goes up the preparation of wrapping tested survey line (5) and control line (6), and the assembling of test strip.
3, the preparation method of tuberculosis antibody gold label test strip as claimed in claim 2 is characterized in that the preparation of described golden labeling antibody layer (7) comprising: the preparation of i Radioactive colloidal gold, and adopt known method to prepare Radioactive colloidal gold; The preparation of ii tuberculosis antigen is got tubercule bacillus type strain TBRV strain culture and is dissolved in the physiological saline centrifugation and gets throw out, throw out is dissolved in centrifugation again behind the physiological saline again, and so repetitive scrubbing is removed the substratum composition for 4-8 time; With the throw out ultrasonication, crushed material through high speed centrifugation, is got supernatant liquor and crossed gel chromatography column then, collect the active peak of tuberculosis antigen; Iii tuberculosis antigen colloid gold label adds the tuberculosis antigen that the above-mentioned ii of 2-10mg prepares in the 100ml colloidal gold solution; In above-mentioned colloidal gold solution, press 0.1-0.6g/100ml again and add animal serum albumin; Adding concentration by 6-12ml/100ml again in above-mentioned colloidal gold solution then is 10% saline solution; The last centrifugal precipitation of going, get supernatant centrifugal again throw out, throw out is dissolved in by 4-10ml/100ml 0.02M, PH7.4Tris-HCl (PBS) contain the 0.1-0.6% animal serum albumin, 0.02% sodium azide gets the tuberculosis antigen colloidal gold solution; Iv is impregnated with glass fibre or non-woven fabrics with the tuberculosis antigen colloidal gold solution, till the immersion amount begins outwards to flow out with immersion liquid, then at 37 ℃ of dry down golden labeling antibody layers (7) that get.
4,, it is characterized in that described gel chromatography column is selected from a kind of among Sephardex G100, Sephardex G200, SepharclyS100, Sepharcly S200, the Sepharcly S300 as the preparation method of claim 2 or 3 described tuberculosis antibody gold label test strips.
5, the preparation method of tuberculosis antibody gold label test strip as claimed in claim 2, it is characterized in that the preparation of described detection layers (4), add fixing agent in the tuberculosis antigen of preparation and rabbit, goat-anti mycobacterium tuberculosis antibody, Membrane jetter is sprayed on tuberculosis antigen and rabbit, goat-anti mycobacterium tuberculosis antibody and forms detection line (5) and control line (6) on the cellulose membrane then; Contain 10% calf serum sealing rinsing in 30 minutes, the dry detection layers (4) that gets with 0.01MPH7.0PBS liquid again.
6,, it is characterized in that described fixing agent is selected from a kind of in formaldehyde, the acetone as the preparation method of claim 2 or 5 described tuberculosis antibody gold label test strips.
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| CN 01131834 CN1425775A (en) | 2001-12-11 | 2001-12-11 | Tuberculosis antibody gold label test strip and its preparing method |
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| CN 01131834 CN1425775A (en) | 2001-12-11 | 2001-12-11 | Tuberculosis antibody gold label test strip and its preparing method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101533015A (en) * | 2009-04-03 | 2009-09-16 | 李克生 | Method for detecting tuberculosis antibody array, array detection kit and preparation method thereof |
| CN101871938A (en) * | 2010-05-12 | 2010-10-27 | 蓝十字生物药业（北京）有限公司 | Rapid test strip with colloid gold label |
| CN101925819A (en) * | 2007-12-28 | 2010-12-22 | 株式会社比尔生命 | Immunodetection assay for mycobacterium tuberculosis complex |
| CN101650365B (en) * | 2008-08-14 | 2012-11-14 | 美艾利尔（上海）诊断产品有限公司 | Method and kit for quickly detecting mycobacterium tuberculosis |
| CN101520458B (en) * | 2008-02-25 | 2014-04-16 | 广州天美生物技术有限公司 | Simple method for detecting HLA-G and antibody thereof by gold-labeled immunoassay |
-
2001
- 2001-12-11 CN CN 01131834 patent/CN1425775A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101925819A (en) * | 2007-12-28 | 2010-12-22 | 株式会社比尔生命 | Immunodetection assay for mycobacterium tuberculosis complex |
| CN101925819B (en) * | 2007-12-28 | 2013-06-12 | 株式会社比尔生命 | Immunodetection assay for mycobacterium tuberculosis complex |
| CN103123353B (en) * | 2007-12-28 | 2014-11-12 | 株式会社比尔生命 | Immunodetection assay for mycobacterium tuberculosis complex |
| CN103123354B (en) * | 2007-12-28 | 2014-11-12 | 株式会社比尔生命 | Immunodetection assay for mycobacterium tuberculosis complex |
| CN101520458B (en) * | 2008-02-25 | 2014-04-16 | 广州天美生物技术有限公司 | Simple method for detecting HLA-G and antibody thereof by gold-labeled immunoassay |
| CN101650365B (en) * | 2008-08-14 | 2012-11-14 | 美艾利尔（上海）诊断产品有限公司 | Method and kit for quickly detecting mycobacterium tuberculosis |
| CN101533015A (en) * | 2009-04-03 | 2009-09-16 | 李克生 | Method for detecting tuberculosis antibody array, array detection kit and preparation method thereof |
| CN101533015B (en) * | 2009-04-03 | 2013-12-25 | 李克生 | Kit for detecting tuberculosis antibody array |
| CN101871938A (en) * | 2010-05-12 | 2010-10-27 | 蓝十字生物药业（北京）有限公司 | Rapid test strip with colloid gold label |
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Open date: 20030625 |