CN102759623A - Colloidal gold test strip for detecting NGAL (Neutrophil Gelatinase Associated Lipocalin) and preparation method thereof - Google Patents
Colloidal gold test strip for detecting NGAL (Neutrophil Gelatinase Associated Lipocalin) and preparation method thereof Download PDFInfo
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- CN102759623A CN102759623A CN2012102315098A CN201210231509A CN102759623A CN 102759623 A CN102759623 A CN 102759623A CN 2012102315098 A CN2012102315098 A CN 2012102315098A CN 201210231509 A CN201210231509 A CN 201210231509A CN 102759623 A CN102759623 A CN 102759623A
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Abstract
The invention discloses a colloidal gold test strip for detecting neutrophil gelatinase associated lipocalin (NGAL) and a preparation method thereof. The colloidal gold test strip comprises a plastic bottom liner, a nitrocellulose membrane, a gold mark pad and a water sucking pad; a quality control line and a detection line which are mutually parallel are arranged on the nitrocellulose membrane; and the quality control line contains a rabbit anti-human IgG antibody, the detection line contains a NGAL (Neutrophil Gelatinase Associated Lipocalin) monoclonal antibody, and the gold mark pad contains another NGAL monoclonal antibody marked by colloidal gold. The colloidal gold test strip has the advantages of being capable of carrying out early prediction on acute renal failure, having strong specificity, being accurate in results, simple in operation and suitable for rapid diagnosis at bedsides.
Description
Technical field
The invention belongs to the clinical diagnose field, be specifically related to a kind of detection neutrophil gelatinase-associated lipocalin (NGAL) colloidal gold strip and preparation method thereof.
Background technology
Be secondary to the renal tubular cell injury acute renal failure (AFR) of (comprising the damage of ischemia injury or renal toxicity); Although in the supportive treatment method, obtained great progress; But remain a general and potential destructive difficult problem in clinical medicine and the nephrology, this disease has the lasting mortality ratio and the characteristics of high incidence.Research shows that neutrophil gelatinase-associated lipocalin (NGAL) is one of important symbol thing of diagnosing acute injury of kidney as one of lipocalin family new member.United States Patent (USP) 2004/0219603 has been described the urine biomarker that neutrophil gelatinase-associated lipocalin (NGAL) detects the renal tubular cell injury early onset thereof the most.
The result of study of U.S.'s clinical chemistry association (AACC) annual meeting is in recent years delivered and is learnt that detecting in the urine the relevant apolipoprotein of neutrophil leucocyte gelatinase has and help the clinician and detect the toxicity that heart transplant patient cyclosporin is induced, and the irreversible injury of regulating ring p0-357 dosage to prevent it that kidney is caused.Kim RW etc. shows that the NGAL in the urine and blood reached highest level in 6 hours after surgery after the openheart surgery, and urine NGAL and blood NGAL level are relevant with postoperative acute injury of kidney AKI and poor prognosis.
At present, the method that detects NGAL mainly contains immunosorbent determination method (ELISA), and Chinese patent CN101566633 relates to the level that latex enhancing immune turbidimetry, sandwich method ELISA, competition law ELISA are measured NGAL.Yet this method has certain requirement to checkout equipment, complex operation, and detection time is long, can not carry out quick early prediction to acute renal failure, can not be used for the bedside quick diagnosis.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art; Provide a kind of high specificity, result accurate, simple to operate; Be fit to detection NGAL colloidal gold strip of bedside quick diagnosis and preparation method thereof, carrying out early prediction for acute renal failure provides a kind of simple detection diagnostic method and instrument.
For addressing this problem, the present invention adopts following technical scheme to be achieved:
A kind of detection NGAL colloidal gold strip comprises plastics end liner, nitrocellulose filter, gold mark pad, sample pad, adsorptive pads, it is characterized in that described nitrocellulose filter is provided with nature controlling line and the detection line that is parallel to each other; Contain rabbit anti-human igg's antibody on the described nature controlling line, said detection line contains a kind of NGAL monoclonal antibody, and the another kind of NGAL monoclonal antibody of golden mark is arranged on the described gold mark pad.
NGAL monoclonal antibody on the described detection line is secreted by hybridoma cell strain 3B1 and hybridoma cell strain 4B2 with the another kind of NGAL monoclonal antibody on the gold mark pad and is produced.Hybridoma cell strain 3B1 and hybridoma cell strain 4C2 derive from Nanjing base egg bio tech ltd; Be deposited in Chinese typical culture collection center (CCTCC); Deposit number is respectively CCTCC NO:C201249 and CCTCC NO:C201250, and preservation date is on May 9th, 2012.
A kind of NGAL monoclonal antibody on the said detection line can be matched with the another kind of NGAL monoclonal antibody of the golden mark that the gold mark fills up each other.
Said a kind of NGAL monoclonal antibody is 3B1, and the another kind of NGAL monoclonal antibody of golden mark is 4C2.
Said a kind of NGAL monoclonal antibody is 4C2, and the another kind of NGAL monoclonal antibody of golden mark is 3B1.
Prepare a kind of preparation method who detects the NGAL colloidal gold strip, its step comprises:
1) a kind of NGAL monoclonal anti body fluid is sprayed onto forms detection line on the nitrocellulose filter, another zone that rabbit anti-human igg's antibody liquid is sprayed onto nitrocellulose filter forms nature controlling line;
2) another kind of NGAL monoclonal antibody is added in the collaurum, obtains collaurum---antibody complex solution, with obtaining gold mark pad on plain film of this complex solution coated glass fiber or the polyester film;
3) plastics end liner, nitrocellulose filter, gold mark pad, sample pad, adsorptive pads are pasted, be assembled into and detect the NGAL colloidal gold strip.
Described nitrocellulose filter encapsulates in the process, and NGAL monoclonal anti body fluid is that PB (sodium hydrogen phosphate, the sodium dihydrogen phosphate) damping fluid with 20mmol/L, pH7.4 is diluted to 2.0mg/ml.
The hybridoma cell strain 3B1 monoclonal antibody that secretion produces with hybridoma cell strain 4C2 has different epi-positions, identifies through the double antibodies sandwich method.
Described nature controlling line encapsulates rabbit anti-human igg's antibody.
Said gold mark pad is polyester film, glass fibre membrane with the material of sample pad.
The present invention utilizes the double antibodies sandwich principle to detect the content of NGAL, comes the content of NGAL in the judgement sample liquid according to band color on the detection line, if there is not the aubergine band to occur on the nature controlling line, then this test strips lost efficacy.
Detection NGAL colloidal gold strip of the present invention has the following advantages: 1) detect the NGAL kit with present ELISA method and compare; Do not need the professional; But Direct observation obtains the result, also can in ten minutes, obtain quantitative result according to FIA8000 series immune quantitative analyser; 2) detection method is simple, fast, is applicable to NGAL bedside quick diagnosis.The present invention carries out quick early prediction to acute renal failure, has a extensive future.
Description of drawings
Fig. 1 is a kind of NGAL colloidal gold strip structural representation that is used to detect.
Fig. 2 is a kind of NGAL colloidal gold strip detecting pattern figure that is used to detect.
Fig. 3 is the range of linearity correlativity of the test strips of the embodiment of the invention 1 preparation.
Fig. 4 is that the test strips and the ARCHITECT of the Abbott Laboratories urine neutrophil gelatinase-associated lipocalin of the embodiment of the invention 1 preparation measured kit testing result correlativity.
Wherein, 1. plastics end liner; 2. nitrocellulose filter; 3. the gold mark fills up; 4. sample pad; 5. adsorptive pads; 6. nature controlling line; 7. detection line; 8. sample cell; A. chromatography direction.
Embodiment:
Below in conjunction with accompanying drawing and embodiment the present invention is done further detailed elaboration.
Below embodiments of the invention are elaborated: present embodiment provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.The experimental technique of not marked actual conditions in the following example is usually according to normal condition.
1, the preparation of collaurum-antibody complex
(1) preparation aqueous solution of chloraurate: with the dissolving of the tri-distilled water of 10g gold chloride 1000ml, be mixed with 1% the WS, place 4 ℃ subsequent use, the term of validity 3 months;
(2) preparation trisodium citrate: dissolve trisodium citrate with tri-distilled water, preparation 1% the WS, with 0.22 μ membrane filtration mistake, place 4 ℃ subsequent use, the term of validity 7 days;
(3) preparation 1% wet chemical: 1g sal tartari is dissolved with tri-distilled water with 100ml, with 0.22 μ membrane filtration mistake, place 4 ℃ subsequent use, the term of validity 7 days;
(4) the golden labeling antibody of preparation is preserved liquid: with the sucrose of 15g, the Sodium azide of 20 μ l, in the 1%BSA of 100mlpH 7.4 dissolving, with 0.22 μ membrane filtration mistake, place 4 ℃ subsequent use, the term of validity 7 days;
(5) preparation colloid gold particle: 1% gold chloride is diluted to 0.01% with tri-distilled water; Place 95 ℃ of reactions 10 minutes, add the 1ml trisodium citrate, continue reaction 15 minutes; Treat the colloidal gold solution color by indigo plant after purple stain is red, it is subsequent use that cooling back adds 2ml 1% solution of potassium carbonate.Outward appearance should be pure, and is bright, do not have deposition and floating thing;
(6) preparation collaurum-antibody complex: regulate collaurum pH value to 7.5 with 1% solution of potassium carbonate; Amount according to 10 μ g streptavidin/ml collaurums adds the abundant mixing of streptavidin solution; Place 25 ℃ of water-baths to add 5%BSA after 30 minutes again, seal after 20 minutes, the centrifuging and taking deposition; Using BSA to recover its final concentration is that 1%, 4 ℃ of preservation is subsequent use.Amount by 50 μ g monoclonal antibody-biotin/ml collaurums adds NGAL monoclonal antibody 3B1 (or NGAL monoclonal antibody 4C2)-biotin in the collaurum; 37 ℃ of stirring reactions 40 minutes add 5%BSA, and sealing was stirred 20 minutes; Centrifugal 20 minutes of 6000r/min; Abandon supernatant, deposition is preserved liquid with golden labeling antibody and is recovered volume, 4 ℃ of preservations.
2, the assembling of immuno-chromatographic test paper strip
(1) processing of nitrocellulose filter
The preparation of detection line: with the PB (sodium hydrogen phosphate of NGAL monoclonal antibody 4C2 (or NGAL monoclonal antibody 3B1) with 20mmol/L pH7.2; Sodium dihydrogen phosphate) damping fluid is diluted to the concentration of 2.0mg/ml; 0.8 μ l/cm rules on nitrocellulose filter, 20 ℃ of forced air drying 12h in drying box.
The preparation of nature controlling line: with the concentration that rabbit anti-human igg's antibody is pressed 4mg/ml, 0.8 μ l/cm draws nature controlling line on nitrocellulose filter, and this line is parallel with detection line, and line-to-line is at a distance from 4mm, 20 ℃ of forced air drying 12h in drying box then, hermetically drying preservation.
(2) preparation of gold mark pad
The pre-service of gold mark pad: material glass cellulose membrane or the polyester film that will make gold mark pad put into gold mark damping fluid (immersion of 20mM PBS+1% casein+1%PVP+1% sucrose+0.2%Triton) 2～4 hours, after the taking-up, 25 ℃ of dryings 8 hours.
Collaurum-the antibody complex that employing is different from the used antibody NGAL monoclonal antibody 3B1 (or NGAL monoclonal antibody 4C2) of detection line is layered on the gold mark pad of handling well uniformly with the metal spraying appearance; Discharge rate 1.0 μ l/cm; 25 ℃ of dryings 4～8 hours, hermetically drying is preserved.
(3) preparation of sample pad
Sample pad was soaked 2～4 hours with 100mM PBS damping fluid, took out back 25 ℃ of dryings 8 hours.
(4) preparation of adsorptive pads
Thieving paper is cut into every of 30*2.7cm.
(5) assembling
Plastics end liner, sample pad and thieving paper are the general parts in this area.With above-mentioned nitrocellulose filter, adsorptive pads, gold mark pad, sample pad sticks on (like accompanying drawing 1) on the plastics end liner.The intermediate that posts is cut into the wide test strips of 5.6cm with cutting machine.
Embodiment 2 detects the use of NGAL colloid gold test paper
1. qualitative detection
Be added in sample drop to be checked on the sample pad of this test paper, when sample to be checked got into test lead, because capillary effect, liquid moved along chromatography direction A.If occur a mauve band respectively, show that containing corresponding antigen in the liquid to be checked is positive at the nature controlling line of test paper and the position of detection line; If only mauve band occurs, show that containing corresponding antigen in the liquid to be checked is negative sample below detecting lower limit in the nature controlling line position of test paper; If all do not have mauve band appearance at the nature controlling line of test paper and the position of detection line, show that this testing result is a null result.
2. detection by quantitative
(1) range of linearity
Adopt the test strips of the present invention's preparation to do the experiment of the detection range of linearity.Get the NGAL standard items; Be diluted to 8 concentration with physiological saline, its concentration range is 20ng/mL-1500ng/mL, each concentration replication 3 times; Mean value and the theoretical concentration of measuring concentration are carried out linear regression analysis; Calculate regression equation y=0.905x-22.17, correlation coefficient r=0.9995 shows test strips of the present invention fine (see figure 3) of correlativity in the 20ng/mL-1500ng/mL range of linearity.
(2) sensitivity
With physiological saline is dummy, measures with test strips of the present invention, repeats 20 times, and calculating reading average V is 23.95, and standard deviation SD is 8.319.Calculating V+2SD is 40.59, is 15.58ng/ml according to regression equation calculation dummy concentration.
(3) sensing range
Whenever get concentration value at a distance from 8% at the linear detection range lower limit, standard items are diluted to 16.8 ng/mL, 18.4 ng/mL; 20 ng/mL, 21.6 ng/mL, 23.2 ng/mL; Utilize FIA8000 series immune quantitative analyser replication 10 times, calculate coefficient of variation CV respectively.16.8 ng/mL, 18.4 ng/mL NGAL measure CV and are respectively 12.65%, 10.89%.And 20 ng/mL, 21.6 ng/mL, 23.2 ng/mL NGAL measure CV about 8%, all less than 10%.This shows that least concentration level 20 ng/mL are for detecting lower limit.
(4) measure the kit correlativity relatively with the ARCHITECT of Abbott Laboratories urine neutrophil gelatinase-associated lipocalin
Get 100 parts of urine samples; With its five equilibrium, a test strips of the present invention's preparation and the necessary instrument FIA8000 series immune quantitative analyser thereof of adopting measured, and the ARCHITECT of another part Abbott Laboratories urine neutrophil gelatinase-associated lipocalin measures kit and necessary instrument ARCHITECT immunity analysis instrument is measured; Measure the result and see Fig. 4; The result shows the fine r=0.9905 of its correlativity, no difference of science of statistics between kind of the method for P＞0.05, two.
Claims (5)
1. one kind is detected the NGAL colloidal gold strip, comprises plastics end liner, nitrocellulose filter, gold mark pad, sample pad, adsorptive pads, it is characterized in that described nitrocellulose filter is provided with nature controlling line and the detection line that is parallel to each other; Contain rabbit anti-human igg's antibody on the described nature controlling line, said detection line contains a kind of NGAL monoclonal antibody, and the another kind of NGAL monoclonal antibody of golden mark is arranged on the described gold mark pad.
2. detection NGAL colloidal gold strip according to claim 1 is characterized in that a kind of NGAL monoclonal antibody on the detection line can be matched with the another kind of NGAL monoclonal antibody that gold is marked the golden mark on the pad each other.
3. detection NGAL colloidal gold strip according to claim 1 is characterized in that a kind of NGAL monoclonal antibody is 3B1, and the another kind of NGAL monoclonal antibody of golden mark is 4C2.
4. detection NGAL colloidal gold strip according to claim 1 is characterized in that a kind of NGAL monoclonal antibody is 4C2, and the another kind of NGAL monoclonal antibody of golden mark is 3B1.
5. preparation method who detects the NGAL colloidal gold strip may further comprise the steps:
1) a kind of NGAL monoclonal anti body fluid is sprayed onto forms detection line on the nitrocellulose filter, another zone that rabbit anti-human igg's antibody liquid is sprayed onto nitrocellulose filter forms nature controlling line;
2) another kind of NGAL monoclonal antibody is added in the collaurum, obtains collaurum---antibody complex solution, with obtaining gold mark pad on plain film of this complex solution coated glass fiber or the polyester film;
3) plastics end liner, nitrocellulose filter, gold mark pad, sample pad, adsorptive pads are pasted, be assembled into and detect the NGAL colloidal gold strip.
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| CN2012102315098A CN102759623A (en) | 2012-07-06 | 2012-07-06 | Colloidal gold test strip for detecting NGAL (Neutrophil Gelatinase Associated Lipocalin) and preparation method thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102955036A (en) * | 2012-11-27 | 2013-03-06 | 南京医科大学第二附属医院 | Nanometer detection test paper strip of high-sensitivity neutrophils gelatinase associated lipocalin (NGAL) and preparation method thereof |
| CN102967714A (en) * | 2012-12-10 | 2013-03-13 | 天津市协和医药科技集团有限公司 | Neutrophil gelatinase associated lipocalin (NGAL) chemiluminescence detection kit |
| CN103226143A (en) * | 2013-04-07 | 2013-07-31 | 南京基蛋生物科技有限公司 | Dry-type immunoassay test strip and preparation method and application thereof |
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| CN108061800A (en) * | 2017-12-08 | 2018-05-22 | 重庆市畜牧科学院 | Hog cholera antibody colloidal-gold detecting-card and preparation method thereof |
| CN109030833A (en) * | 2018-08-06 | 2018-12-18 | 无锡全策生物科技有限公司 | A kind of neutrophil leucocyte gelatinase correlation apolipoprotein detection kit |
| CN112147323A (en) * | 2020-09-07 | 2020-12-29 | 深圳市第二人民医院 | A test strip for detecting 31-kDa Occludin in cerebral hemorrhage after thrombolysis and its preparation method and use |
| CN114075280A (en) * | 2020-08-19 | 2022-02-22 | 东莞市朋志生物科技有限公司 | Monoclonal antibody for resisting NGAL (Next Generation Clay antigen), application thereof and detection kit |
| WO2022253173A1 (en) * | 2021-05-31 | 2022-12-08 | Fresenius Medical Care Deutschland Gmbh | H-ngal for the detection of peritonitis |
| CN117741165A (en) * | 2023-11-29 | 2024-03-22 | 浙江鼎创医疗科技有限公司 | Colloidal gold immunochromatographic test strip for high-specificity and high-sensitivity HNL detection and preparation method and use method thereof |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102955036B (en) * | 2012-11-27 | 2015-11-18 | 南京医科大学第二附属医院 | Nano material test strip of a kind of hypersensitivity neutrophil leucocyte gelatinase relative carrier lipoprotein NGAL and preparation method thereof |
| CN102955036A (en) * | 2012-11-27 | 2013-03-06 | 南京医科大学第二附属医院 | Nanometer detection test paper strip of high-sensitivity neutrophils gelatinase associated lipocalin (NGAL) and preparation method thereof |
| CN102967714A (en) * | 2012-12-10 | 2013-03-13 | 天津市协和医药科技集团有限公司 | Neutrophil gelatinase associated lipocalin (NGAL) chemiluminescence detection kit |
| CN103226143A (en) * | 2013-04-07 | 2013-07-31 | 南京基蛋生物科技有限公司 | Dry-type immunoassay test strip and preparation method and application thereof |
| CN103529223A (en) * | 2013-10-17 | 2014-01-22 | 天津中新科炬生物制药有限公司 | Detecting device and method for simultaneously detecting cystatin C and neutrophil gelatinase related lipid carrier protein |
| CN105988004B (en) * | 2015-01-30 | 2017-12-29 | 江苏众红生物工程创药研究院有限公司 | The collaurum quantitative testing test paper card of human tissue kallikrein 1 |
| CN105988004A (en) * | 2015-01-30 | 2016-10-05 | 江苏众红生物工程创药研究院有限公司 | Colloidal gold quantitative test strip for human tissue kallikrein 1 |
| WO2017121001A1 (en) * | 2016-01-16 | 2017-07-20 | 深圳市华科安测信息技术有限公司 | Gold-labeled antibody test strip for use in early-stage diabetic nephropathy test |
| CN106932573A (en) * | 2017-04-28 | 2017-07-07 | 天津医科大学总医院 | Immunocolloidal gold test strip for detecting thyroglobulin and preparation method thereof |
| CN108061800A (en) * | 2017-12-08 | 2018-05-22 | 重庆市畜牧科学院 | Hog cholera antibody colloidal-gold detecting-card and preparation method thereof |
| CN109030833A (en) * | 2018-08-06 | 2018-12-18 | 无锡全策生物科技有限公司 | A kind of neutrophil leucocyte gelatinase correlation apolipoprotein detection kit |
| CN114075280A (en) * | 2020-08-19 | 2022-02-22 | 东莞市朋志生物科技有限公司 | Monoclonal antibody for resisting NGAL (Next Generation Clay antigen), application thereof and detection kit |
| CN112147323A (en) * | 2020-09-07 | 2020-12-29 | 深圳市第二人民医院 | A test strip for detecting 31-kDa Occludin in cerebral hemorrhage after thrombolysis and its preparation method and use |
| WO2022253173A1 (en) * | 2021-05-31 | 2022-12-08 | Fresenius Medical Care Deutschland Gmbh | H-ngal for the detection of peritonitis |
| CN117741165A (en) * | 2023-11-29 | 2024-03-22 | 浙江鼎创医疗科技有限公司 | Colloidal gold immunochromatographic test strip for high-specificity and high-sensitivity HNL detection and preparation method and use method thereof |
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