CN202837307U - Neutrophil gelatinase-associated lipocalin (NGAL) fluorescence nano immunochromatography quantitative test strip - Google Patents
Neutrophil gelatinase-associated lipocalin (NGAL) fluorescence nano immunochromatography quantitative test strip Download PDFInfo
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- CN202837307U CN202837307U CN 201220392399 CN201220392399U CN202837307U CN 202837307 U CN202837307 U CN 202837307U CN 201220392399 CN201220392399 CN 201220392399 CN 201220392399 U CN201220392399 U CN 201220392399U CN 202837307 U CN202837307 U CN 202837307U
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Abstract
The utility model discloses a neutrophil gelatinase-associated lipocalin (NGAL) fluorescence nano immunochromatography quantitative test strip. A sample pad, a combination pad, a nitrocellulose membrane and a piece of absorbent paper are pasted on a bottom liner of the test strip in sequence in an overlapping joint mode. Fluorescence nano-particle labeled anti-NGAL antibodies A are coated on the combination pad. An anti-NGAL antibody B detection line and a rabbit-anti-rat intravenous gamma globulin (IgG) antibody quality control line are coated on the nitrocellulose membrane. The NGAL fluorescence nano immunochromatography quantitative test strip can achieve rapid quantitative detection, and is simple and convenient to operate and high in sensitivity.
Description
Technical field
The utility model belongs to medical immunology in-vitro diagnosis field, is specifically related to a kind of NGAL fluorescence nano immune quantitative test strip.
Background technology
Neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-associated lipocalin, NGAL) be 1993 found first in neutrophil leucocyte, with inflammation, embryonic development, immune response, chemotaxis, signal transduction and kinds of tumors to betide the processes such as development relevant.Studies show that both at home and abroad NGAL albumen has the characteristics of specific expressed variation in the various diseases generating process, so that NGAL becomes the biomarker that detects disease.
The result of study of in recent years U.S.'s clinical chemistry association (AACC) annual meeting is delivered and is learnt that neutrophil leucocyte gelatinase relative carrier lipoprotein may help the clinician to detect the toxicity that the turnover in patients following heart transplantation cyclosporin is induced in the detection urine, and the irreversible injury of regulating ring p0-357 dosage to prevent it that kidney is caused.Chinese patent CN101163971A studies early stage that matter ephrosis (ATIN) is fallen ill between acute renal failure (ARF), acute tubular necrosis (ATN) and acute tubular, the NGAL level significantly increases in urine and the blood, so NGAL is that the early detection kidney is to the mark of ischemia injury.
At present, detect the NGAL method mainly contain euzymelinked immunosorbent assay (ELISA) (Chinese patent CN101650369A), based on micro-column method of testing (Chinese patent CN101163971A) and the latex immunoturbidimetry (Chinese patent CN102072960A) of antibody mediated immunity.The euzymelinked immunosorbent assay (ELISA) processing ease is subject to the impact that enzymatic activity changes, and the result is not too accurate; Latex turbidimetry atopic is bad, required reagent more complicated.
It is a kind of fluorescence nano and protein molecular coupling to be prepared into the immunochromatography diagnostic reagent that fluorescence nano detects, and the novel method for quick of testing with special fluorescence nano detector.The method has advantages such as detecting quick, easy and simple to handle, suitable bedside detection and real time record saving result, yet there are no NGAL and detects the report that adopts fluorescence nano dry type immunological technique.
Summary of the invention
The NGAL fluorescence nano quantitative testing test paper bar that the utility model purpose provides a kind of fast detecting, easy and simple to handle, highly sensitive, suitable bedside to detect.
The technical solution of the utility model is: a kind of NGAL fluorescence nano immunochromatographiassay assay quantitative detection test paper, sample pad, pad, nitrocellulose filter and thieving paper are pasted in overlap joint ground successively on end liner, be coated with the anti-NGAL antibody A of fluorescent nano particle mark on the described pad, be coated with the detection line of anti-NGAL antibody B and the nature controlling line of rabbit anti-mouse igg antibody on the described nitrocellulose filter.
Described fluorescent nano particle is inorganic light-emitting quantum dot or composite fluorescence Nano particles of silicon dioxide.
The anti-NGAL antibody A of described mark and coated anti-NGAL antibody B are the monoclonal antibody of pairing or the monoclonal antibody of pairing-how anti-.
Described pad material is glass fibre element film or polyester film.
Described sample pad material is glass fibre element film, polyester film or red blood cell filtering membrane.
A kind of preparation method of NGAL fluorescence nano immunochromatographiassay assay quantitative detection test paper comprises the steps:
1). the preparation of coated fluorescent nano particle mark NGAL antibody A pad
Fluorescent nano particle forms fluorescence nano antibody A compound with the NGAL antibody A under the effect of coupling agent; The anti-NGAL antibody A of gained fluorescent nano particle mark is sprayed on the pad.
2). the preparation of nitrocellulose filter
Dilute respectively anti-NGAL antibody B and rabbit anti-mouse igg antibody with coated damping fluid, and two kinds of antibody after will diluting are sprayed on respectively on the nitrocellulose filter abreast, and two kinds of antibody infiltrate the nature controlling line that forms respectively anti-NGAL antibody B detection line and rabbit anti-mouse igg antibody behind the nitrocellulose filter.
3). on end liner, mutually paste sample pad, pad, nitrocellulose filter and thieving paper in turn overlap joint and obtain test paper plate, cut into as requested the test strips of proper width.
The utility model compared with prior art has following advantage:
1) the utility model is easy to make, and volume is little, be easy to carry;
2) the utility model can accurately carry out quantitatively NGAL content by the detection to fluorescence nano-antibody on the test strips-antigen-antibody complex fluorescence signal;
3) the utility model can be mass, and is applicable to bedside quick diagnosis and field quick detection, is easy to preserve and promoting the use of at basic hospital.
Description of drawings
Fig. 1 is the utility model NGAL fluorescence nano immune quantitative test strip structural representation;
Wherein, Fig. 1: 1, end liner; 2, sample pad; 3, pad; 4, nitrocellulose filter; 5, thieving paper; 6, detection line; 7, nature controlling line.
Embodiment
Below in conjunction with the drawings and specific embodiments the utility model is described in further detail.
As shown in Figure 1, a kind of NGAL fluorescence nano immunity Quantitative detection test strips, comprise end liner 1, thieving paper 5, from left to right paste successively sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads 5 on the end liner 1, be coated with the fluorescent nano particle of NGAL monoclonal antibody phase coupling on the pad 3, be coated with the detection line 6 of NGAL antibody and the nature controlling line 7 of rabbit anti-mouse igg antibody on the nitrocellulose filter 4.
Described fluorescent nano particle is inorganic light-emitting quantum dot or composite fluorescence Nano particles of silicon dioxide.
Described pad material is glass fibre element film or polyester film.
Described sample pad material is glass fibre element film, polyester film or red blood cell filtering membrane.
The preparation method is as follows for NGAL fluorescence nano immune quantitative test strip:
1). the preparation of coated fluorescent nano particle mark NGAL antibody A pad 3
CdTe fluorescence quantum and NGAL monoclonal antibody 3B1 obtain CdTe mark NGAL antibody complex under coupling agent EDC effect, the shower nozzle of gained compound with the Bio-Dot instrument is sprayed onto on the glass fibre element film, namely get pad 3.
Described NGAL monoclonal antibody 3B1 is documented in the Chinese patent application numbers 2012102309716, produced by hybridoma cell strain 3B1, described hybridoma cell strain 3B1 depositary institution is Chinese Typical Representative culture collection center (CCTCC), its deposit number is CCTCC NO:C201249, and preservation date is on May 9th, 2012.
2). the preparation of detection line 6: use the PBS (pH7.2) of 20mmol/L to be diluted to the concentration of 3mg/ml NGAL monoclonal antibody 4C2, in nitrocellulose filter 4 line, namely get detection line 6,20 ℃ of forced air drying 12h in drying box by 0.8 μ l/cm.
Described NGAL monoclonal antibody 4C2 is documented in the Chinese patent application numbers 2012102309716, produced by hybridoma cell strain 4C2, described hybridoma cell strain 4C2 depositary institution is Chinese Typical Representative culture collection center (CCTCC), its deposit number is CCTCC NO:C201250, and preservation date is on May 9th, 2012.
3). the preparation of nature controlling line 7: use the PBS (pH7.2) of 20mmol/L to be diluted to the concentration of 3mg/ml rabbit anti-mouse igg antibody, rule at nitrocellulose filter 4 by 0.8 μ l/cm, namely get nature controlling line 7, this line is parallel with detection line 6, then 20 ℃ of forced air drying 12h in drying box, hermetically drying is preserved.
4) preparation of sample pad 2
5) test strips assembling
On end liner 1, mutually paste in turn sample pad 2, glass fibre element film 3, nitrocellulose filter 4 and thieving paper 5 overlap joint and obtain test paper plate, cut into as requested the test strips of proper width.
The preparation method is as follows for NGAL fluorescence nano immune quantitative test strip:
1). the preparation of coated fluorescent nano particle mark NGAL antibody A pad 3
1. contain the silica fluorescent nano particle of covalently bound organic dyestuff and the Streptavidin PBS solution of 0.5mg/ml, pH7.4,4 ℃ of reaction 18-24h with the sealing of 1mol/L glycocoll, washing, namely get Streptavidin-silica fluorescent nano-complex.
2. will resist NGAL monoclonal antibody 3B1 to be diluted to 1mg/ml with 0.1mol/L, pH8.0 sodium bicarbonate buffer liquid, with 0.1mol/L, pH8.0 sodium bicarbonate buffer liquid NGAL antibody 3B1 fully be dialysed alternately; NHSB with 1ml DMSO dissolving 1mg obtains NHSB solution; Add 20 μ l NHSB solution to above-mentioned 1mlNGAL monoclonal antibody 3B1 solution, stirring at room 2-4 hour, continued stirring at room 10 minutes, then with 20mM, the dialysis of pH7.2PBS damping fluid, namely get the anti-NGAL monoclonal antibody of biotinylation 3B1.
3. Streptavidin-silica fluorescent nano-complex and the anti-NGAL monoclonal antibody of biotinylation 3B1 are mixed, react centrifugal after 30 minutes, precipitation returns to original volume with the sodium citrate buffer solution dissolving of the 50mM, the pH5.8 that contain 0.05%BSA.
4. with step 3. gained silica fluorescent Nanoparticle labeling NGAL monoclonal antibody 3B1 be sprayed onto on the glass fibre element film with the shower nozzle of Bio-Dot instrument, namely get pad 3.
2). the preparation of detection line 6: use the PBS (pH7.2) of 20mmol/L to be diluted to the concentration of 3mg/ml the NGAL polyclonal antibody, in nitrocellulose filter 4 line, namely get detection line 6,20 ℃ of forced air drying 12h in drying box by 0.8 μ l/cm.
3). the preparation of nature controlling line 7: the concentration that rabbit anti-mouse igg antibody is diluted to 3mg/ml with the PBS (pH7.2) of 20mmol/L, rule at nitrocellulose filter 4 by 0.8 μ l/cm, namely get nature controlling line 7, this line is parallel with detection line 6, then 20 ℃ of forced air drying 12h in drying box, hermetically drying is preserved.
4) preparation of sample pad 2
5) test strips assembling
On end liner 1, mutually paste in turn sample pad 2, glass fibre element film 3, nitrocellulose filter 4 and thieving paper 5 overlap joint and obtain test paper plate, cut into as requested the test strips of proper width.
Get fresh serum, whole blood or urine 100 μ l, join and carry out immune response, time 5-8min on the test strips sample pad; Reaction is put in special fluorescence signal with test strips and reads in the instrument after finishing, and reads the size of fluorescence signal, carries out quantitative measurement.
Claims (5)
1. NGAL fluorescence nano immune quantitative test strip, sample pad (2), pad (3), nitrocellulose filter (4) and thieving paper (5) are pasted in overlap joint ground successively on end liner (1), it is characterized in that being coated with on the described pad (3) the anti-NGAL antibody A of fluorescent nano particle mark, be coated with the detection line (6) of anti-NGAL antibody B and the nature controlling line (7) of rabbit anti-mouse igg antibody on the described nitrocellulose filter (4).
2. described NGAL fluorescence nano immune quantitative test strip according to claim 1 is characterized in that described fluorescent nano particle is inorganic light-emitting quantum dot or composite fluorescence Nano particles of silicon dioxide.
3. described NGAL fluorescence nano immune quantitative test strip according to claim 1 is characterized in that the anti-NGAL antibody A of described mark and coated anti-NGAL antibody B are the monoclonal antibody of pairing or the monoclonal antibody of pairing-how anti-.
4. described NGAL fluorescence nano immune quantitative test strip according to claim 1 is characterized in that described pad (3) material is glass fibre element film or polyester film.
5. described NGAL fluorescence nano immune quantitative test strip according to claim 1 is characterized in that described sample pad (2) material is glass fibre element film, polyester film or red blood cell filtering membrane.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201220392399 CN202837307U (en) | 2012-08-09 | 2012-08-09 | Neutrophil gelatinase-associated lipocalin (NGAL) fluorescence nano immunochromatography quantitative test strip |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201220392399 CN202837307U (en) | 2012-08-09 | 2012-08-09 | Neutrophil gelatinase-associated lipocalin (NGAL) fluorescence nano immunochromatography quantitative test strip |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104880563A (en) * | 2015-04-08 | 2015-09-02 | 上海盛复源生物医药有限公司 | NGAL rapid detection reagent and preparation method thereof |
| CN105067584A (en) * | 2015-08-11 | 2015-11-18 | 郑州安图生物工程股份有限公司 | Immunochromatographic kit for quantitative detection of RV (rubella virus) IgG (immunoglobulin G) antibody through quantum dots |
| CN105527439A (en) * | 2015-12-30 | 2016-04-27 | 厦门依柯利斯医疗科技有限公司 | An NGAL time-resolved fluoroimmunoassay nanometer immunochromatographic quantitative detection test paper strip and a preparing method thereof |
-
2012
- 2012-08-09 CN CN 201220392399 patent/CN202837307U/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104880563A (en) * | 2015-04-08 | 2015-09-02 | 上海盛复源生物医药有限公司 | NGAL rapid detection reagent and preparation method thereof |
| CN105067584A (en) * | 2015-08-11 | 2015-11-18 | 郑州安图生物工程股份有限公司 | Immunochromatographic kit for quantitative detection of RV (rubella virus) IgG (immunoglobulin G) antibody through quantum dots |
| CN105527439A (en) * | 2015-12-30 | 2016-04-27 | 厦门依柯利斯医疗科技有限公司 | An NGAL time-resolved fluoroimmunoassay nanometer immunochromatographic quantitative detection test paper strip and a preparing method thereof |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: NANJING EGG-BASED BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: SU ENBEN Effective date: 20130619 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20130619 Address after: Liuhe District of Nanjing City, Jiangsu province 211505 Yangtze River Industrial Development Zone Bo Fu Road No. 9 Patentee after: Nanjing Getein Biotechnology Co.,Ltd. Address before: Liuhe District of Nanjing City, Jiangsu province 211505 Yangtze River Industrial Development Zone Bo Fu Road No. 9 Patentee before: Su Enben |
|
| C56 | Change in the name or address of the patentee |
Owner name: GETEIN BIOTECH, INC. Free format text: FORMER NAME: NANJING EGG-BASED BIOTECHNOLOGY CO., LTD. |
|
| CP01 | Change in the name or title of a patent holder |
Address after: Liuhe District of Nanjing City, Jiangsu province 211505 Yangtze River Industrial Development Zone Bo Fu Road No. 9 Patentee after: GETEIN BIOTECH, Inc. Address before: Liuhe District of Nanjing City, Jiangsu province 211505 Yangtze River Industrial Development Zone Bo Fu Road No. 9 Patentee before: Nanjing Getein Biotechnology Co.,Ltd. |
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| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20130327 |