CN102226809A - Time resloved fluoroimmunoassay kit for detecting Galectin-3 - Google Patents
Time resloved fluoroimmunoassay kit for detecting Galectin-3 Download PDFInfo
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- CN102226809A CN102226809A CN2011100368999A CN201110036899A CN102226809A CN 102226809 A CN102226809 A CN 102226809A CN 2011100368999 A CN2011100368999 A CN 2011100368999A CN 201110036899 A CN201110036899 A CN 201110036899A CN 102226809 A CN102226809 A CN 102226809A
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Abstract
The invention discloses a time resloved fluoroimmunoassay (TRF) kit for detecting Galectin-3.The kit comprises: a coating buffer, a blocking solution, a cleaning solution, a loading buffer, a enhancement solution, a assay buffer, coated antibody (monoclonal anti-human Galectin-3 antibody), detection antibody (biotinylated anti-human Galectin-3 antibody), Eu<3+> labeled streptavidin and neomycin, a standard substance (recombinant human Galectin-3). The kit is characterized in that: the kit adopts a double antibody sandwich method and TRF principle for detecting a concentration of the Galectin-3 in a sample. The method is characterized by: coating an ELISA plate through the coated antibody; adding the sample to be detected to the ELISA plate to carry out a incubation after blocking; washing the plate, then adding the detection antibody to the plate to carry out the incubation; washing the plate again, followed by adding the Eu<3+> labeled streptavidin to carry out the incubation; washing the plate, followed by adding the enhancement solution; oscillating for 5 minutes, followed by exciting through light having a excitation wavelength of 337nm; detecting a fluorescence value of the sample at a emission wavelength of 615nm after delaying 200 microseconds; drawing a standard curve through the concentration of the standard substance and the corresponding fluorescence value, such that the concentration of the Galectin-3 in the sample to be detected can be calculated through the fluorescence value and the standard curve.
Description
Technical field
The invention belongs to medical biotechnology field, relate to a kind of time resolution immunofluorescence (Time Resolved Fluoroimmunoassay, TRF) detection method is specially the concentration of using the Galectin-3 albumen in the time resolution immunofluorescence method test sample.There is close association in the generation of the increase of Galectin-3 protein concentration and cancer of pancreas development in the human peripheral, uses this kit to play diagnostic effect to the generation development of cancer of pancreas.
Background technology
Galectin-3 is that the beta galactose glycosides is in conjunction with albumen, belong to the lectin family member, it has the CRD domain, has very high affinity with glycoprotein that contains the beta galactose glycosides and glycolipid, between cell and cell, cell and matrix, play the specific adhesion effect, closely related with transfer, infiltration, growth and the adhesion of tumour.We use the MALDI-TOF-MS mass-spectrometric technique to find that Galectin-3 presents high expressed in the human pancreas cancer tissue
[1], and its expression and cancer of pancreas to reach prognosis relevant by stages.We discover, all do not detect Galectin-3 albumen in normal person, cancer of pancreas and other digestive system tumor periphery serum, and the Galectin-3 positive rate in the Pancreas cancer patients periphery serum is up to being 30%
[2]Therefore, Galectin-3 has clear diagnostic value to cancer of pancreas.
Time resolution immunofluorescence (Time Resolved Fluoroimmunoassay, TRF) detection method is a kind of immune analysis method of maturation, has high special, high-sensitive advantage, being to obtain very using widely in all many-sides such as clinical medicine and laboratory medicines, is a kind of quantitative detecting method that replaces radioimmunoassay at present.Its ultimate principle is to utilize trivalent rare earth ions (as Eu
3+) as tracer, the delay detection life-span to get rid of the interference of non-specific and background fluorescence, is promptly measured the delayed fluorescence intensity that the rare earth ion in the compound is excited than long specificity fluorescence excitation, thereby determine the concentration of testing protein.Use the TRF of fluorescence enhancement solution can detect pik (pg, 10
-12G/ml) albumen of level, therefore, this kit adopts the method for TRF to detect the concentration of Galectin-3, is to have considered many-sided advantage.
At present, also do not have report to be specifically designed to the time resolution immunofluorescence method that detects Galectin-3 in the world, also do not have the reagent corresponding box to provide, therefore, the development and application of this kit is a blank of having filled up this respect.
List of references:
1. Jian-Hua?Chen,?Run-Zhou?Ni☆,?Ming-Bing?Xiao,?et?al. ?Comparative?proteomic?analysis?of?differentially?expressed?proteins?in?human?pancreatic?cancer?tiss
2. Ge Fei, Ni Run continent ☆, Xiao Mingbing, Jiang Feng, Lu Cuihua, Li Liren, Zhu Jing, Li Xiaoyan, Liu Zhaoxiu.The pre-test that serum Galectin-3 is worth diagnosis of pancreatic cancer." Nantong University's journal (medicine) ", 2010,30(1): 32-34
Summary of the invention
The purpose of this invention is to provide kit of Galectin-3 protein concentration in a kind of sensitivity, the special detection sample and preparation method thereof.
The objective of the invention is to realize by the following method: in ELISA Plate, wrap quilt with coated antibody (monoclonal mouse anti human Galectin-3 antibody), the sealing back adds testing sample and hatches, wash plate, add to detect antibody (how anti-biotinylated goat-anti people Galectin-3 is) and hatch, add Eu after washing plate once more
3+The affine mycin of the chain of mark is hatched, and washes plate then and adds fluorescence enhancement solution, and vibrating excited with the 337nm wavelength light after 5 minutes, and 200 microseconds of delaying time detect the emitting fluorescence value at 615nm wavelength place; By the concentration and the corresponding fluorescent value drawing standard curve of standard items, the Galectin-3 concentration in the sample to be checked just can obtain by its fluorescent value and corresponding typical curve.
The present invention detects the time resolution immunofluorescence kit of Galectin-3, and it comprises following composition:
(1) to be cushioned liquid be phosphate buffer (PBS) to bag, and its composition is 140mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 1.8mM KH
2PO
4, the pH value is 7.4;
(2) confining liquid is the PBS that contains 1%BSA, and its composition is PBS, 1%BSA;
(3) cleansing solution is the Tris-HCl damping fluid (TBS) that contains 0.05%Tween-20, and its composition is the Tris-HCl damping fluid (pH 7.8) of 50mM, 0.05%Tween-20;
(4) composition of sample loading buffer is the PBS that contains 1%BSA, and its composition is PBS, 1%BSA;
(5) fluorescence enhancement solution (Enhancement Solution);
(6) detecting damping fluid (Assay Buffer) is the Tris-HCl damping fluid (pH 7.8) of 50mM, wherein contains 1%BSA, 1%bovine globulin, and 0.05%Tween 40, DTPA;
(7) coated antibody is monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody), and storing concentration is 1 μ g/ μ l;
(8) detecting antibody is that biotinylated goat-anti people Galectin-3 resists (Biotinylated Anti-Human Galectin-3 Antibody) more, and storing concentration is 50ng/ μ l;
(9) Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), storing concentration is 0.1 μ g/ μ l;
(10) standard items, recombined human Galectin-3 albumen (Recombinant Human Galectin-3), concentration is 100 μ g/ μ l.
The use step of time resolution immunofluorescence kit that the present invention detects Galectin-3 is as follows:
(1) diluting coated antibody-monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody) to concentration with PBS is 4 μ g/ml, adds 96 hole ELISA Plate with 100 μ l/ holes, and 4 ℃ are spent the night;
(2) discard coated antibody, 300 μ l/ hole cleansing solution washings three times add confining liquid 200 μ l/ holes, hatch 1h for 37 ℃;
(3) discard confining liquid, 300 μ l/ hole cleansing solutions wash three times, add the standard items 100 μ l/ holes of culture supernatant sample to be measured and doubling dilution, hatch 1h for 37 ℃;
(4) discard sample and standard items, 300 μ l/ hole cleansing solution washings three times;
(5) with sample loading buffer dilution detect antibody-biotinylated goat-anti people Galectin-3 how anti-(Biotinylated Anti-Human Galectin-3 Antibody) to concentration be 75ng/ml, add the good detection antibody of dilution with 100 μ l/ holes, hatch 1h for 37 ℃;
(6) discard detection antibody, 300 μ l/ hole cleansing solution washings three times;
(7) with detecting damping fluid (Assay Buffer) dilution Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), final concentration is 500ng/ml, adds ELISA Plate with 100 μ l/ holes, and 37 ℃ of lucifuges are hatched 1h;
(8) discard Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), 300 μ l/ hole cleansing solution washings are four times
(9) add fluorescence enhancement solution (Enhancement Solution) 200 μ l/ holes, the slow level of room temperature lucifuge is shaken 5min;
(10) read plate: set excitation wavelength (Excitation wavelength) and be 337nm, wavelength of transmitted light (Emission wavelength) is 615nm, and 200 microseconds of delaying time are measured the fluorescent value at 615nm place;
(11) utilize the statistics mapping software according to the value drawing standard curve of measuring;
(12) fluorescent value by typical curve and unknown concentration sample calculates the concentration of the Galectin-3 in the testing sample.
This kit employing time resolution immunofluorescence method comes the Galectin-3 protein concentration in the test sample, wherein used double antibody sandwich method, utilize coated antibody-monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody) to catch the Galectin-3 albumen of trace in the sample, how anti-(Biotinylated Anti-Human Galectin-3 Antibody) comes and captive Galectin-3 protein combination with biotinylated goat-anti people Galectin-3 then, because biotin can combine by high-affinity with the affine mycin of chain, we use Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin) come to close with the many resistive connections of biotinylated goat-anti people Galectin-3, form sandwich sample composite structure, the Galectin-3 in the sample is just by Eu
3+On the mark and be fixed in the ELISA Plate, use the excitation of 337nm wavelength at last, (Emission wavelength) is the 615nm place at wavelength of transmitted light, measures fluorescent value; Calculate the concentration of Galectin-3 then according to typical curve.Using the time resolution immunofluorescence technique, to come highly sensitive, the high Galectin-3 concentration that detects specifically in the peripheral blood be one of this kit striking features and innovation.
The present invention also has good stable, and the running time is short, and cost is low.
Description of drawings
Fig. 1 is for detecting the examination criteria curve of Galectin-3 protein concentration.
Embodiment
Some terms and abbreviation among the present invention: TRF: time resolution immunofluorescence; BSA: bovine serum albumin(BSA); Tween-20: polysorbas20; Tween-40: polysorbate40; PBS: phosphate buffer; The TBS:Tris-HCl damping fluid; Bovine globulin: ox globulin; DTPA: diethylene triamine pentacetic acid (DTPA).
Agent prescription:
1. damping fluid:
Bag is cushioned liquid: be phosphate buffer (PBS) that its composition is 140mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 1.8mM KH
2PO
4, the pH value is 7.4;
Cleansing solution: for containing the Tris-HCl damping fluid (TBS) of 0.05%Tween-20, its composition is the Tris-HCl damping fluid (pH 7.8) of 50mM, 0.05%Tween-20;
Confining liquid: for containing the PBS of 1%BSA, its composition is PBS, 1%BSA;
Sample loading buffer: for containing the PBS of 1%BSA, its composition is PBS, 1%BSA;
Detect damping fluid: the Tris-HCl damping fluid (pH 7.8) for 50mM, wherein contain 1%BSA, 1%bovine globulin, 0.05%Tween 40, DTPA;
2. standard items albumen:
Commercial recombined human Galectin-3 albumen (R﹠amp; D company product)
4. ELISA Plate:
Nunc eight hole enzyme mark bars
Operate time resolution immunofluorescence method of the present invention comes the Galectin-3 protein concentration in the test sample, and the use step of its kit is as follows:
(1) diluting coated antibody-monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody) to concentration with PBS is 4 μ g/ml, adds 96 hole ELISA Plate with 100 μ l/ holes, and 4 ℃ are spent the night;
(2) discard coated antibody, 300 μ l/ hole cleansing solution washings three times add confining liquid 200 μ l/ holes, hatch 1h for 37 ℃;
(3) discard confining liquid, 300 μ l/ hole cleansing solutions wash three times, add the standard items 100 μ l/ holes of culture supernatant sample to be measured and doubling dilution, hatch 1h for 37 ℃;
(4) discard sample and standard items, 300 μ l/ hole cleansing solution washings three times;
(5) with sample loading buffer dilution detect antibody-biotinylated goat-anti people Galectin-3 how anti-(Biotinylated Anti-Human Galectin-3 Antibody) to concentration be 75ng/ml, add the good detection antibody of dilution with 100 μ l/ holes, hatch 1h for 37 ℃;
(6) discard detection antibody, 300 μ l/ hole cleansing solution washings three times;
(7) with detecting damping fluid (Assay Buffer) dilution Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), final concentration is 500ng/ml, adds ELISA Plate with 100 μ l/ holes, and 37 ℃ of lucifuges are hatched 1h;
(8) discard Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), 300 μ l/ hole cleansing solution washings are four times
(9) add fluorescence enhancement solution (Enhancement Solution) 200 μ l/ holes, the slow level of room temperature lucifuge is shaken 5min;
(10) read plate: set excitation wavelength (Excitation wavelength) and be 337nm, wavelength of transmitted light (Emission wavelength) is 615nm, and 200 microseconds of delaying time are measured the fluorescent value at 615nm place;
(11) utilize the statistics mapping software according to the value drawing standard curve of measuring;
(12) fluorescent value by typical curve and unknown concentration sample calculates the concentration of the Galectin-3 in the testing sample.
Claims (2)
1. detect the time resolution immunofluorescence kit of Galectin-3, it is characterized in that comprising following composition
(1) to be cushioned liquid be phosphate buffer (PBS) to bag, and its composition is 140mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 1.8mM KH
2PO
4, the pH value is 7.4;
(2) confining liquid is the PBS that contains 1%BSA, and its composition is PBS, 1%BSA;
(3) cleansing solution is the Tris-HCl damping fluid (TBS) that contains 0.05%Tween-20, and its composition is the Tris-HCl damping fluid (pH 7.8) of 50mM, 0.05%Tween-20;
(4) composition of sample loading buffer is the PBS that contains 1%BSA, and its composition is PBS, 1%BSA;
(5) fluorescence enhancement solution (Enhancement Solution);
(6) detecting damping fluid (Assay Buffer) is the Tris-HCl damping fluid (pH 7.8) of 50mM, wherein contains 1%BSA, 1%bovine globulin, and 0.05%Tween 40, DTPA;
(7) coated antibody is monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody), and storing concentration is 1 μ g/ μ l;
(8) detecting antibody is that biotinylated goat-anti people Galectin-3 resists (Biotinylated Anti-Human Galectin-3 Antibody) more, and storing concentration is 50ng/ μ l;
(9) Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), storing concentration is 0.1 μ g/ μ l;
(10) standard items, recombined human Galectin-3 albumen (Recombinant Human Galectin-3), concentration is 100 μ g/ μ l.
2. detect the using method of the time resolution immunofluorescence kit of Galectin-3 according to claim 1, it is characterized in that following steps
(1) diluting coated antibody-monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody) to concentration with PBS is 4 μ g/ml, adds 96 hole ELISA Plate with 100 μ l/ holes, and 4 ℃ are spent the night;
(2) discard coated antibody, 300 μ l/ hole cleansing solution washings three times add confining liquid 200 μ l/ holes, hatch 1h for 37 ℃;
(3) discard confining liquid, 300 μ l/ hole cleansing solutions wash three times, add the standard items 100 μ l/ holes of culture supernatant sample to be measured and doubling dilution, hatch 1h for 37 ℃;
(4) discard sample and standard items, 300 μ l/ hole cleansing solution washings three times;
(5) with sample loading buffer dilution detect antibody-biotinylated goat-anti people Galectin-3 how anti-(Biotinylated Anti-Human Galectin-3 Antibody) to concentration be 75ng/ml, add the good detection antibody of dilution with 100 μ l/ holes, hatch 1h for 37 ℃;
(6) discard detection antibody, 300 μ l/ hole cleansing solution washings three times;
(7) with detecting damping fluid (Assay Buffer) dilution Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), final concentration is 500ng/ml, adds ELISA Plate with 100 μ l/ holes, and 37 ℃ of lucifuges are hatched 1h;
(8) discard Eu
3+The chain of mark is affine mycin (Eu
3+-labeled streptavidin), 300 μ l/ hole cleansing solution washings are four times
(9) add fluorescence enhancement solution (Enhancement Solution) 200 μ l/ holes, the slow level of room temperature lucifuge is shaken 5min;
(10) read plate: set excitation wavelength (Excitation wavelength) and be 337nm, wavelength of transmitted light (Emission wavelength) is 615nm, and 200 microseconds of delaying time are measured the fluorescent value at 615nm place;
(11) utilize the statistics mapping software according to the value drawing standard curve of measuring;
(12) fluorescent value by typical curve and unknown concentration sample calculates the concentration of the Galectin-3 in the testing sample.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643909A (en) * | 2012-04-05 | 2012-08-22 | 天津和泽干细胞科技有限公司 | Detection method for biological efficacy of umbilical cord mesenchymal stem cell |
| CN103293313A (en) * | 2012-02-24 | 2013-09-11 | 上海新波生物技术有限公司 | Carbohydrate antigen 15-3 time-resolved immunofluorescence assay and kit |
| CN104515855A (en) * | 2013-10-05 | 2015-04-15 | 倪润洲 | Galectin-3 detection nanometer magnetic bead sorting-time resolved immunofluorescence kit |
| CN110068685A (en) * | 2019-05-05 | 2019-07-30 | 南通大学附属医院 | The detection device and detection method of a kind of immune-blotting method |
| CN111060689A (en) * | 2019-11-01 | 2020-04-24 | 广州博康生物技术有限公司 | Endometrial cancer detection kit |
| CN112710596A (en) * | 2020-11-30 | 2021-04-27 | 浙江正熙生物医药有限公司 | Method for qualitative/quantitative detection of target antibody concentration using flow cytometer |
| CN115746134A (en) * | 2021-10-15 | 2023-03-07 | 深圳市睿盟创新生物科技有限公司 | Galectin-3 immunoassay |
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2011
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| CN101363051A (en) * | 2008-08-29 | 2009-02-11 | 芮屈生物技术（上海）有限公司 | Kit for GALECTIN-3 gene hybridization in situ, detection method and application thereof |
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| CN103293313A (en) * | 2012-02-24 | 2013-09-11 | 上海新波生物技术有限公司 | Carbohydrate antigen 15-3 time-resolved immunofluorescence assay and kit |
| CN102643909A (en) * | 2012-04-05 | 2012-08-22 | 天津和泽干细胞科技有限公司 | Detection method for biological efficacy of umbilical cord mesenchymal stem cell |
| CN102643909B (en) * | 2012-04-05 | 2013-03-20 | 天津和泽干细胞科技有限公司 | Detection method for biological efficacy of umbilical cord mesenchymal stem cell |
| CN104515855A (en) * | 2013-10-05 | 2015-04-15 | 倪润洲 | Galectin-3 detection nanometer magnetic bead sorting-time resolved immunofluorescence kit |
| CN110068685A (en) * | 2019-05-05 | 2019-07-30 | 南通大学附属医院 | The detection device and detection method of a kind of immune-blotting method |
| CN110068685B (en) * | 2019-05-05 | 2023-12-05 | 南通大学附属医院 | Detection device and detection method for dot immunoblotting detection |
| CN111060689A (en) * | 2019-11-01 | 2020-04-24 | 广州博康生物技术有限公司 | Endometrial cancer detection kit |
| CN112710596A (en) * | 2020-11-30 | 2021-04-27 | 浙江正熙生物医药有限公司 | Method for qualitative/quantitative detection of target antibody concentration using flow cytometer |
| CN115746134A (en) * | 2021-10-15 | 2023-03-07 | 深圳市睿盟创新生物科技有限公司 | Galectin-3 immunoassay |
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