CN102226809B - Time resloved fluoroimmunoassay kit for detecting Galectin-3 - Google Patents

Time resloved fluoroimmunoassay kit for detecting Galectin-3 Download PDF

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CN102226809B
CN102226809B CN201110036899.9A CN201110036899A CN102226809B CN 102226809 B CN102226809 B CN 102226809B CN 201110036899 A CN201110036899 A CN 201110036899A CN 102226809 B CN102226809 B CN 102226809B
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galectin
antibody
concentration
sample
damping fluid
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CN102226809A (en
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倪润洲
倪温慨
肖明兵
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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

Detect the time resolution immunofluorescent reagent box of Galectin-3
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 Applicative time and differentiates the concentration that immunofluorescence method detects the Galectin-3 albumen in sample.In human peripheral there is close association in the increase of Galectin-3 protein concentration and the generation of cancer of pancreas development, uses this kit to play diagnostic effect to the generation development of cancer of pancreas.
Background technology
Galectin-3 is beta-galactoside binding protein, belong to lectin family member, it has CRD domain, there is very high affinity with glycoprotein and glycolipid containing beta galactose glycosides, between cell and cell, cell and matrix, play specific adhesion effect, closely related with transfer, infiltration, growth and the adhesion of tumour.We use MALDI-TOF-MS mass-spectrometric technique to find that Galectin-3 presents high expressed in Tissues of Human Adenocarcinoma of Pancreas [1], and its expression and cancer of pancreas by stages and prognosis relevant.Our research is found, in normal person, cancer of pancreas and other digestive system tumor Peripheral Blood, does not all detect Galectin-3 albumen, and Galectin-3 positive rate in Pancreas cancer patients Peripheral Blood is up to being 30% [2].Therefore, Galectin-3 has clear and definite diagnostic value to cancer of pancreas.
Time resolution immunofluorescence (Time Resolved Fluoroimmunoassay, TRF) detection method is a kind of immune analysis method of maturation, there is high special, high-sensitive advantage, being to obtain applying very widely in all many-sides such as clinical medicine and laboratory medicines, is a kind of quantitative detecting method that replaces at present radioimmunoassay.Its ultimate principle is to utilize trivalent rare earth ions (as Eu 3+) as tracer, postpone to detect longer specificity fluorescence excitation of life-span to get rid of the interference of non-specific and background fluorescence, measure the delayed fluorescence intensity that the rare earth ion in compound is excited, thereby determine the concentration of testing protein.Use the TRF of fluorescence enhancement solution pik (pg, 10 can be detected -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 in the world report to be specifically designed to the time resolution immunofluorescence method that detects Galectin-3, also do not have corresponding kit to provide, therefore, the development and application of this kit, is the 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 tissue. Hepatobiliary Pancreat Dis Int. 2009 Apr;8(2):193-200.(☆:Corresponding author）
2. Ge Fei, Ni Run continent ☆, Xiao Mingbing, Jiang Feng, Lu Cuihua, Li Liren, Zhu Jing, Li little Yan, Liu Zhaoxiu.The pre-test that serum Galectin-3 is worth diagnosis of pancreatic cancer.< < Nantong University journal (medicine) > >, 2010,30(1): 32-34
Summary of the invention
The object of this invention is to provide kit of Galectin-3 protein concentration in a kind of sensitive, special detection sample and preparation method thereof.
The object of the invention is to realize by the following method: coated with coated antibody (monoclonal mouse anti human Galectin-3 antibody) in ELISA Plate, after sealing, add testing sample to hatch, wash plate, add and detect antibody (how anti-biotinylated goat-anti people Galectin-3 is) and hatch, add Eu after again washing plate 3+the affine mycin of chain of mark is hatched, and then washes plate and adds fluorescence enhancement solution, vibrates and by 337nm wavelength light, excites after 5 minutes, and time delay 200 microseconds detect emitting fluorescence value at 615nm wavelength place; By concentration and the corresponding fluorescent value drawing standard curve of standard items, the Galectin-3 concentration in sample to be checked just can obtain by its fluorescent value corresponding typical curve.
The present invention detects the time resolution immunofluorescent reagent box of Galectin-3, and it comprises following composition:
(1) coated damping fluid is phosphate buffer (PBS), and its composition is 140mM NaCl, 2.7mM KCl, 10mM Na 2hPO 4, 1.8mM KH 2pO 4, pH value is 7.4;
(2) confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(3) cleansing solution is the Tris-HCl damping fluid (TBS) containing 0.05%Tween-20, the Tris-HCl damping fluid that its composition is 50mM (pH 7.8), 0.05%Tween-20;
(4) composition of sample loading buffer is the PBS containing 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+affine mycin (the Eu of chain of mark 3+-labeled streptavidin), storing concentration is 0.1 μ g/ μ l;
(10) standard items, recombinant human Galectin-3 albumen (Recombinant Human Galectin-3), concentration is 100 μ g/ μ l.
The use step of time resolution immunofluorescent reagent box that the present invention detects Galectin-3 is as follows:
(1) with PBS, diluting coated antibody-monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody) to concentration is 4 μ g/ml, with 100 μ l/ holes, adds 96 hole ELISA Plate, 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, with 100 μ l/ holes, add the detection antibody having diluted, 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+affine mycin (the Eu of chain of mark 3+-labeled streptavidin), final concentration is 500ng/ml, with 100 μ l/ holes, adds ELISA Plate, and 37 ℃ of lucifuges are hatched 1h;
(8) discard Eu 3+affine mycin (the Eu of chain of mark 3+-labeled streptavidin), 300 μ l/ hole cleansing solution washing is 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) for 337nm, wavelength of transmitted light (Emission wavelength) is 615nm, and time delay 200 microseconds are measured the fluorescent value at 615nm place;
(11) utilize statistics mapping software according to the value drawing standard curve of measuring;
(12) by the fluorescent value of typical curve and unknown concentration sample, calculate the concentration of the Galectin-3 in testing sample.
This kit adopts time resolution immunofluorescence method to detect the Galectin-3 protein concentration in sample, wherein applied 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 sample, then with biotinylated goat-anti people Galectin-3, how anti-(Biotinylated Anti-Human Galectin-3 Antibody) comes and captive Galectin-3 protein combination, because the affine mycin of biotin and chain can be combined by high-affinity, we use Eu 3+affine mycin (the Eu of chain of mark 3+-labeled streptavidin) come and the many anti-bindings of biotinylated goat-anti people Galectin-3, form sandwich sample composite structure, the Galectin-3 in sample is just by Eu 3+on mark and be fixed in ELISA Plate, finally applying the excitation of 337nm wavelength, is 615nm place at wavelength of transmitted light (Emission wavelength), measures fluorescent value, then according to typical curve, calculate the concentration of Galectin-3.Applicative time is differentiated immunofluorescence technique, and to carry out highly sensitive, the high Galectin-3 concentration detecting specifically in peripheral blood be one of this kit striking features and innovation.
The present invention also has good stability, and the running time is short, and cost is low.
Accompanying drawing explanation
Fig. 1 is for detecting the examination criteria curve of Galectin-3 protein concentration.
Embodiment
Some terms and abbreviation in the present invention: TRF: time resolution immunofluorescence; BSA: bovine serum albumin(BSA); Tween-20: polysorbas20; Tween-40: polysorbate40; PBS: phosphate buffer; TBS:Tris-HCl damping fluid; Bovine globulin: ox globulin; DTPA: diethylene triamine pentacetic acid (DTPA).
Agent prescription:
1. damping fluid:
Coated damping fluid: be phosphate buffer (PBS), its composition is 140mM NaCl, 2.7mM KCl, 10mM Na 2hPO 4, 1.8mM KH 2pO 4, pH value is 7.4;
Cleansing solution: be the Tris-HCl damping fluid (TBS) containing 0.05%Tween-20, the Tris-HCl damping fluid that its composition is 50mM (pH 7.8), 0.05%Tween-20;
Confining liquid: be the PBS containing 1%BSA, its composition is PBS, 1%BSA;
Sample loading buffer: be the PBS containing 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 recombinant human Galectin-3 albumen (R & D company product)
4. ELISA Plate:
Nunc eight hole enzyme mark bars
Operate time of the present invention is differentiated immunofluorescence method and is detected the Galectin-3 protein concentration in sample, and the use step of its kit is as follows:
(1) with PBS, diluting coated antibody-monoclonal mouse anti human Galectin-3 antibody (Monoclonal Anti-Human Galectin-3 Antibody) to concentration is 4 μ g/ml, with 100 μ l/ holes, adds 96 hole ELISA Plate, 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, with 100 μ l/ holes, add the detection antibody having diluted, 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+affine mycin (the Eu of chain of mark 3+-labeled streptavidin), final concentration is 500ng/ml, with 100 μ l/ holes, adds ELISA Plate, and 37 ℃ of lucifuges are hatched 1h;
(8) discard Eu 3+affine mycin (the Eu of chain of mark 3+-labeled streptavidin), 300 μ l/ hole cleansing solution washing is 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) for 337nm, wavelength of transmitted light (Emission wavelength) is 615nm, and time delay 200 microseconds are measured the fluorescent value at 615nm place;
(11) utilize statistics mapping software according to the value drawing standard curve of measuring;
(12) by the fluorescent value of typical curve and unknown concentration sample, calculate the concentration of the Galectin-3 in testing sample.

Claims (1)

1. detect the using method of the time resolution immunofluorescent reagent box of Galectin-3, it is characterized in that: the time resolution immunofluorescent reagent box of described detection Galectin-3 comprises following composition
(1) coated damping fluid is phosphate buffer, and its composition is 140mM NaCl, 2.7mM KCl, 10mM Na 2hPO 4, 1.8mM KH 2pO 4, pH value is 7.4;
(2) confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(3) cleansing solution is the Tris-HCl damping fluid containing 0.05%Tween-20, the Tris-HCl damping fluid that its composition is 50mM, and 0.05%Tween-20, pH 7.8;
(4) composition of sample loading buffer is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(5) fluorescence enhancement solution;
(6) detect the Tris-HCl damping fluid that damping fluid is 50mM, pH 7.8, wherein contain 1%BSA, 1%bovine globulin, and 0.05%Tween 40, diethylene triamine pentacetic acid (DTPA);
(7) coated antibody is monoclonal mouse anti human Galectin-3 antibody, and storing concentration is 1 μ g/ μ l;
(8) detecting antibody is that how anti-biotinylated goat-anti people Galectin-3 is, and storing concentration is 50ng/ μ l;
(9) Eu 3+the affine mycin of chain of mark, storing concentration is 0.1 μ g/ μ l;
(10) standard items, recombinant human Galectin-3 albumen, concentration is 100 μ g/ μ l;
Described using method is specially following steps
(1) with PBS, diluting coated antibody-monoclonal mouse anti human Galectin-3 antibody to concentration is 4 μ g/ml, with 100 μ l/ holes, adds 96 hole ELISA Plate, 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 resist more to concentration be 75ng/ml, with 100 μ l/ holes, add the detection antibody having diluted, hatch 1h for 37 ℃;
(6) discard detection antibody, 300 μ l/ hole cleansing solution washings three times;
(7) with detecting damping fluid dilution Eu 3+the affine mycin of chain of mark, final concentration is 500ng/ml, with 100 μ l/ holes, adds ELISA Plate, 37 ℃ of lucifuges are hatched 1h;
(8) discard Eu 3+the affine mycin of chain of mark, 300 μ l/ hole cleansing solution washings four times
(9) add fluorescence enhancement solution 200 μ l/ holes, the slow level of room temperature lucifuge is shaken 5min;
(10) read plate: setting excitation wavelength is 337nm, wavelength of transmitted light is 615nm, and time delay 200 microseconds are measured the fluorescent value at 615nm place;
(11) utilize statistics mapping software according to the value drawing standard curve of measuring;
(12) by the fluorescent value of typical curve and unknown concentration sample, calculate the concentration of the Galectin-3 in testing sample.
CN201110036899.9A 2011-02-13 2011-02-13 Time resloved fluoroimmunoassay kit for detecting Galectin-3 Expired - Fee Related CN102226809B (en)

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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
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
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
CN111060689B (en) * 2019-11-01 2020-12-25 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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