CN104515855A - Galectin-3 detection nanometer magnetic bead sorting-time resolved immunofluorescence kit - Google Patents
Galectin-3 detection nanometer magnetic bead sorting-time resolved immunofluorescence kit Download PDFInfo
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Abstract
The invention relates to a Galectin-3 detection nanometer magnetic bead sorting-time resolved immunofluorescence kit, which comprises superparamagnetic composite micro-particles with a particle size of 30 nm, a coupling liquid, a blocking solution, a washing liquid, a loading buffer, a fluorescence enhancement solution, a detection buffer, a capture antibody, a detection antibody, Eu<3+> labeled streptavidin and a standard substance. The use of the kit comprises: adopting nanometer magnetic bead sorting and time resolved immunofluorescence adopting a double antibody sandwich method as the base to detect the concentration of Galectin-3 protein in a sample, adopting nanometer magnetic beads coupled with the Galectin-3 capture antibody to enrich the Galectin-3 in the sample, adding the detection antibody, incubating, washing, adding the Eu<3+> labeled streptavidin, incubating, washing again, adding the fluorescence enhancement solution, vibrating for 5 min, adopting light with a wavelength of 337 nm to excite, delaying for 200 microseconds, detecting the emitted fluorescence value at a wavelength of 615 nm, and obtaining the concentration of the Galectin-3 in the sample to be detected through the fluorescence value and the corresponding standard curve, wherein the sample to be detected can further be used for detecting the next target protein.
Description
Technical field
The invention belongs to medical biotechnology field, relate to a kind of Series detectors method of nanometer magnetic bead sorting and time resolution immunofluorescence, be specially and use nanometer magnetic bead and Galectin-3 antibody coupling to carry out Galectin-3 albumen in specificity Acquisition Detection sample, then Applicative time resolved immuno fluorometric method detects the concentration of caught Galectin-3 albumen further.In human peripheral there is close association in the increase of Galectin-3 protein concentration and the generation development of cancer of pancreas, use this kit can play diagnostic effect to the generation development of cancer of pancreas, realize reusing serum specimen, and the detection sensitivity to serum Galectin-3 can be improved.
Background technology
Galectin-3 is a kind of galactose agglutinin, is expressed on tenuigenin, cell membrane and mitochondria, and can be secreted into extracellular matrix.It has CRD domain, can identify specificity glycoprotein and glycolipid, participates in multiple physiology and pathologic process, comprises Growth of Cells and the formation of apoptosis, cell adhesion and new vessels.We use MALDI-TOF-MS mass-spectrometric technique to find Galectin-3 presents high expressed in Tissues of Human Adenocarcinoma of Pancreas, and its expression and cancer of pancreas by stages and prognosis relevant.Our research finds, does not all detect Galectin-3 albumen in normal person, cancer of pancreas and other digestive system tumor Peripheral Blood, and Galectin-3 positive rate in Pancreas cancer patients Peripheral Blood is up to being 30%.Therefore, Galectin-3 has clear and definite diagnostic value to cancer of pancreas.
Time resolution immunofluorescent detection method is a kind of immune analysis method of maturation, there is high special, high-sensitive advantage, being obtain applying very widely in all many-sides such as clinical medicine and laboratory medicine, is a kind of quantitative detecting method replacing radioimmunoassay at present.Its ultimate principle utilizes trivalent rare earth ions (as Eu
3+) as tracer, postpone to detect life-span longer specificity fluorescence excitation to get rid of the interference of non-specific and background fluorescence, namely measure the delayed fluorescence intensity that the rare earth ion in compound is excited, thus determine the concentration of testing protein.
But time resolution immunofluorescence needs to consume the serum of some, and the not reproducible use of this serum, the serum that therefore patient provides only can detect limited index, and this is also to the foundation of clinical serum sample storehouse with use formation one to limit greatly.The serum how making patient provide can detect different marks unlimitedly, and this is one, and to clinical and scientific research, all tool is significant studies a question.Therefore, we have developed nanometer magnetic bead sorting-time resolution immunofluorescence Tandem analysis method, first the nanometer magnetic bead by being coated with Galectin-3 carrys out (as 500 HL serum) Galectin-3 albumen in adsorption and enrichment serum, then by the Galectin-3 protein delivery collected to (as 5 microlitres) in the detection liquid of a micro volume, make sample once concentration 100 times, thus detection sensitivity is improve 2 orders of magnitude.Further, for same detection sample, successively can carry out enrichment isolation with the nanometer magnetic bead of different antibodies mark, thus reach the object of high flux detection.
Nanometer magnetic bead sorting and time resolution Immunofluorescence test are together in series by the present invention, tentatively achieve and object is reused to patients serum, and improve highly sensitive, provide good platform for the detection of clinical tumor markers and TRFIA high flux detect.
At present, also do not have relevant report in the world, not more being specifically designed to the nanometer magnetic bead sorting-time resolution immunofluorescent reagent box detecting Galectin-3 provides, and therefore, the development and application of this kit is the blank having filled up this respect.
Summary of the invention
The object of this invention is to provide and a kind ofly can make that detected sample can reuse and sensitive special detection kit, determine Galectin-3 protein concentration in sample with this.
The object of the invention is to realize by the following method: with the super-paramagnetic composite particle of Galectin-3 antibody coupling 30nm particle diameter, detect in samples at 500 microlitres and catch enrichment destination protein, add detection antibody incubation after washing, add Eu subsequently
3+the affine mycin of chain of mark is hatched, and again discharge this magnetic bead after washing in check-out console, and add fluorescence enhancement solution, vibrate and excite by 337nm wavelength light after 5 minutes, time delay 200 microsecond detects 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 measuring samples just can by its fluorescent value and corresponding typical curve obtain.
The present invention detects the nanometer magnetic bead sorting-time resolution immunofluorescent reagent box of Galectin-3, and it comprises following composition:
(1) super-paramagnetic composite particle of 30nm particle diameter;
(2) coupling buffer;
(3) 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;
(4) confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(5) cleansing solution is the Tris-HCl damping fluid (TBS) containing 0.05%Tween-20, and its composition is the Tris-HCl damping fluid (pH 7.8) of 50mM, 0.05%Tween-20;
(6) composition of sample loading buffer is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(7) fluorescence enhancement solution (Enhancement Solution);
(8) the Tris-HCl damping fluid (pH 7.8) that damping fluid (Assay Buffer) is 50mM is detected, wherein containing 1%BSA, 1%bovine globulin, 0.05%Tween 40, DTPA;
(9) 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;
(10) detecting antibody is biotinylated goat-anti people Galectin-3 many anti-(Biotinylated Anti-Human Galectin-3 Antibody), and storing concentration is 50ng/ μ l;
(11) Eu
3+affine mycin (the Eu of chain of mark
3+-labeled streptavidin), storing concentration is 0.1 μ g/ μ l;
(12) standard items, recombined human Galectin-3 albumen (Recombinant Human Galectin-3), concentration is 100 μ g/ μ l.
The use step that the present invention detects the time resolution immunofluorescent reagent box of Galectin-3 is as follows:
(1) dissolve 500 μ g monoclonal mouse anti human Galectin-3 antibody with 1ml coupling liquid, add the super-paramagnetic composite particle of 30nm particle diameter, jog mixes, and is placed in constant-temperature table, 37 DEG C, 180 rpm oscillating reactions 20min.React complete, magnetic resolution 2 min;
(2) in above-mentioned centrifuge tube, add 1 ml cleansing solution, jog mixes, and magnetic resolution, repeats this cleaning operation 1 time;
(3) in centrifuge tube, add 3 ml confining liquids, be placed in constant-temperature table, 37 DEG C, 180 rpm oscillating reactions 2 h.Magnetic resolution, abandons supernatant.
(4) in centrifuge tube, add 1 ml cleansing solution, jog mixes, and magnetic resolution, abandons supernatant.Repeat this cleaning operation 3 times;
(5) in the standard items of 500 μ l culture supernatant sample to be measured and doubling dilution, add the antibody 2 μ l of nanometer magnetic bead coupling, mix, be placed in 37 DEG C of shaking table 180rpm, 1h;
(6) above-mentioned 1.5ml Ep pipe is placed on magnetic separator, magnetic resolution 2min, then 500ul serum is all taken out rear frozen;
(7) above-mentioned 1.5ml Ep pipe is placed on magnetic separator, washs three times with 500 μ l PBS-0.05%Tween20;
(8) the detection antibody Biotinylated Anti-Human Galectin-3 Antibody(Detect Antibody diluted is added) 100 μ l/ manage, mix, and are placed in 37 DEG C of shaking table 180rpm, 1h;
(9) be again placed on magnetic separator by above-mentioned 1.5ml Ep pipe, magnetic resolution 2min, then discards detection antibody, washs three times with 500 μ l Wash Buffer;
(10) add the Eu-labeled streptavidin 100 μ l/ hole with Assay Buffer preparation, mix, be placed in 37 DEG C of shaking table 180rpm, lucifuge, 1h;
(11) be again placed on magnetic separator by above-mentioned 1.5ml Ep pipe, magnetic resolution 2min, then discards Eu-labeled streptavidin, and 500 μ l/ hole Wash Buffer wash four times;
(12) add Enhancement Solution 200 μ l/ hole, after mixing, be added in 96 hole check-out consoles by 200 μ l liquid, room temperature lucifuge hatches 45min and Slowly Shaking 5min;
(13) read plate: setting excitation wavelength (Excitation wavelength) is 337nm, and wavelength of transmitted light (Emission wavelength) is 615nm, time delay 200 microsecond measures the fluorescent value at 615nm place;
(14) utilize statistics mapping software according to the value drawing standard curve measured;
(15) concentration of the Galectin-3 in testing sample is calculated by the fluorescent value of typical curve and unknown concentration sample.
This kit adopts nanometer magnetic bead sorting-time resolution immunofluorescence series process, and use the super-paramagnetic composite particle of 30nm particle diameter, its core is magnetic nanoparticle, and core surface is the shell of gold.By magnetic separator, nanometer gold magnetic particle can be separated from suspending medium.Utilize the character of its surface gold, do not need covalent coupling reagent, can a step by Galectin-3 antibody coupling nanometer gold magnetic particle surface, coupling has the nanometer gold magnetic particle of Galectin-3 antibody can isolate Galectin-3 from serum, by Galectin-3 standard protein serial dilution, linear within the scope of 0.78-100ng/ml, serum after separation can add the second antibody-coupled magnetic beads further, carry out the detection (Fig. 1) of next mark, realize the recycling to same serum specimen, this is a most striking features and the innovation of this kit.
In the present invention, have employed the nanometer magnetic bead sorting technology of antibody labeling, it can go out destination protein by (as 500 microlitres) enrichment isolation from the larger sample of volume, and be again discharged into (as 5 microlitres) in the detection liquid of micro volume, make sample once concentration 100 times, thus detection sensitivity is improve 2 orders of magnitude.
The present invention also has good stability, and the running time is short, and cost is low.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of nanometer magnetic bead sorting-time resolution immunofluorescence joint detection method.
Fig. 2 is the examination criteria curve detecting Galectin-3 protein concentration.
Embodiment
Some terms in the present invention and abbreviation: 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:
Bag is buffered liquid: be phosphate buffer (PBS) that 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, its composition is the Tris-HCl damping fluid (pH 7.8) of 50mM, 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: be the Tris-HCl damping fluid (pH 7.8) of 50mM, wherein containing 1%BSA, 1%bovine globulin, 0.05%Tween 40, DTPA;
2. standard items albumen:
Commercial recombined human Galectin-3 albumen (R & D Products)
4. ELISA Plate:
Coastar 96 hole circle base plate
Concrete operation step:
(1) dissolve 500 μ g monoclonal mouse anti human Galectin-3 antibody with 1ml coupling liquid, add the super-paramagnetic composite particle of 30nm particle diameter, jog mixes, and is placed in constant-temperature table, 37 DEG C, 180 rpm oscillating reactions 20min.React complete, magnetic resolution 2 min;
(2) in above-mentioned centrifuge tube, add 1 ml cleansing solution, jog mixes, and magnetic resolution, repeats this cleaning operation 1 time;
(3) in centrifuge tube, add 3 ml confining liquids, be placed in constant-temperature table, 37 DEG C, 180 rpm oscillating reactions 2 h.Magnetic resolution, abandons supernatant.
(4) in centrifuge tube, add 1 ml cleansing solution, jog mixes, and magnetic resolution, abandons supernatant.Repeat this cleaning operation 3 times;
(5) in the standard items of 500 μ l culture supernatant sample to be measured and doubling dilution, add the antibody 2 μ l of nanometer magnetic bead coupling, mix, be placed in 37 DEG C of shaking table 180rpm, 1h;
(6) above-mentioned 1.5ml Ep pipe is placed on magnetic separator, magnetic resolution 2min, then 500ul serum is all taken out rear frozen;
(7) above-mentioned 1.5ml Ep pipe is placed on magnetic separator, washs three times with 500 μ l PBS-0.05%Tween20;
(8) the detection antibody Biotinylated Anti-Human Galectin-3 Antibody(Detect Antibody diluted is added) 100 μ l/ manage, mix, and are placed in 37 DEG C of shaking table 180rpm, 1h;
(9) be again placed on magnetic separator by above-mentioned 1.5ml Ep pipe, magnetic resolution 2min, then discards detection antibody, washs three times with 500 μ l Wash Buffer;
(10) add the Eu-labeled streptavidin 100 μ l/ hole with Assay Buffer preparation, mix, be placed in 37 DEG C of shaking table 180rpm, lucifuge, 1h;
(11) be again placed on magnetic separator by above-mentioned 1.5ml Ep pipe, magnetic resolution 2min, then discards Eu-labeled streptavidin, and 500 μ l/ hole Wash Buffer wash four times;
(12) add Enhancement Solution 200 μ l/ hole, after mixing, be added in 96 hole check-out consoles by 200 μ l liquid, room temperature lucifuge hatches 45min and Slowly Shaking 5min;
(13) read plate: setting excitation wavelength (Excitation wavelength) is 337nm, and wavelength of transmitted light (Emission wavelength) is 615nm, time delay 200 microsecond measures the fluorescent value at 615nm place;
(14) utilize statistics mapping software according to the value drawing standard curve measured;
(15) concentration of the Galectin-3 in testing sample is calculated by the fluorescent value of typical curve and unknown concentration sample.
Claims (2)
1. detect the nanometer magnetic bead sorting-time resolution immunofluorescent reagent box of Galectin-3, it is characterized in that comprising following composition:
(1) super-paramagnetic composite particle of 30nm particle diameter;
(2) coupling buffer;
(3) 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;
(4) confining liquid is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(5) cleansing solution is the Tris-HCl damping fluid (TBS) containing 0.05%Tween-20, and its composition is the Tris-HCl damping fluid (pH 7.8) of 50mM, 0.05%Tween-20;
(6) composition of sample loading buffer is the PBS containing 1%BSA, and its composition is PBS, 1%BSA;
(7) fluorescence enhancement solution (Enhancement Solution);
(8) the Tris-HCl damping fluid (pH 7.8) that damping fluid (Assay Buffer) is 50mM is detected, wherein containing 1%BSA, 1%bovine globulin, 0.05%Tween 40, DTPA;
(9) 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;
(10) detecting antibody is biotinylated goat-anti people Galectin-3 many anti-(Biotinylated Anti-Human Galectin-3 Antibody), and storing concentration is 50ng/ μ l;
(11) Eu
3+affine mycin (the Eu of chain of mark
3+-labeled streptavidin), storing concentration is 0.1 μ g/ μ l;
(12) 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 nanometer magnetic bead sorting-time resolution immunofluorescent reagent box of Galectin-3 as claimed in claim 1, it is characterized in that following steps:
(1) dissolve 500 μ g monoclonal mouse anti human Galectin-3 antibody with 1ml coupling liquid, add the super-paramagnetic composite particle of 30nm particle diameter, jog mixes, and is placed in constant-temperature table, 37 DEG C, 180 rpm oscillating reactions 20min.React complete, magnetic resolution 2 min;
(2) in above-mentioned centrifuge tube, add 1 ml cleansing solution, jog mixes, and magnetic resolution, repeats this cleaning operation 1 time;
(3) in centrifuge tube, add 3 ml confining liquids, be placed in constant-temperature table, 37 DEG C, 180 rpm oscillating reactions 2 h.Magnetic resolution, abandons supernatant.
(4) in centrifuge tube, add 1 ml cleansing solution, jog mixes, and magnetic resolution, abandons supernatant.Repeat this cleaning operation 3 times;
(5) in the standard items of 500 μ l culture supernatant sample to be measured and doubling dilution, add the antibody 2 μ l of nanometer magnetic bead coupling, mix, be placed in 37 DEG C of shaking table 180rpm, 1h;
(6) above-mentioned 1.5ml Ep pipe is placed on magnetic separator, magnetic resolution 2min, then 500ul serum is all taken out rear frozen;
(7) above-mentioned 1.5ml Ep pipe is placed on magnetic separator, washs three times with 500 μ l PBS-0.05%Tween20;
(8) the detection antibody Biotinylated Anti-Human Galectin-3 Antibody(Detect Antibody diluted is added) 100 μ l/ manage, mix, and are placed in 37 DEG C of shaking table 180rpm, 1h;
(9) be again placed on magnetic separator by above-mentioned 1.5ml Ep pipe, magnetic resolution 2min, then discards detection antibody, washs three times with 500 μ l Wash Buffer;
(10) add the Eu-labeled streptavidin 100 μ l/ hole with Assay Buffer preparation, mix, be placed in 37 DEG C of shaking table 180rpm, lucifuge, 1h;
(11) be again placed on magnetic separator by above-mentioned 1.5ml Ep pipe, magnetic resolution 2min, then discards Eu-labeled streptavidin, and 500 μ l/ hole Wash Buffer wash four times;
(12) add Enhancement Solution 200 μ l/ hole, after mixing, be added in 96 hole check-out consoles by 200 μ l liquid, room temperature lucifuge hatches 45min and Slowly Shaking 5min;
(13) read plate: setting excitation wavelength (Excitation wavelength) is 337nm, and wavelength of transmitted light (Emission wavelength) is 615nm, time delay 200 microsecond measures the fluorescent value at 615nm place;
(14) utilize statistics mapping software according to the value drawing standard curve measured;
(15) concentration of the Galectin-3 in testing sample is calculated by the fluorescent value of typical curve and unknown concentration sample.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911041A (en) * | 2016-06-01 | 2016-08-31 | 章健 | Time-resolved fluorescence detection method |
| CN106053786A (en) * | 2016-06-01 | 2016-10-26 | 章健 | Fluorescence detection method for time resolution fluorescence marker and device of method |
| CN111024940A (en) * | 2019-12-31 | 2020-04-17 | 广州源起健康科技有限公司 | Time-resolved fluorescence immunoassay method based on gold magnetic particles |
| CN111308075A (en) * | 2020-03-18 | 2020-06-19 | 杭州广科安德生物科技有限公司 | Multi-tumor combined detection kit and use method thereof |
| CN111505304A (en) * | 2019-01-31 | 2020-08-07 | 艾维可生物科技有限公司 | Kit for detecting galectin-3 by chemiluminescence method and use method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226809A (en) * | 2011-02-13 | 2011-10-26 | 倪润洲 | Time resloved fluoroimmunoassay kit for detecting Galectin-3 |
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
-
2013
- 2013-10-05 CN CN201310459430.5A patent/CN104515855A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226809A (en) * | 2011-02-13 | 2011-10-26 | 倪润洲 | Time resloved fluoroimmunoassay kit for detecting Galectin-3 |
| CN102507947A (en) * | 2011-11-11 | 2012-06-20 | 南方医科大学 | CEA TRFIA (time-resolved fluoroimmunoassay) kit based on IMB (immunomagnetic beads) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105911041A (en) * | 2016-06-01 | 2016-08-31 | 章健 | Time-resolved fluorescence detection method |
| CN106053786A (en) * | 2016-06-01 | 2016-10-26 | 章健 | Fluorescence detection method for time resolution fluorescence marker and device of method |
| CN105911041B (en) * | 2016-06-01 | 2019-03-05 | 章健 | A kind of time-resolved fluorescence detection method |
| CN111505304A (en) * | 2019-01-31 | 2020-08-07 | 艾维可生物科技有限公司 | Kit for detecting galectin-3 by chemiluminescence method and use method thereof |
| CN111024940A (en) * | 2019-12-31 | 2020-04-17 | 广州源起健康科技有限公司 | Time-resolved fluorescence immunoassay method based on gold magnetic particles |
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