CN115902204A - Detection method and kit for exosomal galectin 9 - Google Patents
Detection method and kit for exosomal galectin 9 Download PDFInfo
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- CN115902204A CN115902204A CN202211163591.5A CN202211163591A CN115902204A CN 115902204 A CN115902204 A CN 115902204A CN 202211163591 A CN202211163591 A CN 202211163591A CN 115902204 A CN115902204 A CN 115902204A
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Abstract
The invention provides a detection method and a kit of exosome-form galectin 9, wherein the method comprises the following steps: incubating the sample with an ELISA plate coated and blocked with a mouse anti-human CD63 antibody; performing enzyme-linked immunosorbent assay using the incubated ELISA plate and galectin 9 antibody, biotin-labeled goat anti-rabbit IgG antibody, streptavidin-coupled horseradish peroxidase; and (3) detecting by using a developing solution and a microplate reader, and calculating the content of the exosome-form galectin 9 in the sample.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a detection method and a kit of exosomal galectin 9.
Background
The combination of the galectin 9 (Gal-9) and a T lymphocyte surface immune checkpoint molecule TIM-3 results in limited immune treatment effect benefits, and if the exosome form Gal-9 can be quickly and accurately predicted through quick diagnosis of a body fluid sample and quantification of the exosome form Gal-9, the T cell immune suppression level of the exosome form Gal-9 in the body fluid of a tumor patient can be quickly and accurately predicted, so that a new reliable strategy is provided for predicting the immune treatment benefits of the patient.
However, in the prior art, the existing ELISA kit for detecting Gal-9 can only detect liquid-soluble Gal-9, and cannot distinguish between galectin 9 in an exosome form (Ex-Gal-9) and soluble galectin 9 (sGal-9), and non-soluble Gal-9 needs to be converted into soluble Gal-9, i.e. if the existing ELISA kit for detecting exosome membrane protein Gal-9 needs to rely on an exosome separation process, the separation process takes about 8-10 hours, and the total time consumption of staining and ELISA process is more than 24 hours, so the consumption time is too long, and the detection is very inconvenient. How to rapidly detect the exosome form Gal-9 in human plasma or body fluid and simultaneously detect the exosome form Gal-9 and soluble Gal-9 are problems to be solved.
To this end, the methods of the present technology provide methods and kits for specifically detecting exosome form Gal-9, as well as methods and kits that can simultaneously detect exosome form Gal-9 and soluble Gal-9.
Disclosure of Invention
In a first aspect, the present invention provides a method for detecting galectin 9 in an exosome form, comprising:
incubating the sample with an ELISA plate coated and sealed with a mouse anti-human CD63 antibody to obtain an incubated ELISA plate;
performing enzyme-linked immunosorbent assay using the incubated ELISA plate and antibodies to galectin 9, biotin-labeled goat anti-rabbit IgG antibodies, streptavidin-coupled horseradish peroxidase (Streptavidin-HRP);
and (3) detecting by using a developing solution and a microplate reader, and calculating the content of the exosome form Gal-9 in the sample.
Optionally, the antibody to galectin 9 is a rabbit anti-human galectin 9 antibody;
optionally, the biotin-labeled IgG antibody is a biotin-labeled goat anti-rabbit IgG antibody.
In a second aspect, the present invention provides a method for detecting galectin 9, comprising:
incubating the sample with an ELISA plate coated and blocked with a murine anti-human CD63 antibody to obtain an incubated ELISA plate comprising soluble Gal-9 (sGal-9) and a treated sample coupled to exosomal galectin 9 (Exo-Gal-9);
incubating an ELISA plate coated and sealed by an anti-Gal-9 antibody with the treated sample, and detecting the content of soluble Gal-9 by an enzyme-linked immunosorbent assay;
performing enzyme-linked immunosorbent assay using the incubated ELISA plate and anti-galectin 9 antibody, biotin-labeled IgG antibody, streptavidin-coupled horseradish peroxidase;
and (3) detecting by using a developing solution and a microplate reader, and calculating the content of the exosome form Gal-9 in the sample.
Alternatively, the anti-galectin 9 antibody is a rabbit anti-human galectin 9 antibody;
optionally, the biotin-labeled IgG antibody is a biotin-labeled goat anti-rabbit IgG antibody;
the method can synchronously realize the detection of the galectin 9 and the soluble galectin 9 in the exosome form.
In a third aspect, the present invention provides a kit for detecting galectin 9, comprising:
mouse anti-human CD63 antibody, ELISA plate, galectin 9 antibody, and biotin-labeled IgG antibody.
Optionally, the kit of the present invention further comprises: anti-Gal-9 antibodies (i.e., antibodies to galectin 9);
optionally, the kit of the present invention further comprises: streptavidin coupled horseradish peroxidase, a developing solution and a developing stop solution.
Alternatively, the antibody to galectin 9 is a rabbit anti-human galectin 9 antibody;
alternatively, the biotin-labeled IgG antibody is a biotin-labeled goat anti-rabbit IgG antibody;
optionally, the ELISA developing solution comprises developing solution a and developing solution B; when used, the solution A and the solution B were mixed at a ratio of 1.
The formula of the solution A comprises: sodium acetate 13.56g
Citric acid 1.6g
30％H 2 O 2 0.3mL
ddH 2 O to 500mL
The formula of the solution B is as follows: ethylene diamine tetraacetic acid 0.2g
Citric acid 0.95g
Glycerol 50mL
TMB 0.15g ddH2O to 500mL
Optionally, the chromogenic stop solution is 2 MH 2 SO 4 。
Further, the kit also comprises a commercially available detection reagent for soluble galectin 9, a corresponding reagent for detecting the soluble galectin 9, an anti-Gal-9 antibody, an enzyme-linked reaction reagent and the like.
Advantages of the present invention include, but are not limited to:
1. the exosome form Gal-9 is detected specifically, quantitatively and quickly for the first time;
2. the method does not depend on an exosome membrane protein quantitative detection technology for exosome separation, can save the reagent material cost of an exosome separation step, can realize the rapid detection and quantification of Gal-9 in exosome within 3-4 hours by reducing the total detection time, saves 5-6 hours compared with the prior method, and obviously improves the efficiency;
3. the simultaneous detection of the exosome form Gal-9 and the soluble exosome form Gal-9 is realized for the first time;
4. the method has high sensitivity and strong specificity; the first antibody (mouse anti-human CD63 antibody) enriches and adsorbs exosomes, and the second antibody (galectin 9 antibody) detects Gal9 on the exosomes, so that the double benefits of reducing background and improving sensitivity are achieved;
5. the method needs few samples and has high detection flux: the ELISA plate type detection only needs 50 microliters of samples to meet the detection requirement;
6. the kit is suitable for rapidly screening the level of the immunosuppressive molecule Gal-9 in a human body fluid or plasma sample, and can use a 96-well plate detection plate to meet the requirement of high-throughput detection. Gal-9 is one of molecules for negatively regulating the functions of CD8+ T cells, and the detection of the membrane type Gal-9 in body fluid samples such as plasma, ascites and the like provides a new fine typing basis for rapidly screening whether a patient is suitable for immunotherapy.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic diagram of a method for detecting galectin 9 in an exosome form according to the present invention;
FIG. 2 is a schematic diagram of a detection method of galectin 9 according to the present invention;
FIG. 3 is a schematic flow chart of a method for detecting galectin 9 in an exosome form according to an embodiment of the present invention;
FIG. 4 is a schematic diagram of an enzyme-linked immunosorbent assay for the detection of galectin 9 in the exosome form according to the present invention;
FIG. 5 is a result of detecting galectin 9 in an exosome form according to a further embodiment of the present invention, wherein A is a color result graph, B is a standard curve graph, C is a standard curve when a rabbit-derived CD63 antibody and a mouse-derived Gal-9 antibody are combined;
FIG. 6 is a graph (A) and a numerical value (B) of a comparative test of the detection method of galectin 9 in the exosome form of the present invention (left column) with the detection after the prior exosomes were isolated (right column).
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it should be understood that they are presented herein only to illustrate and explain the present invention and not to limit the present invention.
The main sources of reagents and instruments involved in the examples of the present invention are as follows:
wherein, the ELISA coated plate (R & D Systems, goods # DY 008) comprises a 96-hole plate, a sealing film, a substrate color development liquid, a stop solution, a coating liquid, a washing liquid and a confining liquid;
the above list of reagents can also be purchased separately:
96 well plates (R & D Systems, cat # DY 990).
Sealing films (R & D Systems, cat # DY 992).
PBS:137mM NaCl,2.7mM KCl,8.1mM Na 2 HPO 4 ,1.5mM KH 2 PO 4 ,
pH 7.2-7.4,0.2 μm filtration (R & D Systems, cat # DY 006).
25 XWash buffer 20mL 25XWash buffer plus 480mL ddH 2 Dilution of O (R)&D Systems, cat # WA 126).
10X confining liquid ddH used before use 2 O10-fold, the working solution was 1% BSA in PBS, pH 7.2-7.4,0.2 μm filtration (R)&D Systems,Catalog#DY995).
1 mixture of developer A (H2O 2) and developer B (tetramethyllbenzidine) of (R & D Systems, cat # DY 999).
The other reagents related to the invention are domestic analytical purifiers unless otherwise specified.
The instruments adopted by the invention are all common commercial products and can be purchased in the market.
Referring to fig. 1, the present invention provides a method for detecting galectin 9 in an exosome form, comprising the steps of:
removing cells and cell debris from a human body fluid sample (plasma or ascites) by centrifugation;
loading the obtained supernatant on an ELISA plate, wherein the ELISA plate is coated and sealed by an anti-CD 63 antibody (preferably a mouse source);
then sequentially adding an antibody (preferably rabbit-derived) of the galectin 9, biotin-labeled IgG and streptavidin-coupled horseradish peroxidase for enzyme-linked immunosorbent assay;
and measuring by using a developing solution and a microplate reader to obtain the content of the exosomal galectin 9.
Referring to fig. 2, the present invention also provides a method for detecting galectin 9, i.e., simultaneously detecting galectin 9 in an exosome form and soluble galectin 9, comprising the steps of:
removing cells and cell debris from a human body fluid sample (plasma or ascites) by centrifugation;
loading the obtained supernatant on an ELISA plate, wherein the ELISA plate is coated and sealed by an anti-CD 63 antibody (preferably a mouse source);
transferring the supernatant into a new EP tube again, and monitoring the supernatant by using the existing commercial soluble galectin 9ELISA kit to obtain the content of the soluble galectin 9;
then sequentially adding an antibody (preferably rabbit source) of the galectin 9, biotin-labeled IgG and streptavidin-coupled horse radish peroxidase for enzyme-linked immunosorbent assay;
and measuring by using a developing solution and a microplate reader to obtain the content of the exosomal galectin 9.
Example 1
Referring to FIGS. 3, 4 and 5, a method for detecting galectin 9 in an exosome form according to an embodiment of the present invention will be described in detail,
1) Coating of ELISA plate: mouse anti-human CD63 antibody was diluted with R & D coating buffer as 1;
2) Blocking of ELISA plate: removing the coated antibody, adding washing solution 300 μ L per well, and washing for 3 times, each for 1 min; then adding 300 mu L of washing solution/hole, and incubating for one hour at 37 ℃;
3) Washing: discarding the confining liquid, washing with 300 μ L of washing solution per well for 1 min each time for 3 times;
4) Adding a sample to be tested: 50 mu L/hole, incubating in an incubator at 37 ℃ for 2 hours, and removing cells and cell debris from a sample by centrifugation in advance, wherein the sample is centrifuged plasma;
5) Washing: discarding the solution to be tested, adding 300 μ L of washing solution into each hole, washing for 1 min for 3 times;
6) Adding an antibody: the rabbit anti-human galectin 9 antibody was diluted with 1X blocking solution 1;
7) Washing: discarding the solution to be tested, adding 300 μ L of washing solution into each hole, washing for 1 min for 3 times;
8) Biotinylated IgG: the biotin-labeled goat anti-rabbit IgG antibody was diluted with 1X blocking solution 1;
9) Washing: discarding the solution to be tested, adding 300 μ L of washing solution into each hole, washing for 1 min for 3 times;
10 Streptavidin-coupled horseradish peroxidase (Streptavidin-HRP): streptavidin-HRP,100 μ Ι/well was diluted with 1 × blocking solution 1; incubating in an incubator at 37 ℃ for 30 minutes;
11 Substrate solution addition: 1, mixing a substrate solution A and a substrate solution B,100 mu L/hole, and carrying out HRP catalytic reaction;
12 Terminate the color development; 50 mu L/well stop solution
13 Enzyme-linked immunosorbent assay; measurement was performed using a deacon microplate reader OD 450.
14 Standard curve drawing: establishing a standard curve according to the OD value of the body fluid sample diluted by multiple times;
15 According to the OD value of the sample to be tested, the sample to be tested is quantified.
See fig. 5B for experimental results.
Example 2
Referring again to FIGS. 3, 4 and 5, substantially the same procedure as described in example 1 was performed except that the murine CD63 antibody was replaced with the rabbit CD63 antibody, the rabbit Gal-9 antibody was replaced with the murine Gal-9 antibody, the sample was ascites after centrifugation, the other experimental procedures and experimental reagents were the same, and the experimental results were as shown in FIG. 5C, respectively.
From the results of examples 1 and 2, it can be seen that the method of the present invention has stable, reliable and repeatable results, R of 5B 2 The value is 0.9997, and the detection of the Gal-9 on the surface of the exosome membrane is time-saving and accurate compared with the detection after the exosome is separated; the experiment can be completed in 3-4 hours from the incubation of the coated and sealed pretreated ELISA plate and the sample; as can be seen by comparing FIGS. 5B and 5C, R of 5B 2 The value was 0.9997, and when the combination of rabbit-derived CD63 antibody and mouse-derived Gal-9 antibody was changed in 5C, the standard curve R was obtained after gradient dilution of exosomes 2 The value is 0.8131, the result is poor in linearity and unstable; therefore, the use of a murine CD63 antibody and a rabbit Gal-9 antibody is more preferable.
Example 3
The detection method for directly measuring the exosomal galectin 9 by using the method of the invention has the comparative test and experimental result of detection after the prior exosomal is separated;
in the first part, the method steps and reagents of the invention are as described in example 1, and the sample is ascites;
the second part, after separating exosome with ascites from the same patient by using a gradient differential centrifugation method, extracting exosome protein by using protein lysate, and then using a commercial soluble galactosidase-9 kit (R & D, DGAL 90), wherein enzyme linked immunosorbent assay and measurement are as described above; NTA detection shows that the particle size of the exosome is in accordance with 30-150nm;
the experimental results are shown in fig. 6A and 6B, respectively; the ascites in the same batch are directly subjected to ELISA (left column) and extracted by the method, and then is subjected to Gal-9 commercial kit (right column); 6A and 6B respectively show pictures and the OD450 values measured by the corresponding microplate readers, and as can be seen from the pictures, the body fluid has higher sensitivity by adopting ELISA.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A method for detecting galectin 9 in an exosome form, comprising:
incubating the sample with an ELISA plate coated and sealed with a mouse anti-human CD63 antibody to obtain an incubated ELISA plate;
performing enzyme-linked immunosorbent assay by using the incubated ELISA plate, an antibody of galectin 9, a biotin-labeled goat anti-rabbit IgG antibody and streptavidin-coupled horseradish peroxidase;
and (3) detecting by using a developing solution and a microplate reader, and calculating the content of the exosome-form galectin 9 in the sample.
2. The method of claim 1, wherein the sample is a human body fluid or plasma from which cells and cell debris have been removed by centrifugation.
3. The method of detecting according to claim 1, wherein the antibody to galectin 9 is a rabbit anti-human galectin 9 antibody.
4. A method for detecting galectin 9, comprising:
incubating the sample with an ELISA plate coated and blocked with a murine anti-human CD63 antibody to obtain an incubated ELISA plate and a treated sample,
incubating the treated sample with an ELISA plate coated and blocked with an anti-galectin 9 antibody, and then detecting the content of soluble galectin 9 by an enzyme-linked immunosorbent assay;
enzyme-linked immunosorbent assay was performed using the incubated ELISA plate and secondary antibody to galectin 9, biotin-labeled goat anti-rabbit IgG antibody, streptavidin-coupled horseradish peroxidase;
and (3) detecting by using a developing solution and a microplate reader, and calculating the content of the exosome-form galectin 9 in the sample.
5. The method of claim 4, wherein the sample is a human body fluid or plasma from which cells and cell debris have been removed by centrifugation.
6. The detection method according to claim 4, wherein the antibody against galectin 9 is a rabbit anti-human galectin 9 antibody.
7. The detection method according to claim 4, wherein the biotin-labeled IgG antibody is a biotin-labeled goat anti-rabbit IgG antibody.
8. A galectin 9 detection kit, comprising:
mouse anti-human CD63 antibody, ELISA plate, rabbit anti-human galectin 9 antibody, and biotin-labeled goat anti-rabbit IgG antibody.
9. The test kit of claim 8, further comprising an anti-Gal-9 antibody.
10. The detection kit according to claim 8 or 9, further comprising streptavidin-coupled horseradish peroxidase, a developing solution and a developing stop solution.
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