CN113624725A - Method for detecting compound blocking receptor-ligand binding capacity by using HTRF technology - Google Patents
Method for detecting compound blocking receptor-ligand binding capacity by using HTRF technology Download PDFInfo
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- 238000005516 engineering process Methods 0.000 title claims abstract description 11
- 230000000903 blocking effect Effects 0.000 title claims abstract description 8
- 238000002868 homogeneous time resolved fluorescence Methods 0.000 title claims abstract 6
- 238000001514 detection method Methods 0.000 claims abstract description 13
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- 102000001307 androgen receptors Human genes 0.000 description 4
- 108010080146 androgen receptors Proteins 0.000 description 4
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- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
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- 229950001379 darolutamide Drugs 0.000 description 2
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- BLIJXOOIHRSQRB-PXYINDEMSA-N n-[(2s)-1-[3-(3-chloro-4-cyanophenyl)pyrazol-1-yl]propan-2-yl]-5-(1-hydroxyethyl)-1h-pyrazole-3-carboxamide Chemical compound C([C@H](C)NC(=O)C=1NN=C(C=1)C(C)O)N(N=1)C=CC=1C1=CC=C(C#N)C(Cl)=C1 BLIJXOOIHRSQRB-PXYINDEMSA-N 0.000 description 2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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Abstract
The invention discloses a method for detecting the receptor-ligand binding blocking capacity of a compound by using an HTRF technology, belonging to the field of biochemical detection. A method for detecting receptor-ligand binding capacity using HTRF technology, the method comprising the steps of: step one, labeling a ligand fluorescence donor; secondly, labeling a receptor fluorescent receptor; incubating the receptor, the ligand and the tested compound together; and step four, detecting a receptor fluorescence signal, and calculating the capacity of the compound for blocking the receptor-ligand combination. The invention has the advantages that: homogeneous reaction, simple operation and high throughput test.
Description
Technical Field
The invention belongs to the field of high-throughput compound screening, and particularly relates to a method for detecting the receptor-ligand binding capacity of a compound by using an HTRF (high-throughput RF) technology.
Background
Receptors (receptors) are cellular protein components formed by cells during evolution, which recognize certain trace chemicals in the surrounding environment, bind to them first, and trigger subsequent physiological or pharmacological effects through intermediary information transduction and amplification systems.
Since Langley proposed the receptor theory for 100 years, the receptor has been proven to be an objectively existing entity, is of a large variety, has many mechanisms of action elucidated, and is now no longer an empty general concept.
The receptor molecules are present in very small amounts in the cells, and 1mg of tissue generally contains only about 10 fmol. Substances that specifically bind to receptors are called ligands (ligand).
The receptor is only a "sensor" and has a very high recognition of the corresponding ligand.
Receptor-ligands are a coupling of life activities, and receptors all have their endogenous ligands, such as neurotransmitters, hormones, self-actives (autocoids), etc.
Ligands that activate receptors are called agonists (agonst) and ligands that block their activity are called antagonists (agonst). Receptors are divided into several subtypes according to their high specificity of binding to ligands, such as adrenergic receptors, which are divided into α 1, α 2, β 1 and β 2 subtypes, which differ in their distribution and function.
The receptor has high affinity with the ligand, and most ligands can cause the pharmacological effect of cells at the concentration of 1 pmol-1 nmol/L. Such sensitivity is mainly due to the amplification, differentiation and integration of subsequent information transduction systems, such as the intracellular second messenger (second messenger).
Enzymes, vectors, ion channels and nucleic acids may also act directly with drugs, but these substances themselves have an effect force and should not be considered as receptors strictly speaking.
Certain cellular protein components can bind to ligands but do not have the ability to trigger an effect, called binders (acceptors).
The cellular protein component can bind to the ligand but has no ability to trigger an effect, called an adaptor.
Receptor kinetics studies the quantitative relationship and regularity with which ligands (drugs, transmitters, hormones, auto-active substances) bind to receptors and cause biological effects. Receptor binding assays often employ certain ligands to detect receptor binding. Receptor kinetics were studied as follows: functional assay, radioligand binding method, affinity chromatography, interface optics method, nuclear magnetic resonance technique, capillary electrophoresis (capillary electrophoresis) method, liquid flash proximity assay technique.
One of the main indicators for the presence of a receptor is the saturation of the receptor, i.e. a saturation curve is plotted for specific binding. The principle is to determine the active substance and the content according to the principle that the active substance and the marked active substance compete for the same receptor, and belongs to the saturation analysis method. The characteristic binding is obtained by subtracting non-specificity from the total binding in the experiment, then a saturation curve and a competition curve are drawn, and the Bmax and Kd values are calculated.
In summary, the existing detection methods all require special instruments and are expensive, and some consumable materials, such as the isotope labels, which need to be customized are difficult to popularize.
Disclosure of Invention
To overcome the above-mentioned deficiencies, the present invention provides a method for detecting the ability of a compound to block receptor-ligand binding using HTRF technology.
A method for detecting the ability of a compound to block receptor-ligand binding using HTRF technology, the method comprising the steps of:
marking a ligand fluorescence donor, wherein the fluorescence donor is generally lanthanide Tb;
marking a receptor fluorescent receptor, wherein the fluorescent receptor is FITC or APC generally;
step three, incubating the receptor, the ligand and the tested compound at room temperature;
detecting a receptor fluorescent signal, and calculating the capacity of the compound for blocking the receptor-ligand combination;
further, in step three, the drug is an agonist or an inhibitor.
HTRF is short for homogeneous phase time-resolved fluorescence technology, which is a further improvement on TR-FRET technology. HTRF is based on two major techniques, time-resolved fluorescence (TRF) and Fluorescence Resonance Energy Transfer (FRET). The TRF utilizes the characteristic of long half-life period of rare earth elements (millisecond level, nanosecond level compared with common fluorescence level, 6 magnitude order difference). The background can be excluded by delaying 50-100 microseconds. FRET utilizes energy transfer by two fluorescent cliques, called energy Donor (Donor) and energy Acceptor (Acceptor), respectively. Donor is excited by an external light source and, if it is close to the Acceptor, can shift the energy resonance to the Acceptor, so that it is excited and emits an emission light with a specific wavelength of 665 nm. Compared with the traditional TR-FRET energy Donor encapsulated in chelate, two kinds of Donor europium (Eu3+ cryptate) and terbium (Lumi4Tb) of HTRF fluorescence energy are permanently embedded in cryptate and are more sensitive and stable. After the europium and the terbium are excited by the laser, the emission spectrum has a peak at 490nm and 620nm respectively. There are also two types of acceptors, one is the APC hinge complex, under the commercial name XL665, and the other is FITC, a small molecule compound. The excitation light of XL665 and FITC overlaps with the emission light of Donor, and forms a specific peak at 665nm or 520nm after excitation.
The technical characteristics of the HTRF detection compound for blocking the receptor-ligand binding capacity are as follows:
the operation is very simple: directly adding sample into the empty plate, detecting after adding the sample, and shortening the time (sample adding and incubation for 2 h);
the system is very stable: the detection can be carried out at any time within 7 days, and the signal is basically unchanged;
is suitable for precious samples: the whole experiment system only needs 20 ul;
homogeneous phase detection system: coating is not needed, and ELISA is free from washing, so that time and labor are saved;
the data is objective and real: the ratio processing can effectively remove background fluorescence, and the false positive rate and the false negative rate are low;
reflecting the actual condition of the sample, the false positive and false negative rate is low, and the background caused by autofluorescence of the solar thermal product can be removed.
The invention has the advantages that:
1. providing a new method: the detection of the ability of the compound to block the receptor-ligand binding can be completed only by the reagent.
2. Greatly saves the experiment workload and the experiment time: the experiment operation is from original coating, sealing and plate washing for many times, only a sample and a detection reagent are needed to be added at present, and the experiment time is shortened from original 1 day to 1-2 hours;
3. greatly improving the flux of the experiment: the detection is performed by passing a single sample through a column by 96-well original ELISA or FACS and the like, and is improved to 384-well or even 1536-well, so that the large-batch sample screening is realized in a short time;
4. the signal is more stable: the fluorescence duration is long, detection can be carried out after overnight, the detection result is not influenced, and the detection results of other methods are limited by time, and the detection result is influenced by overlong time or overlong time.
Drawings
Fig. 1 is a schematic diagram of the principle provided by the present invention.
FIG. 2 is a graph of the ability of Darolutamide to block androgen receptor binding to androgen as measured by HTRF according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The first embodiment is as follows:
step one, an androgen receptor containing a GST tag is labeled with a GST antibody of chimeric lanthanide Tb;
marking androgen by using FITC;
step three, uniformly incubating the gradiently diluted Darolutamide, the marked androgen receptor and androgen at room temperature;
detecting a receptor fluorescent signal, and calculating the capacity of the compound for blocking the receptor-ligand combination;
as shown in figure 2, darolutamine dose-dependently blocked androgen receptor and androgen binding.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, or direct or indirect applications in other related fields, which are made by the contents of the present specification, are included in the scope of the present invention.
Claims (3)
1. The detection of the ability of a compound to block receptor-ligand binding using HTRF techniques, characterized in that the method comprises the steps of: step one, labeling a ligand fluorescence donor; secondly, labeling a receptor fluorescent receptor; incubating the receptor, the ligand and the tested compound together; and step four, detecting a receptor fluorescence signal, and calculating the capacity of the compound for blocking the receptor-ligand combination.
2. The method of detecting the ability of a compound to block receptor-ligand binding using HTRF technology of claim 1, wherein: in step one, the fluorescence donor is Tb chimera and the acceptor is FITC or APC.
3. The method of detecting the ability of a compound to block receptor-ligand binding using HTRF technology of claim 1, wherein: in step three, the compound is an agonist or an inhibitor.
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