CN117214155A - Homogeneous phase immunity detection method using cage type lanthanide series supermolecular marking system - Google Patents
Homogeneous phase immunity detection method using cage type lanthanide series supermolecular marking system Download PDFInfo
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- CN117214155A CN117214155A CN202311038613.XA CN202311038613A CN117214155A CN 117214155 A CN117214155 A CN 117214155A CN 202311038613 A CN202311038613 A CN 202311038613A CN 117214155 A CN117214155 A CN 117214155A
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- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 229910052747 lanthanoid Inorganic materials 0.000 title claims abstract description 21
- 150000002602 lanthanoids Chemical class 0.000 title claims abstract description 21
- 230000036039 immunity Effects 0.000 title claims abstract description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 30
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000004132 cross linking Methods 0.000 claims abstract description 9
- 230000005284 excitation Effects 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 238000005259 measurement Methods 0.000 claims abstract description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 3
- 238000003018 immunoassay Methods 0.000 claims description 12
- 239000003550 marker Substances 0.000 claims description 8
- 238000004020 luminiscence type Methods 0.000 claims description 4
- 229910052693 Europium Inorganic materials 0.000 claims description 3
- OGPBJKLSAFTDLK-UHFFFAOYSA-N europium atom Chemical compound [Eu] OGPBJKLSAFTDLK-UHFFFAOYSA-N 0.000 claims description 3
- 239000013522 chelant Substances 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 238000010828 elution Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 abstract description 3
- 150000003384 small molecules Chemical class 0.000 abstract description 2
- 239000007790 solid phase Substances 0.000 description 8
- 239000012071 phase Substances 0.000 description 5
- 108010048233 Procalcitonin Proteins 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 description 4
- 238000002372 labelling Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 150000000918 Europium Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000005081 chemiluminescent agent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013636 protein dimer Substances 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Abstract
The invention provides a homogeneous phase immunity detection method using cage type lanthanide series supermolecule marking system, which uses cage type lanthanide series supermolecule with carboxyl and amino as donor, and covalent cross-linking is carried out with antibody 1 as reagent 1; adopting small organic molecules as receptors, and carrying out covalent cross-linking with the antibody 2 to obtain a reagent 2; taking a sample to be tested, and adding a reagent 1 and a reagent 2 into the sample to be tested; mixing the above mixture, and incubating at 37deg.C for T time; the mixture to be measured is irradiated with excitation light, the luminous intensities thereof at the two detection wavelengths are recorded, and the measurement result is calculated against a standard curve. The donor and the acceptor adopted by the invention belong to small molecules, the steric hindrance is small, the non-specific adsorption does not exist, the elution process is avoided by a homogeneous detection system, and the detection accuracy, sensitivity and efficiency are improved.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to a homogeneous immunodetection method using a cage type lanthanide series supermolecular marker system.
Background
The immunolabeling technique is one of the most widely used techniques in the current diagnostic field, and generally refers to the technique of labeling an antigen or an antibody with a fluorescent substance, a radioisotope, an enzyme, colloidal gold, a chemiluminescent agent, or the like as a tracer, and directly microscopic observation or automatic measurement of an experimental result by using a fluorescence microscope, a radiometer, an enzyme-labeled instrument, an electron microscope, a luminescent immunoassay instrument, or the like.
Some immunoassay techniques require removal of unbound free or interfering substances from the system after the labeling and immune reaction are completed for further detection. In order to make the cleaning effect more thorough, a solid phase carrier, namely a heterogeneous immunoassay technology, is introduced into the reaction system. Common solid supports are magnetic particles, microplates, etc., which can bind antibodies or antigens to the solid support surface. When an immune reaction occurs, an immune complex of a solid-phase carrier-antibody-antigen-antibody-marker is formed in the solution; then the solid phase carrier and the whole immune complex are adsorbed by a special method (for example, magnetic particles are adsorbed by the change of a magnetic field), the solution is emptied and injected into a new buffer solution, and the detection is carried out after the mixture is uniformly mixed and resuspended. However, the adsorption/elution steps are often repeated for several times, which is tedious and time-consuming, and easily causes breakage or loss of the solid phase carrier, which affects the detection result. In addition, the high cost of expensive solid phase carriers, the potential for solid phase non-specific adsorption interference results, and the like, have limited the application of heterogeneous immunoassay techniques to some extent.
Homogeneous immunoassay techniques have been a research hotspot in recent years. The invention develops a homogeneous phase immunity detection method based on a pair of cage type lanthanide supermolecules and small organic molecules as markers. The concrete steps are as follows: the cage lanthanide supermolecule and the small organic molecule are used as a donor and an acceptor to respectively carry out covalent crosslinking with the monoclonal antibody, and form a donor-antibody-antigen-antibody-acceptor immune complex with an antigen in a sample to be detected during reaction; the reaction solution is irradiated with excitation light, and the donor receives the excitation light and transmits energy to the acceptor, so that the acceptor performs a luminescence reaction. The reaction described above occurs only when the donor-acceptor distance is between 2 and 10nm, i.e. an immune complex must be formed and the binding site is convenient. Therefore, the donor and acceptor with wrong free or binding site in the system can not generate energy transfer and luminescence reaction, and the immune conjugate of the object to be detected can be identified without cleaning and separation.
The cage-type lanthanide supermolecule and the small organic molecule can be combined to different biomolecules, such as protein dimers, DNA complementary chains and the like, besides crosslinking antigen antibodies, and the application market and the prospect are wide.
The homogeneous phase immunoassay technology effectively avoids complex steps such as elution and separation, greatly improves analysis efficiency and cost performance, has higher accuracy and sensitivity, and has the potential of replacing the traditional heterogeneous phase immunoassay.
Disclosure of Invention
The aim of the present invention is to solve at least one of the drawbacks of heterogeneous immunoassay techniques.
Therefore, one object of the present invention is to provide a homogeneous immunoassay method using a cage-type lanthanide supermolecular marker system, comprising the following steps in order:
step S1: adopting cage lanthanide supermolecule with carboxyl and amino as donor, and making covalent cross-linking with antibody 1 as reagent 1;
step S2: adopting small organic molecules as receptors, and carrying out covalent cross-linking with the antibody 2 to obtain a reagent 2;
step S3: taking a sample to be tested, and adding a reagent 1 and a reagent 2 into the sample to be tested;
step S4: mixing the above mixture, and incubating at 37deg.C for T time;
step S5: the mixture to be measured is irradiated with excitation light, the luminous intensities thereof at the two detection wavelengths are recorded, and the measurement result is calculated against a standard curve.
Specifically, the donor in the step S1 is a cage lanthanide supermolecule, preferably a hole trivalent europium chelate, and more preferably a terpyridyl europium derivative; the acceptor in step S2 is a small organic molecule having a molecular weight of not more than 2000, preferably of the alexafiur series, more preferably alexafiur 647.
In any of the above embodiments, the reagent 1 and the reagent 2 described in steps S1, S2, and S3 are preferable, and may be used independently as a double reagent or may be mixed as a single reagent.
In any of the above embodiments, it is preferred that the incubation time described in step S4 is between 8 and 20 minutes.
In any of the above embodiments, it is preferable that the excitation light described in step S5 has a wavelength of 280 to 380nm; the wavelength of the two detection lights is 580-680nm.
In any of the above schemes, it is preferable that steps S3 to S5 are performed in a dedicated complete automated chemiluminescence analyzer.
In any of the above schemes, it is preferable that the standard curve in step S5 has an abscissa representing the sample concentration and an ordinate representing the ratio of the luminescence intensities of the sample at the two detection wavelengths.
Compared with the prior art, the invention has the beneficial effects that: based on the labeling immune technology of cage lanthanide supermolecules and organic small molecules, a fully homogeneous phase washing-free reaction system is established, the conventional solid phase carrier is abandoned, the cost of the reagent is reduced, the problems of errors caused by loss or rupture of the solid phase carrier, pollution and time consumption caused by repeated elution and the like can be avoided, and the accuracy and the sensitivity of detection are improved. In addition, the detection process is completed on a special full-automatic chemiluminescence analyzer, so that the detection efficiency is greatly improved, and the clinical examination work is more efficient and convenient.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a "sample concentration-signal value" standard curve of the results of Procalcitonin (PCT) detection.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
Procalcitonin (PCT) detection process
Reagent 1: crosslinking cage lanthanide supermolecule with antibody 1, and adding preservation buffer to prepare reagent 1.
Reagent 2: alexaFlour647 was cross-linked to antibody 2 and added to a preservation buffer to prepare reagent 2.
The detection system comprises: the time T is 1080s, the excitation wavelength is 320nm, and the detection wavelengths are 665nm and 620nm.
The calibration process comprises the following steps: and (3) placing the sample with known concentration, the reagent 1 and the reagent 2 in the corresponding positions of the full-automatic chemiluminescence analyzer, setting measurement parameters and flow, and starting detection. And (3) taking the ratio of the luminous intensities of the samples at 665nm and 620nm wavelengths as a signal value, and establishing a standard curve of 'sample concentration-signal value'.
The detection process comprises the following steps: and (3) placing the sample to be detected, the reagent 1 and the reagent 2 in the corresponding positions of the full-automatic chemiluminescence analyzer, setting measurement parameters and flow, and starting detection. And (5) comparing the signal value of the standard curve, and calculating and outputting the concentration of the sample to be detected.
Comparing the detection results:
the data show that compared with the Roche detection result, R2= 0.9988 is more than 0.990, and the homogeneous detection method has good clinical consistency.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. A homogeneous phase immunity detection method using cage type lanthanide series supermolecule marking system includes the following steps according to sequence:
(1) Step S1: adopting cage lanthanide supermolecule with carboxyl and amino as donor, and making covalent cross-linking with antibody 1 as reagent 1;
(2) Step S2: adopting small organic molecules as receptors, and carrying out covalent cross-linking with the antibody 2 to obtain a reagent 2;
(3) Step S3: taking a sample to be tested, and adding a reagent 1 and a reagent 2 into the sample to be tested;
(4) Step S4: mixing the above mixture, and incubating at 37deg.C for T time;
(5) Step S5: the mixture to be measured is irradiated with excitation light, the luminous intensities thereof at the two detection wavelengths are recorded, and the measurement result is calculated against a standard curve.
2. The method according to claim 1, wherein the donor in step S1 is a cage-type lanthanide supermolecule, preferably a hole-type trivalent europium chelate, more preferably a europium terpyridyl derivative.
3. A homogeneous immunoassay employing a caged lanthanide supramolecular marker system according to claim 1, wherein the receptor in step S2 is a small organic molecule having a molecular weight not exceeding 2000, preferably of the Alexa-flor series, more preferably Alexa-flor 647.
4. The method for homogeneous phase immunoassay using a cage-type lanthanide series supermolecular marker system according to claim 1, wherein the reagent 1 and the reagent 2 in the steps S1, S2 and S3 may be present as double reagents or may be mixed as single reagent.
5. A homogeneous immunoassay employing a caged lanthanide supramolecular marker system according to claim 1, wherein the incubation time T in step S4 is preferably 8-20 minutes.
6. The method for homogeneous immunodetection using a cage-type lanthanide series supermolecular marker system according to claim 1, wherein said excitation light in step S5 has a wavelength of 280-380nm; the wavelength of the two detection lights is 580-680nm.
7. The method for homogeneous immunodetection using a cage-type lanthanide series supermolecular marker system according to claim 1, wherein steps S3 to S5 are performed in a dedicated complete-set full-automatic chemiluminescence analyzer.
8. The method according to claim 1, wherein the standard curve in step S5 has an abscissa representing the sample concentration and an ordinate representing the ratio of the luminescence intensities of the sample at two detection wavelengths.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311038613.XA CN117214155A (en) | 2023-08-17 | 2023-08-17 | Homogeneous phase immunity detection method using cage type lanthanide series supermolecular marking system |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311038613.XA CN117214155A (en) | 2023-08-17 | 2023-08-17 | Homogeneous phase immunity detection method using cage type lanthanide series supermolecular marking system |
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| CN202311038613.XA Pending CN117214155A (en) | 2023-08-17 | 2023-08-17 | Homogeneous phase immunity detection method using cage type lanthanide series supermolecular marking system |
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- 2023-08-17 CN CN202311038613.XA patent/CN117214155A/en active Pending
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