CN117554445A - Method and kit for reducing electrochemiluminescence detection background signal - Google Patents
Method and kit for reducing electrochemiluminescence detection background signal Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
The application belongs to the technical field of in-vitro detection, and particularly relates to a method and a kit for reducing an electrochemiluminescence detection background signal. According to the method, through exploring the competitive products and analogues of a plurality of terpyridyl ruthenium structures, the fact that the antibody competitive products with certain concentration are added into the magnetic bead solution can reduce the non-specific combination of the ruthenium marker, the solid phase carrier and the protein to reduce the background signal of the electrochemiluminescence detection kit, the background signal value of most electrochemiluminescence reagents can be reduced by about 50%, and the sensitivity and the signal to noise ratio of electrochemiluminescence detection can be effectively improved.
Description
Technical Field
The application belongs to the technical field of in-vitro detection, and particularly relates to a method and a kit for reducing an electrochemiluminescence detection background signal.
Background
Electrochemiluminescence (also known as electrochemiluminescence) refers to the phenomenon that by applying a suitable voltage across the surface of an electrode, specific electric substances are generated, electrons are transferred between these electric substances or between the electric substances and some components in the system through specific reactions, and then an excited state is formed, and these excited state substances return to the ground state from the excited state while releasing energy and accompanying luminescence. Electrochemiluminescence needs to undergo two processes: electrochemical reactions and chemiluminescent reactions. The task of the electrochemical reaction process is mainly to provide intermediates for chemiluminescent reaction, wherein the chemiluminescent reaction is a process of generating excited states by chemiluminescent reaction between some intermediates or between the intermediates and some other components in the system, and the excited state substances are accompanied by light radiation when returning to the ground state.
Modern diagnostic techniques, represented by electrochemiluminescence, currently exhibit great value and role in medical diagnostics. The high sensitivity and the measurement range of the method enable various trace protein markers in the sample to be accurately and rapidly measured. Among them, the magnetic bead Electrochemiluminescence (ECL) kit represented by rogowski diagnosis (Roche Diagnostics) occupies the dominant position in the whole market. The electrochemiluminescence technology using magnetic beads as a carrier and combining with terpyridyl ruthenium as a tracer marker also becomes a hot spot for more and more enterprise technology development.
In practical electrochemiluminescence kit development, higher luminescence signal levels and lower background signal levels are often the main directions of technology development. Background signal level (value) refers to the level of background luminescent signal generated by the kit when there is no target analyte in the sample. The lower the signal, the easier the sensitivity of the kit is to be increased. Otherwise, if the background signal of the kit is higher, the signal generated by the trace target object is easily covered in the stronger background signal, and cannot be distinguished from the background signal, so that the sensitivity is lower.
In the development of an electrochemiluminescence kit using magnetic beads as a carrier, in order to reduce and control background signals, conventional techniques add substances such as Bovine Serum Albumin (BSA), polyethylene glycol (PEG), casein and the like to reduce non-specific binding of antibodies or target proteins to the magnetic beads, thereby reducing the background signals, but such methods alone often cannot sufficiently control the background signals. Therefore, how to further reduce background signals and improve sensitivity is still an important difficulty and direction of development of some kits.
Disclosure of Invention
Based on this, an embodiment of the present application provides a method for reducing the background signal of electrochemiluminescence detection to effectively reduce the background signal and the detection sensitivity of electrochemiluminescence detection.
In one aspect, the present application provides a method for reducing an electrochemiluminescence detection background signal, comprising the steps of adding an antibody competitor to a detection system during electrochemiluminescence detection;
the antibody competitor comprises nickel tris (2, 2' -bipyridine) chloride.
Reagents used for the electrochemiluminescence detection include terpyridyl ruthenium labeled antibodies and magnetic bead solutions.
In one embodiment, the antibody competitor comprises nickel tris (2, 2 '-bipyridine) chloride, and the final concentration of the nickel tris (2, 2' -bipyridine) chloride in the magnetic bead solution is 1ppm to 100ppm.
In one embodiment, the magnetic beads comprise streptavidin-coated magnetic beads.
In one embodiment, the concentration of the streptavidin-coated magnetic beads is 0.5mg/ml to 1.0mg/ml.
In one embodiment, the species source of the terpyridyl ruthenium labeled antibody is at least one of a mouse, a rabbit, and a sheep.
In another aspect, the present application provides a kit for reducing the background signal of electrochemiluminescence detection, said kit comprising an antibody competitor as defined in said method and a chemiluminescent detection reagent.
The application also provides application of the kit in reducing the background signal value of the electrochemiluminescence kit.
According to the method, through exploring the competitive products and analogues of a plurality of terpyridyl ruthenium structures, the fact that the antibody competitive products with certain concentration are added into the magnetic bead solution can reduce the non-specific combination of the ruthenium marker, the solid phase carrier and the protein to reduce the background signal of the electrochemiluminescence detection kit, and the background signal value of most electrochemiluminescence reagents can be reduced by about 50%, so that the sensitivity and the signal to noise ratio of electrochemiluminescence detection are effectively improved.
Detailed Description
The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order to provide a more thorough understanding of the present disclosure. It is also to be understood that this application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but is capable of numerous changes or modifications without departing from the spirit of the application, as equivalent forms are intended to be within the scope of this application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application, it being understood that the present application may be practiced without one or more of these details.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Terminology
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Unless otherwise conflict with the purpose and/or technical solution of the present application, the present application relates to the cited documents which are incorporated by reference in their entirety for all purposes. When reference is made to a cited document in this application, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated by reference. Examples of the relevant technical features and preferred modes to be cited in the present application when the cited documents are referred to in the present application are incorporated by reference in the present application, but are not limited to being able to implement the present application. It should be understood that when a reference is made to the description herein, it is intended to control or adapt the present application in light of the description herein.
The term "electrochemiluminescence" refers to an electrochemiluminescence analysis in which a certain electrochemical signal (including voltage and current) is applied to a chemical system containing a chemiluminescent substance through an electrode to generate a new substance which reacts with the chemical substance existing in the system or itself undergoes a decomposition reaction, the reaction not only provides enough energy, but also generates a suitable luminophor and accepts the energy released by the reaction to form an excited state luminophor, and when the unstable excited state returns to the ground state, an emitted light consistent with the nature of the luminophor is emitted, and the luminescence spectrum or the luminescence intensity is measured by a common optical means such as a photomultiplier tube to perform trace analysis on the substance.
The term "ruthenium terpyridyl is an emerging luminescent agent with good physical and chemical properties. In recent years, it has been widely used in the fields of chemistry, biology, medicine, materials, electronics, and the like. Principle of luminescence of ruthenium terpyridyl: the divalent terpyridyl ruthenium serving as a luminescent substrate loses electrons on the surface of an electrode to be oxidized, and the divalent terpyridyl ruthenium is reduced to be divalent ruthenium by a strong reducing agent and simultaneously releases photons to restore to a ground state luminescent substrate. The method can be used for electrochemiluminescence immunoassay according to the terpyridyl ruthenium luminescence principle. Ruthenium terpyridyl is used as a catalyst in the catalytic oxidation of ethanol, carbonylation of aryl compounds, hydroformylation and the like. Can also be used in the photochemical fields of fluorescent light-emitting devices, photoelectric switches and the like.
The term "streptavidin-coated magnetic beads" Streptavidin (SAV) MagBeads is a target molecule which is formed into a fixed layer by fixing streptavidin (strepavidin) on a polymer magnetic microsphere and by means of the high-affinity binding property of streptavidin-biotin, and the like, and further is separated and interacted with biotinylated molecules, and can be used for various applications such as affinity purification, cell sorting, protein interaction, DNA-protein interaction, probe capture, mRNA separation and the like.
One of the outstanding features of the electrochemiluminescence kit, which is distinguished from other chemiluminescent kits, is the use of an antibody labeled with a terpyridyl ruthenium structure as a capture antibody. The terpyridyl ruthenium structure plays a key role in the final electrochemiluminescence process, and the final luminescence intensity is in linear relation with the quantity of the terpyridyl ruthenium. Aiming at the characteristics of the electrochemiluminescence reagent, one important direction is to reduce the background signal of the kit, namely to reduce the nonspecific binding of the antibody marked with the terpyridyl ruthenium with magnetic beads and proteins. However, common additives such as BSA, etc., mainly reduce the nonspecific binding of the additive protein to the magnetic beads and proteins by competing with the antibody protein portion. Whereas, in addition to the antibody protein moiety, the nonspecific binding of the ruthenium terpyridyl moiety to the beads and protein can be reduced or avoided if it is possible to reduce the background signal from another direction.
The application finds that the terpyridyl ruthenium marker has obvious positive charges, most of magnetic beads have negative charges, certain electrostatic attraction exists between the terpyridyl ruthenium marker and the magnetic beads, and the terpyridyl ruthenium molecule itself also has strong adhesion capability, so that a container or tool stained with the terpyridyl ruthenium is difficult to wash. The background signal of the electrochemiluminescence kit is partly due to the non-specific binding of the ruthenium terpyridyl structure to the magnetic beads or proteins, whereas suitable competitors and analogues of the ruthenium terpyridyl structure are expected to reduce this non-specific binding of ruthenium terpyridyl. At the same time, the competitor or the like is also required to be unable to adversely affect chemiluminescence or immune response.
The application examines the competitors and analogues of a plurality of terpyridyl ruthenium structures, and discovers that the tris (2, 2' -bipyridyl) nickel chloride (terpyridyl nickel for short) can reduce the background of a part of electrochemiluminescence kit and can not generate interference signals under the measurement condition.
In one aspect, the present application provides a method for reducing the background signal of an electrochemiluminescence detection comprising the step of adding an antibody competitor to a detection system during the electrochemiluminescence detection.
The antibody competitor comprises tris (2, 2' -bipyridine) nickel chloride, and the metal complex is combined with the magnetic beads as a whole, so that the charge and the special structure brought by the metal ions play a relatively large role.
Reagents used for the electrochemiluminescence detection include terpyridyl ruthenium labeled antibodies and magnetic bead solutions.
In one embodiment, the antibody competitor comprises nickel tris (2, 2 '-bipyridine) chloride, and the final concentration of the nickel tris (2, 2' -bipyridine) chloride in the magnetic bead solution is 1ppm to 100ppm.
In another aspect, the present application provides a kit for reducing the background signal of electrochemiluminescence detection, said kit comprising an antibody competitor as defined in said method and a chemiluminescent detection reagent.
The application also provides application of the kit in reducing the background signal value of the electrochemiluminescence kit.
Embodiments of the present application will be described in detail below with reference to examples.
It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are preferably referred to in the guidelines given in the present application, may be according to the experimental manual or conventional conditions in the art, may be according to the conditions suggested by the manufacturer, or may be referred to experimental methods known in the art.
In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.
Example 1
Adding a certain concentration of nickel terpyridyl or other analogues into RM (magnetic bead) component of the existing Total Prostate Specific Antigen (TPSA) electrochemiluminescence kit, and keeping other R1 (biotinylated primary antibody) and R2 (terpyridyl ruthenium labeled secondary antibody) components unchanged. Results of measurement of blank samples (horse serum) and high and low serum values.
The specific process is that a plurality of terpyridyl ruthenium analogues are prepared into a 1% concentration aqueous solution. To the bead working solution (RM of the kit) a certain amount of analogue solution was added to reach the indicated concentration. Preserving for more than 24 hours at room temperature, and fully adsorbing the magnetic beads.
The measurement was performed on an electrochemical luminescence immunoassay analyzer of SE1200 of the company (jiangsu trigeminy bioengineering Co., ltd.) using an electrochemical luminescence kit of the company (see the company patent CN111044719a for kit preparation). Background signals are typically measured using horse serum or other blank buffers, and positive signals are clinical serum assays containing higher concentrations of the test substance.
The background signal of the electrochemiluminescence and the change of the positive signal level can be clearly judged through the measurement. The effect of specific nickel terpyridyl on background signal reduction is shown in table 1:
TABLE 1
From the table above, the addition of nickel terpyridyl to the beads at a suitable concentration can significantly reduce the background signal of the kit. While the positive signal is substantially unaffected, thereby significantly improving the signal-to-noise ratio. In other analogues, tris (2, 2' -bipyridine) ruthenium dichloride has the capability of chemiluminescence, and the addition of the component improves the background signal. The results also reflect the tendency of the ruthenium terpyridyl structure to do non-specific binding to the magnetic beads. However, the other analogues of cobalt (II) tris (2, 2' -bipyridyl) have a certain background reducing effect, but the effect is not as obvious as that of nickel terpyridyl.
And tris (2, 2' -bipyridyl) cobalt (II) is much more expensive than nickel terpyridyl. Nickel terpyridyl is therefore preferred as a means of reducing the background signal. The addition of the other bipyridine compounds has no obvious influence on the background signal and the positive signal of the kit, which indicates that the combination of the ruthenium bipyridine and the magnetic beads is not caused by the bipyridine part, but the combination of the metal complex as a whole and the magnetic beads, and the charge and the special structure brought by the metal ions can play a relatively large role.
Example 2
The effect of nickel terpyridyl on reducing background signal in the kit with other multiple indexes is verified, and specifically as shown in table 2, the effect of nickel terpyridyl on reducing background signal in different indexes (concentration 10 ppm) is shown.
TABLE 2
From the above results, it is known that adding a certain amount of nickel terpyridyl (final concentration of 10 ppm) to the magnetic bead component (RM) can effectively reduce the background signal of the electrochemiluminescence kit, thereby improving the signal-to-noise ratio of the reagent and improving the sensitivity of the reagent.
The addition of the terpyridyl nickel has no obvious influence on positive signals, stability and other aspects of the kit. In addition to the magnetic bead component, R1 (primary antibody solution) and R2 (secondary antibody solution) have similar effects. Since the components of R1, R2 are generally complex and the stability of the antibody may be affected by additives, the addition to the magnetic bead component is also recommended.
The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Further, it will be understood that various changes or modifications may be made to the present application by those skilled in the art after reading the foregoing teachings, and equivalents thereof will be within the scope of the present application. It should also be understood that those skilled in the art, based on the technical solutions provided in the present application, can obtain technical solutions through logical analysis, reasoning or limited experiments, all fall within the protection scope of the claims attached to the present application. The scope of the patent application is therefore intended to be indicated by the appended claims, and the description may be used to interpret the contents of the claims.
Claims (7)
1. A method for reducing the background signal of electrochemiluminescence detection is characterized by comprising the step of adding an antibody competitor into a detection system in the electrochemiluminescence detection process;
the antibody competitor comprises nickel tris (2, 2' -bipyridine) chloride;
reagents used for the electrochemiluminescence detection include terpyridyl ruthenium labeled antibodies and magnetic bead solutions.
2. The method of claim 1, wherein the antibody competitor comprises nickel tris (2, 2 '-bipyridine) chloride, the final concentration of the nickel tris (2, 2' -bipyridine) chloride in the magnetic bead solution being 1ppm to 100ppm.
3. The method of claim 1, wherein the magnetic beads comprise streptavidin-coated magnetic beads.
4. A method according to claim 3, wherein the concentration of the streptavidin-coated magnetic beads is 0.5mg/ml to 1.0mg/ml.
5. The method of any one of claims 1-4, wherein the species source of the ruthenium terpyridine labeled antibody is at least one of a mouse, a rabbit, and a sheep.
6. A kit for reducing the background signal of electrochemiluminescence detection, comprising an antibody competitor as defined in any one of claims 1 to 5 and a chemiluminescent detection reagent.
7. Use of the kit of claim 6 for reducing the background signal value of an electrochemiluminescent kit.
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