CN113125415A - Competitive homogeneous phase chemiluminescence detection method and application thereof - Google Patents
Competitive homogeneous phase chemiluminescence detection method and application thereof Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention relates to a competitive homogeneous phase chemiluminescence detection method, which comprises the following steps: contacting the analyte with a reagent containing a first composition and a second composition, a third composition and a fourth composition to form a mixture to be tested; providing exciting light to irradiate the mixture to be detected at least once; the intensity of the chemiluminescent signal produced thereby is then detected to determine the presence and/or concentration of the analyte. The method utilizes 'differential receptor microspheres', so that the method has excellent functional sensitivity and detection range at the same time.
Description
Technical Field
The invention belongs to the technical field of homogeneous phase chemiluminescence detection, and particularly relates to a competitive homogeneous phase chemiluminescence detection method and application thereof.
Background
Competitive immunoassay is a detection method for the quantitative analysis of small molecule haptens. Radioimmunoassay (RIA) was the first established competitive immunoassay and awarded the prize for Nobel's biomedicine in 1974. In radioimmunoassay, a competitive antigen (labeled antigen) labeled with a radionuclide and a limited amount of a specific antibody are contained, and an antigen to be tested in a specimen and a labeled antigen as a reagent are competitively bound to the specific antibody, respectively. Separating the bound label (B) from the free label (F) and determining the radioactivity (or intensity, counts per minute, CPM) of the bound label as an inverse proportional function of the antigen to be detected. A series of calibrators of known concentrations are used to obtain a mathematical functional relationship (calibration function, which can be simply understood as a calibration curve) for the calibrators. And (4) operating the unknown sample according to the same conditions of the calibrator, measuring the radioactivity, and obtaining the concentration value of the sample to be measured through the standard function.
In competitive immunoassays, the amount of competing antigen is directly related to the functional sensitivity of the competitive immunoassay. Furthermore, in competitive immunoassays, the selection of the appropriate specific antibody and the concentration used are also critical. The chemiluminescence analysis has good analysis performance, and the analysis specificity, the analysis sensitivity, the automatic operation and the like can better meet the clinical requirements. However, for some special indexes such as steroid hormones, both the functional sensitivity and the detection range are highly required, and the existing chemiluminescence immunoassay, electrochemiluminescence immunoassay, light-activated chemiluminescence immunoassay and the like have defects, so that the special requirements of the functional sensitivity and the analysis range cannot be effectively met. Therefore, there is a need for a chemiluminescence detection technique that can meet both functional sensitivity and analytical range requirements.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a competitive homogeneous chemiluminescence detection method, and the method has excellent functional sensitivity and detection range when used for detection.
To this end, the present invention provides in a first aspect a competitive homogeneous chemiluminescent assay comprising the steps of: contacting the analyte with a reagent containing a first composition and a second composition, a third composition and a fourth composition to form a mixture to be tested; providing exciting light to irradiate the mixture to be detected at least once; and then detecting the intensity of the chemiluminescent signal produced thereby to determine the presence and/or concentration of the analyte; wherein the content of the first and second substances,
the first composition comprises a first receptor and a first antibody or binding fragment thereof that binds thereto, the first antibody or binding fragment thereof being a detection antibody that specifically binds to an analyte;
the second composition comprises a second receptor and a second antibody or binding fragment thereof that binds to the second receptor, the second antibody or binding fragment thereof being a detection antibody that specifically binds to the analyte;
the receptor can react with singlet oxygen to generate chemiluminescence;
the third composition comprises a competing antigen that competes with the analyte for binding to the detection antibody, the competing antigen binding to one of the specific binding pair members;
the fourth composition comprises a reactive oxygen species-generating donor that binds to the other member of the specific binding pair member;
the first antibody or binding fragment thereof specifically binds to the analyte with a higher affinity than the second antibody or binding fragment thereof specifically binds to the analyte; at the same time, the user can select the desired position,
the mass ratio of the first antibody or binding fragment thereof to the first receptor is lower than the mass ratio of the second antibody or binding fragment thereof to the second antibody.
In some embodiments of the invention, the mass ratio of the first antibody or binding fragment thereof to the first receptor is selected from 1 (100-1000), preferably from 1 (200-800), and more preferably from 1 (300-600).
In other embodiments of the invention, the concentration of the first composition in the agent is higher than the concentration of the second composition in the agent.
In some preferred embodiments of the present invention, the ratio of the mass concentration of the first composition in the reagent to the mass concentration of the second composition in the reagent is (2-50): 1, preferably (2-25): 1, more preferably (2-10): 1.
In some embodiments of the present invention, the mass concentration of the first composition in the reagent is 5-500 ug/ml, preferably 10-250 ug/ml, and more preferably 15-200 ug/ml.
In some embodiments of the invention, the first composition and the second composition are dispersed separately in the same buffer.
In other embodiments of the invention, the first composition and the second composition are mixed and dispersed in a buffer to assemble a reagent.
In some embodiments of the present invention, the first receptor and the second receptor are both receptor microspheres comprising a polymeric carrier, and the average particle size of the first receptor microspheres is the same as the average particle size of the second receptor microspheres.
In other embodiments of the present invention, the average particle size of the first acceptor microspheres is the same as the average particle size of the second acceptor microspheres.
In some embodiments of the invention, the analyte is a small molecule antigen or hapten.
In some embodiments of the invention, the competing antigen is an analyte or analyte analog; analyte analogs are preferred.
In some embodiments of the invention, the analyte is contacted with a reagent comprising a first composition and a second composition, a third composition, and then a fourth composition is added thereto.
In some embodiments of the invention, the analyte is contacted with the reagents comprising the first and second compositions, the third composition and the fourth composition, and incubated at 30-40 ℃ for 1-15 min to form a test mixture.
In a second aspect the present invention provides the use of a method according to the first aspect of the invention in a chemiluminescent analyzer.
The invention has the beneficial effects that: the method of the invention realizes the selective play of the two antibodies with different affinities according to the concentration difference of the antigen to be detected by selecting the antibodies with different affinities and coupling the receptor microspheres with different mass ratios respectively and mixing the two receptor microspheres with a proper proportion, thereby widening the detection range and preventing the occurrence of the hook effect while ensuring the functional sensitivity. In addition, to further improve functional sensitivity, an analog that is structurally similar to the analyte is selected as the competitive antigen marker biotin, ensuring that the analyte is able to preferentially bind to the specific antibody.
Drawings
The invention will be further explained with reference to the drawings.
FIG. 1 is a schematic diagram of the detection of the method of the present invention; wherein the reference numerals have the following meanings: 1, a small amount of first antibody with high affinity is coated on the surface of the first receptor microsphere, but the concentration of the first receptor microsphere is higher, so that the first receptor microsphere can play a role preferentially when a low-concentration E2 sample is detected; 2, a second receptor microsphere and a second antibody combined with the second receptor microsphere, wherein the surface of the second receptor microsphere is coated with more second antibodies with low affinity, but the concentration of the second receptor microsphere is lower, so that the second receptor microsphere can play a role preferentially when a high-concentration E2 sample is detected; 3E 2 bound to biotin; 4E 2 (estradiol) to be tested.
Fig. 2 is a plot of the correlation between the E2 sample and the control kit in example 5, where n is 133.
Detailed Description
In order that the invention may be readily understood, a detailed description of the invention is provided below. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Term (I)
The term "homogeneous" as used herein is defined in english as "homogeneous" and means that the bound antigen-antibody complex and the remaining free antigen or antibody are detected without separation.
The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure.
The term "donor microsphere" as used herein refers to a sensitizer capable of generating a reactive intermediate, such as singlet oxygen, upon activation by energy or an active compound, which reacts with the acceptor microsphere. The donor microspheres may be light activated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor microspheres are polymeric microspheres filled with a photosensitizer, which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. patent No. 5709994, which is incorporated herein by reference in its entirety, and derivatives of these compounds having 1-50 atom substituents that are used to render these compounds more lipophilic or more hydrophilic and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as those described in US patent No. US6406913, which is incorporated herein by reference.
The term "acceptor microsphere" as used herein refers to a compound that is capable of reacting with singlet oxygen to produce a detectable signal. The donor microsphere is induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is captured by a nearby acceptor microsphere, thereby transferring energy to activate the acceptor microsphere. In some embodiments of the present invention, the acceptor microsphere comprises a luminescent composition and a matrix, wherein the luminescent composition is filled in the matrix and/or coated on the surface of the matrix. The "matrix" according to the present invention is microspheres or microparticles known to the skilled person, of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which have any density, but preferably have a density close to that of water, preferably are capable of floating in water, and which are made of a transparent, partially transparent or opaque material. The substrate may or may not have a charge, and when charged, is preferably negatively charged. The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes.
The term "biotin" is widely present in animal and plant tissues, and has two cyclic structures on the molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main part bound with streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known to include proteins, nucleic acids, polysaccharides, lipids, and the like, mediated by a protein crosslinking agent. The "avidin" molecule consists of 4 identical peptide chains, each of which is capable of binding a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby creating a "tentacle effect" that increases assay sensitivity.
The term "epitope" as used herein refers to a specific chemical group in an antigenic molecule that determines the specificity of an antigen. For proteins, an epitope is a specific amino acid sequence (linear epitope) or a spatial conformation (conformational epitope) composed of several specific amino acid sequences. An epitope is not only the minimal structural and functional unit of antibody binding, but is also the basic unit of recognition by lymphocyte (B cell) antigen receptors.
The term "monoclonal antibody" as used herein refers to an antibody prepared by hybridoma fusion technology, which is directed against a single epitope and has a single specificity and a completely uniform structure and function. Firstly, the monoclonal antibody has single specificity, so that cross reaction is avoided, and the specificity of the labeled immunoassay is improved. Secondly, the monoclonal antibody ensures continuous supply and small batch-to-batch variation, effectively reducing the batch-to-batch variation of the immunodiagnostic kit. Again, different monoclonal antibodies recognize different antigenic sites and display different avidity characteristics.
The term "differential receptor microspheres" as used herein refers specifically to receptor microspheres (FG) coupled with monoclonal antibodies of different affinities.
The term "functional sensitivity" refers to the lowest detection line, that is, the lowest content which can be detected by an analysis method after a sample with a known concentration is diluted by multiple proportions, and the precision in a batch cannot be more than 20%. Analytical sensitivity is the true assay gain, also referred to as "functional sensitivity".
The term "detection range" as used herein refers to the effective range of the dose function, e.g., the dilution of a high concentration standard by a multiple ratio, the linear regression analysis of the measurement results of the diluted samples, and the correlation coefficient (R) is greater than 0.990.
Detailed description of the preferred embodiments
The present invention will be described in detail below.
For competitive immunoassays, to obtain a good competitive calibration function (which can be simply understood as a calibration curve), two basic conditions need to be met: firstly, the competitive antigen is homologous with the antigen to be detected and has the same or similar affinity with the specific antibody; secondly, the antibody limiting principle is ensured, and the dosage of the specific antibody is required to be less than the cumulative dosage of the antibodies required by the two antigens, but is required to be more than the cumulative dosage of the antibodies required by the competitive antigen or the antigen to be detected. The competitive homogeneous phase chemiluminescence detection method for quantitatively detecting the content of the analyte is obtained based on a light-activated chemiluminescence technology, and the analysis performance index of the method can meet the industry standard or the basic requirement of a clinical laboratory. The main points are as follows: two antibodies with different affinities for the analyte are selected to be respectively coupled with the receptor microsphere, so that the measured value conformance of a high-end sample and a low-end sample can be improved. In addition, by selecting an analogue having a similar structure to the analyte as a competitive antigen labeled biotin, it is ensured that the analyte to be detected can preferentially bind to a specific antibody, further improving functional sensitivity.
Accordingly, the present invention relates to a competitive homogeneous chemiluminescent assay according to the first aspect comprising the steps of: contacting the analyte with a reagent containing a first composition and a second composition, a third composition and a fourth composition to form a mixture to be tested; providing exciting light to irradiate the mixture to be detected at least once; and then detecting the intensity of the chemiluminescent signal produced thereby to determine the presence and/or concentration of the analyte; wherein the content of the first and second substances,
the first composition comprises a first receptor and a first antibody or binding fragment thereof that binds thereto, the first antibody or binding fragment thereof being a detection antibody that specifically binds to an analyte;
the second composition comprises a second receptor and a second antibody or binding fragment thereof that binds to the second receptor, the second antibody or binding fragment thereof being a detection antibody that specifically binds to the analyte;
the receptor can react with singlet oxygen to generate chemiluminescence;
the third composition comprises a competing antigen that competes with the analyte for binding to the detection antibody, the competing antigen binding to one of the specific binding pair members (e.g., biotin);
the fourth composition comprises a reactive oxygen species-generating donor that binds to the other member of the specific binding pair member (e.g., avidin);
the first antibody or binding fragment thereof specifically binds to the analyte with a higher affinity than the second antibody or binding fragment thereof specifically binds to the analyte; at the same time, the user can select the desired position,
the mass ratio of the first antibody or binding fragment thereof to the first receptor is lower than the mass ratio of the second antibody or binding fragment thereof to the second antibody. I.e., the amount of the first antibody or binding fragment thereof conjugated to the first receptor is less than the amount of the second antibody or binding fragment thereof conjugated to the second receptor.
In the present invention, the receptor is capable of binding the analyte via the first antibody or binding fragment thereof and/or the second antibody or binding fragment thereof.
In some embodiments of the invention, the first antibody and the second antibody are both monoclonal antibodies that specifically bind to the analyte.
In some embodiments of the invention, the mass ratio of the first antibody or binding fragment thereof to the first receptor is selected from 1 (100-1000), preferably from 1 (200-800), and more preferably from 1 (300-600). In some embodiments of the invention, the mass ratio of the first antibody or binding fragment thereof to the first receptor is 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, or 1:1000, etc.
In some embodiments of the invention, the concentration of the first composition in the agent is higher than the concentration of the second composition in the agent. In the present invention, the concentration may be a mass concentration or a molar concentration.
In some preferred embodiments of the present invention, the ratio of the concentration of the first composition in the reagent to the mass concentration of the second composition in the reagent is (2-50): 1, preferably (2-25): 1, more preferably (2-10): 1. In some embodiments of the invention, the ratio of the concentration of the first composition in the kit to the concentration of the second composition in the kit is 2:1, 2.5:1, 5:1, 10:1, 20:1, 30:1, 40:1, or 50:1, etc.
In some embodiments of the present invention, the mass concentration of the first composition in the reagent is 5-500 ug/ml, preferably 10-250 ug/ml, and more preferably 15-200 ug/ml.
In some embodiments of the invention, the first composition and the second composition are dispersed separately in the same buffer.
In other embodiments of the invention, the first composition and the second composition are mixed and dispersed in a buffer to assemble a reagent (i.e., reagent 1).
In some embodiments of the present invention, the first receptor and the second receptor are both receptor microspheres comprising a polymeric carrier, and the average particle size of the first receptor microspheres is the same as the average particle size of the second receptor microspheres. It is worth noting that: the "receptor" of the present invention may include not only polymeric microspheres but also microspheres such as magnetic particles.
In other embodiments of the present invention, the average particle size of the first acceptor microspheres is the same as the average particle size of the second acceptor microspheres.
In some embodiments of the invention, the analyte is a small molecule antigen or hapten.
In some embodiments of the invention, the competing antigen is an analyte or analyte analog; analyte analogs are preferred. In the present invention, the specific binding capacity of the analyte analogue to the detection antibody is lower than that of the analyte to the detection antibody, that is, preferably, the specific binding capacity of the competitive antigen to the detection antibody is lower than that of the antigen to be detected to the detection antibody, so that the functional sensitivity can be further improved. In the present invention, the reagent containing the third composition is also referred to as reagent 2.
In some embodiments of the invention, the analyte is contacted with a reagent comprising a first composition and a second composition, a third composition, and then a fourth composition is added thereto.
In some embodiments of the invention, the analyte is contacted with the release agent, the reagent comprising the first composition and the second composition, the third composition, and then the fourth composition is added thereto.
In some preferred embodiments of the invention, the release agent is selected from one or more of dihydrotestosterone, mesterone, danazol and diethylstilbestrol.
In some embodiments of the invention, the analyte is contacted with the reagents comprising the first and second compositions, the third composition and the fourth composition, and incubated at 30-40 ℃ for 1-15 min to form a test mixture.
For both extreme samples, the first antibody-coupled first receptor microsphere and the second antibody-coupled second receptor microsphere function differently: aiming at a low-concentration sample to be detected, the antibody on the surface of the antigen-binding microsphere to be detected depends on the concentration of the receptor microsphere, namely the concentration of the first receptor microsphere coupled with the first antibody is high, and the microsphere is dominant; aiming at a high-concentration sample to be detected, the antibody combined with the surface of the microsphere by the antigen to be detected does not depend on the concentration of the microsphere any more, but depends on the molecular number of the antibody on the surface of the microsphere, although the concentration of the receptor microsphere coupled by the second antibody is low, the molecular number of the antibody on the surface of the microsphere is large, more antibody molecules are needed for the antigen to be detected at high concentration, and the second receptor microsphere coupled by the second antibody plays a role in determination.
Based on the analysis, the invention adopts antibodies with different affinities to prepare differential receptor microspheres in different modes (coupling mass ratio and/or coupling mode). The differential receptor microspheres are mixed according to a certain proportion to be used as a single solution (reagent 1), two receptor microspheres with different properties in the solution can selectively play a role according to the concentration difference of the antigen to be detected in a sample by utilizing the microsphere liquid phase diffusion principle and the monoclonal antibody affinity difference, and the special requirements of clinical application on functional sensitivity and detection range are met.
In addition, by selecting the analogue with the structure similar to that of the analyte as the competitive antigen labeled biotin, the binding capacity of the competitive antigen and the detection antibody on the receptor microsphere is lower than that of the analyte and the detection antibody on the receptor microsphere, so that the analyte to be detected can be ensured to be preferentially bound with the detection antibody, and the functional sensitivity is further improved.
In some embodiments of the invention, a method for detecting an analyte using the method comprises:
step N1, mixing a sample to be detected, a reagent 1 and a reagent 2 to obtain a first mixture;
step N2, mixing the donor microsphere solution combined with the avidin with the first mixture to obtain a second mixture;
step N3, exciting the donor microsphere in the second mixture with energy or an active compound to generate active oxygen, and reacting the acceptor microsphere with the active oxygen to generate a chemiluminescent signal;
and step N4, detecting the intensity of the chemiluminescence signal in the step N3, and analyzing whether the analyte exists in the sample to be detected and/or the concentration of the analyte.
In the method of the present invention, the reagents may be mixed and incubated as necessary.
In some embodiments of the invention, the method further comprises the step of creating a standard curve of chemiluminescent signal versus analyte concentration using a series of calibrator solutions of known analyte concentration; the standard curve is used for determining the content of the analyte in the sample to be detected.
In other embodiments of the present invention, in step N3, the second mixture is irradiated with excitation light with a wavelength of 600-700nm to excite the donor microsphere in the second mixture to generate active oxygen, and then the acceptor microsphere reacts with the active oxygen in contact therewith to generate emission light with a wavelength of 520-620 nm.
Mixing and incubating a serum sample to be detected, the reagent 1 and the reagent 2, binding the antigen to be detected and a Bio-competitive antigen in the serum sample competitively with a detection antibody (FG-Ab) on an acceptor microsphere to respectively form a complex (FG-Ab-competitive antigen-Bio and FG-Ab-antigen to be detected), then binding SA-GG (donor microsphere bound with avidin) and biotin (Bio), enabling the acceptor microsphere and the donor microsphere to be close to each other, and inducing the generation of a light signal after excitation. Free receptor particles do not gain energy and no light signal is generated. Because the invention adopts a competitive analysis mode, the optical signal intensity and the content of the antigen to be detected in the serum sample to be detected are in an inverse proportional function relationship, and the concentration level of the antigen to be detected in the unknown serum sample can be calculated through a mathematical function formed by the analyte calibration product with the known concentration.
In a second aspect the present invention provides the use of a method according to the first aspect of the invention in a chemiluminescent analyzer.
Examples
In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.
Example 1: preparation of reagents for use in the methods of the invention for which the analyte is estradiol (E2)
(1) Preparation process of monoclonal antibody coupled receptor microsphere solution (reagent 1)
Acceptor microspheres: the surface of the microsphere contains aldehyde group (-CHO), and the microsphere is connected with antibody molecules through the aldehyde group. Chelate compounds containing a luminescent compound (derivative of dimethylthiophene) and a lanthanide (Eu) compound.
Biological raw materials: a high affinity monoclonal antibody that specifically binds to E2 (i.e., HA-McAb) and a low affinity monoclonal antibody that specifically binds to E2 (i.e., LA-McAb).
The preparation process comprises the following steps: the preparation process comprises the following steps:
1) dialyzing a monoclonal antibody (namely HA-McAb) which is specifically bound with E2 and HAs high affinity with a carbonate buffer solution overnight, mixing the monoclonal antibody with a receptor microsphere (FG) at a mass ratio of 1:400, coating for 2 hours, adding a sealing solution, sealing for 1 hour, and preparing a concentrated solution containing FG-HA-McAb-n for later use;
2) dialyzing a monoclonal antibody (namely LA-McAb) which is specifically bound with E2 and HAs low affinity with E2 with carbonate buffer solution overnight, mixing the monoclonal antibody with receptor microspheres (FG) at a mass ratio of 1:80, coating for 2 hours, adding a sealing solution, sealing for 1 hour, and preparing a concentrated solution containing FG-HA-McAb-N for later use;
3) FG-HA-McAb-n is diluted according to the ratio of 1:200 by using a reagent 1 diluent and is numbered as R1-1; FG-LA-McAb-N is diluted according to the ratio of 1:500 by using a reagent 1 diluent and is numbered as R1-2;
4) and mixing the two solutions R1-1 and R1-2 in equal volume to obtain a reagent 1.
(2) Process for preparing competitive antigen (reagent 2) binding to biotin
1ug/ml of Bio-E2 was diluted at a ratio of 1:10000 with a diluent of reagent 2 to prepare an R2 working solution as reagent 2.
The reagent 2 can also be diluted by Bio-E3 of 1ug/ml of reagent 2 diluent slurry according to the ratio of 1:8000 to prepare R2 working solution which is used as the reagent 2.
(3) Preparation of the Release agent
The pure mesterone product is prepared into 100ng/L by using 0.1M phosphate buffer saline solution with pH 7.4 and containing 20 percent inactivated calf serum.
(4) Preparation process of E2 series calibration product with known concentration
Taking the E2 pure product, and preparing 0.5ml of each 0-4800 ng/L series of calibrator solutions by using 0.1M phosphate buffered saline solution with pH 7.4 and containing 20% inactivated calf serum.
Example 2:
the method in example 1 was used to change the antibody type, the coupling mass ratio, the competitive antigen type, and other conditions, and the LiCA500 automated light-activated chemiluminescence analysis system was used to detect the same batch of samples, automatically complete and output homogeneous chemiluminescence signals, and analyze the detection range and detection limit of the detection results.
The detection process using the reagent prepared in example 1 is fully automated by an automatic light-activated chemiluminescence analysis system using LiCA500 and the detection result is output, and the specific steps are as follows:
a. respectively adding 10 mul of sample, calibrator or quality control material into the reaction hole;
b. adding 25 mul of releasing agent, 25 mul of reagent 1 and 25 mul of reagent 2 into the reaction hole in sequence;
c.37 ℃ temperature 15 minutes;
d. add LiCA universal solution (donor microsphere solution combined with avidin) 175. mu.l;
e.37 ℃ temperature 15 minutes;
e. irradiating the micropores by laser and calculating the quantity of light photons emitted by each hole;
f. from the calibration curve, the sample concentration was calculated. The results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the results of coupling the two affinity antibodies to the receptor microsphere are that the lower the mass ratio of the antibody to the receptor microsphere, the better the detection result. When only the receptor microsphere coupled by the high-affinity antibody (HA-McAb) is used for detection, the low value and the detection limit of the linear range are superior to those of the receptor microsphere coupled by only the low-affinity antibody (LA-McAb), and the detection rate can reach 10-20 ng/L; however, the upper limit of the linear range of the receptor microsphere coupled with the low-affinity antibody (LA-McAb) is better than that of the receptor microsphere coupled with the high-affinity antibody (HA-McAb), and the upper limit can reach more than 5000 ng/L. When the high-affinity antibody receptor microsphere and the low-affinity antibody receptor microsphere with the appropriate coupling mass ratio are mixed according to the equal volumes of 1:200 and 1:500, the upper limit, the lower limit and the detection limit of the linear range can reach the optimal result. In addition, the use of the Bio-E3 antigen as a competitive antigen, which is less competitive than the Bio-E2 antigen, is more favorable for the binding of E2 in the sample to the detection antibody, so that the functional sensitivity (detection limit) of detection using the Bio-E3 antigen as a competitive antigen is slightly better than that of detection using the Bio-E2 antigen as a competitive antigen.
Example 3:
the precision of the method using the reagent described in test No. 7 in example 2, in which Bio-E2 was used as a competitive antigen, was examined.
The significance of precision is as follows: the precision is an important index for measuring the variation of the kit between batches, is an important basis for evaluating the effectiveness of the products to be marketed, and generally comprises the intra-batch precision and the inter-batch precision.
The evaluation method of the precision in the batch comprises the following steps: independent analysis of 1 batch of product was performed using low (L), medium (M), and high (H) value samples, the assay was repeated 10 times for each batch, and the average of 10 measurements was calculated
And Standard Deviation (SD), according to the formula
The Coefficient of Variation (CV) was calculated, and the results are shown in table 1.
The method for evaluating the batch precision comprises the following steps: independent analysis of 3 batches of product was performed using low (L), medium (M) and high (H) value samples, the measurements were repeated 20 times for each batch, and the average of the measurements was calculated
And Standard Deviation (SD), according to the formula
The Coefficient of Variation (CV) was calculated, and the results are shown in table 2.
Table 2: test results
As can be seen from Table 2, the precision of the three reagent batches was < 10%, indicating that the measured values of the method have good repeatability and small random errors.
Example 4:
the accuracy of the method using the reagent described in test No. 7 in example 2, in which Bio-E2 was used as a competitive antigen, was examined
The accuracy significance is as follows: the coincidence degree of the measured value and the actual value reflects the magnitude of the system error.
The accuracy evaluation method comprises the following steps: the results of the multi-point dilution of 2 samples containing different levels of E2 with the calibrator matrix solution, the concentration measurement of the diluted samples by the method described in example 2, and the recovery of 2 samples were calculated based on the dilution ratio, respectively, are shown in tables 3 and 4, respectively.
TABLE 3
TABLE 4
As can be seen from tables 3 and 4, the recovery rates were all in the range of 90% to 110% after multi-point dilution with 2 samples of E2 at different levels, indicating that the measured values are close to the actual values and the detection error of the method is small.
Example 5:
the E2 sample was tested by the method using the reagent described in test No. 7 of example 2, in which Bio-E3 was used as a competitive antigen, and the results were compared with those of the same type of imported kit, and the results are shown in FIG. 2.
As can be seen from fig. 2, the correlation r between the measured value of the E2 sample and the measured value of the similar imported kit in the method of the present invention is 0.9888, which is good. The method can accurately detect the content of the estradiol hormone in the sample.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Claims (14)
1. A competitive homogeneous chemiluminescent assay comprising the steps of: contacting the analyte with a reagent containing a first composition and a second composition, a third composition and a fourth composition to form a mixture to be tested; providing exciting light to irradiate the mixture to be detected at least once; and then detecting the intensity of the chemiluminescent signal produced thereby to determine the presence and/or concentration of the analyte; wherein the content of the first and second substances,
the first composition comprises a first receptor and a first antibody or binding fragment thereof that binds thereto, the first antibody or binding fragment thereof being a detection antibody that specifically binds to an analyte;
the second composition comprises a second receptor and a second antibody or binding fragment thereof that binds to the second receptor, the second antibody or binding fragment thereof being a detection antibody that specifically binds to the analyte;
the receptor can react with singlet oxygen to generate chemiluminescence;
the third composition comprises a competing antigen that competes with the analyte for binding to the detection antibody, the competing antigen binding to one of the specific binding pair members;
the fourth composition comprises a reactive oxygen species-generating donor that binds to the other member of the specific binding pair member;
the first antibody or binding fragment thereof specifically binds to the analyte with a higher affinity than the second antibody or binding fragment thereof specifically binds to the analyte; at the same time, the user can select the desired position,
the mass ratio of the first antibody or binding fragment thereof to the first receptor is lower than the mass ratio of the second antibody or binding fragment thereof to the second antibody.
2. The method of claim 1, wherein the mass ratio of the first antibody or binding fragment thereof to the first receptor is selected from 1 (100-1000), preferably from 1 (200-800), and more preferably from 1 (300-600).
3. The method according to claim 1 or 2, wherein the concentration of the first composition in the reagent is higher than the concentration of the second composition in the reagent.
4. The method according to any one of claims 1 to 3, wherein the ratio of the mass concentration of the first composition in the reagent to the mass concentration of the second composition in the reagent is (2-50): 1, preferably (2-25): 1, more preferably (2-10): 1.
5. The method according to any one of claims 1 to 4, wherein the mass concentration of the first composition in the reagent is 5 to 500ug/ml, preferably 10 to 250ug/ml, more preferably 15 to 200 ug/ml.
6. The method of any one of claims 1-5, wherein the first composition and the second composition are separately dispersed in the same buffer.
7. The method of any one of claims 1-5, wherein the first composition and the second composition are mixed and dispersed in a buffer to assemble a reagent.
8. The method of any one of claims 1-7, wherein the first receptor and the second receptor are both receptor microspheres comprising a polymeric support, and wherein the average particle size of the first receptor microspheres is the same as the average particle size of the second receptor microspheres.
9. The method of claim 8, wherein the average particle size of the first acceptor microsphere is the same as the average particle size of the second acceptor microsphere.
10. A method according to any one of claims 1 to 9, wherein the analyte is a small molecule antigen or hapten.
11. The method of any one of claims 1 to 10, wherein the competing antigen is an analyte or analyte analogue; analyte analogs are preferred.
12. A method according to any one of claims 1 to 11 wherein the analyte is contacted with the reagent comprising the first and second compositions, the third composition and then the fourth composition is added thereto.
13. A method according to any one of claims 1 to 12, wherein the analyte is contacted with the reagents comprising the first and second compositions, the third and fourth compositions, and the test mixture is formed after incubation at 30 to 40 ℃ for 1 to 15 min.
14. Use of a method according to any one of claims 1 to 13 in a chemiluminescent analyzer.
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