Competitive homogeneous chemiluminescence assay kit and its application

Abstract

The present invention relates to a competitive homogeneous chemiluminescence assay kit, comprising: a first composition comprising a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to an analyte; a second composition comprising a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to an analyte, and wherein the receptor can react with singlet oxygen to generate chemiluminescence; and a third composition comprising a competing antigen that competes with estradiol for binding to the detection antibody, wherein the affinity of the first antibody or the binding fragment thereof for specific binding to the analyte is higher than the affinity of the second antibody or the binding fragment thereof for specific binding to the analyte; and, at the same time, the mass ratio of the first antibody or the binding fragment thereof to the first receptor is lower than the mass ratio of the second antibody or the binding fragment thereof to the second receptor.

Description

Technical Field

[0001] The invention belongs to the technical field of homogeneous chemiluminescence, and particularly relates to a competitive homogeneous chemiluminescence assay kit and application thereof. Background Art

[0002] Competitive immunoassay is a detection method used for the quantitative analysis of small molecule haptens. Radioimmunoassay (RIA) is the earliest established competitive immunoassay method and was awarded the 1974 Nobel Prize in Physiology or Medicine. In a radioimmunoassay, a competing antigen (labeled antigen) labeled with a radionuclide and a limited amount of specific antibody are contained. The antigen to be tested in the specimen and the labeled antigen as a reagent compete with the specific antibody for binding. The bound label (B) and the free label (F) are separated, and the radioactivity (or intensity, in counts per minute, CPM) of the bound label is measured. The radioactivity is inversely proportional to the antigen to be tested. A series of calibrators of known concentrations are used to obtain the mathematical function relationship of the calibrators (calibration function, which can be simply understood as a calibration curve). The unknown sample is operated under the same conditions as the calibrator, the radioactivity is measured, and the concentration value of the sample to be tested is then obtained using the standard function.

[0003] In competitive immunoassays, the amount of competing antigen used is directly related to the functional sensitivity of the competitive immunoassay. In addition, in competitive immunoassays, the selection of appropriate specific antibodies and the use concentration are also crucial. Chemiluminescence analysis has excellent analytical performance, and its analytical specificity, analytical sensitivity, and automated operation can better meet clinical requirements. However, for certain special indicators such as steroid hormones, there are very high requirements for functional sensitivity and detection range, and the existing chemiluminescence immunoassays, electrochemiluminescence immunoassays, photochemiluminescence immunoassays, etc. still have defects and cannot effectively take into account the special requirements of functional sensitivity and analysis range. Therefore, there is an urgent need for a chemiluminescence detection technology that can take into account the requirements of functional sensitivity and analysis range. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a competitive homogeneous chemiluminescence assay kit, which has excellent functional sensitivity and detection range when used for detection.

[0005] To this end, the first aspect of the present invention provides a competitive homogeneous chemiluminescence assay kit, comprising a first composition, a second composition, and a third composition, wherein the first composition comprises a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to the analyte; the second composition comprises a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to the analyte, wherein the receptor can react with singlet oxygen to produce chemiluminescence; and the third composition comprises a competing antigen that competes with estradiol for binding to the detection antibody;

[0006] The affinity of the first antibody or its binding fragment for specific binding to the analyte is higher than the affinity of the second antibody or its binding fragment for specific binding to the analyte; at the same time,

[0007] The mass ratio of the first antibody or its binding fragment to the first receptor is lower than the mass ratio of the second antibody or its binding fragment to the second receptor.

[0008] In some embodiments of the present invention, the mass ratio of the first antibody or its binding fragment to the first receptor is selected from 1:(100-1000), preferably selected from 1:(200-800), and more preferably selected from 1:(300-600).

[0009] In some embodiments of the invention, the concentration of the first composition in the kit is higher than the concentration of the second composition in the kit.

[0010] In some preferred embodiments of the present invention, the mass concentration ratio of the first composition in the kit to the mass concentration ratio of the second composition in the kit is (2-50):1, preferably (2-25):1, and more preferably (2-10):1.

[0011] In some specific embodiments of the present invention, the mass concentration of the first composition in the kit is 5 to 500 ug / ml, preferably 10 to 250 ug / ml, and more preferably 15 to 200 ug / ml.

[0012] In some embodiments of the present invention, the first composition and the second composition are separately dispersed in the same buffer.

[0013] In other embodiments of the present invention, the first composition and the second composition are mixed and dispersed in a buffer solution to assemble into a reagent.

[0014] In some embodiments of the present invention, the first receptor and the second receptor are both receptor microspheres containing a high molecular weight polymer carrier, and the average particle size of the first receptor microspheres is the same as the average particle size of the second receptor microspheres.

[0015] 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.

[0016] In some embodiments of the present invention, the analyte is a small molecule antigen or a hapten.

[0017] In some embodiments of the present invention, the competing antigen is the analyte or an analyte analog; preferably, it is an analyte analog; more preferably, the competing antigen binds to one of the specific binding pair members.

[0018] The second aspect of the present invention provides a use of the kit as described in the first aspect of the invention in a chemiluminescence analyzer.

[0019] The beneficial effects of the present invention are as follows: By selecting antibodies with different affinities and coupling them to receptor microspheres at different mass ratios, and then mixing the two receptor microspheres in an appropriate proportion, the kit achieves the selective effect of the two antibodies with different affinities depending on the concentration of the antigen to be detected. This ensures functional sensitivity while broadening the detection range. The kit is a homogeneous immunoassay, without any separation or washing steps, which not only saves detection time but also avoids errors caused by washing, and has high precision and accuracy. In addition, to further improve functional sensitivity, an analog with a similar structure to the analyte is selected as a competing antigen labeled with biotin to ensure that the analyte can preferentially bind to the specific antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below with reference to the accompanying drawings.

[0021] Figure 1 Schematic diagram of the detection principle of the method of the present invention; wherein, the meanings of the reference numerals are as follows: 1. First receptor microspheres and the first antibody bound thereto; the surface of the first receptor microspheres is coated with a small amount of high-affinity first antibody, but the concentration of the first receptor microspheres is relatively high, ensuring that the first receptor microspheres have priority when detecting low-concentration E2 samples; 2. Second receptor microspheres and the second antibody bound thereto; the surface of the second receptor microspheres is coated with a large amount of low-affinity second antibody, but the concentration of the second receptor microspheres is relatively low, ensuring that the second receptor microspheres have priority when detecting high-concentration E2 samples; 3. E2 bound to biotin; 4. E2 to be detected (estradiol).

[0022] Figure 2 This is a correlation diagram of the measured values ​​of the E2 sample and the measured values ​​of the comparison kit in Example 5, where n=133. DETAILED DESCRIPTION

[0023] To facilitate understanding of the present invention, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0024] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of the range and any other specified or intervening values ​​in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the present invention, subject to any express exclusions in the specified range. Where a specified range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the present invention.

[0025] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. 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, preferred methods and materials are now described.

[0026] I. Terminology

[0027] The term "homogeneous" as used in the present invention is defined in English as "homogeneous", which means that detection can be performed without separating the bound antigen-antibody complex from the remaining free antigen or antibody.

[0028] The term "specific binding" as used in the present invention refers to the mutual recognition and selective binding reaction between two substances, which refers to the conformational correspondence between the corresponding reactants from a three-dimensional structural perspective.

[0029] The term "donor microspheres" as used herein refers to sensitizers that can generate active intermediates such as singlet oxygen that react with acceptor microspheres after being activated by energy or active compounds. The donor microspheres can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.). In some specific embodiments of the present invention, the donor microspheres are polymer microspheres filled with photosensitizers. The photosensitizers can be photosensitizers known in the art, preferably compounds that are relatively stable to light and do not react effectively with singlet oxygen. Non-limiting examples include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll disclosed in U.S. Pat. No. 5,709,994 (which is incorporated herein by reference in its entirety), as well as derivatives of these compounds having 1-50 atom substituents, the substituents being used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups for attachment to specific binding pair members. Examples of other photosensitizers known to those skilled in the art can also be used in the present invention, such as those described in U.S. Pat. No. 6,406,913, which is incorporated herein by reference.

[0030] The term "acceptor microspheres" described in the present invention refers to compounds that can react with singlet oxygen to produce a detectable signal. The donor microspheres are activated by energy or active compounds and release high-energy singlet oxygen, which is captured by the acceptor microspheres in close proximity, thereby transferring energy to activate the acceptor microspheres. In some specific embodiments of the present invention, the acceptor microspheres contain a luminescent composition and a matrix, and the luminescent composition is filled in the matrix and / or coated on the surface of the matrix. The "matrix" described in the present invention is a microsphere or particle known to those skilled in the art, which can be of any size, can be organic or inorganic, can be expandable or non-expandable, can be porous or non-porous, and has any density, but preferably has a density close to that of water, preferably can float in water, and is composed of transparent, partially transparent or opaque materials. The matrix may or may not have an electric charge, and when it has an electric charge, it is preferably a negative charge. The matrix can 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.

[0031] The term "biotin," as used herein, is widely found in plant and animal tissues. Its molecule consists of two rings: an imidazolone ring and a thiophene ring. The imidazolone ring is the primary binding site for streptavidin. Activated biotin can be coupled to virtually all known biomacromolecules, including proteins, nucleic acids, polysaccharides, and lipids, using protein crosslinkers. Avidin molecules consist of four identical peptide chains, each capable of binding to a single biotin molecule. Therefore, each antigen or antibody can be coupled to multiple biotin molecules simultaneously, creating a "tentacle effect" that enhances analytical sensitivity.

[0032] As used herein, the term "epitope" refers to a specific chemical group within an antigen molecule that determines its specificity. For proteins, an epitope is a specific amino acid sequence (linear epitope) or a spatial conformation of several specific amino acid sequences (conformational epitope). An epitope is not only the smallest structural and functional unit for antibody binding but also the fundamental unit of antigen receptor recognition on lymphocytes (B cells).

[0033] The term "monoclonal antibody" as used herein refers to an antibody produced using hybridoma fusion technology that targets a single antigenic epitope, possesses a single specificity, and is completely homogeneous in structure and function. First, the single specificity of monoclonal antibodies eliminates cross-reactivity and improves the specificity of labeled immunoassays. Second, monoclonal antibodies ensure a continuous supply and minimize batch-to-batch variability, effectively reducing batch-to-batch variability in immunodiagnostic kits. Third, different monoclonal antibodies recognize different antigenic sites and exhibit varying affinity characteristics.

[0034] The term "differential receptor microspheres" described in the present invention specifically refers to receptor microspheres (FG) coupled with monoclonal antibodies of different affinities.

[0035] The term "functional sensitivity" as used herein refers to the minimum detection limit, i.e., the lowest concentration that can be detected by an analytical method after serial dilution of a sample of known concentration, with an intra-batch precision of no greater than 20%. Analytical sensitivity is obtained through actual measurement and is also referred to as "functional sensitivity."

[0036] The term "detection range" used in the present invention refers to the effective range of the dose function, such as when a high-concentration sample is diluted in multiples, the correlation coefficient (R) of the linear regression analysis of the measurement results of the diluted samples is greater than 0.990.

[0037] II. Specific implementation plan

[0038] The present invention will be described in detail below.

[0039] Regarding competitive immunoassay, in order to obtain a high-quality competitive calibration function (which can be simply understood as a calibration curve), it is necessary to meet two basic conditions: first, the competing antigen and the antigen to be detected are homologous and have the same or similar affinity as the specific antibody; second, to ensure the principle of antibody limit, the amount of specific antibody used needs to be less than the cumulative amount of antibodies required for the two antigens, but must be greater than the cumulative amount of antibodies required for the competing antigen or the antigen to be detected. The present invention is based on photochemiluminescence technology to obtain a competitive homogeneous chemiluminescence assay kit for quantitatively detecting the content of the analyte by photochemical method. The analytical performance indicators of the kit can meet the basic requirements of industry standards or clinical laboratories. It is mainly manifested in: selecting two antibodies with different affinities for the analyte to couple the receptor microspheres respectively, which can improve the measurement compliance of high-end samples and low-end samples. In addition, by selecting an analogue with a similar structure to the analyte as a competing antigen to label biotin, it is ensured that the analyte to be tested can preferentially bind to the specific antibody, thereby further improving the functional sensitivity.

[0040] Therefore, the competitive homogeneous chemiluminescence assay kit involved in the first aspect of the present invention comprises a first composition, a second composition, and a third composition. The first composition comprises a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to the analyte; the second composition comprises a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to the analyte, wherein the receptor is capable of reacting with singlet oxygen to produce chemiluminescence; and the third composition comprises a competing antigen that competes with estradiol for binding to the detection antibody.

[0041] The affinity of the first antibody or its binding fragment for specific binding to the analyte is higher than the affinity of the second antibody or its binding fragment for specific binding to the analyte; at the same time,

[0042] The mass ratio of the first antibody or its binding fragment to the first receptor is lower than the mass ratio of the second antibody or its binding fragment to the second receptor. In other words, the amount of the first antibody or its binding fragment coupled to the first receptor is lower than the amount of the second antibody or its binding fragment coupled to the second receptor.

[0043] In the present invention, the receptor is capable of binding to the analyte via the first antibody or its binding fragment and / or the second antibody or its binding fragment.

[0044] In some embodiments of the present invention, both the first antibody and the second antibody are monoclonal antibodies that specifically bind to the analyte.

[0045] In some embodiments of the present invention, the mass ratio of the first antibody or its binding fragment to the first receptor is selected from 1:(100-1000), preferably selected from 1:(200-800), and more preferably selected from 1:(300-600). In some specific embodiments of the present invention, the mass ratio of the first antibody or its binding fragment 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.

[0046] In other embodiments of the present invention, the concentration of the first composition in the kit is higher than the concentration of the second composition in the kit. In the present invention, the concentration can be either mass concentration or molar concentration.

[0047] In some preferred embodiments of the present invention, the ratio of the mass concentration of the first composition in the kit to the mass concentration of the second composition in the kit is (2-50):1, preferably (2-25):1, more preferably (2-10):1. In some specific embodiments of the present invention, the ratio of the mass concentration of the first composition in the kit to the mass 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.

[0048] In some specific embodiments of the present invention, the mass concentration of the first composition in the kit is 5 to 500 ug / ml, preferably 10 to 250 ug / ml, and more preferably 15 to 200 ug / ml.

[0049] In some embodiments of the present invention, the first composition and the second composition are separately dispersed in the same buffer.

[0050] In other embodiments of the present invention, the first composition and the second composition are mixed and dispersed in a buffer solution to assemble into a reagent (ie, reagent 1).

[0051] In some embodiments of the present invention, the first and second receptors are both receptor microspheres containing a polymer 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 "receptors" described in the present invention include not only polymer microspheres but also microspheres such as magnetic particles.

[0052] 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.

[0053] In some embodiments of the present invention, the analyte is a small molecule antigen or a hapten.

[0054] In some embodiments of the present invention, the competing antigen is an analyte or an analyte analog; preferably, it is an analyte analog; more preferably, the competing antigen binds to a member of a specific binding pair (e.g., biotin). In the present invention, the specific binding ability of the analyte analog to the detection antibody is lower than the specific binding ability of the analyte to the detection antibody, that is, preferably, the specific binding ability of the competing antigen to the detection antibody is lower than the specific binding ability of the antigen to be detected to the detection antibody, which can further improve functional sensitivity. In the present invention, the reagent comprising the third composition is also referred to as reagent 2.

[0055] In other embodiments of the present invention, the kit further comprises a column calibrator solution with a known analyte concentration.

[0056] In some embodiments of the present invention, the kit further comprises a fourth composition comprising donor microspheres bound to the other member of the specific pair (eg, avidin).

[0057] In some preferred embodiments of the present invention, the kit further comprises a releasing agent; preferably, the releasing agent is selected from one or more of dihydrotestosterone, mesterolone, danazol and diethylstilbestrol.

[0058] For the two extreme samples, the first receptor microspheres coupled to the first antibody and the second receptor microspheres coupled to the second antibody work differently: for low-concentration test samples, the antibodies on the surface of the antigen-binding microspheres depend on the concentration of the receptor microspheres, that is, the concentration of the first receptor microspheres coupled to the first antibody is high, and this type of microspheres is dominant; for high-concentration test samples, the antibodies on the surface of the antigen-binding microspheres no longer depend on the concentration of the microspheres, but on the number of antibody molecules on the surface of the microspheres. Although the concentration of the receptor microspheres coupled to the second antibody is low, the number of antibody molecules on the surface of the microspheres is large. High-concentration test antigens require more antibody molecules, and at this time, the second receptor microspheres coupled to the second antibody play a decisive role.

[0059] Based on the above analysis, the present invention uses antibodies with different affinities and different methods (coupling mass ratio and / or coupling method) to prepare differential receptor microspheres. These differential receptor microspheres are mixed in a specific ratio to form a single solution (reagent 1). The two different receptor microspheres in this solution, utilizing the principles of microsphere liquid-phase diffusion and the differences in monoclonal antibody affinity, can selectively act based on the concentration of the target antigen in the sample, meeting the specific clinical requirements for functional sensitivity and detection range.

[0060] In addition, by selecting an analog with a similar structure to the analyte as a competing antigen labeled with biotin, the binding ability of the competing antigen to the detection antibody on the receptor microspheres is lower than the binding ability of the analyte to the detection antibody on the receptor microspheres, thereby ensuring that the analyte to be tested can preferentially bind to the detection antibody, further improving the functional sensitivity.

[0061] In some embodiments of the present invention, the method of detecting an analyte using the kit includes:

[0062] Step N1, mixing the sample to be tested, reagent 1, and reagent 2 to obtain a first mixture;

[0063] Step N2, mixing the donor microsphere solution bound to avidin with the first mixture to obtain a second mixture;

[0064] Step N3, using energy or an active compound to excite the donor microspheres in the second mixture to generate reactive oxygen species, and then the acceptor microspheres react with the reactive oxygen species to generate chemiluminescent signals;

[0065] Step N4: detecting the intensity of the chemiluminescent signal in step N3, and analyzing whether the analyte exists in the sample to be tested and / or the concentration of the analyte.

[0066] In the method of the present invention, the reagents can be incubated as needed after mixing. Specifically, the incubation temperature can be 35-45°C and the time can be 10-50 minutes; preferably, the incubation temperature can be selected from 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C or 44°C; and the incubation time can be selected from 10 minutes, 20 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes or 50 minutes.

[0067] In some embodiments of the present invention, the method further comprises the step of preparing a standard curve of chemiluminescent signal-analyte concentration using a series of calibrator solutions with known analyte concentrations; the standard curve is used to determine the content of the analyte in the sample to be tested.

[0068] In other embodiments of the present invention, in step N3, the second mixture is irradiated with excitation light of a wavelength of 600-700 nm to excite the donor microspheres in the second mixture to produce reactive oxygen species, and then the acceptor microspheres react with the reactive oxygen species they come into contact with to generate emission light of 520-620 nm.

[0069] The serum sample to be tested, reagent 1, and reagent 2 are mixed and incubated. The test antigen and Bio-competing antigen in the serum sample competitively bind to the detection antibody (FG-Ab) on the acceptor microspheres, forming complexes (FG-Ab-competing antigen-Bio, FG-Ab-test antigen), respectively. Subsequently, SA-GG (donor microspheres bound to avidin) binds to biotin (Bio), and the acceptor microspheres and donor microspheres approach each other, inducing the generation of a light signal after excitation. Free acceptor particles cannot obtain energy, and no light signal is generated. Because the present invention adopts a competitive analysis mode, the light signal intensity is inversely proportional to the content of the test antigen in the test serum sample. The concentration level of the test antigen in the unknown serum sample can be calculated using a mathematical function formed by a known concentration of the analyte calibrator.

[0070] The second aspect of the present invention provides a use of the kit as described in the first aspect of the invention in a chemiluminescence analyzer.

[0071] Example

[0072] To make the present invention easier to understand, the present invention will be further described in detail below with reference to the following examples. These examples are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

[0073] Example 1: Preparation of the kit of the present invention for estradiol (E2) as the analyte

[0074] (1) Preparation of monoclonal antibody-coupled receptor microsphere solution (reagent 1)

[0075] Acceptor microspheres: The surface of the microspheres contains aldehyde groups (-CHO), which are connected to the antibody molecules through the aldehyde groups. They contain a luminescent compound (a derivative of dimethylthiophene) and a chelate of a lanthanide (Eu) compound.

[0076] Biological materials: a monoclonal antibody with high affinity that specifically binds to E2 (ie, HA-McAb) and a monoclonal antibody with low affinity that specifically binds to E2 (ie, LA-McAb).

[0077] Preparation process: Preparation process:

[0078] 1) A high-affinity monoclonal antibody that specifically binds to E2 (i.e., HA-McAb) was dialyzed overnight against carbonate buffer, mixed with receptor microspheres (FG) at a mass ratio of 1:400, and coated for 2 hours. A blocking solution was added and blocked for 1 hour to prepare a concentrated solution containing FG-HA-McAb-n for later use;

[0079] 2) A low-affinity monoclonal antibody that specifically binds to E2 (i.e., LA-McAb) was dialyzed overnight against carbonate buffer and mixed with receptor microspheres (FG) at a mass ratio of 1:80. The solution was coated for 2 hours, and then blocked with blocking solution for 1 hour to prepare a concentrated solution containing FG-HA-McAb-N for later use.

[0080] 3) Dilute FG-HA-McAb-n at 1:200 with Reagent 1 diluent, numbered R1-1; dilute FG-LA-McAb-N at 1:500 with Reagent 1 diluent, numbered R1-2;

[0081] 4) Mix equal volumes of solutions R1-1 and R1-2 to obtain reagent 1.

[0082] (2) Preparation of competing antigen (reagent 2) bound to biotin

[0083] Dilute 1ug / ml Bio-E2 at a ratio of 1:10000 with Reagent 2 diluent to prepare R2 working solution, which is used as Reagent 2.

[0084] Alternatively, 1ug / ml Bio-E3 can be diluted 1:8000 with Reagent 2 diluent to prepare R2 working solution, which is used as Reagent 2.

[0085] (3) Preparation of release agent

[0086] Pure mesterolone was prepared at a concentration of 100 ng / L using 0.1 M pH 7.4 phosphate buffered saline solution containing 20% ​​inactivated calf serum.

[0087] (4) Preparation process of E2 series calibrators with known concentrations

[0088] Take pure E2 and prepare 0.5 ml of each series of calibration solutions ranging from 0 to 4800 ng / L using 0.1 M pH 7.4 phosphate buffered saline solution containing 20% ​​inactivated calf serum.

[0089] Example 2:

[0090] Using the method in Example 1, the conditions such as antibody type, coupling mass ratio, and competitive antigen type were changed respectively. The same batch of samples was detected using the LiCA500 automatic photochemiluminescence analysis system, which automatically completed and output homogeneous chemiluminescence signals, and analyzed the detection range and detection limit of the detection results.

[0091] The detection process using the kit prepared in Example 1 is fully automatically completed by the LiCA500 automatic photochemiluminescence analysis system and the detection results are output. The specific steps are as follows:

[0092] a. Add 10 μl of sample, calibrator, or quality control material to each reaction well;

[0093] b. Add 25 μl of release agent, 25 μl of reagent 1, and 25 μl of reagent 2 to the reaction wells in sequence;

[0094] c. Incubate at 37°C for 15 minutes;

[0095] d. Add 175 μl of LiCA universal solution (donor microsphere solution bound to avidin);

[0096] e. Incubate at 37°C for 15 minutes;

[0097] e. Laser irradiation of microwells and calculation of the amount of photons emitted from each well;

[0098] f. Calculate the sample concentration based on the calibration curve. The results are shown in Table 1.

[0099] Table 1

[0100]

[0101] As shown in Table 1, for both affinity antibody-coupled receptor microspheres, the lower the mass ratio of antibody to receptor microspheres, the better the detection results. When using only high-affinity antibody (HA-McAb)-coupled receptor microspheres for detection, the lower linear range and detection limit are better than those using only low-affinity antibody (LA-McAb)-coupled receptor microspheres, reaching 10-20 ng / L. However, the upper linear range limit of the low-affinity antibody (LA-McAb)-coupled receptor microspheres is better than that of the high-affinity antibody (HA-McAb)-coupled receptor microspheres, reaching over 5000 ng / L. When high-affinity antibody receptor microspheres and low-affinity antibody receptor microspheres with appropriate coupling mass ratios are mixed at equal volumes of 1:200 and 1:500, the upper and lower linear range limits and detection limits are optimized. In addition, Bio-E3 antigen is used as a competing antigen. Since its competitive ability is weaker than that of Bio-E2 antigen, it is more conducive to the binding of E2 in the sample with the detection antibody. Therefore, the functional sensitivity (detection limit) of detection using Bio-E3 antigen as a competing antigen is slightly better than that using Bio-E2 antigen as a competing antigen.

[0102] Example 3:

[0103] The precision of the kit used in Experiment No. 7 in Example 2 (in which Bio-E2 was used as a competing antigen) was examined.

[0104] Significance of precision: Precision is an important indicator to measure the intra-batch and inter-batch variation of a test kit. It is an important basis for evaluating the effectiveness of products to be marketed. It usually includes intra-batch precision and inter-batch precision.

[0105] Intra-batch precision evaluation method: Use low (L), medium (M), and high (H) value samples to independently analyze a batch of products, repeat the measurement 10 times for each batch, and calculate the average of the 10 measurement results and standard deviation (SD), according to the formula The coefficient of variation (CV) was calculated and the results are shown in Table 1.

[0106] Inter-batch precision assessment method: Use low (L), medium (M), and high (H) value samples to independently analyze three batches of products, repeat the measurement 20 times for each batch, and calculate the average value of the measurement results and standard deviation (SD), according to the formula The coefficient of variation (CV) was calculated and the results are shown in Table 2.

[0107] Table 2: Test results

[0108]

[0109]

[0110] As shown in Table 2, the intra-batch and inter-batch precisions of the three batches of reagents were all <10%, indicating that the test kit has good repeatability and small random errors.

[0111] Example 4:

[0112] Detection of the accuracy of the kit used in test No. 7 in Example 2 (where Bio-E2 is used as a competing antigen)

[0113] The meaning of accuracy: the degree of consistency between the measured value and the true value, reflecting the size of the system error.

[0114] Accuracy assessment method: Two samples containing different E2 levels were diluted at multiple points with the calibrator matrix solution. The concentrations of the diluted samples were measured using the method described in Example 2. The recoveries of the two samples were then calculated based on the dilution ratios. The results are shown in Tables 3 and 4, respectively.

[0115] Table 3

[0116]

[0117] Table 4

[0118]

[0119] As shown in Tables 3 and 4, after multi-point dilution using two E2 samples with different levels, the recovery rates were all within the range of 90% to 110%, indicating that the measured values ​​were close to the true values ​​and the detection error of the kit was small.

[0120] Example 5:

[0121] The E2 sample was tested using the kit used in test number 7 in Example 2 (in which Bio-E3 was used as a competing antigen), and the test results were compared with those of similar imported kits. The results were as follows: Figure 2 shown.

[0122] from Figure 2 It can be seen that the correlation between the values ​​measured by the kit of the present invention and those measured by similar imported kits for E2 samples is r=0.9888, which is a good correlation, indicating that the kit of the present invention can accurately detect the content of estradiol hormone in the sample.

[0123] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A competitive homogeneous chemiluminescence assay kit comprising a first composition, a second composition, and a third composition, wherein the first composition comprises a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to an analyte; the second composition comprises a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to an analyte, wherein the receptor can react with singlet oxygen to produce chemiluminescence; the third composition comprises a competing antigen that competes with estradiol for binding to the detection antibody; the analyte is estradiol, and the first and second receptors are both receptor microspheres, characterized in that: The affinity of the first antibody or its binding fragment for specific binding to the analyte is higher than the affinity of the second antibody or its binding fragment for specific binding to the analyte; at the same time, The mass ratio of the first antibody or its binding fragment to the first receptor is lower than the mass ratio of the second antibody or its binding fragment to the second receptor, and the mass ratio of the first antibody or its binding fragment to the first receptor is selected from 1:(100-1000); The concentration of the first composition in the kit is higher than the concentration of the second composition in the kit, and the ratio of the mass concentration of the first composition in the kit to the mass concentration of the second composition in the kit is (2-50):1; The competing antigen is bound to one member of the specific binding pair; the kit further comprises a fourth composition comprising donor microspheres, the donor microspheres being bound to the other member of the specific binding pair.

2. The kit according to claim 1, wherein The mass ratio of the first antibody or its binding fragment to the first receptor is selected from 1:(200-800).

3. The kit according to claim 2, wherein The mass ratio of the first antibody or its binding fragment to the first receptor is selected from 1:(300-600).

4. The kit according to claim 1, wherein The mass concentration ratio of the first composition in the kit to the mass concentration of the second composition in the kit is (2-25):

1.

5. The kit according to claim 4, characterized in that The mass concentration ratio of the first composition in the kit to the mass concentration of the second composition in the kit is (2-10):

1.

6. The kit according to claim 1, wherein The mass concentration of the first composition in the kit is 5 to 500 ug / ml.

7. The kit according to claim 6, characterized in that The mass concentration of the first composition in the kit is 10 to 250 ug / ml.

8. The kit according to claim 7, characterized in that The mass concentration of the first composition in the kit is 15 to 200 ug / ml.

9. The kit according to claim 1, wherein The first composition and the second composition are separately dispersed in the same buffer solution.

10. The kit according to claim 1, wherein The first composition and the second composition are mixed and dispersed in a buffer solution to assemble into a reagent.

11. The kit according to claim 1, wherein The first receptor and the second receptor are both receptor microspheres containing a high molecular polymer carrier.

12. The kit according to claim 1, wherein The average particle size of the first acceptor microspheres is the same as the average particle size of the second acceptor microspheres.

13. The kit according to claim 1, wherein The competing antigen is estradiol or an estradiol analogue.

14. The kit according to claim 13, characterized in that The competing antigen is an estradiol analogue.

15. Use of the kit according to any one of claims 1 to 14 in a chemiluminescence analyzer.

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