A competitive homogeneous chemiluminescence assay kit and its application

Abstract

The present invention relates to a competitive homogeneous chemiluminescence assay kit, which comprises a first composition, a second composition and a third composition. The first composition contains a detection antibody that specifically binds to an analyte; the second composition contains a first receptor and a first antigen bound thereto, and the first antigen competes with the analyte for binding to the detection antibody; the third composition contains a second receptor and a second antigen bound thereto, and the second antigen competes with the analyte for binding to the detection antibody; the receptor can react with singlet oxygen to generate chemiluminescence; the affinity of the first antigen for specific binding to the detection antibody is higher than that of the second antigen for specific binding to the detection antibody; meanwhile, the mass ratio of the first antigen to the first receptor is higher than the mass ratio of the second antigen to the second receptor. The kit of the present invention has both high functional sensitivity and a wide detection range.

Description

Technical Field

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

[0002] Competitive immunoassay is a detection method for the quantitative analysis of small molecule haptens. Radioimmunoassay (RIA) is the earliest established competitive immunoassay method and won the Nobel Prize in Physiology or Medicine in 1974. In radioimmunoassay, there are a radiolabeled competitive antigen (labeled antigen) and a limited amount of specific antibody. The antigen to be detected in the specimen and the labeled antigen as a reagent competitively bind to the specific antibody respectively. The bound label (B) and the free label (F) are separated, and the radioactivity (or intensity, counts per minute, CPM) of the bound label is measured. The radioactivity has an inverse proportional function relationship with the antigen to be detected. A series of calibration standards with known concentrations are used to obtain the mathematical function relationship (calibration function, which can be simply understood as a calibration curve) of the calibration standards. The unknown specimen is operated under the same conditions as the calibration standards, the radioactivity is measured, and then the concentration value of the specimen to be detected is obtained through the standard function.

[0003] In competitive immunoassay, the dosage of the competitive antigen is directly related to the functional sensitivity of competitive immunoassay. In addition, in competitive immunoassay, it is also crucial to select a suitable specific antibody and its working concentration. Chemiluminescence analysis has good analytical performance, and its analytical specificity, analytical sensitivity, and automated operation can better meet the clinical requirements. However, for some special indicators such as steroid hormones, there are high requirements for both functional sensitivity and detection range, while the existing chemiluminescence immunoassay, electrochemiluminescence immunoassay, photochemiluminescence immunoassay, etc. still have defects and cannot effectively balance the special requirements of functional sensitivity and analytical range. Therefore, there is an urgent need for a chemiluminescence detection technology that can balance the requirements of functional sensitivity and analytical 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. When detecting using this kit, it has excellent functional sensitivity and detection range at the same time.

[0005] To this end, the first aspect of the present invention provides a competitive homogeneous chemiluminescence assay kit, which comprises a first composition, a second composition and a third composition. The first composition contains a detection antibody that specifically binds to the analyte; the second composition contains a first receptor and a first antigen that binds thereto, and the first antigen competes with the analyte for binding to the detection antibody; the third composition contains a second receptor and a second antigen that binds thereto, and the second antigen competes with the analyte for binding to the detection antibody; the receptor is capable of generating chemiluminescence by reacting with singlet oxygen;

[0006] The affinity of the first antigen for specifically binding to the detection antibody is higher than the affinity of the second antigen for specifically binding to the detection antibody; meanwhile,

[0007] The mass ratio of the first antigen to the first receptor is higher than the mass ratio of the second antigen to the second receptor.

[0008] In some embodiments of the present invention, the mass ratio of the second antigen to the second receptor is 1:(10 - 200), preferably 1:(50 - 150), more preferably 1:(80 - 120).

[0009] In some other embodiments of the present invention, the mass ratio of the first antigen to the first receptor is 1:(1 - 20), preferably 1:(5 - 15), more preferably 1:(8 - 12).

[0010] In some embodiments of the present invention, the concentration of the second composition in the kit is lower than the concentration of the third composition in the kit.

[0011] In some preferred embodiments of the present invention, the mass concentration ratio of the second composition to the third composition in the kit is 1:(10 - 100), preferably 1:(20 - 80), more preferably 1:(40 - 60).

[0012] In some specific embodiments of the present invention, the mass concentration of the third composition in the kit is 5 - 500 μg / ml, preferably 10 - 250 μg / ml, more preferably 15 - 200 μg / ml.

[0013] In some embodiments of the present invention, the second composition and the third composition are separately dispersed in the same buffer.

[0014] In some other embodiments of the present invention, the second composition and the third composition are combined and dispersed in a buffer to form a reagent.

[0015] In some embodiments of the present invention, both the first receptor and the second receptor are receptor microspheres containing a polymer carrier, and the average particle size of the first receptor microspheres is the same as that of the second receptor microspheres.

[0016] In other embodiments of the present invention, the average particle size of the first receptor microspheres is the same as that of the second receptor microspheres.

[0017] In some embodiments of the present invention, the analyte is a small molecule antigen or hapten.

[0018] In other embodiments of the present invention, the first antigen and the second antigen are the analyte and / or an analyte analogue; preferably, the first antigen is the analyte and the second antigen is an analyte analogue.

[0019] In some embodiments of the present invention, the detection antibody binds to one of the specific binding partner members.

[0020] In other embodiments of the present invention, the kit further includes a series of calibrator solutions with known analyte concentrations.

[0021] A second aspect of the present invention provides an application of the kit as described in the first aspect of the present invention in a chemiluminescence analyzer.

[0022] The beneficial effects of the present invention are as follows: By coupling competitive antigens with different affinities for the detection antibody to receptor microspheres at different mass ratios and then mixing the two receptor microspheres in an appropriate proportion, the kit of the present invention enables two competitive antigens with different affinities for the detection antibody to selectively play roles according to the concentration difference of the analyte to be detected. While ensuring the functional sensitivity, the detection range is broadened to prevent the occurrence of the hook effect. Moreover, the kit belongs to homogeneous immunoassay, and there is no separation and washing process throughout, which not only saves the detection time but also avoids the errors caused by washing, and has high precision and accuracy. In addition, in order to further improve the functional sensitivity, an analogue similar to the analyte structure is selected as one of the competitive antigens to ensure that when detecting low-end samples, the analyte to be detected can preferentially bind to the detection antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below in conjunction with the drawings.

[0024] Figure 1The detection principle diagram of the kit according to the present invention; wherein, the meanings of the reference numerals are as follows: 1. The second receptor microspheres and the second antigen bound thereto. A small amount of the second antigen with low affinity for the detection antibody is coated on the surface of the second receptor microspheres, but the concentration of the second receptor microspheres is relatively high to ensure that the second receptor microspheres play a dominant role when detecting low-concentration testosterone samples; 2. The first receptor microspheres and the first antigen bound thereto. A relatively large amount of the first antigen with high affinity for the detection antibody is coated on the surface of the first receptor microspheres, but the concentration of the first receptor microspheres is relatively low to ensure that the first antigen on the first receptor microspheres plays a dominant role when detecting high-concentration testosterone samples; 3. The detection antibody bound to biotin, and the detection antibody can specifically bind to testosterone; 4. The testosterone to be detected.

[0025] Figure 2 It is a correlation diagram of the measured values of the kit using R1-1 as reagent 1 and the Beckman measured values.

[0026] Figure 3 It is a correlation diagram of the measured values of the kit using R1-2 as reagent 1 and the Beckman measured values.

[0027] Figure 4 It is a correlation diagram of the measured values of the kit using R1-3 as reagent 1 and the Beckman measured values. Detailed implementation mode

[0028] To make the present invention easy to understand, 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 implementation modes described. It should also be understood that the terms used herein are only for describing the specific implementation modes and do not represent any limitation.

[0029] When a numerical range is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified or intermediate value in the specified range is encompassed within the present invention. The upper and lower limits of these smaller ranges can be independently included in the smaller ranges and are also encompassed within the present invention, subject to any explicit exclusions in the specified range. When the specified range includes one or both of the limits, the ranges excluding either or both of the included limits are also included in the present invention.

[0030] 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 implementation or testing of the present invention, the preferred methods and materials are now described.

[0031] Ⅰ. Terms

[0032] As used herein, the term "homogeneous" is defined in English as "homogeneous", which means that the detection can be carried out without separating the bound antigen-antibody complex from the remaining free antigen or antibody.

[0033] As used herein, the term "specific binding" refers to the mutual discrimination and selective binding reaction between two substances, which, in terms of three-dimensional structure, means the conformational correspondence between the corresponding reactants.

[0034] As used herein, the term "donor microsphere" refers to a sensitizer that can generate reactive intermediates such as singlet oxygen that can react with acceptor microspheres after activation by energy or active compounds. The donor microsphere 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 microsphere is a polymer microsphere filled with a photosensitizer, and the photosensitizer can be a photosensitizer known in the art, preferably a compound that is relatively light-stable and does not react effectively with singlet oxygen. Non-limiting examples thereof include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll disclosed in U.S. Patent No. 5,709,994 (the entire patent document is hereby incorporated by reference in its entirety), and derivatives of these compounds having 1-50 atomic substituents, which are used to make these compounds more lipophilic or more hydrophilic and / or as a linking group for linking to specific binding partner members. Examples of other photosensitizers known to those skilled in the art can also be used in the present invention, such as the content described in U.S. Patent No. 6,406,913, which is incorporated herein by reference.

[0035] As used herein, the term "acceptor microsphere" refers to a compound that can react with singlet oxygen to generate a detectable signal. The donor microsphere is induced and activated by energy or active compounds to release high-energy singlet oxygen, and the high-energy singlet oxygen is captured by the nearby acceptor microsphere, thereby transferring energy to activate the acceptor microsphere. In some specific embodiments of the present invention, the acceptor microsphere comprises a luminescent composition and a matrix, and the luminescent composition is filled in the matrix and / or coated on the surface of the matrix. As used herein, the "matrix" is a microsphere or microparticle well-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, has any density, but preferably has a density close to that of water, preferably can float in water, and is composed of a transparent, partially transparent or opaque material. The matrix can have or not have a charge, and when charged, it is preferably a negative charge. The matrix can be a latex particle or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, small oil droplets, silicon particles, metal sols, cells, and microcrystalline dyes.

[0036] The term "biotin" in the present invention is widely present in animal and plant tissues. It has two ring structures on its molecule, namely an imidazolone ring and a thiophene ring, and the imidazolone ring is the main site for binding to streptavidin. Activated biotin can be coupled to almost all known biological macromolecules under the mediation of a protein cross-linking agent, including proteins, nucleic acids, polysaccharides, lipids, etc. The "avidin" molecule is composed of 4 identical peptide chains, and each peptide chain can bind a biotin. Therefore, each antigen or antibody can be conjugated with multiple biotin molecules simultaneously, thereby producing a "tentacle effect" to improve the analysis sensitivity.

[0037] The term "epitope" in the present invention refers to the special chemical group that determines the antigen specificity in an antigen molecule. For proteins, the antigen epitope is a specific amino acid sequence (linear epitope), or it can also be a spatial conformation formed by several specific amino acid sequences (conformational epitope). The antigen epitope is not only the smallest structural and functional unit for antibody binding, but also the basic unit recognized by the antigen receptor of lymphocytes (B cells).

[0038] The term "monoclonal antibody" in the present invention refers to an antibody prepared by using the hybridoma fusion technology, which is directed against a single antigen epitope, has a single specificity, and is completely homogeneous in structure and function. First, monoclonal antibodies have a single specificity, eliminating cross-reactions and improving the specificity of labeled immunoassays. Second, monoclonal antibodies ensure continuous supply and have small batch-to-batch differences, effectively reducing the batch-to-batch differences of immunoassay kits. Third, different monoclonal antibodies recognize different antigen sites and show different affinity characteristics.

[0039] The term "differential receptor microspheres" in the present invention specifically refers to receptor microspheres (FG) conjugated with competitive antigens having different affinities for the detection antibody.

[0040] The term "functional sensitivity" in the present invention refers to the lowest detection limit, that is, after serially diluting a specimen with a known concentration, the lowest content that the analytical method can detect, and the within-batch precision should not be greater than 20%. The analytical sensitivity is obtained from actual measurements and is also called "functional sensitivity".

[0041] The term "detection range" in the present invention refers to the effective range of the dose function. For example, if a high-concentration specimen is serially diluted and the measurement results of the diluted specimens are subjected to linear regression analysis, the correlation coefficient (R) is greater than 0.990.

[0042] II. Specific implementation examples

[0043] The present invention will be described in detail below.

[0044] Regarding competitive immunoassay, to obtain a high-quality competitive calibration function (which can be simply understood as a calibration curve), it needs to meet two basic conditions: First, the competitive antigen and the analyte to be detected are homologous and have the same or similar affinity with the detection antibody; Second, the principle of limited antibody is ensured. The amount of the detection antibody used needs to be less than the cumulative amount of antibody required for the two antigens, but must be greater than the cumulative amount of antibody required for the competitive antigen or the analyte to be detected. Based on the technology of photochemiluminescence, the present invention obtains a homogeneous chemiluminescence detection kit for quantitatively detecting the level of an analyte by the photochemiluminescence method. The analytical performance indicators of this kit can meet the industry standards or the basic requirements of clinical laboratories. It is mainly manifested in: Selecting two competitive antigens with different affinities for the detection antibody and conjugating them to receptor microspheres respectively can improve the measurement value compliance of high-end samples and low-end samples. In addition, by selecting an analog similar to the structure of the analyte as one of the competitive antigens to label biotin, it is ensured that the analyte to be detected can preferentially bind to the detection antibody during the detection of low-end samples, further improving the functional sensitivity.

[0045] Therefore, the competitive homogeneous chemiluminescence assay kit involved in the first aspect of the present invention includes a first composition, a second composition, and a third composition. The first composition contains a detection antibody that specifically binds to the analyte; the second composition contains a first receptor and a first antigen that binds thereto, and the first antigen competes with the analyte for binding to the detection antibody; the third composition contains a second receptor and a second antigen that binds thereto, and the second antigen competes with the analyte for binding to the detection antibody; the receptor can generate chemiluminescence by reacting with singlet oxygen;

[0046] The affinity of the first antigen for specifically binding to the detection antibody is higher than the affinity of the second antigen for specifically binding to the detection antibody; at the same time,

[0047] The mass ratio of the first antigen to the first receptor is higher than the mass ratio of the second antigen to the second receptor. That is, the coupling amount of the first antigen on the first receptor microsphere is higher than the coupling amount of the second antigen on the second receptor microsphere.

[0048] In some specific embodiments of the present invention, the detection antibody is a monoclonal antibody that specifically binds to the analyte.

[0049] In some embodiments of the present invention, the mass ratio of the second antigen to the second receptor is 1:(10 - 200), preferably 1:(50 - 150), more preferably 1:(80 - 120). In some specific embodiments of the present invention, the mass ratio of the second antigen to the second microsphere is 1:10, 1:30, 1:50, 1:80, 1:100, 1:120, 1:150, 1:180, or 1:200, etc.

[0050] In some other embodiments of the present invention, the mass ratio of the first antigen to the first receptor is 1:(1-20), preferably 1:(5-15), more preferably 1:(8-12). In some specific embodiments of the present invention, the mass ratio of the second antigen to the second microsphere is 1:1, 1:3, 1:5, 1:8, 1:10, 1:12, 1:15, 1:18 or 1:20, etc.

[0051] In some embodiments of the present invention, the concentration of the second composition in the kit is lower than the concentration of the third composition in the kit. In the present invention, the concentration can be either a mass concentration or a molar concentration.

[0052] In some preferred embodiments of the present invention, the mass concentration ratio of the second composition to the third composition in the kit is 1:(10-100), preferably 1:(20-80), more preferably 1:(40-60). In some specific embodiments of the present invention, the mass concentration ratio of the second composition to the third composition in the kit is 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90 or 1:100, etc.

[0053] In some specific embodiments of the present invention, the mass concentration of the third composition in the kit is 5-500 μg / ml, preferably 10-250 μg / ml, more preferably 15-200 μg / ml.

[0054] In some embodiments of the present invention, the second composition and the third composition are separately dispersed in the same buffer.

[0055] In some other embodiments of the present invention, the second composition and the third composition are combined and dispersed in a buffer to form a reagent (i.e., Reagent 1).

[0056] In some embodiments of the present invention, both the first receptor and the second receptor are receptor microspheres containing a polymer carrier, and the average particle size of the first receptor microsphere is the same as that of the second receptor microsphere. It should be noted that the "receptor" in the present invention not only includes polymer microspheres but also can include microspheres such as magnetic particles.

[0057] In some other embodiments of the present invention, the average particle size of the first receptor microsphere is the same as that of the second receptor microsphere.

[0058] In some embodiments of the present invention, the analyte is a small molecule antigen or hapten.

[0059] In some other embodiments of the present invention, the first antigen and the second antigen are analytes and / or analyte analogs; preferably, the first antigen is an analyte and the second antigen is an analyte analog. In the present invention, the affinity of the analyte analog for specific binding to the detection antibody is lower than the affinity of the analyte for specific binding to the detection antibody.

[0060] In some embodiments of the present invention, the detection antibody binds to one member of the specific binding pair (such as biotin). The reagent including the first composition in the present invention is also called Reagent 2.

[0061] In some other embodiments of the present invention, the kit further includes a column calibration solution with a known analyte concentration.

[0062] In some embodiments of the present invention, the kit further includes a fourth composition, which includes donor microspheres that specifically bind to the other member of the specific binding pair (such as avidin).

[0063] In some embodiments of the present invention, the kit may further include a releasing agent, and the releasing agent includes dimethoxyestradiol. In some specific embodiments of the present invention, the releasing agent is composed of: 40 ng / ml of dimethoxyestradiol, which is diluted and prepared with 50 mM HEPES pH 6.0. The releasing agent can release the analyte bound to the protein in the sample to be tested.

[0064] The principle of the two receptor microspheres in the kit of the present invention to function intelligently is as follows:

[0065] For low-concentration analyte specimens (female specimens), there are few analyte molecules to be tested, and it is not easy for the analyte to be tested to bind to the detection antibody. In Reagent 1, the number of second receptor microspheres bound to the second antigen is dominant, and the chance of binding to the detection antibody (Bio-McAb) is much higher than that of the first receptor microspheres bound to the first antigen, that is, the second receptor microspheres with a high microsphere concentration are dominant. The number of second antigen molecules on the surface of the second receptor microspheres is small, and the affinity for the detection antibody is lower than that of the analyte molecules to be tested in the specimen. At this time, it is ensured that the analyte to be tested has a stronger ability to bind to the detection antibody, so as to obtain better functional sensitivity.

[0066] For high-concentration analyte specimens (male specimens), there are many analyte molecules to be detected, and the analyte to be detected easily binds to the detection antibody in the same liquid phase. In Reagent 1, although the number of second receptor microspheres bound to the second antigen is dominant, and the chance of binding to the detection antibody (Bio-McAb) is much higher than that of the first receptor microspheres bound to the first antigen, due to the relatively small number of second antigen molecules on the surface of the second receptor microspheres, the contribution of the number of microspheres to the competitive reaction is not obvious at this time. On the contrary, the number of first receptor microspheres is small, but the number of first antigen molecules on the surface of the microspheres is large, and the affinity of the antibody is the same as that of the analyte molecules to be detected in the specimen. The binding strength of the first antigen to the antibody can balance the high-concentration analyte to be detected, thereby obtaining an ideal detection range.

[0067] In some embodiments of the present invention, the method for detecting an analyte using the kit includes:

[0068] Step N1, mixing the sample to be tested, Reagent 1, and Reagent 2 to obtain a first mixture;

[0069] Step N2, mixing the donor microsphere solution bound to avidin with the first mixture to obtain a second mixture;

[0070] 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 receptor microspheres react with the reactive oxygen species they come into contact with to generate a chemiluminescence signal;

[0071] Step N4, detecting the intensity of the chemiluminescence signal in Step N3, and analyzing whether there is an analyte and / or the concentration of the analyte in the sample to be tested.

[0072] In the method of the present invention, after the reagents are mixed, incubation can be carried out as needed. Specifically, the incubation temperature can be 35-45°C, and the time can be 10-50 min; 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; the incubation time can be selected from 10 min, 20 min, 30 min, 35 min, 40 min, 45 min, or 50 min.

[0073] In some embodiments of the present invention, the method further includes the step of making a standard curve of chemiluminescence 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.

[0074] In some other embodiments of the present invention, in step N3, the second mixture is irradiated with excitation light having a wavelength of 600 - 700 nm 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 they come into contact with to generate emission light with a wavelength of 520 - 620 nm.

[0075] The serum sample to be tested, reagent 1, and reagent 2 are mixed and incubated. The analyte in the serum sample and the competitive antigen conjugated to the acceptor microspheres competitively bind to the detection antibody (Bio-McAb) to respectively form complexes (Bio-McAb-competitive antigen-FG, Bio-McAb-analyte-FG). Subsequently, SA-GG (donor microspheres conjugated to avidin) binds to biotin (Bio), the acceptor microspheres and the donor microspheres approach each other, and light signals are induced after excitation. The free acceptor microparticles cannot obtain energy and no light signals are generated. Since the present invention adopts a competitive analysis mode, the light signal intensity has an inverse proportional function relationship with the content of the analyte in the serum sample to be tested. Through the mathematical function formed by the calibration standard of the analyte with a known concentration, the concentration level of the analyte in the unknown serum specimen can be calculated.

[0076] The second aspect of the present invention provides an application of the kit as described in the first aspect of the present invention in a chemiluminescence analyzer.

[0077] Examples

[0078] To make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples. These examples are only illustrative and are not limited to the application scope of the present invention. The raw materials or components used in the present invention can be obtained through commercial channels or conventional methods without special instructions.

[0079] Example 1: Preparation of the kit according to the present invention with the analyte being testosteroneReagents and instruments:

[0080] Testosterone antibody conjugated to biotin, acceptor microspheres, BSA-conjugated dihydrotestosterone (BSA-DHT), BSA-conjugated testosterone (BSA-T), phosphate buffer (0.02M PBS, pH 7.2), BSA, Tween-20, LiCA 500 (Beijing KeMei Biotechnology Co., Ltd.), Hitachi high-speed refrigerated centrifuge.

[0081] Preparation process:

[0082] (1) Preparation of the solution of competitive antigen conjugated to acceptor microspheres (reagent 1)

[0083] 1) In two 2 mL centrifuge tubes, 2 mg of acceptor microspheres are respectively taken, centrifuged at 10000 rpm for 15 min at 4°C, and washed once.

[0084] 2) Ultrasonic dispersion is uniform. Add 0.2 mg of BSA-T to one centrifuge tube and 0.02 mg of BSA-DHT to the other tube, mix well and coat overnight at 4°C;

[0085] 3) Add 20 μL of 10 mg / mL BSA to each of the two centrifuge tubes to block the receptor microspheres, and rotate at room temperature for 2 h;

[0086] 4) Centrifuge and wash the receptor microspheres three times with PBS buffer solution containing 0.5% Tween-20, and then dilute the receptor microspheres in the two centrifuge tubes to 0.1 mg / ml with pH 7.2, 0.05 M PBS solution, and label them as FG-NT (R1-1) and FG-nDHT (R1-2) respectively and store them.

[0087] 5) Mix R1-1 and R1-2 so that the mixing ratio of the microsphere concentrations of the two is FG-NT:FG-nDHT = 1:50, and label it as R1-3, which is used as Reagent 1.

[0088] (2) Preparation of the detection antibody (Reagent 2) conjugated with biotin

[0089] Dilute the testosterone antibody conjugated with biotin 20,000 times with the diluent of Reagent 2 until the antibody concentration is 0.05 μg / ml, which is used as Reagent 2.

[0090] (3) Preparation of the releasing agent

[0091] The releasing agent consists of: 40 ng / ml of dimethoxyestradiol, which is diluted and prepared with 50 mM HEPES pH 6.0.

[0092] (4) Preparation process of the known concentration T series calibration standards

[0093] Add the testosterone solution with a concentration of 1 mg / mL to horse serum to prepare calibration standards 1-6 with concentrations of 0 ng / dl, 50 ng / dl, 150 ng / dl, 400 ng / dl, 800 ng / dl, and 1600 ng / dl respectively.

[0094] Example 2

[0095] Use the kits with R1-1, R1-2, and R1-3 prepared in Example 1 as Reagent 1 to detect the samples containing testosterone respectively, and compare the detection results with the Beckman measurements. The results are as follows Figure 2-4 shown.

[0096] The detection process is fully automated by the LiCA500 automatic photochemiluminescence analysis system and the detection results are output. The specific steps are as follows:

[0097] a. Add 20 μl of the sample, calibrator, or quality control product into the reaction wells respectively;

[0098] b. Sequentially add 20 μl of the releasing agent, 25 μl of Reagent 1, and 25 μl of Reagent 2 into the reaction wells;

[0099] c. Incubate at 37 °C for 15 minutes;

[0100] d. Add 175 μl of the LiCA universal solution (donor microsphere solution conjugated with avidin);

[0101] e. Incubate at 37 °C for 15 minutes;

[0102] e. Irradiate the microplate with laser and calculate the amount of luminescent photons per well;

[0103] f. Calculate the sample concentration according to the calibration curve.

[0104] From Figure 1-3 It can be seen that when FG-nDHT is used as the competitive antigen, the low-value samples have a better correlation with Beckman, but the high-value samples have a poor correlation; when FG-NT is used as the competitive antigen, the low-value functional sensitivity (detection limit) is poor, but the high-value correlation is good; when the two competitive antigens are used in combination, the low-value functional sensitivity of testosterone and the detection range can be taken into account. From the perspective of the correlation with the measured values of Beckman, the best results are obtained when the two competitive antigens FG-nDHT and FG-NT are used in combination.

[0105] Example 3: Precision detection

[0106] Intra-assay precision detection: Use the kit described in the present invention to perform precision detection on high, medium, and low samples: Each batch of the kit is measured 10 times, and the average value X and standard deviation SD of the 10 measurement results are calculated. The coefficient of variation CV is obtained according to the formula CV = SD / X × 100%. The results are shown in Tables 1 and 3.

[0107] Inter-assay precision detection: Detect high, medium, and low samples with three batches of the kit described in the present invention, each repeated 10 times, calculate the average value X and standard deviation SD of the 30 measurement results, and obtain the coefficient of variation CV according to the formula CV = SD / X × 100%. The results are shown in Tables 2 and 3.

[0108] Table 1: Original data of intra-assay precision of the LiCA T kit (photochemiluminescence immunoassay)

[0109]

[0110] Table 2: Original data of inter-assay precision of three batches of the LiCA T kit (photochemiluminescence immunoassay)

[0111]

[0112]

[0113] Table 3: Analysis Precision of the Kit (Luminescence Immunoassay) Described in the Present Invention

[0114]

[0115]

[0116] As can be seen from Table 3, the within-batch and between-batch precisions of the three batches of kits are both < 2%, indicating that the measured values of the kit described in the present invention have good repeatability and small random errors.

[0117] Example 4: Accuracy Detection

[0118] Significance of accuracy: The degree of conformity between the measured value and the true value, reflecting the magnitude of systematic error.

[0119] Accuracy evaluation method: Detection was performed using reference sera with concentrations of 0.40 ng / mL, 5.97 ng / mL, and 8.15 ng / mL respectively, and the recovery rate was calculated according to Formula 1. The results are shown in Table 4.

[0120] B = Xi / T × 100% Formula 1

[0121] In the formula: B - recovery rate; X i - Measured concentration of the sample; T - target value of the sample.

[0122] Table 4: Accuracy of the Kit (Luminescence Immunoassay) Described in the Present Invention

[0123]

[0124] As can be seen from Table 4, after detection with samples of different concentrations, the recovery rates are all in the range of 100% - 110%, indicating that the measured values are close to the true values and the detection error of the kit described in the present invention is small.

[0125] Example 5:

[0126] Using the method in Example 1, conditions such as the type of competitive antigen, the mass ratio of conjugation, and the concentration of receptor microspheres were changed respectively. The same batch of samples was detected using the LiCA500 automatic luminescence immunoassay system, and the homogeneous chemiluminescence signal was automatically completed and output, and the detection range and detection limit of the detection results were analyzed. The results are shown in Table 5.

[0127] Table 5

[0128]

[0129]

[0130] As can be seen from Table 5, when the competitive antigen is FG-nDHT, it is beneficial for the detection limit, but the measured values of high-value samples are on the low side and the linear range is narrow; when the competitive antigen is FG-NT, the low-value functional sensitivity (detection limit) is poor, but the high-value correlation is good and the linear range is wide; when the two are mixed as the competitive antigen, the low-value functional sensitivity of testosterone and the detection range can be taken into account.

[0131] It should be noted that the above-described embodiments are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described by referring to the typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words rather than limiting words. Modifications can be made to the present invention within the scope of the claims of the present invention as provided, and the present invention can 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, which comprises a first composition, a second composition and a third composition. The first composition contains a detection antibody that specifically binds to an analyte; the second composition contains a first receptor and a first antigen bound thereto, and the first antigen competes with the analyte for binding to the detection antibody; the third composition contains a second receptor and a second antigen bound thereto, and the second antigen competes with the analyte for binding to the detection antibody; the receptor can generate chemiluminescence by reacting with singlet oxygen; and it is characterized in that both the first receptor and the second receptor are receptor microspheres; the affinity of the first antigen for specifically binding to the detection antibody is higher than that of the second antigen for specifically binding to the detection antibody; and at the same time, the mass ratio of the first antigen to the first receptor is higher than the mass ratio of the second antigen to the second receptor; the mass ratio of the first antigen to the first receptor is 1:(1 - 20), and the mass ratio of the second antigen to the second receptor is 1:(10 - 200); the mass concentration ratio of the second composition in the kit to the mass concentration of the third composition in the kit is 1:(10 - 100).

2. The kit according to claim 1, wherein The mass ratio of the second antigen to the second receptor is 1:(50 - 150).

3. The kit according to claim 2, wherein The mass ratio of the second antigen to the second receptor is 1:(80 - 120).

4. The kit according to claim 1, characterized in that, The mass ratio of the first antigen to the first receptor is 1:(5 - 15).

5. The kit according to claim 4, wherein The mass ratio of the first antigen to the first receptor is 1:(8 - 12).

6. The kit according to claim 1, wherein The mass concentration ratio of the second composition in the kit to the mass concentration of the third composition in the kit is 1:(20 - 80).

7. The kit according to claim 6, wherein The mass concentration ratio of the second composition in the kit to the mass concentration of the third composition in the kit is 1:(40 - 60).

8. The kit according to claim 1, wherein The mass concentration of the third composition in the kit is 5 - 500 μg / ml.

9. The kit according to claim 8, characterized in that, The mass concentration of the third composition in the kit is 10 - 250 μg / ml.

10. The kit according to claim 9, wherein The mass concentration of the third composition in the kit is 15 - 200 μg / ml.

11. The kit according to claim 1, wherein The second composition and the third composition are separately dispersed in the same buffer.

12. The kit according to claim 1, characterized in that, The second composition and the third composition are mixed and dispersed in a buffer to assemble a reagent.

13. The kit according to claim 1, wherein The first receptor and the second receptor are respectively a first receptor microsphere and a second receptor microsphere containing a polymer carrier.

14. The kit according to claim 13, characterized in that, The average particle size of the first receptor microsphere is the same as that of the second receptor microsphere.

15. The kit according to claim 1, characterized in that, The analyte is a small molecule antigen or hapten.

16. The kit according to claim 1, wherein The first antigen and the second antigen are the analyte and / or an analyte analogue.

17. The kit according to claim 1, characterized in that, The first antigen is the analyte, and the second antigen is an analyte analogue.

18. The kit according to claim 1, wherein The detection antibody binds to one of the members of the specific binding pair.

19. The kit according to claim 1, characterized in that, The kit further includes a series of calibration solution with known analyte concentration.

20. Use of a kit according to any one of claims 1 - 19 in a chemiluminescence analyzer.

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