A competitive homogeneous chemiluminescence detection method and its application

Abstract

The present invention relates to a competitive homogeneous chemiluminescent detection method comprising the following steps: contacting an analyte with a first composition, a reagent comprising a second composition, a third composition, and a fourth composition to form a test mixture; illuminating the test mixture at least once with excitation light; and then detecting the intensity of the resulting chemiluminescent signal to determine the presence and / or concentration of the analyte. The method utilizes "differential receptor microspheres," resulting in excellent functional sensitivity and detection range.

Description

Technical Field

[0001] The present invention belongs to the technical field of homogeneous chemiluminescence, and in particular relates to a competitive homogeneous chemiluminescence detection method 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 detection method, 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 detection method, which comprises the following steps: contacting an analyte with a first composition, a reagent comprising a second composition and a third composition, and a fourth composition to form a test mixture; providing excitation light to irradiate the test mixture at least once; and then detecting the intensity of the chemiluminescent signal generated thereby to determine the presence and / or concentration of the analyte; wherein,

[0006] The first composition comprises a detection antibody that specifically binds to the analyte, wherein the detection antibody binds to one of the specific binding pair members;

[0007] The second composition comprises a first receptor and a first antigen bound thereto, wherein the first antigen competes with the analyte for binding to the detection antibody;

[0008] The third composition comprises a second receptor and a second antigen bound thereto, wherein the second antigen competes with the analyte for binding to the detection antibody; the receptor can react with singlet oxygen to produce chemiluminescence;

[0009] The fourth composition comprises a donor capable of generating reactive oxygen species, the donor being bound to the other member of the specific binding pair;

[0010] The affinity of the specific binding of the first antigen to the detection antibody is higher than the affinity of the specific binding of the second antigen to the detection antibody; at the same time,

[0011] 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.

[0012] 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), and more preferably 1:(80-120).

[0013] In 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), and more preferably 1:(8-12).

[0014] In some embodiments of the present invention, the concentration of the second composition in the reagent is lower than the concentration of the third composition in the reagent.

[0015] In some preferred embodiments of the present invention, the ratio of the mass concentration of the second composition in the reagent to the mass concentration of the third composition in the reagent is 1:(10-100), preferably 1:(20-80), and more preferably 1:(40-60).

[0016] In some specific embodiments of the present invention, the mass concentration of the third composition in the reagent is 5 to 500 ug / ml, preferably 10 to 250 ug / ml, and more preferably 15 to 200 ug / ml.

[0017] In some embodiments of the present invention, the second composition and the third composition are separately dispersed in the same buffer.

[0018] In other embodiments of the present invention, the second composition and the third composition are combined and dispersed in a buffer solution to assemble into a reagent.

[0019] 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.

[0020] 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.

[0021] In some embodiments of the present invention, the analyte is a small molecule antigen or a hapten.

[0022] In other embodiments of the present invention, the first antigen and the second antigen are analytes and / or analyte analogs; preferably, the first antigen is the analyte and the second antigen is an analyte analog.

[0023] In some embodiments of the present invention, the analyte is first contacted with the first composition, the reagent comprising the second composition and the third composition, and then the fourth composition is added thereto.

[0024] In other embodiments of the present invention, the analyte is contacted with the first composition, the reagent comprising the second composition and the third composition, and the fourth composition, and then incubated at 30-40° C. for 1-15 minutes to form a test mixture.

[0025] The second aspect of the present invention provides an application of the method according to the first aspect of the present invention in a chemiluminescence analyzer.

[0026] The beneficial effects of the present invention are as follows: the method of the present invention selects competing antigens with different affinities with the detection antibody to couple receptor microspheres in different mass ratios, and then mixes the two receptor microspheres in an appropriate proportion, so that the two competing antigens with different affinities with the detection antibody can selectively act according to the difference in the concentration of the antigen to be detected. While ensuring functional sensitivity, the detection range is broadened to prevent the occurrence of the hook effect. The kit belongs to homogeneous immunoassay and there is no separation and washing process throughout the process. It not only saves detection time, but also avoids errors caused by washing, and has high precision and accuracy. In addition, in order to further improve functional sensitivity, an analogue with a similar structure to the analyte is selected as one of the competing antigens to ensure that the analyte to be detected can preferentially bind to the detection antibody when detecting low-end samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings.

[0028] Figure 1 Figure 2 is a diagram of the detection principle of the method of the present invention; wherein the meanings of the reference numerals are as follows: 1. a second receptor microsphere and a second antigen bound thereto; the surface of the second receptor microsphere is coated with a small amount of the second antigen having a low affinity for the detection antibody, but the concentration of the second receptor microsphere is relatively high, ensuring that the second receptor microsphere plays a dominant role when detecting low-concentration testosterone samples; 2. a first receptor microsphere and a first antigen bound thereto; the surface of the first receptor microsphere is coated with a large amount of the first antigen having a high affinity for the detection antibody, but the concentration of the first receptor microsphere is relatively low, ensuring that the first antigen on the first receptor microsphere plays a dominant role when detecting high-concentration testosterone samples; 3. a detection antibody bound to biotin, wherein the detection antibody can specifically bind to testosterone; 4. the testosterone to be detected.

[0029] Figure 2 This is a correlation diagram between the measured values ​​using R1-1 as reagent 1 and the Beckman measured values.

[0030] Figure 3 This is a correlation diagram between the measured values ​​using R1-2 as reagent 1 and the Beckman measured values.

[0031] Figure 4 This is a correlation diagram between the measured values ​​using R1-3 as reagent 1 and the Beckman measured values. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] 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.

[0035] I. Terminology

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] The term "differential receptor microspheres" described in the present invention specifically refers to receptor microspheres (FG) coupled with competing antigens having different affinities with the detection antibody.

[0044] 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 intra-batch precision no greater than 20%. Analytical sensitivity is achieved through actual measurement and is also referred to as "functional sensitivity."

[0045] 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.

[0046] II. Specific implementation plan

[0047] The present invention will be described in detail below.

[0048] 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 detection antibody; second, to ensure the principle of antibody limit, the amount of detection antibody 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 homogeneous chemiluminescence detection method for quantitatively detecting the level of analytes using photochemical methods. The analytical performance indicators of this method can meet the basic requirements of industry standards or clinical laboratories. It is mainly manifested in: selecting two competing antigens with different affinities with the detection antibody to couple 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 one of the competing antigens to label biotin, it is ensured that the analyte to be detected can preferentially bind to the detection antibody when detecting low-end samples, thereby further improving the functional sensitivity.

[0049] The first aspect of the present invention relates to a competitive homogeneous chemiluminescence detection method, which comprises the following steps: contacting an analyte with a first composition, a reagent comprising a second composition and a third composition, and a fourth composition to form a test mixture; providing excitation light to illuminate the test mixture at least once; and then detecting the intensity of the chemiluminescent signal generated thereby to determine the presence and / or concentration of the analyte; wherein,

[0050] The first composition comprises a detection antibody that specifically binds to the analyte, wherein the detection antibody is bound to one member of a specific binding pair (e.g., biotin);

[0051] The second composition comprises a first receptor and a first antigen bound thereto, wherein the first antigen competes with the analyte for binding to the detection antibody;

[0052] The third composition comprises a second receptor and a second antigen bound thereto, wherein the second antigen competes with the analyte for binding to the detection antibody; the receptor can react with singlet oxygen to produce chemiluminescence;

[0053] The fourth composition comprises a donor capable of generating reactive oxygen species, the donor being bound to the other member of the specific binding pair (e.g., avidin);

[0054] The affinity of the specific binding of the first antigen to the detection antibody is higher than the affinity of the specific binding of the second antigen to the detection antibody; at the same time,

[0055] 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.

[0056] In some embodiments of the present invention, the detection antibody is a monoclonal antibody that specifically binds to the analyte.

[0057] 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), and 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.

[0058] In 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), and 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.

[0059] In some embodiments of the present invention, the concentration of the second composition in the reagent is lower than the concentration of the third composition in the reagent. In the present invention, the concentration can be either mass concentration or molar concentration.

[0060] In some preferred embodiments of the present invention, the ratio of the mass concentration of the second composition in the reagent to the mass concentration of the third composition in the reagent is 1:(10-100), preferably 1:(20-80), and more preferably 1:(40-60). In some specific embodiments of the present invention, the ratio of the mass concentration of the second composition in the kit to the mass concentration of 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.

[0061] In some specific embodiments of the present invention, the mass concentration of the third composition in the reagent is 5 to 500 ug / ml, preferably 10 to 250 ug / ml, and more preferably 15 to 200 ug / ml.

[0062] In some embodiments of the present invention, the second composition and the third composition are separately dispersed in the same buffer.

[0063] In other embodiments of the present invention, the second composition and the third composition are combined and dispersed in a buffer solution to assemble into a reagent (ie, reagent 1).

[0064] 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.

[0065] 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.

[0066] In some embodiments of the present invention, the analyte is a small molecule antigen or a hapten.

[0067] In other embodiments of the present invention, the first antigen and the second antigen are analytes and / or analyte analogs; preferably, the first antigen is the analyte and the second antigen is an analyte analog. In the present invention, the affinity of the specific binding of the analyte analog to the detection antibody is lower than the affinity of the specific binding of the analyte to the detection antibody.

[0068] In the present invention, the reagent comprising the first composition is also referred to as reagent 2.

[0069] In some embodiments of the present invention, the analyte is first contacted with the first composition, the reagent comprising the second composition and the third composition, and then the fourth composition is added thereto.

[0070] In some embodiments of the present invention, the analyte is first contacted with the releasing agent, the first composition, and the reagent comprising the second composition and the third composition, and then the fourth composition is added thereto.

[0071] In some embodiments of the present invention, the release agent comprises dimethoxyestradiol. In some specific embodiments of the present invention, the release agent comprises 40 ng / ml dimethoxyestradiol diluted in 50 mM HEPES, pH 6.0. The release agent can release protein-bound analytes from the sample being tested.

[0072] In other embodiments of the present invention, the analyte is contacted with the first composition, the reagent comprising the second composition and the third composition, and the fourth composition, and then incubated at 30-40° C. for 1-15 minutes to form a test mixture.

[0073] The method of the present invention utilizes two types of receptor microspheres to intelligently function as follows:

[0074] For low-concentration analyte specimens (female specimens), the number of analyte molecules to be detected is small, and the analyte to be detected is not easy to bind to the detection antibody. In Reagent 1, the number of second receptor microspheres bound to the second antigen is predominant, 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. In other words, the second receptor microspheres with high microsphere concentration are predominant. The number of second antigen molecules on the surface of the second receptor microspheres is small, and the affinity with the detection antibody is lower than that of the analyte molecules to be detected in the specimen. This ensures that the analyte to be detected has a stronger ability to bind to the detection antibody, thereby achieving better functional sensitivity.

[0075] 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, the contribution of the number of microspheres to the competitive reaction is not obvious at this time because the number of second antigen molecules on the surface of the second receptor microspheres is small. 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 with the antibody is the same as that of the analyte molecules to be detected in the specimen. The strength of the first antigen binding antibody can be balanced with the high concentration of the analyte to be detected, thereby obtaining an ideal detection range.

[0076] In some specific embodiments of the present invention, the method for detecting the analyte using the method includes:

[0077] Step N1, mixing the sample to be tested, reagent 1, and reagent 2 to obtain a first mixture;

[0078] Step N2, mixing the donor microsphere solution bound to avidin with the first mixture to obtain a second mixture;

[0079] 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;

[0080] 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.

[0081] In the method of the present invention, the reagents can be incubated as needed after mixing.

[0082] 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.

[0083] 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.

[0084] The serum sample to be tested, reagent 1, and reagent 2 are mixed and incubated. The analyte in the serum sample and the competing antigen coupled to the receptor microspheres competitively bind to the detection antibody (Bio-McAb) to form complexes (Bio-McAb-competing antigen-FG, Bio-McAb-analyte-FG), respectively. Subsequently, SA-GG (donor microspheres bound to avidin) binds to biotin (Bio), and the receptor microspheres and donor microspheres approach each other, inducing the generation of light signals after excitation. Free receptor 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 analyte content in the serum sample to be tested. The analyte concentration level of an unknown serum sample can be calculated using a mathematical function formed by a calibrator of known analyte concentration.

[0085] The second aspect of the present invention provides an application of the method according to the first aspect of the present invention in a chemiluminescence analyzer.

[0086] Example

[0087] 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.

[0088] Example 1: Preparation of Reagents Used in the Method of the Present Invention Where the Analyte is Testosterone Reagents and Instruments:

[0089] Biotin-conjugated testosterone antibody, receptor microspheres, BSA-modified dihydrotestosterone (BSA-DHT), BSA-modified testosterone (BSA-T), phosphate buffer (0.02 M PBS, pH 7.2), BSA, Tween-20, LiCA 500 (Beijing Kemei Biotechnology Co., Ltd.), and Hitachi high-speed refrigerated centrifuge.

[0090] Preparation process:

[0091] (1) Preparation of competitive antigen-coupled receptor microsphere solution (reagent 1)

[0092] 1) Place 2 mg of receptor microspheres in two 2 mL centrifuge tubes, centrifuge at 10,000 rpm for 15 min at 4°C, and wash once.

[0093] 2) Ultrasonic dispersion was performed, and 0.2 mg BSA-T was added to one centrifuge tube and 0.02 mg BSA-DHT was added to the other tube. The mixture was mixed thoroughly and coated at 4°C overnight.

[0094] 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 hours;

[0095] 4) Wash the acceptor microspheres three times by centrifugation with PBS buffer solution containing 0.5% Tween-20, and then dilute the acceptor microspheres in two centrifuge tubes to 0.1 mg / ml with pH 7.2, 0.05 M PBS solution, respectively. Label them as FG-NT (R1-1) and FG-nDHT (R1-2) and store them.

[0096] 5) R1-1 and R1-2 were mixed to make the microsphere concentration ratio of FG-NT:FG-nDHT=1:50, marked as R1-3, and used as reagent 1.

[0097] (2) Preparation of detection antibody conjugated to biotin (reagent 2)

[0098] The testosterone antibody conjugated to biotin was diluted 20,000-fold using the diluent of reagent 2 to an antibody concentration of 0.05 μg / ml, which was used as reagent 2.

[0099] (3) Preparation of release agent

[0100] The release agent component is: 40 ng / ml dimethoxyestradiol, which is diluted with 50 mM HEPES pH 6.0.

[0101] (4) Preparation process of T series calibrators with known concentrations

[0102] A 1 mg / mL testosterone solution was added to horse serum to prepare calibrators 1 to 6 with concentrations of 0 ng / dl, 50 ng / dl, 150 ng / dl, 400 ng / dl, 800 ng / dl, and 1600 ng / dl, respectively.

[0103] Example 2

[0104] The testosterone-containing samples were tested using R1-1, R1-2, and R1-3 prepared in Example 1 as reagent 1, and the test results were compared with the Beckman test values. The results were as follows: Figure 2-4 shown.

[0105] The detection process is fully automated by the LiCA500 automatic photochemiluminescence analysis system and the test results are output. The specific steps are as follows:

[0106] a. Add 20 μl of sample, calibrator, or control into each reaction well;

[0107] b. Add 20 μl of release agent, 25 μl of reagent 1, and 25 μl of reagent 2 to the reaction wells in sequence;

[0108] c. Incubate at 37°C for 15 minutes;

[0109] d. Add 175 μl of LiCA universal solution (donor microsphere solution bound to avidin);

[0110] e. Incubate at 37°C for 15 minutes;

[0111] e. Laser irradiation of microwells and calculation of the amount of photons emitted from each well;

[0112] f. Calculate the sample concentration based on the calibration curve.

[0113] from Figure 1-3 As shown, when FG-nDHT was used as a competing antigen, the correlation with Beckman's method was good for low-value samples, but poor for high-value samples. When FG-NT was used as a competing antigen, the functional sensitivity (detection limit) for low-value values ​​was poor, but the correlation for high-value values ​​was good. When a combination of the two competing antigens was used, both the functional sensitivity for low-value testosterone and the detection range were achieved. In terms of correlation with Beckman's methods, the best results were achieved when FG-nDHT and FG-NT were used in combination.

[0114] Example 3: Precision testing

[0115] Intra-batch precision testing: Using the reagents used in the method described herein and high, medium, and low-dose samples, precision testing was performed. Each batch of reagent was measured 10 times. The mean (X) and standard deviation (SD) of the 10 measurements were calculated. The coefficient of variation (CV) was calculated using the formula (CV = SD / X × 100%). The results are shown in Tables 1 and 3.

[0116] Inter-batch precision testing: Three batches of the reagent used in the method described herein were used to test high, medium, and low-dose samples, each replicated 10 times. The mean (X) and standard deviation (SD) of the 30 measurements were calculated, and the coefficient of variation (CV) was calculated using the formula (CV = SD / X × 100%). The results are shown in Tables 2 and 3.

[0117] Table 1: Raw data of intra-batch precision of LiCAT reagent (photochemiluminescence)

[0118]

[0119]

[0120] Table 2: Raw data of inter-batch precision of three batches of LiCAT reagent (photochemiluminescence)

[0121]

[0122]

[0123] Table 3: Analytical precision of reagents used in the method of the present invention (photochemiluminescence method)

[0124]

[0125] As shown in Table 3, the intra-batch and inter-batch precisions of the three batches of reagents were all <2%, indicating that the measured values ​​of the reagents used in the method of the present invention have good repeatability and small random errors.

[0126] Example 4: Accuracy detection

[0127] The meaning of accuracy: the degree of consistency between the measured value and the true value, reflecting the size of the system error.

[0128] Accuracy assessment method: Reference serum with concentrations of 0.40 ng / mL, 5.97 ng / mL, and 8.15 ng / mL was used for testing. The recovery rate was calculated according to Formula 1. The results are shown in Table 4.

[0129] B=Xi / T×100% Formula 1

[0130] Where: B-recovery rate; X i - measured concentration of the sample; T - target value of the sample.

[0131] Table 4: Accuracy of reagents used in the method of the present invention (photochemiluminescence method)

[0132]

[0133] As can be seen from Table 4, after testing with samples of different concentrations, the recovery rates are all within the range of 100% to 110%, indicating that the measured values ​​are close to the true values ​​and the detection error of the reagents used in the method of the present invention is small.

[0134] Example 5:

[0135] Using the method described in Example 1, the same batch of samples was tested using the LiCA500 automated photochemiluminescence analysis system, with varying conditions such as the type of competing antigen, the mass ratio of the coupling, and the concentration of the receptor microspheres. The system automatically generated and output homogeneous chemiluminescence signals, and analyzed the detection range and detection limit. The results are shown in Table 5.

[0136] Table 5

[0137]

[0138] As can be seen from Table 5, when FG-nDHT is used as the competing antigen, it is more favorable for the detection limit, but the high-value sample measurement value is low and the linear range is narrow; when FG-NT is used as the competing antigen, 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 competing antigens, the low-value functional sensitivity and detection range of testosterone can be taken into account.

[0139] 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 chemiluminescent detection method comprising the following steps: contacting an analyte with a first composition, a reagent comprising a second composition and a third composition, and a fourth composition to form a test mixture; irradiating the test mixture with excitation light at least once; and then detecting the intensity of the chemiluminescent signal generated thereby to determine the presence and / or concentration of the analyte; wherein: The first composition comprises a detection antibody that specifically binds to the analyte, wherein the detection antibody binds to one of the specific binding pair members; The second composition comprises a first receptor and a first antigen bound thereto, wherein the first antigen competes with the analyte for binding to the detection antibody; the third composition comprises a second receptor and a second antigen bound thereto, wherein the second antigen competes with the analyte for binding to the detection antibody; the receptor is capable of reacting with singlet oxygen to produce chemiluminescence; the first receptor and the second receptor are both receptor microspheres; The fourth composition comprises a donor capable of generating reactive oxygen species, the donor being bound to the other member of the specific binding pair; The affinity of the specific binding of the first antigen to the detection antibody is higher than the affinity of the specific binding of the second antigen to the detection antibody; 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 ratio of the mass concentration of the second composition in the reagent to the mass concentration of the third composition in the reagent is 1:(10-100).

2. The method according to claim 1, characterized in that The mass ratio of the second antigen to the second receptor is 1:(50-150).

3. The method according to claim 2, characterized in that The mass ratio of the second antigen to the second receptor is 1:(80-120).

4. The method according to claim 1, wherein The mass ratio of the first antigen to the first receptor is 1:(5-15).

5. The method according to claim 4, characterized in that The mass ratio of the first antigen to the first receptor is 1:(8-12).

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

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

8. The method according to claim 1, characterized in that The mass concentration of the third composition in the reagent is 5 to 500 ug / ml.

9. The method according to claim 8, characterized in that The mass concentration of the third composition in the reagent is 10 to 250 ug / ml.

10. The method according to claim 9, characterized in that The mass concentration of the third composition in the reagent is 15 to 200 ug / ml.

11. The method according to claim 1, characterized in that The second composition and the third composition are separately dispersed in the same buffer solution.

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

13. The method according to claim 1, wherein The first receptor and the second receptor are respectively first receptor microspheres and second receptor microspheres containing a high molecular weight polymer carrier.

14. The method according to claim 13, characterized in that The average particle size of the first acceptor microspheres is the same as the average particle size of the second acceptor microspheres.

15. The method according to claim 1, wherein The analyte is a small molecule antigen or a hapten.

16. The method according to claim 1, wherein The first antigen and the second antigen are analytes and / or analyte analogs.

17. The method according to claim 1, wherein The first antigen is an analyte, and the second antigen is an analyte analog.

18. The method according to claim 1, wherein The analyte is first contacted with the first composition, the reagent comprising the second composition and the third composition, and then the fourth composition is added thereto.

19. The method according to claim 1, wherein The analyte is contacted with the first composition, the reagent comprising the second composition and the third composition, and the fourth composition, and then incubated at 30-40° C. for 1-15 minutes to form a test mixture.

20. Use of the method according to any one of claims 1 to 19 in a chemiluminescence analyzer.

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