Detection reagents, kits, and methods for simultaneously improving sensitivity and broadening detection range in competitive immunoassays.

Abstract

This application relates to a detection reagent, kit, and method that simultaneously improves sensitivity and broadens the detection range in competitive immunoassay. The reagent includes a detection antibody bound to luminescent microspheres that specifically binds to analyte molecules in the sample, and two sets of labeled antigens containing the same competing antigen and labeled molecules of different molecular weights bound to it. The competing antigen competes with the analyte molecule for binding to the detection antibody, and the affinity of the competing antigen for binding to the detection antibody is higher than that of the analyte molecule for binding to the detection antibody. The technical solution of this application, by using two sets of labeled antigens with different affinities, ensures that the labeled antigens that play a dominant role in samples with varying concentrations of analyte molecules differ. This is suitable for competitive immunoassay of small molecule antigens or haptens in both low-concentration and high-concentration samples, and can broaden the detection range while ensuring high functional sensitivity.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to a detection reagent, kit, and method that simultaneously improves sensitivity and broadens the detection range in competitive immunoassays. Background Technology

[0002] Small molecules, such as steroid hormones (estradiol, testosterone, progesterone, etc.), typically possess only a single antigenic epitope and are classified as haptens, meaning they cannot simultaneously bind to two antibody molecules. This limits the application of traditional sandwich assays in the immunoassay of small molecules. Therefore, the immunoassay of small molecules primarily utilizes competitive immunoassay methods.

[0003] Competitive immunoassay is an analytical technique based on antigen-antibody specific reactions. In this method, a competing antigen labeled with a tracer (labeled antigen) and the target antigen in the sample compete for binding with a specific antibody, forming a target antigen-specific antibody complex and a competing antigen-specific antibody complex. After separating the competing antigen-specific antibody complex and the free competing antigen, the tracer signal intensity on the competing antigen-specific antibody complex is measured, and the concentration of the target antigen in the sample is obtained using a dose-response standard function.

[0004] The competitive assay curve is an inverted "S" curve, with a flattened head and tail and an almost straight central portion, providing an ideal detection range, i.e., a linear interval, but this interval is very narrow. When clinical tests require a wide detection range and high functional sensitivity, traditional competitive assay reagents cannot meet the corresponding testing needs. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a detection reagent, kit, and method that simultaneously improves sensitivity and broadens the detection range in competitive immunoassay. This method can intelligently select labeled antigens with high or low affinity to compete with the analyte molecules for binding to detection antibodies based on the concentration differences of the analyte molecules. It is suitable for both low-concentration and high-concentration samples, and can broaden the detection range while ensuring high functional sensitivity, thus meeting clinical testing requirements.

[0006] The first aspect of this application provides a photo-induced chemiluminescence detection reagent, comprising:

[0007] A luminescent composition comprising luminescent microspheres and a detection antibody bound thereto, the detection antibody being capable of specifically binding to a target molecule in a test sample;

[0008] A first labeled antigen and a second labeled antigen, comprising the same competing antigen and the same labeled molecule of different molecular weights bound to the competing antigen, wherein the competing antigen is capable of binding to the detection antibody;

[0009] The competitive antigen has a higher affinity for the detection antibody than the analyte has for the detection antibody.

[0010] In some embodiments, the first labeled antigen comprises a first labeled molecule, and the second labeled antigen comprises a second labeled molecule; the molecular weight of the first labeled molecule is lower than the molecular weight of the second labeled molecule.

[0011] In some embodiments, the molecular weight of the second labeled molecule is at least 2.5 times that of the first labeled molecule; preferably 3 to 40 times.

[0012] In some embodiments, the molecular weight of the first labeled molecule is ≤800 Da.

[0013] In some embodiments, the molecular weight of the second labeled molecule is ≥2000 Da.

[0014] In some embodiments, the first and second labeled molecules are selected from biotin containing hydrophilic groups; the biotin is bound to hydrophilic polymers of different molecular weights; the polymers are selected from PEG or dextran.

[0015] In some embodiments, the concentration of the first labeled antigen in the reagent is less than or equal to the concentration of the second labeled antigen.

[0016] In some embodiments, the mass ratio of the first labeled antigen to the second labeled antigen is 1:(1 to 10).

[0017] In some embodiments, the molar ratio of competing antigens in the two sets of labeled antigens to the corresponding labeled molecules is the same.

[0018] In some embodiments, the first labeled antigen and the second labeled antigen are mixed and dispersed in a buffer solution to assemble a reagent.

[0019] A second aspect of this application provides a photo-induced chemiluminescence detection kit, comprising:

[0020] R1 reagent comprises luminescent microspheres and a detection antibody bound thereto; the detection antibody is capable of specifically binding to the analyte molecule in the test sample.

[0021] The R2 reagent comprises a first labeled antigen and a second labeled antigen; the first labeled antigen and the second labeled antigen contain the same competing antigen and the same labeled molecule with different molecular weights that bind to it; the affinity of the competing antigen to the detection antibody is higher than the affinity of the analyte molecule to the detection antibody.

[0022] R3 reagent contains a releasing agent.

[0023] A third aspect of this application provides a method for simultaneously improving sensitivity and broadening the detection range in competitive immunoassays, wherein the same competing antigen is labeled simultaneously with homolabeled molecules of different molecular weights during detection.

[0024] In some embodiments, the molecular weight of the second labeled molecule is at least 2.5 times that of the first labeled molecule; preferably 3 to 40 times.

[0025] In some embodiments, the molecular weight of the first labeled molecule is ≤800 Da.

[0026] In some embodiments, the molecular weight of the second labeled molecule is ≥2000 Da.

[0027] The fourth aspect of this application provides the application of the above-described reagents, kits, and methods in the detection of small molecule antigens or haptens.

[0028] In some embodiments, the small molecule antigen or hapten is a steroid hormone; preferably estradiol, testosterone, androstenedione, progesterone, estrone, or cortisol.

[0029] It is worth noting that the application described is for purposes other than disease diagnosis.

[0030] The technical solution provided in this application may include the following beneficial effects:

[0031] By labeling the same antigen with the same marker of different molecular weights, two sets of labeled antigens with different affinities when binding to the detection antibody are obtained. The labeled antigen with high affinity provides a wider detection range, while the labeled antigen with low affinity provides higher sensitivity, thus balancing high functional sensitivity and a wide detection range. Specifically, the concentration of the analyte molecule differs between low-concentration and high-concentration samples. When the two labeled antigens are mixed and used as a single reagent, the labeled antigen that plays the dominant role differs in samples of different concentrations. This method is suitable for detecting both high-concentration and low-concentration samples, balancing high functional sensitivity and a wide detection range, and eliminating the need for multiple tests, thus saving detection costs and time. This competitive detection method can be applied to the detection of small molecule antigens or haptens, meeting the clinical testing needs of small molecule substances.

[0032] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0033] Figure 1 The figure shows the correlation between the concentrations of samples with concentrations of 0–3.15 ng / mL detected using reagent R2-14 and the values ​​measured using the control reagent, where the sample size n = 40.

[0034] Figure 2 The figure shows the correlation between the concentrations of samples (3.15–40 ng / mL) detected using reagent R2-14 and the values ​​measured using the control reagent, where the sample size n = 40.

[0035] Figure 3 The figure shows the correlation between the concentrations of samples with concentrations of 0–40 ng / mL detected using reagent R2-14 and the values ​​measured using the control reagent, where the sample size n = 80. Detailed Implementation

[0036] To facilitate understanding of the present invention, it 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 describing specific embodiments only and is not intended to be restrictive.

[0037] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0038] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0039] I. Terminology

[0040] The term "molecule to be tested" as used herein refers to molecules whose various properties (such as structure, content, activity, etc.) are to be studied or detected. In this application, these molecules specifically refer to detectable biomacromolecules and small biomolecules. Biomacromolecules include proteins, nucleic acids, and bioactive polypeptides. Specifically, proteins may include immunoglobulins such as IgG and IgM, cytokines such as interleukins and interferons, and tumor marker proteins such as alpha-fetoprotein and carcinoembryonic antigen. Nucleic acids may specifically include DNA and RNA. Polypeptides may specifically include insulin and growth hormone-releasing peptide. Small biomolecules include hormones, vitamins, and small metabolic molecules. Hormones may include thyroid hormones such as thyroxine and triiodothyronine, sex hormones such as estradiol, progesterone, and testosterone, and adrenocortical hormones such as cortisol and aldosterone.

[0041] The term "sample / test sample" as used in this article refers to a mixture that may contain the analyte molecule. Typical (test) samples include bodily fluids such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion. Before use, the test sample may be diluted with a diluent or buffer solution to obtain a solution that may contain the analyte molecule. For example, to avoid the hook effect, the analyte molecule can be diluted with a sample diluent before detection on the instrument. In this case, the diluted solution that may contain the analyte molecule is collectively referred to as the test sample.

[0042] The term "antibody" as used herein is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins containing the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other parts, such as biotin or avidin.

[0043] The antigens described herein refer to substances capable of inducing antibody production, and can be classified into complete antigens and incomplete antigens (haptens). These antigens can be natural antigens extracted from pathogens or animal tissues, or recombinant antigens with specific antigenic properties prepared through genetic engineering. Where necessary, antigens can be further conjugated to other components, such as biotin or avidin.

[0044] The labeled molecules / tracers mentioned in this article refer to substances that can be used to identify and track specific substances or processes. Labeled molecules / tracers can be attached to antigens / antibodies to enhance the functional sensitivity of immune responses; examples include isotopes, fluorescein, biotin, enzymes, colloidal gold, and ferritin.

[0045] The trapping molecules described in this article are substances that can interact with labeled molecules through covalent or non-covalent bonds, such as avidin, which can bind to biotin.

[0046] Biotin, as described in this article, is a small molecule widely found in animal and plant tissues. Its molecule has two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the primary site for binding to avidin or streptavidin. Activated biotin can be coupled to almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents. Biotin can be further modified through coupling or genetic engineering techniques to obtain derivatives with activating functional groups, such as biotin derivatives containing amino (-NH2), carboxyl (-COOH), NHS-activated esters, maleimide, thiol, azide, or alkyne groups. Biotin can also bind to polymers containing hydrophilic groups, such as the aforementioned -NH2 and carboxyl-COOH groups. Examples of such polymers include PEG (polyethylene glycol) and dextran.

[0047] The streptavidin described herein is a protein secreted by Streptomyces. The streptavidin molecule consists of four identical peptide chains, each capable of binding one biotin, with a molecular weight of 65 kDa. Each antigen or antibody can simultaneously couple multiple biotin molecules, thereby creating a "tentacle effect" with streptavidin to enhance analytical sensitivity. Where necessary, any reagent used in this invention, including antigens, antibodies, receptors, or donors, can be conjugated to any member of the biotin-streptavidin combination, depending on the specific requirements.

[0048] The releasing agent is a substance that can promote the release of the analyte molecule from the binding protein, facilitating accurate measurement of the analyte content in the sample. After mixing with the sample, the releasing agent disrupts the binding between the analyte molecule and the binding protein, allowing the analyte molecule to exist in the sample in a free form. The analyte molecules for which a releasing agent is required in this invention can be small biological molecules, particularly small molecule antigens or haptens, such as sex hormones and adrenocortical hormones.

[0049] The binding / coupling described herein refers to the union between two substances caused by interactions such as covalent, electrostatic, hydrophobic, ionic and / or hydrogen bonding, or interactions including but not limited to salt bridges and water bridges.

[0050] The specific binding described in this article refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, is the conformational correspondence between the reactants in the response.

[0051] The affinity described in this article refers to the binding strength between the antigen-binding site of an intact antibody molecule and several corresponding antigenic epitopes, and is composed of the sum of multiple affinities. Affinity refers to the strength of a single binding interaction between an antigenic epitope and a monovalent antibody.

[0052] The luminescent microspheres described herein refer to polymeric microparticles filled with luminescent substances, capable of reacting with reactive oxygen species to generate detectable light signals. Luminescent microspheres may also be called acceptor microspheres or luminescent microparticles. In some specific embodiments of the invention, the luminescent substance undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkylacridinium, aryl vinyl ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. In other specific embodiments of the invention, the luminescent substance may further comprise europium complexes; more preferably, the europium complex is MTTA-EU. 3+ .

[0053] The photosensitive microspheres described herein refer to polymeric microparticles filled with photosensitizers that can generate reactive oxygen species upon photoexcitation. These can also be called donor microspheres or photosensitive microparticles. Solutions containing such photosensitive microspheres can be called photosensitive solutions or universal solutions. The photosensitizers can be those known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, but are not limited to these. The photosensitive microspheres can also be filled with other sensitizers; non-limiting examples include certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species.

[0054] The reactive oxygen species (ROS) discussed in this article refer to a general term for oxygen-containing and reactive substances in the body or natural environment. They are primarily excited-state oxygen molecules, including the one-electron reduction product superoxide anion (O2·-), the two-electron reduction product hydrogen peroxide (H2O2), the three-electron reduction product hydroxyl radical (·OH), as well as nitric oxide and singlet oxygen. 1 O2), etc.

[0055] The microparticles described herein can be of any size and shape, expandable or non-expandable, porous or non-porous, and have any density, but preferably close to that of water. They are preferably buoyant in water and are composed of transparent, partially transparent, or opaque materials. The microparticles can be solids (such as polymers, metals, glass, organic or inorganic substances such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and siliceous fluids), vesicles (such as synthetic phospholipids, or natural substances such as cells and organelles). A non-limiting example of microparticles suitable for use in this invention is carboxylated polystyrene latex microspheres.

[0056] The functional sensitivity / sensitivity mentioned in this article refers to the limit of detection, which is the lowest concentration that an analytical method can detect after serially diluting a sample of known concentration, and the intra-assay precision cannot exceed 20%. Analytical sensitivity is obtained from actual measurements and is also known as "functional sensitivity".

[0057] The detection range mentioned in this article refers to the effective range of the dose function. For example, if a high-concentration sample is serially diluted, the results of the determination of the diluted sample are analyzed by linear regression, and the correlation coefficient (R) is greater than 0.990.

[0058] The inhibition rate described in this article refers to the ratio of the current signal value shown by the analysis system to the zero-point signal value, which is the signal value of the calibrator at a concentration of 0 ng / mL. The inhibition rate indicates how sensitive the reagent is to changes in the concentration of the analyte; a low inhibition rate indicates high sensitivity.

[0059] The overall discrimination ratio described in this article refers to the multiple between the signal value of the 0 ng / mL calibrator and the signal value of the highest concentration. The low-end discrimination ratio described in this article refers to the multiple between the signal value of the 0 ng / mL calibrator and the signal value of the 1 ng / mL concentration.

[0060] II. Specific Implementation Plan

[0061] This application will now be described in more detail.

[0062] Photocatalytic chemiluminescence (PRC) analysis is a next-generation immunoassay technique based on nanoscale polymer particles. Its core principle is the generation and transfer of reactive oxygen species (ROS): energy transfer between photosensitive microspheres (GG) and luminescent microspheres (FG) generates high-energy red light during energy level transitions. A single-photon counter and mathematical fitting convert the photon count into a relative light signal. In the presence of the target analyte (e.g., the analyte molecule / antibody), the antibody / antigen modified on the surfaces of the two microspheres undergoes an antigen-antibody specific binding reaction, shortening the spatial distance between the two microspheres. The light signal generated by the ROS transfer between the two microspheres enables qualitative or quantitative analysis of the analyte molecule / antibody.

[0063] The Heidelberg curve is a theoretical curve describing the relationship between precipitate formation and the ratio of antigen to antibody in antigen-antibody reactions. The formation of antigen-antibody immune complexes initially increases and then gradually decreases with increasing antigen concentration, exhibiting a parabolic curve. Studies have also found that the starting and ending points of the Heidelberg curves differ depending on whether the antibody binds to antigens with different competitive abilities. The competitive ability of the antigen directly affects the detection capability of the reagent. Antigens with weak competitive ability are suitable for low-concentration samples, providing high functional sensitivity; antigens with strong competitive ability are suitable for high-concentration samples, providing a wide detection range. Developing two separate reagents for different antigens to detect low-concentration and high-concentration samples would significantly increase reagent costs and the workload of testing personnel, and weak signal intensity would affect detection precision and detection capability.

[0064] To achieve optimal detection capability, reagents can improve detection sensitivity by reducing the competitive ability of competing antigens. Currently, there are two main approaches to reducing the competitive ability of antigens: one is to use structural analogs as competing antigens. These analogs need to exhibit some degree of cross-reactivity with specific antibodies and have weak affinity, ensuring that the antigen preferentially binds to the antibody even at low concentrations. However, the high structural similarity between the structural analog and the antigen can easily cause interference, affecting the accuracy of the detection results. Furthermore, their weak affinity results in weak signal intensity, impacting the reagent's detection capability and precision. Another method involves conjugating a large protein (such as bovine serum albumin (BSA) or ovalbumin (OVA)) to the antigen to obtain a competing antigen. This competing antigen has a higher molecular weight, which slows down the diffusion rate of the competing antigen and reduces the probability of collision with specific antibodies, ensuring that the antigen to be tested can preferentially bind to the antibody. However, the conjugation method varies greatly from batch to batch, making it difficult to accurately control the conjugation ratio of antigen and large protein between batches, resulting in inconsistent detection capabilities between reagents from batch to batch.

[0065] Moreover, reducing the amount of competing antigens, using antigens with weaker competitive ability, or conjugating large molecular proteins to improve the sensitivity of the detection function will simultaneously lead to weak detection signal intensity, affecting detection precision and detection range. It is difficult to balance functional sensitivity and detection range, thus limiting its clinical application.

[0066] The inventors of this application address the inherent performance limitations of current competitive immunoassays, which cannot simultaneously achieve high functional sensitivity and a wide detection range. By labeling the same antigen with two different molecular weight markers, two sets of labeled antigens with different binding affinities are obtained, enabling specific binding to detection antibodies. These antigens can be used in photo-induced chemiluminescence immunoassay platforms with luminescent and photosensitive microspheres to detect analytes, particularly small molecule antigens or haptens. In the detection system, the different labeled antigens in the two sets play a dominant role at varying antigen concentrations, satisfying the detection needs of both high- and low-concentration samples, as well as the competitive quantitative analysis requirements of small molecule haptens, ensuring high functional sensitivity and a wide detection range.

[0067] The photoluminescent detection reagent involved in this application includes a luminescent composition, a first labeled antigen, and a second labeled antigen. The luminescent composition comprises luminescent microspheres and a detection antibody bound to the surface of the luminescent microspheres. The detection antibody can specifically bind to the analyte molecule in the sample. The first labeled antigen and the second labeled antibody each contain the same competing antigen and the same labeled molecule bound to the competing antigen. The competing antigen can compete with the analyte molecule for binding to the detection antibody. The labeled molecules on the two sets of labeled antigens have different molecular weights; and the affinity of the competing antigen for binding to the detection antibody is higher than the affinity of the analyte molecule for binding to the detection antibody.

[0068] In this application, two groups of labeled molecules with different molecular weights bind to the same competing antigen. The resulting two groups of labeled antigens have different molecular weights and therefore different affinities for binding to the detection antibody. The Heidelberg curves for the binding of the two groups of labeled antigens with different affinities to the detection antibody are different.

[0069] High-affinity labeled antigens are suitable for competitive binding to detection antibodies when the concentration of the analyte molecule is high; low-affinity labeled antigens are suitable for competitive binding to detection antibodies when the concentration of the analyte molecule is low. The distribution density of the analyte molecule differs between high-concentration and low-concentration samples; the former has a higher density, and the latter a lower density.

[0070] In high-concentration samples, the high density of analyte molecules makes it easier for them to contact and bind to the detection antibody molecules on the surface of the luminescent microspheres. In this case, the labeled antigen with relatively high affinity plays a dominant role, binding more quickly to the detection antibody molecules on the luminescent microsphere surface. This reduces the number of analyte molecules bound to the detection antibody on the luminescent microsphere surface, resulting in a wider detection range. Conversely, in low-concentration samples, the low density of analyte molecules reduces the probability of contact and binding between the analyte molecules and the detection antibody molecules on the luminescent microsphere surface. In this case, the labeled antigen with relatively low affinity plays a dominant role, making it easier for the detection antibody on the luminescent microsphere surface to bind to the analyte molecules in the sample compared to competing antigens, resulting in higher sensitivity. Therefore, by using two labeled antigens with different competitive abilities, it is possible to ensure that one labeled antigen plays a dominant role regardless of the concentration of analyte molecules in the sample, while simultaneously achieving high functional sensitivity and a wide detection range.

[0071] In some embodiments of this application, the first labeled antigen and the second labeled antigen are separately dispersed in the same buffer solution.

[0072] In other embodiments of this application, the first labeled antigen and the second labeled antigen are mixed and dispersed in a buffer solution to assemble a reagent (reagent R2). The mixed reagent contains both the first labeled antigen and the second labeled antigen. The labeled antigen that plays the main role varies depending on the concentration of the analyte in the sample, thus meeting the detection requirements of samples with different concentrations.

[0073] High-affinity labeled antigens can be defined as HA-antigens; low-affinity labeled antigens can be defined as LA-antigens. When these two are mixed and assembled into reagent R2, the dominant antigen differs depending on the concentration of the analyte in the sample. This R2 reagent is suitable for detecting analytes in both low- and high-concentration samples.

[0074] In some embodiments of this application, the first labeled antigen comprises a first labeled molecule, and the second labeled antigen comprises a second labeled molecule, wherein the molecular weight of the first labeled molecule is lower than that of the second labeled molecule. Therefore, the first labeled antigen has a relatively lower molecular weight, diffuses relatively faster in the detection system, has a high affinity for the detection antibody, and can provide a wide detection range; the second labeled antigen has a relatively higher molecular weight, diffuses relatively slower in the detection system, has a low affinity for the detection antibody, and can provide high sensitivity.

[0075] In some preferred embodiments of this application, the molecular weight of the second labeled antigen is at least 2.5 times that of the first labeled antigen; preferably 3 to 40 times.

[0076] In some specific embodiments of this application, the molecular weight of the first labeled molecule is ≤800 Da.

[0077] In some specific embodiments of this application, the molecular weight of the second labeled molecule is ≥2000 Da.

[0078] Both the first and second labeled antigens are homologous to the analyte molecule; that is, the antigens of the first and second labeled antigens are the analyte molecule or its structural analogues. By adjusting the molecular weight of the labeled molecules on the two labeled antigens, the binding affinity between the competing antigens and the detection antibody can be modulated, reducing batch-to-batch variability and ensuring the consistency of test results.

[0079] In some embodiments of this application, the molar ratio of competing antigens in the two groups of labeled antigens to their corresponding bound labeled molecules is the same.

[0080] The difference between the two groups of labeled antigens in this application lies in the different molecular weights of the two labeled molecules. By using labeled molecules of different molecular weights, the affinity between the two groups of labeled antigens and the detection antibody is different, so that they can be used for the detection of both high-concentration and low-concentration samples.

[0081] In some preferred embodiments of this application, the first labeling molecule and the second labeling molecule are selected from biotin containing hydrophilic groups, such as hydroxyl, amino, carboxyl, aldehyde, etc.

[0082] In some preferred embodiments of this application, biotin is bound to hydrophilic polymers of different molecular weights, such as biotin bound to PEG or dextran.

[0083] For example, when the biotin is selected from biotin conjugated with PEG, i.e., Biotin-(PEG). n When -NH2, the first labeled molecule can be selected from biotin with a molecular weight of 588 Da, and the second labeled molecule can be selected from biotin with a molecular weight of 20 kDa.

[0084] For example, when the biotin is selected from dextran-bound biotin, i.e., dextran-Biotin, the first labeling molecule can be selected from biotin with a molecular weight of 540 Da, and the second labeling molecule can be selected from biotin with a molecular weight of 20 kDa.

[0085] In some preferred embodiments of this application, biotin is selected from biotin conjugated with PEG.

[0086] In some embodiments of this application, the concentration of the first labeled antigen in the reagent is less than or equal to the concentration of the second labeled antigen.

[0087] In some embodiments of this application, the mass ratio of the first labeled antigen to the second labeled antigen is 1:(1 to 10). In some specific embodiments of this application, the mass ratio of the first labeled antigen to the second labeled antigen is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, etc.

[0088] In some embodiments of this application, the mass concentration of the first labeled antigen in the reagent is 0.05 to 10 ng / mL.

[0089] In some embodiments of this application, the mass concentration of the second labeled antigen in the reagent is 0.05 to 10 ng / mL.

[0090] In this application, by further adjusting the proportion of the first labeled antigen and the second labeled antigen in the reagent, the two groups of labeled antigens can better exert the main role of one of the labeled antigens in samples of different concentrations, while reducing interference, significantly improving the detection capability of the reagent, and improving high sensitivity and broadening the detection range.

[0091] In some embodiments of this application, the mass ratio of the detection antibody to the luminescent microspheres is 1:(5-100); preferably 1:(10-50). In some specific embodiments of this application, the mass ratio of the detection antibody to the luminescent microspheres is 1:5, 1:10, 1:20, 1:30, 1:50, 1:100, etc. In this application, the reagent comprising the detection antibody and the luminescent microspheres is referred to as reagent R1.

[0092] In some embodiments of this application, the molecule to be tested can be a small molecule hormone such as estradiol, testosterone, androgen, progesterone, estrone, or cortisol. The detection antibody is a corresponding antibody to the aforementioned small molecule hormone. The antigen in the competing antigen can be the aforementioned small molecule hormone or its analogue.

[0093] In some embodiments of this application, the reagent further includes a releasing agent for promoting the release of the analyte molecule. Specifically, the releasing agent is selected from one or more of dihydrotestosterone, mesostearin, 8-anilino-1-naphthalenesulfonic acid, and diethylstilbestrol. In this application, the reagent containing the releasing agent is referred to as reagent R3.

[0094] In some embodiments of this application, the reagent further includes photosensitive microspheres and a trapping molecule bound thereto, the trapping molecule being capable of specifically binding to the labeled molecule. Preferably, the trapping molecule can be avidin, which is capable of specifically binding to biotin. In this application, the reagent comprising the photosensitive microspheres and the trapping molecule is referred to as reagent R4.

[0095] In some embodiments of this application, the reagent further includes a series of calibrator solutions of the analyte molecule with known concentrations; preferably, the concentration of the analyte molecule in the series of calibrator solutions is 0–4800 ng / L.

[0096] The photo-induced chemiluminescence detection kit involved in this application includes:

[0097] The R1 reagent contains luminescent microspheres and a detection antibody bound to them; the detection antibody can specifically bind to the analyte molecule in the test sample.

[0098] The R2 reagent contains a first labeled antigen and a second labeled antigen; the two groups of labeled antigens contain the same competing antigen and labeled molecules of different molecular weights that bind to it; the affinity of the competing antigen to the detection antibody is higher than the affinity of the analyte molecule to the detection antibody.

[0099] R3 reagent contains a releasing agent.

[0100] In some embodiments of this application, the kit further includes an R4 reagent comprising photosensitive microspheres and a capture molecule bound thereto, the capture molecule being capable of specifically binding to the labeled molecule.

[0101] The primary labeled antigens differ between low-concentration and high-concentration samples. For high-concentration samples, labeled antigens with low molecular weight but high affinity compete more with the analyte molecules in the sample, binding more readily to the detection antibody molecules on the surface of the luminescent microspheres. This reduces the likelihood of the detection antibody molecules on the microsphere surface binding to the analyte molecules in the sample. In this case, the low-molecular-weight, high-affinity labeled antigen plays the dominant role, providing a wider detection range. Conversely, for low-concentration samples, labeled antigens with high molecular weight but low affinity compete less with the analyte molecules in the sample. The detection antibody molecules on the surface of the luminescent microspheres bind more readily to the analyte molecules in the sample. In this case, the high-molecular-weight, low-affinity labeled antigen plays the dominant role, providing higher functional sensitivity.

[0102] The method disclosed in this application for simultaneously improving sensitivity and broadening the detection range in competitive immunoassay involves using homologous marker molecules of different molecular weights to label the same competing antigen during detection.

[0103] In some embodiments of this application, the molecular weight of the first labeled molecule is lower than that of the second labeled molecule.

[0104] In some preferred embodiments of this application, the molecular weight of the second labeling molecule is at least 2.5 times that of the first labeling molecule; more preferably, it is 3 to 40 times.

[0105] In some specific embodiments of this application, the molecular weight of the first labeled molecule is ≤800 Da.

[0106] In some specific embodiments of this application, the molecular weight of the second labeled molecule is ≥2000 Da.

[0107] Based on the above analysis, this application uses a mixture of labeled antigens with different affinities as a single reagent. When the concentration of the analyte molecule in the sample varies, the labeled antigen that plays the main role is different, thus meeting the clinical testing requirements for functional sensitivity and detection range.

[0108] The method described in this application is applicable to competitive quantitative analysis and detection of analyte molecules, and is particularly applicable to competitive detection and analysis of small molecule antigens or haptens.

[0109] The reagents, kits, or methods described in this application can be used to detect small molecule antigens or haptens, specifically including the detection of steroid hormone concentrations. These steroid hormones include, but are not limited to, estradiol, testosterone, androgens, progesterone, estrone, and cortisol.

[0110] In some specific embodiments of this application, the method for detecting steroid hormones includes:

[0111] Step S1: Mix the sample to be tested, reagent R1, reagent R2 and optional reagent R3 to obtain the first mixture;

[0112] Step S2: Mix reagent R4 with the first mixture to obtain the second mixture;

[0113] Step S3: Energy or an active compound is used to excite the photosensitive microspheres in the second mixture to generate active oxygen, and then the luminescent microspheres react with the active oxygen they come into contact with to generate a chemiluminescent signal.

[0114] Step S4: Detect the intensity of the chemiluminescence signal mentioned in step S3, and analyze whether the test molecule exists and / or the concentration of the test molecule in the sample to be tested.

[0115] In the method described in this application, the reagents can be incubated as needed after mixing. Specifically, the incubation temperature can be 35-45°C, and the incubation time can be 8-50 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; and the incubation time can be selected from 8 min, 10 min, 15 min, 20 min, 30 min, 35 min, 40 min, 45 min, 50 min, etc.

[0116] In some embodiments of this application, the method further includes the step of preparing a standard curve of chemiluminescence signal-analyte molecule concentration using a series of calibrator solutions of known concentrations; the standard curve is used to determine the content of the analyte molecule in the sample to be tested.

[0117] In some other embodiments of this application, in step S3, the second mixture is irradiated with excitation light of 600-700 nm wavelength to excite the photosensitive microspheres in the second mixture to generate active oxygen, and then the luminescent microspheres react with the active oxygen they come into contact with to generate emission light of 520-620 nm.

[0118] Because this application uses a competitive analysis mode, the light signal intensity is inversely proportional to the content of the analyte in the serum sample. By using the mathematical function relationship (i.e., calibration curve) formed by a calibrator with a known concentration, the concentration level of the analyte in the unknown serum sample can be calculated by measuring the light signal intensity of the competitive antigen-specific antibody complex under the same conditions as the calibrator.

[0119] III. Specific Implementation Examples

[0120] To make the present invention easier to understand, the present application will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not limited to the scope of application of the present application. Unless otherwise specified, the raw materials or components used in the present application can be obtained commercially or by conventional methods.

[0121] Next, taking serum progesterone (Prog) as the analyte, we used biotin of different molecular weights as labeling molecules to label the progesterone antigen. We then mixed the two biotinylated antigens in an appropriate ratio as a single reagent and applied it on a competitive photochemiluminescence analysis platform to determine the effect of competing antigens labeled with different molecular weights of biotin on improving the reagent's detection capability.

[0122] Example 1: Preparation of Progesterone Detection Reagent

[0123] 1. The main experimental materials and equipment are shown in Table 1.

[0124] Table 1

[0125]

[0126] 2. Reagent Preparation

[0127] 2.1 Preparation of reagent R1

[0128] Progesterone (Prog) antibody was mixed with luminescent microspheres at a mass ratio of 1:20 to coat the FG microspheres. After reacting for 18 hours, blocking solution was added and reacted for 3 hours to prepare luminescent microspheres FG-Prog coated with progesterone antibody.

[0129] Dilute 10 mg / mL FG-Prog with PBS buffer at a volume ratio of 1:400 to prepare reagent R1.

[0130] 2.2 Preparation of reagent R2

[0131] Weigh 0.1 mg of progesterone (Prog) antigen, add 9 μL of 30 mg / mL EDC solution (prepared with pure water), then add DMSO to make the total volume 0.1 mL, and let stand at room temperature for 30 min to activate Prog.

[0132] 0.26 μmol of the labeled molecules Biotin-(PEG)n-NH2 (588 Da), Biotin-(PEG)n-NH2 (3.4 kDa), and Biotin-(PEG)n-NH2 (20 kDa) were dissolved in DMSO, then added to the activated Prog antigen solution, mixed, and allowed to stand at 2–8 °C for 18 h to prepare biotinylated antigens Prog-(PEG)n-Biotin with different molecular weights.

[0133] Dilute Prog-(PEG)n-Biotin of different molecular weights to 1 ng / mL with Tris buffer, and mix the diluted solutions according to the volume ratio shown in Table 2 (for two solutions of the same concentration, the volume ratio is the mass ratio of the solute) to prepare reagent R2.

[0134] Table 2. Composition of Reagent R2

[0135] Number Component R2-1 Prog-(PEG)n-Biotin(588Da) R2-2 Prog-(PEG)n-Biotin(3.4kDa) R2-3 Prog-(PEG)n-Biotin(20kDa) R2-4 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(3.4kDa) = 20:1 R2-5 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(3.4kDa) = 10:1 R2-6 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(3.4kDa) = 5:1 R2-7 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(3.4kDa) = 1:1 R2-8 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(3.4kDa) = 1:5 R2-9 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(3.4kDa) = 1:10 R2-10 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(3.4kDa) = 1:20 R2-11 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(20kDa) = 20:1 R2-12 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(20kDa) = 10:1 R2-13 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(20kDa) = 5:1 R2-14 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(20kDa) = 1:1 R2-15 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(20kDa) = 1:5 R2-16 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(20kDa) = 1:10 R2-17 Prog-(PEG)n-Biotin(588Da):Prog-(PEG)n-Biotin(20kDa) = 1:20

[0136] Note: Prog-(PEG)n-Biotin (588 Da) refers to the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 during its preparation process being 588 Da; Prog-(PEG)n-Biotin (3.4 kDa) refers to the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 during its preparation process being 3.4 kDa; Prog-(PEG)n-Biotin (20 kDa) refers to the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 during its preparation process being 20 kDa. The same applies below.

[0137] 2.3 Preparation of reagent R3

[0138] Prepare a solution of 8-anilino-1-naphthalenesulfonic acid at a concentration of 5 μg / mL using 0.25 M citrate buffer.

[0139] 2.4 Preparation process of progesterone series calibrators with known concentrations

[0140] Take pure progesterone antigen and prepare a series of calibrator solutions ranging from 0 to 120 ng / L using 0.25 M citrate buffer.

[0141] Example 2: Performance Test of Progesterone Detection

[0142] Using the reagents prepared in Example 1, the same batch of samples (calibrators) were detected using a photo-induced chemiluminescence detection system. The detection results are shown in the table below.

[0143] 1. Detection Method

[0144] Mix 10 μL of the sample to be tested, 25 μL of reagent R3, 25 μL of reagent R1, and 25 μL of reagent R2 sequentially, and incubate at 37°C for 15 min; add 175 μL of general reagent or reagent R4 (containing photosensitive microparticles coated with streptavidin), and incubate at 37°C for 10 min; read the signal value by photoexcitation.

[0145] 2. Experimental Results

[0146] 2.1 Detection results of reagent R2 prepared using Prog-(PEG)n-Biotin labeled antigen with different molecular weights.

[0147] Table 3. Test signal values ​​and inhibition rates using reagents R2-1 to R2-3.

[0148]

[0149] 2.2 Detection results of reagent R2 prepared by mixing different molecular weight labeled antigens Prog-(PEG)n-Biotin (labeled molecules with molecular weights of 588 Da and 3.4 kDa) in different volume ratios.

[0150] Table 4 shows the test signal values ​​and inhibition rates using reagents R2-4 to R2-10.

[0151]

[0152] 2.3 Detection results of reagent R2 prepared by mixing different molecular weight labeled antigens Prog-(PEG)n-Biotin (labeled molecules with molecular weights of 588 Da and 20 kDa) in different volume ratios.

[0153] Table 5. Test signal values ​​and inhibition rates using reagents R2-11 to R2-17.

[0154]

[0155]

[0156] 3. Results Analysis

[0157] 3.1 Experimental Data Analysis

[0158] Table 3 shows that the R2-1 reagent exhibited the highest overall discrimination of 154.19, but the discrimination of low-value calibrators was lower and inconsistent with the concentration dilution factor. As the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 increased from 588 kDa to 3.4 kDa, the trend of luminescent signal changes remained largely consistent with the R2-1 reagent data. At a calibrator concentration of 1 ng / mL, the inhibition rate decreased by 1.5 times, low-end sensitivity improved, and the overall discrimination of the reagent decreased by 1.31 times. When the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 continued to increase from 3.4 kDa to 20 kDa, the signal value changes remained largely consistent with the R2-1 reagent data. At a calibrator concentration of 1 ng / mL, the inhibition rate decreased by 1.64 times, low-end sensitivity further improved, and the overall discrimination of the reagent decreased by 1.45 times.

[0159] Table 4 shows that when different molecular weights of Prog-(PEG)n-Biotin (labeled molecules Biotin-(PEG)n-NH2 with molecular weights of 588 Da and 3.4 kDa) were mixed, the signal value trends were basically consistent with those of reagent R2-1, compared to the detection data of reagent R2-1. As the mass of Prog-(PEG)n-Biotin (588 Da) in reagent R2 decreased and the mass of Prog-(PEG)n-Biotin (3.4 kDa) increased, the inhibition rate gradually decreased at a calibrator concentration of 1 ng / mL, the low-end sensitivity improved, and the overall discrimination of the reagent was further improved. Within the mass ratio of Prog-(PEG)n-Biotin (588 Da) to Prog-(PEG)n-Biotin (3.4 kDa) of 1:1 to 1:10, the inhibition rate at a calibrator concentration of 1 ng / mL was approximately 1.46 times lower than that of reagent R2-1, and the overall discrimination of the reagent was approximately 1.28 times higher than that of reagent R2-2. It achieves a balance between detection capability and linear range.

[0160] Table 5 shows that when different molecular weights of Prog-(PEG)n-Biotin (labeled molecules Biotin-(PEG)n-NH2 with molecular weights of 588 Da and 20 kDa) were mixed, the signal value trends were basically consistent with those of reagent R2-1, compared to the detection data. As the mass of Prog-(PEG)n-Biotin (588 Da) in reagent R2 decreased and the mass of Prog-(PEG)n-Biotin (20 kDa) increased, the inhibition rate gradually decreased at a calibrator concentration of 1 ng / mL, the low-end sensitivity improved, and the overall discrimination of the reagent was further improved. Within the mass ratio of Prog-(PEG)n-Biotin (588 Da) to Prog-(PEG)n-Biotin (20 kDa) of 1:1 to 1:10, the inhibition rate at a calibrator concentration of 1 ng / mL was approximately 1.54 times lower than that of reagent R2-1, and the overall discrimination of the reagent was approximately 1.44 times higher than that of reagent R2-3. It achieves a balance between detection capability and linear range.

[0161] 3.3 Experimental Conclusions

[0162] The molecular weight of progesterone antigen Prog is 387 Da. When progesterone antigen is labeled with three different molecular weight markers, the theoretical molecular weights of the biotinylated progesterone antigens are 967 Da (the molecular weight of the marker molecule Biotin-(PEG)n-NH2 is 588 Da), 3787 Da (the molecular weight of the marker molecule Biotin-(PEG)n-NH2 is 3.4 kDa), and 20387 Da (the molecular weight of the marker molecule Biotin-(PEG)n-NH2 is 20 kDa).

[0163] Prog-(PEG)n-Biotin (967Da), using a biotinylated progesterone antigen labeled with a molecular weight of 588Da, has the smallest molecular weight and the strongest competitiveness; it can be called HA-Bio-Prog, which is beneficial for broadening the linear range. Prog-(PEG)n-Biotin (20387Da), using a biotinylated progesterone antigen labeled with a molecular weight of 20kDa, has the largest molecular weight and the weakest competitiveness; it can be called LA-Bio-Prog, which is beneficial for improving sensitivity.

[0164] When biotinylated progesterone antigen Prog-(PEG)n-Biotin prepared with biotin labels of different molecular weights are used in combination, both detection capability and detection range can be taken into account.

[0165] Example 3: Performance Test of Progesterone Detection

[0166] Following the method in Example 2, reagent R2-14 was used, with a commercially available imported Prog-like chemiluminescence reagent (Roche reagent) as a control. Samples with concentrations ranging from 0 to 40 ng / mL were detected in a photo-induced chemiluminescence detection system, and the consistency of the measured values ​​was compared. The detection results are as follows: Figures 1 - 3 As shown.

[0167] from Figure 1 It can be seen that, for low-concentration samples, the correlation between the progesterone measurement value of the detection reagent described in this application and the measurement value of similar imported reagents is r = 0.9456. From... Figure 2 It can be seen that, for high-concentration samples, the correlation r between the detection reagent described in this application and similar imported reagents for progesterone sample measurements is 0.989. From... Figure 3 It can be seen that the overall correlation (r = 0.9949) between the detection reagent described in this application and the detection values ​​of similar imported reagents for progesterone samples is good. This indicates that the reagent described in this application can accurately detect the progesterone content in the sample.

[0168] Example 4: Adaptability of Different Molecular Weight Labeled Antigens to the Detection of Different Items

[0169] 1. Reagent Preparation

[0170] Following the method described in Example 1, reagent R1 containing estradiol E2 antibody and dehydroepiandrosterone sulfate (DHEA-s) antibody, and reagent R2 containing estradiol E2 antigen and DHEA-s antigen were prepared respectively. The labeling molecules used in reagent R2 were Biotin-(PEG)n-NH2 (588 Da) and Biotin-(PEG)n-NH2 (20 kDa), which were diluted to 1 ng / mL with Tris buffer and then mixed at a 1:1 volume ratio.

[0171] 2. Detection Method

[0172] Using a commercially available imported chemiluminescence reagent of the same type (Roche reagent) as a control reagent, the samples were tested according to the method shown in Example 2, and the consistency of the sample measurements was compared. The test results are shown in the table below.

[0173] 3. Experimental Results

[0174] 3.1 The labeled antigen E2-(PEG)n-Biotin was prepared by labeling estradiol E2 antigen with the labeled molecules Biotin-(PEG)n-NH2 (588 Da) and Biotin-(PEG)n-NH2 (20 kDa), and the detection results of the two labeled antigens mixed at a volume ratio of 1:1 with the control reagent for low-value samples were compared.

[0175] Table 6 Comparison of measured values ​​of E2-(PEG)n-Biotin with different molecular weights and control reagents

[0176]

[0177] Note: E2-(PEG)n-Biotin (588Da) refers to the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 during its preparation process being 588Da; E2-(PEG)n-Biotin (20kDa) refers to the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 during its preparation process being 20kDa.

[0178] 3.2 The labeled antigen E2-(PEG)n-Biotin was prepared by labeling estradiol E2 antigen with the labeled molecules Biotin-(PEG)n-NH2 (588 Da) and Biotin-(PEG)n-NH2 (20 kDa), and the detection results of high-value samples were obtained by mixing the two labeled antigens at a volume ratio of 1:1 with the control reagent.

[0179] Table 7 Comparison of measured values ​​of E2-(PEG)n-Biotin with different molecular weights and control reagents

[0180]

[0181] 3.3 The labeled antigen (DHEA-s)-(PEG)n-Biotin was prepared by labeling the dehydroepiandrosterone sulfate (DHEA-s) antigen with the labeled molecules Biotin-(PEG)n-NH2 (588 Da) and Biotin-(PEG)n-NH2 (20 kDa), and the detection results of the two labeled antigens mixed at a volume ratio of 1:1 with the control reagent for low-value samples were obtained.

[0182] Table 8 Comparison of measured values ​​of (DHEA-s)-(PEG)n-Biotin with different molecular weights and control reagents

[0183]

[0184] Note: (DHEA-s)-(PEG)n-Biotin (588Da) refers to the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 during its preparation process being 588Da; (DHEA-s)-(PEG)n-Biotin (20kDa) refers to the molecular weight of the labeled molecule Biotin-(PEG)n-NH2 during its preparation process being 20kDa.

[0185] 3.4 The labeled antigen (DHEA-s)-(PEG)n-Biotin was prepared by labeling the dehydroepiandrosterone sulfate (DHEA-s) antigen with the labeled molecules Biotin-(PEG)n-NH2 (588 Da) and Biotin-(PEG)n-NH2 (20 kDa), and the detection results of high-value samples were obtained by mixing the two labeled antigens at a volume ratio of 1:1 with the control reagent.

[0186] Table 9 Comparison of measured values ​​of different molecular weights of (DHEA-s)-(PEG)n-Biotin with the control reagent

[0187]

[0188] 4. Results Analysis

[0189] Data from Tables 6 to 9 show that when estradiol E2 antigen labeled with Biotin-PEG-NH2 (588 Da) and Biotin-PEG-NH2 (20 KDa) are used together, and when dehydroepiandrosterone sulfate (DHEA-s) antigen labeled with Biotin-PEG-NH2 (588 Da) and Biotin-PEG-NH2 (20 KDa) is used together to detect low-value and high-value samples, the results show good consistency with the control reagents, small deviation, and a good balance between detection capability and linear range.

[0190] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A photochemiluminescent detection reagent, characterized by comprising: include: A luminescent composition comprising luminescent microspheres and a detection antibody bound thereto, the detection antibody being capable of specifically binding to a target molecule in a test sample; A first labeled antigen and a second labeled antigen, comprising the same competing antigen and the same labeled molecule of different molecular weights bound to the competing antigen, wherein the competing antigen is capable of binding to the detection antibody; The competitive antigen has a higher affinity for the detection antibody than the analyte has for the detection antibody.

2. The reagent according to claim 1, characterized in that, The first labeled antigen contains a first labeled molecule, the second labeled antigen contains a second labeled molecule, and the molecular weight of the first labeled molecule is lower than the molecular weight of the second labeled molecule; Preferably, the molecular weight of the second labeled molecule is at least 2.5 times that of the first labeled molecule; more preferably, it is 3 to 40 times. Preferably, the molecular weight of the first labeled molecule is ≤800 Da; Preferably, the molecular weight of the second labeled molecule is ≥2000 Da.

3. The reagent according to claim 2, characterized in that, The first and second labeled molecules are selected from biotin containing hydrophilic groups; the biotin is bound to hydrophilic polymers of different molecular weights; the polymers are selected from PEG or dextran.

4. The reagent according to any one of claims 1 to 3, characterized in that, The concentration of the first labeled antigen in the reagent is less than or equal to the concentration of the second labeled antigen.

5. The reagent according to any one of claims 1 to 4, characterized in that, The mass ratio of the first labeled antigen to the second labeled antigen is 1:(1-10).

6. The reagent according to any one of claims 1 to 5, characterized in that, The molar ratio of the competing antigens in the two groups of labeled antigens to the corresponding labeled molecules is the same.

7. A photo-induced chemiluminescence detection kit, characterized in that, include: R1 reagent comprises luminescent microspheres and a detection antibody bound thereto; the detection antibody is capable of specifically binding to the analyte molecule in the test sample. The R2 reagent comprises a first labeled antigen and a second labeled antigen; the first labeled antigen and the second labeled antigen contain the same competing antigen and the same labeled molecule with different molecular weights that bind to it; the affinity of the competing antigen to the detection antibody is higher than the affinity of the analyte molecule to the detection antibody. R3 reagent contains a releasing agent.

8. A method for simultaneously improving sensitivity and broadening the detection range in competitive immunoassays, characterized in that, During detection, the same competing antigen is labeled simultaneously using the same marker molecules with different molecular weights.

9. The method according to claim 8, characterized in that, The molecular weight of the second labeled molecule is at least 2.5 times that of the first labeled molecule; preferably 3 to 40 times. Preferably, the molecular weight of the first labeled molecule is ≤800 Da; Preferably, the molecular weight of the second labeled molecule is ≥2000 Da.

10. The application of the reagent according to any one of claims 1 to 6, or the kit according to claim 7, or the method according to any one of claims 8 to 9 in the detection of small molecule antigens or haptens; preferably, the small molecule antigen or hapten is a steroid hormone; more preferably, the steroid hormone is estradiol, testosterone, androgen, progesterone, estrone, or cortisol.

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