Homogeneous chemiluminescence assay kit and its application

Abstract

The present invention relates to a homogeneous chemiluminescent immunoassay kit, which comprises at least two compositions, wherein the first composition comprises a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to a first epitope of an analyte; the second composition comprises a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to a second epitope of the analyte; the receptor can generate chemiluminescence when reacting with active oxygen, and the first epitope and the second epitope have no overlapping portion; the affinity of the first antibody or the binding fragment thereof for specific binding to the analyte is higher than the affinity of the second antibody or the binding fragment thereof for specific binding to the analyte; and at the same time, the mass ratio of the first antibody or the binding fragment thereof to the first receptor is lower than the mass ratio of the second antibody or the binding fragment thereof to the second receptor.

Description

Technical Field

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

[0002] Since the development of radioimmunoassays in 1959, enzyme immunoassays, fluorescence immunoassays, and chemiluminescence immunoassays (including electrochemiluminescence and photochemiluminescence) have been developed. In particular, chemiluminescence immunoassays, characterized by their nanospheres, full automation, and optical signaling, have reached considerable maturity and are widely used. Regardless of the type of labeled immunoassay, they all derive from the fundamental principles of immunochemistry and labeled analysis. "Immunochemistry" refers to the specific binding of antigens and antibodies, which gives these assays their high specificity; "labeled analysis" refers to the use of highly sensitive signaling molecules (radionuclides, fluoresceins, luminescent agents, etc.), which ensure their high sensitivity. For macromolecules (proteins), which possess multiple epitopes (also sites for antibody binding), a double antibody sandwich format is often employed.

[0003] Luminescent immunoassays offer excellent analytical performance, with specificity, sensitivity, and automated operation meeting clinical requirements. However, for certain specific indicators, such as hCG, HBs-Ag, and AFP, high requirements are placed on both functional sensitivity and detection range. Existing chemiluminescent immunoassays, such as electrochemiluminescent immunoassays and photochemiluminescent immunoassays, have limitations and cannot effectively address these specific requirements. Therefore, a chemiluminescent detection technology that can achieve both functional sensitivity and analytical range is urgently needed. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a homogeneous chemiluminescence assay kit, which includes "differential receptor microspheres" (a mixture of receptor microspheres coupled to antibodies with different affinities), thereby enabling the kit to have excellent functional sensitivity and detection range.

[0005] To this end, the first aspect of the present invention provides a homogeneous chemiluminescent immunoassay kit, which includes at least two compositions, wherein the first composition comprises a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to a first epitope of the analyte; the second composition comprises a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to a second epitope of the analyte; the first antibody or the binding fragment thereof and the second antibody or the binding fragment thereof have different affinities for the analyte; the receptor can generate chemiluminescence by reacting with reactive oxygen species.

[0006] The first epitope and the second epitope have no overlapping portion;

[0007] The affinity of the first antibody or its binding fragment for specific binding to the analyte is higher than the affinity of the second antibody or its binding fragment for specific binding to the analyte; at the same time,

[0008] The mass ratio of the first antibody or its binding fragment to the first receptor is lower than the mass ratio of the second antibody or its binding fragment to the second receptor.

[0009] In some embodiments of the present invention, the mass ratio of the first antibody or its binding fragment to the first receptor is 1:(10-200), preferably 1:(20-180), and more preferably 1:(40-160).

[0010] In other embodiments of the present invention, the concentration of the first composition in the kit is higher than the concentration of the second composition in the kit.

[0011] In some preferred embodiments of the present invention, the mass concentration ratio of the first composition in the kit to the mass concentration ratio of the second composition in the kit is (5-100):1, preferably (10-50):1, and more preferably (15-25):1.

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

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

[0014] In other embodiments of the present invention, the first composition and the second composition are mixed and dispersed in a buffer solution to assemble into a reagent.

[0015] 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.

[0016] In other embodiments of the invention, the average particle size of the first acceptor microspheres is different from the average particle size of the second acceptor microspheres.

[0017] In some embodiments of the present invention, the analyte is a macromolecular antigen; preferably, the macromolecular antigen has three or more epitopes.

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

[0019] The beneficial effects of the present invention are as follows: the kit of the present invention selects antibodies with different affinities to couple to receptor microspheres in different mass ratios, and then mixes the two receptor microspheres in an appropriate proportion, so that the two antibodies with different affinities can selectively exert their effects depending on 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. Moreover, the kit belongs to homogeneous chemiluminescence analysis and there is no separation and washing process throughout the whole process, which not only saves detection time but also avoids errors caused by washing, and has high precision and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 1 The following is a diagram showing the detection principle of the kit of the present invention; wherein the reference numerals have the following meanings: 1. first receptor microspheres and first antibodies or binding fragments thereof bound thereto; the surface of the first receptor microspheres is coated with a small amount of high-affinity first antibodies or binding fragments thereof, but the concentration of the first receptor microspheres is relatively high, ensuring that the first receptor microspheres take precedence when detecting low-concentration β-hCG samples; 2. second receptor microspheres and second antibodies or binding fragments thereof bound thereto; the surface of the second receptor microspheres is coated with a larger amount of low-affinity second antibodies or binding fragments thereof, but the concentration of the second receptor microspheres is relatively low, ensuring that the second receptor microspheres take precedence when detecting high-concentration β-hCG samples; 3. third antibodies or binding fragments thereof bound to biotin; 4. β-hCG (human chorionic gonadotropin) to be detected.

[0022] Figure 2 This is a correlation diagram between the sample measurement values ​​of three batches in Example 6 and the Beckmann measurement values. DETAILED DESCRIPTION

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

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

[0025] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0026] I. Terminology

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

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

[0029] The term "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.

[0030] 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.

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

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

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

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

[0035] The term "functional sensitivity" as used in this application 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."

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

[0037] II. Specific implementation plan

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

[0039] The inventors of this application prepared differential receptor microspheres by coupling two or more antibodies with different specific binding affinities to the analyte at different "mass ratios" to receptor microspheres. The mass ratio was used to control the number of antibody molecules on the microsphere surface and in the local environment. At the same time, the receptor microspheres coupled to antibodies with different affinities were mixed in an "appropriate ratio" as a single solution (reagent 1). By controlling the number of microspheres per unit volume of solution (microsphere concentration), the probability of mutual collision with the analyte can be controlled, thereby achieving selective action of antibodies with different affinities depending on the analyte concentration. While ensuring functional sensitivity, it also broadens the detection range and prevents the occurrence of the hook effect.

[0040] Therefore, the homogeneous chemiluminescent immunoassay kit involved in the first aspect of the present invention comprises at least two compositions, wherein the first composition comprises a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to a first epitope of the analyte; the second composition comprises a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to a second epitope of the analyte; the first antibody or the binding fragment thereof and the second antibody or the binding fragment thereof have different affinities for the analyte; the receptor can generate chemiluminescence by reacting with reactive oxygen species.

[0041] The first epitope and the second epitope have no overlapping portion;

[0042] The affinity of the first antibody or its binding fragment for specific binding to the analyte is higher than the affinity of the second antibody or its binding fragment for specific binding to the analyte; at the same time,

[0043] The mass ratio of the first antibody or its binding fragment to the first receptor is lower than the mass ratio of the second antibody or its binding fragment to the second receptor. In other words, the amount of the first antibody or its binding fragment coupled to the first receptor is lower than the amount of the second antibody or its binding fragment coupled to the second receptor.

[0044] In the present invention, the receptor is capable of binding to the analyte via the first antibody or its binding fragment and / or the second antibody or its binding fragment.

[0045] In some embodiments of the present invention, both the first antibody and the second antibody are monoclonal antibodies that specifically bind to the analyte.

[0046] In some embodiments of the present invention, the mass ratio of the first antibody or its binding fragment to the first receptor is 1:(10-200), preferably 1:(20-180), and more preferably 1:(40-160). In some specific embodiments of the present invention, the mass ratio of the first antibody or its binding fragment to the first receptor is 1:10, 1:20, 1:30, 1:40, 1:80, 1:120, 1:160, 1:180 or 1:200, etc.

[0047] In other embodiments of the present invention, the concentration of the first composition in the kit is higher than the concentration of the second composition in the kit. In the present invention, the concentration can be either mass concentration or molar concentration.

[0048] In some preferred embodiments of the present invention, the ratio of the mass concentration of the first composition in the kit to the mass concentration of the second composition in the kit is (5-100):1, preferably (10-50):1, more preferably (15-25):1. In some specific embodiments of the present invention, the ratio of the mass concentration of the first composition in the kit to the mass concentration of the second composition in the kit is 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 50:1, 80:1 or 100:1, etc.

[0049] In some specific embodiments of the present invention, the mass concentration of the first composition in the kit is 5 to 500 ug / ml, preferably 10 to 250 ug / ml, and more preferably 15 to 200 ug / ml.

[0050] In some embodiments of the present invention, the first composition and the second composition are separately dispersed in the same buffer.

[0051] In other embodiments of the present invention, the first composition and the second composition are mixed and dispersed in a buffer solution to assemble into a reagent (ie, reagent 1).

[0052] For the two extreme samples, the receptor microspheres coupled to the first antibody and the receptor microspheres coupled to the second antibody work differently: for low-concentration test samples, the antibodies on the surface of the microspheres bound to the antigen to be tested depend on the concentration of the receptor microspheres, that is, the concentration of the receptor microspheres coupled to the first antibody is high, and this type of microspheres is dominant; for high-concentration test samples, the antibodies on the surface of the microspheres bound to the antigen to be tested no longer depend on the concentration of the microspheres, but on the number of antibody molecules on the surface of the microspheres. Although the concentration of the receptor microspheres coated with the second antibody is low, the number of antibody molecules on the surface of the microspheres is large. High-concentration test antigens require more antibody molecules, and at this time, the receptor microspheres coated with the second antibody play a decisive role.

[0053] Based on the above analysis, the present invention uses antibodies with different affinities (e.g., monoclonal antibodies) to prepare differential receptor microspheres using different methods (coating amount and / or coupling method). The differential receptor microspheres are mixed in a specific ratio to form a single solution (reagent 1). The two different receptor microspheres in this solution, utilizing the principle of microsphere liquid phase diffusion and differences in antibody affinity, can selectively act based on the concentration of the target antigen in the sample, meeting the specific requirements of the analyte for functional sensitivity and detection range.

[0054] 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.

[0055] In other embodiments of the invention, the average particle size of the first acceptor microspheres is different from the average particle size of the second acceptor microspheres.

[0056] In some embodiments of the present invention, the kit further comprises a third composition comprising a third antibody or a binding fragment thereof, wherein the third antibody or binding fragment thereof is a capture antibody that specifically binds to the analyte and whose binding site does not overlap with the first and second epitopes; the third antibody or binding fragment thereof is bound to a member of a specific binding pair (e.g., biotin). The third composition is reagent 2.

[0057] In some embodiments of the present invention, the affinity of the third antibody or its binding fragment specifically binding to the analyte is between the affinity of the first antibody or its binding fragment specifically binding to the analyte and the affinity of the second antibody or its binding fragment specifically binding to the analyte.

[0058] In some embodiments of the present invention, the third antibody is a monoclonal antibody that specifically binds to the analyte.

[0059] In other embodiments of the present invention, the kit further comprises a column calibrator solution with known analyte concentration.

[0060] In some embodiments of the present invention, the kit further comprises a donor that binds to the other member of the specific binding pair (e.g., avidin), wherein the donor is capable of generating reactive oxygen species when excited. In some specific embodiments of the present invention, the donor is a donor microsphere containing a high molecular weight polymer carrier.

[0061] In some embodiments of the present invention, the analyte is a macromolecular antigen; preferably, the macromolecular antigen has three or more epitopes.

[0062] In some embodiments of the present invention, the method for using the kit comprises:

[0063] Step N1, mixing the sample to be tested, reagent 1 and reagent 2 to obtain a first mixture;

[0064] Step N2, mixing the donor microsphere solution bound to avidin with the first mixture to obtain a second mixture;

[0065] 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 produce a chemiluminescent signal;

[0066] 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.

[0067] In the above method, the reagents can be incubated as needed after mixing. Specifically, the incubation temperature can be 35-45°C and the time can be 10-50 minutes. Preferably, the incubation temperature can be selected from 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C or 44°C; and the incubation time can be selected from 10 minutes, 20 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes or 50 minutes.

[0068] The second aspect of the present invention provides a use of the kit according to the first aspect of the present invention in a chemiluminescence analyzer.

[0069] Example

[0070] 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.

[0071] Example 1: Preparation of the Homogeneous Chemiluminescence Assay Kit of the Present Invention for β-hCG Analyte (1) Preparation of Monoclonal Antibody-Conjugated Receptor Microsphere Solution (Reagent 1)

[0072] Acceptor microspheres (FG): The microspheres have aldehyde groups (-CHO) on their surface, which are linked to antibody molecules. They contain a luminescent compound (a derivative of dimethylthiophene) and a chelate of a lanthanide (Eu) compound.

[0073] Biological materials: a monoclonal antibody that specifically binds to β-hCG with high affinity (i.e., hCG(A)) and a monoclonal antibody that specifically binds to β-hCG with low affinity (i.e., hCG(a)).

[0074] Preparation process:

[0075] 1) Wash the acceptor microspheres with coating buffer (pH 7.2-8.0 phosphate buffer);

[0076] 2) hCG(A) was coupled to the receptor microspheres at a mass ratio of 1:200 and incubated at 37°C overnight to obtain FG-An; hCG(a) was coupled to the receptor microspheres at a mass ratio of 1:20 and incubated at 37°C overnight to obtain FG-aN;

[0077] 3) Block FG-An and FG-aN with blocking agent and keep at 37°C for 2 hours;

[0078] 4) Clean FG-An and FG-aN with cleaning solution;

[0079] 5) Store FG-An and FG-aN in a hepes buffer system;

[0080] 6) Dilute FG-An and FG-aN at 1:200 and 1:3000, respectively, with the buffer of Reagent 1 to prepare Reagent 1, which is set aside. The concentration ratio of FG-An to FG-aN in Reagent 1 is 15:1.

[0081] (2) Preparation of a solution of anti-β-hCG antibody bound to biotin (reagent 2)

[0082] Biological Materials: Activated biotin and a monoclonal antibody that specifically binds to β-hCG with medium affinity.

[0083] Preparation process: Transfer 0.5 mg of antibody to a 14KD dialysis bag and dialyze with labeling buffer (0.1 M NaHCO3) for 2 hours each time, changing the solution once; add 10 ul of 5 mg / ml biotin solution, mix quickly, replenish labeling buffer to 500 μl, mix overnight at 2-8°C, and the labeling ratio is 1:30 (antibody: biotin-molar ratio); transfer the labeled Bio-Ab reagent to a 14KD dialysis bag and dialyze with dialysis buffer (0.1 M Tris-HCl) for 2 hours each time, changing the solution once; dilute to 5 μg / ml with pH 8.0, 0.1 M Tris-HCl solution.

[0084] (3) Preparation process of a series of β-hCG calibrators with known concentrations

[0085] Prepare 0.5 ml of each of the 0, 5, 50, 500, 5000, and 10000 IU / L series of calibrator solutions using 0.1 M pH 7.4 phosphate buffered saline solution containing 20% ​​inactivated calf serum.

[0086] Example 2: Detection of the linear range of the kit of the present invention

[0087] Linear range meaning: The linearity of an analytical method is the ability to obtain test results that are proportional to the concentration of the test substance in the sample within a given range.

[0088] Linear range assessment method: Serum with a high concentration near the upper limit of the linear range (10,000 IU / L) was diluted to six concentrations at specific ratios. Samples with low concentrations must be close to the lower limit of the linear range (0.5 IU / L). Each concentration was tested three times according to the kit instructions below. The average value was calculated. The average value and the dilution ratio were then fitted using the least squares method to calculate the correlation coefficient, r, which should be greater than or equal to 0.9900. The test results are shown in Tables 1 and 2.

[0089] The detection process using the kit prepared in Example 1 is fully automated by the LiCA500 automatic photochemiluminescence analysis system and outputs the detection results. The specific steps are:

[0090] a. Add 10 μl of sample, calibrator, or quality control material to each reaction well.

[0091] b. Add 25 μl of reagent 1 and 25 μl of reagent 2 to the reaction wells in sequence;

[0092] c. Incubate at 37°C for 15 minutes;

[0093] d. Add 175 μl of LiCA universal solution (donor microsphere solution bound to avidin);

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

[0095] e. Laser irradiation of microwells and calculation of the amount of photons emitted from each well;

[0096] f. Calculate the sample concentration based on the calibration curve.

[0097] Table 1: Raw data of linearity test of the kit of the present invention

[0098]

[0099]

[0100] Table 2: Linear range of the kit of the present invention for quantitative detection of serum β-hCG

[0101]

[0102] As shown in Table 2, the kit of the present invention has a wide linear range for the quantitative detection of serum β-hCG and a high linear correlation coefficient.

[0103] Example 3: Detection of the precision of the kit of the present invention

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

[0105] Intra-batch precision assessment method: 1 batch of products was analyzed independently using low (L), medium (M), and high (H) value samples. Each batch was measured 10 times using the method described in Example 2, and the average of the 10 measurement results was calculated. and standard deviation (SD), according to the formula The coefficient of variation (CV) was calculated and the results are shown in Tables 3 and 4.

[0106] Inter-batch precision assessment method: 3 batches of products were analyzed independently using low (L), medium (M), and high (H) value samples. Each batch was measured 10 times using the method described in Example 2, and the average of the 30 measurement results was calculated. and standard deviation (SD), according to the formula The coefficient of variation (CV) was calculated and the results are shown in Tables 3 and 5.

[0107] Table 3: Raw data of precision test of the kit of the present invention

[0108]

[0109]

[0110] Table 4: Intra-batch precision of the kit of the present invention for serum β-hCG detection

[0111]

[0112] Table 5: Inter-batch precision of the kit of the present invention for serum β-hCG detection

[0113]

[0114] As can be seen from Tables 4 and 5, the intra-batch and inter-batch precisions of the three batches of reagents were all <5%, indicating that the test kit of the present invention has good repeatability and small random error.

[0115] Example 4: Detection of the Accuracy of the Kit of the Present Invention

[0116] The meaning of accuracy: the degree of consistency between the measured value and the true value, reflecting the size of the system error.

[0117] Accuracy Assessment Method: Two samples containing different β-hCG levels were diluted at multiple points in the calibrator matrix solution. The concentrations of the diluted samples were measured using the method described in Example 2. The results are shown in Table 6. The recoveries of the two samples were then calculated based on the dilution ratios. The results are shown in Tables 7 and 8, respectively.

[0118] Table 6: Raw data of the accuracy test of the kit of the present invention

[0119]

[0120] Table 7: Accuracy of the kit of the present invention for detecting β-hCG in sample 1

[0121]

[0122] Table 8: Accuracy of the kit of the present invention for detecting β-hCG in sample 2

[0123]

[0124]

[0125] As shown in Tables 7 and 8, after multi-point dilution using two β-hCG samples with different levels, the recoveries were all within the range of 90% to 110%, indicating that the measured values ​​were close to the true values ​​and that the detection error of the kit of the present invention was small.

[0126] Example 5: Detection of the functional sensitivity and linear range of the kit of the present invention

[0127] Comparative kit 1: The difference from the kit of the present invention is that reagent 1 is a solution of receptor microspheres coupled with a monoclonal antibody with low affinity that specifically binds to β-hCG; the mass ratios of the antibody to the receptor microspheres are 1:20, 1:40, 1:80, and 1:160, respectively.

[0128] Comparative kit 2: The difference from the kit of the present invention is that reagent 1 is a solution of receptor microspheres coupled with a monoclonal antibody with high affinity that specifically binds to β-hCG; the mass ratios of the antibody to the receptor microspheres are 1:20, 1:40, 1:80, and 1:160, respectively.

[0129] The kit of the present invention: the kit prepared in Example 1, wherein the concentration ratios of FG-An and FG-aN in reagent 1 are 5:1, 10:1, 15:1, 20:1, and 25:1, respectively.

[0130] The above kit was used to perform detection according to the method described in Example 2. The results are shown in Table 9.

[0131] Table 9

[0132]

[0133]

[0134] The above results indicate that kits containing a receptor microsphere solution coupled to a monoclonal antibody with low affinity for β-hCG have a wider detection range but relatively low functional sensitivity; whereas kits containing a receptor microsphere solution coupled to a monoclonal antibody with high affinity for β-hCG have higher functional sensitivity but a narrower detection range. When these two receptor microsphere solutions are mixed in different ratios to prepare differentiated receptor microsphere solutions, the kits containing these differentiated receptor microsphere solutions exhibit both a wider detection range and higher functional sensitivity.

[0135] Example 6: Comparison of sample measurement values ​​of the kit of the present invention and Beckman test values

[0136] The three batches of samples were tested using the method described in Example 2, and the test results were compared with the Beckman test values. The results are shown in Tables 10-12. Figure 2 shown.

[0137] Table 10

[0138]

[0139]

[0140] Table 11

[0141]

[0142] Table 12

[0143]

[0144]

[0145] From Table 10-12 and Figure 2 It can be seen that the correlation between the values ​​measured by the kit of the present invention for the three batches of samples and the Beckman test value is r=0.9920, which is a good correlation, indicating that the kit of the present invention can accurately detect the content of human chorionic gonadotropin in samples.

[0146] 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 homogeneous chemiluminescent assay kit comprising at least two compositions, wherein the first composition comprises a first receptor and a first antibody or a binding fragment thereof bound thereto, wherein the first antibody or the binding fragment thereof is a detection antibody that specifically binds to a first epitope of an analyte; the second composition comprises a second receptor and a second antibody or a binding fragment thereof bound thereto, wherein the second antibody or the binding fragment thereof is a detection antibody that specifically binds to a second epitope of the analyte; the first antibody or the binding fragment thereof and the second antibody or the binding fragment thereof have different affinities for the analyte; the receptor can generate chemiluminescence by reacting with reactive oxygen species, wherein: The first receptor and the second receptor are both receptor microspheres; The first epitope and the second epitope have no overlapping portion; The affinity of the first antibody or its binding fragment for specific binding to the analyte is higher than the affinity of the second antibody or its binding fragment for specific binding to the analyte, and the ratio of the mass concentration of the first composition in the kit to the mass concentration of the second composition in the kit is (5-100):1; at the same time, The mass ratio of the first antibody or its binding fragment to the first receptor is lower than the mass ratio of the second antibody or its binding fragment to the second receptor, and the mass ratio of the first antibody or its binding fragment to the first receptor is 1:(10-200).

2. The kit according to claim 1, wherein The kit further comprises a third composition comprising a third antibody or a binding fragment thereof, wherein the third antibody or binding fragment thereof is a capture antibody that specifically binds to the analyte and whose binding site does not overlap with the first and second epitopes, and the third antibody or binding fragment thereof binds to one of the specific binding pair members.

3. The kit according to claim 2, wherein The affinity of the third antibody or its binding fragment for specific binding to the analyte is between the affinity of the first antibody or its binding fragment and the affinity of the second antibody or its binding fragment for specific binding to the analyte.

4. The kit according to claim 1, wherein The mass ratio of the first antibody or its binding fragment to the first receptor is 1:(20-180).

5. The kit according to claim 1, wherein The mass ratio of the first antibody or its binding fragment to the first receptor is 1:(40-160).

6. The kit according to claim 1, wherein The mass concentration ratio of the first composition in the kit to the mass concentration of the second composition in the kit is (10-50):

1.

7. The kit according to claim 1, wherein The mass concentration ratio of the first composition in the kit to the mass concentration of the second composition in the kit is (15-25):

1.

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

9. The kit according to claim 1, wherein The mass concentration of the first composition in the kit is 10 to 250 ug / ml.

10. The kit according to claim 1, wherein The mass concentration of the first composition in the kit is 15 to 200 ug / ml.

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

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

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

14. The kit according to claim 1, wherein The average particle size of the first acceptor microspheres is different from the average particle size of the second acceptor microspheres.

15. The kit according to claim 1, wherein The analyte is a macromolecular antigen.

16. The kit according to claim 15, characterized in that The macromolecular antigen has three or more epitopes.

17. Use of the kit according to any one of claims 1 to 16 in a chemiluminescence analyzer.

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