Immunoassay kit, assay method and system

Abstract

The invention relates to an immunoassay kit, an immunoassay method and an immunoassay system. The kit comprises a reagent 1 and a reagent 2 which are the same in component, the total content of specific capture molecules in the reagent 1 is different from the total content of specific capture molecules in the reagent 2, and the specific capture molecules can be specifically combined with target molecules to be detected. According to the present invention, with the immunoassay method using the kit, the HOOK effect sample problem can be solved, the method is not limited by the detection range, the high-value sample concentration reaching the level of 106 ng / ml can be directly measured, and the repeatability is good.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to an immunoassay kit, assay method, and system. Background Technology

[0002] Immunological testing is based on the principle of antigen-antibody specific reaction. Because it can use isotopes, enzymes, chemiluminescent substances, etc. to display or amplify the signal of the analyte, it is often used to detect trace amounts of bioactive substances such as proteins and hormones.

[0003] Photocatalytic chemiluminescence (PRC) is a commonly used method in chemiluminescence analysis, used to study interactions between biomolecules and primarily for disease detection in clinical practice. This technology integrates research in related fields such as polymer microparticle technology, organic synthesis, protein chemistry, and clinical testing. The technical principle of PRC is as follows: under laser irradiation, a sensitizer excites oxygen molecules in the surrounding environment into singlet oxygen molecules. These singlet oxygen molecules react with a luminescent composition approximately 200 nm away, generating a light signal of a specific wavelength. When the sample contains the antigen or antibody to be tested, the immune reaction of this antigen and antibody allows donor particles containing the sensitizer to bind to receptor particles containing the luminescent composition, thereby generating a light signal of a specific wavelength. Detecting this light signal allows for the determination of the content of the antigen or antibody to be tested.

[0004] In the antigen-antibody dose-response curve, when the antibody dose is fixed, the reaction signal first rises and then falls as the antigen dose increases. The region where the reaction signal rises with increasing antigen dose is called the "pre-band" region, and the region where the reaction signal falls with increasing antigen dose is called the "post-band" region. The region connecting the pre-band and post-band is called the "equivalence band".

[0005] In an immune response, the reactivity initially increases and then decreases as the ratio of antigen to antibody rises; this phenomenon is known as the "hook effect." Clinically, the hook effect can lead to false negative results for high-value samples.

[0006] Current immunoassay methods typically utilize the front band of a dose-response curve to calculate the analyte concentration based on the linear relationship between the analyte concentration and the reaction signal. However, this method has several drawbacks, such as: Narrow detection range: Traditional immunoassay reagents can only detect samples within the early band of the dose-response curve, resulting in a narrow detection concentration range. Samples outside this range require dilution before testing, which is complex, time-consuming, and demands high precision in dilution. HOOK effect: Traditional immunoassay reagents lack the means to identify the HOOK effect. Clinicians often need to combine the patient's clinical manifestations with the method of diluting serum samples to identify whether the sample has a HOOK effect. This operation is complicated, time-consuming and prone to false negatives. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide an immunoassay kit, assay method, and system. The method and system for immunoassay using the described kit enable simple, rapid, and accurate calculation of the analyte concentration.

[0008] To achieve the above and other related objectives, the present invention adopts the following technical solution: To this end, the first aspect of the present invention provides an immunoassay kit comprising reagent 1 and reagent 2 having identical components, wherein the total content of specific capture molecules in reagent 1 is different from the total content of specific capture molecules in reagent 2, and the specific capture molecules are capable of specifically binding to the target molecule to be tested; preferably, the specific capture molecules include a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecule to be tested.

[0009] In some embodiments of the present invention, reagent 1 comprises luminescent microparticles coated with a first antibody (or antigen) at a concentration of α1, reagent 2 comprises luminescent microparticles coated with a first antibody (or antigen) at a concentration of β1, and α1 is greater than β1.

[0010] In some other embodiments of the present invention, reagent 1 further comprises a second antibody (or antigen) labeled with a marker at a concentration of α2, and reagent 2 further comprises a second antibody (or antigen) labeled with a marker at a concentration of β2, wherein α2 is not less than β2; preferably, α2 is greater than β2.

[0011] A second aspect of the present invention provides a method for performing an immunoassay using a kit as described in the first aspect of the present invention, comprising the following steps: S1, the test sample containing the target molecule is subjected to two parallel immune response tests with reagent 1 and reagent 2 in the kit, respectively, and the test results of the two parallel immune responses are activated and recorded; wherein, the reading of the test using reagent 1 is the first measurement value, the reading of the test using reagent 2 is the second measurement value, and the ratio of the content of specific capture molecules to the content of the target molecule corresponding to the first measurement value in the two parallel immune response tests is greater than the ratio of the content of specific capture molecules to the content of the target molecule corresponding to the second measurement value; S2, calculate the ratio of the first measurement to the second measurement.

[0012] In some specific embodiments of the present invention, the ratio of the content of specific capture molecules to the content of target molecules corresponding to the first measurement in two parallel immune response assays is made greater than the ratio of the content of specific capture molecules to the content of target molecules corresponding to the second measurement by any of the following methods: Method 1: The amount of test sample containing the target molecule used in two parallel immunoassays is the same, and the amounts of reagent 1 and reagent 2 used are equal. Method 2: In two parallel immunoassays, the amount of test sample containing the target molecule is different when using reagent 1 and when using reagent 2, and the amounts of reagent 1 and reagent 2 used are equal. Method 3: In two parallel immune response assays, the amount of test sample containing the target molecule detected using reagent 1 is different from the amount of test sample containing the target molecule detected using reagent 2, and the amount of reagent 1 used is also different from the amount of reagent 2 used. Method 4: The amount of test sample containing the target molecule is the same in two parallel immunoassays, and the amount of reagent 1 used is different from that of reagent 2.

[0013] In some embodiments of the present invention, the method further includes the following steps: A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed. The results of the two parallel immune reactions are stimulated and recorded, and are respectively denoted as value a and value a'. The detection method of value a is the same as that of the first value of the sample to be tested, and the detection method of value a' is the same as that of the second value of the sample to be tested. A2, calculate the ratio of measured value a to measured value a'; A3. Create a standard curve showing the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance, and save it.

[0014] In other embodiments of the present invention, the method further includes the following steps: The stored correlation standard curve is retrieved, and the ratio of the first measured value to the second measured value of the test sample containing the target molecule is substituted into the standard curve for calculation to determine the concentration of the sample.

[0015] In some embodiments of the present invention, the method further includes the following steps: B1, a series of standard substances of different concentrations with known content of the target molecule are tested, wherein two parallel immune reactions are performed on each standard substance, and the results of the two parallel immune reactions are stimulated and recorded, which are respectively denoted as measurement value b and measurement value b'. The detection method of measurement value b is the same as that of the first measurement value of the sample to be tested, and the detection method of measurement value b' is the same as that of the second measurement value of the sample to be tested. B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it; B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it; B4. Take a point in the overlapping area of ​​the standard substance concentrations corresponding to the front zone of reaction curve A and the back zone of reaction curve B, record the ratio of the measured value b to the measured value b' at that point as the critical point c, and store it.

[0016] In other embodiments of the present invention, the method further includes the following steps: Retrieve the stored reaction curves A and B, and the critical point, and determine the magnitude of the ratio of the first measured value to the second measured value of the test sample and the critical point c; when the ratio of the first measured value to the second measured value of the test sample is less than or equal to the critical point c, the concentration of the target molecule in the test sample is calculated using the front band region of reaction curve A; when the ratio of the first measured value to the second measured value of the test sample is greater than the critical point c, the concentration of the target molecule in the test sample is calculated using the back band region of reaction curve B.

[0017] A third aspect of the present invention provides a system for performing an immunoassay as described in the second aspect of the present invention, comprising: An immunoassay apparatus comprising two or more reaction containers for simultaneously performing two parallel immunoassays on the same test sample in two of the reaction containers; wherein reaction container 1 is filled with reagent 1 of the kit, and reaction container 2 is filled with reagent 2 of the kit; A chemiluminescent immunoassay excitation and counting device is used to excite and record chemiluminescence readings, and to record two parallel immunoassay readings of the same sample as the first measurement value and the second measurement value, respectively. The first measurement value is derived from reaction container 1 and the second measurement value is derived from reaction container 2. The processor calculates the ratio of the first measurement to the second measurement, and calculates the concentration of the sample to be tested based on the ratio.

[0018] In some embodiments of the present invention, the processor stores a standard curve showing the correlation between the ratio of the measured value a / measured value a' and the concentration of the standard substance, a reaction curve A between the measured value b and the concentration of the standard substance, a reaction curve B between the measured value b' and the concentration of the standard substance, and the critical point c, for calculating the concentration of the sample to be tested.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method and system for detection using the kit described in this invention can solve the HOOK effect problem and avoid missed detection caused by the HOOK effect. The method and system are not limited by the detection range. (2) The method and system for detection using the kit described in this invention directly use the classical dose-response curve for calculation, with good repeatability, no need for multiple dilutions, and accurate measurement of HOOK effect samples can be obtained in a single test, with fast measurement speed; (3) The detection range of the method and system using the kit described in this invention is much greater than that of conventional detection methods. Attached Figure Description

[0020] Figure 1 This is the dose-response curve of the antigen-antibody interaction.

[0021] Figure 2 This is a schematic diagram of calculation method 1 when performing immunoassay using the kit of the present invention.

[0022] Figure 3 This is a schematic diagram of calculation method 2 when performing an immune test using the kit of the present invention.

[0023] Figure 4 The graph shows the reaction curves between the concentration of the standard substance and the signals of reagent 1 and reagent 2 in Example 2. Detailed Implementation

[0024] 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.

[0025] 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.

[0026] 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 similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0027] The immunoassay kit according to the first aspect of the present invention includes reagent 1 and reagent 2 with the same components, wherein the total content of specific capture molecules in reagent 1 is different from the total content of specific capture molecules in reagent 2, and the specific capture molecules are capable of specifically binding to the target molecules to be tested.

[0028] In some embodiments of the present invention, there is only one specific capture molecule; in a preferred embodiment of the present invention, there is more than one specific capture molecule, including a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecule to be tested.

[0029] In some embodiments of the present invention, reagent 1 comprises luminescent microparticles coated with a first antibody (or antigen) at a concentration of α1, reagent 2 comprises luminescent microparticles coated with a first antibody (or antigen) at a concentration of β1, and α1 is greater than β1.

[0030] In this invention, the luminescent microparticles contain luminescent groups that can rapidly absorb singlet oxygen and then emit light of a certain wavelength (e.g., 500-615 nm).

[0031] In other embodiments of the present invention, reagent 1 further includes a second antibody (or antigen) labeled with a marker at a concentration of α2, and reagent 2 further includes a second antibody (or antigen) labeled with a marker at a concentration of β2, wherein α2 is not less than β2; preferably, α2 is greater than β2. In the present invention, the marker may be biotin.

[0032] That is, in some preferred embodiments of the present invention, the kit includes reagent 1 and reagent 2 with the same components, and reagent 1 contains luminescent microparticles coated with a first antibody (or antigen) at a concentration of α1 and a biotin-labeled second antibody (or antigen) at a concentration of α2; reagent 2 contains luminescent microparticles coated with a first antibody (or antigen) at a concentration of β1 and a biotin-labeled second antibody (or antigen) at a concentration of β2; and α1 is greater than β1, and α2 is greater than β2.

[0033] In some specific embodiments of the present invention, the target molecule to be tested is selected from antigens or antibodies. According to some embodiments of the present invention, the antigen refers to any substance having immunogenicity, including but not limited to substances listed in the examples of the aforementioned target molecules having immunogenicity. According to some embodiments of the present invention, the antibody is used in the broadest sense herein and explicitly covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two complete antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab regions, Fc regions, single-chain antibodies).

[0034] The second aspect of this invention relates to a method for immunoassay using a kit as described in the first aspect of this invention. This method uses reagents 1 and 2 from the kit to perform two parallel tests on each sample. The ratio of the content of the target molecule to the content of the specific capture molecule in the detection reagent differs between the two tests, ultimately generating two different signals: a first measurement and a second measurement (the ratio of the specific capture molecule content to the target molecule content corresponding to the first measurement is greater than the ratio corresponding to the second measurement). As the content of the target molecule increases, the ratio of the first measurement to the second measurement continuously increases and exhibits a certain linear relationship. Based on this principle, the method for immunoassay using the kit described in this invention provides the following two methods for calculating the concentration of the target molecule, as follows: Calculation Method 1: Directly calculate the concentration of the target molecule from the ratio of the first measured value to the second measured value. like Figure 2 As shown, based on the correlation curve between the ratio of the first measured value to the second measured value and the concentration of the standard substance, the concentration of the target molecule can be calculated by substituting the ratio of the first measured value to the second measured value into the curve.

[0035] Calculation Method 2: Calculate the concentration of the target molecule using reaction curve A or reaction curve B: In the dose-response curve of antigen-antibody ( Figure 1 In this study, when the antibody level is fixed, the reaction signal initially rises and then falls as the antigen level increases. The region where the reaction signal rises with increasing antigen level is called the pre-zone region, and the region where the reaction signal falls with increasing antigen level is called the post-zone region. The region connecting the pre-zone and post-zone is called the equivalence band.

[0036] like Figure 3 As shown, reaction curves A and B are obtained based on the first and second measured values ​​and the concentration of the standard substance, respectively. There is a concentration overlap between the front band region of reaction curve A and the back band region of reaction curve B (e.g., ...). Figure 3 (The part within the dashed box) Take a point in the overlapping area of ​​the above concentrations, and take the ratio of the first measurement value to the second measurement value at that point (A / B=15 in the figure) as the critical point c.

[0037] When the ratio of the first measured value to the second measured value of the sample is less than or equal to the critical point c, the concentration is calculated using the front zone of the reaction curve A.

[0038] When the ratio of the first measured value to the second measured value of the sample is greater than the critical point c, the concentration is calculated using the back zone of the reaction curve B.

[0039] Corresponding to the above calculation method 1, the method for performing immunoassay using the kit described in this invention specifically includes the following steps: First, a standard curve of the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance is obtained by a method including the following steps: A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed. The results of the two parallel immune reactions are stimulated and recorded, and are respectively denoted as value a and value a'. The detection method of value a is the same as that of the first value of the sample to be tested, and the detection method of value a' is the same as that of the second value of the sample to be tested. A2, calculate the ratio of measured value a to measured value a'; A3. Create a standard curve showing the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance, and save it.

[0040] Then, the concentration of the target molecule in the sample is determined by a method including the following steps: S1, the test sample containing the target molecule is subjected to two parallel immune response tests with reagent 1 and reagent 2 in the kit, respectively, and the test results of the two parallel immune responses are activated and recorded; wherein, the reading of the test using reagent 1 is the first measurement value, the reading of the test using reagent 2 is the second measurement value, and the ratio of the content of specific capture molecules to the content of the target molecule corresponding to the first measurement value in the two parallel immune response tests is greater than the ratio of the content of specific capture molecules to the content of the target molecule corresponding to the second measurement value; S2, calculate the ratio of the first measured value to the second measured value; S3, retrieve the stored correlation standard curve, substitute the ratio of the first measured value to the second measured value of the sample to be tested into the correlation standard curve for calculation, so as to determine the concentration of the target molecule in the sample to be tested.

[0041] Corresponding to calculation method 2 above, the method for performing immunoassay using the kit described in this invention specifically includes the following steps: First, reaction curve A, reaction curve B, and critical point c are obtained using a method that includes the following steps: B1, a series of standard substances of different concentrations with known content of the target molecule are tested, wherein two parallel immune reactions are performed on each standard substance, and the results of the two parallel immune reactions are stimulated and recorded, which are respectively denoted as measurement value b and measurement value b'. The detection method of measurement value b is the same as that of the first measurement value of the sample to be tested, and the detection method of measurement value b' is the same as that of the second measurement value of the sample to be tested. B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it; B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it; B4. Take a point in the overlapping area of ​​the standard substance concentrations corresponding to the front zone of reaction curve A and the back zone of reaction curve B, record the ratio of the measured value b to the measured value b' at that point as the critical point c, and store it.

[0042] Then, the concentration of the target molecule in the sample is determined by a method including the following steps: S1, the test sample containing the target molecule is subjected to two parallel immune response tests with reagent 1 and reagent 2 in the kit, respectively, and the test results of the two parallel immune responses are activated and recorded; wherein, the reading of the test using reagent 1 is the first measurement value, the reading of the test using reagent 2 is the second measurement value, and the ratio of the content of specific capture molecules to the content of the target molecule corresponding to the first measurement value in the two parallel immune response tests is greater than the ratio of the content of specific capture molecules to the content of the target molecule corresponding to the second measurement value; S2, calculate the ratio of the first measured value to the second measured value; S3, retrieve the stored reaction curves A and B and the critical point c, and determine the ratio of the first measured value to the second measured value of the sample and the magnitude of the critical point c; when the processor determines that the ratio of the first measured value to the second measured value of the sample is ≤ c, calculate the sample concentration using the front band region of the reaction curve A; when the processor determines that the ratio of the first measured value to the second measured value of the sample is > c, calculate the sample concentration using the back band region of the reaction curve B.

[0043] In this invention, the ratio of the content of specific capture molecules in reagent 1 to the content of the target molecules in two parallel immunoassays is greater than the ratio of the content of specific capture molecules in reagent 2 to the content of the target molecules in two parallel immunoassays, achieved through any of the following methods: Method 1: The amount of test sample containing the target molecule used in two parallel immunoassays is the same, and the amounts of reagent 1 and reagent 2 used are equal. Method 2: In two parallel immunoassays, the amount of test sample containing the target molecule is different when using reagent 1 and when using reagent 2, and the amounts of reagent 1 and reagent 2 used are equal. Method 3: In two parallel immune response assays, the amount of test sample containing the target molecule detected using reagent 1 is different from the amount of test sample containing the target molecule detected using reagent 2, and the amount of reagent 1 used is also different from the amount of reagent 2 used. Method 4: The amount of test sample containing the target molecule is the same in two parallel immunoassays, and the amount of reagent 1 used is different from that of reagent 2.

[0044] The "standard substance" mentioned in this invention refers to a solution of target molecules whose content of the target molecules is known or whose content of the target molecules can be quantitatively determined and assigned a value.

[0045] A third aspect of the present invention relates to a system for performing an immunoassay as described in the second aspect of the present invention, comprising: An immunoassay apparatus comprising two or more reaction containers for simultaneously performing two parallel immunoassays on the same test sample in two of the reaction containers; wherein reaction container 1 is filled with reagent 1 of the kit, and reaction container 2 is filled with reagent 2 of the kit; A chemiluminescent immunoassay excitation and counting device is used to excite and record chemiluminescence readings, and to record two parallel immunoassay readings of the same sample as the first measurement value and the second measurement value, respectively. The first measurement value is derived from reaction container 1 and the second measurement value is derived from reaction container 2. The processor calculates the ratio of the first measurement to the second measurement, and calculates the concentration of the sample to be tested based on the ratio.

[0046] In this invention, there is no specific limitation on the shape of the reaction container. In some specific embodiments of this invention, the reaction container may be a reaction orifice, etc.; the chemiluminescent immunoassay excitation and counting device may include a photon counting module and a light-emitting diode; the processor may be a computer to process, plot, and store the readings, etc.

[0047] In some embodiments of the present invention, the processor stores a standard curve showing the correlation between the ratio of the measured value a / measured value a' and the concentration of the standard substance, a reaction curve A between the measured value b and the concentration of the standard substance, a reaction curve B between the measured value b' and the concentration of the standard substance, and the critical point c. The processor retrieves the stored data as needed to calculate the concentration of the sample to be tested.

[0048] In some embodiments of the present invention, the correlation standard curve between the ratio of the measured value a / measured value a' stored in the process and the concentration of the standard substance is obtained by performing a method comprising the following steps: A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed. The results of the two parallel immune reactions are stimulated and recorded, and are respectively denoted as value a and value a'. The detection method of value a is the same as that of the first value of the sample to be tested, and the detection method of value a' is the same as that of the second value of the sample to be tested. A2, calculate the ratio of measured value a to measured value a'; A3. Create a standard curve showing the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance, and save it.

[0049] In other embodiments of the present invention, the stress curve A, reaction curve B, and critical point c stored in the process are obtained by performing a method comprising the following steps: B1, a series of standard substances of different concentrations with known content of the target molecule are tested, wherein two parallel immune reactions are performed on each standard substance, and the results of the two parallel immune reactions are stimulated and recorded, which are respectively denoted as measurement value b and measurement value b'. The detection method of measurement value b is the same as that of the first measurement value of the sample to be tested, and the detection method of measurement value b' is the same as that of the second measurement value of the sample to be tested. B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it; B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it; B4. Take a point in the overlapping area of ​​the standard substance concentrations corresponding to the front zone of reaction curve A and the back zone of reaction curve B, record the ratio of the measured value b to the measured value b' at that point as the critical point c, and store it.

[0050] Example To make the present invention easier to understand, specific embodiments using the AFP project as an example are provided below for further detailed explanation. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. Unless otherwise specified, the raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0051] Alpha-fetoprotein (AFP) is a glycoprotein, also known as fetal alpha-globulin, belonging to the albumin family. The serum AFP levels in patients with primary liver cancer (PLC) vary wildly, with normal values ​​differing from pathological values ​​by up to seven orders of magnitude. One report states that a direct measurement using IEMA of a primary liver cancer patient's pre-existing serum AFP concentration was 29 ng / mL; however, after a series of dilutions, the actual calculated AFP concentration was 5.9 × 10⁻⁶. 6 ng / mL. This shows that conventional techniques for AFP detection still have significant limitations.

[0052] Example 1: Detection of AFP samples using conventional methods and reagent kits. The standard reagent kit used was the Alpha-fetoprotein (AFP) Detection Kit (Chemiluminescence Method) (batch number: L2001) manufactured by Komeiboyang Diagnostic Technology (Shanghai) Co., Ltd., whose main components are: Reagent 1: Luminescent microparticles coated with AFP antibody; Reagent 2: Biotin-labeled AFP antibody.

[0053] Test method: 1. Add 25 μl of the sample to be tested, 25 μl of reagent 1, and 25 μl of reagent 2 to the reaction wells respectively, and incubate at 37℃ for 15 min; 2. Add 175 μl of universal solution for photo-induced chemiluminescence analysis system (donor reagent) to the reaction well, incubate at 37°C for 10 min, and use LiCA... ® The analyzer takes readings.

[0054] Test samples (collected from clinical serum samples): Sample 1: Negative serum sample (actual value approximately 5 ng / mL) Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL) Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 (ng / mL) The test results are shown in Table 1.

[0055] Table 1

[0056] The results shown in Table 1 are from direct testing using a standard kit. Sample 3 had a value of only 11.79 ng / mL, which could easily be misjudged as a weakly positive sample without considering clinical manifestations. Knowing that Sample 3 was a strongly positive sample, it was diluted 50-fold with diluent and retested, with the results shown in Table 2.

[0057] Table 2

[0058] As shown above, sample 3, after a 50-fold dilution, had a measured value >1000 ng / mL, confirming it as a hook sample, but a specific measured value still could not be obtained. The diluted sample was then diluted another 50 times with diluent, and the measured values ​​are shown in Table 3 below.

[0059] Table 3

[0060] As shown above, the measured value of the sample after 2500-fold dilution was 849.51 ng / mL. By reverse calculation, the true concentration of sample 3 can be obtained as approximately 2.12 × 10⁻⁶. 6 ng / mL.

[0061] Example 2: Detection of AFP samples using the kit and immunoassay method of the present invention. Reagent Kit: The main components of the two-reagent reagent kit of the present invention are as follows: Reagent 1: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL). Reagent 2: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0062] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL) Test samples (collected from clinical serum samples): Sample 1: Negative serum sample (actual value approximately 5 ng / mL) Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL) Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 (ng / mL) Test Method 1: For both reaction vessels, which are from the same sample test group, repeat the following liquid addition steps 1 and 2 for different sample test groups: 1. Add 10 μl of the sample to be tested and 25 μl of reagent 1 to reaction well 1; 2. Add 10 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively; 3. Incubate each reaction well at 37°C for 15 minutes simultaneously; 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction well, incubate at 37°C for 10 min, and use LiCA... ® The analyzer takes readings.

[0063] The AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 4.

[0064] Based on the values ​​in Table 4, plot reaction curves A and B respectively between the concentrations of the standard substance and the signals from reagent 1 and reagent 2. Figure 4 It can be seen that standard substances 1-11 correspond to the front zone of reaction curve A, and standard substances 9-20 correspond to the back zone of reaction curve B. Therefore, the concentration overlap between the front zone of reaction curve A and the back zone of reaction curve B is the concentration range of standard substances 9-11, and its corresponding A / B signal ratio is 15.05-24.31. The midpoint A / B signal ratio of 19 is taken as the critical point.

[0065] Based on the values ​​in Table 4, construct a standard curve showing the correlation between the A / B signal ratio and the concentration of the standard substance.

[0066] Store the above standard material test results in LiCA. ® Analyzer.

[0067] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 5. Calculation method 1 is the test result obtained by calling the correlation standard curve, and calculation method 2 is the test result obtained by calling the critical point.

[0068] Table 4

[0069] Table 5

[0070] As shown in Table 5, the immunoassay method using the kit of this invention can avoid the problem of low sample values ​​caused by the HOOK effect, and can directly obtain high values ​​of up to 2×10⁻⁶. 6 The detection results are in ng / mL. The method is not limited by the detection range, and both calculation methods are feasible.

[0071] Example 3: Detection of AFP samples using the kit and immunoassay method of the present invention. The experimental materials were the same as in Example 2.

[0072] Test Method 2: For both reaction vessels, which are from the same sample test group, repeat the following liquid addition steps 1 and 2 for different sample test groups: 1. Add 10 μl of the sample to be tested and 25 μl of reagent 1 to reaction well 1; 2. Add 20 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively; 3. Incubate each reaction well at 37°C for 15 minutes simultaneously; 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction well, incubate at 37°C for 10 min, and use LiCA... ® The analyzer takes readings.

[0073] The serially diluted AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 6. A standard curve was plotted based on the correlation between the A / B signal ratio and the concentration of the standard substances according to the values ​​in Table 6. The test results of the above standard substances were stored in the LiCA® analyzer.

[0074] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 7 below.

[0075] Table 6

[0076] Table 7

[0077] The results showed that the kit and immunoassay method of the present invention can detect samples with extremely high AFP values, have a wide detection range, and can easily, quickly, and accurately calculate the concentration of the analyte.

[0078] Example 4: Detection of AFP samples using the kit and immunoassay method of the present invention. The experimental materials were the same as in Example 2.

[0079] Test Method 3: For both reaction vessels, which are from the same sample test group, repeat the following liquid addition steps 1 and 2 for different sample test groups: 1. Add 10 μl of the sample to be tested and 50 μl of reagent 1 to reaction well 1; 2. Add 20 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively; 3. Incubate each reaction well at 37°C for 15 minutes simultaneously; 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction well, incubate at 37°C for 10 min, and use LiCA... ® The analyzer takes readings.

[0080] The serially diluted AFP standard reference materials (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 8. A standard curve was plotted based on the correlation between the A / B signal ratio and the concentration of the standard reference materials according to the values ​​in Table 8. The test results of the above standard reference materials were stored in the LiCA® analyzer. Three groups of samples were tested according to the above test method, and the test results are shown in Table 9.

[0081] Table 8

[0082] Table 9

[0083] The results showed that the kit and immunoassay method of the present invention can detect samples with extremely high AFP values, have a wide detection range, and can easily, quickly, and accurately calculate the concentration of the analyte.

[0084] Example 5: Detection of AFP samples using the kit and immunoassay method of the present invention. The experimental materials were the same as in Example 2.

[0085] Test Method 4: For both reaction vessels, which are from the same sample test group, repeat the following liquid addition steps 1 and 2 for different sample test groups: 1. Add 10 μl of the sample to be tested and 50 μl of reagent 1 to reaction well 1; 2. Add 10 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively; 3. Incubate each reaction well at 37°C for 15 minutes simultaneously; 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction well, incubate at 37°C for 10 min, and use LiCA... ® The analyzer takes readings.

[0086] The serially diluted AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 10. A standard curve was plotted based on the correlation between the A / B signal ratio and the concentration of the standard substances according to the values ​​in Table 10. The test results of the above standard substances were stored in the LiCA® analyzer.

[0087] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 11 below.

[0088] Table 10

[0089] Table 11

[0090] The results showed that the kit and immunoassay method of the present invention can detect samples with extremely high AFP values, have a wide detection range, and can easily, quickly, and accurately calculate the concentration of the analyte.

[0091] Example 6: Verification of the Precision of Ultra-High-End Measurements The experimental materials were the same as in Example 2.

[0092] Test method: For two reaction wells belonging to the same sample test group, repeat the following liquid addition steps 1 and 2 for different sample test groups: 1. Add 10 μl of the sample to be tested and 25 μl of reagent 1 to reaction well 1; 2. Add 10 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively; 3. Incubate each reaction well at 37°C for 15 minutes simultaneously; 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction well, incubate at 37°C for 10 min, and use LiCA... ® The analyzer takes readings.

[0093] The test results are shown in Table 12 below (calculated using calculation method 1 of the present invention).

[0094] Table 12

[0095] The results showed that, using the assay method of the kit described in this invention, the CV of the three high-value samples were all within 10% after 10 repeated measurements, indicating that the precision results were good.

[0096] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on 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 terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. An immunoassay kit comprising reagent 1 and reagent 2 having identical components, wherein the total content of specific capture molecules in reagent 1 is different from the total content of specific capture molecules in reagent 2, and the specific capture molecules are capable of specifically binding to the target molecule to be tested.

2. The reagent kit according to claim 1, characterized in that, The specific capture molecules include a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecule to be tested.

3. The reagent kit according to claim 1, characterized in that, Reagent 1 contains luminescent microparticles coated with a first antibody (or antigen) at a concentration of α1, and reagent 2 contains luminescent microparticles coated with a first antibody (or antigen) at a concentration of β1, wherein α1 is greater than β1.

4. The reagent kit according to claim 3, characterized in that, The reagent 1 further contains a second antibody (or antigen) labeled with a marker at a concentration of α2, and the reagent 2 further contains a second antibody (or antigen) labeled with a marker at a concentration of β2, wherein α2 is not less than β2; preferably, α2 is greater than β2.

5. A method for performing an immunoassay using the kit described in any one of claims 1-4, comprising the following steps: S1, the test sample containing the target molecule is subjected to two parallel immune responses with reagents 1 and 2 in the kit, respectively, and the results of the two parallel immune responses are activated and recorded; wherein, The reading of the tester using reagent 1 is the first measurement value, and the reading of the tester using reagent 2 is the second measurement value. In the two parallel immune reaction tests, the ratio of the content of specific capture molecules to the content of target molecules corresponding to the first measurement value is greater than the ratio of the content of specific capture molecules to the content of target molecules corresponding to the second measurement value. S2, calculate the ratio of the first measurement to the second measurement.

6. The method according to claim 5, characterized in that, Achieve a ratio greater than the ratio of specific capture molecule content to target molecule content corresponding to the first measurement in two parallel immune response assays by any of the following methods: Method 1: The amount of test sample containing the target molecule used in two parallel immunoassays is the same, and the amounts of reagent 1 and reagent 2 used are equal. Method 2: In two parallel immunoassays, the amount of test sample containing the target molecule is different when using reagent 1 and when using reagent 2, and the amounts of reagent 1 and reagent 2 used are equal. Method 3: In two parallel immune response assays, the amount of test sample containing the target molecule detected using reagent 1 is different from the amount of test sample containing the target molecule detected using reagent 2, and the amount of reagent 1 used is also different from the amount of reagent 2 used. Method 4: The amount of test sample containing the target molecule is the same in two parallel immunoassays, and the amount of reagent 1 used is different from that of reagent 2.

7. The method according to claim 5 or 6, characterized in that, The method further includes the following steps: A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed. The results of the two parallel immune reactions are stimulated and recorded, and are respectively denoted as value a and value a'. The detection method of value a is the same as that of the first value of the sample to be tested, and the detection method of value a' is the same as that of the second value of the sample to be tested. A2, calculate the ratio of measured value a to measured value a'; A3. Create a standard curve showing the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance, and save it.

8. The method according to claim 7, characterized in that, The method further includes the following steps: The stored correlation standard curve is retrieved, and the ratio of the first measured value to the second measured value of the test sample containing the target molecule is substituted into the standard curve for calculation to determine the concentration of the sample.

9. A system for performing an immunoassay by the method of any one of claims 5-8, comprising: An immunoassay apparatus comprising two or more reaction containers for simultaneously performing two parallel immunoassays on the same test sample in two of the reaction containers; wherein reaction container 1 is filled with reagent 1 of the kit, and reaction container 2 is filled with reagent 2 of the kit; A chemiluminescent immunoassay excitation and counting device is used to excite and record chemiluminescence readings, and to record two parallel immunoassay readings of the same sample as the first measurement value and the second measurement value, respectively. The first measurement value is derived from reaction container 1 and the second measurement value is derived from reaction container 2. The processor calculates the ratio of the first measurement to the second measurement, and calculates the concentration of the sample to be tested based on the ratio.

10. The system according to claim 9, characterized in that, The processor stores a standard curve showing the correlation between the ratio of the measured value a / measured value a' and the concentration of the standard substance, which is used to calculate the concentration of the sample to be tested.

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