A method for using an immunoassay kit and its detection system

Abstract

This invention relates to an immunoassay kit and its detection system. The kit includes reagent 1 and reagent 2. The method of using the kit includes the following steps: adding a test sample containing the target molecule to containers 1 and 2 respectively; then adding an α dose of reagent 1 and reagent 2 to container 1 and a β dose of reagent 1 and reagent 2 to container 2 to perform two parallel immunoassays on the test sample; wherein the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 1 to the content of the target molecule is different from the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 2 to the content of the target molecule. The method of using the kit of this invention can directly measure up to 10 6 High sample concentrations at the ng / ml level.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a method of using an immunoassay kit and its detection 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:

[0007] 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.

[0008] 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

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for using an immunoassay kit and a detection system thereof. The method for using the immunoassay kit described in this invention enables simple, rapid, and accurate calculation of the analyte concentration, and the detection system described in this invention can implement the method for using the kit.

[0010] To achieve the above and other related objectives, the present invention adopts the following technical solution:

[0011] The first aspect of the present invention provides a method of using an immunoassay kit, the kit comprising reagent 1 and reagent 2, wherein reagent 1 comprises luminescent microparticles coated with a first antibody (or antigen), and reagent 2 comprises a second antibody (or antigen) labeled with a marker; the method of using the kit comprises the following steps:

[0012] The test sample containing the target molecule is added to container 1 and container 2 respectively. Then, α doses of reagent 1 and reagent 2 are added to container 1, and β doses of reagent 1 and reagent 2 are added to container 2 to perform two parallel immune reaction detections on the test sample. The ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 1 to the contents of the target molecule is different from the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 2 to the contents of the target molecule.

[0013] In some embodiments of the present invention, the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to container 1 to the content of the target molecule is achieved by any of the following methods:

[0014] Method 1: The amount of test sample containing the target molecule added to container 1 and container 2 is the same, and α is not equal to β;

[0015] Method 2: The amount of test sample containing the target molecule added to container 1 is different from the amount of test sample containing the target molecule added to container 2, and α equals β;

[0016] Method 3: The amount of test sample containing the target molecule added to container 1 is different from the amount of test sample containing the target molecule added to container 2, and α is not equal to β.

[0017] In some embodiments of the present invention, the method further includes the following steps:

[0018] The detection results of two parallel immune reactions are stimulated and recorded, and are respectively recorded as the first value and the second value. The ratio of the first value to the second value is calculated. The first value is from container 1, the second value is from container 2, and the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first value to the contents of the target molecule is greater than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second value to the contents of the target molecule.

[0019] In some embodiments of the present invention, the method further includes the following steps:

[0020] 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.

[0021] A2, calculate the ratio of measured value a to measured value a';

[0022] 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.

[0023] In some embodiments of the present invention, the method further includes the following steps:

[0024] 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.

[0025] In some embodiments of the present invention, the method further includes the following steps:

[0026] 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.

[0027] B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it;

[0028] B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it;

[0029] 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.

[0030] In some embodiments of the present invention, the method further includes the following steps:

[0031] 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.

[0032] In some embodiments of the present invention, the target molecule to be tested is an antigen or an antibody.

[0033] In other embodiments of the present invention, the first antibody (or antigen) and the second antibody (or antigen) are capable of specifically binding to the target molecule to be tested.

[0034] A second aspect of the present invention provides a detection system for performing an immunoassay as described in the first aspect of the present invention, comprising:

[0035] An immunoassay apparatus comprising two or more containers for simultaneously performing two parallel immunoassays on the same test sample in two containers; wherein container 1 contains α doses of reagent 1 and reagent 2, and container 2 contains β doses of reagent 1 and reagent 2;

[0036] 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 from container 1 and the second measurement value is from container 2.

[0037] 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.

[0038] 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.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] (1) The method of using the kit described in this invention can solve the HOOK effect problem and avoid false detection caused by the HOOK effect. The method and system are not limited by the detection range.

[0041] (2) The method of using the kit described in this invention is to directly use the classic dose-response curve for calculation, which has good repeatability, does not require multiple dilutions, and can obtain accurate values ​​of HOOK effect samples in a single test, with fast measurement speed;

[0042] (3) The detection range of the kit described in this invention is much greater than that of conventional detection methods. Attached Figure Description

[0043] Figure 1 This is the dose-response curve of the antigen-antibody interaction.

[0044] Figure 2 This is a schematic diagram of the calculation method 1 for the use of the reagent kit of the present invention.

[0045] Figure 3 This is a schematic diagram of calculation method 2 for the use of the reagent kit of the present invention.

[0046] Figure 4 The graph shows the reaction curves between the concentration of the standard substance and the signals from reaction well 1 and reaction well 2 in Example 2. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The immunoassay kit used in the first aspect of this invention comprises reagent 1 and reagent 2. Reagent 1 contains luminescent microparticles coated with a first antibody (or antigen), and reagent 2 contains a second antibody (or antigen) labeled with a marker. In the method of using the kit, two parallel tests are performed on each sample. The ratio of the content of the target molecule to the content of the specific capture molecules (such as luminescent microparticles coated with a first antibody (or antigen) or a second antibody (or antigen) labeled with a marker) differs between the two tests, ultimately generating two different signals: a first measurement and a second measurement. The ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first measurement to the content of the target molecule is greater than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second measurement to the content of the target molecule. 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 of using the kit of this invention provides the following two methods for calculating the concentration of the target molecule, as follows:

[0051] Calculation Method 1: Directly calculate the concentration of the target molecule from the ratio of the first measured value to the second measured value.

[0052] 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.

[0053] Calculation Method 2: Calculate the concentration of the target molecule using reaction curve A or reaction curve B:

[0054] 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.

[0055] like Figure 3As 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.

[0056] 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.

[0057] 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.

[0058] Corresponding to the above calculation method 1, the method of using the reagent kit of the present invention includes the following steps:

[0059] 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:

[0060] 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.

[0061] A2, calculate the ratio of measured value a to measured value a';

[0062] 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.

[0063] Then, the concentration of the target molecule in the sample is determined by a method including the following steps:

[0064] S1, the test sample containing the target molecule is added to container 1 and container 2 respectively. Then, α doses of reagent 1 and reagent 2 are added to container 1, and β doses of reagent 1 and reagent 2 are added to container 2 to perform two parallel immune reaction detections on the test sample. The ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 1 to the contents of the target molecule is different from the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 2 to the contents of the target molecule.

[0065] S2, trigger and record the detection results of two parallel immune reactions, which are respectively recorded as the first measurement value and the second measurement value, and calculate the ratio of the first measurement value to the second measurement value; wherein the first measurement value comes from container 1, the second measurement value comes from container 2, and the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first measurement value to the contents of the target molecule is greater than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second measurement value to the contents of the target molecule.

[0066] 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.

[0067] Corresponding to calculation method 2 above, the method of using the reagent kit of the present invention includes the following steps:

[0068] First, reaction curve A, reaction curve B, and critical point c are obtained using a method that includes the following steps:

[0069] 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.

[0070] B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it;

[0071] B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it;

[0072] B4, there is a concentration overlap between the front zone of reaction curve A and the back zone 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, record the ratio of the measured value b to the measured value b' at that point as the critical point c, and store it.

[0073] Then, the concentration of the target molecule in the sample is determined by a method including the following steps:

[0074] S1, the test sample containing the target molecule is added to container 1 and container 2 respectively. Then, α doses of reagent 1 and reagent 2 are added to container 1, and β doses of reagent 1 and reagent 2 are added to container 2 to perform two parallel immune reaction detections on the test sample. The ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 1 to the contents of the target molecule is different from the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagents added to container 2 to the contents of the target molecule.

[0075] S2, trigger and record the detection results of two parallel immune reactions, which are respectively recorded as the first measurement value and the second measurement value, and calculate the ratio of the first measurement value to the second measurement value; wherein the first measurement value comes from container 1, the second measurement value comes from container 2, and the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first measurement value to the contents of the target molecule is greater than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second measurement value to the contents of the target molecule.

[0076] 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.

[0077] In this invention, the amount of sample containing the target molecule and the amount of reagent added in container 1 and container 2 can be changed individually or simultaneously, ultimately resulting in different ratios of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in container 1 and container 2 to the contents of the target molecule.

[0078] In some specific embodiments of the present invention, the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to container 1 to the content of the target molecule is different from the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to container 2 to the content of the target molecule by any of the following methods:

[0079] Method 1: The amount of test sample containing the target molecule added to container 1 and container 2 is the same, and α is not equal to β;

[0080] Method 2: The amount of test sample containing the target molecule added to container 1 is different from the amount of test sample containing the target molecule added to container 2, and α equals β;

[0081] Method 3: The amount of test sample containing the target molecule added to container 1 is different from the amount of test sample containing the target molecule added to container 2, and α is not equal to β.

[0082] In some 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 intact antibodies (e.g., bispecific antibodies), and antibody fragments (e.g., Fab regions, Fc regions, single-chain antibodies).

[0083] In other embodiments of the present invention, the first antibody (or antigen) and the second antibody (or antigen) are capable of specifically binding to the target molecule to be tested.

[0084] In some specific embodiments of the present invention, the marker may be biotin.

[0085] 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).

[0086] 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.

[0087] The second aspect of the present invention relates to a detection system for performing an immunoassay as described in the first aspect of the present invention, comprising:

[0088] An immunoassay apparatus comprising two or more containers for simultaneously performing two parallel immunoassays on the same test sample in two containers; wherein container 1 contains α doses of reagent 1 and reagent 2, and container 2 contains β doses of reagent 1 and reagent 2;

[0089] 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 from container 1 and the second measurement value is from container 2.

[0090] 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.

[0091] In this invention, there is no specific limitation on the shape of the container. In some specific embodiments of this invention, the 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.

[0092] 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.

[0093] 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:

[0094] 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.

[0095] A2, calculate the ratio of measured value a to measured value a';

[0096] 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.

[0097] In other embodiments of the 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:

[0098] 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.

[0099] B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it;

[0100] B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it;

[0101] B4. In the region where the concentrations of the front band of reaction curve A and the back band of reaction curve B overlap, take a point in the region where the concentrations overlap, and record the ratio of the measured value b to the measured value b' at that point as the critical point c, and store it.

[0102] Example

[0103] 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.

[0104] 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 drastically, 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.

[0105] Example 1: Detection of AFP samples using the standard usage method of the kit

[0106] The kit used was an alpha-fetoprotein (AFP) detection kit (chemiluminescence method) (batch number: L2001) manufactured by Komeiboyang Diagnostic Technology (Shanghai) Co., Ltd., whose main components are:

[0107] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0108] Reagent 2: Biotin-labeled AFP antibody.

[0109] The instructions for using the kit are as follows:

[0110] 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;

[0111] 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 then use... The analyzer takes readings.

[0112] Test samples (collected from clinical serum samples):

[0113] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0114] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0115] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0116] The test results are shown in Table 1.

[0117] Table 1

[0118] Preliminary values Measured value ng / mL Sample 1 7.41 Sample 2 109.43 Sample 3 11.79

[0119] 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.

[0120] Table 2

[0121] 50-fold dilution Measured value ng / mL Diluted sample >1000

[0122] 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.

[0123] Table 3

[0124] 2500 times dilution Measured value ng / mL Diluted sample 849.51

[0125] 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.

[0126] Example 2: Detection of AFP samples using the kit method of the present invention

[0127] The kit used was an alpha-fetoprotein (AFP) detection kit (chemiluminescence method) (batch number: L2001) manufactured by Komeiboyang Diagnostic Technology (Shanghai) Co., Ltd., whose main components are:

[0128] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0129] Reagent 2: Biotin-labeled AFP antibody.

[0130] Instructions for using the kit:

[0131] For both containers, which are from the same sample test group, repeat steps 1 and 2 below for different sample test groups:

[0132] 1. Add 10 μl of the sample to be tested, 50 μl of reagent 1, and 50 μl of reagent 2 to reaction well 1 respectively;

[0133] 2. Add 10 μL of the sample to be tested, 5 μL of reagent 1, and 5 μL of reagent 2 to reaction well 2 respectively;

[0134] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0135] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0136] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0137] Test samples (collected from clinical serum samples):

[0138] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0139] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0140] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0141] The AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 4.

[0142] Based on the values ​​in Table 4, plot reaction curves A and B respectively, showing the relationship between the standard substance concentration and the signals from reaction well 1 and reaction well 2. Figure 4 It can be seen that standard substance 1-11 corresponds to the front zone of reaction curve A, and standard substance 9-20 corresponds 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 substance 9-11, and its corresponding A / B signal ratio is 14.90-25.53. The midpoint A / B signal ratio = 19 is taken as the critical point.

[0143] 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.

[0144] Store the above standard substance test results. Analyzer.

[0145] 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.

[0146] Table 4

[0147]

[0148] Table 5

[0149]

[0150] As shown in Table 5, the method of using the kit of the present invention can avoid the problem of low sample measurement values ​​caused by the HOOK effect, and can directly obtain 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.

[0151] Example 3: Detection of AFP samples using the kit method of the present invention

[0152] The kit used was an alpha-fetoprotein (AFP) detection kit (chemiluminescence method) (batch number: L2001) manufactured by Komeiboyang Diagnostic Technology (Shanghai) Co., Ltd., whose main components are:

[0153] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0154] Reagent 2: Biotin-labeled AFP antibody.

[0155] Instructions for using the kit:

[0156] For both containers, which are from the same sample test group, repeat steps 1 and 2 below for different sample test groups:

[0157] 1. Add 10 μl of the sample to be tested, 50 μl of reagent 1, and 50 μl of reagent 2 to reaction well 1 respectively;

[0158] 2. Add 20 μL of the sample to be tested, 50 μL of reagent 1, and 50 μL of reagent 2 to reaction well 2 respectively;

[0159] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0160] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0161] Test standard material: concentration range of 0 ng / mL - 4 × 106 Purified AFP antigen solution (ng / mL)

[0162] Test samples (collected from clinical serum samples):

[0163] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0164] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0165] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0166] 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 database / system / etc.]. Analyzer.

[0167] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 7 below (calculated using the calculation method 1 of this invention).

[0168] Table 6

[0169]

[0170] Table 7

[0171]

[0172] The results show that the method of using the kit of the present invention can detect samples with ultra-high AFP values, has a wide detection range, and can easily, quickly and accurately calculate the concentration of the analyte.

[0173] Example 4: Detection of AFP samples using the kit method of the present invention

[0174] The kit used was an alpha-fetoprotein (AFP) detection kit (chemiluminescence method) (batch number: L2001) manufactured by Komeiboyang Diagnostic Technology (Shanghai) Co., Ltd., whose main components are:

[0175] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0176] Reagent 2: Biotin-labeled AFP antibody.

[0177] Instructions for using the kit:

[0178] For both containers, which are from the same sample test group, repeat steps 1 and 2 below for different sample test groups:

[0179] 1. Add 10 μl of the sample to be tested, 50 μl of reagent 1, and 50 μl of reagent 2 to reaction well 1 respectively;

[0180] 2. Add 20 μL of the sample to be tested, 5 μL of reagent 1, and 5 μL of reagent 2 to reaction well 2 respectively;

[0181] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0182] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0183] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0184] Test samples (collected from clinical serum samples):

[0185] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0186] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0187] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0188] 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 8. 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 8. The test results of the above standard substances were stored in [the database / system / etc.]. Analyzer.

[0189] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 9 below (calculated using the calculation method 1 of this invention).

[0190] Table 8

[0191]

[0192]

[0193] Table 9

[0194]

[0195] The results show that the method of using the kit of the present invention can detect samples with ultra-high AFP values, has a wide detection range, and can easily, quickly and accurately calculate the concentration of the analyte.

[0196] Example 5: Verification of the Precision of Ultra-High-End Measurements

[0197] The kit used was an alpha-fetoprotein (AFP) detection kit (chemiluminescence method) (batch number: L2001) manufactured by Komeiboyang Diagnostic Technology (Shanghai) Co., Ltd., whose main components are:

[0198] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0199] Reagent 2: Biotin-labeled AFP antibody.

[0200] Instructions for using the kit:

[0201] 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:

[0202] 1. Add 10 μl of the sample to be tested, 50 μl of reagent 1, and 50 μl of reagent 2 to reaction well 1 respectively;

[0203] 2. Add 10 μL of the sample to be tested, 5 μL of reagent 1, and 5 μL of reagent 2 to reaction well 2 respectively;

[0204] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0205] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0206] The test results are shown in Table 10 below (calculated using the calculation method 1 of this invention).

[0207] Table 10

[0208]

[0209]

[0210] 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.

[0211] 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. A method of using an immunoassay kit, the kit comprising reagent 1 and reagent 2, wherein reagent 1 comprises luminescent microparticles coated with a first antibody or antigen, and reagent 2 comprises a second antibody or antigen labeled with a marker; The method of using the kit includes the following steps: adding the test sample containing the target molecule to container 1 and container 2 respectively; then adding α doses of reagent 1 and reagent 2 to container 1 and β doses of reagent 1 and reagent 2 to container 2 to perform two parallel immune reaction detections on the test sample; wherein the ratio of the sum of the contents of the first antibody or antigen and the second antibody or antigen in the reagents added to container 1 to the contents of the target molecule is different from the ratio of the sum of the contents of the first antibody or antigen and the second antibody or antigen in the reagents added to container 2 to the contents of the target molecule. The method further includes the following steps: stimulating and recording the detection results of two parallel immune reactions, respectively recorded as a first measurement value and a second measurement value, and calculating the ratio of the first measurement value to the second measurement value; wherein the first measurement value originates from container 1, the second measurement value originates from container 2, and the ratio of the sum of the contents of the first antibody or antigen and the second antibody or antigen corresponding to the first measurement value to the contents of the target molecule is greater than the ratio of the sum of the contents of the first antibody or antigen and the second antibody or antigen corresponding to the second measurement value to the contents of the target molecule. The method further includes: The concentration of the target molecule in the test sample is determined based on reaction curve A or reaction curve B, where reaction curve A and reaction curve B are reaction curves obtained based on the first and second measured values ​​and the concentration of the standard substance, respectively. A point is selected in the overlapping region of the concentrations in the front band of reaction curve A and the back band of reaction curve B, and the ratio of the first measured value to the second measured value at this point is taken as 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 of reaction curve A; when the ratio of the first measured value to the second measured value of the test sample is greater than or equal to the critical point c, the concentration of the target molecule in the test sample is calculated using the back band of reaction curve B.

2. The method of use according to claim 1, characterized in that, The ratio of the sum of the contents of the first antibody or antigen and the second antibody or antigen in the reagent added to container 1 to the contents of the target molecule is made different from the ratio of the sum of the contents of the first antibody or antigen and the second antibody or antigen in the reagent added to container 2 to the contents of the target molecule by any of the following methods: Method 1: The amount of test sample containing the target molecule added to container 1 and container 2 is the same, and α is not equal to β; Method 2: The amount of test sample containing the target molecule added to container 1 is different from the amount of test sample containing the target molecule added to container 2, and α equals β; Method 3: The amount of test sample containing the target molecule added to container 1 is different from the amount of test sample containing the target molecule added to container 2, and α is not equal to β.

3. The method of use according to claim 1 or 2, characterized in that, 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.

4. The method of use according to claim 3, characterized in that, The method further includes the following steps: 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 to be tested and the magnitude of the critical point c.

5. The method of use according to claim 1, characterized in that, The target molecule to be tested is an antigen or an antibody.

6. The method of use according to claim 1, characterized in that, The first antibody or antigen and the second antibody or antigen can specifically bind to the target molecule to be tested.

7. A detection system for implementing the method of use as described in any one of claims 1-6, comprising: An immunoassay apparatus comprising two or more containers for simultaneously performing two parallel immunoassays on the same test sample in two of the containers. Container 1 contains α doses of reagent 1 and reagent 2, and container 2 contains β doses of reagent 1 and reagent 2. 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 from container 1 and the second measurement value is from 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.

8. The system according to claim 7, characterized in that, The processor stores the reaction curve A between the measured value b and the concentration of the standard substance, the reaction curve B between the measured value b' and the concentration of the standard substance, and the critical point c, which are used to calculate the concentration of the sample to be tested.

Images