An immunoassay device for detecting the concentration of a target molecule.

Abstract

This invention relates to an immunoassay device for detecting the concentration of a target molecule, comprising an immunoassay assembly including at least two reaction containers and a reagent arm. The reagent arm is used to add the same detection reagent to each of the two reaction containers. The two reaction containers are used to perform two parallel immunoassays on the same sample. The ratio of the concentration of a specific capture molecule in reaction container 1 to the concentration of the target molecule in the sample differs from the ratio in reaction container 2. The device of this invention can solve the problem of hook effect samples, is not limited by the detection range, and has good repeatability.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to an immunoassay device for detecting the concentration of a target molecule. 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 an immunoassay device for detecting the concentration of target molecules. The device of the present invention effectively avoids the hook effect and allows for simple, rapid, and accurate calculation of the analyte concentration.

[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 an immunoassay device for detecting the concentration of a target molecule, comprising an immunoassay assembly, the immunoassay assembly comprising at least two reaction containers and a reagent arm, wherein the reagent arm is used to add the same detection reagent to the two reaction containers respectively, and the two reaction containers are used to perform two parallel immunoassays on the same sample.

[0012] The ratio of the content of specific captured molecules in reaction vessel 1 to the content of target molecules in the same sample is different from the ratio of the content of specific captured molecules in reaction vessel 2 to the content of target molecules in the same sample.

[0013] In some embodiments of the present invention, the ratio of the content of specifically captured molecules in reaction vessel 1 to the content of target molecules in the test sample is different from the ratio of the content of specifically captured molecules in reaction vessel 2 to the content of target molecules in the test sample by any of the following methods:

[0014] Method 1: The amount of sample to be tested added to reaction vessel 1 and reaction vessel 2 is the same, but the amount of detection reagent added is different;

[0015] Method 2: The amount of sample to be tested added to reaction vessel 1 is different from the amount of sample to be tested added to reaction vessel 2, but the amount of detection reagent added is the same;

[0016] Method 3: The amount of sample to be tested added to reaction vessel 1 is different from the amount of sample to be tested added to reaction vessel 2, and the amount of detection reagent added is also different.

[0017] In other embodiments of the present invention, the device includes an immunoassay component for recording the detection results of two parallel immunoreactions of the same test sample, namely a first measurement value and a second measurement value; wherein, the reading meter in reaction container 1 is the first measurement value, and the reading meter in reaction container 2 is the second measurement value; preferably, the ratio of the content of specific capture molecules corresponding to the first measurement value to the content of the target molecules to be tested in the two parallel immunoreaction detections is greater than the ratio of the content of specific capture molecules corresponding to the second measurement value to the content of the target molecules to be tested.

[0018] In some embodiments of the present invention, the device includes a processor for calculating the ratio of a first measurement to a second measurement and determining the concentration of the target molecule in the sample to be tested.

[0019] In some specific embodiments of the present invention, the detection reagent includes reagent 1 and reagent 2, wherein 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.

[0020] In some specific embodiments of the present invention, the target molecule to be tested is an antigen or an antibody; and / or

[0021] The first antibody (or antigen) and the second antibody (or antigen) can specifically bind to the target molecule to be tested.

[0022] In some embodiments of the present invention, the device performs the following steps:

[0023] 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 recorded as measured value a and measured value a', respectively. Measured value a is detected in the same way as the first measured value of the sample to be tested, and measured value a' is detected in the same way as the second measured value of the sample to be tested.

[0024] A2: Calculate the ratio of measured value a to measured value a';

[0025] 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.

[0026] In other embodiments of the present invention, the device performs the following steps:

[0027] The processor retrieves the stored correlation standard curve, substitutes the ratio of the first measured value to the second measured value of the sample into the correlation standard curve for calculation, and determines the concentration of the target molecule in the sample.

[0028] In some embodiments of the present invention, the device performs the following steps:

[0029] B1: A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed, and the results of the two parallel immune reactions are recorded as measured value b and measured value b', respectively. Measured value b is detected in the same way as the first measured value of the sample to be tested, and measured value b' is detected in the same way as the second measured value of the sample to be tested.

[0030] B2: Plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it;

[0031] B3: Plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it;

[0032] 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.

[0033] In other embodiments of the present invention, the device performs the following steps:

[0034] The processor retrieves the stored reaction curves A and B and the critical point c, and judges the ratio of the first measured value to the second measured value of the test sample and the magnitude of 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 the 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 the reaction curve B.

[0035] A second aspect of the present invention provides a computer-readable storage medium having a computer program product stored thereon, the computer program product causing the apparatus as described in the first aspect of the present invention to perform corresponding steps.

[0036] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0037] (1) When performing immunoassay using the device of the present invention, the HOOK effect problem can be solved, the HOOK effect can be avoided, and the detection range is not limited.

[0038] (2) When performing immunoassays using the device of the present invention, the classic dose-response curve can be used directly for calculation, and the repeatability is good;

[0039] (3) When using the device of the present invention to perform immune testing, the detection range is much greater than that of conventional detection methods. Attached Figure Description

[0040] Figure 1 This is the dose-response curve of the antigen-antibody interaction.

[0041] Figure 2 This is a schematic diagram of calculation method 1 for performing immunoassays using the device of the present invention.

[0042] Figure 3 This is a schematic diagram of calculation method 2 for performing an immune test using the device of the present invention.

[0043] Figure 4 This is a graph showing the reaction between the concentration of the standard substance and the signals from reaction vessel 1 and reaction vessel 2 in Example 2.

[0044] Figure 5 This is a schematic diagram of the structure of the immunoassay device described in this invention. Detailed Implementation

[0045] 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.

[0046] 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.

[0047] 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.

[0048] The immunoassay device for detecting the concentration of a target molecule according to the first aspect of this invention can perform two parallel tests on each sample and calculate the concentration of the target molecule in the sample based on the results of the two parallel tests. The device has no detection range limitation and can avoid missed detections caused by the hook effect.

[0049] In some embodiments of the present invention, the immunoassay device for detecting the concentration of the target molecule includes an immunoassay assembly, the immunoassay assembly including at least two reaction containers and a reagent arm, wherein the reagent arm is used to add the same detection reagent to the two reaction containers respectively, and the two reaction containers are used to perform two parallel immunoassays on the same sample.

[0050] The ratio of the content of specific captured molecules in reaction vessel 1 to the content of target molecules in the same sample is different from the ratio of the content of specific captured molecules in reaction vessel 2 to the content of target molecules in the same sample.

[0051] In this invention, the amount of sample containing the target molecule and the amount of detection reagent added in reaction vessel 1 and reaction vessel 2 can be changed individually or simultaneously, ultimately resulting in different ratios of the content of the specifically captured molecule / the content of the target molecule in reaction vessel 1 and reaction vessel 2.

[0052] In some embodiments of the present invention, the ratio of the content of specifically captured molecules in reaction vessel 1 to the content of target molecules in the test sample is different from the ratio of the content of specifically captured molecules in reaction vessel 2 to the content of target molecules in the test sample by any of the following methods:

[0053] Method 1: The amount of sample to be tested added to reaction vessel 1 and reaction vessel 2 is the same, but the amount of detection reagent added is different;

[0054] Method 2: The amount of sample to be tested added to reaction vessel 1 is different from the amount of sample to be tested added to reaction vessel 2, but the amount of detection reagent added is the same;

[0055] Method 3: The amount of sample to be tested added to reaction vessel 1 is different from the amount of sample to be tested added to reaction vessel 2, and the amount of detection reagent added is also different.

[0056] This invention does not specifically limit the shape of the reaction vessel. In some specific embodiments of this invention, the reaction vessel may be a reaction orifice, a reaction cup, a reaction tank, etc.

[0057] In some embodiments of the present invention, the device includes an immunoassay component for recording the detection results of two parallel immunoreactions of the same test sample, namely a first measurement value and a second measurement value; wherein, the reading meter in reaction container 1 is the first measurement value, and the reading meter in reaction container 2 is the second measurement value; preferably, the ratio of the content of specific capture molecules corresponding to the first measurement value to the content of the target molecules to be tested in the two parallel immunoreaction detections is greater than the ratio of the content of specific capture molecules corresponding to the second measurement value to the content of the target molecules to be tested.

[0058] In some specific embodiments of the invention, the immunoassay assembly may include a photon counting module and a light-emitting diode, which are then used to excite and record the detection results of two parallel immune responses to the same test sample.

[0059] In some embodiments of the present invention, the device includes a processor for calculating the ratio of a first measurement to a second measurement and determining the concentration of the target molecule in the sample to be tested.

[0060] In some specific embodiments of the present invention, the processor may be a computer, for processing, plotting, and storing the detection results.

[0061] In some specific embodiments of the present invention, the detection reagent includes reagent 1 and reagent 2, wherein 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.

[0062] In some specific embodiments of the present invention, the target molecule to be tested is an antigen or an antibody. 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 term "antibody" is used in the broadest sense herein and explicitly encompasses 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).

[0063] 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.

[0064] In some specific embodiments of the present invention, the marker may be biotin.

[0065] 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).

[0066] In the immunoassay device of this invention, during two parallel tests on each sample, the ratio of the content of the target molecule to the content of the specific capture molecules in the detection reagent (e.g., luminescent microparticles coated with a first antibody (or antigen), or a labeled second antibody (or antigen)) differs, 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 (concentration) of the target molecule increases, the ratio of the first measurement to the second measurement continuously rises and exhibits a certain linear relationship. Based on this principle, the processor of the assay device of this invention can calculate the concentration of the target molecule in the sample by executing the following two methods, as follows:

[0067] Calculation Method 1: Directly calculate the concentration of the target molecule in the sample from the ratio of the first measured value to the second measured value.

[0068] 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 processor substitutes the ratio of the first measured value to the second measured value of the target molecule into the curve to calculate its concentration.

[0069] Calculation Method 2: Calculate the concentration of the target molecule in the sample using reaction curve A or reaction curve B.

[0070] 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.

[0071] 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 use the ratio of the first measurement value to the second measurement value corresponding to this point (A / B = 15 in the figure) as the critical point c, and store it.

[0072] When the processor determines that 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, it uses the front band region of the reaction curve A to calculate the concentration.

[0073] When the processor determines that the ratio of the first measured value to the second measured value of the sample is greater than the critical point c, it uses the back band region of the reaction curve B to calculate its concentration.

[0074] Corresponding to the above calculation method 1, the immunoassay device provided by the present invention performs the following steps:

[0075] First, the device obtains a standard curve showing the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance by performing the following steps:

[0076] A1: The reagent arm adds the test reagents (reagent 1 and reagent 2) to reaction vessel 1 and reaction vessel 2, which contain the same concentration of the target molecule, respectively, so as to perform two parallel immune reaction detections for each of the series of standard substances with different concentrations of known target molecule content.

[0077] A2: The immunoassay component records the detection results of two parallel immune reactions, which are respectively denoted as value a and value a'. Value a is greater than value a', and the detection method of value a is the same as that of the first value of the sample to be tested, while the detection method of value a' is the same as that of the second value of the sample to be tested.

[0078] A3: The processor calculates the ratio of measured value a / measured value a', and based on the calculated ratio of measured value a / measured value a', plots a standard curve showing the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance, and stores the curve.

[0079] The device then determines the concentration of the target molecule in the sample by performing the following steps:

[0080] S1: The reagent arm adds the test reagents (reagent 1 and reagent 2) into reaction container 1 and reaction container 2, which contain the sample to be tested, respectively, to perform two parallel immune reaction tests on the same sample;

[0081] S2: The immunoassay assembly records the detection results of two parallel immunoassays, which are recorded as the first measurement and the second measurement, respectively. The reading in reaction container 1 is recorded as the first measurement, and the reading in reaction container 2 is recorded as the second measurement. In the two parallel immunoassays, the ratio of the content of specific capture molecules to the content of target molecules corresponding to the first measurement is greater than the ratio of the content of specific capture molecules to the content of target molecules corresponding to the second measurement.

[0082] S3: The processor calculates the ratio of the first measured value to the second measured value of the sample to be tested, and retrieves the stored correlation standard curve; then, it substitutes the ratio of the first measured value to the second measured value of the sample to be tested into the correlation standard curve to calculate and determine the concentration of the target molecule in the sample to be tested.

[0083] Corresponding to calculation method 2 above, the immunoassay device provided by the present invention performs the following steps:

[0084] First, the device obtains reaction curve A, reaction curve B, and critical point c by performing the following steps:

[0085] B1: The reagent arm adds the test reagents (reagent 1 and reagent 2) to reaction vessel 1 and reaction vessel 2, which contain the same concentration of the target molecule, respectively, so as to perform two parallel immune reactions for each of the series of standard substances with different concentrations of known target molecule content.

[0086] B2: The immunoassay kit records the detection results of two parallel immune reactions, which are respectively denoted as value b and value b'. The detection method of value b is the same as that of the first value of the sample to be tested, and the detection method of value b' is the same as that of the second value of the sample to be tested.

[0087] B3: The processor generates and stores the reaction curve A between the measured value b and the concentration of the standard substance based on a series of known measured values ​​b of different concentrations of standard substances; at the same time, it generates and stores the reaction curve B between the measured value b' and the concentration of the standard substance based on a series of known measured values ​​b' of different concentrations of standard substances.

[0088] 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) The processor selects a point in the overlapping concentration area, records the ratio of the measured value b to the measured value b' at that point as the critical point c, and stores it.

[0089] The device then determines the concentration of the target molecule in the sample by performing the following steps:

[0090] S1: The reagent arm adds the test reagents (reagent 1 and reagent 2) into reaction container 1 and reaction container 2, which contain the sample to be tested, respectively, to perform two parallel immune reaction tests on the same sample;

[0091] S2: The immunoassay assembly records the detection results of two parallel immunoassays, which are recorded as the first measurement and the second measurement, respectively. The reading in reaction container 1 is recorded as the first measurement, and the reading in reaction container 2 is recorded as the second measurement. In the two parallel immunoassays, the ratio of the content of specific capture molecules to the content of target molecules corresponding to the first measurement is greater than the ratio of the content of specific capture molecules to the content of target molecules corresponding to the second measurement.

[0092] S3: The processor retrieves the stored reaction curves A and B and the critical point c, and judges 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, the sample concentration is calculated 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, the sample concentration is calculated using the back band region of the reaction curve B.

[0093] 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.

[0094] Example

[0095] 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.

[0096] 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.

[0097] Example 1: Detection of AFP samples using a conventional immunoassay apparatus

[0098] Test samples (collected from clinical serum samples):

[0099] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0100] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0101] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0102] 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:

[0103] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0104] Reagent 2: Biotin-labeled AFP antibody.

[0105] Test method:

[0106] 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;

[0107] 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.

[0108] The test results are shown in Table 1.

[0109] Table 1

[0110] Preliminary values Measured value ng / mL Sample 1 7.41 Sample 2 109.43 Sample 3 11.79

[0111] The results shown in Table 1 are from direct detection using conventional equipment. 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 times with diluent and retested, and the results are shown in Table 2.

[0112] Table 2

[0113] 50-fold dilution Measured value ng / mL Diluted sample >1000

[0114] 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.

[0115] Table 3

[0116] 2500 times dilution Measured value ng / mL Diluted sample 849.51

[0117] 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.

[0118] Example 2: Detection of AFP samples using the immunoassay device of the present invention

[0119] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0120] Test samples (collected from clinical serum samples):

[0121] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0122] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0123] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0124] 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:

[0125] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0126] Reagent 2: Biotin-labeled AFP antibody.

[0127] The detection steps performed by the measuring device are as follows:

[0128] Set up two reaction vessels as the same sample test group, and repeat the following liquid addition steps 1 and 2 for different sample test groups:

[0129] 1. Add 10 μl of the same test sample to both reaction vessel 1 and reaction vessel 2;

[0130] 2. Using the reagent arm, add 50 μL of reagent 1 and 50 μL of reagent 2 to reaction vessel 1;

[0131] 3. Using the reagent arm, add 5 μL of reagent 1 and 5 μL of reagent 2 to reaction vessel 2;

[0132] 4. Incubate each reaction vessel simultaneously at 37°C for 15 minutes;

[0133] 5. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction vessel and incubate at 37°C for 10 min.

[0134] 6. Record the detection results of the immune reaction using the immunoassay kit, and record the results of two parallel immune reactions of the same sample as the first measurement value and the second measurement value, respectively; wherein, the reading in reaction container 1 is recorded as the first measurement value, and the reading in reaction container 2 is recorded as the second measurement value;

[0135] 7. The processor calculates the ratio of the first measured value to the second measured value of the same sample, retrieves the stored data for judgment, and then calculates the concentration of the target molecule in the sample.

[0136] The AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 4.

[0137] 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.

[0138] 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.

[0139] The test results of the above standard substances are stored in the immunoassay device.

[0140] 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.

[0141] Table 4

[0142]

[0143]

[0144] Table 5

[0145]

[0146] As can be seen from the results shown in Table 5, the device 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 The detection results are in ng / mL. The method is not limited by the detection range, and both calculation methods are feasible.

[0147] Example 3: Detection of AFP samples using the immunoassay device of the present invention

[0148] The experimental materials were the same as in Example 2.

[0149] The detection steps performed by the measuring device are as follows:

[0150] Set up two reaction vessels as the same sample test group, and repeat the following liquid addition steps 1 and 2 for different sample test groups:

[0151] 1. Add 10 μl of the sample to be tested to reaction vessel 1 and 20 μl of the sample to be tested to reaction vessel 2;

[0152] 2. Using the reagent arm, add 50 μL of reagent 1 and 50 μL of reagent 2 to reaction vessel 1;

[0153] 3. Using the reagent arm, add 50 μL of reagent 1 and 50 μL of reagent 2 to reaction vessel 2;

[0154] 4. Incubate each reaction vessel simultaneously at 37°C for 15 minutes;

[0155] 5. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction vessel and incubate at 37°C for 10 min.

[0156] 6. Record the detection results of the immune reaction using the immunoassay kit, and record the results of two parallel immune reactions of the same sample as the first measurement value and the second measurement value, respectively; wherein, the reading in reaction container 1 is recorded as the first measurement value, and the reading in reaction container 2 is recorded as the second measurement value;

[0157] 7. The processor calculates the ratio of the first measured value to the second measured value of the same sample, retrieves the stored data for judgment, and then calculates the concentration of the target molecule in the sample.

[0158] 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 immunoassay device.

[0159] 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).

[0160] Table 6

[0161]

[0162] Table 7

[0163]

[0164]

[0165] The results show that the device of the present invention can detect samples with ultra-high AFP values, has a wide detection range, and can calculate the concentration of the analyte easily, quickly, and accurately.

[0166] Example 4: Detection of AFP samples using the immunoassay device of the present invention

[0167] The experimental materials were the same as in Example 2.

[0168] The detection steps performed by the measuring device are as follows:

[0169] Set up two reaction vessels as the same sample test group, and repeat the following liquid addition steps 1 and 2 for different sample test groups:

[0170] 1. Add 10 μl of the sample to be tested to reaction vessel 1 and 20 μl of the sample to be tested to reaction vessel 2;

[0171] 2. Using the reagent arm, add 50 μL of reagent 1 and 50 μL of reagent 2 to reaction vessel 1;

[0172] 3. Using the reagent arm, add 5 μL of reagent 1 and 5 μL of reagent 2 to reaction vessel 2;

[0173] 4. Incubate each reaction vessel simultaneously at 37°C for 15 minutes;

[0174] 5. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction vessel and incubate at 37°C for 10 min.

[0175] 6. Record the detection results of the immune reaction using the immunoassay kit, and record the results of two parallel immune reactions of the same sample as the first measurement value and the second measurement value, respectively; wherein, the reading in reaction container 1 is recorded as the first measurement value, and the reading in reaction container 2 is recorded as the second measurement value;

[0176] 7. The processor calculates the ratio of the first measured value to the second measured value of the same sample, retrieves the stored data for judgment, and then calculates the concentration of the target molecule in the sample.

[0177] 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 immunoassay device.

[0178] 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).

[0179] Table 8

[0180]

[0181] Table 9

[0182]

[0183] The results show that the device of the present invention can detect samples with ultra-high AFP values, has a wide detection range, and can calculate the concentration of the analyte easily, quickly, and accurately.

[0184] Example 5: Verification of the Precision of Ultra-High-End Measurements

[0185] 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:

[0186] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0187] Reagent 2: Biotin-labeled AFP antibody.

[0188] The detection steps performed by the measuring device are the same as in Example 2.

[0189] The test results are shown in Table 10 below (calculated using the calculation method 1 of this invention).

[0190] Table 10

[0191]

[0192] The results showed that the CV of the measuring device of the present invention was within 10% for 10 repeated measurements of the three high-value samples, indicating that its precision results were good.

[0193] 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 device for detecting the concentration of a target molecule using photo-induced chemiluminescence, comprising: An immunoassay assembly includes at least two reaction containers and a reagent arm, wherein the reagent arm is used to add the same detection reagent to each of the two reaction containers, and the two reaction containers are used to perform two parallel immunoassays on the same test sample; wherein the ratio of the content of specific capture molecules in reaction container 1 to the content of target molecules in the test sample is different from the ratio of the content of specific capture molecules in reaction container 2 to the content of target molecules in the test sample. An immunoassay kit is used to record the detection results of two parallel immune reactions of the same test sample, namely a first measurement value and a second measurement value. The processor retrieves stored reaction curves A and B, and a critical point c. It then compares the ratio of the first measured value to the second measured value of the sample with the critical point c. When the ratio of the first measured value to the second measured value is less than or equal to the critical point c, the concentration of the target molecule in the sample is calculated using the front region of reaction curve A. When the ratio of the first measured value to the second measured value is greater than the critical point c, the concentration of the target molecule in the sample is calculated using the back region of reaction curve B. Reaction curves A and B are obtained from the detection results of two parallel immunoreactions using a standard substance with a known content of the same target molecule. A point is taken where the concentrations overlap between the front region of reaction curve A and the back region of reaction curve B; the ratio of the first measured value to the second measured value of the standard substance at this point is the critical point c.

2. The apparatus according to claim 1, characterized in that, The ratio of the content of specifically captured molecules in reaction vessel 1 to the content of target molecules in the test sample is different from the ratio of the content of specifically captured molecules in reaction vessel 2 to the content of target molecules in the test sample by any of the following methods: Method 1: The amount of sample to be tested added to reaction vessel 1 and reaction vessel 2 is the same, but the amount of detection reagent added is different; Method 2: The amount of sample to be tested added to reaction vessel 1 is different from the amount of sample to be tested added to reaction vessel 2, but the amount of detection reagent added is the same; Method 3: The amount of sample to be tested added to reaction vessel 1 is different from the amount of sample to be tested added to reaction vessel 2, and the amount of detection reagent added is also different.

3. The apparatus according to claim 1, characterized in that, In two parallel immunoassays, the ratio of the content of specific capture molecules to the content of target molecules corresponding to the first measurement is greater than the ratio of the content of specific capture molecules to the content of target molecules corresponding to the second measurement.

4. The apparatus according to claim 1, characterized in that, The detection reagent includes reagent 1 and reagent 2, wherein 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.

5. The apparatus according to claim 4, characterized in that, The target molecule to be tested is an antigen or antibody; and / or The first antibody or antigen and the second antibody or antigen can specifically bind to the target molecule to be tested.

6. The apparatus according to any one of claims 1-5, characterized in that, The device performs the following steps: B1: A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed, and the results of the two parallel immune reactions are recorded as measured value b and measured value b', respectively. Measured value b is detected in the same way as the first measured value of the sample to be tested, and measured value b' is detected in the same way as the second measured 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.

7. A computer-readable storage medium storing a computer program product thereon, characterized in that, The computer program product causes the apparatus as described in any one of claims 1-6 to perform the corresponding steps.

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