A method, system, and application for identifying HOOK effect samples in immunoassays.

Abstract

This invention relates to a method and system for identifying hook effect samples in immunoassays. The method includes the following steps: S1, performing two parallel immunoassays on a sample containing the target molecule, and recording the results of the two parallel immunoassays as a first value and a second value, respectively; S2, calculating the ratio of the first value to the second value; S3, obtaining the critical point c of a standard substance, wherein the results of the two parallel immunoassays of the standard substance corresponding to the critical point c are recorded as value a and value a', respectively, wherein value a is greater than value a'; S4, comparing the ratio of the first value to the second value with the critical point c, thereby identifying whether the sample is a hook effect sample. This method can solve the problem that traditional detection reagents are difficult to distinguish hook effect samples and is not limited by the detection range.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a method, system, and application for identifying HOOK effect samples in immunoassays. 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] Photoluminescence immunoassay (PLIA) 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 PLACI analysis is as follows: A sensitizer, under laser irradiation, 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 the donor particles containing the sensitizer to bind to the 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] Traditional immunoassay reagents lack methods to identify the HOOK effect, often requiring clinicians to combine the patient's clinical presentation with diluted serum samples to determine whether the HOOK effect exists. This process is complex, time-consuming, and prone to false negatives. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a method, system, and application for identifying hook effect samples in immunoassays. The method and system described herein enable a simple, rapid, and accurate identification of whether a sample to be tested is a hook effect sample.

[0009] To achieve the above and other related objectives, the present invention adopts the following technical solution:

[0010] The first aspect of this invention provides a method for identifying HOOK effect samples in an immunoassay, comprising the following steps:

[0011] S1, Perform two parallel immune response tests on the test sample containing the target molecule, and record the results of the two parallel immune responses as the first and second values, respectively.

[0012] S2, calculate the ratio of the first measured value to the second measured value;

[0013] S3, obtain the critical point c of the standard substance, and the detection results of two parallel immune reactions of the standard substance corresponding to the critical point c are respectively recorded as measured value a and measured value a', wherein measured value a is greater than measured value a';

[0014] S4, compare the ratio of the first measured value to the second measured value with the critical point c, and then identify whether the sample to be tested is a HOOK effect sample.

[0015] In some embodiments of the present invention, when the ratio of the first measured value to the second measured value is less than the critical point c, the sample to be tested is a non-HOOK effect sample;

[0016] When the ratio of the first measured value to the second measured value is greater than the critical point c, and the first measured value is greater than the measured value a, the sample to be tested is a non-HOOK effect sample.

[0017] In some other embodiments of the present invention, when the ratio of the first measured value to the second measured value is greater than the critical point c, and the first measured value is less than the measured value a, the sample to be tested is a HOOK effect sample.

[0018] In some embodiments of the present invention, the ratio of the content of the target molecule to the content of the specific capture molecule is different in the two parallel immune response assays; wherein the specific capture molecule is capable of specifically binding to the target molecule.

[0019] In some embodiments of the present invention, in the two parallel immune response assays, the immune response with a larger ratio of the content of the specific capture molecule to the content of the target molecule is recorded as the first measurement or measurement a, and the other is recorded as the second measurement or measurement a'.

[0020] In some embodiments of the present invention, step S2, the method for obtaining the critical point c includes the following steps:

[0021] A1. A series of standard substances with different concentrations of known target molecules were tested. For each standard substance, two parallel immunoassays were performed, and the results of the two parallel immunoassays were recorded as measured value a and measured value a', respectively.

[0022] A2, plot the reaction curve A between the measured value a and the concentration of the standard substance, and store it;

[0023] A3, plot the reaction curve B between the measured value a' and the concentration of the standard substance, and store it;

[0024] A4. 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 a / measured value a' at that point as the critical point c, and store it.

[0025] In some embodiments of the present invention, the ratio of the content of the target molecule to the content of the specific capture molecule is different in the two parallel immune response assays; wherein the specific capture molecule is capable of specifically binding to the target molecule.

[0026] In other embodiments of the present invention, in the two parallel immune response assays, the result of the immune response with a larger ratio of the content of the specific capture molecule to the content of the target molecule is counted as the first value, and the other is counted as the second value.

[0027] In some embodiments of the present invention, the target molecule to be tested is selected from antigens or antibodies; and / or

[0028] The specific capture molecules include a first capture molecule that binds to a solid phase substance and a second capture molecule that is labeled with a marker.

[0029] In other embodiments of the present invention, the content of the specific capturing molecule is the content of the first capturing molecule, the content of the second capturing molecule, or the sum of the contents of the first capturing molecule and the second capturing molecule; preferably, it is the content of the first capturing molecule.

[0030] A second aspect of the present invention provides a system for implementing the method as described in the first aspect of the present invention, comprising:

[0031] An immunoassay apparatus, wherein the immunoassay apparatus has a reaction cup, the reaction cup being used to load the sample to be tested and the detection reagent for an immunoassay;

[0032] An immunoassay device, wherein the immunoassay device is equipped with a data acquisition device;

[0033] The controller is used to move two reaction cups loaded with the same test sample and corresponding detection reagent in the immunoassay device to the immunoassay device. The data acquisition device collects data from two parallel immunoassays of the same test sample and evaluates the collected data to obtain the detection results of two parallel immunoassays, which are respectively recorded as the first measurement value and the second measurement value.

[0034] A processor is used to calculate the ratio of a first measurement to a second measurement and to identify whether the sample to be tested is a HOOK effect sample.

[0035] In some embodiments of the present invention, the two reaction cups for loading the same test sample and corresponding detection reagent are placed at any designated position on the immunoassay apparatus; preferably, the two reaction cups for loading the same test sample and corresponding detection reagent are positioned adjacent to each other on the immunoassay apparatus.

[0036] In other embodiments of the present invention, the immunoassay device sequentially collects data from two parallel immunoreaction tests on the same sample; preferably, the immunoassay device simultaneously collects data from two parallel immunoreaction tests on the same sample.

[0037] In some embodiments of the present invention, the processor stores reaction curve A, reaction curve B and critical point c, which are used to identify whether the sample to be tested is a HOOK effect sample.

[0038] A third aspect of the present invention provides the application of the method described in the first aspect or the system described in the second aspect of the present invention in chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), and immunoturbidimetric immunoassay; preferably in chemiluminescent immunoassay; and more preferably in photo-induced chemiluminescent immunoassay.

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

[0040] The method and system described in this invention can easily, quickly and accurately identify whether a sample to be tested is a HOOK effect sample, avoiding missed detections caused by the HOOK effect. The method and system are not limited by the detection range. Attached Figure Description

[0041] Figure 1 This is the dose-response curve of the antigen-antibody interaction.

[0042] Figure 2 This is a schematic diagram of the method described in this invention.

[0043] Figure 3 The graph shows the reaction curves between the concentration of the standard substance and the signals of reagent 1 and reagent 2 in Example 2.

[0044] Figure 4 This is a schematic diagram of the system 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 method for identifying HOOK effect samples in immunoassays according to the first aspect of this invention involves performing two parallel tests on each sample. The ratio of the content of the target molecule to the content of the specifically captured molecule differs between the two tests, ultimately generating two distinct signals: a first measurement and a second measurement. As the content of the target molecule increases, the ratio of the first measurement to the second measurement continuously increases and exhibits a certain linear relationship. Based on this principle, the method for identifying HOOK effect samples in immunoassays provided by this invention is as follows:

[0049] Use the critical point c to determine whether the sample to be tested is a HOOK effect sample:

[0050] 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.

[0051] like Figure 2As 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 2 (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.

[0052] When the ratio of the first measured value to the second measured value is less than the critical point c, the sample to be tested is a non-HOOK effect sample.

[0053] When the ratio of the first measured value to the second measured value is greater than the critical point c, and the first measured value is greater than the measured value a, the sample to be tested is a non-HOOK effect sample.

[0054] When the ratio of the first measured value to the second measured value is greater than the critical point c, and the first measured value is less than the measured value a, the sample to be tested is a HOOK effect sample.

[0055] In some embodiments of the present invention, the method for identifying HOOK effect samples in immunoassays provided by the present invention specifically includes the following steps:

[0056] First, the critical point c is obtained using a method that includes the following steps:

[0057] A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immunoassays are performed, and the results of the two parallel immunoassays are recorded as value a and value a', respectively. 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.

[0058] A2, plot the reaction curve A between the measured value a and the concentration of the standard substance, and store it;

[0059] A3, plot the reaction curve B between the measured value a' and the concentration of the standard substance, and store it;

[0060] A4. 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 a / measured value a' at that point as the critical point c, and store it.

[0061] Then, the method comprising the following steps is used to determine whether the test sample is a HOOK effect sample:

[0062] S1, Perform two parallel immune response tests on the test sample containing the target molecule, and record the results of the two parallel immune responses as the first and second values, respectively.

[0063] S2, calculate the ratio of the first measured value to the second measured value;

[0064] S3, compare the ratio of the first measured value to the second measured value with the critical point c. When the ratio of the first measured value to the second measured value is less than the critical point c, the sample to be tested is a non-HOOK effect sample; when the ratio of the first measured value to the second measured value is greater than the critical point c, and the first measured value is greater than the measured value a, the sample to be tested is a non-HOOK effect sample; when the ratio of the first measured value to the second measured value is greater than the critical point c, and the first measured value is less than the measured value a, the sample to be tested is a HOOK effect sample.

[0065] According to some embodiments of the present invention, in the two parallel immune response assays, there is a difference in the ratio of the content of the specific capture molecule to the content of the target molecule. This objective can be achieved by referring to, but is not limited to, the following methods:

[0066]

[0067]

[0068] In some embodiments of the present invention, in the two parallel immune response assays, the immune response with a larger ratio of the content of the specific capture molecule to the content of the target molecule is counted as the first value, and the other is counted as the second value.

[0069] In other embodiments of the present invention, the content of the specific capturing molecule is the content of the first capturing molecule, the content of the second capturing molecule, or the sum of the contents of the first capturing molecule and the second capturing molecule; preferably, it is the content of the first capturing molecule.

[0070] The target molecule described in this invention refers to any inorganic or organic molecule that can be detected by immunoassay, including any target biological substance. Examples of the target molecule include cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, oligosaccharides, polysaccharides, lipopolysaccharides, cell metabolites, haptens, hormones, pharmaceutical substances, alkaloids, steroid compounds, vitamins, amino acids, and sugars.

[0071] The specific capture molecule described in this invention refers to a molecule capable of binding to another molecule (the target molecule) due to intermolecular attraction. Examples of such specific capture molecules include, but are not limited to, proteins, nucleic acids, carbohydrates, lipids, and small organic molecules. The specific capture molecule is defined as a capture molecule capable of recognizing and binding to a specific target molecule, rather than any arbitrary target molecule.

[0072] According to 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 above-described target molecules having immunogenicity. According to some embodiments of the present invention, the specific capture molecule is selected from one member of a specific binding pair, such as an antibody, and the target molecule to be tested is the other member of that specific binding pair, such as its paired antigen. The term "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).

[0073] According to some embodiments of the present invention, the specific capture molecule used in the immunoassay includes a first capture molecule bound to a solid-phase substance and a second capture molecule labeled with a marker. The first and second capture molecules may be the same or different, and may be of the same or different types, but both can specifically bind to the target molecule to be tested.

[0074] According to some embodiments of the present invention, the solid material to which the first capturing molecule is bound is selected from particles, microparticles, beads, electrodes, and porous plates. In some embodiments, the first capturing molecule is bound to luminescent microparticles containing luminescent groups, which can rapidly absorb singlet oxygen and then emit light of a certain wavelength (e.g., 500-615 nm).

[0075] 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.

[0076] The "content" of a substance in this invention refers to the mass concentration of the substance in its corresponding solution.

[0077] The second aspect of the present invention relates to a system for implementing the method as described in the first aspect of the present invention, comprising: an immune reaction device, an immunoassay device, a controller, and a processor, capable of performing two parallel tests on each sample and identifying whether the sample to be tested is a HOOK effect sample based on the results of the two parallel tests.

[0078] In this invention, the immunoassay apparatus includes reaction cups for loading the test sample and detection reagents for an immunoassay. The form of the reaction cups is not particularly limited; they can be single or assembled together to form a strip (row) or plate-type reaction cup group. In this invention, two reaction cups used to load the same test sample and corresponding detection reagent can be positioned at any designated location on the immunoassay apparatus. In some embodiments of this invention, two reaction cups used to load the same test sample and corresponding detection reagent are positioned adjacent to each other on the immunoassay apparatus.

[0079] The immunoassay device of this invention is equipped with a data acquisition unit. This data acquisition unit can be a device for acquiring optical signals, which can be emitted by the luminescent composition after being irradiated with excitation light, emitted by the luminescent composition after being excited by chemical energy, generated after an electrochemical reaction occurs on the electrode surface, or transmitted through a medium of suspended particles, etc. Therefore, the system of this invention can be applied to photo-induced chemiluminescence immunoassay, fluorescence immunoassay, electrochemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), and turbidimetric immunoassay, etc. The data acquisition unit of this invention can also evaluate the acquired data.

[0080] In some specific embodiments of the present invention, the system is used for photo-induced chemiluminescence immunoassay. In this case, the data acquisition device is a photon counter. Furthermore, the immunoassay device also includes a photoexciter for emitting excitation light and irradiating the reaction vessel, thereby exciting the complex formed by the immunoreaction to emit a light signal. The photon counter receives the light signal and records the reading.

[0081] The controller of the present invention moves two reaction cups containing the same test sample and corresponding detection reagent in the immunoassay device to the immunoassay device according to the set instructions. Then the data acquisition device collects data from two parallel immunoassays of the same test sample.

[0082] In some embodiments of the present invention, the immunoassay device sequentially collects data from two parallel immunoreaction tests on the same sample; in other embodiments of the present invention, the immunoassay device simultaneously collects data from two parallel immunoreaction tests on the same sample.

[0083] After evaluating the collected data, the data acquisition device obtains two parallel detection results of immune response, which are recorded as the first measurement value and the second measurement value, respectively, and transmits the measurement values ​​to the processor.

[0084] The processor of the present invention stores reaction curve A, reaction curve B and critical point c. The processor can calculate the ratio of the first measured value to the second measured value of the sample to be tested, and compare the ratio with the stored critical point c, thereby identifying whether the sample to be tested is a HOOK effect sample.

[0085] In some embodiments of the present invention, the reaction curve A, reaction curve B, and critical point c stored in the processor are obtained by performing a method comprising the following steps:

[0086] A1. A series of known standard substances with different concentrations containing the target molecule are tested. For each standard substance with the same target molecule concentration, two parallel immunoassays are performed, and the results of the two parallel immunoassays are recorded as value a and value a', respectively. The detection method of value a is the same as that of the first value of the test sample, and the detection method of value a' is the same as that of the second value of the test sample.

[0087] A2, plot the reaction curve A between the measured value a and the concentration of the standard substance, and store it;

[0088] A3, plot the reaction curve B between the measured value a' and the concentration of the standard substance, and store it;

[0089] A4. 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 a / measured value a' at that point as the critical point c, and store it.

[0090] The third aspect of the present invention relates to the application of the method as described in the first aspect or the system as described in the second aspect of the present invention in chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), and immunoturbidimetric immunoassay; preferably in chemiluminescent immunoassay; and more preferably in photo-induced chemiluminescent immunoassay.

[0091] Example

[0092] 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.

[0093] 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.

[0094] Example 1: Detection of AFP samples using conventional methods

[0095] 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:

[0096] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0097] Reagent 2: Biotin-labeled AFP antibody.

[0098] Test method:

[0099] 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;

[0100] 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.

[0101] Test samples (collected from clinical serum samples):

[0102] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0103] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0104] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0105] The test results are shown in Table 1.

[0106] Table 1

[0107] Initial measurement value Measured value ng / mL Sample 1 7.41 Sample 2 109.43 Sample 3 11.79

[0108] The results shown in Table 1 are those obtained by direct detection using conventional methods. The measured value of sample 3 was only 11.79 ng / mL. Without considering clinical manifestations, it would be very easy to misjudge it as a weakly positive sample and it would be impossible to identify it as a HOOK effect sample.

[0109] Given that sample 3 was a strong positive sample, it was diluted 50 times with diluent and retested. The test results are shown in Table 2.

[0110] Table 2

[0111] 50-fold dilution Measured value ng / mL Diluted sample >1000

[0112] As shown above, the measured value of sample 3 after 50-fold dilution is >1000ng / mL, which confirms that it is a HOOK sample.

[0113] Example 2: Identification of HOOK effect samples using the method of the present invention

[0114] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0115] Test samples (collected from clinical serum samples):

[0116] Sample 1: Negative (actual measurement approximately 5 ng / mL)

[0117] Sample 2: Low positive value (actual measured value approximately 100 ng / mL)

[0118] Sample 3: Strongly positive (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0119] The main components of the reagent kit used are as follows:

[0120] Reagent 1: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0121] Reagent 2: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0122] Test method:

[0123] For both reaction vessels, which are from the same sample test group, repeat the following liquid addition steps 1 and 2 for different sample test groups:

[0124] 1. Add 10 μl of the sample to be tested and 25 μl of reagent 1 to reaction well 1;

[0125] 2. Add 10 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively;

[0126] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0127] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each reaction well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0128] The AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 3.

[0129] Based on the values ​​in Table 3, plot reaction curves A and B respectively between the concentrations of the standard substance and the signals from reagent 1 and reagent 2. Figure 3 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 15.05-24.31. The midpoint A / B signal ratio = 19.18 is taken as the critical point.

[0130] Store the above standard substance test results. Analyzer.

[0131] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 4.

[0132] Table 3

[0133]

[0134] Table 4

[0135]

[0136] Test Result Analysis:

[0137] The ratio of the first measurement to the second measurement of sample 1 (7.87) is less than the critical point c (19.18), and the ratio of the first measurement to the second measurement of sample 2 (11.18) is less than the critical point c (19.18). Therefore, both sample 1 and sample 2 are normal samples (non-HOOK effect samples).

[0138] The ratio of the first measurement to the second measurement in sample 3 (339.41) is greater than the critical point c (19.18), and the first measurement (427658) is less than the measurement a (7932206). Therefore, sample 3 is a sample of the HOOK effect.

[0139] Therefore, the method of the present invention can directly, easily, quickly and accurately determine whether the sample to be tested is a HOOK effect sample, thereby avoiding missed detection or detection errors caused by the HOOK effect.

[0140] Example 4: Identification System for HOOK Effect Samples

[0141] The main components of the reagent kit used are as follows:

[0142] Reagent 1: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0143] Reagent 2: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0144] system:

[0145] Two centrally symmetrical reaction cups on the immunoassay apparatus are designated as the same sample test group. The value with the larger ratio of the content of specific capture molecules in the detection reagent to the content of target molecules in the sample is designated as the first value, and the other is designated as the second value.

[0146] The detection steps performed by the system are as follows:

[0147] 1. The controller moves two reaction cups containing the same sample and corresponding test reagent from the immunoassay apparatus to the immunoassay device;

[0148] 2. The photoexciter emits a laser beam and irradiates the reaction vessel, thereby exciting the complex formed by the target molecule in the sample and the specific capture molecule in the detection reagent to emit a light signal;

[0149] 3. The counter receives the light signal and simultaneously acquires data from two parallel immune response tests, recording the immune response test results as the first and second measured values, respectively;

[0150] 4. The processor calculates the ratio of the first measurement to the second measurement of the same test sample and retrieves the stored data to determine whether it is a HOOK effect sample.

[0151] The test results are shown in Tables 5 and 6 below.

[0152] Table 5: Measurement results of the critical point c of the standard substances

[0153]

[0154] Table 6: Measurement results of the samples to be tested

[0155]

[0156] Results analysis: The ratio of the first measurement to the second measurement of samples 03 and 08 is less than the critical point c (15.18), so they are normal samples (non-HOOK effect samples); The ratio of the first measurement to the second measurement of sample 11 is greater than the critical point c (15.18), and the first measurement (7974192) is greater than the measurement a (6848780), so it is also a normal sample (non-HOOK effect sample); The ratio of the first measurement to the second measurement of sample 15 is greater than the critical point c (15.18), and the first measurement (4321968) is less than the measurement a (6848780), so it is a HOOK effect sample.

[0157] Therefore, the method of the present invention can directly, easily, quickly and accurately determine whether the sample to be tested is a HOOK effect sample, thereby avoiding missed detection or detection errors caused by the HOOK effect.

[0158] 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 for identifying HOOK effect samples in an immunoassay, comprising the following steps: S1, Perform two parallel immune response tests on the test sample containing the target molecule, and record the results of the two parallel immune responses as the first and second values, respectively. S2, calculate the ratio of the first measured value to the second measured value; S3, the critical point c of the standard substance is obtained through the following method. A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immunoassays are performed, and the results of the two parallel immunoassays are recorded as value a and value a', respectively. The detection method of value a is the same as that of the first value of the sample to be tested, and the detection method of value a' is the same as that of the second value of the sample to be tested. A2, plot the reaction curve A between the measured value a and the concentration of the standard substance; A3, plot the reaction curve B between the measured value a' and the concentration of the standard substance; A4. Take a point in the overlapping part of the standard substance concentrations corresponding to the front zone of reaction curve A and the back zone of reaction curve B, and record the ratio of the measured value a / measured value a' at that point as the critical point c. S4, compare the ratio of the first measured value to the second measured value with the critical point c. When the ratio of the first measured value to the second measured value is greater than the critical point c and the first measured value is less than the measured value a, the sample to be tested is a HOOK effect sample. In the two parallel immune response assays, the ratio of the content of the target molecule to the content of the specific capture molecule is different, wherein the specific capture molecule can specifically bind to the target molecule.

2. The method according to claim 1, characterized in that, When the ratio of the first measured value to the second measured value is less than the critical point c, the sample to be tested is a non-HOOK effect sample. When the ratio of the first measured value to the second measured value is greater than the critical point c, and the first measured value is greater than the measured value a, the sample to be tested is a non-HOOK effect sample.

3. The method according to claim 1, characterized in that, In the two parallel immune response assays, the immune response with a larger ratio of the content of the specific capture molecule to the content of the target molecule is recorded as the first measurement or measurement a, and the other is recorded as the second measurement or measurement a'.

4. The method according to claim 1, characterized in that, The target molecule to be tested is selected from antigens or antibodies; and / or The specific capture molecules include a first capture molecule that binds to a solid phase substance and a second capture molecule that is labeled with a marker.

5. The method according to claim 4, characterized in that, The content of the specific capture molecule is the content of the first capture molecule, the content of the second capture molecule, or the sum of the contents of the first capture molecule and the second capture molecule.

6. The method according to claim 4, characterized in that, The content of the specific capture molecule is the content of the first capture molecule.

7. A system for implementing the method according to any one of claims 1-6, comprising: An immunoassay apparatus, wherein the immunoassay apparatus has a reaction cup, the reaction cup being used to load the sample to be tested and the detection reagent for an immunoassay; An immunoassay device, wherein the immunoassay device is equipped with a data acquisition device; The controller is used to move two reaction cups loaded with the same test sample and corresponding detection reagent in the immunoassay device to the immunoassay device. The data acquisition device collects data from two parallel immunoassays of the same test sample and evaluates the collected data to obtain the detection results of two parallel immunoassays, which are respectively recorded as the first measurement value and the second measurement value. The processor stores reaction curve A, reaction curve B, and critical point c, and is used to calculate the ratio of the first measurement to the second measurement and to identify whether the sample to be tested is a HOOK effect sample.

8. The system according to claim 7, characterized in that, The two reaction cups used to hold the same sample to be tested and the corresponding test reagent are placed at any designated position on the immunoassay apparatus.

9. The system according to claim 8, characterized in that, The two reaction cups used to load the same sample and corresponding test reagent are positioned adjacent to each other on the immunoassay apparatus.

10. The system according to claim 8, characterized in that, The immunoassay device collects data from two parallel immunoreaction tests on the same sample in sequence.

11. The system according to claim 8, characterized in that, The immunoassay device simultaneously collects data from two parallel immunoreaction tests on the same sample.

12. The application of the method according to any one of claims 1-6 or the system according to any one of claims 7-11 in chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), and immunoturbidimetric immunoassay.

13. The application according to claim 12, characterized in that, The application is in chemiluminescent immunoassay.

14. The application according to claim 13, characterized in that, The application is in photo-induced chemiluminescence immunoassay.

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