A system for immunoassay

Abstract

This invention provides a system for immunoassay, comprising: an immunoreaction device with reaction cups for loading a test sample and a detection reagent for an immunoreaction; an immunoassay device with a data acquisition unit; a controller for transferring two reaction cups loaded with the same test sample and corresponding detection reagent from the immunoreaction device to the immunoassay device; the data acquisition unit acquiring and evaluating data from two parallel immunoreactions of the same test sample to obtain two parallel immunoreaction results, designated as a first value and a second value; and a processor for calculating the ratio of the first value to the second value and determining the concentration of the target molecule in the test sample. This system can solve the problem of hook effect samples, is not limited by the detection range, has good repeatability, and is fast.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and specifically relates to a system for immunoassay. 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 cause high-value samples to produce erroneous low-value results, or even negative results, which is called a "false negative."

[0006] Current immunoassay methods typically utilize the front band of a dose-response curve to calculate the analyte concentration based on the linear relationship between the analyte concentration and the reaction signal. However, this method has several drawbacks, such as:

[0007] Narrow detection range: Traditional immunoassay reagents can only detect samples within the early band of the dose-response curve, resulting in a narrow detection concentration range. Samples outside this range require dilution before testing, which is complex, time-consuming, and demands high precision in dilution.

[0008] HOOK effect: Traditional immunoassay reagents lack the means to identify the HOOK effect. Clinicians often need to combine the patient's clinical manifestations with the method of diluting serum samples to identify whether the sample has a HOOK effect. This operation is complicated, time-consuming and prone to false negatives. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a novel system for immunoassay. The assay system of the present invention enables simple, rapid, and accurate calculation of the concentration of the analyte.

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

[0011] A first aspect of the present invention provides a system for an immunoassay, comprising:

[0012] 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;

[0013] An immunoassay device, wherein the immunoassay device is equipped with a data acquisition device;

[0014] 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 two parallel immunoassay results, namely the first measurement value and the second measurement value.

[0015] 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.

[0016] 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.

[0017] 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.

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

[0019] In other embodiments of the present invention, in the two parallel immune response assays, the immune response with the larger ratio of the content of the specific capture molecule in the detection reagent to the content of the target molecule in the sample to be tested is counted as the first value, and the other is counted as the second value.

[0020] In some embodiments of the present invention, the target molecule to be tested is selected from antigens or antibodies.

[0021] In other embodiments of the invention, the specific capture molecule includes a first capture molecule bound to a solid phase substance and a second capture molecule labeled with a marker.

[0022] In some specific 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.

[0023] In some embodiments of the present invention, the system performs the following steps:

[0024] A1: A series of standard substances of different concentrations with known content of the target molecule are tested, wherein two parallel immune reactions are performed on each standard substance, and the results of the two parallel immune reactions are recorded as measured value a and measured value a', respectively; preferably, in the two parallel immune reaction tests, the immune reaction with a larger ratio of the content of the specific capture molecule to the content of the target molecule is recorded as measured value a.

[0025] A2: Calculate the ratio of measured value a to measured value a';

[0026] 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.

[0027] In other embodiments of the present invention, the system performs the following steps:

[0028] The stored correlation standard curve is retrieved, and the ratio of the first measured value to the second measured value of the sample to be tested is substituted into the correlation standard curve for calculation to determine the concentration of the target molecule in the sample to be tested.

[0029] In some embodiments of the present invention, the system performs the following steps:

[0030] B1: A series of standard substances of different concentrations with known content of the target molecule are tested, wherein two parallel immune reactions are performed on each standard substance, and the results of the two parallel immune reactions are recorded as measured value b and measured value b', respectively; preferably, in the two parallel immune reaction tests, the immune reaction with a larger ratio of the content of the specific capture molecule to the content of the target molecule is recorded as measured value b.

[0031] B2: Plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it;

[0032] B3: Plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it;

[0033] 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.

[0034] In other embodiments of the present invention, the system performs the following steps:

[0035] Retrieve the stored reaction curves A and B and the critical point c, and determine the ratio of the first measured value to the second measured value of the 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.

[0036] 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 system of the first aspect of the present invention to perform corresponding steps.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) When using the system of the present invention for immune testing, the HOOK effect problem can be solved and the HOOK effect can be avoided to prevent false negatives. The method is not limited by the detection range.

[0039] (2) When using the system of the present invention for immune testing, the classic dose-response curve can be directly used for calculation, with good repeatability and fast measurement speed;

[0040] (3) When using the system of the present invention for immune testing, the detection range is much greater than that of conventional detection methods. Attached Figure Description

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

[0042] Figure 2 This is a schematic diagram of calculation method 1 for performing immunoassays using the system of the present invention.

[0043] Figure 3 This is a schematic diagram of calculation method 2 for performing immune testing using the system of the present invention.

[0044] Figure 4 The graph shows the reaction curves between the concentration of the standard substance and the signals of reagent A and reagent B in Example 2.

[0045] Figure 5 This is a schematic diagram of the structure of the system for immunoassay described in this invention. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] 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.

[0049] This invention provides a system for immunoassay, comprising: an immunoassay apparatus, an immunoassay apparatus, a controller, and a processor. The system is capable of performing two parallel tests on each sample and calculating the concentration of the target molecule in the sample based on the results of the two parallel tests. The system has no detection range limitation and avoids missed detections caused by the hook effect.

[0050] The immunoassay apparatus of this invention includes reaction cups for loading test samples and detection reagents for immunoassay. In this invention, the form of the reaction cups is not particularly limited; the reaction cups can be single or multiple cups assembled together to form strip (row) or plate-type reaction cup groups. In this invention, two reaction cups used to load the same test sample and corresponding detection reagent can be located at any designated position 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] After evaluating the collected data, the data acquisition device obtains two parallel detection results of the immune response, which are recorded as the first measurement value and the second measurement value, respectively. After the measurement value is transmitted to the processor, the processor calculates the ratio of the first measurement value to the second measurement value and determines the concentration of the target molecule in the sample to be tested.

[0056] The system for immunoassay of this invention can perform two parallel tests on each sample. The ratio of the concentration of the target molecule in the sample to the concentration of the specifically captured molecule in the detection reagent differs between the two tests, ultimately generating two distinct signals: a first measurement and a second measurement. As the 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 system for immunoassay of this invention can calculate the concentration of the target molecule in the sample by executing the following two methods, as follows:

[0057] 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.

[0058] 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.

[0059] Calculation Method 2: Calculate the concentration of the target molecule in the sample using reaction curve A or reaction curve B.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] Corresponding to the calculation method 1 above, the system for immunoassay provided by the present invention performs the following steps:

[0065] First, the system 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:

[0066] A1: The controller moves two reaction cups containing the same concentration of the target molecule and the corresponding detection reagent in the immunoassay device to the immunoassay device. The data acquisition device collects data from two parallel immunoassays of the same concentration of the target molecule and records the detection results of the two parallel immunoassays, which are respectively recorded as value a and value a'.

[0067] A2: The processor calculates the ratio of measured value a to measured value a';

[0068] A3: The processor generates a standard curve showing the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance, based on the measured values ​​a and a' of a series of standard substances with known content of the target molecule, and stores the curve.

[0069] Throughout the testing process, the measurement value 'a' can be set as the value of the specific capture molecule content in the detection reagent / the target molecule content in the sample to be tested; the other value can be set as 'a'.

[0070] The system then determines the concentration of the target molecule in the sample by performing the following steps:

[0071] S1: The controller moves two reaction cups containing the same test sample and corresponding detection reagent from the immunoassay device to the immunoassay device. The data acquisition device collects data from two parallel immunoassays of the same concentration of the target molecule and records the detection results of the two parallel immunoassays, which are the first measurement value and the second measurement value, respectively. In some embodiments of the present invention, the measurement value with the larger ratio of the content of the specific capture molecule in the detection reagent to the content of the target molecule in the test sample is counted as the first measurement value, and the other is counted as the second measurement value.

[0072] S2: The processor calculates the ratio of the first measured value to the second measured value of the sample under test;

[0073] S3: The processor retrieves the stored correlation standard curve, substitutes the ratio of the first measured value to the second measured value of the sample to be tested into the correlation standard curve for calculation, and determines the concentration of the target molecule in the sample to be tested.

[0074] Throughout the testing process, the measurement value with the larger ratio of the content of the specific capture molecule to the content of the target molecule can be set as the first measurement value, and the other as the second measurement value; the reverse is also possible.

[0075] Corresponding to the above calculation method 2, the system for immunoassay provided by the present invention performs the following steps:

[0076] First, the system obtains reaction curve A, reaction curve B, and critical point c by performing the following steps:

[0077] B1: The controller moves two reaction cups containing the same concentration of the target molecule and the corresponding detection reagent in the immunoassay device to the immunoassay device. The data acquisition device collects data from two parallel immunoassays of the same concentration of the target molecule and records the detection results of the two parallel immunoassays, which are respectively recorded as value b and value b'.

[0078] B2: The processor generates a reaction curve A between the measured value b and the concentration of the standard substance based on a series of measured values ​​b of a known target molecule content and a series of measured values ​​b of a series of standard substances, and stores the curve.

[0079] B3: The processor generates and stores the reaction curve B between the measured value b' and the concentration of the standard substance based on a series of measured values ​​b' of a known target molecule content.

[0080] B4: There is a concentration overlap between the front zone of reaction curve A and the back zone of reaction curve B (e.g., Figure 3 (The part within the dashed box) Take a point in the overlapping area of ​​the above concentrations, record the ratio of the measured value b to the measured value b' at that point as the critical point c, and store it.

[0081] Throughout the testing process, the measurement value b can be set as the one with the larger ratio of the content of the specific capture molecule to the content of the target molecule, and the other as b'; the reverse is also possible.

[0082] The system then determines the concentration of the target molecule in the sample by performing the following steps:

[0083] S1: The controller moves two reaction cups containing the same test sample and corresponding detection reagent from the immunoassay device to the immunoassay device. The data acquisition device collects data from two parallel immunoassays of the same concentration of the target molecule and records the detection results of the two parallel immunoassays, which are the first measurement value and the second measurement value, respectively. In some embodiments of the present invention, the measurement value with the larger ratio of the content of the specific capture molecule in the detection reagent to the content of the target molecule in the test sample is counted as the first measurement value, and the other is counted as the second measurement value.

[0084] S2: The processor calculates the ratio of the first measured value to the second measured value of the sample under test;

[0085] 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.

[0086] Throughout the testing process, the measurement value with the larger ratio of the content of the specific capture molecule to the content of the target molecule can be set as the first measurement value, and the other as the second measurement value; the reverse is also possible.

[0087] In some embodiments of the present invention, the ratio of the content of specific capture molecules in the detection reagent to the content of target molecules in the test sample differs between the two parallel immunoassays. A similar effect can be achieved by referring to, but is not limited to, the following methods:

[0088]

[0089] 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.

[0090] 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.

[0091] In some embodiments of the present invention, the target molecule to be tested is selected from antigens or antibodies. According to some embodiments of the present invention, the antigen refers to any substance having immunogenicity, including but not limited to substances listed in the examples of the aforementioned target molecules having immunogenicity. According to some embodiments of the present invention, the 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).

[0092] In other 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.

[0093] In 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 microspheres containing luminescent groups, which can rapidly absorb singlet oxygen and then emit light of a certain wavelength (e.g., 500-615 nm).

[0094] 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.

[0095] A second aspect of the present invention relates to a computer-readable storage medium storing a computer program product thereon, the computer program product causing the system to perform the aforementioned steps related to the immune detection.

[0096] In some preferred embodiments of the present invention, the system further includes the aforementioned computer-readable storage medium.

[0097] Example

[0098] To make the present invention easier to understand, the following specific embodiments, using the photo-induced chemiluminescence immunoassay system for detecting AFP as an example, are provided to further illustrate the invention. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the invention. Unless otherwise specified, all raw materials or components used in the present invention can be obtained commercially or by conventional methods.

[0099] The technical principle of photoluminescence analysis is as follows: under laser irradiation, the sensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules. The singlet oxygen molecules can react with the luminescent composition about 200 nm away to generate a light signal of a certain wavelength. When the sample contains the antigen or antibody to be tested, the immune reaction of the antigen and antibody can enable the donor particles containing the sensitizer to bind to the acceptor particles containing the luminescent composition, thereby generating a light signal of a specific wavelength. The content of the antigen or antibody to be tested can be detected by detecting this light signal.

[0100] 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.

[0101] Example 1: Detection of AFP samples using a conventional photo-induced chemiluminescence immunoassay system

[0102] Test samples (collected from clinical serum samples):

[0103] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0104] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0105] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

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

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

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

[0109] Test system: Analyzer.

[0110] The test results are shown in Table 1.

[0111] Table 1

[0112] Initial measurement value Measured value ng / mL Sample 1 7.41 Sample 2 109.43 Sample 3 11.79

[0113] The results shown in Table 1 are from direct detection using a conventional system. 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 a diluent and retested. The results are shown in Table 2.

[0114] Table 2

[0115] 50-fold dilution Measured value ng / mL Diluted sample >1000

[0116] 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 could not yet be obtained. The diluted sample was then diluted 50 times again with diluent, and the measured values ​​are shown in Table 3 below.

[0117] Table 3

[0118] 2500 times dilution Measured value ng / mL Diluted sample 849.51

[0119] 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.

[0120] Example 2: System 1 detects AFP samples

[0121] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0122] Test samples (collected from clinical serum samples):

[0123] Sample 1: Negative (actual measurement approximately 5 ng / mL)

[0124] Sample 2: Low positive value (actual measured value approximately 100 ng / mL)

[0125] Sample 3: Strongly positive (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0126] The main components of the reagent kit used:

[0127] Reagent A: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0128] Reagent B: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0129] System 1:

[0130] Two adjacent reaction cups on the immunoassay apparatus are designated as the same sample test group. The value with the larger ratio of the content of the specific capture molecule in the detection reagent to the content of the target molecule in the sample is designated as the first value, and the other is designated as the second value.

[0131] The detection steps performed by the system are as follows:

[0132] 1. The controller moves two reaction cups containing the same sample and corresponding test reagent from the immunoassay apparatus to the immunoassay device;

[0133] 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;

[0134] 3. The counter receives the light signal and simultaneously acquires data from two parallel immune response detections, recording the detection results of the immune response as the first and second measured values, respectively;

[0135] 4. 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 calculates the concentration of the target molecule in the sample.

[0136] The above system was used to test the serially diluted AFP standard materials (numbered 1-20), and the test results are shown in Table 4.

[0137] The processor generates reaction curves A and B based on the values ​​in Table 4, respectively, for the concentration of the standard substance and the signals from reagent A and reagent B. Figure 4 It can be seen that standard substances 1-11 correspond to the front band region of reaction curve A, and standard substances 9-20 correspond to the back band region of reaction curve B. Therefore, the concentration overlap between the front band region of reaction curve A and the back band region of reaction curve B is the concentration range of standard substances 9-11, and its corresponding A / B signal ratio is 15.05-24.31. The midpoint A / B signal ratio = 19 is set as the critical point and stored in the computer-readable storage medium of the system.

[0138] The three groups of samples were tested, and the results are shown in Table 5.

[0139] Table 4

[0140]

[0141] Table 5

[0142]

[0143] As shown in Table 5, the immunoassay method using System 1 of the present invention can avoid the problem of low sample values ​​caused by the HOOK effect, and can directly obtain high values ​​of up to 2×10⁻⁶. 6 The detection result of ng / mL is not limited by the detection range of the immunoassay method using System 1 of the present invention.

[0144] Example 3: System 2 detects AFP samples

[0145] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0146] Test samples (collected from clinical serum samples):

[0147] Sample 1: Negative (actual measurement approximately 5 ng / mL)

[0148] Sample 2: Low positive value (actual measured value approximately 100 ng / mL)

[0149] Sample 3: Strongly positive (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0150] The main components of the reagent kit used:

[0151] Reagent A: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0152] Reagent B: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0153] System 2:

[0154] 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.

[0155] The detection steps performed by the system are as follows:

[0156] 1. The controller moves two reaction cups containing the same sample and corresponding test reagent from the immunoassay apparatus to the immunoassay device;

[0157] 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;

[0158] 3. The counter receives the light signal and simultaneously acquires data from two parallel immune response detections, recording the detection results of the immune response as the first and second measured values, respectively;

[0159] 4. 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 calculates the concentration of the target molecule in the sample.

[0160] The above system was used to test the serially diluted AFP standard materials (numbered 1-20), and the test results are shown in Table 6.

[0161] The processor generates a standard curve of the correlation between the A / B signal ratio and the concentration of the standard substance based on the values ​​in Table 6, and stores the standard curve in a computer-readable storage medium of the system.

[0162] The three groups of samples were tested, and the results are shown in Table 7.

[0163] Table 6

[0164]

[0165] Table 7

[0166]

[0167]

[0168] As shown in Table 7, the immunoassay method using System 2 of the present invention can avoid the problem of low sample values ​​caused by the HOOK effect, and can directly obtain high values ​​of up to 2×10⁻⁶. 6 The detection result of ng / mL is not limited by the detection range of the immunoassay method using System 2 of the present invention.

[0169] Example 4: Precision Verification of Ultra-High Value Sample Measurements

[0170] The main components of the reagent kit used:

[0171] Reagent A: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0172] Reagent B: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0173] Test system: System 1 in Example 2

[0174] The test results are shown in Table 8 below.

[0175] Table 8

[0176]

[0177] As can be seen from the results in Table 8, when the system described in this invention was used, the CV of the three high-value samples was within 10% after 10 repeated measurements, indicating that the precision results were good.

[0178] 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 system for immunoassay, used for photo-induced chemiluminescence immunoassay, said system 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 is provided with a data acquisition device, which is a photon counter; the immunoassay device also includes a photoexciter for emitting excitation light and irradiating the reaction cup, thereby stimulating the complex formed by the immune reaction to emit a light signal, and the photon counter receives the light signal and records the reading. The controller is used to transfer two reaction cups loaded with the same test sample and corresponding detection reagent from 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 two parallel immunoassay results, which are respectively recorded as the first measurement value and the second measurement value. In the two parallel immunoassays, the ratio of the content of the target molecule in the test sample to the content of the specific capture molecule in the detection reagent is different, wherein the specific capture molecule can specifically bind to the target molecule. The processor is configured to determine the concentration of a target molecule in a test sample based on reaction curve A or reaction curve B. Reaction curve A and reaction curve B are reaction curves obtained from the detection results of two parallel immunoreactions of a standard substance with a known content of the same target molecule. A point is selected where the concentrations overlap between the front band of reaction curve A and the back band of reaction curve B; the ratio of the first measured value to the second measured value of the standard substance at this point is taken as a critical point c. When the processor determines that the ratio of the first measured value to the second measured value of the test sample is ≤ critical point c, the concentration of the target molecule in the test sample is calculated using the front band of reaction curve A. When the processor determines that the ratio of the first measured value to the second measured value of the test sample is > critical point c, the concentration of the target molecule in the test sample is calculated using the back band of reaction curve B.

2. The system according to claim 1, characterized in that, Two reaction cups for loading the same sample and corresponding reagent are placed at any designated position on the immunoassay apparatus.

3. The system according to claim 2, 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.

4. The system according to claim 1, characterized in that, The immunoassay device collects data from two parallel immunoreaction tests on the same sample in sequence.

5. The system according to claim 1, characterized in that, The immunoassay device simultaneously collects data from two parallel immunoreaction tests on the same sample.

6. The system according to claim 1, characterized in that, In the two parallel immune response tests, the immune response with the larger ratio of the content of the specific capture molecule in the test reagent to the content of the target molecule in the test sample is counted as the first value, and the other is counted as the second value.

7. The system according to claim 1, characterized in that, The target molecule to be tested is selected from antigens or antibodies.

8. The system according to claim 1, characterized in that, 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.

9. The system according to claim 8, 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.

10. The system according to claim 8, characterized in that, The content of the specific capture molecule is the content of the first capture molecule.

11. The system according to any one of claims 1-8, characterized in that, The system performs the following steps: B1: 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 b and measured value b', respectively. 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.

12. The system according to claim 11, characterized in that, The system performs the following steps: Retrieve the stored reaction curves A and B and the critical point c, and determine the ratio of the first measured value to the second measured value of the sample to be tested and the magnitude of the critical point c.

13. The system according to claim 11, characterized in that, In the two parallel immune response assays, the immune response with the larger ratio of the content of the specific capture molecule to the content of the target molecule is recorded as value b.

14. A computer-readable storage medium storing a computer program product thereon, characterized in that, The computer program product causes the system according to any one of claims 1-13 to perform the corresponding steps.

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