An immunoassay kit, assay method and system

Abstract

The present application relates to an immunoassay kit, method and system. The kit comprises reagent 1 and reagent 2 with the same components, and the total amount of specific capture molecules in reagent 1 is different from that in reagent 2, which can specifically bind to the target molecules to be detected. The method for performing immunoassay using the kit of the present application can solve the problem of the hook effect sample, the method is not limited by the detection range, and can directly measure the high value sample concentration up to 10 6 ng / ml level with good repeatability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of immunoassay, and particularly relates to an immunoassay kit, an assay method and a system. BACKGROUND

[0002] Immunological detection is based on the principle of antigen-antibody specific reaction. Since it can utilize isotopes, enzymes, chemiluminescent substances and the like to display or amplify the signal of the measured substance, it is often used to detect trace biological active substances such as proteins and hormones.

[0003] Photostimulated chemiluminescence is one of the commonly used methods of chemiluminescence analysis technology, which can be used to study the interaction between biological molecules, and is mainly used for disease detection in clinical practice. This technology integrates research in related fields such as polymer microparticle technology, organic synthesis, protein chemistry and clinical detection. The technical principle of photostimulated chemiluminescence analysis technology is that the sensitizing agent can excite the oxygen molecules in the surrounding environment into singlet oxygen molecules under laser irradiation, and the singlet oxygen molecules can react with the luminescent composition within a distance of about 200 nm to produce light signals of a certain wavelength; when the sample contains the antigen or antibody to be measured, the immune reaction of the antigen and antibody can make the donor particles containing the sensitizing agent combine with the acceptor particles containing the luminescent composition, thereby producing light signals of a specific wavelength, and the content of the antigen or antibody to be measured can be detected by detecting the light signals.

[0004] In the dose-response curve of antigen-antibody, when the amount of antibody is fixed, the reaction signal will first increase and then decrease with the increase of the amount of antigen. The region where the reaction signal increases with the increase of the antigen dose is called the "front band" region, the region where the reaction signal decreases with the increase of the antigen dose is called the "rear band" region, and the region connecting the front band and the rear band is called the "equivalent band".

[0005] In the immune reaction, the reactivity of the antigen and the antibody increases first and then decreases with the increase of the ratio of the antigen and the antibody, which is called "hook effect" or "HOOK effect". In clinical practice, hook effect can cause false negative results of high value samples, that is, "false negative".

[0006] The current immunological detection method usually utilizes the front band region of the dose-response curve to calculate the concentration of the measured substance through the linear relationship between the content of the measured substance and the reaction signal. However, this detection method has many defects, for example:

[0007] Narrow detection range: the traditional immunoassay reagent can only detect the front band section of the dose-response curve, and the detection concentration range is narrow. Samples outside the detection range need to be diluted for detection, which is complex, time-consuming and requires high precision of dilution;

[0008] HOOK effect: Because of lacking means to identify the HOOK effect, the traditional immunoassay reagent often needs the clinician to combine the patient's clinical manifestations to dilute the serum sample to identify whether the sample has the HOOK effect, which is complex in operation, time-consuming and easy to cause missed detection. SUMMARY

[0009] In view of the deficiencies in the prior art, the purpose of the present application is to provide an immunoassay kit, a determination method and a system. The method and system for immunoassay using the kit can simply, quickly and accurately calculate the concentration of the measured substance.

[0010] In order to achieve the above-mentioned purpose and other related purposes, the present application adopts the following technical solutions:

[0011] Therefore, the first aspect of the present application provides an immunoassay kit, which comprises reagent 1 and reagent 2 with the same components, and the total content of specific capture molecules in the reagent 1 is different from the total content of specific capture molecules in the reagent 2, and the specific capture molecules can specifically bind to the target molecules to be measured; preferably, the specific capture molecules comprise a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecules to be measured.

[0012] In some embodiments of the present application, the reagent 1 comprises a first antibody (or antigen) coated luminescent microparticle with an alpha 1 concentration, the reagent 2 comprises a first antibody (or antigen) coated luminescent microparticle with a beta 1 concentration, and the alpha 1 is greater than the beta 1.

[0013] In other embodiments of the present application, the reagent 1 further comprises a second antibody (or antigen) labeled with a marker with an alpha 2 concentration, and the reagent 2 further comprises a second antibody (or antigen) labeled with a marker with a beta 2 concentration, and the alpha 2 is not less than the beta 2; preferably, the alpha 2 is greater than the beta 2.

[0014] The second aspect of the present application provides a method for immunoassay using the kit as described in the first aspect of the present application, which comprises the following steps:

[0015] S1, the sample containing the target molecules to be measured is subjected to two parallel immunoassay detections with the reagent 1 and the reagent 2 in the kit respectively, and the detection results of the two parallel immunoassay detections are excited and recorded; wherein the reading of the detection using the reagent 1 is the first measured value, the reading of the detection using the reagent 2 is the second measured value, and the ratio of the content of the specific capture molecules corresponding to the first measured value to the content of the target molecules to be measured is greater than the ratio of the content of the specific capture molecules corresponding to the second measured value to the content of the target molecules to be measured;

[0016] S2, the ratio of the first measured value to the second measured value is calculated.

[0017] In some embodiments of the present application, the ratio of the amount of specific capture molecules corresponding to the first measurement to the amount of target molecules to be detected in the two parallel immunoassay detections is greater than the ratio of the amount of specific capture molecules corresponding to the second measurement to the amount of target molecules to be detected in any of the following ways:

[0018] Method 1: The amount of the sample containing the target molecules to be detected used in the two parallel immunoassay detections is the same, and the amount of reagent 1 and reagent 2 used is the same.

[0019] Method 2: In the two parallel immunoassay detections, the amount of the sample containing the target molecules to be detected using reagent 1 is different from the amount of the sample containing the target molecules to be detected using reagent 2, and the amount of reagent 1 and reagent 2 used is the same.

[0020] Method 3: In the two parallel immunoassay detections, the amount of the sample containing the target molecules to be detected using reagent 1 is different from the amount of the sample containing the target molecules to be detected using reagent 2, and the amount of reagent 1 and reagent 2 used is also different.

[0021] Method 4: The amount of the sample containing the target molecules to be detected used in the two parallel immunoassay detections is the same, and the amount of reagent 1 and reagent 2 used is different.

[0022] In some embodiments of the present application, the method further comprises the following steps:

[0023] A1, detecting a series of standard substances with different concentrations of the target molecules to be detected, wherein two parallel immunoassay detections are performed on each standard substance, and the detection results of the two parallel immunoassay detections are recorded as measurement a and measurement a', respectively, and the detection method of measurement a is the same as the first measurement of the sample to be detected, and the detection method of measurement a' is the same as the second measurement of the sample to be detected.

[0024] A2, calculating the ratio of measurement a to measurement a';

[0025] A3, making a correlation standard curve of the ratio of measurement a to measurement a' and the concentration of the standard substance, and storing it.

[0026] In some embodiments of the present application, the method further comprises the following steps:

[0027] Retrieve the stored correlation standard curve, and substitute the ratio of the first measurement to the second measurement of the sample containing the target molecules to be detected into the standard curve for calculation to determine the concentration of the sample.

[0028] In some embodiments of the present application, the method further comprises the following steps:

[0029] B1, detecting a series of standard substances with different concentrations of the target molecule to be detected, wherein two parallel immunoassay detections are performed for each standard substance, and the detection results of the two parallel immunoassays are recorded as measurement value b and measurement value b', respectively, and the measurement value b is detected in the same way as the first measurement value of the sample to be detected, and the measurement value b' is detected in the same way as the second measurement value of the sample to be detected;

[0030] B2, making a reaction curve A of measurement value b and standard substance concentration, and storing it;

[0031] B3, making a reaction curve B of measurement value b' and standard substance concentration, and storing it;

[0032] B4, taking a point in the overlapping part of the front band region of reaction curve A and the back band region of reaction curve B, and recording the ratio of measurement value b / measurement value b' of the point as critical point c, and storing it.

[0033] In some embodiments of the present application, the method further comprises the following steps:

[0034] Retrieve the stored reaction curve A, reaction curve B and critical point c, and judge the ratio of the first measurement value / second measurement value of the sample to be detected to the size of the critical point c; when the ratio of the first measurement value / second measurement value of the sample to be detected is ≤ the critical point c, use the front band region of the reaction curve A to calculate the concentration of the target molecule to be detected in the sample to be detected; when the ratio of the first measurement value / second measurement value of the sample to be detected is > the critical point c, use the back band region of the reaction curve B to calculate the concentration of the target molecule to be detected in the sample to be detected.

[0035] The third aspect of the present application provides a system for immunoassay of the method according to the second aspect of the present application, which comprises:

[0036] An immunoassay device, which comprises two or more reaction vessels to simultaneously perform two parallel immunoassay detections on the same sample to be detected in the two reaction vessels; wherein reaction vessel 1 is added with reagent 1 of the kit, and reaction vessel 2 is added with reagent 2 of the kit;

[0037] A chemiluminescence immunoassay excitation and counting device for exciting and recording chemiluminescence readings, and recording the readings of the two parallel immunoassay detections of the same sample to be detected as first measurement value and second measurement value, respectively, wherein the first measurement value is derived from reaction vessel 1, and the second measurement value is derived from reaction vessel 2;

[0038] The processor calculates the ratio of the first measurement value and the second measurement value, and calculates the concentration of the sample to be measured according to the ratio.

[0039] In some embodiments of the present application, the processor stores the correlation standard curve of the ratio of the measurement value a / measurement value a' and the standard substance concentration, the reaction curve A of the measurement value b and the standard substance concentration, the reaction curve B of the measurement value b' and the standard substance concentration, and the critical point c, for calculating the concentration of the sample to be measured.

[0040] Compared with the prior art, the present application has the following beneficial effects:

[0041] (1) The method and system for detecting using the kit of the present application can solve the problem of the hook effect, avoid the missed detection caused by the hook effect, and are not limited by the detection range.

[0042] (2) The method and system for detecting using the kit of the present application directly use the classical dose-response curve for calculation, have good repeatability, do not need multiple dilutions, can obtain accurate measurement values of the hook effect sample through single detection, and have fast determination speed.

[0043] (3) The method and system for detecting using the kit of the present application have a detection range much larger than that of the conventional detection method. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 is the dose-response curve of antigen-antibody.

[0045] Figure 2 is the principle diagram of calculation method 1 when the kit of the present application is used for immunoassay.

[0046] Figure 3 is the principle diagram of calculation method 2 when the kit of the present application is used for immunoassay.

[0047] Figure 4 is the reaction curve diagram of the standard substance concentration and the signal of reagent 1 and the signal of reagent 2 in Example 2. DETAILED DESCRIPTION

[0048] In order to make the present application easy to understand, the present application will be described in detail below. However, before the present application is described in detail, it should be understood that the present application is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments, and are not intended to be limiting.

[0049] Where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the that that range and any other stated or intervening values in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are now described.

[0051] The immunoassay kit according to the first aspect of the present application comprises reagent 1 and reagent 2 which are identical in composition, and the total amount of specific capture molecules in reagent 1 is different from that in reagent 2, wherein the specific capture molecules are capable of specifically binding to the target molecules to be detected.

[0052] In some embodiments of the present application, there is only one kind of specific capture molecules; in preferred embodiments of the present application, there are more than one kind of specific capture molecules, including a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecules to be detected.

[0053] In some embodiments of the present application, reagent 1 comprises a first antibody (or antigen)-coated luminescent microparticle at a concentration of a1, reagent 2 comprises a first antibody (or antigen)-coated luminescent microparticle at a concentration of β1, and a1 is greater than β1.

[0054] In the present application, the luminescent microparticle contains a luminescent group, which is capable of rapidly absorbing singlet oxygen and then emitting light at a certain wavelength (e.g. 500-615 nm).

[0055] In some embodiments of the present application, reagent 1 further comprises a second antibody (or antigen) labeled with a label at a concentration of a2, and reagent 2 further comprises a second antibody (or antigen) labeled with a label at a concentration of β2, and a2 is not less than β2; preferably, a2 is greater than β2. In the present application, the label can be biotin.

[0056] That is, in some preferred embodiments of the present invention, the kit includes reagent 1 and reagent 2 with the same components, and reagent 1 contains luminescent microparticles coated with a first antibody (or antigen) at a concentration of α1 and a biotin-labeled second antibody (or antigen) at a concentration of α2; reagent 2 contains luminescent microparticles coated with a first antibody (or antigen) at a concentration of β1 and a biotin-labeled second antibody (or antigen) at a concentration of β2; and α1 is greater than β1, and α2 is greater than β2.

[0057] In some specific embodiments of the present invention, the target molecule to be tested is selected from antigens or antibodies. According to some embodiments of the present invention, the antigen refers to any substance having immunogenicity, including but not limited to substances listed in the examples of the aforementioned target molecules having immunogenicity. According to some embodiments of the present invention, the antibody is used in the broadest sense herein and explicitly encompasses 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).

[0058] The second aspect of this invention relates to a method for immunoassay using a kit as described in the first aspect of this invention. This method uses reagents 1 and 2 from the kit to perform two parallel tests on each sample. The ratio of the content of the target molecule to the content of the specific capture molecule in the detection reagent differs between the two tests, ultimately generating two different signals: a first measurement and a second measurement (the ratio of the specific capture molecule content to the target molecule content corresponding to the first measurement is greater than the ratio corresponding to the second measurement). As the content of the target molecule increases, the ratio of the first measurement to the second measurement continuously increases and exhibits a certain linear relationship. Based on this principle, the method for immunoassay using the kit described in this invention provides the following two methods for calculating the concentration of the target molecule, as follows:

[0059] Calculation Method 1: Directly calculate the concentration of the target molecule from the ratio of the first measured value to the second measured value.

[0060] like Figure 2 As shown, based on the correlation curve between the ratio of the first measured value to the second measured value and the concentration of the standard substance, the concentration of the target molecule can be calculated by substituting the ratio of the first measured value to the second measured value into the curve.

[0061] Calculation Method 2: Calculate the concentration of the target molecule using reaction curve A or reaction curve B:

[0062] In the dose-response curve of antigen-antibody ( Figure 1In 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.

[0063] like Figure 3 As shown, reaction curves A and B are obtained based on the first and second measured values ​​and the concentration of the standard substance, respectively. There is a concentration overlap between the front band region of reaction curve A and the back band region of reaction curve B (e.g., ...). Figure 3 (The part within the dashed box) Take a point in the overlapping area of ​​the above concentrations, and take the ratio of the first measurement value to the second measurement value at that point (A / B = 15 in the figure) as the critical point c.

[0064] When the ratio of the first measured value to the second measured value of the sample is less than or equal to the critical point c, the concentration is calculated using the front zone of the reaction curve A.

[0065] When the ratio of the first measured value to the second measured value of the sample is greater than the critical point c, the concentration is calculated using the back zone of the reaction curve B.

[0066] Corresponding to the above calculation method 1, the method for performing immunoassay using the kit described in this invention specifically includes the following steps:

[0067] First, a standard curve of the correlation between the ratio of measured value a / measured value a' and the concentration of the standard substance is obtained by a method including the following steps:

[0068] A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed. The results of the two parallel immune reactions are stimulated and recorded, and are respectively denoted as value a and value a'. The detection method of value a is the same as that of the first value of the sample to be tested, and the detection method of value a' is the same as that of the second value of the sample to be tested.

[0069] A2, calculate the ratio of measured value a to measured value a';

[0070] 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.

[0071] Then, the concentration of the target molecule in the sample is determined by a method including the following steps:

[0072] S1, respectively, the test sample containing the target molecule to be detected and reagent 1 and reagent 2 in the kit are subjected to two parallel immunoassay detections, and the detection results of the two parallel immunoassays are excited and recorded; wherein the reading count of the detection using reagent 1 is the first measured value, the reading count of the detection using reagent 2 is the second measured value, and the ratio of the content of the specific capture molecule corresponding to the first measured value to the content of the target molecule to be detected is greater than the ratio of the content of the specific capture molecule corresponding to the second measured value to the content of the target molecule to be detected;

[0073] S2, the ratio of the first measured value to the second measured value is calculated;

[0074] S3, the stored correlation standard curve is called, and the ratio of the first measured value to the second measured value of the test sample is substituted into the correlation standard curve for calculation to determine the concentration of the target molecule to be detected in the test sample.

[0075] Corresponding to the above-mentioned calculation method 2, the method for immunoassay using the kit of the present application specifically comprises the following steps:

[0076] First, the reaction curve A, the reaction curve B and the critical point c are obtained by the method comprising the following steps:

[0077] B1, a series of standard substances with different concentrations of the target molecule to be detected are detected, wherein two parallel immunoassay detections are performed on each standard substance, and the detection results of the two parallel immunoassays are excited and recorded, respectively, as measured value b and measured value b', and the detection method of the measured value b is the same as that of the first measured value of the test sample, and the detection method of the measured value b' is the same as that of the second measured value of the test sample;

[0078] B2, a reaction curve A of measured value b and standard substance concentration is made and stored;

[0079] B3, a reaction curve B of measured value b' and standard substance concentration is made and stored;

[0080] B4, a point is taken in the overlapping part of the front band region of the reaction curve A and the rear band region of the reaction curve B corresponding to the standard substance concentration, and the ratio of the measured value b to the measured value b' corresponding to the point is recorded as the critical point c and stored.

[0081] Then, the concentration of the target molecule to be detected in the test sample is determined by the method comprising the following steps:

[0082] S1, the sample containing the target molecule to be detected is respectively detected with reagent 1 and reagent 2 in the kit to excite and record the detection results of two parallel immunoassays; wherein the first measurement value is obtained by using reagent 1 for detection, and the second measurement value is obtained by using reagent 2 for detection, and the ratio of the content of the specific capture molecule corresponding to the first measurement value to the content of the target molecule to be detected is greater than the ratio of the content of the specific capture molecule corresponding to the second measurement value to the content of the target molecule to be detected in the two parallel immunoassays;

[0083] S2, the ratio of the first measurement value to the second measurement value is calculated;

[0084] S3, the stored reaction curve A, reaction curve B and critical point c are called, and the ratio of the first measurement value to the second measurement value of the sample to be detected is judged; when the processor determines that the ratio of the first measurement value to the second measurement value of the sample to be detected 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 measurement value to the second measurement value of the sample to be detected is > c, the sample concentration is calculated using the rear band region of the reaction curve B.

[0085] In the present application, the ratio of the content of the specific capture molecule in reagent 1 to the content of the target molecule to be detected in the two parallel immunoassays is greater than the ratio of the content of the specific capture molecule in reagent 2 to the content of the target molecule to be detected in the following any one of the following ways:

[0086] Method 1: the amount of the sample containing the target molecule to be detected used in the two parallel immunoassays is the same, and the amount of reagent 1 and reagent 2 used is the same;

[0087] Method 2: in the two parallel immunoassays, the amount of the sample containing the target molecule to be detected detected by reagent 1 is different from the amount of the sample containing the target molecule to be detected detected by reagent 2, and the amount of reagent 1 and reagent 2 used is the same;

[0088] Method 3: in the two parallel immunoassays, the amount of the sample containing the target molecule to be detected detected by reagent 1 is different from the amount of the sample containing the target molecule to be detected detected by reagent 2, and the amount of reagent 1 and reagent 2 used is also different;

[0089] Method 4: the amount of the sample containing the target molecule to be detected used in the two parallel immunoassays is the same, and the amount of reagent 1 and reagent 2 used is different.

[0090] The "standard substance" in the present application refers to a solution of the target molecule to be detected, the content of which is known or can be quantitatively determined and assigned.

[0091] A third aspect of the present invention relates to a system for performing an immunoassay as described in the second aspect of the present invention, comprising:

[0092] An immunoassay apparatus comprising two or more reaction containers for simultaneously performing two parallel immunoassays on the same test sample in two of the reaction containers; wherein reaction container 1 is filled with reagent 1 of the kit, and reaction container 2 is filled with reagent 2 of the kit;

[0093] A chemiluminescent immunoassay excitation and counting device is used to excite and record chemiluminescence readings, and to record two parallel immunoassay readings of the same sample as the first measurement value and the second measurement value, respectively. The first measurement value is derived from reaction container 1 and the second measurement value is derived from reaction container 2.

[0094] The processor calculates the ratio of the first measurement to the second measurement, and calculates the concentration of the sample to be tested based on the ratio.

[0095] In this invention, there is no specific limitation on the shape of the reaction container. In some specific embodiments of this invention, the reaction container may be a reaction orifice, etc.; the chemiluminescent immunoassay excitation and counting device may include a photon counting module and a light-emitting diode; the processor may be a computer to process, plot, and store the readings, etc.

[0096] In some embodiments of the present invention, the processor stores a standard curve showing the correlation between the ratio of the measured value a / measured value a' and the concentration of the standard substance, a reaction curve A between the measured value b and the concentration of the standard substance, a reaction curve B between the measured value b' and the concentration of the standard substance, and the critical point c. The processor retrieves the stored data as needed to calculate the concentration of the sample to be tested.

[0097] In some embodiments of the present invention, the correlation standard curve between the ratio of the measured value a / measured value a' stored in the process and the concentration of the standard substance is obtained by performing a method comprising the following steps:

[0098] A1. A series of standard substances with different concentrations of known target molecules are tested. For each standard substance, two parallel immune reactions are performed. The results of the two parallel immune reactions are stimulated and recorded, and are respectively denoted as value a and value a'. The detection method of value a is the same as that of the first value of the sample to be tested, and the detection method of value a' is the same as that of the second value of the sample to be tested.

[0099] A2, calculate the ratio of measured value a to measured value a';

[0100] 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.

[0101] In other embodiments of the invention, the stress curve A, reaction curve B, and critical point c stored in the process are obtained by performing a method comprising the following steps:

[0102] B1, a series of standard substances of different concentrations with known content of the target molecule are tested, wherein two parallel immune reactions are performed on each standard substance, and the results of the two parallel immune reactions are stimulated and recorded, which are respectively denoted as measurement value b and measurement value b'. The detection method of measurement value b is the same as that of the first measurement value of the sample to be tested, and the detection method of measurement value b' is the same as that of the second measurement value of the sample to be tested.

[0103] B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it;

[0104] B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it;

[0105] 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.

[0106] Example

[0107] 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.

[0108] 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.

[0109] Example 1: Detection of AFP samples using conventional methods and reagent kits.

[0110] The standard reagent kit used was the Alpha-fetoprotein (AFP) Detection Kit (Chemiluminescence Method) (batch number: L2001) manufactured by Komeiboyang Diagnostics Technology (Shanghai) Co., Ltd., whose main components are:

[0111] Reagent 1: Luminescent microparticles coated with AFP antibody;

[0112] Reagent 2: Biotin-labeled AFP antibody.

[0113] Test method:

[0114] 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;

[0115] 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.

[0116] Test samples (collected from clinical serum samples):

[0117] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0118] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0119] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0120] The test results are shown in Table 1.

[0121] Table 1

[0122] Initial value Measured value ng / mL Sample 1 7.41 Sample 2 109.43 Sample 3 11.79

[0123] The results shown in Table 1 are from direct testing using a standard kit. Sample 3 had a value of only 11.79 ng / mL, which could easily be misjudged as a weakly positive sample without considering clinical manifestations. Knowing that Sample 3 was a strongly positive sample, it was diluted 50-fold with diluent and retested, with the results shown in Table 2.

[0124] Table 2

[0125] 50-fold dilution Measured value ng / mL Diluted sample >1000

[0126] As shown above, sample 3, after a 50-fold dilution, had a measured value >1000 ng / mL, confirming it as a hook sample, but a specific measured value still could not be obtained. The diluted sample was then diluted another 50 times with diluent, and the measured values ​​are shown in Table 3 below.

[0127] Table 3

[0128] 2500-fold dilution Measured value ng / mL Diluted sample 849.51

[0129] 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.

[0130] Example 2: Detection of AFP samples using the kit and immunoassay method of the present invention.

[0131] Reagent Kit: The main components of the two-reagent reagent kit of the present invention are as follows:

[0132] Reagent 1: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0133] Reagent 2: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0134] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0135] Test samples (collected from clinical serum samples):

[0136] Sample 1: Negative serum sample (actual value approximately 5 ng / mL)

[0137] Sample 2: Low-value positive serum sample (actual measured value approximately 100 ng / mL)

[0138] Sample 3: Strongly positive serum sample (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0139] Test Method 1:

[0140] 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:

[0141] 1. Add 10 μl of the sample to be tested and 25 μl of reagent 1 to reaction well 1;

[0142] 2. Add 10 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively;

[0143] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0144] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0145] The AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 4.

[0146] Based on the values ​​in Table 4, plot reaction curves A and B respectively between the concentrations of the standard substance and the signals from reagent 1 and reagent 2. Figure 4 It can be seen that standard 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 is taken as the critical point.

[0147] Based on the values ​​in Table 4, construct a standard curve showing the correlation between the A / B signal ratio and the concentration of the standard substance.

[0148] Store the above standard substance test results. Analyzer.

[0149] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 5. Calculation method 1 is the test result obtained by calling the correlation standard curve, and calculation method 2 is the test result obtained by calling the critical point.

[0150] Table 4

[0151]

[0152]

[0153] Table 5

[0154]

[0155] As shown in Table 5, the immunoassay method using the kit of this invention can avoid the problem of low sample values ​​caused by the HOOK effect, and can directly obtain high values ​​of up to 2×10⁻⁶. 6 The detection results are in ng / mL. The method is not limited by the detection range, and both calculation methods are feasible.

[0156] Example 3: Detection of AFP samples using the kit and immunoassay method of the present invention.

[0157] The experimental materials were the same as in Example 2.

[0158] Test Method 2:

[0159] 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:

[0160] 1. Add 10 μl of the sample to be tested and 25 μl of reagent 1 to reaction well 1;

[0161] 2. Add 20 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively;

[0162] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0163] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0164] The serially diluted AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 6. A standard curve was plotted based on the correlation between the A / B signal ratio and the concentration of the standard substances according to the values ​​in Table 6. The test results of the above standard substances were stored in [the database / system / etc.]. Analyzer.

[0165] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 7 below.

[0166] Table 6

[0167]

[0168] The results showed that the kit and immunoassay method of the present invention can detect samples with extremely high AFP values, have a wide detection range, and can easily, quickly, and accurately calculate the concentration of the analyte.

[0169] Example 4: Detection of AFP samples using the kit and immunoassay method of the present invention.

[0170] The experimental materials were the same as in Example 2.

[0171] Test Method 3:

[0172] 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:

[0173] 1. Add 10 μl of the sample to be tested and 50 μl of reagent 1 to reaction well 1;

[0174] 2. Add 20 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively;

[0175] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0176] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0177] The serially diluted AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 8. A standard curve was plotted based on the correlation between the A / B signal ratio and the concentration of the standard substances according to the values ​​in Table 8. The test results of the above standard substances were stored in [the database / system / etc.]. Analyzer. The three groups of samples were tested according to the above testing method, and the test results are shown in Table 9 below.

[0178] Table 8

[0179]

[0180] Table 9

[0181]

[0182] The results showed that the kit and immunoassay method of the present invention can detect samples with extremely high AFP values, have a wide detection range, and can easily, quickly, and accurately calculate the concentration of the analyte.

[0183] Example 5: Detection of AFP samples using the kit and immunoassay method of the present invention.

[0184] The experimental materials were the same as in Example 2.

[0185] Test Method 4:

[0186] 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:

[0187] 1. Add 10 μl of the sample to be tested and 50 μl of reagent 1 to reaction well 1;

[0188] 2. Add 10 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively;

[0189] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0190] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0191] The serially diluted AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 10. A standard curve was plotted based on the correlation between the A / B signal ratio and the concentration of the standard substances according to the values ​​in Table 10. The test results of the above standard substances were stored in [the database / system / etc.]. Analyzer.

[0192] The three groups of samples were tested according to the above testing method, and the test results are shown in Table 11 below.

[0193] Table 10

[0194]

[0195]

[0196] Table 11

[0197]

[0198] The results showed that the kit and immunoassay method of the present invention can detect samples with extremely high AFP values, have a wide detection range, and can easily, quickly, and accurately calculate the concentration of the analyte.

[0199] Example 6: Verification of the Precision of Ultra-High-End Measurements

[0200] The experimental materials were the same as in Example 2.

[0201] Test method:

[0202] For two reaction wells belonging to the same sample test group, repeat the following liquid addition steps 1 and 2 for different sample test groups:

[0203] 1. Add 10 μl of the sample to be tested and 25 μl of reagent 1 to reaction well 1;

[0204] 2. Add 10 μl of the sample to be tested and 25 μl of reagent 2 to reaction well 2 respectively;

[0205] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0206] 4. Add 175 μl of the universal solution (donor reagent) for the photo-induced chemiluminescence analysis system to each well, incubate at 37°C for 10 min, and then use... The analyzer takes readings.

[0207] The test results are shown in Table 12 below (calculated using the calculation method 1 of this invention).

[0208] Table 12

[0209]

[0210]

[0211] The results showed that, using the assay method of the kit described in this invention, the CV of the three high-value samples were all within 10% after 10 repeated measurements, indicating that the precision results were good.

[0212] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. An immunoassay method using a photo-induced chemiluminescence immunoassay kit, wherein the kit comprises reagent 1 and reagent 2 with identical components, and the total content of specific capture molecules in reagent 1 is different from the total content of specific capture molecules in reagent 2, wherein the specific capture molecules are capable of specifically binding to the target molecule to be tested; The method for performing immunoassay using the kit includes: performing two parallel immunoassay tests on each sample using reagents 1 and 2 from the kit; wherein the two parallel immunoassay tests generate two different signals, namely a first measurement value and a second measurement value, and the ratio of the content of the specific capture molecule to the content of the target molecule corresponding to the first measurement value is greater than the ratio of the content of the specific capture molecule to the content of the target molecule corresponding to the second measurement value. The method for performing immunoassay using the kit further includes: calculating the ratio of a first measured value to a second measured value, and determining the concentration of the target molecule in the sample to be tested based on reaction curve A or reaction curve B; wherein reaction curve A and reaction curve B are reaction curves obtained from the detection results of two parallel immunoassays of a standard substance with known content of the same target molecule; taking a point where the concentration overlaps between the front band region of reaction curve A and the back band region of reaction curve B, and using the ratio of the first measured value to the second measured value of the two parallel immunoassays of the standard substance corresponding to this point as the critical point c; when the ratio of the first measured value to the second measured value of the sample to be tested is ≤ the critical point c, the concentration of the target molecule in the sample to be tested is calculated using the front band region of reaction curve A; when the ratio of the first measured value to the second measured value of the sample to be tested is > the critical point c, the concentration of the target molecule in the sample to be tested is calculated using the back band region of reaction curve B.

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

3. The immunoassay method according to claim 1, characterized in that, The specific capture molecules include a first antibody or antigen and a second antibody or antigen capable of specifically binding to the target molecule to be tested.

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

5. The immunoassay method according to claim 4, characterized in that, The α2 is greater than the β2.

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

7. The immunoassay method according to claim 1, characterized in that, The method further includes the following steps: B1, a series of standard substances of different concentrations with known content of the target molecule are tested, wherein two parallel immune reactions are performed on each standard substance, and the results of the two parallel immune reactions are stimulated and recorded, which are respectively denoted as measurement value b and measurement value b'. The detection method of measurement value b is the same as that of the first measurement value of the sample to be tested, and the detection method of measurement value b' is the same as that of the second measurement value of the sample to be tested. B2, plot the reaction curve A between the measured value b and the concentration of the standard substance, and store it; B3, plot the reaction curve B between the measured value b' and the concentration of the standard substance, and store it; B4. Take a point in the overlapping area of ​​the standard substance concentrations corresponding to the front zone of reaction curve A and the back zone of reaction curve B, record the ratio of the measured value b to the measured value b' at that point as the critical point c, and store it.

8. The immunoassay method according to claim 7, characterized in that, The method further includes the following steps: Retrieve the stored reaction curves A and B and the critical point, 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.

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