An immunoassay method and its use

Abstract

The application provides an immunoassay method, which comprises the following steps: S1: detecting two parallel immuno-reactions of a sample to be tested containing target molecules to be tested, recording the detection results of the two parallel immuno-reactions, and taking the first detection result and the second detection result as the first measured value and the second measured value respectively; S2: calculating the ratio of the first measured value / second measured value; and S3: determining the concentration of the target molecules to be tested in the sample to be tested. The determination method of the application can solve the problem of the sample with the hook effect, the method is not limited by the detection range, and the concentration of the sample with a high value of up to 10 6 ng / ml level can be directly measured; the repeatability is good, and the detection speed is fast.

Description

TECHNICAL FIELD

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

[0002] Immunological detection is based on the principle of antigen-antibody specific reaction, and can be used for detecting trace biological active substances such as proteins and hormones due to the use of isotopes, enzymes, chemiluminescent substances and the like for displaying or amplifying signals.

[0003] Photostimulated chemiluminescence is one of the commonly used methods of chemiluminescence analysis technology, which can be used for studying the interaction between biological molecules, and is mainly used for detecting diseases in clinic. The technology integrates researches in the fields of high molecular particle technology, organic synthesis, protein chemistry and clinical detection. The technical principle of photostimulated chemiluminescence analysis technology is as follows: under laser irradiation, a sensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules, and the singlet oxygen molecules can react with luminescent compositions within a distance of about 200 nm to generate light signals of a certain wavelength; when the sample contains an antigen or an antibody to be detected, the immunoreaction of the antigen and the antibody can make the donor particles containing the sensitizer combine with the acceptor particles containing the luminescent composition, so as to generate light signals of a specific wavelength, and the content of the antigen or the antibody to be detected 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 in which the reaction signal increases with the increase of the antigen dose is called the "front band" region, the region in which 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 immunoreaction, the reactivity of antigen and antibody increases first and then decreases with the increase of the ratio of antigen and antibody, which is called "hook effect" or "HOOK effect". In clinic, the hook effect can cause false negative results of high value samples, that is, "false negative".

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

[0007] Narrow detection range: the traditional immunodetection reagent can only utilize the front band section of the dose-response curve for detection, and the detection concentration range is narrow. Samples exceeding the detection range need to be detected after dilution, 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, 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 method. The determination method of the present application can simply, quickly and accurately calculate the concentration of the target molecule.

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

[0011] The first aspect of the present application provides an immunoassay method, which comprises the following steps:

[0012] S1: Two parallel immunoassay detections are performed on the sample containing the target molecule to be detected, and the detection results of the two parallel immunoassay detections are recorded, respectively, as the first measured value and the second measured value;

[0013] S2: Calculate the ratio of the first measured value / second measured value;

[0014] S3: Determine the concentration of the target molecule to be detected in the sample.

[0015] According to some embodiments of the present application, in the two parallel immunoassay detections, the ratio of the content of the target molecule to be detected / the content of the specific capture molecule is different; wherein the specific capture molecule can specifically bind to the target molecule to be detected.

[0016] According to some embodiments of the present application, in the two parallel immunoassay detections, the detection result of the immunoassay with a larger ratio of the content of the specific capture molecule / the content of the target molecule to be detected is counted as the first measured value, and the other is the second measured value.

[0017] According to some embodiments of the present application, the target molecule to be detected is selected from an antigen or an antibody.

[0018] According to some embodiments of the present application, the specific capture molecule comprises a first capture molecule combined with a solid phase material and a second capture molecule labeled with a label.

[0019] According to some embodiments of the present application, 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; preferably the content of the first capture molecule.

[0020] According to some embodiments of the present application, the method for determining the concentration of the target molecule in the sample to be tested is to substitute the ratio of the first measurement value / second measurement value of the sample to be tested into the correlation standard curve for calculation.

[0021] According to some embodiments of the present application, the method for obtaining the correlation standard curve comprises the following steps:

[0022] A1: detecting a series of standard substances with different concentrations of the target molecule to be tested, 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 a and measurement value a', respectively; preferably, the detection result of the immunoassay with a larger ratio of the content of the specific capture molecule to the content of the target molecule to be tested is counted as measurement value a in the two parallel immunoassay detections;

[0023] A2: calculating the ratio of measurement value a / measurement value a';

[0024] A3: making a correlation standard curve of the ratio of measurement value a / measurement value a' and the concentration of the standard substance.

[0025] According to some embodiments of the present application, the method for determining the concentration of the target molecule in the sample to be tested is to substitute the ratio of the first measurement value / second measurement value of the sample to be tested into the correlation standard curve for calculation.

[0026] When the ratio of the first measurement value / second measurement value of the sample to be tested is ≤ critical point c, the concentration of the target molecule in the sample to be tested is calculated using the pre-zone of reaction curve A; when the ratio of the first measurement value / second measurement value of the sample to be tested is > critical point c, the concentration of the target molecule in the sample to be tested is calculated using the post-zone of reaction curve B.

[0027] According to some embodiments of the present application, the method further comprises the following steps:

[0028] B1: detecting a series of standard substances with different concentrations of the target molecule to be tested, 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; preferably, the detection result of the immunoassay with a larger ratio of the content of the specific capture molecule to the content of the target molecule to be tested is counted as measurement value b in the two parallel immunoassay detections;

[0029] B2: making reaction curve A of measurement value b and the concentration of the standard substance;

[0030] B3: making reaction curve B of measurement value b' and the concentration of the standard substance;

[0031] B4: taking a point in the part where the pre-zone of reaction curve A and the post-zone of reaction curve B correspond to the concentration of the target molecule to be tested, and recording the ratio of measurement value b / measurement value b' corresponding to the point as critical point c.

[0032] The second aspect of the present application provides an application of the method according to the first aspect of the present application in chemiluminescence immunoassay, enzyme-linked immunoassay and immune turbidimetric immunoassay.

[0033] According to some embodiments of the present application, the method is applied in chemiluminescence immunoassay.

[0034] According to further preferred embodiments of the present application, the method is applied in photochemical chemiluminescence immunoassay.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] (1) The method of the present application can solve the problem of the hook effect and avoid the missed detection caused by the hook effect, and the method is not limited by the detection range;

[0037] (2) The method of the present application directly uses the classical dose-response curve calculation, has good repeatability, and fast determination speed;

[0038] (3) The detection range of the method of the present application is much larger than that of the conventional detection method, and when detecting high-value samples with a concentration higher than 10 3 ng / mL, there is no need for secondary dilution, and the high-value sample concentration up to 10 6 ng / mL level can be directly measured. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is an antigen-antibody dose-response curve.

[0040] Figure 2 is a schematic diagram of the determination method according to the calculation method 1 of the present application.

[0041] Figure 3 is a schematic diagram of the determination method according to the calculation method 2 of the present application.

[0042] Figure 4 is a reaction curve diagram of the standard substance concentration and the A reagent signal and the B reagent signal in Example 2. DETAILED DESCRIPTION

[0043] In order to make the present application easy to understand, the present application will be described in detail below. However, before the detailed description of the present application, 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 the purpose of describing the specific embodiments and are not intended to be limiting.

[0044] Where a numerical range is provided, it is understood that every intervening value between the lower and upper limit of that range and any other stated or intervening values in that stated range, is encompassed within the application. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the application, 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 in the application.

[0045] 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.

[0046] The present application is based on a single sample double measurement method, that is, two parallel tests are performed on each sample, the ratio of the content of the target molecule to be tested to the content of the specific capture molecule is different in the two tests, and finally two different signals are generated, which are the first measurement value and the second measurement value respectively. With the increase of the content of the target molecule to be tested, the ratio of the first measurement value / second measurement value continues to rise and shows a certain linear relationship. According to this principle, the present application provides the following two methods for calculating the concentration of the target molecule to be tested, which are as follows:

[0047] Calculation method 1, directly calculating the concentration of the target molecule to be tested from the ratio of the first measurement value / second measurement value:

[0048] As shown in Figure 2 , according to the correlation standard curve of the ratio of the first measurement value / second measurement value and the concentration of the standard substance, the concentration of the target molecule to be tested can be calculated by substituting the ratio of the first measurement value / second measurement value of the target molecule to be tested into the curve.

[0049] Calculation method 2, using reaction curve A or reaction curve B to calculate the concentration of the target molecule to be tested:

[0050] In the dose-response curve of antigen-antibody Figure 1 , when the amount of antibody is fixed, the reaction signal will show a phenomenon of first rising and then falling with the increase of the amount of antigen. The region where the reaction signal rises with the increase of the amount of antigen is called the front band region, the region where the reaction signal falls with the increase of the amount of antigen is called the rear band region, and the region connecting the front band and the rear band is called the equivalent band.

[0051] As shown in Figure 3 , reaction curve A and reaction curve B are reaction curves obtained from the first measurement value and the second measurement value and the concentration of the standard substance respectively. There is a concentration overlapping part between the front band region of reaction curve A and the rear band region of reaction curve B (as shown in Figure 3In the part of the middle dotted line box), take a point in the part of the above concentration overlap, the ratio of the first measured value / second measured value corresponding to the point (such as A / B=15 in the figure) as the critical point c.

[0052] When the ratio of the first measured value / second measured value of the sample to be measured is ≤ the critical point c, the concentration of the sample to be measured is calculated using the front band region of the reaction curve A.

[0053] When the ratio of the first measured value / second measured value of the sample to be measured is > the critical point c, the concentration of the sample to be measured is calculated using the rear band region of the reaction curve B.

[0054] Corresponding to the above calculation method 1, the immunoassay method provided by the application specifically comprises the following steps:

[0055] First, the correlation standard curve of the ratio of the measured value a / measured value a' and the concentration of the standard substance is obtained by the method comprising the following steps:

[0056] A1: Detect a series of different concentrations of standard substances with known contents of target molecules to be measured, wherein two parallel immunoassays are performed for each standard substance, and the detection results of the two parallel immunoassays are recorded as measured value a and measured value a', respectively;

[0057] A2: Calculate the ratio of the measured value a / measured value a';

[0058] A3: According to the measured value a and the measured value a' of the series of different concentrations of standard substances, a correlation standard curve of the ratio of the measured value a / measured value a' and the concentration of the standard substance is drawn.

[0059] In the whole test process, the measured value of the specific capture molecule content / target molecule content ratio can be set as measured value a, and the other as a'; vice versa.

[0060] Then, the concentration of the target molecule to be measured in the sample to be measured is determined by the method comprising the following steps:

[0061] S1: Two parallel immunoassays are performed on the sample to be measured containing the target molecule to be measured, and the detection results of the two parallel immunoassays are recorded as the first measured value and the second measured value; in the immunoassay, the measured value of the specific capture molecule content / target molecule content ratio is the first measured value, and the other is the second measured value;

[0062] S2: Calculate the ratio of the first measured value / second measured value of the sample to be measured;

[0063] S3: The ratio of the first measured value / second measured value of the sample to be measured is substituted into the correlation standard curve for calculation to determine the concentration of the target molecule to be measured in the sample to be measured.

[0064] In the whole test procedure, the test value of the specific capture molecule content / target molecule content ratio can be set as the first test value, and the other as the second test value.

[0065] Corresponding to the above calculation method 2, the immunoassay method provided by the application specifically comprises the following steps:

[0066] Firstly, the reaction curve A, the reaction curve B and the critical point c are obtained by the method comprising the following steps:

[0067] B1: detecting a series of standard substances with different concentrations of known target molecule content, wherein two parallel immunoassay detections are performed on each standard substance, and the detection results of the two parallel immunoassay detections are recorded, respectively, as test value b and test value b';

[0068] B2: according to the test value b of the series of standard substances with different concentrations, a reaction curve A of test value b and standard substance concentration is drawn;

[0069] B3: according to the test value b' of the series of standard substances with different concentrations, a reaction curve B of test value b' and standard substance concentration is drawn;

[0070] B4: there is a concentration overlap between the front band region of the reaction curve A and the rear band region of the reaction curve B (such as Figure 3 the part in the dashed box in the middle), and a point in the above concentration overlap is taken, and the ratio of the test value b and the test value b' corresponding to the point is recorded as the critical point c.

[0071] In the whole test procedure, the test value of the specific capture molecule content / target molecule content ratio can be set as the test value b, and the other as the test value b'; vice versa.

[0072] Then, the concentration of the target molecule in the test sample is determined by the method comprising the following steps:

[0073] S1: two parallel immunoassay detections are performed on the test sample containing the target molecule, and the detection results of the two parallel immunoassay detections are recorded, respectively, as the first test value and the second test value; in the immunoassay detection, the test value of the specific capture molecule content / target molecule content ratio is the first test value, and the other is the second test value;

[0074] S2: calculating the ratio of the first test value / second test value of the test sample;

[0075] S3: when the ratio of the first test value / second test value of the test sample is ≤c, the front band region of the reaction curve A is used to calculate the sample concentration; when the ratio of the first test value / second test value of the test sample is >c, the rear band region of the reaction curve B is used to calculate the sample concentration.

[0076] Throughout the test procedure, the test value with a greater ratio of the amount of specific capture molecules to the amount of target molecules to be detected can be defined as the first test value, and the other as the second test value; vice versa.

[0077] According to some embodiments of the present application, in the two parallel immunoassay detections, the ratio of the amount of specific capture molecules to the amount of target molecules to be detected is different. The following methods can be used to achieve this purpose, but are not limited thereto:

[0078]

[0079] The target molecules to be detected according to the present application refer to any inorganic or organic molecules that can be detected by immunoassay, including any target biological substances. Examples of the target molecules to be detected include cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, peptides, polypeptides, nucleic acids, oligosaccharides, polysaccharides, lipopolysaccharides, cellular metabolites, haptens, hormones, pharmaceutical substances, alkaloids, steroids, vitamins, amino acids, and sugars.

[0080] The specific capture molecules according to the present application refer to molecules that can be bonded to another molecule (target molecules to be detected) due to the mutual attraction between molecules. Examples of the specific capture molecules include, but are not limited to, proteins, nucleic acids, carbohydrates, lipids, and small organic molecules. The specific capture molecules refer to capture molecules that can recognize and bind to specific target molecules to be detected, but not to any target molecules.

[0081] According to some embodiments of the present application, the target molecules to be detected are selected from antigens or antibodies. According to some embodiments of the present application, the antigen refers to any substance with immunogenicity. Examples of the above-mentioned target molecules with immunogenicity are included, but are not limited thereto. According to some embodiments of the present application, the specific capture molecules are selected from one member of a specific binding pair, such as an antibody, and the target molecules to be detected are the other member of the specific binding pair, such as its paired antigen. The term "antibody" is used in the broadest sense herein and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments (e.g., Fab regions, Fc regions, single-chain antibodies).

[0082] According to some embodiments of the present application, the specific capture molecules used in the immunoassay detection include a first capture molecule bound to a solid phase material and a second capture molecule labeled with a label. The first capture molecule and the second capture molecule can be the same or different, and can be the same or different capture molecules, but can specifically bind to the target molecules to be detected.

[0083] According to some embodiments of the present application, the solid phase material to which the first capture molecule binds is selected from the group consisting of particles, microparticles, beads, electrodes and multi-well plates. In some embodiments, the first capture molecule binds to a luminescent microsphere which contains a luminescent group capable of rapidly absorbing singlet oxygen and then emitting light at a certain wavelength (e.g. 500-615 nm).

[0084] The "standard substance" according to 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.

[0085] The present application also provides the use of the assay method according to the present application in chemiluminescent immunoassay, enzyme-linked immunoassay and immune turbidimetric immunoassay.

[0086] According to some embodiments of the present application, the method is used in chemiluminescent immunoassay.

[0087] According to further preferred embodiments of the present application, the method is used in photoactivated chemiluminescent immunoassay.

[0088] Examples

[0089] In order to make the present application more easily understood, the present application will be further described in detail below by means of specific examples taking AFP as an example. These examples are only illustrative and do not limit the scope of application of the present application. The raw materials or components used in the present application can be obtained by commercial means or conventional methods if not otherwise specified.

[0090] Alpha-fetoprotein (AFP) is a glycoprotein also known as fetal albumin, which belongs to the albumin family. The AFP (alpha-fetoprotein) value in the serum of patients with primary liver cancer (PLC) varies greatly, and the difference between the normal value and the pathological value can reach 7 orders of magnitude. According to a literature report, the AFP concentration of a primary liver cancer patient was directly measured by IEMA to be 29 ng / mL, but after a series of dilutions of the serum sample, the actual AFP concentration was calculated to be 5.9 x 10 6 ng / mL. It can be seen that the detection of AFP in conventional technology still has great defects.

[0091] Example 1: Detection of AFP samples by conventional method

[0092] Test samples (collected from clinical serum samples):

[0093] Sample 1: negative serum sample (true value about 5 ng / mL)

[0094] Sample 2: low-value positive serum sample (true value about 100 ng / mL)

[0095] Sample 3: strongly positive serum sample (true value about 2 x 10 6 ng / mL)

[0096] The kit used is AFP detection kit (chemiluminescence method) produced by Keminboyang Diagnostics Technology (Shanghai) Co., Ltd. (batch number: L2001), and its main components are:

[0097] Reagent 1: luminescent microparticles coated with AFP antibody;

[0098] Reagent 2: biotin-labeled AFP antibody.

[0099] Test method:

[0100] 1. Add 25 μl of the sample to be tested, 25 μl of reagent 1, and 25 μl of reagent 2 to the reaction well, respectively, and incubate at 37°C for 15 min;

[0101] 2. Add 175 μl of light-activated chemiluminescence analysis system general solution (donor reagent) to the reaction well, incubate at 37°C for 10 min, and use the analyzer to read.

[0102] The detection results are shown in Table 1.

[0103] Table 1

[0104] Initial value Measured value ng / mL Sample 1 7.41 Sample 2 109.43 Sample 3 11.79

[0105] The results shown in Table 1 are the results of direct detection by the conventional reagent, and the sample 3 value is only 11.79 ng / mL, which is easily misjudged as a weak positive sample without combining with the clinical manifestations. In the case of knowing that sample 3 is a strongly positive sample, it is re-detected after being diluted 50 times with the diluent, and the detection results are shown in Table 2.

[0106] Table 2

[0107] 50-fold dilution Measured value ng / mL Diluted sample >1000

[0108] As above, the sample 3 value after 50 times dilution is >1000 ng / mL, which can be determined as a HOOK sample, and the specific value cannot be obtained. After the diluted sample is diluted again with the diluent for 50 times, the value is shown in Table 3.

[0109] Table 3

[0110] 2500-fold dilution Measured value ng / mL Diluted sample 849.51

[0111] As above, the sample value after 2500 times dilution is 849.51 ng / mL, and the true concentration of sample 3 can be calculated to be about 2.12 x 10 6 ng / mL. ​

[0112] Example 2: The method of the present invention for detecting AFP samples

[0113] Test standard material: concentration range of 0 ng / mL - 4 × 10 6 Purified AFP antigen solution (ng / mL)

[0114] Test samples (collected from clinical serum samples):

[0115] Sample 1: Negative (actual measurement approximately 5 ng / mL)

[0116] Sample 2: Low positive value (actual measured value approximately 100 ng / mL)

[0117] Sample 3: Strongly positive (actual measured value approximately 2 × 10⁻⁶) 6 ng / mL)

[0118] The main components of the reagent kit used:

[0119] Reagent A: Luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0120] Reagent B: Luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0121] Test method:

[0122] 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:

[0123] 1. Add 10 μl of the sample to be tested and 25 μl of reagent A to reaction well 1;

[0124] 2. Add 10 μl of the sample to be tested and 25 μl of reagent B to reaction well 2 respectively;

[0125] 3. Incubate each reaction well at 37°C for 15 minutes simultaneously;

[0126] 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.

[0127] The AFP standard substances (numbered 1-20) were tested according to the above test method, and the test results are shown in Table 4.

[0128] Based on the values ​​in Table 4, plot reaction curves A and B respectively between the concentration of the standard substance and the signals of reagent A and reagent B. Figure 4; it can be seen that the front band region of the reaction curve A corresponds to the standard substance 1-11, and the rear band region of the reaction curve B corresponds to the standard substance 9-20, therefore, the concentration range of the standard substance 9-11 exists in the overlapping part of the front band region of the reaction curve A and the rear band region of the reaction curve B, and the A / B signal ratio corresponding to the concentration range is 15.05-24.31, and the A / B signal ratio of the middle point is taken as a critical point.

[0129] The correlation standard curve of the A / B signal ratio and the concentration of the standard substance is drawn according to the values in Table 4.

[0130] The test results of the above standard substances are stored in the analyzer.

[0131] Three groups of samples are tested according to the above test method, and the test results are shown in Table 5. The calculation method 1 is the test result obtained by calling the correlation standard curve, and the calculation method 2 is the test result obtained by calling the critical point.

[0132] Table 4

[0133]

[0134]

[0135] Table 5

[0136]

[0137] It can be seen from the results shown in Table 5 that the detection method of the present application can avoid the problem of low sample test value caused by the hook effect, and can directly obtain a detection result as high as 2x10 6 ng / mL. The method is not limited by the detection range, and both calculation methods are feasible.

[0138] Example 3: Precision verification of ultra-high value sample test value

[0139] The main components of the kit used are:

[0140] Reagent A: luminescent microparticles coated with AFP antibody (concentration 100 μg / mL), biotin-labeled AFP antibody (concentration 2 μg / mL);

[0141] Reagent B: luminescent microparticles coated with AFP antibody (concentration 20 μg / mL), biotin-labeled AFP antibody (concentration 0.4 μg / mL).

[0142] Test method:

[0143] Two reaction wells are the same sample test group, and the following liquid addition steps 1 and 2 are repeated for different sample test groups:

[0144] 1. Add 10 μl of sample to be tested and 25 μl of reagent A to reaction well 1, respectively;

[0145] 2. Add 10 μl of sample to be tested and 25 μl of reagent B to reaction well 2, respectively;

[0146] 3. Incubate each reaction well at 37°C for 15 min;

[0147] 4. Add 175 μl of general solution of light-activated chemiluminescence analysis system (donor reagent) to each reaction well, incubate at 37°C for 10 min, and use analyzer to read.

[0148] The test results are shown in Table 6 (calculated using the calculation method 1 of the present application).

[0149] Table 6

[0150]

[0151] According to the results in Table 6, it can be seen that the CV of the measured values of the three high-value samples determined 10 times using the determination method of the present application is within the range of 10%, indicating that the precision result is good.

[0152] Example 4: Test speed test

[0153] Determination method A (determination method of the prior art):

[0154] The kit used is an alpha-fetoprotein (AFP) test kit (chemiluminescence method) produced by Keminboyang Diagnostics Technology (Shanghai) Co., Ltd. (batch number: L2001), and the main components thereof are:

[0155] Reagent 1: luminescent microparticles coated with AFP antibody;

[0156] Reagent 2: biotin-labeled AFP antibody.

[0157] Test method:

[0158] 1. Add 25 μl of sample to be tested, 25 μl of reagent 1, and 25 μl of reagent 2 to the reaction well, respectively, and incubate at 37°C for 15 min;

[0159] 2. Add 175 μl of general solution of light-activated chemiluminescence analysis system (donor reagent) to the reaction well, incubate at 37°C for 10 min, and use an analyzer to read.

[0160] Determination method B (determination method of the present application):

[0161] Main components of the kit used:

[0162] Reagent A: AFP antibody coated luminescent microparticles (concentration 100 μg / mL), biotin labeled AFP antibody (concentration 2 μg / mL);

[0163] Reagent B: AFP antibody coated luminescent microparticles (concentration 20 μg / mL), biotin labeled AFP antibody (concentration 0.4 μg / mL).

[0164] Test method:

[0165] Two reaction wells are the same sample test group, different sample test group repeats the following liquid addition steps 1 and 2:

[0166] 1. Add 10 μl of the sample to be tested and 25 μl of reagent A to reaction well 1, respectively;

[0167] 2. Add 10 μl of the sample to be tested and 25 μl of reagent B to reaction well 2, respectively;

[0168] 3. Incubate each reaction well at 37°C for 15 min;

[0169] 4. Add 175 μl of the general solution of the light-activated chemiluminescence analysis system to each reaction well, incubate at 37°C for 10 min, and use the analyzer to read the results.

[0170] The detection results are shown in Table 7.

[0171] Table 7

[0172]

[0173] According to the test results in Table 7, whether it is the first sample test time or the total sample test time, the test time using the determination method of the present application is less than or equal to the test time of the prior art, and in particular, the first sample test time of a single detection item is significantly faster than the test speed of the prior art.

[0174] It should be noted that the above-described embodiments are only used to explain the present application and do not constitute any limitation on the present application. The present application is described by referring to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified as specified within the scope of the claims of the present application, and the present application can be revised within the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein, on the contrary, the present application can be extended to all other methods and applications with the same function.​

Claims

1. An immunoassay method applied to photochemiluminescence immunoassay, so the method comprises the following steps: S1: two parallel immunoassay detections are performed on a sample to be tested containing a target molecule to be tested, and the detection results of the two parallel immunoassays are recorded, respectively as a first measurement value and a second measurement value; in the two parallel immunoassay detections, the ratio of the content of the target molecule to be tested to the content of a specific capture molecule is different, wherein the specific capture molecule can specifically bind to the target molecule to be tested; S2: calculating the ratio of the first measurement value to the second measurement value; S3: determining the concentration of the target molecule to be tested in the sample to be tested; The method for determining the concentration of the target molecule in the sample to be measured is: when the ratio of the first measurement value / second measurement value of the sample to be measured is less than or equal to the critical point c, the concentration of the target molecule in the sample to be measured is calculated using the front band region of the reaction curve A; when the ratio of the first measurement value / second measurement value of the sample to be measured is greater than the critical point c, the concentration of the target molecule in the sample to be measured is calculated using the rear band region of the reaction curve B; wherein, The reaction curve A and the reaction curve B are respectively obtained from the detection results of two parallel immunoassays of a standard substance with a known content of the same target molecule to be tested; a point is taken in the overlapping part of the front band region of the reaction curve A and the back band region of the reaction curve B, and the ratio of the first measurement value to the second measurement value of the two parallel immunoassays of the standard substance corresponding to the point is taken as a critical point c.

2. The method of claim 1, wherein, In the two parallel immunoassay detections, the detection result of the immunoassay with a larger ratio of the content of the specific capture molecule to the content of the target molecule to be tested is taken as the first measurement value, and the other is taken as the second measurement value.

3. The method of claim 1, wherein, The target molecule to be tested is selected from an antigen or an antibody; and / or The specific capture molecule comprises a first capture molecule combined with a solid phase material and a second capture molecule labeled with a label.

4. The method of claim 3, wherein, 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.

5. The method of claim 3, wherein, The content of the specific capture molecule is the content of the first capture molecule.

6. The method of claim 1, wherein, The method further comprises the following steps: B1: a series of standard substances with different concentrations and a known content of the target molecule to be tested are detected, wherein two parallel immunoassay detections are performed on each standard substance, and the detection results of the two parallel immunoassays are recorded, respectively as a measurement value b and a measurement value b'; B2: a reaction curve A of the measurement value b and the concentration of the standard substance is made; B3: a reaction curve B of the measurement value b' and the concentration of the standard substance is made; B4: a point is taken in the overlapping part of the standard substance concentration corresponding to the front band region of the reaction curve A and the back band region of the reaction curve B, and the ratio of the measurement value b to the measurement value b' corresponding to the point is taken as a critical point c.

7. The method of claim 6, wherein, In the two parallel immunoassay detections, the detection result of the immunoassay with a larger ratio of the content of the specific capture molecule to the content of the target molecule to be tested is taken as the measurement value b.

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