Immunological detection method for substance to be detected in whole blood sample

Abstract

The invention provides an immunological detection method for an object to be detected in a whole blood sample. The immunological detection method comprises the following steps: a1) mixing a whole blood sample with a first diluent to obtain a detection diluent; a2) mixing the detection diluent with an immunodetection reagent to obtain a detection system, and incubating the detection system to obtain an incubation compound; the osmotic pressure of the detection system is 1000-1700 mOSm; a3) performing signal detection on the incubation compound, and obtaining the concentration of the to-be-detected substance through the signal intensity; the detection time of the immunological detection method is controlled to be less than or equal to 10min. The problem that in the prior art, the erythrocyte state influences the detection result in the whole blood detection process can be solved, and the method is suitable for the field of whole blood sample detection.

Description

Technical Field

[0001] The present invention relates to the field of whole blood sample detection, and in particular to an immunological detection method for an object to be detected in a whole blood sample. Background Art

[0002] In the field of in vitro diagnosis, especially in environments where rapid testing is required, test results need to be obtained in the shortest possible time. Therefore, whole blood samples need to be used instead of plasma / serum samples that require pre-treatment. Direct use of whole blood samples for testing can greatly shorten sample processing time, which requires the detection method to be not only accurate and reliable, but also efficient and fast. However, in actual operation, the detection of whole blood samples often faces many challenges.

[0003] Whole blood samples contain a variety of cells and biomolecules, such as red blood cells, white blood cells, platelets, proteins, nucleic acids, etc. These components may interfere with the detection process and affect the accuracy of the results. How to ensure the accuracy of whole blood sample detection while ensuring detection efficiency has become an urgent problem to be solved in the field of rapid detection. In order to reduce the interference in whole blood cells, the traditional practice is to pre-treat the whole blood with a sample diluent with added surfactants. However, although conventional sample diluents can reduce interference to a certain extent, they still cannot effectively meet the needs of rapid detection in rapid detection scenarios. In view of the above problems, it is urgent to develop a whole blood detection method that can improve detection speed and accuracy. Summary of the invention

[0004] The main purpose of the present invention is to provide an immunological detection method for an object to be detected in a whole blood sample, so as to solve the problem of slow detection speed for whole blood samples in the prior art.

[0005] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, there is provided an immunological detection method for an object to be detected in a whole blood sample, the above-mentioned immunological detection method comprising: a1) mixing the whole blood sample with a first diluent to obtain a detection diluent; a2) mixing the detection diluent and an immunoassay reagent to obtain a detection system, and incubating the detection system to obtain an incubation complex; the osmotic pressure of the detection system is 1000-1700mOSm; a3) performing signal detection on the incubation complex, and obtaining the concentration of the object to be detected by the signal intensity; and controlling the detection time of the immunological detection method to be ≤10min.

[0006] Furthermore, the first dilution solution contains NaCl. Preferably, the concentration of NaCl in the first dilution solution is 7-15 wt %; preferably, the incubation time is 1-6 min.

[0007] Furthermore, the first diluent contains a surfactant; preferably, the surfactant includes one or more of casein sodium salt, polyethylene glycol or dextran sulfate sodium salt.

[0008] Furthermore, the first diluent contains any one or more of the following: a pH buffer, bovine serum albumin, glycine or a preservative; preferably, the pH buffer includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or Tris-HCl.

[0009] Furthermore, the immunological detection method also includes a step of correcting the concentration of the analyte: including correcting the concentration of the analyte using hematocrit; the hematocrit detection method includes: b1) mixing the whole blood sample with a second diluent to obtain a correction diluent; b2) detecting the absorbance of the correction diluent and calculating the hematocrit of the correction diluent; preferably, the hematocrit detection method is performed before a1)-a3), after a1)-a3), or simultaneously with a1)-a3); preferably, the osmotic pressure of the correction diluent is 600-2600mOSm; preferably, the wavelength used to detect the absorbance in b2) is 540-620nm.

[0010] Furthermore, the second dilution liquid also contains NaCl; preferably, the concentration of NaCl in the second dilution liquid is 2-8wt%; preferably, the second dilution liquid also contains any one or more of the following: a pH buffer, an anticoagulant or a preservative; preferably, the pH buffer includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or Tris-HCl.

[0011] Furthermore, the immunoassay reagent includes a signal molecule-protein complex and a magnetic microsphere-protein complex, and the magnetic microsphere-protein complex includes magnetic microspheres and a first affinity protein coated on the magnetic microspheres.

[0012] Furthermore, the signal molecule-protein complex includes a second affinity protein labeled with a luminescent marker; the second affinity protein is a protein that can specifically bind to the analyte.

[0013] Furthermore, the immunoassay reagent is loaded in a one-step method, and the signal molecule-protein complex and the magnetic microsphere-protein complex are mixed with the detection diluent at the same time to obtain a detection system.

[0014] Furthermore, the immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

[0015] By applying the technical solution of the present invention, in the above-mentioned immunological detection method, by controlling the osmotic pressure in the detection system to 1000-1700mOSm, the whole blood sample is detected in a liquid with high osmotic pressure, and the detection time of the immunological detection method is controlled to be ≤10min. The above-mentioned immunological detection method not only shortens the detection time and meets the demand for rapid detection in practical applications, but also reduces the influence of the red blood cell status on the detection results during the detection process, thereby improving the accuracy of the detection. DETAILED DESCRIPTION

[0016] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0017] Terminology explanation:

[0018] Osmotic pressure: The attraction of solute particles on water molecules in a solution. This attraction makes water molecules tend to move from a low concentration area (such as pure water or a solution with a lower concentration) through a semipermeable membrane to a high concentration area (such as a solution with a higher concentration) until a dynamic equilibrium state is reached. In this process, the higher the concentration of solute particles, the greater the attraction on water molecules, that is, the greater the osmotic pressure. Osmotic pressure is an important property of a solution, and plays a vital role in maintaining the stability of the internal environment of an organism, the normal function of cells, and a variety of physiological processes.

[0019] As mentioned in the background technology, in the field of medical testing, especially in the whole blood test project, it is crucial to ensure that the osmotic pressure of the test system is maintained within the optimal physiological range of blood cells (especially red blood cells). Red blood cells, as the main cell component in the blood, have a semipermeable outer membrane that allows specific solutes to pass through and are directly affected by the osmotic pressure of the surrounding solution. Osmotic pressure is an important indicator for measuring the pressure exerted by the concentration of solutes in the solution on the solvent molecules through the semipermeable membrane. Its stability is crucial for maintaining the morphology, function and integrity of red blood cells. Ideally, whole blood testing should be carried out in an osmotic pressure environment close to or equivalent to 0.9% sodium chloride solution (i.e., isotonic solution), because this concentration matches the osmotic pressure of normal whole blood, which can minimize the volume change, morphological distortion and even hemolysis of red blood cells caused by osmotic pressure differences, thereby ensuring the accuracy and reliability of the test results. However, in actual operation, the inventors found that due to the complexity of the test samples, the diversity of the test reagents and the uncontrollability of the operating conditions, it is difficult to accurately control the osmotic pressure of the system to the optimal physiological level of red blood cells in all test projects. For example, some test items may require the addition of reagents of a specific concentration to trigger a specific biochemical reaction or achieve a specific test purpose. The addition of these reagents often changes the osmotic pressure of the original solution, causing the test system to deviate from the isotonic state, resulting in insufficient test accuracy. A test method is urgently needed to ensure the accuracy of the test for whole blood samples. Therefore, in this application, the inventors attempt to develop a new test method for whole blood samples, and on this basis, propose a series of protection schemes of this application.

[0020] In a first typical embodiment of the present application, an immunological detection method for an object to be detected in a whole blood sample is provided, and the above-mentioned immunological detection method comprises: a1) mixing the whole blood sample with a first diluent to obtain a detection diluent; a2) mixing the detection diluent and an immunoassay reagent to obtain a detection system, and incubating the detection system to obtain an incubation complex; the osmotic pressure of the detection system is 1000-1700mOSm; a3) performing signal detection on the incubation complex, and obtaining the concentration of the object to be detected by the signal intensity; and controlling the detection time of the immunological detection method to be ≤10min.

[0021] In the above immunological detection method, a whole blood sample, a first diluent, and an immunological detection reagent are mixed to obtain a detection system with a high osmotic pressure. After the detection system is incubated to obtain an incubation complex, the incubation complex is subjected to signal detection using an immunological detection method in the prior art, thereby detecting the concentration of the analyte in the whole blood sample through the signal intensity.

[0022] Ideally, whole blood testing should be performed in an osmotic pressure environment close to that of an isotonic solution, thereby reducing changes in red blood cell volume, morphological distortion, and even hemolysis caused by osmotic pressure differences, thereby ensuring the accuracy and reliability of the test results. However, in the present application, the inventor unexpectedly discovered that when the time required for the overall test is controlled within 10 minutes, it not only meets the demand for obtaining test results in a short time in practical applications, but also uses the above method to accurately detect the content of the test substance in a whole blood sample under a high osmotic pressure system, which is more accurate than testing conducted under isotonic or near-isotonic conditions.

[0023] Hyperosmotic solutions generally contain a variety of salt ions and other chemical substances. In the present application, the inventors found that, compared with low osmotic solutions, hyperosmotic solutions composed of a variety of salt ions can promote the rapid binding of the antigen and antibody to be tested and increase the rate of immune response. The reaction between antigen and antibody is mainly affected by the hydrophobic interaction force, van der Waals force, electrostatic interaction, hydrogen bond, etc. between the two. It is speculated that the main influence is the hydration layer on the antigen surface, the exposure of the antigen epitope and its binding with the antibody caused by the action of salt ions.

[0024] It should be noted that the above immunological detection method is an improvement on the immunological detection method of the object to be detected in the whole blood sample in the prior art. For a feasible immunological detection method in the prior art, the osmotic pressure of the diluted whole blood sample is increased, and the detection is completed within 10 minutes, which can improve the stability and accuracy of the detection method by ensuring the stability of red blood cells in the detection system. And the above osmotic pressure conditions do not affect the detection of antigen-antibody binding, chemiluminescence, etc. in the immunological detection method.

[0025] The osmotic pressure of the above detection system includes but is not limited to 1000, 1100, 1200, 1300, 1400, 1500, 1600 or 1700 mOSm; the detection time is ≤10 min, preferably ≤8 min, or even ≤6 min.

[0026] In a preferred embodiment, the first dilution contains NaCl; preferably, the concentration of NaCl in the first dilution is 7-15wt%, including but not limited to 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt% or 15wt%.

[0027] The first diluent contains NaCl, and NaCl is used to achieve the main condition effect on osmotic pressure. Before using the diluent in the prior art to perform the above immunological detection method, NaCl can be optionally added to the diluent in the prior art to obtain the first diluent that meets the requirements of the immunological detection method of this application.

[0028] In a preferred embodiment, the incubation time is 1-6 min, more preferably 4 min; preferably, the incubation temperature is 37°C.

[0029] Under the above incubation conditions, the test object can be fully contacted and combined with the immunological detection reagent, and the probability of swelling, damage, hemolysis, cell membrane dissolution and other phenomena in the red blood cells in the whole blood sample can be reduced, thereby making the detection more accurate. In particular, the above incubation time, the inventors found that compared with the methods and parameters in the prior art, shortening the incubation time to 3-6 minutes can still ensure the accuracy of the test results, and it is convenient to control the time required for the overall detection.

[0030] In a preferred embodiment, the first diluent contains a surfactant; preferably, the surfactant includes one or more of casein sodium salt, polyethylene glycol or dextran sulfate sodium salt.

[0031] The surfactant in the first diluent is a surfactant that does not cause hemolysis, thereby further ensuring the integrity of red blood cells in the detection system.

[0032] In a preferred embodiment, the first diluent contains any one or more of the following: a pH buffer, bovine serum albumin, glycine or a preservative; preferably, the pH buffer includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or Tris-HCl.

[0033] The above components are reagents commonly used in the prior art for whole blood sample detection, which can ensure the stability of the detection system and the normal progress of the detection. Those skilled in the art can also flexibly adjust the reagents based on the prior art.

[0034] In a preferred embodiment, the detection method further includes the step of correcting the concentration of the analyte: including correcting the concentration of the analyte using the hematocrit; the hematocrit detection method includes: b1) mixing the whole blood sample with the second diluent to obtain a correction diluent; b2) detecting the absorbance of the correction diluent and calculating the hematocrit (HCT value) of the correction diluent. Preferably, the hematocrit detection method is performed before a1)-a3), or after a1)-a3), or simultaneously with a1)-a3).

[0035] In the existing detection method for whole blood samples, the whole blood sample detection value is preferably converted into the plasma value of the analyte, using the HCT value (hematocrit) for conversion, and the conversion formula is: plasma value = whole blood sample detection value ÷ (1-HCT).

[0036] In a preferred embodiment, the detection diluent used in a1) is derived from the calibration diluent. After obtaining the HCT value, a buffer is added to the calibration diluent to obtain the detection diluent, or the calibration diluent is used as the detection diluent, and then a2) and a3) are performed; preferably, the osmotic pressure of the calibration diluent is 600-2600mOSm, including but not limited to 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600mOSm; preferably, the wavelength used for detecting absorbance in b2) is 540-620nm, including but not limited to 540, 550, 560, 570, 580, 590, 600, 610 or 620nm. The wavelength used for the above-mentioned detection of absorbance is more preferably 540-562nm, including but not limited to 540, 545, 550, 555, 560 or 562nm.

[0037] The HCT calculation formula is:

[0038] As described above for the HCT value, when the whole blood sample is subjected to immunological detection to achieve the quantification of the object to be detected, the HCT value can be optionally detected. The whole blood sample used in the detection of the HCT value is the same as the sample used in the immunological detection, and is a sample obtained from the same sample and the same batch of blood collection. In an optional detection method, the HCT value of the whole blood sample is first detected. Since the HCT value detection is not destructive to the sample, the immunological detection can be continued with the detection object (i.e., the correction diluent) as the substrate after the HCT value detection, and finally the HCT value is used to correct the amount of the object to be detected. The detection diluent in a1) can be the product of the correction diluent and the buffer solution. In the mixed liquid, the solute is the whole blood sample, and the solution is the first diluent obtained by mixing the correction diluent and the buffer solution; the correction diluent can also be directly used as the detection diluent, without the need to add additional buffer, and directly mixed with the immunoassay reagent. In another optional detection method, the above-mentioned immunological detection (from a1) to a3) steps) and the HCT value detection step are performed simultaneously or separately.

[0039] In the detection of the above-mentioned HCT value, the inventors found that using light with a wavelength of 540-620nm to detect the absorbance of the sample can reduce the influence of bilirubin and human hemoglobin solution in the sample on the absorbance result, thereby improving the ability of the detection method to resist bilirubin and human hemoglobin interference, and has better accuracy in detecting the HCT value of special types of whole blood samples such as jaundice and hemolysis, and has broad application prospects.

[0040] In a preferred embodiment, the second dilution further contains NaCl; preferably, the concentration of NaCl in the second dilution is 2-8wt%, including but not limited to 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt% or 8wt%; preferably, the second dilution further contains any one or more of the following: a pH buffer, an anticoagulant or a preservative; preferably, the pH buffer includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or Tris-HCl; preferably, the anticoagulant includes EDTA-2K.

[0041] In a preferred embodiment, the immunoassay reagent comprises a signal molecule-protein complex and a magnetic microsphere-protein complex, and the magnetic microsphere-protein complex comprises magnetic microspheres and a first affinity protein coated on the magnetic microspheres.

[0042] In a preferred embodiment, the signal molecule-protein complex includes a second affinity protein labeled with a luminescent marker; the second affinity protein is a protein that can specifically bind to the analyte.

[0043] In a preferred embodiment, the immunoassay reagent is added in a one-step method, and the signal molecule-protein complex and the magnetic microsphere-protein complex are mixed with the detection diluent at the same time to obtain a detection system.

[0044] In order to further control the time required for the overall immunological detection method and improve the detection efficiency, the immunoassay reagent adopts a one-step addition method, adding the signal molecule-protein complex and the magnetic microsphere-protein complex to the detection diluent in one step, and mixing to obtain the detection system. The time required for the two-step addition method (first mixing the magnetic microsphere-protein complex with the detection diluent, and then mixing the signal molecule-protein complex with the magnetic microsphere after magnetic separation) is reduced.

[0045] In a preferred embodiment, the immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

[0046] Preferably, the luminescent marker includes, but is not limited to, any one or more of the following: isoluminol and its derivatives, acridinium ester and its derivatives, or terpyridine ruthenium.

[0047] In the above-mentioned immunological detection method, technicians in this field can flexibly select detection methods known in the prior art to conduct experiments, all of which can achieve this immunological detection, realize rapid detection of the analyte in the whole blood sample, and significantly improve the accuracy of the components to be detected in the whole blood sample.

[0048] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0049] In this application, a YASN Osmo310 single sample osmometer was used to measure the osmotic pressure of the solution.

[0050] Example 1 Detection of analytes in whole blood samples

[0051] The test was performed using the Maglumi X3 biochemiluminescent immunoassay analyzer and the self-produced NT-proBNP STAT luminescent kit. The components in the kit are diluents for adjusting salt ion concentration and osmotic pressure, which are used to detect the consistency of whole blood samples and plasma samples. The test steps are as follows:

[0052] 1. Preparation of basic reagents:

[0053] Detection reagents for the substances to be detected in the sample:

[0054] 1) The sample diluent (first diluent) component uses the components in the NT-proBNP STAT luminescence kit produced by Xinye Biotechnology. The diluent component (i.e., the first diluent) includes 50mM Tris buffer, 1M glycine, 0.5wt% bovine serum albumin, 0.15wt% Proclin300 preservative, 0.05wt% casein sodium salt and 1wt% NaCl.

[0055] 2) The magnetic ball-protein complex component uses the magnetic ball component in the NT-proBNP STAT luminescence detection kit produced by Xinye Biotechnology, and the diluent component contains 0.02wt% potassium dihydrogen phosphate, 0.29wt% sodium dihydrogen phosphate, 0.5wt% bovine serum albumin, 0.15wt% Proclin300 preservative and 0.5wt% NaCl.

[0056] 3) The marker-protein complex component uses the ABEI component in the NT-proBNP STAT luminescence kit produced by Xinye Biotechnology, and the diluent component contains 50mM Tris buffer, 1M glycine, 10wt% mannitol, 0.5wt% bovine serum albumin, 0.15wt% Proclin300 preservative and 0.5wt% NaCl.

[0057] HCT detection reagents:

[0058] Components and preparation of the sample diluent (second diluent): The components include 0.2 wt % Tris buffer, 0.02 wt % EDTA-2K, 0.15 wt % Proclin 300 preservative and 8 wt % NaCl.

[0059] 2. Detection steps

[0060] Whole blood samples from 9 healthy subjects were collected for testing, and it was ensured that the samples were free of hemolysis and jaundice interference. Each sample was divided into two parts, one was a whole blood sample, and the other was a plasma sample collected after centrifugation.

[0061] The whole blood sample test for the NT-proBNP STAT project was performed using the configured reagents and the chemiluminescent immunoassay analyzer MAGLUMI X3 produced by New Industry Bio was used for the test. The test steps were as follows: add 20 μL of sample, add 50 μL of sample diluent, then add 50 μL of magnetic ball reagent, 20 μL of ABEI component, incubate for 4 minutes, wash the unbound components, add the substrate that excites the marker to emit light, and detect the light signal.

[0062] The concentration of the analyte in the whole blood sample was converted to the concentration in the plasma sample.

[0063] Plasma concentration = whole blood test concentration / (1-HCT);

[0064] HCT was detected by spectrophotometry, and the calculation formula was:

[0065]

[0066] Absolute deviation = |plasma sample test value - whole blood sample test value| / plasma sample test value.

[0067] The HCT detection steps are as follows: add 4 μL of sample and 496 μL of sample diluent (second diluent), mix well in the reaction cup, transfer to the instrument measurement room for detection after mixing, and convert the sample absorbance at the detection wavelength into HCT.

[0068] Detection of the target substance in plasma: The above plasma samples were tested using Roche immunoassay analyzer cobas e401 and supporting reagent PBNPX (REF: (240) 09315268190) to determine the value of the target substance in plasma. The detection steps were carried out according to the preset steps of the instrument and the kit.

[0069] The osmotic pressure was tested by a YASN Osmo310 single sample osmometer, where the osmotic pressure of the HCT test reagent and the sample after mixing was 2550.

[0070] 3. Experimental Group Setting

[0071] Using the basic reagent in step 1 as the control group, the osmotic pressure of the final detection system was adjusted as shown in Table 1 by adding NaCl to the first dilution solution, and the whole blood samples were tested according to the following experimental groups.

[0072] Table 1

[0073]

[0074]

[0075] 4. Test results

[0076] The collected samples were tested and the experiment was repeated three times. The mean absolute deviations of the above control group and experimental group and the plasma values ​​measured by Roche instrument and kit were calculated. The results are shown in Table 2 below:

[0077] Table 2

[0078] Group Control group 1 Control group 2 Experimental Group 1 Experimental Group 2 Average Deviation 8.63％ 24.13％ 25.08％ 13.27％ Experimental Group 3 Experimental Group 4 Experimental Group 5 Experimental Group 6 Experimental Group 7 7.44％ 9.59％ 16.97％ 8.93％ 6.93％

[0079] The results showed that the osmotic pressure of the whole blood samples tested in experimental groups 2-4, that is, the control system, was 1048-1681 mOSm. When the detection time was 4 minutes, the detection accuracy was basically the same as that of the control group 1. The deviation between the whole blood samples and the plasma values ​​was low, indicating that the reagent scheme of the high osmotic pressure reaction system of this scheme can adapt to the needs of rapid detection of whole blood samples, and the detection time can be shortened to 1-6 minutes.

[0080] Example 2 HCT detection of whole blood samples

[0081] Based on the HCT detection in Example 1, the influence of osmotic pressure in the HCT detection system was further verified.

[0082] The control base components were 0.2wt% Tris buffer, 0.02wt% EDTA-2K, and 0.15wt% Proclin300 preservative. The reagents were prepared according to the control group 4 and the experimental group in Table 3. The HCT values ​​of the whole blood samples were tested. 14 fresh whole blood samples with known HCT values ​​were collected for testing. The test results are shown in Table 4 below:

[0083] Table 3

[0084]

[0085] Table 4

[0086]

[0087]

[0088] The results showed that the control group used 4 dilution schemes to dilute whole blood and detect HCT. The deviation of the low HCT sample detection results was basically within 10%, but the deviation of the high HCT samples exceeded 10%, and even exceeded 15%, indicating that the control whole blood dilution scheme had insufficient detection linearity; while the experimental group detected whole blood samples with different levels of HCT with deviations within ±10%, indicating that the whole blood dilution scheme had better accuracy and better linearity in detecting different HCT samples.

[0089] Example 3

[0090] In the process of in vitro diagnosis, sometimes there are abnormal samples, such as jaundice, lipemia, hemolysis, etc., which cause great interference to the HCT results. This experiment simulates abnormal samples, prepares different concentrations of bilirubin and human hemoglobin interference solutions, and performs HCT value detection according to the above experimental group 8 (detection wavelength 450nm). The detection wavelength is changed to experimental group 11: 540nm, experimental group 12: 562nm, and experimental group 13: 620nm. The results are shown in Table 5 below.

[0091] Table 5 Effects of different types of interfering substances on HCT results

[0092]

[0093] The results showed that the detection light wavelength was further adjusted based on the high osmotic pressure diluent scheme. After adjusting to between 540-620nm, no significant increase was observed in the HCT of bilirubin and human hemoglobin solutions, which was basically close to the sample diluent. This shows that the detection wavelength scheme has better resistance to interference from bilirubin and human hemoglobin, and is more accurate in detecting HCT of special types of whole blood samples such as jaundice and hemolysis.

[0094] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: in the above immunological detection method, by controlling the osmotic pressure in the detection system to 1000-1700mOSm, the whole blood sample is detected in a high osmotic pressure liquid, and the detection time of the immunological detection method is controlled to be ≤10min, which shortens the detection time, meets the demand for rapid detection in practical applications, and reduces the influence of the red blood cell status on the detection results during the detection process, thereby improving the accuracy of the detection. By optimizing the detection wavelength in the HCT value, the accuracy of the overall detection is further improved.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An immunological detection method for an analyte in a whole blood sample, characterized in that: The immunological detection method comprises: a1) mixing the whole blood sample with the first diluent to obtain a test diluent; a2) mixing the detection diluent and the immunoassay reagent to obtain a detection system, and incubating the detection system to obtain an incubation complex; the osmotic pressure of the detection system is 1000-1700 mOSm; a3) performing signal detection on the incubation complex, and obtaining the concentration of the analyte according to the signal intensity; The detection time of the immunological detection method is controlled to be ≤10min.

2. The immunological detection method according to claim 1, characterized in that The first dilution solution contains NaCl; Preferably, the concentration of NaCl in the first dilution solution is 7-15wt%; Preferably, the incubation time is 1-6 min.

3. The immunological detection method according to claim 1 or 2, characterized in that: The first dilution liquid contains a surfactant; Preferably, the surfactant comprises one or more of casein sodium salt, polyethylene glycol or dextran sulfate sodium salt.

4. The immunological detection method according to any one of claims 1 to 3, characterized in that: The first diluent contains any one or more of the following: a pH buffer, bovine serum albumin, glycine or a preservative; Preferably, the pH buffer comprises one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or Tris-HCl.

5. The immunological detection method according to any one of claims 1 to 4, characterized in that: The immunological detection method further comprises the step of correcting the concentration of the analyte: comprising correcting the concentration of the analyte using the hematocrit; The method for detecting the hematocrit comprises: b1) mixing the whole blood sample with a second diluent to obtain a calibration diluent; b2) detecting the absorbance of the calibration dilution and calculating the hematocrit of the calibration dilution; Preferably, the hematocrit detection method is performed before a1)-a3), after a1)-a3), or simultaneously with a1)-a3); Preferably, the osmotic pressure of the calibration diluent is 600-2600 mOSm; Preferably, the wavelength used for detecting the absorbance in b2) is 540-620 nm.

6. The immunological detection method according to claim 5, characterized in that: The second dilution solution further contains NaCl; Preferably, the concentration of NaCl in the second dilution solution is 2-8wt%; Preferably, the second diluent further contains any one or more of the following: a pH buffer, an anticoagulant or a preservative; Preferably, the pH buffer comprises one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate or Tris-HCl.

7. The immunological detection method according to any one of claims 1 to 6, characterized in that: The immunodetection reagent comprises a signal molecule-protein complex and a magnetic microsphere-protein complex. The magnetic microsphere-protein complex comprises a magnetic microsphere and a first affinity protein coated on the magnetic microsphere.

8. The immunological detection method according to claim 7, characterized in that: The signal molecule-protein complex comprises a second affinity protein marked with a luminescent marker; the second affinity protein is a protein that can specifically bind to the object to be detected.

9. The immunological detection method according to claim 7, characterized in that: The immunoassay reagent is added in a one-step method, and the signal molecule-protein complex and the magnetic microsphere-protein complex are mixed with the detection diluent at the same time to obtain the detection system.

10. The immunological detection method according to any one of claims 1 to 8, characterized in that: The immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

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