Immunological detection method of analyte in whole blood sample

Abstract

The application provides an immunological detection method for a to-be-detected substance in a whole blood sample. The immunological detection method comprises the following steps: a1) mixing the whole blood sample with a first diluent to obtain a detection diluent; a2) mixing the detection diluent with an immunological detection reagent to obtain a detection system, and incubating the detection system to obtain an incubated complex; the osmotic pressure of the detection system is 1000-1700 mOSm; a3) detecting a signal of the incubated complex, and obtaining the concentration of the to-be-detected substance through the signal intensity; and the detection time of the immunological detection method is controlled to be less than or equal to 10 min. The method can solve the problem that the state of red blood cells affects the detection result in the whole blood detection process in the prior art, and is suitable for the field of whole blood sample detection.

Description

Technical Field

[0001] This invention relates to the field of whole blood sample testing, and more specifically, to an immunological detection method for analytes in whole blood samples. Background Technology

[0002] In the field of in vitro diagnostics, especially in environments requiring rapid testing, results must be obtained in the shortest possible time. Therefore, whole blood samples are often used instead of plasma / serum samples that require pretreatment. Direct use of whole blood samples can significantly shorten sample processing time, demanding that the testing method be not only accurate and reliable but also highly efficient and rapid. However, in practice, testing whole blood samples often faces numerous challenges.

[0003] Whole blood samples contain various cells and biomolecules, such as red blood cells, white blood cells, platelets, proteins, and nucleic acids. These components can interfere with the testing process, affecting the accuracy of the results. Ensuring both testing efficiency and accuracy in whole blood sample analysis has become a pressing issue in the field of rapid testing. To reduce interference from whole blood cells, the traditional approach is to pretreat the whole blood with a sample diluent containing surfactants. However, while conventional sample diluents can reduce interference to some extent, they still cannot effectively meet the demands of rapid testing in fast-paced scenarios. Therefore, there is an urgent need to develop a whole blood testing method that can improve both testing speed and accuracy. Summary of the Invention

[0004] The main objective of this invention is to provide an immunological detection method for analytes in whole blood samples, in order to solve the problem of slow detection speed in the prior art for whole blood samples.

[0005] To achieve the above objectives, according to a first aspect of the present invention, an immunological detection method for an analyte in a whole blood sample is provided, the immunological detection method comprising: a1) mixing the whole blood sample with a first diluent to obtain a detection diluent; a2) mixing the detection diluent with 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 being 1000-1700 mSm; a3) detecting the signal of the incubation complex and obtaining the concentration of the analyte by the signal intensity; and controlling the detection time of the immunological detection method to be ≤10 min.

[0006] Furthermore, the first diluent contains NaCl, preferably with a concentration of 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 sodium casein salt, polyethylene glycol, or sodium dextran sulfate.

[0008] Further, 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 a whole blood sample with a second diluent to obtain a corrected diluent; b2) detecting the absorbance of the corrected diluent and calculating the hematocrit of the corrected 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 corrected diluent is 600-2600 mSm; preferably, the wavelength used to detect the absorbance in b2) is 540-620 nm.

[0010] Furthermore, the second diluent also contains NaCl; preferably, the concentration of NaCl in the second diluent is 2-8 wt%; preferably, the second diluent also contains any one or more of the following: pH buffer, anticoagulant or 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, wherein 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 capable of specifically binding to the analyte.

[0013] Furthermore, the immunoassay reagent is added using a one-step method, where the signal molecule-protein complex and the magnetic microsphere-protein complex are simultaneously mixed with the detection diluent to obtain the 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 this invention, in the above-mentioned immunological detection method, by controlling the osmotic pressure in the detection system to 1000-1700 mOSm, whole blood samples are detected in a high-osmotic-pressure liquid, and the detection time of the immunological detection method is controlled to be ≤10 min. This 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 red blood cell status on the detection results during the detection process, thereby improving the accuracy of the detection. Detailed Implementation

[0016] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0017] Terminology Explanation:

[0018] Osmotic pressure is the attractive force exerted by solute particles on water molecules in a solution. This attraction causes water molecules to tend to move from areas of low concentration (such as pure water or solutions with low concentration) through a semipermeable membrane to areas of high concentration (such as solutions with high concentration), until a dynamic equilibrium is reached. In this process, the higher the concentration of solute particles, the greater the attraction to water molecules, i.e., the greater the osmotic pressure. Osmotic pressure is an important property of solutions, playing a crucial role in maintaining the stability of the internal environment of organisms, normal cell function, and various physiological processes.

[0019] As mentioned in the background section, in the field of medical testing, especially in whole blood testing, ensuring that the osmotic pressure of the testing system is maintained within the optimal physiological range for blood cells (especially red blood cells) is crucial. Red blood cells, as the main cellular component of blood, have semi-permeable outer membranes that allow specific solutes to pass through and are directly affected by the osmotic pressure of the surrounding solution. Osmotic pressure, as an important indicator of the pressure exerted by solute concentration on solvent molecules passing through a semi-permeable membrane, is essential for maintaining the morphology, function, and integrity of red blood cells. Ideally, whole blood testing should be conducted in an osmotic environment close to or equivalent to that of a 0.9% sodium chloride solution (i.e., an isotonic solution), because this concentration matches the osmotic pressure of normal whole blood, minimizing changes in red blood cell volume, morphological distortion, and even hemolysis caused by osmotic pressure differences, thus ensuring the accuracy and reliability of the test results. However, in practice, the inventors have found that due to the complexity of the test samples, the diversity of test reagents, and the uncontrollability of operating conditions, it is difficult to precisely control the osmotic pressure of the system to the optimal physiological level for red blood cells in all testing items. For example, some tests may require the addition of reagents at specific concentrations to trigger specific biochemical reactions or achieve specific detection objectives. The addition of these reagents often alters the osmotic pressure of the original solution, causing the detection system to deviate from an isotonic state, thus resulting in insufficient detection accuracy. There is an urgent need for a detection method that ensures accuracy for whole blood samples. Therefore, in this application, the inventors attempt to develop a novel detection method for whole blood samples, and based on this, propose a series of protection schemes.

[0020] In a first typical embodiment of this application, an immunological detection method for an analyte in a whole blood sample is provided. The immunological detection method includes: a1) mixing the whole blood sample with a first diluent to obtain a detection diluent; a2) mixing the detection diluent with 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 1000-1700 mSm; a3) detecting the signal of the incubation complex and obtaining the concentration of the analyte by the signal intensity; and controlling the detection time of the immunological detection method to be ≤10 min.

[0021] In the aforementioned immunological detection method, whole blood samples, a first diluent, and immunoassay reagents are mixed to obtain a detection system with high osmotic pressure. After incubating the detection system to obtain an incubation complex, the incubation complex is detected using existing immunological detection methods, thereby enabling the detection of the concentration of the analyte in the whole blood sample through signal intensity.

[0022] Ideally, whole blood testing should be performed in an osmotic environment close to that of an isotonic solution to reduce 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 this application, the inventors unexpectedly discovered that when the overall testing time is controlled to within 10 minutes, not only is the need for obtaining test results in a short time in practical applications met, but also, using the above method, the content of analytes in whole blood samples can be accurately detected in a high-osmotic-pressure system, resulting in higher accuracy compared to testing under isotonic or near-isotonic conditions.

[0023] Hyperosmolar solutions generally contain a variety of salt ions and other chemical substances. In this application, the inventors discovered that, compared to hypoosmolar solutions, hyperosmolar solutions composed of a variety of salt ions can promote the rapid binding of antigens and antibodies to the analyte, thereby increasing the rate of the immune response. The antigen-antibody reaction is mainly affected by hydrophobic interactions, van der Waals forces, electrostatic interactions, hydrogen bonds, etc. between the two. It is speculated that the hydration layer on the antigen surface, the exposure of antigen epitopes, and their binding with antibodies caused by the action of salt ions have the main influence.

[0024] It should be noted that the above-described immunological detection method is an improvement upon existing immunological detection methods for analytes in whole blood samples. For a feasible existing immunological detection method, increasing the osmotic pressure of the diluted whole blood sample and completing the detection within 10 minutes improves the stability and accuracy of the detection method by ensuring the stability of red blood cells in the detection system. Furthermore, the aforementioned osmotic pressure conditions do not affect the antigen-antibody binding, chemiluminescence, or other detection processes 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 mSm; the detection time is ≤10 min, preferably ≤8 min, or even ≤6 min.

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

[0027] The first diluent contains NaCl, which is used to achieve the main conditional effect on osmotic pressure. Optionally, before using the existing diluent for the above-mentioned immunological detection method, additional NaCl can be added to the existing diluent to obtain a 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 minutes, more preferably 4 minutes; preferably, the incubation temperature is 37°C.

[0029] Under the aforementioned incubation conditions, the analyte and immunological reagents can fully contact and bind, while the probability of erythrocytes in whole blood samples swelling, rupturing, hemolysis, or cell membrane lysis is low, thus improving detection accuracy. In particular, regarding the incubation time, the inventors found that compared to existing methods and parameters, shortening the incubation time to 3-6 minutes still ensures the accuracy of the test results and facilitates control over the overall testing time.

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

[0031] The surfactant in the first diluent is a non-hemolytic surfactant, 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-mentioned components are reagents commonly used in the prior art for whole blood sample testing, which can ensure the stability of the testing system and the normal operation of the test. 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 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 a whole blood sample with a second diluent to obtain a corrected diluent; b2) detecting the absorbance of the corrected diluent and calculating the hematocrit (HCT value) of the corrected diluent. Preferably, the hematocrit detection method is performed before a1)-a3), after a1)-a3), or simultaneously with a1)-a3).

[0035] In existing methods for testing whole blood samples, it is preferable to convert the whole blood sample test value into the plasma value of the analyte using the HCT value (hematocrit). The conversion formula is: plasma value = whole blood sample test 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 solution is added to the calibration diluent to obtain the detection diluent, or the calibration diluent is used as the detection diluent before performing a2) and a3). Preferably, the osmotic pressure of the calibration diluent is 600-2600 ms, including but not limited to 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, and 2600 ms. Preferably, the wavelength used for detecting absorbance in b2) is 540-620 nm, including but not limited to 540, 550, 560, 570, 580, 590, 600, 610, or 620 nm. More preferably, the wavelength used for detecting absorbance is 540-562 nm, including but not limited to 540, 545, 550, 555, 560, or 562 nm.

[0037] The formula for calculating HCT is:

[0038] As described above regarding the HCT value, when performing immunological testing on whole blood samples to quantify the analyte, HCT value testing can optionally also be performed. The whole blood sample used for HCT value testing is the same as the sample used for immunological testing, meaning it comes from the same sample and the same batch of blood collected. In one optional testing method, the whole blood sample is first tested for HCT value. Since HCT value testing is non-destructive to the sample, immunological testing can continue after HCT value testing using the analyte (i.e., calibration diluent) as a substrate. Finally, the amount of the analyte is corrected using the HCT value. The detection diluent in a1) can be the product of mixing calibration diluent and buffer solution, where the solute in the mixed liquid is the whole blood sample, and the solution is the first diluent obtained by mixing calibration diluent and buffer solution; alternatively, the calibration diluent can be directly used as the detection diluent without adding additional buffer solution, and directly mixed with the immunoassay reagent. In another optional testing method, the steps of immunological testing (from a1) to a3) and HCT value testing are performed simultaneously or separately.

[0039] In the above-mentioned HCT value detection, the inventors discovered 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 results, thereby improving the detection method's ability to resist bilirubin and human hemoglobin interference. It 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 diluent further contains NaCl; preferably, the concentration of NaCl in the second diluent is 2-8 wt%, including but not limited to 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, or 8 wt%; 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 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 includes a signal molecule-protein complex and a magnetic microsphere-protein complex, wherein the magnetic microsphere-protein complex includes 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 capable of specifically binding to the analyte.

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

[0044] To further control the time required for the overall immunological detection method and improve detection efficiency, the immunoassay reagent adopts a one-step sample addition method. The signal molecule-protein complex and the magnetic microsphere-protein complex are added to the detection diluent in one step, and then mixed to obtain the detection system. This reduces the time required for the two-step sample addition method (first mixing the magnetic microsphere-protein complex with the detection diluent, then magnetically separating it, and finally mixing the signal molecule-protein complex with the magnetic microspheres).

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

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

[0047] In the above-mentioned immunological detection methods, those skilled in the art can flexibly select known detection methods in the prior art to conduct experiments, all of which can achieve this immunological detection, realize the rapid detection of the analyte in whole blood samples, and significantly improve the accuracy of the analyte in whole blood samples.

[0048] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0049] In this application, the osmotic pressure of the solution was determined using a YASN Osmo310 single-sample osmoremeter.

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

[0051] The assay was performed using the Maglumi X3 chemiluminescence immunoassay analyzer from New Industries Biomedical and its proprietary NT-proBNP STAT chemiluminescence reagent kit. The kit contained diluents to adjust salt ion concentration and osmotic pressure, used to detect the consistency between whole blood and plasma samples. The assay steps are as follows:

[0052] 1. Preparation of basic reagents:

[0053] Detection reagents for analytes in samples:

[0054] 1) The sample diluent (first diluent) uses the components from the NT-proBNP STAT chemiluminescence kit produced by New Industries Biopharmaceuticals. The diluent components (i.e., the first diluent) contain 50 mM Tris buffer pair, 1 M glycine, 0.5 wt% bovine serum albumin, 0.15 wt% Proclin 300 preservative, 0.05 wt% sodium caseinate and 1 wt% NaCl.

[0055] 2) The magnetic bead-protein complex component uses the magnetic bead component from the NT-proBNP STAT luminescent detection kit produced by New Industries Biopharmaceuticals. The diluent component contains 0.02 wt% potassium dihydrogen phosphate, 0.29 wt% disodium hydrogen phosphate, 0.5 wt% bovine serum albumin, 0.15 wt% Proclin 300 preservative and 0.5 wt% NaCl.

[0056] 3) The marker-protein complex component used was the ABEI component from the NT-proBNP STAT chemiluminescence kit produced by New Industries Biopharmaceuticals. The diluent component contained 50 mM Tris buffer pair, 1 M glycine, 10 wt% mannitol, 0.5 wt% bovine serum albumin, 0.15 wt% Proclin 300 preservative and 0.5 wt% NaCl.

[0057] HCT test reagents:

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

[0059] 2. Testing Procedures

[0060] Nine whole blood samples were collected from healthy individuals for testing, ensuring that the samples were free from hemolysis and jaundice interference. Each sample was divided into two parts: one part was a whole blood sample, and the other part was a plasma sample collected after centrifugation.

[0061] Whole blood samples were tested for the NT-proBNP STAT assay using the prepared reagents. The assay was performed using a MAGLUMI X3 chemiluminescence immunoassay analyzer manufactured by New Industries Biopharmaceuticals. The assay steps were as follows: 20 μL of sample was added, followed by 50 μL of sample diluent, then 50 μL of magnetic bead reagent and 20 μL of ABEI component. The mixture was incubated for 4 minutes, unbound components were washed off, and then substrate to excite the luminescence of the marker was added. The light signal was then detected.

[0062] The concentration of the analyte in the whole blood sample is converted into the concentration in the plasma.

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

[0064] HCT is detected using spectrophotometry, and the calculation formula is as follows:

[0065]

[0066] Absolute deviation = |Plasma sample test value - Whole blood sample test value| / Plasma sample test value.

[0067] The HCT detection procedure is as follows: Add 4 μL of sample and 496 μL of sample diluent (second diluent), mix them in a reaction vessel, and then transfer them to the instrument measurement chamber for detection. The sample absorbance at the detection wavelength is converted into HCT.

[0068] Detection of analytes in plasma: The aforementioned plasma samples were tested using the Roche Cobas E401 immunoassay analyzer and the matching reagent PBNPX (REF: (240)09315268190) to determine the value of the target substance in the plasma. The detection steps were performed according to the preset steps of the instrument and the reagent kit.

[0069] Osmometry was measured using a YASN Osmo310 single-sample osmoremeter, with the osmometry of the HCT test reagent mixed with the sample being 2550.

[0070] 3. Experimental group setup

[0071] Using the basic reagents in step 1 as the control group, the osmotic pressure of the final detection system was adjusted by adding NaCl to the first diluent, as shown in Table 1 below. Whole blood samples were then 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 deviation of the plasma values ​​measured by the control group and experimental group compared with those measured by Roche instruments and kits was 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 whole blood samples tested in experimental groups 2-4, i.e., the osmotic pressure of the control system was 1048-1681 mOSm, and the detection time was 4 minutes, had a detection accuracy that was basically the same as that in control group 1. The deviation between the whole blood sample and the plasma value was low, indicating that the reagent scheme of the high osmotic pressure reaction system of this method can meet the needs of rapid detection of whole blood samples, and the detection time can be reduced to 1-6 minutes.

[0080] Example 2: Hematocrit (HCT) detection in 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 basic components were controlled to be 0.2 wt% Tris buffer pair, 0.02 wt% EDTA-2K, and 0.15 wt% Proclin 300 preservative. Reagents were prepared according to the control group 4 and experimental group in Table 3. The hematocrit (HCT) values ​​of whole blood samples were detected. Fourteen fresh whole blood samples with known HCT values ​​were collected for testing. The results are shown in Table 4 below.

[0083] Table 3

[0084]

[0085] Table 4

[0086]

[0087]

[0088] The results showed that when whole blood was diluted and HCT was detected using the control group's 4-dilution regimen, the deviation of the detection results for low HCT samples was generally within 10%, but the deviation for high HCT samples exceeded 10%, even exceeding 15%, indicating that the linearity of the control whole blood dilution regimen was insufficient. In contrast, the deviation of the experimental group in detecting whole blood samples with different levels of HCT was within ±10%, indicating that the whole blood dilution regimen had better accuracy and linearity in detecting different HCT samples.

[0089] Example 3

[0090] In in vitro diagnostics, abnormal samples, such as jaundice, lipemia, and hemolysis, can sometimes significantly interfere with HCT results. This experiment simulated these abnormal samples by preparing solutions of different concentrations of bilirubin and human hemoglobin interfering agents. HCT values ​​were then measured according to experimental group 8 (detection wavelength 450 nm). The detection wavelengths were changed to experimental groups 11 (540 nm), 12 (562 nm), and 13 (620 nm). 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 after further adjusting the detection wavelength to between 540-620 nm based on the high osmotic dilution solution, no significant increase was observed in the HCT of bilirubin and human hemoglobin solutions, which was basically close to that of the sample dilution solution. This indicates that the detection wavelength solution has better resistance to interference from bilirubin and human hemoglobin, and is more accurate in detecting HCT in special types of whole blood samples such as jaundice and hemolysis.

[0094] As can be seen from the above description, the 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 1000-1700 mOSm, whole blood samples are detected in a high-osmotic-pressure liquid, and the detection time of the immunological detection method is controlled to ≤10 min, thus shortening the detection time, meeting the needs of rapid detection in practical applications, reducing the influence of red blood cell status on the detection results during the detection process, and improving the accuracy of the detection. Furthermore, by optimizing the detection wavelength in the HCT value, the overall accuracy of the detection is further improved.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An immunological detection method for a analyte in a whole blood sample for non-diagnostic or therapeutic purposes, characterized in that, The immunological detection method includes: a1) Mix the whole blood sample with the first diluent to obtain the test diluent; a2) The detection diluent and the immunoassay reagent are mixed to obtain a detection system, and the detection system is incubated to obtain an incubation complex; the osmotic pressure of the detection system is 1000-1700 mSm; a3) The incubation complex is subjected to signal detection, and the concentration of the analyte is obtained by the signal intensity; The detection time of the immunological detection method is controlled to be ≤10 min, and the incubation time is 1-4 min; The immunoassay reagent includes a signal molecule-protein complex and a magnetic microsphere-protein complex, wherein the magnetic microsphere-protein complex includes magnetic microspheres and a first affinity protein coated on the magnetic microspheres; The first diluent contains NaCl, and the concentration of NaCl in the first diluent is 10-13 wt%. The immunological detection method further includes a step of correcting the concentration of the analyte: including correcting the concentration of the analyte using hematocrit. The method for detecting hematocrit includes: b1) Mix the whole blood sample with the second diluent to obtain a calibration diluent; b2) Detect the absorbance of the correction dilution and calculate the hematocrit of the correction dilution; The osmotic pressure of the corrective diluent is 600-2600 mSm; The second diluent also contains NaCl, and the concentration of NaCl in the second diluent is 2-8 wt%.

2. The immunological detection method according to claim 1, characterized in that, The first diluent contains a surfactant.

3. The immunological detection method according to claim 2, characterized in that, The surfactant includes one or more of sodium casein, polyethylene glycol, or sodium dextran sulfate.

4. The immunological detection method according to claim 1, characterized in that, The first diluent contains any one or more of the following: pH buffer, bovine serum albumin, glycine, or preservative.

5. The immunological detection method according to claim 4, characterized in that, The pH buffer includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or Tris-HCl.

6. The immunological detection method according to claim 1, characterized in that, The method for detecting hematocrit is performed before, after, or simultaneously with a1)-a3).

7. The immunological detection method according to claim 1, characterized in that, The wavelength used to detect absorbance in b2) is 540-620nm.

8. The immunological detection method according to claim 1, characterized in that, The second diluent also contains any one or more of the following: pH buffer, anticoagulant, or preservative.

9. The immunological detection method according to claim 8, characterized in that, The pH buffer includes one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or Tris-HCl.

10. The immunological detection method according to claim 1, characterized in that, The signal molecule-protein complex includes a second affinity protein labeled with a luminescent marker; the second affinity protein is a protein capable of specifically binding to the analyte.

11. The immunological detection method according to claim 1, characterized in that, The immunoassay reagent is added using a one-step method, in which the signal molecule-protein complex and the magnetic microsphere-protein complex are simultaneously mixed with the detection diluent to obtain the detection system.

12. The immunological detection method according to any one of claims 1-11, characterized in that, The immunological detection method is selected from any one of chemiluminescence immunoassay, enzyme-linked immunosorbent assay, or radioimmunoassay.

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