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: a) mixing a whole blood sample with a polyethylene glycol diluent to obtain a treating fluid; b) mixing the treating fluid and an immunodetection reagent, and incubating to obtain an incubated compound; wherein the immunodetection reagent comprises a signal molecule and a first affinity protein which is specifically combined with an object to be detected; and c) carrying out signal detection on the incubation compound, and obtaining the amount of the to-be-detected substance through the signal intensity. The problem of poor detection accuracy of the whole blood sample in the prior art 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 analyte in a whole blood sample. Background Art

[0002] The significant advantage of whole blood testing is that it can achieve instant sampling and analysis. However, despite the speed advantage of whole blood testing, its complex composition brings many challenges to the testing process.

[0003] Whole blood contains a variety of blood cells, including red blood cells, white blood cells, and platelets, as well as various components in plasma, such as proteins, electrolytes, and metabolites. This highly complex biological environment makes the detection process extremely susceptible to interference from a variety of factors. Among them, the agglutination of blood cells causes blood cells to clump together, affecting the full contact between the detection reagent and the substance to be tested, thereby interfering with the accuracy of the test results. In addition, the rupture of blood cells, especially the hemoglobin released after the rupture of red blood cells, may not only mask the signal of the target analyte, but may also directly participate in chemical reactions, resulting in false positive or false negative results.

[0004] In summary, the complex components of whole blood samples, especially the presence of blood cells, significantly interfere with the immune response detection process, resulting in large deviations between the test results of whole blood samples and those of serum / plasma samples, affecting the clinical application of whole blood samples. Therefore, there is an urgent need for a detection reagent suitable for whole blood samples to improve the accuracy and reliability of the test results. Summary of the Invention

[0005] The main purpose of the present invention is to provide an immunological detection method for an analyte in a whole blood sample, so as to solve the problem of poor detection accuracy of whole blood samples in the prior art.

[0006] To achieve the above objectives, according to a first aspect of the present invention, there is provided an immunological detection method for an analyte in a whole blood sample, the method comprising: a) mixing the whole blood sample with a polyethylene glycol diluent to obtain a treated solution; b) mixing and incubating the treated solution with an immunoassay reagent to obtain an incubation complex; wherein the immunoassay reagent comprises a signal molecule and a first affinity protein that specifically binds to the analyte; and c) performing signal detection on the incubation complex to obtain the amount of the analyte based on the signal intensity.

[0007] Furthermore, the polyethylene glycol diluent includes a first polyethylene glycol diluent or a second polyethylene glycol diluent, and the whole blood sample includes a first whole blood sample or a second whole blood sample. Correspondingly, a) includes a1) or a2); a1) includes: mixing the first whole blood sample with the first polyethylene glycol diluent to obtain a first treatment liquid; in the first treatment liquid, the volume of the first whole blood sample accounts for 50%-90%, and the molecular weight of the polyethylene glycol in the first polyethylene glycol diluent is 8000-20000 Da; a2) includes: mixing the second whole blood sample with the second polyethylene glycol diluent to obtain a second treatment liquid; in the second treatment liquid, the volume of the second whole blood sample accounts for 3%-20%, and the molecular weight of the polyethylene glycol in the second polyethylene glycol diluent is 800-5000 Da.

[0008] Furthermore, the mass content of polyethylene glycol in the first polyethylene glycol dilution solution is 0.05-1 wt %; the mass content of polyethylene glycol in the second polyethylene glycol dilution solution is 0.1-4 wt %.

[0009] Furthermore, the whole blood sample includes a fresh blood sample or an old blood sample.

[0010] Furthermore, the second polyethylene glycol diluent further contains dextran sulfate sodium; preferably, the concentration of dextran sulfate sodium in the polyethylene glycol diluent is 0.01-0.1 wt %; preferably, the molecular weight of dextran sulfate sodium is 7000-20000 Da.

[0011] Furthermore, the second polyethylene glycol diluent further contains casein sodium salt; preferably, the concentration of casein sodium salt in the polyethylene glycol diluent is 0.05-0.1 wt %.

[0012] Furthermore, the immunological detection method is selected from any one of chemiluminescence immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

[0013] Furthermore, the immunological detection method is a chemiluminescent immunoassay; the signal molecule includes a second affinity protein labeled with a luminescent marker; preferably, the immunoassay reagent also includes magnetic microspheres; the magnetic microspheres are coated with a first affinity protein that specifically binds to the analyte.

[0014] Furthermore, the immunological detection method of a1) includes: high-sensitivity troponin I detection; the immunological detection method of a2) includes: procalcitonin detection.

[0015] Furthermore, the polyethylene glycol diluent further contains any one or more of the following: pH buffer, NaCl, 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.

[0016] By adopting the above-mentioned immunological detection method, the present application can significantly improve the detection efficiency and accuracy of the components to be detected in whole blood samples. The use of polyethylene glycol diluent effectively reduces the interference of interfering substances in the whole blood sample on the detection accuracy, improving the accuracy while ensuring the detection efficiency. Utilizing relevant immunological detection reagents such as signal molecules in the prior art, combined with known corresponding detection methods and detection systems, it is possible to achieve high sensitivity and high specificity detection of the components to be detected in whole blood samples. DETAILED DESCRIPTION

[0017] 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 with reference to the embodiments.

[0018] Explanation of terms:

[0019] PEG: polymer formed by ring-opening polymerization of ethylene oxide;

[0020] DSS: a polyanionic derivative of dextran, formed by the esterification reaction of dextran and chlorosulfonic acid;

[0021] RLU: relative light units.

[0022] As mentioned in the background technology, whole blood testing is currently widely used in multiple scenarios. However, when directly processing whole blood samples, traditional chemiluminescence immunoassay technology often faces challenges such as unstable test results and significant deviations between the test results and serum plasma. These problems not only affect the accuracy and timeliness of diagnosis, but also limit the application potential of whole blood testing technology in more fields. Therefore, in this application, the inventors attempted to optimize the detection method of whole blood samples and proposed a series of protection schemes for this application.

[0023] In a first typical embodiment of the present application, an immunological detection method for an analyte in a whole blood sample is provided, the immunological detection method comprising: a) mixing the whole blood sample with a polyethylene glycol diluent to obtain a treated liquid; b) mixing and incubating the treated liquid with an immunodetection reagent to obtain an incubation complex; wherein the immunodetection reagent comprises a signal molecule and a first affinity protein that specifically binds to the analyte; and c) performing signal detection on the incubation complex, and obtaining the amount of the analyte based on the signal intensity.

[0024] In the above-mentioned immunological detection method, to detect the component to be detected in a whole blood sample, the whole blood sample is first mixed with a diluent containing polyethylene glycol to obtain a treated solution. The treated solution is then mixed and incubated with an immunoassay reagent, wherein the immunoassay reagent includes a signal molecule and a first affinity protein that can specifically bind to the analyte. The first affinity protein binds to the component to be detected in the whole blood sample, and the amount of the component to be detected is determined by the signal molecule reaction. After incubation, the incubated complex containing the component to be detected and the signal molecule is subjected to signal detection. The amount of the analyte is determined based on the signal intensity, thereby determining the content of the component to be detected in the whole blood sample.

[0025] In the immunological detection method of the present application, those skilled in the art can flexibly select any immunological detection method known in the prior art, including but not limited to enzyme-linked immunosorbent assay (ELISA); immunofluorescence technique (IFA), radioimmunoassay (RIA) and other methods.

[0026] In a preferred embodiment, the polyethylene glycol diluent includes a first polyethylene glycol diluent or a second polyethylene glycol diluent, and the whole blood sample includes a first whole blood sample or a second whole blood sample, and correspondingly, a) includes a1) or a2); a1) includes: mixing the first whole blood sample with the first polyethylene glycol diluent to obtain a first processing solution; in the first processing solution, the volume proportion of the first whole blood sample is 50%-90% (including but not limited to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90%), and the molecular weight of the polyethylene glycol in the first polyethylene glycol diluent is 8000-20000 Da (including but not limited to 8000 Da, 9000 Da, 10000 Da, 11000 Da, 12000 Da, 13000 Da, 14000 Da, 15000 Da). , 16000Da, 17000Da, 18000Da, 19000Da or 20000Da); a2) comprising: mixing a second whole blood sample with a second polyethylene glycol diluent to obtain a second treatment solution; in the second treatment solution, the volume proportion of the second whole blood sample is 3%-20% (including but not limited to 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%), and the molecular weight of the polyethylene glycol in the second polyethylene glycol diluent is 800-5000Da (including but not limited to 800Da, 1000Da, 1500Da, 200Da, 2500Da, 3000Da, 3500Da, 4000Da, 4500Da or 5000Da).

[0027] In the above-mentioned immunological detection method, different pre-detection treatments are preferably performed for projects with large sample volumes (the sample volume of the whole blood sample exceeds 50% of the reaction system) and for projects with small sample volumes (the sample volume of the whole blood sample is less than 20% of the reaction system).

[0028] In a preferred embodiment, the immunological detection method of a1) includes but is not limited to: high-sensitivity troponin I detection; the immunological detection method of a2) includes but is not limited to: procalcitonin detection.

[0029] For projects with large sample volumes (including but not limited to high-sensitivity troponin I (hs-CtnI) testing), a diluent containing high-molecular-weight (8,000-20,000 Da) polyethylene glycol is added to the first whole blood sample. The large-molecule PEG, through its strong hydrophilicity and steric hindrance effect, can effectively promote the rapid sedimentation of blood cells in the reaction system, thereby reducing direct contact between blood cells and detection reagents, significantly reducing the interference of blood cell aggregation, rupture, and nonspecific adhesion on the immune response, and improving the consistency of whole blood and plasma testing.

[0030] For projects with small sample volumes (including but not limited to procalcitonin (PCT) testing), a diluent containing low-molecular-weight (800-5000Da) polyethylene glycol is added to the second whole blood sample. While ensuring moderate dispersion of blood cells, the small-molecule PEG will not significantly affect the sedimentation of blood cells, thereby avoiding a decrease in detection sensitivity caused by excessive sedimentation of blood cells, thereby maintaining the accuracy of the test results.

[0031] The terms "first whole blood sample" and "second whole blood sample" in this application are used solely to distinguish between whole blood samples used in large-volume and small-volume projects. These "first whole blood sample" and "second whole blood sample" include, but are not limited to, blood samples from the same individual or different individuals, or whole blood samples collected from the same individual from the same or different batches. The "first whole blood sample" and "second whole blood sample" are each independently selected from fresh blood samples or aged blood samples.

[0032] The "affinity protein" in this application refers to a protein that can specifically bind to the analyte in immunoassay, including antigens, antibodies, binding proteins, etc.

[0033] In a preferred embodiment, the mass content of polyethylene glycol in the first polyethylene glycol diluent is 0.05%-1%; the mass content of polyethylene glycol in the second polyethylene glycol diluent is 0.1%-4%.

[0034] In a preferred embodiment, the whole blood sample includes a fresh blood sample or an old blood sample.

[0035] Fresh blood samples (referred to as fresh samples) refer to whole blood samples that have been stored for no more than eight hours after collection. Aged blood samples (testing aged blood samples) refer to whole blood samples that have been stored at 2-8°C for more than five days. The above-mentioned immunological test method can rapidly test both fresh and aged blood samples, providing accurate and reliable results.

[0036] The above-mentioned immunological detection method can achieve accurate detection of old blood samples, which is convenient for retrospective research, forensic identification, blood storage quality testing, drug research, genetic research and other fields to achieve accurate detection of old blood samples, avoiding the limitations of detection caused by the need to use fresh blood samples for testing.

[0037] In a preferred embodiment, the second polyethylene glycol diluent further contains dextran sulfate sodium; preferably, the concentration of dextran sulfate sodium in the polyethylene glycol diluent is 0.01-0.1 wt %; preferably, the molecular weight of dextran sulfate sodium is 7000-20000 Da.

[0038] In the diluent, further adding dextran sulfate sodium (DSS) on the basis of polyethylene glycol can improve the uniformity and repeatability of the whole blood sample detection. The inventor speculates that the reason may be due to the presence of a large number of sialic acid molecules on the cell surface, which expose sulfate groups after plasma ionization and show electronegativity, so that the red blood cell surface has a negative charge, forming mutual repulsion between red blood cells and other cells, avoiding meaningless cell aggregation and interaction in the blood circulation. The shedding of sialic acid molecules will greatly aggravate the abnormal aggregation of red blood cells. The addition of DSS supplements the surface charge of red blood cells and enhances the mutual repulsion of red blood cells in whole blood samples, thereby improving the uniformity and repeatability of the whole blood sample detection. The addition of DSS can also reduce the abnormal deviation of whole blood and plasma results in old whole blood samples.

[0039] In a preferred embodiment, the second polyethylene glycol diluent further contains casein sodium salt; preferably, the concentration of casein sodium salt in the polyethylene glycol diluent is 0.05-0.1 wt %.

[0040] In the diluent, further adding casein sodium salt on the basis of polyethylene glycol can reduce the non-specific adsorption of the marker and improve the detection repeatability.

[0041] More preferably, the polyethylene glycol diluent contains both dextran sulfate sodium and casein sodium salt, which can further improve the accuracy of detection.

[0042] In a preferred embodiment, the immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

[0043] In a preferred embodiment, the immunological detection method is a chemiluminescent immunoassay; the signal molecule includes a second affinity protein labeled with a luminescent marker.

[0044] In a preferred embodiment, the immunoassay reagent further comprises a magnetic microsphere reagent; the magnetic microspheres are coated with a first affinity protein that specifically binds to the analyte.

[0045] In a preferred embodiment, the polyethylene glycol diluent further includes, but is not limited to, any one or more of the following: a pH buffer, NaCl, 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.

[0046] In a preferred embodiment, 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 of the analyte in the whole blood sample, those skilled in the art can flexibly select the detection methods, detection reagents, detection parameters, etc. known in the prior art to conduct experiments, and can realize this immunological detection, realize the detection of the analyte in the whole blood sample, and significantly improve the detection efficiency and 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 with reference to specific embodiments.

[0049] Example 1

[0050] The test was performed using the Maglumi X3 biochemiluminescence immunoassay analyzer and the accompanying self-produced hs-CTnI luminescence kit. The sample diluent in the kit was replaced with a diluent containing polyethylene glycol (PEG). The consistency of whole blood samples and plasma samples, as well as the repeatability of the test, were tested. The test steps are as follows:

[0051] 1. Preparation of diluent:

[0052] Control group 1: The composition was a basic diluent consisting of 50 mM Tris buffer, 0.2% NaCl, 0.5% bovine serum albumin, and 0.015% Proclin 300 preservative.

[0053] Experimental group 1: PEG with MW (molecular weight, Da) = 8000 was added to the basic diluent of the control group to a final PEG concentration of 0.1 wt%;

[0054] Experimental group 2: PEG with MW = 10000 was added to the basic diluent of the control group to a final PEG concentration of 0.1 wt%;

[0055] Experimental group 3: PEG with MW = 20,000 was added to the basic diluent of the control group to a final PEG concentration of 0.1 wt%;

[0056] Experimental group 4: PEG with MW=10000 was added to the basic diluent of the control group to a final PEG concentration of 0.05 wt%.

[0057] Experimental group 5: PEG with MW=10000 was added to the basic diluent of the control group to a final PEG concentration of 0.5 wt%.

[0058] Experimental group 6: PEG with MW=10000 was added to the basic diluent of the control group to a final PEG concentration of 1 wt%.

[0059] 2. Sample preparation:

[0060] Collect one fresh whole blood sample (sample F). Each sample F was divided into two blood collection tubes. One was centrifuged to obtain plasma for later use and the hs-CTnI concentration in plasma was measured. The other was left untreated and the hs-CTnI concentration in whole blood was measured.

[0061] Another 5-day-old sample (sample S) was taken and divided into two blood collection tubes. The plasma was obtained by centrifugation according to the above steps and the other sample was directly tested without treatment.

[0062] Repeat the above steps to collect 9 sets of samples for future use.

[0063] 3. Detection Reagents

[0064] In addition to the sample diluent, other components of the hs-CTnI luminescence kit (Cat. No. 130256014M) produced by New Industry Biotechnology were selected for the detection of the above samples. The other components include:

[0065] The first reagent is a magnetic ball reagent coated with an antibody that specifically binds to hs-CTnI. The liquid component contains 0.04% potassium dihydrogen phosphate, 0.4% potassium dihydrogen phosphate, 0.8% NaCl, 0.5% bovine serum albumin, and 0.15% Proclin 300 preservative.

[0066] The second reagent: a labeling reagent for labeling another antibody of ABEI that specifically binds to hs-CTnI, comprising 70 mM Tris buffer, 0.4% NaCl, 0.5% bovine serum albumin, 1 M glycine, and 0.15% Proclin 300 preservative.

[0067] 4. Sample testing

[0068] The whole blood sample was tested using the Maglumi X3 biochemiluminescence immunoassay analyzer from Xinye Biotechnology. The steps are as follows:

[0069] The whole blood sample from step 2 was tested using the sample diluents prepared for the control and experimental groups and the first and second reagents produced by New Industry Biotechnology. 150 μL of sample was added to a reaction cup, and the following sample addition order was followed: first reagent, experimental diluent from step 1, and second reagent. After sampling, the mixture was added to the reaction cup and mixed thoroughly. After mixing, the mixture was incubated at 37°C for 14 minutes. This allowed the target analyte in the sample to form a double-antibody sandwiched ternary immune complex with the magnetic bead-coated specific antibody and the ABEI-labeled specific antibody in the first reagent. After incubation, unbound substances were removed by magnetic separation and washing. Finally, hydrogen peroxide and sodium hydroxide, substrates for the fully automated immunoassay system, were added to initiate the chemiluminescence reaction, generating a light signal. The relative light intensity (RLU) was measured by a photomultiplier tube. The above steps were repeated for the whole blood sample to obtain the RLU value, and the concentration of the target substance hs-CTnI in the sample was calculated. The test results were converted to the concentration of the target substance in plasma using HCT detection.

[0070] Plasma concentration = whole blood test concentration / (1-HCT);

[0071] HCT was detected by spectrophotometry and the calculation formula was:

[0072]

[0073] Absolute deviation = |plasma sample test value - whole blood sample test value| / plasma sample test value.

[0074] The aforementioned plasma samples were tested using Beckman Analyzer Access2 81600N and the supporting reagent hs-CTnI (REF: B52699) to determine the values ​​of the target substances in the plasma.

[0075] Based on the above test results, the absolute deviation of the whole blood sample and plasma sample tests (referred to as deviation in the table) was calculated as (plasma sample test value - whole blood sample test value) / plasma sample test value.

[0076] The test results are shown in Tables 1 and 2 below:

[0077] Table 1 hs-CTnI detection results in plasma and whole blood of sample F

[0078]

[0079] Table 2: hs-CTnI detection results in plasma and whole blood of sample S

[0080]

[0081] The test was repeated 10 times for each sample and the CV value was calculated. The results are shown in Table 3 below:

[0082] Table 3: Verification of test repeatability of samples F and S

[0083]

[0084] The results showed that when sample F was tested using the diluents from experimental groups 1-2, the deviations between the whole blood sample and the plasma values ​​were reduced to varying degrees compared to the control group, all within 15%. This indicates that the whole blood dilution method can reduce the interference of blood cells on the immune response and improve the consistency of whole blood and plasma samples in projects with large sample volumes. The average deviation of the nine samples in experimental group 5 was only 4.7%, indicating that the accuracy of whole blood testing was highest when 0.5wt% PEG with a molecular weight of 10,000 was added. For the old blood sample S, the deviation between the concentration of the analyte in the whole blood sample and the blood sample even reached 46% when tested using the diluent from the control group. The detection deviations in the experimental groups were all within 20%, indicating that the diluents of this method can greatly improve the consistency of whole blood and plasma testing in old blood samples. The average deviation of experimental group 5 was only 5.05%. Experimental group 5 was further selected for verification of repeatability data. The results showed that the repeatability of whole blood samples tested using the diluents of this method was also greatly improved.

[0085] Example 2

[0086] The test was performed using the Maglumi X3 biochemiluminescence immunoassay analyzer and the accompanying self-produced PCT magnetic microparticle chemiluminescence detection kit. The diluent in the kit was replaced with polyethylene glycol with a molecular weight between 800 and 5000 to test the repeatability and accuracy of the test on old and fresh whole blood samples. The test steps are as follows:

[0087] 1. Preparation of diluent:

[0088] Control group 2: The control group is a basic diluent containing 50 mM Tris buffer, 0.2% NaCl, 0.5% bovine serum albumin, and 0.15% Proclin 300 preservative;

[0089] Experimental group 7: PEG (MW = 800) was added to the base dilution of the control group to a final PEG concentration of 0.1 wt%;

[0090] Experimental group 8: PEG (MW = 4000) was added to the basal diluent of the control group to a final PEG concentration of 0.1 wt%;

[0091] Experimental group 9: PEG (MW = 5000) was added to the control group's basal diluent to a final PEG concentration of 0.1 wt%;

[0092] Experimental group 10: PEG (MW = 4000) was added to the basal diluent of the control group to a final PEG concentration of 0.5 wt%;

[0093] Experimental group 11: PEG (MW = 4000) was added to the control group's basal dilution to a final PEG concentration of 2 wt%;

[0094] Experimental group 12: PEG with MW=4000 was added to the basic diluent of the control group to a final PEG concentration of 4 wt%.

[0095] 2. Sample preparation:

[0096] Collect one fresh whole blood sample (sample F) and divide each sample F into two blood collection tubes. Centrifuge one sample (3000 rpm, 5 min) to obtain plasma for later use and test the PCT concentration in plasma. The other sample remains untreated and is used to test the PCT concentration in whole blood.

[0097] Another 5-day-old sample (sample S) was taken and divided into two blood collection tubes. The plasma was obtained by centrifugation according to the above steps and the other sample was directly tested without treatment.

[0098] Repeat the above steps to collect 9 sets of samples for future use.

[0099] 3. Detection Reagents

[0100] In addition to the sample diluent, other components of the PCT luminescence kit produced by Xinye Biotechnology were selected to detect the above samples. The other components include:

[0101] The first reagent is a magnetic ball reagent coated with an antibody that specifically binds to PCT. The liquid component contains 0.04% potassium dihydrogen phosphate, 0.4% potassium dihydrogen phosphate, 0.8% NaCl, 0.5% bovine serum albumin, and 0.15% Proclin 300 preservative.

[0102] The second reagent: a labeling reagent for labeling another strain of ABEI antibody that specifically binds to PCT, comprising 70 mM Tris buffer, 0.4% NaCl, 0.5% bovine serum albumin, and 0.15% Proclin 300 preservative.

[0103] 4. Sample testing

[0104] Samples were tested using the immunosandwich assay. A 20 μL whole blood sample was added to a reaction cup. Reagents were sampled in the following order: first reagent, experimental group diluent from step 1, and second reagent. The sample was then added to the reaction cup and mixed thoroughly. After mixing, the mixture was incubated at 37°C for 4 minutes. This allowed the target analyte in the sample to form a double-antibody sandwich immune complex with the magnetic bead-coated specific antibody and the ABEI-labeled specific antibody in the first reagent. After incubation, unbound material was removed by magnetic separation and washing. Finally, hydrogen peroxide and sodium hydroxide, substrates used in the automated immunoassay system, were added to initiate the chemiluminescence reaction, generating a light signal. The relative light intensity (RLU) was measured using a photomultiplier tube. Repeating these steps for each sample tested yielded the RLU value, which was then used to calculate the concentration of the target substance, PCT, in the sample.

[0105] The concentration value of the target substance PCT in the sample is calculated, and the test results are converted into the concentration value of the target substance in plasma through HCT detection.

[0106] Plasma concentration = whole blood test concentration / (1-HCT);

[0107] HCT was detected by spectrophotometry and the calculation formula was:

[0108]

[0109] Absolute deviation = |plasma sample test value - whole blood sample test value| / plasma sample test value.

[0110] 5. Use Roche immunoassay analyzer and supporting reagents to test the above plasma to determine the value of the target substance in the plasma.

[0111] The test results are shown in Tables 4 and 5 below:

[0112] Table 4 PCT test results in plasma and whole blood of sample F

[0113]

[0114] Table 5 PCT test results in plasma and whole blood of sample S

[0115]

[0116] The control group 2 and the experimental group 11 were selected, and the test was repeated 10 times for each whole blood sample and the CV value was calculated. The results are shown in Table 6 below:

[0117] Table 6 Verification of test repeatability of samples F and S

[0118]

[0119]

[0120] The results showed that for the PCT project with a sample volume of 20 μL, when the sample F was tested with the sample diluent of the experimental group 7-12, the deviation between the whole blood sample and the plasma value was significantly lower than that of the control group 2. Among them, the deviations of the experimental groups 8 and 10-12 were both within 5%, indicating that the whole blood dilution after this scheme can reduce the interference of blood cells on the immune response and improve the consistency of the whole blood sample and plasma sample for projects with small sample volumes. For the old blood sample S, the control group diluent was used for testing, and the analyte in the whole blood sample was The maximum concentration deviation of the concentration and blood sample reached 22.61%; the detection deviations of experimental groups 7-12 were all within 17%, and the deviations of experimental groups 8, 10-12 were all within 10%. Among them, the deviation of experimental group 11 was only 4.6%, indicating that the diluent of this scheme can greatly improve the consistency of whole blood and plasma detection in old blood samples, and further verify the repeatability of sample detection in experimental group 11. From the test repeatability data, the repeatability of whole blood samples detected using the diluent of this scheme is also greatly improved.

[0121] Example 3

[0122] Based on Example 2, DSS and / or casein sodium salt were further added to the reagent to verify the repeatability of whole blood sample detection.

[0123] 1. Preparation of diluent:

[0124] Control group: The basic dilution of control group 2 in the embodiment was used as the control.

[0125] Experimental group 13: Based on the dilution of experimental group 11, a dilution containing 0.05 wt% casein sodium salt was prepared for use;

[0126] Experimental group 14: Based on the dilution of experimental group 11, a dilution containing 0.1 wt% casein sodium salt was prepared for use;

[0127] Experimental group 15: Based on the dilution of experimental group 11, a dilution containing 1 wt% casein sodium salt was prepared for use;

[0128] Experimental group 16: Based on the dilution of experimental group 11, a dilution containing 0.05 wt% DSS was prepared for use;

[0129] Experimental group 17: Based on the dilution of experimental group 11, a 0.05 wt% MW = 10000 DSS dilution was prepared for use;

[0130] Experimental group 18: Based on the dilution of experimental group 11, a 0.05 wt% DSS dilution with MW = 20,000 was prepared for use;

[0131] Experimental group 19: Based on the dilution of experimental group 11, a 0.01 wt% DSS dilution with MW = 10000 was prepared for use;

[0132] Experimental group 20: Based on the dilution of experimental group 11, a 0.1 wt% DSS dilution with MW = 10000 was prepared for use;

[0133] Experimental group 21: Based on the dilution of experimental group 11, 0.1 wt% casein sodium salt and 0.01 wt% DSS dilution with MW=10000 were prepared for use;

[0134] Experimental Group 22: Based on the dilution of Experimental Group 11, a dilution of 1 wt% casein sodium salt and 0.1 wt% DSS (MW = 10,000) was prepared for use;

[0135] The experimental steps in Example 2 were repeated to test the repeatability. The test results are shown in Table 7 below:

[0136] Table 7:

[0137]

[0138] The results in Table 7 show that the repeatability of samples F and S tested using dilutions 11-21 in the experimental groups improved to varying degrees compared to control group 2, with CV values ​​remaining within 5%. When both DSS and sodium casein were added, the CV for fresh whole blood samples was within 2%, and the CV for aged blood samples was within 3%. This suggests that the addition of DSS and / or sodium casein to the sample diluents can further improve the accuracy of whole blood sample testing.

[0139] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the detection method developed in this application can significantly improve the detection efficiency and accuracy of the components to be detected in the whole blood sample.

[0140] In response to the problems of inaccurate results and large deviations from plasma in the reagents of existing chemiluminescent immunoassay products when testing whole blood samples, this solution provides an immunological detection method for the analyte in whole blood samples. It not only solves the key problem of the accuracy of whole blood test results, but also can achieve accurate detection of old samples, further assisting various application scenarios in the field of medical testing. Among them, the use of polyethylene glycol diluent effectively reduces interfering substances in whole blood samples, improving detection accuracy and repeatability. The introduction of signal molecules such as chemiluminescent markers, combined with the chemiluminescent detection system, achieves high sensitivity and high specificity detection. In addition, this method is simple to operate and suitable for automated detection, greatly improving the convenience and practical application value of detection, especially in clinical diagnosis and disease monitoring. For example, in the diagnosis of cardiovascular diseases, tumors, infectious diseases, etc., this method can quickly and accurately detect markers in whole blood samples, providing important basis for emergency medical treatment, rapid disease assessment, etc. In this application, the system is further optimized for different sample addition amounts, and polyethylene glycol of different molecular weights is selected to be mixed with whole blood samples, which can improve the accuracy and repeatability of detection.

[0141] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection 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: a) mixing the whole blood sample with a polyethylene glycol diluent to obtain a treatment solution; b) mixing and incubating the treatment solution and the immunodetection reagent to obtain an incubation complex; Wherein, the immunoassay reagent comprises a signal molecule and a first affinity protein that specifically binds to the object to be detected; c) performing signal detection on the incubation complex and obtaining the amount of the analyte according to the signal intensity.

2. The immunological detection method according to claim 1, characterized in that: The polyethylene glycol diluent includes a first polyethylene glycol diluent or a second polyethylene glycol diluent, the whole blood sample includes a first whole blood sample or a second whole blood sample, and correspondingly, a) includes a1) or a2); The a1) includes: mixing the first whole blood sample with a first polyethylene glycol diluent to obtain a first processing solution; In the first treatment solution, the volume proportion of the first whole blood sample is 50%-90%, and the molecular weight of the polyethylene glycol in the first polyethylene glycol diluent is 8000-20000Da; The a2) includes: mixing the second whole blood sample with a second polyethylene glycol diluent to obtain a second processing solution; In the second processing solution, the volume proportion of the second whole blood sample is 3%-20%, and the molecular weight of the polyethylene glycol in the second polyethylene glycol diluent is 800-5000 Da.

3. The immunological detection method according to claim 2, characterized in that: The mass content of polyethylene glycol in the first polyethylene glycol diluent is 0.05-1wt%; The mass content of polyethylene glycol in the second polyethylene glycol diluent is 0.1-4wt%.

4. The immunological detection method according to claim 1, characterized in that: The whole blood sample includes a fresh blood sample or an old blood sample.

5. The immunological detection method according to claim 2, characterized in that: The second polyethylene glycol diluent further contains dextran sulfate sodium; Preferably, in the polyethylene glycol diluent, the concentration of dextran sulfate sodium is 0.01-0.1wt%; Preferably, the molecular weight of the dextran sulfate sodium is 7000-20000 Da.

6. The immunological detection method according to claim 2, characterized in that: The second polyethylene glycol diluent further contains casein sodium salt; Preferably, in the polyethylene glycol diluent, the concentration of the casein sodium salt is 0.05-0.1 wt %.

7. The immunological detection method according to any one of claims 1 to 6, characterized in that: The immunological detection method is selected from any one of chemiluminescent immunoassay, enzyme-linked immunosorbent assay or radioimmunoassay.

8. The immunological detection method according to claim 7, characterized in that: The immunological detection method is the chemiluminescent immunoassay; The signal molecule includes a second affinity protein labeled with a luminescent marker; Preferably, the immunodetection reagent further comprises magnetic microspheres; The magnetic microspheres are coated with the first affinity protein that specifically binds to the object to be detected.

9. The immunological detection method according to claim 2, characterized in that: The immunological detection method of a1) includes: high-sensitivity troponin I detection; The immunological detection method of a2) includes: procalcitonin detection.

10. The immunological detection method according to any one of claims 1 to 9, characterized in that: The polyethylene glycol diluent further contains any one or more of the following: pH buffer, NaCl, 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.

Images