CN118425131A - Method for reducing blood cell interference in whole blood detection - Google Patents

Abstract

The present invention discloses a method for reducing the interference of blood cells in whole blood detection, comprising the following steps: S0: aspirating a whole blood sample into a hematocrit pool, then aspirating a whole blood treatment agent and adding it into the hematocrit pool, and measuring the hematocrit (HCT%) of the sample to be tested; S1: adding corresponding R1\R2\M reagents to mix them; S2: washing the reacted magnetic beads in a magnetic field with a cleaning solution, removing excess reagents and unbound substances, and obtaining washed magnetic beads; S3: adding the washed magnetic beads into a luminescent substrate solution, and detecting the luminescent signal value; in the present invention, anti-erythrocyte antibodies can effectively reduce the interference factors of the sample. In addition, the addition of a respiratory inhibitor can significantly inhibit the respiration of blood cells; so that the interference of blood cells on the immune response is greatly reduced, so that the detection results of the whole blood sample have a better correlation with the detection results of the serum or plasma sample.

Description

A method for reducing blood cell interference in whole blood testing

Technical Field

The invention belongs to the technical field of chemiluminescence detection, and specifically relates to a method for reducing blood cell interference in whole blood detection.

Background technique

Chemiluminescent immunoassay is a highly sensitive detection technology that is widely used in clinical diagnosis, especially in the detection of inflammatory markers, myocardial injury markers, etc. However, traditional chemiluminescent immunoassays mainly rely on serum or plasma samples. The acquisition of these samples requires the separation of blood cells from whole blood samples through a centrifugation process. This process is not only time-consuming, but also requires a large amount of blood sample. In addition, the centrifugation process may be affected by factors such as equipment and operator skills, resulting in inconsistencies in sample processing, which in turn affects the accuracy and repeatability of the test results.

As an alternative, whole blood testing is more likely to be chosen clinically because of its rapid and simple characteristics. Whole blood testing does not require centrifugation, which can reduce the number of operation steps and time, and is especially suitable for rapid diagnosis of emergency and critically ill patients. However, the complexity of whole blood samples brings new challenges to immunoassays. The presence of blood cells may interfere with the immune response. For example, blood cells may adsorb target molecules and hinder the binding of biorecognition molecules to the analyte, or the electrons and free radicals produced by the aerobic respiration of blood cells may affect the affinity between the biorecognition molecules and the analyte, or even destroy the structure of the biomolecules, resulting in inaccurate test results.

Summary of the invention

In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method for reducing the interference of blood cells in whole blood testing, which effectively solves the problem of interference of blood cells in whole blood testing.

To achieve the above object, the present invention provides the following technical solution: a method for reducing blood cell interference in whole blood detection, comprising the following steps:

S0: aspirate the whole blood sample into the hematocrit pool, then aspirate the whole blood treatment agent and add it into the hematocrit pool to measure the hematocrit (HCT%) of the sample to be tested;

S1: Add the corresponding R1\R2\M reagents and mix them;

S2: washing the magnetic beads after the reaction with a washing solution in a magnetic field to remove excess reagents and unbound substances to obtain washed magnetic beads;

S3: Add the washed magnetic beads to the luminescent substrate solution for chemiluminescent detection.

Preferably, the whole blood treatment agent comprises anti-erythrocyte antibodies, respiratory inhibitors and cosolvents.

Preferably, the anti-red blood cell antibody (P-RBC) content is 0.1-0.5 mg/ml.

Preferably, the respiratory inhibitor comprises rotenone, and the content of rotenone is 15 ml/L.

Preferably, the co-solvent comprises acetamide, and the acetamide content is 0.5 ml/L.

Preferably, the R1 reagent is a 50 mM Tris buffer containing 1% BSA and a pH of 7.2.

Preferably, the preparation method of the R2 reagent is: add 120 μl of 0.05-0.5M Na2CO3-NAHCO3 buffer with a pH of 8.0-10.0 into a centrifuge tube, then add 0.2 mg of NT-proBNP monoclonal antibody, and mix; then add 5 μl of 6.5 mmol/l SM-DMSO solution, react for 1 hour in the dark, add AP enzyme and NT-proBNP monoclonal antibody in a mass ratio of 1.5:1, shake and mix, and react at room temperature for 1 hour; then add S-NHS and EDC in a mass ratio of 0.5 times the amount of NT-proBNP monoclonal antibody, and react for 25 minutes; after centrifugation and purification for 3 times, recover the antibody with 50 mm Tris-Hcl buffer with a pH of 8.0; use enzyme label diluent to prepare R2 reagent containing 0.5 mg/ml enzyme-labeled antibody and a dilution of 1:200.

Preferably, the M reagent is a diluent of magnetic microparticles coated with biotin-conjugated antibodies.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention adopts chemiluminescence technology and the signal amplification effect of biotin-streptavidin, which greatly improves the sensitivity and obtains a wider linear range, thus solving the problem of sensitivity.

(2) This invention provides a blood cell processing agent to solve the interference of blood cells in whole blood testing.

(3) A hematocrit measurement module is provided, which adds a HCT% measurement coefficient to make the whole blood measurement result more accurate.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

The present invention provides a method for reducing the interference of blood cells in chemiluminescence detection. The method can be directly used for whole blood testing without centrifugation to separate blood cells, thereby simplifying the operation process and improving the detection speed. It is suitable for the rapid diagnosis needs of emergency and critically ill patients. The specific steps are as follows:

S0: aspirate the whole blood sample into the hematocrit pool, then aspirate the whole blood treatment agent and add it into the hematocrit pool to measure the hematocrit (HCT%) of the sample to be tested;

S1: Add the corresponding R1\R2\M reagents to mix. In this step, the antigen-antibody complex signal value generated by the combination of R2 and M reagents with the antigen in the whole blood sample is strong and sensitive, which helps to improve the accuracy of the test. The whole blood treatment agent processes the blood cells in the whole blood sample to reduce the subsequent non-specific binding with the reagents.

S2: The magnetic beads after the reaction are washed with a washing solution in a magnetic field to remove excess reagents and unbound substances to obtain washed magnetic beads.

S3: Add the washed magnetic beads to the luminescent substrate solution for chemiluminescent detection. The luminescent substrate solution reacts with the specific reaction products on the magnetic beads to generate detectable light signals, thereby obtaining the detection results.

Specific Example 1 is the preparation and detection method of the chemiluminescent kit for detecting NT-proBNP, which is as follows:

NT-proBNP chemiluminescence kit

(1) Preparation of NT-proBNP magnetic microparticle reagent: The specific preparation process is as follows: Mix the streptavidin magnetic beads for 30 minutes to ensure that the magnetic beads are evenly dispersed, and then take 0.6-1.8 ml of magnetic beads into a new centrifuge tube. Then, after adsorbing the magnetic beads with a magnet, remove the supernatant, and then rinse the magnetic beads with PBS (pH 6.0) buffer. Repeat 3 times. Then add biotin-labeled anti-NT-proBNP monoclonal antibody, incubate at 25°C for 0.5h, and then add 0.3-0.9 ml of blocking solution to block for 0.5h. After removing the magnetic beads, remove the supernatant and wash them 3 times with PBS (pH6.0) buffer. After washing, resuspend them with magnetic microparticle washing solution and store them at a fixed volume.

(2) Preparation of alkaline phosphatase-labeled NT-proBNP monoclonal antibody

Add 120 μl of 0.05-0.5 M Na2CO3-NaHCO3 buffer solution with a pH of 8.0-10.0 into a centrifuge tube, then add 0.2 mg of NT-proBNP monoclonal antibody and mix well; then add 5 μl of 6.5 mmol/l SM-DMSO solution, react for 1 h in the dark, add AP enzyme and NT-proBNP monoclonal antibody at a mass ratio of 1.5:1, shake and mix well, and react at room temperature for 1 h.

Then add S-NHS and EDC with a mass ratio of 0.5 times the amount of NT-proBNP monoclonal antibody, and react for 25 minutes; after centrifugation and purification for 3 times, use 50mm Tris-Hcl buffer with a pH value of 8.0 to recover the antibody. Use enzyme labeling diluent to prepare R2 reagent containing 0.5mg/ml enzyme labeling antibody and a dilution of 1:200.

(3) Preparation of NT-proBNP calibrators: Pure NT-proBNP was diluted to concentrations of 0, 20, 200, 1000, 5000, and 20000 pg/ml using a calibrator buffer containing 20% whole blood. The calibrator buffer was 10 mM PBS containing 1% BSA, pH 7.4.

(4) Detection Take 50 μl of whole blood treatment agent and 50 μl of calibration material and mix them evenly, then add 50 μl of magnetic microparticle suspension coupled with NT-proBNP antibody (M reagent), 50 μl of NT-proBNP antibody labeled with alkaline phosphatase (R2 reagent), and 50 μl of R1 buffer solution to the reaction tube in sequence, shake and mix evenly, incubate at 37°C for 5 to 10 minutes, separate, wash, and test in an automatic luminometer, and obtain the corresponding results based on the calibrated standard curve; the sample detection method is the same as the preparation of the standard curve. The buffer solution is: a tris buffer solution with a concentration of 50 mM and containing 1% BSA, and a pH value of 7.2.

(5) The above-mentioned cleaning buffer is a PBS buffer with a pH of 7.0 and a concentration of 5 mmol/l.

The method for eliminating blood cell interference in magnetic microparticle chemiluminescence detection provided by the present invention is to add a whole blood treatment agent to the whole blood sample to be tested, then add R1/R2/M reagents to mix, and after incubation, use a cleaning solution to clean the remaining unbound components, and finally add the washed magnetic beads bound to the antigen-antibody complex to the luminescent substrate solution to perform a test reaction. The improved detection method fully prevents the interference of non-plasma substances in whole blood on the test results.

In the whole blood sample processing process of the present invention, the anti-erythrocyte antibody can effectively bind to the antibody site on the surface of the blood cells, effectively preventing the direct contact between the blood cells and other biological recognition molecules and the target object to be detected, thereby reducing the interference factors of the sample. In addition, the addition of a respiratory inhibitor can significantly inhibit the respiration of blood cells. This effect not only reduces the generation of electrons and free radicals, but also further protects the structural integrity of the biological recognition molecules and the object to be detected, ensuring their biological activity during the detection process. Through this unique processing method, the interference of blood cells on the immune response is greatly reduced, so that the test results of the whole blood sample have a better correlation with the test results of the serum or plasma sample. The improvement of this correlation undoubtedly enhances the reliability and accuracy of the test results.

Therefore, the test results of whole blood samples treated with this whole blood treatment agent have a good correlation with the test results of serum or plasma samples, providing more reliable data support for clinical diagnosis and scientific research. The development and application of this whole blood treatment agent undoubtedly provides an efficient and reliable new method for the treatment of whole blood samples.

It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

1. A method for reducing blood cell interference in whole blood detection, characterized in that it comprises the following steps: S0: aspirate the whole blood sample into the hematocrit pool, then aspirate the whole blood treatment agent and add it into the hematocrit pool to measure the hematocrit (HCT%) of the sample to be tested; S1: Add the corresponding R1\R2\M reagents and mix them; S2: washing the magnetic beads after the reaction with a washing solution in a magnetic field to remove excess reagents and unbound substances to obtain washed magnetic beads; S3: Add the washed magnetic beads to the luminescent substrate solution to detect the luminescent signal value. 2. A method for reducing blood cell interference in whole blood detection according to claim 1, characterized in that the whole blood treatment agent includes anti-red blood cell antibodies, respiratory inhibitors and cosolvents. 3. A method for reducing blood cell interference in whole blood detection according to claim 2, characterized in that the anti-red blood cell antibody (P-RBC) content is 0.1-0.5 mg/ml. 4. A method for reducing blood cell interference in whole blood detection according to claim 2, characterized in that the respiratory inhibitor comprises rotenone, and the content of rotenone is 15 ml/L. 5. A method for reducing blood cell interference in whole blood detection according to claim 2, characterized in that the co-solvent comprises acetamide, and the acetamide content is 0.5 ml/L. 6. A method for reducing blood cell interference in whole blood detection according to claim 1, characterized in that the R1 reagent is a tris buffer solution with a concentration of 50 mM and containing 1% BSA, and a pH value of 7.2. 7. A method for reducing blood cell interference in whole blood detection according to claim 1, characterized in that the preparation method of the R2 reagent is: add 120μl of 0.05-0.5M Na2CO3-NAHCO3 buffer with a pH value of 8.0-10.0 into a centrifuge tube, then add 0.2mg of NT-proBNP monoclonal antibody, and mix; then add 5μl 6.5mmol/l SM-DMSO solution, react for 1h in the dark; add AP enzyme and NT-proBNP monoclonal antibody in a mass ratio of 1.5:1, shake and mix, and react at room temperature for 1h; then add S-NHS and EDC with a mass ratio of 0.5 times the amount of NT-proBNP monoclonal antibody, and react for 25min; after centrifugation and purification for 3 times, use 50mm Tris-Hcl buffer with a pH value of 8.0 to recover the antibody; use enzyme label diluent to prepare R2 reagent containing 0.5mg/ml enzyme-labeled antibody and diluted 1:200. 8. A method for reducing blood cell interference in whole blood detection according to claim 1, characterized in that the M reagent is a magnetic particle diluent coated with a biotin-coupled antibody.