CN113176404A - Kit for multi-index joint inspection of whole blood sample and use method thereof - Google Patents

Kit for multi-index joint inspection of whole blood sample and use method thereof Download PDF

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CN113176404A
CN113176404A CN202110399683.2A CN202110399683A CN113176404A CN 113176404 A CN113176404 A CN 113176404A CN 202110399683 A CN202110399683 A CN 202110399683A CN 113176404 A CN113176404 A CN 113176404A
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antibody
whole blood
kit
reagent
blood sample
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徐兵
杨宝君
王昱琳
马永波
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Beijing Gaugene Biological Technology Co ltd
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Beijing Gaugene Biological Technology Co ltd
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Abstract

The application relates to the technical field of clinical immunoassay, and particularly discloses a kit for multi-index joint inspection of a whole blood sample and a using method thereof. The kit comprises a hemolytic agent for breaking red blood cells in a whole blood sample, an R1 reagent containing antibody-coated magnetic fluorescent coding microspheres, an R2 reagent containing fluorescent labeling antibody/antigen, a buffer solution and a calibrator; the magnetic fluorescent coding microspheres have coding and decoding performance; the hemolytic agent is a buffer solution containing a surfactant, and the fluorescence labeling antibody is an antibody matched with the magnetic fluorescence coding microsphere surface antibody. The kit can be used for multi-index joint inspection of whole blood samples, and has the advantages of high detection sensitivity and capability of simultaneously detecting multiple indexes by one-time immunoreaction.

Description

Kit for multi-index joint inspection of whole blood sample and use method thereof
Technical Field
The application relates to the technical field of clinical immunoassay, in particular to a kit for multi-index joint inspection of a whole blood sample and a using method thereof.
Background
In the field of clinical immunoassays, the samples used generally include whole blood samples, plasma samples, and serum samples; the final analysis purpose is realized by analyzing various targets in the sample, such as antigens, antibodies, various target substances and the like. The plasma sample and the serum sample are prepared to remove various cells (such as leukemia, red blood cells, platelets, plasma fibrin, etc.) in the whole blood. Compared with a plasma sample and a serum sample, the whole blood sample as an analysis sample has the following advantages: on one hand, the whole blood sample does not need to be prepared, so that the clinical requirement of rapid detection and analysis can be met; on the other hand, whole blood samples eliminate the risk of potential contamination and infection during the preparation process. Therefore, the clinical test is more favored to use whole blood samples for the analytical detection of the relevant target substance.
In addition, to further increase the speed of detection analysis, simultaneous detection analysis of multiple targets is often employed, namely: and realizing multi-index detection by one-time reaction. The advantages of the multi-target simultaneous detection and analysis method are as follows: the detection speed is faster than that of one-time single-item detection; in addition, for severe patients and infants, the detection of multiple index items can be realized by using less blood volume samples.
The clinical inflammation related index and the ratio index of various types of white blood cells in the whole blood of a febrile patient are important analysis indexes, and meanwhile, some related targets, such as four conventional infections, need to be detected and analyzed in a matching way to diagnose whether the patient is infected by virus or bacteria (in the case of bacterial infection, the total number of white blood cells in the conventional blood detection is usually higher than 10 x 109Per liter, the proportion of neutrophils exceeds 70 percent; when infected with a virus, the total number of leukocytes usually does not change much, but stranguria is markedThe barytes can typically exceed 30%).
While other inflammation indicators, such as C-reactive protein (CRP), are one of the acute phase reaction proteins, CRP can be increased to a medium or high degree when the bacteria are infected; due to the systemic inflammatory reaction caused by bacterial infection, the concentration of Procalcitonin (PCT) can be increased within 2-3h of the early inflammatory reaction, and the concentration of PCT is in positive correlation with the severity of infection; the time duration of the increase of the interleukin 6 content is obvious and is far longer than the time duration of the increase of the CRP and PCT contents, so the method can be used for early auxiliary diagnosis of acute infection. Serum Amyloid A (SAA) is an acute phase reaction protein produced by the liver, when a human body has acute inflammatory injury, the concentration of the SAA is obviously increased and is increased to more than 1000 times of a normal value within 4-6 hours; and no matter whether the infection is virus infection, bacterial infection or protozoan infection, the SAA can be obviously increased in a short time, and the SAA can be rapidly reduced after the inflammation is controlled, so the SAA is one of the indexes which reflect the sensitivity of human inflammation.
Wherein CRP and SAA are mainly used for testing serum or whole blood by adopting an enhanced immunoturbidimetric method; due to the requirement of sensitivity, PCT and IL-6 are basically detected by serum samples and matched with the leucocyte classification detection of whole blood samples. Therefore, it is of great significance to develop a high-sensitivity multi-index detection method for whole blood samples. Clinically, the detection of some markers needs to obtain the detection result (such as myocardial performance index) quickly in a short time so as to perform the next urgent intervention. In the case of infants and critically ill patients, it is difficult to obtain the amount of blood required for the measurement when a multi-index measurement analysis is required for such patients.
In the related art, indexes such as PCT, CRP, IL-6, SAA and the like in a serum sample can be tested by using a coding microsphere method, which uses four coding fluorescent microspheres to connect specific antibodies, and uses biotinylated corresponding antibodies as detection antibodies, but this method cannot use whole blood as a detection sample to detect the content of the related indexes therein. There are many methods for detecting and analyzing markers in a serum sample, such as an enhanced immunoturbidimetric immunoassay, a glow immunochemiluminescence method, a flash immunochemiluminescence method, an enzyme-linked immunoassay, and a homogeneous immunoassay, and the detection and analysis of the relevant markers cannot be performed by using whole blood as a detection sample. The method can not eliminate background interference caused by red blood cells and fragments thereof, and obvious false positive often appears in a detection result. The immune lateral chromatography realizes whole blood analysis by separating cells through a sedimentation pad, but the method has low sensitivity, cannot detect multiple indexes simultaneously, and cannot meet the requirements of clinical automation and sensitivity.
In addition, in the related art, there is an enzyme-linked immunosorbent assay diluent for whole blood, and a relevant marker is detected and analyzed based on the enzyme-linked immunosorbent assay diluent by an enzyme-linked immunosorbent assay method. However, this method is a solid-phase immunoassay method, and has problems of low sensitivity and false positive.
Therefore, obtaining a whole blood, multi-index and high-sensitivity detection method is of great significance.
Disclosure of Invention
In order to carry out detection and analysis on multiple indexes of a whole blood sample with high sensitivity, the application provides a kit and a detection method for the multiple-index joint detection of the whole blood sample.
In a first aspect, the present application provides a kit for multi-index joint detection of a whole blood sample, which adopts the following technical scheme:
a kit for multi-index joint detection of a whole blood sample comprises a hemolytic agent for breaking red blood cells in the whole blood sample, an R1 reagent containing antibody-coated magnetic fluorescent coding microspheres, an R2 reagent containing fluorescent labeling antibodies, a buffer solution and a calibrator;
the antibody-coated magnetic fluorescent coding microspheres are obtained by coupling antibodies of specific targets to the magnetic fluorescent coding microspheres, and the antibody-coated magnetic fluorescent coding microspheres are a mixture of multiple microspheres coated with multiple specific antibodies, namely, each microsphere with different fluorescent intensity is coated with one specific antibody; the fluorescence labeling antibody is used for labeling a fluorescence labeling substance on a pairing antibody, and the fluorescence labeling antibody is a mixture of fluorescent substances labeled by a plurality of specific antibodies; the calibrator is a composite calibrator comprising a plurality of targets; the specific antibodies are matched with the magnetic fluorescent coding microspheres; the hemolytic agent contains surfactant or salt substance for dissolving erythrocyte in blood.
When substances in whole blood are detected, each detection substance is a detection index; the kit can realize simultaneous detection and analysis of various indexes. The R1 reagent is obtained by connecting different antibodies (the antibodies can also be antigens) with different magnetic fluorescent coding microspheres. The fluorescence intensities of different magnetic fluorescence coding microspheres are different, and different detection indexes are identified according to different fluorescence intensities, so that the purpose of coding different detection indexes is achieved. That is, after different detection indexes in the whole blood sample are combined with the corresponding magnetic fluorescent encoding microspheres (based on the specific combination between antigen and antibody), the different detection indexes can be respectively encoded according to the fluorescence intensity of the magnetic fluorescent encoding microspheres in the later detection. Preparation of R2 reagent simultaneously: different antibodies or antigens are connected with the fluorescent marker, so that the content of a certain index can be detected by the fluorescence intensity at the later stage, and the quantitative analysis of the certain detection index can be realized.
In the first mode of the kit, firstly, hemolytic agents are used for crushing impurities in cells such as red blood cells and white blood cells in a whole blood sample, so that the hemolyzed whole blood sample contains different indicators and cell fragments to be detected. The antigen (the antigen is the detection index) of the corresponding index in the sample is simultaneously connected with the magnetic fluorescent coding microsphere coated by the antibody and the fluorescent labeled antibody to form a magnetic fluorescent coding microsphere-antigen-fluorescent labeled antibody compound coated by the antibody; then, the magnetic fluorescent coding microsphere-antigen-fluorescent marker antibody compound coated by the antibody is separated from cell debris by a magnetic separation technology, so that when a whole blood sample is detected, the final detection result cannot be influenced even if red blood cells, white blood cells and other cells exist in an initial sample. In the second mode, in the whole blood sample, the antigen (the antigen is the detection index) corresponding to the index is simultaneously connected with the antibody-coated magnetic fluorescent coding microsphere and the fluorescent labeled antibody to form an antibody-coated magnetic fluorescent coding microsphere-antigen-fluorescent labeled antibody compound, impurities in red blood cells, white blood cells and other cells in the whole blood sample are crushed by using a hemolytic agent, and then the antibody-coated magnetic fluorescent coding microsphere-antigen-fluorescent labeled antibody compound is separated from cell debris by using a magnetic separation technology, so that when the whole blood sample is detected, the final detection result cannot be influenced even if the red blood cells, the white blood cells and other cells exist in the initial sample. In addition, when the kit is adopted to carry out multi-index joint detection on a whole blood sample, the principle similar to the chemiluminescence principle is that magnetic microspheres react in a liquid phase, and the detection advantage that the chemiluminescence detection method has high sensitivity is achieved.
Preferably, the magnetic fluorescent coding microspheres are prepared by a method comprising the following steps:
I. preparing magnetic particle microspheres:
I-I, preparing a pre-solution: uniformly mixing cyclohexane, n-hexane and a nano ferroferric oxide toluene solution for later use;
I-II, respectively weighing magnetic particle microspheres with different particle sizes, respectively adding the pre-solution into the magnetic particle microspheres with different particle sizes, and obtaining a solution containing the magnetic particle microspheres with different particle sizes after ultrasonic dispersion and stirring;
II. Preparing magnetic coding microspheres:
II-I, preparing quantum dot solutions with different concentrations;
II-II, respectively adding the solutions containing the magnetic particle microspheres with different particle sizes into the quantum dot solutions with different concentrations, stirring at the temperature of 60-90 ℃ for 8-16 h, then rapidly cooling to room temperature, and respectively filtering, washing and drying to obtain magnetic encoding microspheres with different quantum dot concentrations and different particle sizes;
III, functional group coating
III-I, dissolving the magnetic coding microspheres with different particle sizes in absolute ethyl alcohol, performing ultrasonic dispersion, adding 3-aminopropyl triethoxysilane, and mechanically stirring for 10-14 hours; then filtering and washing to obtain the magnetic fluorescent coding microspheres wrapped with the silane layer;
III-II, dissolving the magnetic fluorescent coding microsphere coated with the silane layer in absolute ethyl alcohol, adding succinic anhydride, stirring at room temperature, filtering, washing and drying to obtain the magnetic fluorescent coding microsphere containing the carboxyl functional group.
The application selects the self-made magnetic fluorescent coding microspheres to realize that different detection indexes are respectively distributed in different areas on detection equipment, thereby realizing the effective separation of the different detection indexes; secondly, the magnetic fluorescent coding microspheres have the characteristic of magnetic adsorption, and are prepared for removing red blood cells and white blood cell fragments in the later period.
Preferably, the hemolytic agent is one of a cationic surfactant, an anionic surfactant, a nonionic surfactant, an amphoteric surfactant, or ammonium chloride.
The hemolytic agent selected in the present application may be one capable of disrupting cells in a whole blood sample, and mainly acts on a cell membrane, so that the cell membrane is broken to achieve the effect of disrupting cells.
Preferably, the hemolytic agent is a salt buffer containing a surfactant selected from any one of tween20, tween80 and triton-100.
The hemolytic agent has an excellent effect of breaking cells; the cells are broken up quickly and they do not affect the detection of the target component (i.e., target) at a later stage.
Preferably, the addition amount of the surfactant in the buffer solution is 0.05% to 0.15%.
Preferably, the hemolytic agent is phosphate buffer containing triton-100.
Further preferably, the addition amount of the triton-100 is 0.05% of the volume of the phosphate buffer solution.
Preferably, the pH of the hemolytic agent is 6-8.
Immune reaction is generally carried out under the condition close to neutrality, and strong acid and strong base are not beneficial to immune reaction and protein stability. When the hemolytic agent within the pH6-8 range is adopted to treat whole blood, the range for smooth implementation of immunoreaction can be achieved, influence on subsequent immunodetection reaction can not be caused, and therefore the pH range of the hemolytic agent can be selected to be 6-8.
Preferably, the R1 reagent is prepared by a method comprising the following steps:
placing a magnetic fluorescent-encoded microsphere and a magnetic microsphere activator in a buffer solution A to activate the magnetic fluorescent-encoded microsphere; then adding any one antibody to coat the antibody on the magnetic fluorescent coding microsphere; then adding a buffer solution B for standing treatment to prepare a solution containing one of the antibody-coated magnetic fluorescent coding microspheres;
then preparing other solutions containing the magnetic coding microspheres coated by the antibody corresponding to a certain index to be detected one by one according to the steps;
and subsequently, mixing a plurality of solutions containing the antibody-coated coding microspheres corresponding to the plurality of combined detection items together to obtain the R1 reagent.
In preparing the R1 reagent, the reagent is prepared according to the specific combined test item. Since the kit of the present application is used for multiple joint tests of whole blood samples, the number of indexes to be detected is at least two. In the R1 reagent, at least two kinds of antibody coated magnetic fluorescent coding microspheres are contained. In the specific process of preparing the R1 reagent, for example, if two joint tests of a whole blood sample are performed, two antibodies (i.e., two antibodies) corresponding to two detection indexes are required to be selected, then two magnetic fluorescent-encoded microspheres are selected arbitrarily, one antibody is coated on one of the magnetic fluorescent-encoded microspheres, the other antibody is coated on the other magnetic fluorescent-encoded microsphere, and finally the solutions of the two antibody-coated magnetic fluorescent-encoded microspheres are mixed to prepare the R1 reagent. And (3) preparing a plurality of antibody-coated magnetic fluorescent coding microspheres respectively by carrying out joint inspection on a plurality of indexes, and mixing the microspheres to obtain the R1 reagent.
Preferably, after the activated magnetic fluorescent coding microspheres and the antibody are added into the buffer solution B, the standing treatment temperature is 20-25 ℃, and the standing treatment time is 0.5-1.5 hours.
By adopting the technical scheme, the antibody can be effectively loaded on the magnetic fluorescent coding microsphere.
Preferably, the content of the antibody-coated magnetic fluorescent coding microspheres in the R1 reagent is 0.4-0.6 mg/mL.
By adopting the technical scheme, the magnetic fluorescent coding microspheres coated by the antibody can stably exist for a long time under the concentration.
Preferably, the buffer solution A is 2-morpholine ethanesulfonic acid solution.
Preferably, the buffer B is selected from any one of a BSA phosphate buffer and a casein phosphate buffer.
The R2 reagent is prepared by a method comprising the following steps: crosslinking an antibody and a fluorescent marker in a buffer solution C by using a crosslinking agent to obtain a solution containing the fluorescent marker antibody;
then preparing other solutions containing the fluorescence labeled antibody corresponding to a certain index to be detected one by one according to the steps;
and subsequently, mixing a plurality of solutions containing the fluorescence labeled antibodies corresponding to the joint detection items together to obtain the R2 reagent.
Preferably, the fluorescent marker is any one selected from Phycoerythrin (PE) and Fluorescein Isothiocyanate (FITC).
The fluorescent marker is generally common to phycoerythrin, is not limited to phycoerythrin, and can also be Fluorescein Isothiocyanate (FITC). The fluorescent marker is mainly used for carrying out fluorescent marking on the index to be detected, and then calculating the concentration value of the corresponding index to be detected according to the intensity of a fluorescent signal so as to meet the requirement of high-sensitivity detection.
Preferably, the fluorochrome is Phycoerythrin (PE).
Preferably, the buffer is a salt ion buffer with pH 6-8.
Further preferably, the buffer is phosphate buffer with pH 7.40.02mol/L.
Preferably, the concentration of the fluorescence labeled antibody in the R2 reagent is 0.3-0.8 mu g/mL.
By adopting the technical scheme, the fluorescence labeled antibody can stably exist for a long time under the concentration.
Preferably, the targets of the paired antibodies in the whole blood sample are 2-14.
Preferably, the antibodies are selected from at least two of the antibodies corresponding to procalcitonin, interleukin 6, cardiac troponin I, myoglobin, creatine kinase isozyme, N-terminal pro-B-type natriuretic peptide, lipoprotein-associated phospholipase a2, cardiac fatty acid binding protein, luteinizing hormone, progesterone, respectively.
When multi-index joint inspection in a whole blood sample is carried out, different indexes can be selected to be simultaneously detected and analyzed according to actual detection and analysis requirements. Wherein Procalcitonin (PCT) and interleukin 6(IL-6) can be detected together, and the corresponding antibody can be PCT antibody and IL-6 antibody; the combined detection of cardiac troponin I (cTnI), Myoglobin (MYO), creatine kinase isozyme (CK-MB), N-terminal B-type natriuretic peptide precursor (NT-pro-BNP), lipoprotein-associated phospholipase A2(Lp-PLA 2) and cardiac fatty acid binding protein (H-FABP) can also be carried out, and the corresponding antibodies can be cTn I antibody, MYO antibody, CK-MB antibody, NT-pro-BNP antibody, Lp-PLA 2 antibody and H-FABP antibody; luteinizing Hormone (LH) and progesterone (P) can also be tested in combination, and the corresponding antibodies can be LH antibody and P antibody.
Preferably, the targets are 5 of Proinflammin (PCT), interleukin 6(IL-6), C-reactive protein (CRP), serum amyloid a (saa), and Heparin Binding Protein (HBP) associated with inflammation.
The kit can carry out joint inspection on multiple different detection indexes, has wide application range, and can be used for simultaneous detection and analysis of multiple combined indexes.
In a second aspect, the application provides a use method of the kit for multi-index joint inspection, which adopts the following technical scheme:
the use method of the kit for the multi-index joint inspection comprises the following steps:
preparing an R1 reagent and an R2 reagent according to indexes to be detected;
taking a whole blood sample, adding the hemolytic agent for hemolysis, then adding the R1 reagent and the R2 reagent for reaction, separating, then discarding a liquid phase, and detecting a solid phase;
or the use method comprises the following steps:
preparing an R1 reagent and an R2 reagent according to indexes to be detected;
taking a whole blood sample, adding an R1 reagent and an R2 reagent, reacting, adding the hemolytic agent for hemolysis, separating, discarding a liquid phase, and detecting a solid phase. When the kit provided by the application is used for carrying out multi-index detection analysis on a whole blood sample, a flow cytometer can be finally adopted for sample detection. The reaction modes can be two. Two different reaction modes are illustrated herein by taking the example of detecting an indicator.
A first reaction mode: an antibody capable of specifically binding with an index (i.e., antigen) to be detected is labeled on the magnetic fluorescent encoding microsphere as a capture factor A. The whole blood sample is diluted with a hemolytic agent, a large number of red blood cells are disrupted, and the obtained whole blood sample containing disrupted cells is used as a sample S. Another antibody capable of specifically binding to the marker to be detected (i.e., antigen) is labeled on a fluorescent label as a tracer A'. After the three are mixed, A-S-A' forms A typical sandwich immunoassay mode. Magnetic fluorescent encoded microspheres were magnetically adsorbed and unbound a' was removed after washing. The resulting solid phase was used for final detection.
And (2) reaction mode II: an antibody capable of specifically binding with an index (i.e., antigen) to be detected is labeled on the magnetic fluorescent encoding microsphere as a capture factor A. The whole blood sample was used as the sample. Another antibody capable of specifically binding to the marker to be detected (i.e., antigen) is labeled on a fluorescent label as a tracer A'. A-S-A 'forms A typical sandwich immunoassay mode, hemolytic agent is added to destroy blood cells, magnetic fluorescent coding microspheres are adsorbed by magnetic force, and unbound A' and broken blood cells are eliminated after washing. The solid phase obtained finally is used for final detection.
And detecting reaction results of the obtained solid phases by using a flow cytometry analyzer respectively, and circling magnetic fluorescent coding microspheres on an FSC-SSC (forward scattering-side scattering) scatter diagram, and analyzing the fluorescent signal intensity of the circled different coding microspheres, namely the concentration of the corresponding detection index. Although most of the cell debris is removed by magnetic separation, some of the cell debris remains. The residual cell fragments are defined and removed by a gate when the flow cytometry is used for detection, so that broken blood cells and adhesion substances in whole blood do not influence the result of final detection analysis, and the influence factors can be eliminated by the gate.
When the multi-index detection is carried out, different indexes can be distinguished through the codes of the magnetic fluorescent coding microspheres, and the requirement of multi-index joint detection can be met. Taking the detection of two indexes as an example for explanation, two kinds of magnetic fluorescent coding microspheres are selected, namely a magnetic fluorescent coding microsphere A and a magnetic fluorescent coding microsphere B; respectively coupling two specific binding antibodies corresponding to indexes (namely antigens) to be detected to obtain two capture factors: capture factor a and capture factor B. The whole blood sample is treated with a hemolytic agent to obtain a sample S. The specific binding antibodies corresponding to two indexes (namely antigens) to be detected are respectively coupled with the fluorescent markers to be used as tracers A 'and B'. And performing incubation reaction on A/B + S + A '/B', performing magnetic adsorption washing, and performing machine detection. Due to the fact that magnetic force is adopted to adsorb the A and the B, the combined tracer antibody can be left after sandwich reaction, the unbound tracer antibody can be washed away, and after computer detection, a cluster of magnetic coding microspheres is formed on the FSC-SSC scatter diagram and is not affected by the antibody, the antigen and the tracer. After the clustering is circled, two independent clustering peaks appear on the decoding channel and respectively represent the magnetic fluorescent coding microsphere A (loaded with the index A to be detected) and the magnetic fluorescent coding microsphere B (loaded with the index B to be detected), namely, two items are decoded, the clustering A and the clustering B are respectively circled, and the respective fluorescence intensity is respectively displayed on the signal channel, so that the concentration detection analysis of two targets is realized.
The magnetic fluorescent coding microspheres have the same magnetism as magnetic particle immunochemiluminescence, so that the magnetic fluorescent coding microspheres are convenient to automatically process and wash, the contained fluorescent signals are used for realizing the coding of different items, the positions of the magnetic fluorescent coding microspheres in a fluorescent decoding channel are determined through different concentrations of the same fluorescent dye or quantum dots and the like, the decoding of double signals can also be realized through double dyes or quantum dots, and the simultaneous detection and decoding analysis of more items are realized, for example, common serum detection cell factors are generally 6 factors, 7 factors and even 14 factors.
Preferably, in S3, after the hemolytic agent is added to the whole blood sample, the amount of the hemolytic agent added is 10% to 90% of the total volume of the solution after the hemolytic agent is added.
After the hemolytic agent with the content is added into the whole blood sample, the red blood cells (and white blood cells) in the whole blood sample can be effectively crushed, so that the influence of the whole cells on the later-stage experiment result and the experiment sensitivity is avoided.
In summary, the present application has the following beneficial effects:
1. according to the kit, a hemolytic agent is used for breaking erythrocyte membranes in a whole blood sample, cell fragments are removed through a magnetic separation technology at a later stage, unbound fluorescent marker antibodies are removed through washing, a compound of magnetic fluorescent coding microspheres-antibodies-antigens-fluorescent markers is finally obtained, and then the compound is detected through a flow cytometer so as to realize high-sensitivity joint detection of multiple indexes in the whole blood sample.
Drawings
FIG. 1 is a graph of the results of flow cytometry differentiation and delineation of cell debris populations and target test populations for the combined assay of PCT and IL-6 in whole blood samples;
FIG. 2 is a graph showing the results of PCT and IL-6, respectively, as specific detection indexes distinguished by the code channel when PCT and IL-6 in a whole blood sample are analyzed by joint detection to distinguish a target test group;
FIG. 3 is a graph showing the results of flow cytometry separation and delineation of cell debris populations and target test populations for the co-detection analysis of six indices cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2, and H-FABP in whole blood samples;
FIG. 4 is a graph showing the results of the joint test analysis of cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP six indices in a whole blood sample, wherein the specific detection indices distinguished by the encoding channel when distinguishing the target test group are cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP, respectively;
FIG. 5 is a graph of the results of flow cytometry differentiation and delineation of cell debris populations and target test populations for the combined analysis of LH and P dual indices in a whole blood sample;
FIG. 6 is a diagram showing the results of the joint analysis of LH and P dual indicators in a whole blood sample, wherein the specific detection indicators distinguished by the encoding channel when distinguishing the target test group are LH and P, respectively;
FIG. 7 is a graph showing the results of flow cytometry distinguishing and delineating cell debris populations and target test populations when combined assays were performed on six indices cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2, and H-FABP in whole blood samples in comparative examples;
FIG. 8 is a graph showing the results of the combined assay of cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP in the comparative examples, wherein the specific detection indicators distinguished by the coding channel when the target test group is distinguished by the combined assay of cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP, respectively.
Detailed Description
When the relevant indexes are detected by using the fluorescent coding microspheres, the high-sensitivity joint detection of multiple indexes by using whole blood as a detection sample is difficult to realize. Therefore, when the method is used for multi-index detection, the method has the conditions of long time consumption, low detection sensitivity and false positive. In particular, when the whole blood sample is derived from an infant or a critically ill patient, it is difficult to obtain a whole blood sample necessary for sufficient detection at a time for a large number of detection indexes, and therefore, it is difficult to perform a multi-index and high-sensitivity joint detection.
Based on the background, the application provides a kit for multi-index joint detection in a whole blood sample and a using method thereof.
A kit for multi-index joint detection in a whole blood sample comprises a hemolytic agent for breaking red blood cells in the whole blood sample, an R1 reagent containing antibody-coated magnetic fluorescent coding microspheres and an R2 reagent containing fluorescent labeling antibodies;
the antibody-coated magnetic fluorescent coding microspheres are obtained by coating corresponding antibodies on the magnetic fluorescent coding microspheres, and the fluorescent labeled antibodies are obtained by labeling fluorescent markers on the corresponding antibodies; the number of antibodies is multiple, the number of magnetic fluorescent coding microspheres is multiple, one magnetic fluorescent coding microsphere is correspondingly coated with one antibody, the number of corresponding antibody-coated magnetic fluorescent coding microspheres is multiple, and the number of fluorescent labeling antibodies is multiple;
the hemolytic agent is phosphate buffer containing surfactant, and the surfactant is selected from any one of tween20, tween80 and triton-100.
The hemolytic agent selected in the present application mainly relies on a surfactant to reduce the surface tension of the buffer and change the solubilization properties of the solution; the effect of the surfactant on the cell is to change the permeability of the cell membrane; when the surfactant is added to the phosphate buffer, an excellent cell disruption effect can be finally achieved: cell disruption was efficient and complete. The hemolytic agent of the present application can also crush not only red blood cells but also cells present in whole blood such as white blood cells in a whole blood sample. And the crushing effect can meet the detection requirement in the later period.
The R1 reagent is prepared by the method comprising the following steps:
placing a magnetic fluorescent-encoded microsphere and a magnetic microsphere activator in a buffer solution A to activate the magnetic fluorescent-encoded microsphere; then adding any one antibody to coat the antibody on the magnetic fluorescent coding microsphere; then adding a buffer solution B for standing treatment to prepare the magnetic fluorescent coding microsphere coated by one of the antibodies.
Then preparing the magnetic coding microspheres coated with the antibodies corresponding to other indexes to be detected one by one according to the steps;
and then mixing a plurality of solutions containing different antibody coated coding microspheres corresponding to a plurality of combined detection items together to obtain a corresponding reagent R1.
The buffer solution A can be 2-morpholine ethanesulfonic acid buffer solution, and the concentration of the 2-morpholine ethanesulfonic acid buffer solution can be 0.05-0.15 mol/L.
The buffer B may be 1% BSA phosphate buffer, or casein phosphate buffer.
The temperature during standing treatment can be 20-25 ℃, 21-24 ℃ and 22-23 ℃; specifically, the temperature may be 20.5 ℃, 21.5 ℃, 22.5 ℃, 23.5 ℃ and 24.5 ℃. The standing treatment time can be 0.5-1.5 h, also can be 0.75-1.25 h, and also can be 0.9-1.1 h; specifically, the reaction time can be 0.6h, 0.8h, 1.0h, 1.2h and 1.4 h.
The R1 reagent containing the magnetic fluorescent coding microspheres coated by different antibodies is prepared by the method; finally, the final concentration of the antibody-coated magnetic fluorescent coding microspheres in the solution, which is regulated by the buffer solution B, can be 0.4-0.6 mg/mL, specifically 0.45mg/mL, 0.5mg/mL, 0.55 mg/mL.
The R2 reagent is prepared by the method comprising the following steps:
crosslinking an antibody and a fluorescent marker in a buffer solution C by using a crosslinking agent to obtain a fluorescent marker antibody; then preparing other solutions containing the fluorescence labeled antibodies corresponding to different indexes to be detected one by one according to the steps;
and subsequently, mixing a plurality of fluorescence labeled antibodies corresponding to the plurality of combined detection items together to obtain the R2 reagent.
The buffer C is used for adjusting the final concentration of the fluorescence labeled antibody in the solution to be 0.3-0.8 mu g/mL, specifically 0.41 mu g/mL, 0.52 mu g/mL, 0.61 mu g/mL, 0.69 mu g/mL and 0.73 mu g/mL.
It should be noted that, in the present application, only one magnetic fluorescent coding microsphere correspondingly codes one index to be detected. Different magnetic fluorescent coding microspheres can code different detection indexes and decode the detection indexes during detection.
In addition, the kit of the application also comprises a calibrator used for preparing a standard curve related to the corresponding index to be detected.
The use method of the kit for the multi-index joint inspection of the whole blood sample comprises the following steps: preparing an R1 reagent and an R2 reagent according to indexes to be detected;
taking a whole blood sample, adding the hemolytic agent for hemolysis, adding the R1 reagent and the R2 reagent for reaction, separating, then discarding a liquid phase, and detecting a solid phase.
Wherein, the addition amount of the surfactant can be 0.05-0.15% of the volume of the phosphate buffer solution; the volume of the phosphate buffer solution can be 0.075-0.125%, and the volume of the phosphate buffer solution can be 0.08-0.11%. Specifically, the content may be 0.06%, 0.09%, or 0.13%.
The present application will be described in further detail with reference to the following drawings and examples.
The raw material information used in the present application is shown in table 1.
TABLE 1
Figure BDA0003019945510000121
Figure BDA0003019945510000131
The sample prepared by the kit provided by the application is detected and analyzed by a flow cytometer of which the product model is CytoPOC and which is manufactured by Beijing finger Biotechnology Limited.
Preparation example
Preparation of magnetic fluorescent coding microsphere
The preparation method of the magnetic fluorescent coding microsphere comprises the following steps:
I. preparing magnetic particle microspheres:
I-I, preparing a pre-solution: uniformly mixing cyclohexane, n-hexane and a nano ferroferric oxide toluene solution for later use; wherein the volume ratio of the cyclohexane to the normal hexane to the nano ferroferric oxide toluene solution is as follows: 20:20:16, wherein the concentration of the nano ferroferric oxide in the nano ferroferric oxide toluene solution is 5 mg/mL.
I-II, respectively weighing polystyrene mesoporous microspheres with different particle sizes, respectively adding the pre-solution, performing ultrasonic dispersion, and mechanically stirring at room temperature to obtain magnetic particle microspheres with different particle sizes; wherein, the particle diameters of the polystyrene mesoporous microspheres are respectively as follows: 3 μm and 5 μm.
II. Preparing magnetic coding microspheres:
II-I, preparing quantum dot QDS700 solutions with different concentrations; the quantum dot QDS700 solution comprises the following components in parts by volume: 1/3000, 2/3000, 4/3000, 8/3000, 16/3000, 32/3000.
II-II, respectively adding 50mg of magnetic particle microspheres with different particle sizes into the quantum dot solutions with different concentrations, mechanically stirring for 12 hours at 80 ℃, then rapidly cooling to room temperature, filtering the cooling liquid by qualitative filter paper, washing by absolute ethyl alcohol, and finally drying for 24 hours at 60 ℃ to obtain the magnetic fluorescence encoding microspheres with different quantum dot concentrations and different particle sizes. The addition relationship between the magnetic particle microspheres and the concentrations of the quantum dots QDS700 is not limited, and solutions with different concentrations of the quantum dots QDS700 can be mixed with the magnetic particle microspheres with the above two particle sizes. The different types of magnetic fluorescently encoded microspheres described herein differ in the intensity of fluorescence of each type of magnetic fluorescently encoded microsphere.
III, functional group coating
III-I, dissolving 20mg of the magnetic fluorescent coding microspheres with different particle sizes in 20mL of absolute ethyl alcohol, adding 40 mu L of 3-aminopropyltriethoxysilane after ultrasonic dispersion, and then mechanically stirring for 12 hours; then filtering the stirred solution by qualitative filter paper and washing by absolute ethyl alcohol to obtain the magnetic fluorescent coding microspheres wrapped with the silane layer;
III-II, dissolving the magnetic fluorescent coding microspheres wrapped with the silane layer in 20mL of absolute ethyl alcohol, adding 0.5g of succinic anhydride, mechanically stirring at room temperature for 12h, filtering the mechanically stirred solution by qualitative filter paper, washing by absolute ethyl alcohol, and drying at 60 ℃ for 24h to obtain the magnetic fluorescent coding microspheres with carboxyl functional groups on the surfaces.
Examples
Effect of different kinds of hemolytic agents on hemolysis time
Hemolytic agent with hemolytic effect and containing different surfactants
Taking three parts of 0.02mol/L phosphate buffer solution with pH value of 7.4, adding different surfactants into the three parts of the phosphate buffer solution respectively, wherein the added surfactants are tween20, tween80 and triton-100 which are respectively 0.1 percent of the volume of the phosphate buffer solution, and are respectively named as solution 1, solution 2 and solution 3. The hemolysis time was then tested after adding different proportions of whole blood samples and the results are shown in table 2.
TABLE 2
Figure BDA0003019945510000141
As shown in Table 2, the hemolytic agent containing Triton X-100 can achieve complete hemolysis within 100s when different amounts of whole blood samples are processed, and can rapidly and sufficiently hemolysis. When the hemolytic agent containing triton X-100 was added to the whole blood, and when the whole blood accounted for 10% of the total volume after the hemolytic agent was added (i.e., the amount of the hemolytic agent added was 90% of the total volume of the solution after the hemolytic agent was added), the hemolysis time was the shortest and was only 3s, so in the subsequent experiments, solution 3 was selected as the hemolytic agent for the related experiments.
In addition, the data results of table 1 show that: the larger the proportion of the hemolytic agent is, the faster the hemolysis speed is, and if the higher hemolysis time is required, the higher the amount of the surfactant added can be to accelerate the hemolysis speed. Here, a hemolytic agent containing 0.1% Triton X-100 has been able to be used: the hemolysis can be completely hemolyzed, the hemolysis speed is high, and the sensitivity of later detection is not influenced after the hemolysis.
Example 1 dual-index combined test kit for Procalcitonin (PCT) and interleukin 6(IL-6) in a whole blood sample a method of using a kit for multi-index combined test in a whole blood sample, comprising the steps of:
s1 preparation of antibody-coated magnetic fluorescent coding microspheres
In 0.1 mol/L2-morpholine ethanesulfonic acid buffer solution, after 5.0 mu m magnetic fluorescence coding microspheres are activated by EDC, the PCT antibody is fixed on the magnetic fluorescence coding microspheres A1Then adding 1% BSA phosphate buffer solution to treat for 1h at room temperature, and obtaining the magnetic fluorescent coding microspheres coated with the PCT antibody.
IL-6 antibody is coated on another magnetic fluorescent coding microsphere B by the same method1At the top, mostFinally obtaining the IL-6 antibody coated magnetic fluorescent coding microspheres, respectively preparing the PCT antibody coated magnetic fluorescent coding microspheres and the IL-6 antibody coated magnetic fluorescent coding microspheres into solutions with the concentrations of 0.5mg/mL by using phosphate buffer solution containing 1% BSA and 0.5% bovine serum with the pH value of 7.2, and mixing to obtain the R1 reagent.
Wherein, the magnetic fluorescent coding microsphere A1And magnetic fluorescent encoding microsphere B1The difference lies in the difference of the fluorescence intensity of the two magnetic fluorescence-encoded microspheres.
S2 preparation of fluorescent labeled antibody
Connecting the PCT antibody with Phycoerythrin (PE) through glutaraldehyde to obtain a PE-labeled PCT antibody;
connecting the IL-6 antibody with Phycoerythrin (PE) through glutaraldehyde to obtain a PE-labeled IL-6 antibody; and preparing solutions of the PE-labeled PCT antibody and the PE-labeled IL-6 antibody according to the concentrations of 0.5 mu g/mL and 0.4 mu g/mL respectively by using a pH7.2 protein-containing phosphate buffer solution, and mixing the solutions to obtain the R2 reagent.
S3, adding a hemolytic agent into the whole blood sample, wherein the hemolytic agent is a solution 3 obtained by adding triton-100 with the volume of 0.1% of phosphate buffer solution into 0.02mol/L of phosphate buffer solution with pH 7.4; after adding the hemolytic agent to the whole blood sample, the whole blood sample accounts for 60% of the total volume of the liquid after adding the hemolytic agent. Adding the coated PCT antibody magnetic fluorescent coding microspheres and the coated IL-6 antibody magnetic fluorescent coding microspheres, and simultaneously adding the PE-labeled PCT antibody and the PE-labeled IL-6 antibody for reaction for 20 min. Then, after magnetic separation, a solid phase is left, and finally, the obtained solid phase is washed and then is added with PBS for testing by a flow cytometer.
The test results are shown in FIGS. 1-2 and Table 3. The results in FIG. 1 show that when the PCT and IL-6 are analyzed in a combined test on whole blood samples, the flow cytometer can clearly distinguish and circle the fragment population (i.e., the cell fragment population including red blood cell fragments and white blood cell fragments) and the target population (i.e., PCT and IL-6 containing the index to be detected) after the test sample is added into the flow cytometer. In the results of fig. 2, different specific detection indicators in the target population, PCT and IL-6 respectively, can be distinguished significantly by the encoding channel.
TABLE 3
Figure BDA0003019945510000161
The data in table 3 show that: the PCT and IL-6 project combined detection curve, the detection sensitivity and the linearity in the whole blood detection all meet the reagent using target. During PCT detection, the PCT content and the fluorescence intensity are fitted by five parameters, and the correlation coefficient R1 20.9962; during IL-6 detection, the IL-6 content and the fluorescence intensity are fitted by five parameters, and the correlation coefficient is as follows: r2 2＝0.9999。
When the pH value of hemolytic agent treatment samples with different pH values on PCT and IL-6 detection influencing conventional antigen-antibody reaction is basically in the range of 6-8, 0.02mol/L phosphate buffer solution containing 0.1% triton X-100 is prepared, the pH values are respectively 6.0, 7.4 and 8.0, the volume ratio of whole blood samples (5 groups, respectively, sample A to sample E) to hemolytic agent is treated according to the ratio of 1:1, and then the content of PCT in the samples is determined by adopting the method, and the test results are shown in Table 4.
TABLE 4
Figure BDA0003019945510000162
The results in Table 4 show that when the hemolytic agent is in the pH range of 6-8, the whole blood treated without affecting the subsequent immunoassay reaction, and therefore the pH range of the hemolytic agent can be selected from 6-8.
Example 2 Whole blood samples cardiac troponin I (cTn I), Myoglobin (MYO), creatine kinase isozyme (CK-MB), N-terminal B-type natriuretic peptide precursor (NT-pro-BNP), lipoprotein-associated phospholipase A2(Lp-PLA 2), and cardiac fatty acid binding protein (H-FABP) six-index Combined test kit
The use method of the kit for the multi-index joint detection in the whole blood sample comprises the following steps:
s1 preparation of antibody-coated magnetic fluorescent coding microspheres
In 0.1 mol/L2-morpholine ethanesulfonic acid buffer solution, 5.0 mu m magnetic fluorescence coding microspheres are activated by EDC, cTn I antibody is fixed on the magnetic fluorescence coding microspheresA2Then treating the surface of the microsphere by using 1% BSA phosphate buffer solution for 1h at room temperature to obtain the cTn I antibody-coated magnetic fluorescent coding microsphere. Magnetic fluorescent coding microspheres (with different codes) respectively coated with MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP antibodies are obtained by the same method, and the magnetic fluorescent coding microspheres coated with different antibodies are prepared into mixed liquid with the concentration of 0.5mg/mL by using pH7.2 protein-containing phosphate buffer solution, namely R1 reagent.
Wherein, the difference of the different magnetic fluorescent coding microspheres is that the fluorescence intensity of each magnetic fluorescent coding microsphere is different.
S2 preparation of fluorescent labeled antibody
Connecting the cTn I antibody with Phycoerythrin (PE) through glutaraldehyde to obtain a PE-labeled cTn I antibody, sequentially obtaining a PE-labeled MYO antibody, a PE-labeled CK-MB antibody, a PE-labeled NT-pro-BNP antibody, a PE-labeled Lp-PLA 2 antibody and a PE-labeled H-FABP antibody by the above method, respectively preparing the PE-labeled antibodies into mixed solutions with the concentrations of 0.4 mu g/mL, 0.6 mu g/mL, 0.3 mu g/mL, 0.8 mu g/mL, 0.6 mu g/mL and 0.7 mu g/mL by using a protein-containing phosphate buffer solution with the pH value of 7.2, and mixing to obtain the R2 reagent.
S3, taking a whole blood sample, adding a magnetic fluorescent coding microsphere coated with a cTn I antibody, a magnetic fluorescent coding microsphere coated with a MYO antibody, a magnetic fluorescent coding microsphere coated with a CK-MB antibody, a magnetic fluorescent coding microsphere coated with an NT-pro-BNP antibody, a magnetic fluorescent coding microsphere coated with an Lp-PLA 2 antibody and a magnetic fluorescent coding microsphere coated with an H-FABP antibody, and reacting for 20min after adding a PE-labeled cTn I antibody, a PE-labeled MYO antibody, a PE-labeled CK-MB antibody, a PE-labeled NT-pro-BNP antibody, a PE-labeled Lp-PLA 2 antibody and a PE-labeled H-FABP antibody. Adding 100 mu L of hemolytic agent, wherein the hemolytic agent is solution 3 obtained by adding 0.1% triton-100 in volume of phosphate buffer solution to 0.02mol/L of phosphate buffer solution with pH 7.4. Then, after magnetic separation, a solid phase is left, and finally, the obtained solid phase is washed and then is added with PBS for testing by a flow cytometer.
The test results are shown in FIGS. 3-4 and Table 5. The results in FIG. 3 show that when the cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP are analyzed in a combined test in a whole blood sample, the flow cytometer can clearly distinguish and circle the fragment group (i.e., the cell fragment group including the red cell fragments and the white cell fragments) and the target group (i.e., the cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP containing the index to be detected) after the test sample is added into the flow cytometer. Subsequently, in the results shown in FIG. 4, different specific detection indexes in the target group can be distinguished significantly through the coding channels, namely cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP.
TABLE 5
Figure BDA0003019945510000181
Figure BDA0003019945510000191
The data in table 5 show that: six items of combined detection curves, detection sensitivity and linearity of cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP in whole blood detection all meet the reagent using target. After five-parameter fitting, the correlation coefficient R between the content (cTn I, MYO, CK-MB, NT-pro-BNP, Lp-PLA 2 and H-FABP) of each item and the fluorescence intensity2Respectively as follows: 0.9992, 0.9991, 0.9998, 0.9995, 0.9998, 0.9996.
EXAMPLE 3 Dual-indicator Combined assay kit for Luteinizing Hormone (LH) and progesterone (P) in Whole blood samples
The use method of the kit for the multi-index joint detection in the whole blood sample comprises the following steps:
s1 preparation of antibody-coated magnetic fluorescent coding microspheres
In 0.1 mol/L2-morpholine ethanesulfonic acid buffer solution, activating 5.0 μm magnetic fluorescence coding microsphere with EDC, fixing LH antibody on magnetic fluorescence coding microsphere A3Then treating the surface of the microsphere with 1% BSA phosphate buffer solution at room temperature for 1h to obtain a LH antibody-coated magnetic fluorescent coding microsphere, and coating the P antibody on another magnetic fluorescent coding microsphere B by the same method3In combination with a protein-containing phosphate of pH7.2And respectively preparing the LH antibody-coated magnetic fluorescent coding microspheres and the P antibody-coated magnetic fluorescent coding microspheres into mixed solutions with the concentration of 0.5mg/mL by using a buffer solution, and then mixing to obtain the R1 reagent.
S2 preparation of fluorescent labeled antibody/antigen
Connecting the LH antibody and the P antigen with Phycoerythrin (PE) through glutaraldehyde to obtain a PE-labeled LH antibody and a PE-labeled P antigen, preparing the PE-labeled LH antibody and the PE-labeled P antigen into mixed solutions with the concentrations of 0.8 mu g/mL and 0.1 mu g/mL respectively by using a protein-containing phosphate buffer solution with the pH value of 7.2, and mixing the two mixed solutions to obtain the R2 reagent.
S3, taking a whole blood sample, adding the magnetic fluorescent coding microspheres coated with the LH antibody and the magnetic fluorescent coding microspheres coated with the P antibody, and reacting for 15min after adding the PE-labeled LH antibody and the PE-labeled P antibody. Adding 100 mu L of hemolytic agent, wherein the hemolytic agent is solution 3 obtained by adding triton-100 with the volume of 0.1% of phosphate buffer solution into 0.02mol/L of phosphate buffer solution with pH of 7.4; then, after magnetic separation, a solid phase is left, and finally, the obtained solid phase is washed, added with PBS and tested by a flow cytometer.
The test results are shown in FIGS. 5-6 and Table 6. The results in fig. 5 show that, when the sample to be tested is added to the flow cytometer during the joint test analysis of LH and P in the whole blood sample, the flow cytometer can clearly distinguish and circle the fragment population (i.e. the cell fragment population including the red cell fragments and the white cell fragments) and the target population (i.e. LH and P containing the index to be tested). In the results of fig. 6, different specific detection indexes in the target group, i.e. LH and P, can be distinguished significantly by the encoding channel.
TABLE 6
Figure BDA0003019945510000201
The data in table 6 show that: the double-item combined detection curve, the detection sensitivity and the linearity of LH and P in whole blood detection all meet the use target of the reagent. Wherein, the content of LH and P and the fluorescence intensity are subjected to curve correlation coefficient R after five-parameter fitting2Respectively as follows: 0.9998 and 0.9913.
Comparative example
The difference between this comparative example and example 2 is that the whole blood sample is directly tested after adding the hemolytic agent after adding the magnetic fluorescent-encoded microspheres and the PE-labeled antibody for reaction, and the magnetic separation operation is not performed (i.e., the finally disrupted cell debris is not removed).
After testing, the target magnetic fluorescent coding microspheres are found to be adhered into adherends of various sizes by cell fragments, as shown in a scatter diagram in fig. 7, different detection indexes can be distinguished by all point circle gates as shown in fig. 8, but certain cell fragments are mixed in a low-value coding microsphere group and can bring interference to later analysis, so that the direct testing mode is not adopted.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims (10)

1. A kit for multi-index joint inspection of a whole blood sample is characterized by comprising a hemolytic agent for breaking red blood cells in the whole blood sample, an R1 reagent containing antibody-coated magnetic fluorescent coding microspheres, an R2 reagent containing fluorescent labeling antibodies, a buffer solution and a calibrator;
the antibody-coated magnetic fluorescent coding microspheres are obtained by coupling antibodies of specific targets to the magnetic fluorescent coding microspheres, and the antibody-coated magnetic fluorescent coding microspheres are a mixture of multiple microspheres coated with multiple specific antibodies, namely, each microsphere with different fluorescent intensity is coated with one specific antibody; the fluorescence labeling antibody is used for labeling a fluorescence labeling substance on a pairing antibody, and the fluorescence labeling antibody is a mixture of fluorescent substances labeled by a plurality of specific antibodies; the calibrator is a composite calibrator comprising a plurality of targets; the specific antibodies are matched with the magnetic fluorescent coding microspheres; the hemolytic agent contains surfactant or salt substance for dissolving erythrocyte in blood.
2. The kit for multi-index joint examination according to claim 1, wherein the hemolytic agent is one of cationic surfactant, anionic surfactant, nonionic surfactant, amphoteric surfactant, or ammonium chloride.
3. The kit for multi-index combined test according to claim 2, wherein the hemolytic agent is phosphate buffer containing a surfactant selected from any one of tween20, tween80 and triton-100.
4. The kit for multi-index joint examination according to claim 3, wherein the hemolytic agent is a buffer solution containing triton-100, and the addition amount of the triton-100 in the buffer solution is 0.05% -0.15%.
5. The kit for multi-index joint examination according to claim 4, wherein the amount of triton-100 added in the buffer is 0.05%.
6. The kit of claim 1, wherein the content of each antibody-coated magnetic fluorescent-encoded microsphere in the R1 reagent is 0.4-0.6 mg/mL.
7. The kit for multi-index joint detection according to any one of claims 1-6, wherein the targets of the paired antibodies in the whole blood sample are 2-14.
8. The kit of claim 7, wherein the targets are 5 of Proinflammin (PCT), interleukin 6(IL-6), C-reactive protein (CRP), Serum Amyloid A (SAA), and heparin-binding protein (HBP) associated with inflammation.
9. The use method of the kit for the multi-index joint inspection according to any one of claims 1 to 6, characterized by comprising the following steps:
preparing an R1 reagent and an R2 reagent according to indexes to be detected;
taking a whole blood sample, adding the hemolytic agent for hemolysis, then adding the R1 reagent and the R2 reagent for reaction, separating, then discarding a liquid phase, and detecting a solid phase;
or the use method comprises the following steps:
preparing an R1 reagent and an R2 reagent according to indexes to be detected;
taking a whole blood sample, adding an R1 reagent and an R2 reagent, reacting, adding the hemolytic agent for hemolysis, separating, discarding a liquid phase, and detecting a solid phase.
10. The method for using the kit for multi-index joint examination in the whole blood sample according to claim 9, wherein the hemolytic agent is added to the whole blood sample in an amount of 10% to 90% of the total volume of the hemolytic agent solution.
CN202110399683.2A 2021-04-14 2021-04-14 Kit for multi-index joint inspection of whole blood sample and use method thereof Pending CN113176404A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114660305A (en) * 2022-03-31 2022-06-24 中国人民解放军军事科学院军事医学研究院 Reagent for rapidly detecting blood type antibody titer, preparation method, test strip and application

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106566879A (en) * 2016-10-24 2017-04-19 深圳市液芯生物科技有限公司 Encoding microspheres used for biomolecular screening or detection, and preparation method and application of encoding microspheres
CN108303544A (en) * 2017-01-13 2018-07-20 深圳开立生物医疗科技股份有限公司 A kind of whole blood c reactive protein detection kit
CN109541220A (en) * 2018-10-09 2019-03-29 温州启星生物技术有限公司 The preparation method and application of C reactive protein, Procalcitonin, heparin-binding protein and serum amyloid A protein 1
CN110261602A (en) * 2019-06-03 2019-09-20 苏州百源基因技术有限公司 A kind of detection method and detection kit based on fluorescence-encoded magnetic bead
CN110646620A (en) * 2019-09-29 2020-01-03 湖北新纵科病毒疾病工程技术有限公司 Method for simultaneously detecting four inflammation markers and kit thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106566879A (en) * 2016-10-24 2017-04-19 深圳市液芯生物科技有限公司 Encoding microspheres used for biomolecular screening or detection, and preparation method and application of encoding microspheres
CN108303544A (en) * 2017-01-13 2018-07-20 深圳开立生物医疗科技股份有限公司 A kind of whole blood c reactive protein detection kit
CN109541220A (en) * 2018-10-09 2019-03-29 温州启星生物技术有限公司 The preparation method and application of C reactive protein, Procalcitonin, heparin-binding protein and serum amyloid A protein 1
CN110261602A (en) * 2019-06-03 2019-09-20 苏州百源基因技术有限公司 A kind of detection method and detection kit based on fluorescence-encoded magnetic bead
CN110646620A (en) * 2019-09-29 2020-01-03 湖北新纵科病毒疾病工程技术有限公司 Method for simultaneously detecting four inflammation markers and kit thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
任继欣 等: "外周血 HBP，IL-6，CD64 指数，PCT，CRP 和 SAA 水平检测在血流感染诊断中的应用价值研究", 《现代检验医学杂志》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114660305A (en) * 2022-03-31 2022-06-24 中国人民解放军军事科学院军事医学研究院 Reagent for rapidly detecting blood type antibody titer, preparation method, test strip and application

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Application publication date: 20210727