CN112014573B - Preparation method of high-sensitivity assay kit for troponin I in human whole blood sample and kit - Google Patents

Preparation method of high-sensitivity assay kit for troponin I in human whole blood sample and kit Download PDF

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CN112014573B
CN112014573B CN202010862364.6A CN202010862364A CN112014573B CN 112014573 B CN112014573 B CN 112014573B CN 202010862364 A CN202010862364 A CN 202010862364A CN 112014573 B CN112014573 B CN 112014573B
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reagent
troponin
solution
enzyme
concentration
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黄丹
来祥兵
赵愿安
舒芹
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Wuhan Life Origin Biotech Joint Stock Co ltd
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Abstract

The invention provides a preparation method of a high-sensitivity assay kit for troponin I in a human whole blood sample, belonging to the technical field of immunodetection, comprising the following steps: coating a mouse anti-human troponin I monoclonal antibody by using magnetic microspheres to obtain a reagent M; alkaline phosphatase sequentially reacts with an enzyme cross-linking agent 1 and an enzyme cross-linking agent 2, and the cross-linked alkaline phosphatase is used for marking troponin I goat polyclonal antibody to obtain a reagent R1; the enzyme cross-linking agent 1 is glutaraldehyde solution, and the enzyme cross-linking agent 2 is amino acid solution or polypeptide solution; the reagent R2 solution is prepared according to the formula of the reagent R2. The kit prepared by the method has high sensitivity on troponin I detection, and can detect the whole blood sample without pretreatment on the whole blood sample, thereby improving the working efficiency of detection personnel. The invention also provides a high-sensitivity assay kit for troponin I in a human whole blood sample.

Description

Preparation method of high-sensitivity assay kit for troponin I in human whole blood sample and kit
Technical Field
The invention belongs to the technical field of immunodetection, and relates to a preparation method of a high-sensitivity assay kit for troponin I in a human whole blood sample and the kit.
Background
Troponin (Tn) is a regulator of muscle tissue contraction, located on the filament of contractile proteins, playing an important role in regulation during muscle contraction and relaxation; contains 3 subtypes: fast-response, slow-response, and cardiac troponin (cTn). The former two are related to skeletal muscle, while cardiac troponin exists only in cardiac muscle cells, and is a complex composed of three subunits of troponin T (cTnT), troponin I (cTnI) and troponin C (cTnC). cTnT and cTnI are antigens specific to cardiomyocytes, and have a molecular weight of about 23.88KDa and degrade from myocardial fibers upon myocardial cell injury. The increase of cTn in serum reflects the damage of myocardial cells, and the specificity and the sensitivity of the cTn are higher than those of the myocardial zymogram which is commonly used in the past.
Troponin is a marker of myocardial injury necrosis and has important clinical significance (100% diagnostic sensitivity, 91% specificity and long duration) for diagnosis and risk stratification of acute myocardial infarction. The elevation of troponin value indicates myocardial injury, which can be seen in acute myocardial infarction, unstable angina, pulmonary infarction, heart failure shock and other diseases causing myocardial injury such as pancreatitis, severe diabetic ketoacidosis, connective tissue diseases and the like, the higher the value is, the wider the injury range is, the release is started in 3-6 hours of patients suffering from acute myocardial infarction, the peak is reached in 10-24 hours, and the recovery normal time cTnT and cTnI are respectively 10-15 days and 5-7 days; elevation may also occur in some patients with renal insufficiency.
With the continuous update of detection technology, the sensitivity of cardiac troponin (cTnI) detection has been continuously improved, and the concept of high-sensitivity troponin (hs-cTnI) has been developed. As a high-sensitivity detection index, the high-sensitivity troponin (hs-cTnI) is helpful for detecting the tiny myocardial injury which is easy to be missed in the past, the hs-cTn can detect the clinically significant increase 1-3 h after the myocardial injury, 3-6 h earlier than the traditional method, and compared with the common cTnI detection, the high-sensitivity troponin (hs-cTnI) detection has higher predictive value on myocardial infarction, can reduce troponin blind area time and detect acute myocardial infarction earlier. Meanwhile, hs-cTnI is more reasonable for screening cardiovascular high-risk patients, and clinical treatment decisions and prognosis evaluation are optimized. Global cardiologists recommend hs-cTn as the preferred myocardial injury marker in the 2018 fourth edition of general definition of myocardial infarction. The international union of clinical chemistry and test medicine IFCC (lnternational Federation of Clinical Chemistry and laboratory medicine) cardiac injury marker standardization committee updated troponin assay specific quality control standards at the 2018 academy of AACC (american society of clinical chemistry) that were promulgated to help guide troponin commercial reagent manufacturers and the clinical laboratories that applied the assays. The criteria emphasizes that 99 th percentile values for sex differentiation between men and women are required to provide high sensitivity troponin agents, and that the detected proportion in both men and women apparent healthy populations is higher than 50%.
The current methods for detecting the high-sensitivity troponin I include an immunonephelometry (CN 107422129A), a microfluidic immunoassay (CN 110773246A), a chemiluminescent method (CN 109580954A, CN 110082541A) and the like. Wherein, the immunoturbidimetry method can not meet the requirement because of the limitation of the coupling technology on the detection sensitivity; the microfluidic immunoassay method has high technical barriers and high consumable cost, and can increase medical burden. Chemiluminescent immunoassay has been developed from the beginning of the 80 th century, and technology has tended to mature. The chemiluminescent immunoassay technology has the advantages of high sensitivity, strong specificity, good reagent stability, long effective period, wide detection linear range, simple operation, stable and rapid method, multiple detection items, high automation degree and the like, and is a mainstream method of the conventional immunoassay technology and widely applied to various fields of clinical examination.
Patent CN109580954a relates to a hypersensitive quantitative determination kit of human troponin I and a detection method thereof, the patent adopts a plurality of different antibodies (four monoclonal antibodies) which can identify more sites, and the invention has higher detection sensitivity and specificity compared with the prior art. However, the invention describes the coating or marking process of four antibodies, the production process is complex, the production period is long, and the invention is not suitable for wide popularization. Meanwhile, the invention does not mention the ability to test whole blood samples in human body fluids.
Patent CN110082541a describes a high sensitivity troponin I kit and sample processing fluid, which is a single-person detection kit that is ready to use and capable of detecting whole blood samples. However, according to the latest definition of a hypersensitive troponin reagent: the high-sensitivity troponin reagent needs to use 99 th percentile of sex distinction of men and women, meanwhile, the detection proportion in apparent healthy people of men and women is higher than 50%, and the recognized reference interval of the high-sensitivity troponin is: male (. Ltoreq.0.034 ng/mL, 99 th percentile), female (. Ltoreq.0.016 ng/mL, 99 th percentile), all populations (. Ltoreq.0.026 ng/mL, 99 th percentile). The verification of the patent CN110082541A aiming at the high-sensitivity troponin detection kit only verifies that the upper limit of the 99 th percentile is 0.06ng/mL, namely 60ng/L, and the detection rate in the apparent healthy population of men and women is not verified, and the requirements of the latest high-sensitivity troponin reagent cannot be met.
Disclosure of Invention
In order to solve the technical problem that the sensitivity of the existing high-sensitivity troponin I reagent is poor, the invention provides a preparation method of a high-sensitivity detection kit for troponin I in a human whole blood sample.
The invention also provides a high-sensitivity assay kit for troponin I in a human whole blood sample.
The invention is realized by the following technical scheme:
a preparation method of a high-sensitivity assay kit for troponin I in a human whole blood sample comprises the following steps:
coating a mouse anti-human troponin I monoclonal antibody by using magnetic microspheres to obtain a reagent M;
the alkaline phosphatase sequentially carries out a crosslinking reaction with an enzyme crosslinking agent 1 and an enzyme crosslinking agent 2, and the alkaline phosphatase after the crosslinking reaction is used for marking troponin I sheep polyclonal antibody to obtain a reagent R1;
the enzyme cross-linking agent 1 is glutaraldehyde solution, and the enzyme cross-linking agent 2 is amino acid solution or polypeptide solution;
preparing a reagent R2 solution according to a reagent R2 formula, wherein the reagent R2 formula comprises:
tris:10-100mmol/L; high molecular polymer: 0.1% -1%; hemolysis agent: 0.5% -3%; trehalose: 1% -5%; proclin300:0.01% -0.1%; pH:6.5-8.0.
Preferably, the formulation of the reagent R2 comprises:
tris:20-50mmol/L; high molecular polymer: 0.2% -0.5%; hemolysis agent: 1% -2%; trehalose: 2% -3%; proclin300:0.05%; pH:7.0-7.5;
the hemolytic agent comprises any one of sodium dodecyl benzene sulfonate, saponin, choline salt, cetyltrimethylammonium bromide, lecithin, phosphatidyl acetamide, phosphatidyl choline sodium and choline salicylic acid;
The high polymer comprises any one of polyethylene glycol, polyvinylpyrrolidone, sodium dodecyl benzene sulfonate, lauric acid sulfonated succinic acid monoester disodium, potassium monolauryl phosphate and potassium laureth phosphate;
and (3) filtering the solution of the reagent R2 by using a microporous filter membrane with the diameter of 0.22 mu m after the preparation of the solution of the reagent R2 is finished, so as to obtain the reagent R2, and preserving the reagent R2 at the temperature of 2-8 ℃.
Further, the mass ratio of the magnetic microsphere to the mouse anti-human troponin I monoclonal antibody is 1: (0.05-0.2), the magnetic microsphere is activated by adopting a magnetic microsphere activator 1 and a magnetic microsphere activator 2 before being coated with the mouse anti-human troponin I monoclonal antibody, the magnetic microsphere activator 1 is 20mg/mL NHS, the magnetic microsphere activator 2 is 20mg/mL EDC, the mass ratio of the magnetic microsphere to the NHS is 1: (2-15), and the mass ratio of the magnetic microsphere to the EDC is 1: (1-10).
Further, the activation process of the magnetic microspheres is carried out in a magnetic microsphere activation buffer solution, the magnetic microspheres are cleaned before and after activation, the activated and cleaned magnetic microspheres are resuspended by adopting a magnetic microsphere coating buffer solution, the magnetic microspheres are coated with a mouse anti-human troponin I monoclonal antibody and then are sealed by adopting a magnetic microsphere sealing solution, and the supernatant is removed and then stored in a magnetic microsphere storage solution, so that the reagent M is obtained.
Further, the magnetic microsphere activation buffer solution adopts 20-100 mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution, and the pH value is 4.5-6.5;
the magnetic microsphere coating buffer solution adopts 10-50 mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution, and the pH value is 4.5-6.5;
the magnetic microsphere sealing liquid adopts a mixed liquid of glycine and bovine serum albumin, wherein the concentration of glycine is 0.5-2%, and the concentration of bovine serum albumin is 1-5%;
the magnetic microsphere preservation solution comprises the chemical components of 4- (2-hydroxyethyl) piperazine-1-2-hydroxy propane sulfonic acid, bovine serum albumin, proclin300 and tween 20, wherein the concentration of the 4- (2-hydroxyethyl) piperazine-1-2-hydroxy propane sulfonic acid is 10-100mmol/L, the concentration of the bovine serum albumin is 0.5-3%, the concentration of the Proclin300 is 0.05%, the concentration of the tween 20 is 0.1-1%, and the pH value of the solution is 7.1-8.5.
Further, the concentration of glutaraldehyde solution is 0.1-10%, the concentration of amino acid solution or polypeptide solution is 1% -10%, the molar ratio of alkaline phosphatase to glutaraldehyde is 1:1-5, and the molar ratio of alkaline phosphatase to amino acid is 1:1-10;
the amino acid is an amino acid with at least two active amino groups;
the molar ratio of the alkaline phosphatase to the troponin I sheep polyclonal antibody is as follows: 1 to 0.1-0.5.
Further, the amino acids include one or more of lysine, arginine, asparagine, glutamine, tryptophan, and histidine;
the polypeptide is a polymeric peptide of one or more of the amino acids.
Further, the specific process of sequentially reacting alkaline phosphatase with the enzyme cross-linking agent 1 and the enzyme cross-linking agent 2 comprises the following steps:
adding alkaline phosphatase into enzyme marking buffer solution, adding enzyme cross-linking agent 1, and uniformly mixing and activating;
after dialysis, adding an enzyme crosslinking agent 2 for light-shielding crosslinking reaction;
the crosslinked alkaline phosphatase is dialyzed and then labeled with troponin I sheep polyclonal antibody;
and (3) after the alkaline phosphatase marks the troponin I goat polyclonal antibody, adding an enzyme blocking agent for light-shielding reaction, purifying after the blocking is finished, and storing in an enzyme preservation solution.
Further, the enzyme labeling buffer is 5-50mmol/LPBS buffer, and the pH value is 6.0-7.5;
the enzyme blocking agent is 1% -5% bovine serum albumin;
the chemical components of the enzyme preservation solution comprise PBS solution, bovine serum albumin, glycerol and Proclin300, wherein the concentration of the PBS solution is 20-100mmol/L, the concentration of BSA is 0.2% -2%, the concentration of the glycerol is 1% -10%, and the concentration of the Proclin300 is 0.05%.
A reagent kit for the high-sensitivity determination of troponin I in a human whole blood sample comprises a reagent M, a reagent R1 and a reagent R2, wherein the reagent M, the reagent R1 and the reagent R2 are prepared by a preparation method of the reagent kit for the high-sensitivity determination of troponin I in the human whole blood sample.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
according to the preparation method of the high-sensitivity determination kit for troponin I in the human whole blood sample, the cTnI sheep multi-enzyme labeling reagent R1 labeling process is optimized, alkaline phosphatase is firstly activated by amino or carboxyl on enzyme by using a compound with a double-functional group, then antibody labeling is carried out, the detection sensitivity of the kit can be improved, the detection sensitivity of the kit is improved, and the reagent R2 component is optimized to reduce interference generated when the kit detects the whole blood sample.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a blank interval evaluation histogram of comparative example 1;
FIG. 2 is a blank interval evaluation histogram of example 2;
FIG. 3 is a blank interval evaluation histogram of example 3;
FIG. 4 is a blank interval evaluation histogram of example 4;
FIG. 5 is a linear range evaluation correlation plot;
FIG. 6 is a correlation of control reagents with the test samples of the reagents of example 1;
FIG. 7 is a correlation of serum samples with EDTA plasma samples;
FIG. 8 is a correlation of serum samples with heparin lithium plasma samples
FIG. 9 is a correlation of serum samples with EDTA whole blood samples;
fig. 10 is a correlation of serum samples with lithium heparin whole blood samples.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The technical scheme provided by the embodiment of the invention aims to solve the technical problems, and the overall thought is as follows:
in the existing troponin I detection kit, the cTnI reagent marked by the common enzyme marking method cannot reach the sensitivity of the high-sensitivity troponin reagent, such as the method for marking the antibody by alkaline phosphatase as mentioned in patent CN 109580954A. According to the invention, the detection sensitivity of the kit is improved by optimizing the marking process of the cTnI sheep multi-antibody enzyme-labeled reagent R1. For a whole blood sample, compared with serum and plasma components, the reagent is more complex, contains a large amount of red blood cells and has larger volume, and if the whole blood sample is directly added, errors are caused in detection, and the reagent R2 component is optimized to reduce interference generated when the reagent kit detects the whole blood sample.
Specifically, the preparation method of the high-sensitivity assay kit for troponin I in human whole blood samples comprises the following steps:
coating a mouse anti-human troponin I monoclonal antibody by using magnetic microspheres to obtain a reagent M;
The alkaline phosphatase sequentially carries out a crosslinking reaction with an enzyme crosslinking agent 1 and an enzyme crosslinking agent 2, and the alkaline phosphatase after the crosslinking reaction is used for marking troponin I sheep polyclonal antibody to obtain a reagent R1;
the enzyme cross-linking agent 1 is glutaraldehyde solution, and the enzyme cross-linking agent 2 is amino acid solution or polypeptide solution;
preparing a reagent R2 solution according to a reagent R2 formula, wherein the reagent R2 formula comprises:
tris:10-100mmol/L; high molecular polymer: 0.1% -1%; hemolysis agent: 0.5% -3%; trehalose: 1% -5%; proclin300:0.01% -0.1%; pH:6.5-8.0;
the hemolytic agent comprises any one of sodium dodecyl benzene sulfonate, saponin, choline salt, cetyltrimethylammonium bromide, lecithin, phosphatidyl acetamide, phosphatidyl choline sodium and choline salicylic acid;
the high polymer comprises any one of polyethylene glycol, polyvinylpyrrolidone, sodium dodecyl benzene sulfonate, lauric acid sulfonated succinic acid monoester disodium, potassium monolauryl phosphate and potassium laureth phosphate;
and (3) filtering the solution of the reagent R2 by using a microporous filter membrane with the diameter of 0.22 mu m after the preparation of the solution of the reagent R2 is finished, so as to obtain the reagent R2, and preserving the reagent R2 at the temperature of 2-8 ℃.
Further, the concentration of glutaraldehyde solution is 0.1-10%, the concentration of amino acid solution or polypeptide solution is 1% -10%, the molar ratio of alkaline phosphatase to glutaraldehyde is 1:1-5, and the molar ratio of alkaline phosphatase to amino acid is 1:1-10;
In the invention, glutaraldehyde is used for activating carboxyl groups on alkaline phosphatase so as to be convenient for amino acid to be combined with the alkaline phosphatase, and if glutaraldehyde is too little, the carboxyl groups on the alkaline phosphatase can be insufficiently activated to cause too little bridged amino acid; if glutaraldehyde is too much, residual glutaraldehyde in the activated alkaline phosphatase affects bridging of subsequent amino acids. Too small an amount of amino acids can result in a small amount of antibody labeling affecting reagent performance; too much amino acid can result in binding of the antibody to the amino acid affecting the efficiency of the comparison.
The amino acid is an amino acid with at least two active amino groups;
the molar ratio of the alkaline phosphatase to the troponin I sheep polyclonal antibody is as follows: 1 to 0.1-0.5.
Further, the amino acids include one or more of lysine, arginine, asparagine, glutamine, tryptophan, and histidine;
the polypeptide is a polymeric peptide of one or more of the amino acids.
In the invention, when the alkaline phosphatase and the antibody are labeled, the alkaline phosphatase is firstly activated by amino or carboxyl on the enzyme by using a compound (amino acid or polypeptide) with a bifunctional group, and then the antibody is labeled, so that the detection sensitivity can be improved. However, because the enzyme belongs to a small molecular substance and the antibody contains more free amino groups and carboxyl groups, the uncertain antibody label can be randomly combined with alkaline phosphatase when the antibody is labeled, thereby bringing steric hindrance and influencing the combination of the Fab end of the antibody and antigen to cause detection errors.
Specifically, the enzyme is a small molecular weight substance, the specific surface area is large, and when the antibody is combined with the enzyme, the antibody itself contains a large amount of free amino groups and carboxyl groups, so that the combination of the antibody and the carboxyl groups or amino groups on the surface of the enzyme is random, and the antibody can be combined with the enzyme at different angles. The closer the adjacent antibodies are, the more steric hindrance is formed, resulting in the difficulty in recognizing the antibodies and antigens. After linking carbon chains on the enzyme, the invention indirectly increases the volume of the enzyme, reduces the specific surface area, has larger space for combining the antibody and the enzyme, has smaller steric hindrance, and is easier for the antibody to recognize antigen.
In addition, the kit provided by the invention can eliminate interference caused by a whole blood sample and accurately detect the content of troponin I while improving the detection sensitivity. The reagent R2 is introduced into the invention, and the components comprise Tris-hydroxymethyl-aminomethane (Tris), high molecular polymer, hemolytic agent, trehalose and Proclin300. Wherein, the tris-hydroxymethyl-aminomethane provides a pH environment within a certain range, the high polymer can promote the dissolution of the blood cell lysate, can absorb fragments generated after the blood cell lysis, prevent the influence on detection, the hemolytic agent can dissolve erythrocytes in a blood sample, reduce detection errors, the trehalose is a protective agent, can ensure the solution to be stored stably, and the Proclin300 acts as a preservative.
Further, the mass ratio of the magnetic microsphere to the mouse anti-human troponin I monoclonal antibody is 1: (0.05-0.2), the magnetic microsphere is activated by adopting a magnetic microsphere activator 1 and a magnetic microsphere activator 2 before being coated with the mouse anti-human troponin I monoclonal antibody, the magnetic microsphere activator 1 is 20mg/mL NHS, the magnetic microsphere activator 2 is 20mg/mL EDC, the molar ratio of alkaline phosphatase to glutaraldehyde is 1: (1-5), and the molar ratio of alkaline phosphatase to amino acid is 1:1-10; .
In the invention, the surface groups of the magnetic microsphere are carboxyl groups, and the NHS and the EDC are used for activation, so that the insufficient activation of the carboxyl groups on the surface of the magnetic microsphere can influence the antibody coupling efficiency, and the excessive amount of the NHS and the EDC can cause residues and antibody reaction. When the antibody is coupled with the magnetic microsphere, if the antibody is too little, the reactivity of the reagent is low and the required sensitivity is not achieved; if the antibody is in excess, competition will form when it binds to the carboxyl groups on the magnetic microspheres resulting in the formation of microsphere-antibody-microsphere structures, affecting the binding of the antibody to the antigen.
Further, the activation process of the magnetic microspheres is carried out in a magnetic microsphere activation buffer solution, the magnetic microspheres are cleaned before and after activation, the activated and cleaned magnetic microspheres are resuspended by adopting a magnetic microsphere coating buffer solution, the magnetic microspheres are coated with a mouse anti-human troponin I monoclonal antibody and then are sealed by adopting a magnetic microsphere sealing solution, and the supernatant is removed and then stored in a magnetic microsphere storage solution, so that the reagent M is obtained.
The kit comprises a magnetic microsphere reagent M coated with a mouse anti-human troponin I (cTnI) monoclonal antibody, an enzyme-labeled reagent R1 marked with a troponin I (cTnI) sheep polyclonal antibody and a whole blood treatment reagent R2, when detection is carried out, a sample is firstly taken and mixed with the reagent R2, then the mixture is added into the reagent M and the reagent R1, incubation is carried out for a certain time, and after 3 times of cleaning, free unbound sample and enzyme are removed, and the concentration result of a final sample is obtained through instrument detection.
Compared with the patent CN109580954A, the invention can detect various blood sample types such as human serum, plasma, whole blood and the like, can meet different requirements of different people, and has higher sensitivity. Compared with CN110082541A, the invention can detect the whole blood sample without pretreatment of the whole blood sample treatment liquid on the premise of meeting the standards of the high-sensitivity troponin reagent proposed by IFCC, improves the detection sensitivity of the reagent, improves the working efficiency of operators, and is easy to realize detection automation.
The method for preparing a kit for the high sensitivity measurement of troponin I in a whole blood sample of a human of the present application will be described in detail with reference to examples, comparative examples and experimental data.
Example 1
1. The key materials used in the embodiment of the invention are as follows:
(1) Magnetic microspheres: the magnetic microsphere groups can be carboxyl magnetic microspheres, amino magnetic microspheres, toluenesulfonic acid magnetic microspheres and hydroxyl magnetic microspheres, preferably carboxyl magnetic microspheres and amino magnetic microspheres; the magnetic microsphere particle size is selected from magnetic microsphere particle sizes of 0.6 μm, 1.0 μm, 1.5 μm, 1.8 μm, 3.0 μm, 5.0 μm, preferably 1.0 μm, 1.5 μm, 1.8 μm.
The magnetic microsphere chosen for the experiment was MS160/Carboxyl (Carboxyl magnetic microsphere with particle size of 1.5 μm from JSR company).
(2) Enzyme: the reagent R1 is marked in the kit, and alkaline phosphatase or horseradish peroxidase is selected.
The experiment selects alkaline phosphatase P0114 (available from Merck Co.).
(3) Troponin I (cTnI) antibodies: the coating of reagent M and the labeling of reagent R1 in the kit, the recognition site selects murine anti-human, chicken anti-human etc. troponin I monoclonal antibodies in the interval 10-100 (preferably interval 20-50), wherein murine anti-human troponin I monoclonal antibodies are preferred; polyclonal antibodies such as goat anti-human and rabbit anti-human, and preferably goat anti-human polyclonal antibodies.
The experimental selection was 4T21-3C7 (from Hytest company, recognition site 25-40, murine anti-human monoclonal antibody) and 4T21/2 (from Hytest company, sheep anti-human polyclonal antibody).
(4) Amino acid: the reagent R1 used in the kit may be labeled with one or more polymeric peptides selected from lysine, arginine, asparagine, glutamine, tryptophan, and histidine, preferably lysine, arginine, asparagine, and glutamine (i.e., amino acids having at least two double-active amino groups or polymeric peptides thereof), wherein the polymeric peptide has the structure: NH2CH3 (CH 2) n (NH 2) iCOOH (n.gtoreq.0, i.gtoreq.1), preferably an amino acid having 3 to 10 carbons or a polymeric peptide thereof.
(5) High molecular polymer: for the preparation of reagent R2 in the kit, surfactants or other nonionic high molecular polymers are generally selected, preferably polyethylene glycol (PEG, PEG 6000/8000/20000), polyvinylpyrrolidone (PVP, PVP 10000/30000/40000/360000), sodium dodecyl benzene sulfonate (SDS), disodium monolaurate sulfonate (DLS), potassium monolauryl phosphate (MAPK) and potassium laureth phosphate (MAEPK). Preference is given to polyvinylpyrrolidone (PVP, PVP 10000/30000), disodium monolaurate sulfosuccinate (DLS), potassium monolauryl phosphate (MAPK), potassium laureth phosphate (MAEPK).
(6) Hemolysis agent: the reagent R2 used in the kit is prepared from sodium dodecyl benzene sulfonate (SDS), saponin, choline salt, cetyltrimethylammonium bromide, lecithin, phosphatidyl acetamide, phosphatidyl choline sodium, choline salicylic acid and the like. Saponins, choline salts, phosphatidylacetamides and phosphatidylcholine sodium are preferred.
2. Solution preparation
(1) Magnetic microsphere activation buffer: 20-100 mmol/L2- (N-morpholino) ethanesulfonic acid (MES) buffer, preferably 30-50mmol/L; the pH is 4.5-6.5, preferably 5.0-5.5.
(2) Magnetic microsphere coating buffer solution: 10-50 mmol/L2- (N-morpholino) ethanesulfonic acid (MES) buffer, preferably 15-30mmol/L; the pH is 4.5-6.5, preferably 5.2-5.8.
(3) Magnetic microsphere sealing liquid: glycine (Gly) and Bovine Serum Albumin (BSA) mixture, wherein the glycine concentration is 0.5% -2%, preferably 0.75% -1.25%; the concentration of Bovine Serum Albumin (BSA) solution is 1% -5%, preferably 1.5% -3%.
(4) Magnetic microsphere preservation solution: 4- (2-hydroxyethyl) piperazine-1-2-hydroxypropanesulfonic acid (HEPSO) +bovine serum albumin (BSA) +proclin300+ Tween 20 solution, wherein the HEPSO concentration is 10-100mmol/L, preferably 20-50mmol/L; BSA concentration is 0.5% -3%, preferably 1% -2%; proclin300 concentration was 0.05%; tween 20 concentration is 0.1% -1%, preferably 0.3% -0.6%, and PH of the solution is 7.1-8.5, preferably 7.5-8.0.
(5) Enzyme labeling buffer: 5-50mmol/LPBS buffer, preferably at a concentration of 20-30mmol/L; the pH is 6.0-7.5, preferably 6.8-7.2.
(6) Enzyme blocking agent: 1% -5% Bovine Serum Albumin (BSA), preferably 2% -3%.
(7) Enzyme preservation solution: PBS solution+bovine serum albumin (BSA) +glycerol+proclin 300; wherein the concentration of the PBS solution is 20-100mmol/L, preferably 40-70mmol/L; BSA concentration is 0.2% -2%, preferably 0.5% -1%; glycerol concentration is 1% -10%, preferably 3% -5%, proclin300 concentration is 0.05%;
(8) Magnetic microsphere activator: EDC and NHS activator are weighed and prepared according to the concentration of 20mg/ml, and are weighed and prepared for use in a laboratory to prevent the activation of the activator.
(9) Enzyme crosslinker 1:0.1-10% glutaraldehyde, preferably 0.5% -2%.
(10) Enzymatic crosslinker 2:1% -10% amino acid, preferably 2% -5%.
3. Reagent preparation
3.1 preparation of reagent M
(1) And (3) cleaning magnetic beads: taking 1mL of magnetic microsphere MS160/Carboxyl (purchased from JSR company, carboxyl magnetic microsphere with particle size of 1.5 μm and concentration of 10%), placing on a magnetic separation frame for 2min, removing supernatant, adding 2mL of magnetic microsphere activation buffer (concentration of 30-50mmol/L, pH of 5.0-5.5), uniformly mixing, re-suspending, and repeating the steps for 3 times;
(2) Activation of magnetic beads: adding 0.25mL of 20mg/mLNHS and 0.25mL of 20mg/mL magnetic microsphere activator into 2mL of the magnetic beads washed in the step (1), uniformly mixing by vortex, and then uniformly mixing for 30min at 25-30 ℃;
(3) And (3) cleaning magnetic beads: after the activation of the magnetic beads is finished, placing the activated magnetic bead solution on a magnetic frame for 2min, removing supernatant, adding 2mL of magnetic microsphere coating buffer solution (with the concentration of 15-30mmol/L and the pH of 5.2-5.8), uniformly mixing and re-suspending, and repeating the steps for 3 times;
(4) And (3) coating magnetic beads: adding 0.1mg of monoclonal antibody 4T21-3C7 (purchased from Hytest company) into 0.5mL of magnetic microsphere coating buffer solution (with the concentration of 15-30mmol/L and the pH of 5.2-5.8), mixing uniformly by vortex, and then placing the mixture at the temperature of 25-30 ℃ for uniform mixing for 2 hours;
(5) And (3) magnetic bead sealing: after the coating of the magnetic beads is finished, 0.5mL of magnetic microsphere sealing liquid (glycine concentration is 0.75-1.25 percent, BSA concentration is 1.5-3 percent) is added, and the mixture is uniformly mixed by vortex and then is uniformly mixed for 30min at 25-30 ℃;
(6) And (3) magnetic bead preservation: after the magnetic bead is closed, placing the magnetic bead solution on a magnetic rack for standing for 2min, removing supernatant, adding 2mL of magnetic microsphere preservation solution (HEPSO concentration is 20-50mmol/L, BSA concentration is 1% -2%, proclin300 concentration is 0.05%, tween 20 concentration is 0.3% -0.6%, pH value of the solution is 7.5-8.0), uniformly mixing and resuspending, and preserving at 2-8 ℃ after repeating the steps for 3 times.
3.2 preparation of reagent R1
(1) Enzyme activation: 200. Mu.L of alkaline phosphatase P0114 (purchased from Merck company) is added into 300. Mu.L of enzyme labeling buffer (PBS buffer preferably has the concentration of 20-30mmol/L; pH value is preferably 6.8-7.2), then 500. Mu.L of enzyme cross-linking agent 1 (glutaraldehyde preferably has the concentration of 0.5-2%) is added, uniformly mixed by vortex, and uniformly mixed for 1h at 20-25 ℃ in the dark;
(2) Enzyme dialysis: dialyzing the activated enzyme solution in enzyme labeling buffer solution (PBS buffer solution preferably with concentration of 20-30mmol/L; pH value preferably with pH value of 6.8-7.2) (dialysis bag aperture: 100 kDa), dialyzing at 2-8deg.C for 24 hr, and changing solution for 4 times;
(3) Enzyme-amino acid cross-linking: after the enzyme solution is dialyzed, 200 mu L of enzyme cross-linking agent 2 (the amino acid concentration is preferably 2% -5%) is added into the enzyme solution, and the enzyme solution is uniformly mixed for 1h at 20-25 ℃ in a dark place;
(4) Enzyme dialysis: dialyzing the crosslinked enzyme solution in enzyme labeling buffer solution (PBS buffer solution preferably with concentration of 20-30mmol/L; pH value preferably of 6.8-7.2) (dialysis bag aperture: 100 kDa), dialyzing at 2-8deg.C for 24 hr, and changing solution for 4 times;
(5) Enzyme-antibody labeling: after the step (4) is completed, adding 0.2mg of polyclonal antibody 4T21/2 (purchased from Hytest company) into the enzyme solution, uniformly mixing by vortex, and uniformly mixing for 4 hours at 2-8 ℃ in a dark place;
(6) Enzyme blocking: after the step (5) is finished, adding 300ul of enzyme blocking agent (preferably 2% -3% of BSA concentration) into the enzyme solution, and uniformly mixing for 1h at 2-8 ℃ in a dark place;
(7) Enzyme purification: after enzyme sealing is completed, the enzyme solution is purified and concentrated by an ultrafiltration tube (purchased from Millipore,4mL,100 kDa) to make the final volume of the enzyme solution be 450-550uL;
(8) Preserving enzyme solution: the purified enzyme solution was diluted 1mL with enzyme preservation solution (PBS solution concentration preferably 40-70mmol/L; BSA concentration preferably 0.5% -1%; glycerol concentration preferably 3% -5%, proclin300 concentration 0.05%) and stored at-18℃to-25 ℃.
3.3 preparation of reagent R2
The preparation of reagent R2 was carried out according to the following formulation:
tris concentration is 10-100mmol/L, preferably 20-50mmol/L; the concentration of the high molecular polymer is 0.1% -1%, preferably 0.2% -0.5%; the concentration of the hemolytic agent is 0.5% -3%, preferably 1% -2%; trehalose concentration is 1% -5%, preferably 2% -3%; proclin300 concentration was 0.05%; the pH of the solution is from 6.5 to 8.0, preferably from 7.0 to 7.5.
Filtering with 0.22 μm microporous membrane after solution preparation, and storing at 2-8deg.C.
The reagent M, the reagent R1 and the reagent R2 of the kit of the invention are completed by the steps described above.
In particular to the embodiment, the high-sensitivity assay kit for troponin I in a human whole blood sample is prepared by the following method:
1. preparation of reagent M
(1) And (3) cleaning magnetic beads: taking 1mL of magnetic microsphere MS160/Carboxyl (purchased from JSR company, carboxyl magnetic microsphere with particle size of 1.5 μm and concentration of 10%), placing the mixture on a magnetic separation frame for 2min, removing supernatant, adding 2mL of magnetic microsphere activation buffer (concentration of 30mmol/L, pH of 5.0), uniformly mixing and re-suspending, and repeating the steps for 3 times;
(2) Activation of magnetic beads: adding 0.25mL of 20mg/mLNHS and 0.25mL of 20mg/mL magnetic microsphere activator into 2mL of the magnetic beads washed in the step (1), uniformly mixing by vortex, and then uniformly mixing for 30min at 25-30 ℃;
(3) And (3) cleaning magnetic beads: after the activation of the magnetic beads is finished, placing the activated magnetic bead solution on a magnetic frame for 2min, removing supernatant, adding 2mL of magnetic microsphere coating buffer solution (with the concentration of 20mmol/L and the pH of 5.5), uniformly mixing and re-suspending, and repeating the steps for 3 times;
(4) And (3) coating magnetic beads: adding 0.1mg of monoclonal antibody 4T21-3C7 (purchased from Hytest company) into 0.5mL of magnetic microsphere coating buffer solution (concentration 20mmol/L, pH 5.5), mixing with vortex, and mixing at 25-30deg.C for 2 hr;
(5) And (3) magnetic bead sealing: after the coating of the magnetic beads is finished, 0.5mL of magnetic microsphere sealing liquid (glycine concentration is 1.0 percent, BSA concentration is 1.75 percent) is added, the mixture is uniformly mixed by vortex, and then the mixture is uniformly mixed for 30 minutes at the temperature of 25-30 ℃;
(6) And (3) magnetic bead preservation: after the magnetic bead is closed, placing the magnetic bead solution on a magnetic rack for standing for 2min, removing the supernatant, adding 2mL of magnetic microsphere preservation solution (HEPS 0 concentration is 50mmol/L; BSA concentration is 2%, proclin300 concentration is 0.05%, tween 20 concentration is 0.5%, pH value of the solution is 7.8), uniformly mixing and re-suspending, and preserving at 2-8 ℃ after repeating the steps for 3 times.
2. Preparation of reagent R1
(1) Enzyme activation: 200. Mu.L of alkaline phosphatase P0114 (purchased from Merck company) is added into 300. Mu.L of enzyme labeling buffer (PBS buffer preferably has the concentration of 20mmol/L; pH value is preferably 6.8), then 500. Mu.L of enzyme cross-linking agent 1 (glutaraldehyde preferably has the concentration of 1%) is added, the mixture is uniformly mixed by vortex, and the mixture is uniformly mixed for 1 hour at 20-25 ℃ in the absence of light;
(2) Enzyme dialysis: dialyzing the activated enzyme solution in enzyme labeling buffer solution (PBS buffer solution preferably with concentration of 20mmol/L; pH value preferably of 6.8) (dialysis bag aperture: 100 kDa), dialyzing at 2-8deg.C for 24 hr, and changing solution for 4 times;
(3) Enzyme-amino acid cross-linking: after the enzyme solution is dialyzed, 200 mu L of enzyme cross-linking agent 2 (amino acid is selected to be lysine, and the concentration of lysine is 2.5%) is added into the enzyme solution, and the mixture is uniformly mixed for 1h at 20-25 ℃ in a dark place;
(4) Enzyme dialysis: dialyzing the crosslinked enzyme solution in enzyme labeling buffer solution (PBS buffer solution preferably with concentration of 20mmol/L; pH value preferably of 6.8) (dialysis bag aperture: 100 kDa), dialyzing at 2-8deg.C for 24 hr, and changing solution for 4 times;
(5) Enzyme-antibody labeling: after the step (4) is completed, adding 0.2mg of polyclonal antibody 4T21/2 (purchased from Hytest company) into the enzyme solution, uniformly mixing by vortex, and uniformly mixing for 4 hours at 2-8 ℃ in a dark place;
(6) Enzyme blocking: after the step (5) is finished, adding 300ul of enzyme blocking agent (preferably 2% of BSA concentration) into the enzyme solution, and uniformly mixing for 1h at 2-8 ℃ in a dark place;
(7) Enzyme purification: after enzyme blocking was completed, the enzyme solution was purified and concentrated using an ultrafiltration tube (available from Millipore,4ml,100 kda) to a final volume of 500uL;
(8) Preserving enzyme solution: the purified enzyme solution was diluted to 1mL with an enzyme preservation solution (PBS solution concentration 55mmol/L; BSA concentration 0.75%; glycerol concentration 5%, proclin300 concentration 0.05%) and stored at-18℃to-25 ℃.
3. Preparation of reagent R2
The preparation of reagent R2 was carried out according to the following formulation:
tris concentration 50 mmol/L; the concentration of the high molecular polymer (PVP 10000 is selected) is 0.3 percent; the concentration of hemolytic agent (select saponin) is 1%; trehalose concentration was 2%; proclin300 concentration was 0.05%; the pH of the solution was 7.0. Filtering with 0.22 μm microporous membrane after solution preparation, and storing at 2-8deg.C.
Example 2
This example is based on example 1, except that the reagent R2 component is removed, and other processes and components are the same as in example 1.
Example 3
This example was carried out in the same manner as in example 1 except that the concentration of lysine in the enzyme crosslinking agent 2 was changed to 3.5% based on example 1 and that the component R2 was removed.
Example 4
This example is based on example 1 with the amino acid in enzyme cross-linker 2 replaced with arginine, while the R2 component of the reagent is removed, other processes and components being the same as in example 1.
Comparative example 1
Preparing a reagent according to the method for preparing the high-sensitivity troponin kit in the patent CN 109580954A;
magnetic separation reagent: tris (6.05 g/L), naCl (9 g/L), BSA (1 g/L), tween-20 (1 g/L), primary antibody (Hytest, cat. No. 916:0.15 mg/mL), secondary antibody (Hytest, cat. 3C7:0.15 mg/mL), pH 7.2.
Enzyme-labeled antibody reagent: tris (6.05 g/L), BSA (20 g/L), mgCl2 (0.2 g/L), znCl2 (0.2 g/L), third antibody (Hytest, cat# 560:0.2 mg/mL), second antibody (Hytest, cat# MF4:0.2 mg/mL), pH 7.4.
Comparative example 2
Preparing a reagent according to the method of the high-sensitivity troponin I kit and the sample treatment fluid in the patent CN 110082541A; the preparation method of the reagent comprises the following steps:
(1) Preparing a magnetic bead coating: the magnetic bead coating is a mixture of magnetic particles coated with anti-cardiac troponin I antibodies, tris-HCI, bovine serum albumin, sodium chloride, proclin 300 and Tween-20, wherein the magnetic particles are carboxylated magnetic particles; the anti-cardiac troponin I antibody comprises two kinds of monoclonal antibodies which respectively recognize amino acids 41 to 49 of cardiac troponin I and recognize amino acids 87 to 91. In the magnetic bead coating, each component and the corresponding final concentration are respectively as follows: 0.2mg/mL of magnetic particles coated with anti-cardiac troponin I antibody, 50mM Tris-HC1.1.0% (w/v) bovine serum albumin, 0.9% (w/v) sodium chloride, 0.48% (w/v) Proclin 300, 0.1% (w/v) Tween-20 (pH 7.4).
(2) Preparing an enzyme label: the enzyme label is a mixture of anti-cardiac troponin I antibody, MES, ovalbumin, sodium chloride, zinc chloride, magnesium chloride, proclin300 and Triton X-100 (pH 6.0) for marking alkaline phosphatase, and the anti-cardiac troponin I antibody is a monoclonal antibody for recognizing 27 th to 40 th amino acids of cardiac troponin I. The respective components and the corresponding final concentrations in the enzyme markers are respectively: 4ug/mL of labeled alkaline phosphatase anti-cardiac troponin I antibody, 50mM MES, 1.0% ovalbumin, 0.9% (w/v) sodium chloride, 0.1mM zinc chloride, 5mM magnesium chloride, 0.48% (w/v) Proclin300, 0.1% (w/v) Triton X-100 (pH 6.0).
(3) Preparing a sample treatment solution: the sample treatment solution was a mixture of Tris-HCI, bovine serum albumin, sodium chloride, dextran sodium sulfate, proclin300 and PluronicF-68 (pH 7.4). The sample treatment fluid comprises the following components and corresponding final concentrations: 50mM Tris-HC1.1.0% (w/v) bovine serum albumin, 0.9% (w/v) sodium chloride, 1.5% (w/v) dextran sodium sulfate, 0.48% (w/v) Proclin300 and 0.1% (w/v) Pluronic F-68 (pH 7.4).
(4) Preparing a cleaning solution: the washing solution was a mixture of Tris-HCI, sodium chloride, triton X-405, procin 300 and Brij-35 (pH 7.4). The components and the corresponding final concentrations in the cleaning liquid are respectively as follows: 50mM Tris-HC1, 0.9% (w/v) sodium chloride, 0.1% (w/v) Triton X-405, 0.48% (w/v) Proclin 300.0.1% Brij-35 (pH 7.4).
(5) Preparing a substrate solution: the substrate solution was a mixture of APS-5, tris-HC1, sodium chloride, proclin 300, triton X-100. PluronicF-68 (pH 9.7). Each component in the substrate solution had a final concentration of 0.01% APS-5, 50mM Tris-HCI, 0.9% sodium chloride, 0.48% (w/v) Proclin 300, and 0.1% (w/v) Triton X-100 (pH 9.7), respectively.
The performance of examples 1 to 6 and comparative examples 1 and 2 was evaluated by the following methods:
1. evaluation scheme one (Performance index evaluation)
After calibration on a matched chemiluminescent analyzer, the prepared reagents of control group 1 and examples 2-4 were used to evaluate the blank (LoB), precision, accuracy, linear range and other indicators of the reagents. The evaluation method of each index is as follows:
(1) Blank (LoB) evaluation: the saline was divided into 10 aliquots and tested for 6 days, 1 per sample per day, giving 60 values in total, and the 60 values were statistically analyzed:
if the test results meet the normal distribution, loB =μ B +1.645δ B ；
If the test result is a non-normal distribution, loB =pct B 100-α，Pct B ＝NB(95/100)+0.5。
The test results are shown in tables 1-4 and figures 1-4:
TABLE 1 blank evaluation data-control 1 (Unit: ng/L)
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According to the statistical analysis of the data, p=0.200 > 0.005, the normal distribution was met, and LoB of the control group 1 was obtained according to the formula: loB =12.70+1.645×1.473=15.12 ng/L.
TABLE 2 blank evaluation data-example 2 (Unit: ng/L)
According to the statistical analysis of the data, p=0.016 > 0.005, the normal distribution was met, and LoB for example 2 was given according to the formula: loB =1.97+1.645×0.45=2.71 ng/L.
TABLE 3 blank evaluation data-example 3 (Unit: ng/L)
According to the statistical analysis of the data, p=0.200 > 0.005, which is consistent with a normal distribution, loB for example 3 is given according to the formula: loB =1.81+1.645×0.486=2.61 ng/L.
TABLE 4 blank evaluation data-control group 4 (Unit: ng/L)
According to the statistical analysis of the data, p=0.200 > 0.005, which is in accordance with the normal distribution, loB for example 4 is given according to the formula: loB =1.97+1.645×0.581=2.93 ng/L.
As can be seen from the above results, the reagent blank (LoB) (. Ltoreq.2.93 ng/L) of examples 2, 3, 4 was lower than that of control 1 (LoB) (. Ltoreq.15.12 ng/L).
(2) Precision: the serum samples with the concentration of 15ng/L, 30ng/L and 500ng/L are tested, each sample is tested for 10 times, and the average value, the Coefficient of Variation (CV) and the relative deviation (Bias) of the measured results are calculated, so that the use requirements can be met when the CV is less than 10% and the deviation is less than 10%;
TABLE 5 precision evaluation data (Unit: ng/L)
According to the results of testing three concentration samples, it can be seen that when the reagent of the control group 1 tests low-value samples (15 ng/L and 30 ng/L), the test variation Coefficient (CV) and the relative deviation are large (> 10%), and cannot be satisfied, and the reagents of examples 2, 3 and 4 test samples with 3 concentrations, and the test variation Coefficient (CV) and the relative deviation are small (.ltoreq.10%), so as to satisfy the use requirement. Examples 2, 3, 4 test precision (CV) was due to control group 1.
(3) Accuracy: 3 samples of national standard substances (GBW 09229,0.223 g/g) with theoretical concentrations of 30ng/L, 500ng/L and 5000ng/L are diluted according to a proportion, the 3 samples are tested, each sample is tested for 3 times, the relative deviation between the result of the 3 tests and the theoretical concentration is calculated respectively, and the relative deviation meets < + -20% and can meet the use requirement.
TABLE 6 accuracy evaluation data (Unit: ng/L)
From the test results, it can be seen that the sample deviation of 30ng/L of the reagent test of the control group 1 exceeds + -20%, and the relative deviation of 3 samples tested in examples 2, 3 and 4 is within + -20%. The test accuracy of examples 2, 3, 4 was higher than that of control group 1.
(4) Linear range: taking a low-concentration clinical serum sample (L) with the concentration of 1ng/L and a high-concentration clinical serum sample (H) with the concentration of 50000ng/L, carrying out equal-proportion dilution to obtain 15 samples with concentration gradients, testing the 15 concentration gradient samples by using a reagent, testing each sample for 3 times, calculating the average value, taking the theoretical concentration (xi) obtained by dilution as an independent variable, taking the detection result average value (yi) as the dependent variable to obtain a linear regression equation, and calculating the actual concentration of the diluted sample by using the regression equation. Calculating a correlation coefficient (r) of the linear regression according to the following formula, wherein the result accords with |r| and is more than or equal to 0.9900; the relative deviation between the measured concentration and the actual concentration was calculated and the result of the relative deviation was less than or equal to.+ -. 10%, and the results are shown in Table 7 and FIG. 5.
TABLE 7 Linear Range evaluation data (Unit: ng/L)
As can be seen from the test results, when the sample concentration ranges from 1 to 50000ng/L, the linear regression coefficients of examples 2, 3 and 4 are all 0.9999, the relative deviation of the test concentration from the sample dilution concentration is not more than +/-10% and the concentration range is 4.1 to 50000ng/L, while the control group 1 is in the range of from 1 to 50000ng/L, the linear regression coefficient is 0.9885, the relative deviation of the test concentration from the sample dilution concentration is not more than +/-10% and the concentration range is 25.4 to 12500.8ng/L. The test linearity ranges of examples 2, 3, 4 are wider than the control group.
Evaluation protocol one demonstrates that the high sensitivity troponin I kit of this patent claims to have a higher sensitivity and a wider linearity than patent CN109580954 a.
2. Evaluation scheme II (detection rate of kit)
After calibration on a corresponding matched chemiluminescent analyzer using the reagents prepared in control group 2 and example 1, the collected blood samples were evaluated for detection rate. The evaluation method is as follows: collecting fresh apparent healthy human serum samples, 210 men and 208 women, carrying out statistical analysis on 99 th percentile detection rate of the male samples and the female samples by using the reagents prepared in the control group 2 and the example 1, analyzing 99 th percentile values of sexes of men and women, and analyzing the detection rate.
The detection results are as follows:
table 8 control group 2 test results (Unit: ng/L)
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Control group 2 apparent health crowd statistics (Unit: ng/L)
TABLE 9 example 1 test results (Unit: ng/L)
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Control group 2 apparent health crowd statistics (Unit: ng/L)
According to statistical analysis, compared with the patent CN110082541A, the reagent has higher detection rate for detecting apparent healthy crowd samples, can detect samples with lower concentration, has higher sensitivity, and the test result of the patent CN110082541A does not accord with the standard of the existing IFCC to the high-sensitivity troponin I detection reagent, and accords with the standard.
3. Evaluation scheme III (sample correlation detection)
100 human serum samples are collected, a detection reagent is prepared for detection according to the method of the embodiment 1, the detection result is compared with the detection result of a control reagent (Abbott, STAT High Sensitive Troponin-l reagent kit), the correlation between the embodiment 1 and the control reagent sample is analyzed, the correlation r is more than or equal to 0.975, the reliable detection result is indicated, and the results are shown in the table 10 and fig. 6.
The detection results are as follows:
TABLE 10 control reagents and example 1 test results
Examples and control reagents 100 samples were tested, and the correlation coefficient r=0.9970 > 0.975 of the detection results, the linear regression equation being y= 1.0049x-12.144, was subjected to significance analysis, and P < 0.05.
4. Evaluation scheme IV (different types of sample accuracy detection)
And collecting 40 human serum samples, and plasma samples and whole blood samples (heparin lithium anticoagulant and EDTA anticoagulant) from the same sample, wherein the analysis is carried out by using the detection reagent of the embodiment 1, and the relative deviation between the analysis and the detection result of the serum samples is within +/-10%, namely the use requirement is met, and the results are shown in Table 11 and figures 7-10.
TABLE 11 different types of sample test results (ng/L)
According to the analysis of the test results of the reagent test serum sample and the plasma whole blood sample in the embodiment 1, the same serum sample, EDTA plasma, the whole blood sample, heparin lithium plasma and the whole blood sample are tested, the relative deviation is within +/-10%, and the correlation R2 of the test sample is more than 0.99, so that the use requirement is met.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (6)

1. A method for preparing a high sensitivity assay kit for troponin I in a sample of human whole blood, comprising:
coating a mouse anti-human troponin I monoclonal antibody by using magnetic microspheres to obtain a reagent M;
the alkaline phosphatase sequentially carries out a crosslinking reaction with an enzyme crosslinking agent 1 and an enzyme crosslinking agent 2, and the alkaline phosphatase after the crosslinking reaction is used for marking troponin I sheep polyclonal antibody to obtain a reagent R1;
the enzyme cross-linking agent 1 is glutaraldehyde solution, and the enzyme cross-linking agent 2 is amino acid solution;
the amino acid is lysine or arginine;
The concentration of glutaraldehyde solution is 0.5-2% glutaraldehyde, the concentration of amino acid solution is 2% -5%, and the molar ratio of alkaline phosphatase to glutaraldehyde is 1: (1-5), the molar ratio of alkaline phosphatase to amino acid is 1: (1-10); the molar ratio of alkaline phosphatase to troponin I sheep polyclonal antibody is 1: (0.1 to 0.5);
preparing a reagent R2 solution according to a reagent R2 formula, wherein the reagent R2 formula is as follows:
tris:20-50 mmol/L; PVP10000:0.2% -0.5%; saponins: 1% -2%; trehalose: 2% -3%; proclin300:0.05%; pH:7.0-7.5;
filtering the solution of the reagent R2 with a microporous filter membrane of 0.22 mu m after the preparation of the solution of the reagent R2 is finished, obtaining the reagent R2, and preserving the reagent R2 at the temperature of 2-8 ℃;
the mass ratio of the magnetic microsphere to the mouse anti-human troponin I monoclonal antibody is as follows: 1, (0.05-0.2), before coating the mouse anti-human troponin I monoclonal antibody, the magnetic microsphere is activated by adopting a magnetic microsphere activator 1 and a magnetic microsphere activator 2, wherein the magnetic microsphere activator 1 is NHS of 20mg/mL, the magnetic microsphere activator 2 is EDC of 20mg/mL, and the mass ratio of the magnetic microsphere to the NHS is 1: (2-15), the mass ratio of the magnetic microsphere to EDC is 1 (1-10).
2. The method for preparing the high-sensitivity assay kit for troponin I in human whole blood samples according to claim 1, wherein the activation process of the magnetic microspheres is carried out in a magnetic microsphere activation buffer solution, the magnetic microspheres are washed before and after activation, the activated and washed magnetic microspheres are resuspended by adopting a magnetic microsphere coating buffer solution, the magnetic microspheres are coated with a mouse anti-human troponin I monoclonal antibody and then are sealed by adopting a magnetic microsphere sealing solution, and the supernatant is removed and then the magnetic microspheres are stored in a magnetic microsphere storage solution, so that the reagent M is obtained.
3. The method for preparing a kit for assaying troponin I in a human whole blood sample according to claim 2, wherein the magnetic microsphere activation buffer is 20-100mmol/L of 2- (N-morpholin) ethanesulfonic acid buffer having a pH of 4.5-6.5;
the magnetic microsphere coating buffer solution adopts 10-50 mmol/L2- (N-morpholine) ethanesulfonic acid buffer solution, and the pH value is 4.5-6.5;
the magnetic microsphere sealing liquid adopts a mixed liquid of glycine and bovine serum albumin, wherein the concentration of glycine is 0.5-2%, and the concentration of bovine serum albumin is 1-5%;
the magnetic microsphere preservation solution comprises the chemical components of 4- (2-hydroxyethyl) piperazine-1-2-hydroxy propane sulfonic acid, bovine serum albumin, proclin300 and tween 20, wherein the concentration of the 4- (2-hydroxyethyl) piperazine-1-2-hydroxy propane sulfonic acid is 10-100mmol/L, the concentration of the bovine serum albumin is 0.5-3%, the concentration of the Proclin300 is 0.05%, the concentration of the tween 20 is 0.1-1%, and the pH value of the solution is 7.1-8.5.
4. The method for preparing the kit for the high sensitivity measurement of troponin I in human whole blood according to claim 1, wherein the specific process of sequentially reacting alkaline phosphatase with enzyme cross-linking agent 1 and enzyme cross-linking agent 2 comprises the following steps:
adding alkaline phosphatase into enzyme marking buffer solution, adding enzyme cross-linking agent 1, and uniformly mixing and activating;
After dialysis, adding an enzyme crosslinking agent 2 for light-shielding crosslinking reaction;
the crosslinked alkaline phosphatase is dialyzed and then labeled with troponin I sheep polyclonal antibody;
and (3) after the alkaline phosphatase marks the troponin I goat polyclonal antibody, adding an enzyme blocking agent for light-shielding reaction, purifying after the blocking is finished, and storing in an enzyme preservation solution.
5. The method for preparing a kit for assaying troponin I in a sample of human whole blood according to claim 4, wherein the enzyme-labeled buffer is 5 to 50mmol/LPBS buffer and the pH is 6.0 to 7.5;
the enzyme blocking agent is 1% -5% bovine serum albumin;
the chemical components of the enzyme preservation solution comprise PBS solution, bovine serum albumin, glycerol and Proclin300, wherein the concentration of the PBS solution is 20-100mmol/L, the concentration of BSA is 0.2% -2%, the concentration of the glycerol is 1% -10%, and the concentration of the Proclin300 is 0.05%.
6. A kit for the high sensitivity measurement of troponin I in a sample of human whole blood, comprising a reagent M, a reagent R1 and a reagent R2, wherein the reagent M, the reagent R1 and the reagent R2 are prepared by a method for preparing a kit for the high sensitivity measurement of troponin I in a sample of human whole blood according to any one of claims 1 to 5.
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