CN113238049A - Lipoprotein-associated phospholipase A2 kit and preparation method thereof - Google Patents
Lipoprotein-associated phospholipase A2 kit and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
Abstract
Lipoprotein-associated phospholipase A2 kit and a preparation method thereof, belonging to the technical field of kits. Solves the problems of low precision, low sensitivity, poor reagent stability, long time consumption and low working efficiency of the enzyme-linked immunosorbent assay in the prior art. The kit comprises a reagent 1, a reagent 2, a calibrator, a low value of a quality control material and a high value of the quality control material; wherein, the reagent 1 comprises a buffer solution, an enhancer, salt ions and a preservative; the reagent 2 comprises buffer solution, latex microspheres combined with anti-human Lp-PLA2 antibody, salt ions, a blocking agent and a preservative; the calibrator, the low value of the quality control product and the high value of the quality control product are respectively and independently dilution buffer containing Lp-PLA2 recombinant antigen or extracted pure natural Lp-PLA2 antigen. The kit has the advantages of strong anti-interference capability, high precision, high sensitivity, wide linear range, good reagent stability, short detection time, high efficiency, convenient use and good monitoring effect on cardiovascular and cerebrovascular diseases.
Description
Technical Field
The invention belongs to the technical field of kits, and particularly relates to a lipoprotein-associated phospholipase A2 kit and a preparation method thereof.
Background
Lipoprotein-associated phospholipase A2(Lp-PLA2) belongs to the phospholipase A2 superfamily, is a protein consisting of 441 amino acids and has a molecular weight of 45 kDa. Lp-PLA2 has the effect of degrading Platelet Activating Factor (PAF) activity and is therefore also called PAF-AH. The platelet activating factor has effects of promoting platelet aggregation, chemotaxis of neutrophil and monocyte, and promoting release of inflammatory mediator such as leukotriene, thereby promoting thrombosis and inflammatory reaction.
There are two types of existing Lp-PLA 2: plasma-type Lp-PLA2 and cell-type Lp-PLA 2. Plasma Lp-PLA2 has a molecular weight of 45 kDa; the cell type Lp-PLA2 includes Lp-PLA2II and Lp-PLA2 Ib. Human plasma Lp-PLA2 is produced mainly by secretion of mature macrophages, monocytes, T lymphocytes, mast cells, hepatocytes and the like, and is regulated by inflammatory mediators. The main roles of Lp-PLA2 include: the method has the advantages of generating eicosanoid inflammatory mediators, participating in phospholipid reconstruction and stable balance of biological membranes, metabolizing lipoprotein, transmitting cell signals, reacting hosts, promoting self-disappearance of necrotic tissues of organisms and the like.
Lp-PLA2 is a vascular specific inflammatory enzyme, has the advantage of having very small correlation with other risk factors compared with other inflammatory factors such as CRP, is concerned with clinical diseases, and the detection of the indexes has important clinical significance for risk assessment of disease conditions, monitoring during treatment, prevention of relapse and the like. For example, the level and activity of human serum Lp-PLA2 are positively correlated with the risk of cardiovascular diseases, and the level or activity of Lp-PLA2 in the serum is obviously increased, so that the serum can be regarded as an independent risk factor for diseases such as cardiovascular diseases and cerebral arterial thrombosis. Therefore, Lp-PLA2 can be used as a new index for early diagnosis, risk assessment and clinical detection of various cardiovascular diseases.
In the prior art, the determination method of Lp-PLA2 mainly comprises a mass method and an enzyme activity method. The enzyme activity method is mainly a continuous monitoring method, the detection principle is that the specific recognition of the difference of enzyme and substrate active sites is utilized, the Lp-PLA2 catalytic capability is directly reflected, the method can detect serum and plasma, but compared with a quality method, the enzyme activity method has the advantages of relatively weak anti-interference capability, relatively low precision, relatively poor reagent stability and relatively low detection efficiency of an integrated assembly line. Therefore, the Lp-PLA2 quality method is recommended by the international guidelines and the expert advice on clinical application of lipoprotein-related phospholipase A2. The most common quality method is enzyme-linked immunosorbent assay, which is the earliest domestic Lp-PLA2 detection method, can detect Lp-PLA2 quality by adopting serum or plasma and utilizing an antigen-antibody specificity identification mode, but has low precision, poor stability of the reagent after opening the bottle, long detection time and low working efficiency.
Disclosure of Invention
The invention aims to solve the problems of low precision, low sensitivity, poor reagent stability, long time consumption and low working efficiency of an enzyme-linked immunosorbent assay in the prior art, and provides a lipoprotein-associated phospholipase A2 kit and a preparation method thereof.
The technical scheme adopted by the invention for achieving the purpose is as follows.
The lipoprotein-associated phospholipase A2 kit comprises a reagent 1, a reagent 2, a calibrator, a low value of a quality control product and a high value of the quality control product;
the reagent 1 comprises a buffer solution, an enhancer, salt ions and a preservative;
the reagent 2 comprises buffer solution, latex microspheres combined with anti-human Lp-PLA2 antibody, salt ions, a blocking agent and a preservative;
the calibrator, the quality control product low value and the quality control product high value are respectively and independently solutions containing Lp-PLA2 recombinant antigen or extracted pure natural Lp-PLA2 antigen, and the solvent is a dilution buffer solution.
Preferably, in the reagent 1 and the reagent 2, the buffer solution is one or more selected from a Tris buffer solution, a phosphate buffer solution, a glycine buffer solution, a Hepes buffer solution, a borate buffer solution, a citrate buffer solution, an acetate buffer solution or an ammonium chloride buffer solution, and the concentration is 10-200 mM.
Preferably, in the reagent 1 and the reagent 2, the enhancer is PEG6000, the salt ion is sodium chloride, the preservative is PC300, and the blocking agent is BSA.
Preferably, in the reagent 2, the anti-human Lp-PLA2 antibody is a polyclonal antibody or a monoclonal antibody; more preferably, the polyclonal antibody is a rabbit anti-human Lp-PLA2 antibody, a goat anti-human Lp-PLA2 antibody, a chicken anti-human Lp-PLA2 antibody or a mouse anti-human Lp-PLA2 antibody; the monoclonal antibody is a mouse anti-human Lp-PLA2 antibody.
Preferably, in the reagent 2, the latex microspheres are one or more of polystyrene latex particles, acrylic latex particles or fluorescent latex particles; more preferably, the latex microspheres are one or a combination of polystyrene latex particles with the particle size of 50-100 nm and polystyrene latex particles with the particle size of 120-500 nm.
Preferably, in the reagent 2, the mass percentage of the latex microspheres to which the anti-human Lp-PLA2 antibody is bound is 0.1-1%.
Preferably, in the reagent 2, in the latex microspheres combined with the anti-human Lp-PLA2 antibody, the combination mode of the anti-human Lp-PLA2 antibody and the latex microspheres is chemical crosslinking or physical adsorption; more preferably, the anti-human Lp-PLA2 antibody and the latex microsphere are chemically cross-linked, and the surface of the latex microsphere is modified with one or more of carboxyl, amino and hydroxyl; particularly preferably, the surface of the latex microsphere is modified with carboxyl, and the latex microsphere and the anti-human Lp-PLA2 antibody are chemically crosslinked by a carboxyl and amino covalent coupling method.
Preferably, the preparation method of the reagent 2 comprises the following steps:
firstly, adding latex microspheres into an activation buffer solution containing an activating agent, activating for 30-60 min, centrifuging at 15000rmp/min for 45-60 min, and discarding the supernatant; then dropwise adding a coupling buffer solution, and performing ultrasonic dispersion and activation under the ultrasonic conditions: the power is 500W, the ultrasound is 5s, the interval is 5s, and the ultrasound time is 5 min; then, dropwise adding an anti-human Lp-PLA2 antibody, reversing the mixer at 37 ℃ and uniformly mixing, and carrying out coupling reaction for 2.0-3.0 h; finally, dropwise adding a preservation buffer solution, and reversing and uniformly mixing the mixture for 30-60 min by using a mixer at 37 ℃; carrying out ultrasound under the conditions of 500W of power, 5s of ultrasound, 5s of interval and 30min of ultrasound time to obtain a reagent 2;
the activating buffer solution and the coupling buffer solution are the same, and the preparation process comprises the following steps: purified water 1L, MES 25mM, pH6.0 (25. + -. 0.1 ℃), 0.22 μm filter;
the preparation process of the preservation buffer solution comprises the following steps: purified water 1L, Hepes25mM, sodium chloride 0.9%, BSA 0.6%, PC 3000.1%, pH8.5 + -0.1 (25 + -0.1 deg.C), 0.22 μm filter membrane filtration.
Preferably, the dilution buffer comprises: 25mM citric acid monohydrate, 0.9% sodium chloride, 3.0% BSA, 0.5% trehalose, 0.1% Proclin300 and 3.0% mannitol.
Preferably, the concentration value of the calibrator is 800ng/mL, the concentration value of the low value of the quality control substance is 200 +/-15% ng/mL, and the concentration value of the high value of the quality control substance is 400 +/-15% ng/mL.
The detection principle of the lipoprotein-associated phospholipase A2 kit provided by the invention is as follows: latex particles bound with anti-human Lp-PLA2 antibody, when mixed with a sample containing Lp-PLA2 antigen, undergo an agglutination reaction, causing a change in absorbance, the magnitude of which is proportional to the Lp-PLA2 antigen content of the sample. Comparing the absorbance change with a calibrator with a known concentration, the content of Lp-PLA2 in the sample can be quantitatively obtained.
Compared with the prior art, the invention has the beneficial effects that:
1) the lipoprotein-associated phospholipase A2 kit adopts a latex immunoturbidimetry method and adopts an EDC cross-linking agent one-step method to realize efficient covalent coupling of functional groups on the surfaces of latex microspheres and amino groups on immunocompetent molecules, so that the stability of the reagent is enhanced;
2) the lipoprotein-associated phospholipase A2 kit avoids the binding site of LDL and Lp-PLA2 by optimizing an antibody, reduces the non-specific reaction of a reagent, and optimizes and improves the preparation process of the binding reagent, so that the prepared reagent has stable performance, simple and rapid operation and batch controllability.
3) The lipoprotein-associated phospholipase A2 kit has strong specificity, has no significant difference when being compared with Lp-PLA2 for data statistical analysis of the detection kit, has reliable and stable detection results, effectively avoids the condition of inaccurate measured value caused by unqualified anti-interference of the reagent, and can meet the requirements of clinical application.
4) The lipoprotein-associated phospholipase A2 kit has good reagent performance, high detection and analysis sensitivity, and good reagent reliability, repeatability and stability (the storage at 2-8 ℃ is more than or equal to 12 months), is suitable for a biochemical analyzer, and is easier to integrate into a production line to improve the detection efficiency.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a process of the test method of Lp-PLA2 in an embodiment of the present invention;
FIG. 2 is raw material screening test data for example 1;
FIG. 3 is a graph of data from a latex microsphere raw material screening test of example 2 in accordance with the present invention;
FIG. 4 is a graph of the activation ratio versus the amount of antibody dosed in example 3 of the present invention;
FIG. 5 is the linear detection correlation of Lp-PLA2 in example 5 of the present invention;
FIG. 6 is an accelerated stability study of Lp-PLA2 reagent of example 8 of the invention;
FIG. 7 is a real-time stability study of Lp-PLA2 reagent of example 8 of the present invention;
FIG. 8 shows the unsealing stability of Lp-PLA2 reagent in example 8 of the present invention;
FIG. 9 shows the detection result of the Lp-PLA2 kit of the invention of example 9;
FIG. 10 shows the detection result of the Lp-PLA2 kit of the prior art in example 9 of the present invention.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention, but it is to be understood that the description is intended to illustrate further features and advantages of the invention, and not to limit the scope of the claims.
The lipoprotein-associated phospholipase A2 kit comprises a reagent 1, a reagent 2, a calibrator, a low value of a quality control product and a high value of the quality control product; the reagent 1 comprises a buffer solution, an enhancer, salt ions and a preservative; the reagent 2 comprises buffer solution, latex microspheres combined with anti-human Lp-PLA2 antibody, salt ions, a blocking agent and a preservative; the calibrator, the low value of the quality control product and the high value of the quality control product are respectively and independently solutions containing Lp-PLA2 recombinant antigen or extracted pure natural Lp-PLA2 antigen, and the solvent is a dilution buffer solution.
According to the technical scheme, the buffer solution of the reagent 1 is one or more selected from Tris buffer solution, phosphate buffer solution, glycine buffer solution, Hepes buffer solution, borate buffer solution, citrate buffer solution, acetate buffer solution or ammonium chloride buffer solution, and the concentration of the buffer solution is 10-200 mM; preferably Tris buffer, at a concentration of 70 mM. The reinforcing agent in the reagent 1 is mainly used for promoting latex aggregation in the reaction process of the reagent 1, the reagent 2 and a sample, so that an antibody is more easily combined with an antigen, the specific reaction of the antigen and the antibody is enhanced, and the reactivity of the reagent is further improved; in order to avoid non-specific reaction and latex microsphere aggregation caused by excessive addition, the mass percent of the reinforcing agent is preferably 0.75%; the enhancer is preferably PEG 6000. Preferably, the salt ions are sodium chloride, and the mass percent of the sodium chloride is 0.9%. Preferably, the preservative is PC300, and the mass percent of the preservative is 0.1%. Preferably, the pH of reagent 1 is 7.35. + -. 0.1 (25. + -. 0.1 ℃ C.).
According to the technical scheme, the buffer solution of the reagent 2 is one or more selected from Tris buffer solution, phosphate buffer solution, glycine buffer solution, Hepes buffer solution, borate buffer solution, citrate buffer solution, acetate buffer solution or ammonium chloride buffer solution, and the concentration of the buffer solution is 10-200 mM; preferably Hepes buffer, at a concentration of 25 mM. The blocking agent is preferably BSA, which is mainly used for blocking carboxyl sites on the latex microspheres which are not combined with the antibody, so as to prevent latex from agglutinating and avoid competitive combination with the antibody caused by excessive use, and the concentration of the BSA is preferably 0.45 wt%. Preferably, the salt ions are sodium chloride, and the mass percent of the sodium chloride is 0.9%. Preferably, the preservative is PC300, and the mass percent of the preservative is 0.1%. The anti-human Lp-PLA2 antibody is a polyclonal antibody or a monoclonal antibody; the polyclonal antibody is rabbit anti-human Lp-PLA2 antibody, goat anti-human Lp-PLA2 antibody, chicken anti-human Lp-PLA2 antibody or mouse anti-human Lp-PLA2 antibody; the monoclonal antibody is a murine anti-human Lp-PLA2 antibody. The latex microspheres are one or more of polystyrene latex microspheres, acrylic latex microspheres or acrylic acid related ester latex microspheres; preferably, the latex microspheres are one or a combination of polystyrene latex particles with the particle size of 50-100 nm and polystyrene latex particles with the particle size of 120-500 nm. In the reagent 2, the mass percentage of the latex microspheres against the human Lp-PLA2 antibody is preferably 0.1-1%. In the latex microspheres combined with the anti-human Lp-PLA2 antibody, the combination mode of the anti-human Lp-PLA2 antibody and the latex microspheres is chemical crosslinking or physical adsorption; preferably, the combination mode of the anti-human Lp-PLA2 antibody and the latex microsphere is chemical crosslinking, and the surface of the latex microsphere is modified with one or more of carboxyl, amino and hydroxyl; if the surface of the latex microsphere is modified with carboxyl, the latex microsphere and the anti-human Lp-PLA2 antibody are chemically crosslinked by a carboxyl and amino covalent coupling method. Preferably, reagent 2 has a pH of 8.5. + -. 0.1 (25. + -. 0.1 ℃ C.).
In the above technical solution, the dilution buffer preferably includes: 25mM citric acid monohydrate, 0.9% sodium chloride, 3.0% BSA, 0.5% trehalose, 0.1% Proclin300 and 3.0% mannitol; the concentration value of the calibrator is preferably 800ng/mL, the concentration of the low value of the quality control substance is preferably 200 +/-15% ng/mL, and the concentration of the high value of the quality control substance is preferably 400 +/-15% ng/mL; the pH is preferably 7.35. + -. 0.1 (25. + -. 0.1 ℃ C.), independently of one another.
In the present invention, the reagent 1 and the reagent 2 are free from any influence on the binding site of Lp-PLA2 antigen or Lp-PLA2 antibody, because the steric hindrance of LDL can be eliminated by preferably selecting an anti-human lipoprotein-associated phospholipase A2 antibody without adding any special dissociation agent. Avoids sample-to-sample differences caused by improper use of the dissociation agent in the enzymatic method. And the result of false negative of enzyme method detection caused by human gene mutation can not be generated.
In the preparation of the kit, the preparation of the reagent 1, the calibrator, the low value of the quality control material and the high value of the quality control material can be prepared according to the requirements without special limitation, and the preparation method of the reagent 2 comprises the following steps:
firstly, adding latex microspheres into an activation buffer solution containing an activating agent, activating for 30-60 min, centrifuging at 15000rmp/min for 45-60 min, and discarding the supernatant; then dropwise adding a coupling buffer solution, and performing ultrasonic dispersion and activation under the ultrasonic conditions: the power is 500W, the ultrasound is 5s, the interval is 5s, and the ultrasound time is 5 min; then, dropwise adding an anti-human Lp-PLA2 antibody, reversing the mixer at 37 ℃ and uniformly mixing, and carrying out coupling reaction for 2.0-3.0 h; finally, dropwise adding a preservation buffer solution, and reversing and uniformly mixing the mixture for 30-60 min by using a mixer at 37 ℃; carrying out ultrasound under the conditions of 500W of power, 5s of ultrasound, 5s of interval and 30min of ultrasound time to obtain a reagent 2;
wherein, the activating buffer solution and the coupling buffer solution are the same, and the preparation process comprises the following steps: purified water 1L, MES 25mM, pH6.0 (25. + -. 0.1 ℃), 0.22 μm filter;
the preparation process of the preservation buffer solution comprises the following steps: purified water 1L, Hepes25mM, sodium chloride 0.9%, BSA 0.6%, PC 3000.1%, pH8.5 + -0.1 (25 + -0.1 deg.C), 0.22 μm filter membrane filtration.
The amounts of the respective raw materials in the reagent 2 are calculated from actual conditions so as to satisfy the above-described limitations on the respective substances in the reagent kit 2.
The preparation method of the reagent 2 adopts a unique latex preparation process, so that the stability and the repeatability verification accuracy of the Lp-PLA2 detection reagent are higher, the clinical application is facilitated, the medical cost is reduced, the reagent is mutually beneficial to medical treatment and patients, and the resource waste is avoided.
The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. In order to make those skilled in the art better understand the technical solution of the present invention, the present invention will be further described in detail with reference to the following embodiments.
In the following examples, various procedures and methods not described in detail are conventional methods well known in the art. Materials, reagents, devices, instruments, apparatuses and the like used in the following examples are commercially available unless otherwise specified.
Reagents used in examples:
reagent 1: purified water 1L, Tris 70mM, sodium chloride 0.9%, PEG 60000.75%, PC 3000.1%, pH7.35 + -0.1 (25 + -0.1 deg.C), filtering with 0.22 μm filter membrane, and storing at 2-8 deg.C for use.
Preparation of reagent 2: firstly, 0.075g of carboxyl latex microspheres with the microsphere particle size of 147nm (JSR, P0112) are added into 50mL of activation buffer solution containing 75mM 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide activation, and activation is carried out for 30 min; using a high-speed low-temperature centrifuge to act at 15000rpm/min for 45min, discarding supernatant, dropwise adding 12mL of coupling buffer solution, and ultrasonically dispersing activated latex microspheres under the ultrasonic condition of 500W of power, 5s of ultrasonic, 5s of interval and 5min of ultrasonic time; 7.5mg of anti-human Lp-PLA2 antibody is dripped, the mixture is evenly mixed by inverting the mixer at 37 ℃, and the coupling reaction is carried out for 2.0-3.0 h; dripping 38mL of preservation buffer solution containing 0.6% BSA, and reversing and mixing the solution evenly for 30min at 37 ℃; performing ultrasound under the conditions of 500W of power, 5s of ultrasound, 5s of interval and 30min of ultrasound time; wherein, the activating buffer solution and the coupling buffer solution are prepared as follows: purified water 1L, MES 25mM, pH6.0(25 + -0.1 deg.C), 0.22 μm filter membrane filtering, and storing at 2-8 deg.C; preparing a preservation buffer solution: purified water 1L, Hepes25mM, sodium chloride 0.9%, BSA 0.6%, PC 3000.1%, pH8.5 + -0.1 (25 + -0.1 deg.C), filtering with 0.22 μm filter membrane, and storing at 2-8 deg.C.
In the examples, Lp-PLA2 was measured by using a Dirui biochemical analyzer CS 1200, the procedure is as shown in FIG. 1, and the measurement conditions are as shown in Table 1.
Table 1 measurement conditions of Lp-PLA2 in examples
| Measuring temperature | 37℃ | R1 dosage | 160μL | |
| Dominant wavelength | 546nm | R2 dosage | 40μL | |
| Absorbance range | 0A～3.2A | Sample volume | 8μL | |
| Optical path of cuvette | 1.0 | Reaction time | 300s | |
| Measurement mode | Two-point end point method | Reaction direction | Positive reaction |
Example 1
Lp-PLA2 antibody raw material selection
Reagent performance, clinical reactivity and raw material cost are preferably considered in the antibody screening stage, LDL and Lp-PLA2 binding sites are avoided when selecting antibodies, and the antibody screening stage is combined with a reagent preparation process system for verification. Preparing a process system, and screening antibody raw materials according to the volume ratio of 1.5 to 1.5, 70 mu g/mg of antibody feeding, an activation buffer solution, a coupling buffer solution, a reagent 1 to 2 to a sample amount S of 160 mu L to 40 mu L to 8 mu L, wherein the test results are as follows.
The reactivity of the Lp-PLA2 raw material antibody 1 (rabbit source, pAb), the antibody 2 (rabbit source, pAb) and the antibody 3 (mouse source, mAb) with the Lp-PLA2 antigen is verified by a Dirui full-automatic biochemical analyzer test, raw material screening test data are shown in figure 2, the raw material antibody 1 and the raw material antibody 2 are preferentially selected, and the clinical applicability of the raw material antibody 2 used alone is better in subsequent clinical reactivity verification. In combination with analysis data, the invention proposes to select the preferred antibody to continue reagent reaction system optimization, thereby evaluating reagent performance and clinical application risk.
Example 2
Selection of latex microspheres
Considerations in the selection of latex microspheres include manufacturer, particle size, and carboxyl density. Preparing a process system, and screening the latex microsphere raw material according to the volume ratio of 1.0 to 90 mu g/mg of antibody feeding, the volume ratio of an activation buffer solution to a coupling buffer solution to 1 to 2 reagents to sample amount S of 160 mu L to 40 mu L to 8 mu L, wherein the test result is as follows.
The method is characterized in that a Dirui full-automatic biochemical analyzer is used for testing and verifying the reactivity of latex microspheres 1(JSR, P0011), microspheres 2(JSR, P0115) and microspheres 3(JSR, P0112) and Lp-PLA2 antigen, the raw material screening test data of the latex microspheres are shown in figure 3, microspheres 3 are preferentially selected, and the method is intended to use the microspheres 3 for further verifying the reagent reactivity.
Example 3
Optimization of activation ratio and antibody feeding amount
According to the activation ratio, the antibody feeding and the microsphere concentration of 1.5, 100 mu g/mg and 0.2 percent respectively; 1.5, 70 mug/mg, 0.2%; 1.0, 100 mug/mg, 0.2%; 1.0, 100. mu.g/mg, 0.15% material relationship was coated to verify reagent reactivity.
The verification data is shown in fig. 4, and it can be seen from fig. 4 that too high dosing of the antibody may result in too high sensitivity and narrow linearity, and that appropriate reduction of dosing helps to broaden linearity; reducing the activation ratio or the concentration of microspheres in reagent 2 also contributes to a proper reduction in the reaction sensitivity, broadening the linearity. In addition, in the preparation process of the reagent 2, sufficient ultrasonic conditions are necessarily ensured, and in order to facilitate the reagent production, the coupling process is generally adopted without ultrasonic waves, and after the coupling, the latex reagent is ultrasonically dispersed when the reagent 2 is added to store a buffer solution. The specific ultrasonic time and power need to be found through experiments.
Example 4
Preparation of Lp-PLA2 calibrator and quality control product
The dilution buffer formulation is described in detail below: preparing 1L of basic buffer solution, wherein the buffer solution is 25mM citric acid monohydrate; the salt ion component is 0.9% sodium chloride; the protein stabilizer component is 3.0% BSA; the sugar component is trehalose 0.5%; the preservative component is 0.1% Proclin 300; the excipient is 3.0% mannitol.
The calibrator, the low value of the quality control product and the high value of the quality control product are prepared according to the required concentration. Wherein the required concentration of the calibrator is 800ng/mL, 0.08mg of Lp-PLA2 recombinant antigen is accurately weighed, added into 100mL of dilution buffer solution, and prepared for standby application according to the process after full dissolution; accurately weighing 0.02mg of Lp-PLA2 recombinant antigen with the required low-value concentration of the quality control product of 200 +/-15% ng/mL, adding the Lp-PLA2 recombinant antigen into 100mL of dilution buffer solution, and preparing the Lp-PLA2 recombinant antigen for later use according to the process after the Lp-PLA2 recombinant antigen is fully dissolved; the high-value required concentration of the quality control product is 400 +/-15% ng/mL, 0.04mg of Lp-PLA2 recombinant antigen is accurately weighed and added into 100mL of dilution buffer solution, and the preparation is carried out according to the process for standby after full dissolution. And (3) performing multiple dilution on the calibrator according to the required concentration, calibrating the full-automatic biochemical analyzer, verifying the measurement program, and applying both the low value and the high value of the quality control material to the performance evaluation of a quality system.
Example 5
Lp-PLA2 linear range detection
The high value samples near the upper end of the linear range are diluted in a proportion of at least 5 concentrations (see table 2), where the low value samples must be near the lower end of the linear range. The test was repeated 3 times for each concentration sample using reagents 1 and 2 prepared as exemplified in the reagent preparation examples in the examples, the average value was calculated (see Table 2), and the results were obtainedThe mean value and the dilution ratio are subjected to line fitting by using a least square method, and a linear coefficient R is calculated2(see FIG. 5 and Table 2).
TABLE 2 Linear Range test results (in ng/mL)
As can be seen from Table 2, the deviation between the measured linear dilution value and the estimated value is less than + -5% within 6.0ng/mL-800ng/mL, the equation in FIG. 5 is a linear regression equation, and the linear correlation coefficient R20.999, indicating a good linear correlation coefficient.
Example 6
High dose hook Effect test
The reagent 1 and the reagent 2 prepared by the example of reagent preparation in the embodiment are adopted, samples with the theoretical concentration exceeding the linear range are tested, the test is repeated for 3 times, the average value is taken as the test value, and no false result occurs in the test result.
TABLE 3 high dose hook Effect test results (unit ng/mL)
| Serial number | Theoretical value | Test value | Remarks for |
| 1 | 6.25 | 6.32 | In the |
| 2 | 12.50 | 11.39 | In the |
| 3 | 25.00 | 23.89 | In the |
| 4 | 50.00 | 48.95 | In the |
| 5 | 100.00 | 99.46 | In the linear range |
| 6 | 200.00 | 198.69 | In the linear range |
| 7 | 400.00 | 402.69 | In the linear range |
| 8 | 800.00 | 796.75 | In the linear range |
| 9 | 1600.00 | 1500.69 | Super-linear |
| 10 | 2000.00 | 1987.65 | Super-linear |
| 11 | 2400.00 | 1890.68 | Super-linear |
As can be seen from Table 3, the present invention can satisfy the antigen surplus requirement, and when the concentration value of the detected sample is high, the high-value sample can be measured after being diluted by physiological saline, and the result is multiplied by the dilution factor.
Example 7
Lp-PLA2 reagent repeatability examination
According to the reagent 1 and the reagent 2 prepared by the example of reagent preparation in the examples, the measurement is repeated 10 times for the low value and the high value of the quality control material of 2 samples with different concentrations, the average value (M) and the Standard Deviation (SD) of the measurement results of 10 times are calculated, and the coefficient of variation is obtained according to the following formula. The specific validation data was analyzed as follows:
CV＝SD/M×100％
in the formula: CV is the coefficient of variation;
SD is the standard deviation of 10 measurements;
m is the average of 10 measurements.
TABLE 4 repeatability test results
As can be seen from Table 4, the relative deviation of the detection results of the invention is less than 5%, and the repeatability index meets the requirements.
Example 8
Lp-PLA2 reagent stability study
The reagent preparation is carried out according to the reagent preparation example in the embodiment, and the preparation amount needs to meet the requirements of real-time expiration and accelerated stability performance index investigation and verification.
The results are shown in fig. 6-8, and it can be seen from the graphs that the stability of Lp-PLA2 reagent is verified by using a diry full-automatic biochemical analyzer, and the real-time stability can reach 12 months as shown by verification data; the high temperature can be stabilized for 14 days by the accelerated stability verification of the reagent; the reagent unsealing stability is verified to be stable for 30 days. The deviation of the verification data and the zero-time test data is small, and the performance verification can meet the clinical application requirements.
Example 9
Lp-PLA2 kit application
According to research data, by analyzing data of 100 samples (positive 70% and negative 30%), the Lp-PLA2 latex immunoturbidimetric assay kit and the human plasma lipoprotein phospholipase A2 assay kit (immunoturbidimetric assay) of Shenzhen Jinrui biotechnology limited are clinically compared and analyzed, the detection result is used as the same methodology for comparison, the statistical data correlation and the positive and negative coincidence rate are good, no false positive/negative verification result exists, and the correlation equation is as follows: the correlation coefficient R of Y is 0.986X-0.886 which is more than or equal to 0.975 (figure 9); clinical comparison analysis is carried out with a lipoprotein-associated phospholipase A2 detection kit (rate method) of Yuan Biotechnology Limited in Chongqing, detection results are compared as different methodologies, the statistical data correlation and the yin-yang coincidence rate are good, and the correlation equation is as follows: the correlation coefficient R is not less than 0.975 (figure 10) when Y is 0.411X-76.65. The verification data indicate that the performance index of the Lp-PLA2 kit can meet the clinical application requirement.
In conclusion, the reagent prepared by the Lp-PLA2 process system has stable performance, good repeatability, high detection sensitivity, good correlation between clinical comparison and mainstream competitive product manufacturers (the correlation coefficient R is more than or equal to 0.975), no non-specific comparison data is found, the reagent performance can meet the market application requirements, the accuracy of clinical measurement values is favorably ensured, and doctors are better assisted in making reasonable diagnosis. In the future IVD industry, the method has strong competitive advantages.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. The kit is characterized by comprising a reagent 1, a reagent 2, a calibrator, a low quality control product value and a high quality control product value;
the reagent 1 comprises a buffer solution, an enhancer, salt ions and a preservative;
the reagent 2 comprises buffer solution, latex microspheres combined with anti-human Lp-PLA2 antibody, salt ions, a blocking agent and a preservative;
the calibrator, the quality control product low value and the quality control product high value are respectively and independently solutions containing Lp-PLA2 recombinant antigen or extracted pure natural Lp-PLA2 antigen, and the solvent is a dilution buffer solution.
2. The lipoprotein-associated phospholipase A2 kit of claim 1, wherein in the reagent 1 and the reagent 2, the buffer solution is one or more selected from Tris buffer, phosphate buffer, glycine buffer, Hepes buffer, borate buffer, citrate buffer, acetate buffer or ammonium chloride buffer, the enhancer is PEG6000, the salt ion is sodium chloride, the preservative is PC300, and the blocking agent is BSA.
3. The lipoprotein-associated phospholipase A2 kit of claim 1, wherein in reagent 1 and reagent 2, the buffer solution has a concentration of 10-200 mM, the enhancer has a concentration of 0.75 wt%, the salt ion has a concentration of 0.9 wt%, the preservative has a concentration of 0.1 wt%, and the blocking agent has a concentration of 0.45 wt%.
4. The lipoprotein-associated phospholipase A2 kit of claim 1, wherein in the reagent 2, the anti-human Lp-PLA2 antibody is a polyclonal antibody or a monoclonal antibody; the polyclonal antibody is a rabbit anti-human Lp-PLA2 antibody, a goat anti-human Lp-PLA2 antibody, a chicken anti-human Lp-PLA2 antibody or a mouse anti-human Lp-PLA2 antibody; the monoclonal antibody is a mouse anti-human Lp-PLA2 antibody.
5. The lipoprotein-associated phospholipase A2 kit of claim 1, wherein in the reagent 2, the latex microspheres are one or more of polystyrene latex particles, acrylic latex particles or fluorescent latex particles.
6. The lipoprotein-associated phospholipase A2 kit of claim 5, wherein the latex microsphere is one or a combination of polystyrene latex particles with a particle size of 50-100 nm and polystyrene latex particles with a particle size of 120-500 nm.
7. The lipoprotein-associated phospholipase A2 kit of claim 1, wherein the reagent 2 comprises 0.1-1% by weight of latex microspheres conjugated with anti-human Lp-PLA2 antibody.
8. The kit according to claim 1, wherein the reagent 2 comprises latex microspheres conjugated with anti-human Lp-PLA2 antibody, and the binding mode of the anti-human Lp-PLA2 antibody to the latex microspheres is chemical crosslinking or physical adsorption.
9. The lipoprotein-associated phospholipase A2 kit of claim 1, wherein the preparation method of reagent 2 comprises the following steps:
firstly, adding latex microspheres into an activation buffer solution containing an activating agent, activating for 30-60 min, centrifuging at 15000rmp/min for 45-60 min, and discarding the supernatant; then dropwise adding a coupling buffer solution, and performing ultrasonic dispersion and activation under the ultrasonic conditions: the power is 500W, the ultrasound is 5s, the interval is 5s, and the ultrasound time is 5 min; then, dropwise adding an anti-human Lp-PLA2 antibody, reversing the mixer at 37 ℃ and uniformly mixing, and carrying out coupling reaction for 2.0-3.0 h; finally, dropwise adding a preservation buffer solution, and reversing and uniformly mixing the mixture for 30-60 min by using a mixer at 37 ℃; carrying out ultrasound under the conditions of 500W of power, 5s of ultrasound, 5s of interval and 30min of ultrasound time to obtain a reagent 2;
the activating buffer solution and the coupling buffer solution are the same, and the preparation process comprises the following steps: purified water 1L, MES 25mM, pH6.0 (25. + -. 0.1 ℃), 0.22 μm filter;
the preparation process of the preservation buffer solution comprises the following steps: purified water 1L, Hepes25mM, sodium chloride 0.9%, BSA 0.6%, PC 3000.1%, pH8.5 + -0.1 (25 + -0.1 deg.C), 0.22 μm filter membrane filtration.
10. The lipoprotein-associated phospholipase A2 kit of claim 1,
the dilution buffer comprises: 25mM citric acid monohydrate, 0.9% sodium chloride, 3.0% BSA, 0.5% trehalose, 0.1% Proclin300 and 3.0% mannitol;
the concentration value of the calibrator is 800ng/mL, the concentration of the low value of the quality control product is 200 +/-15% ng/mL, and the concentration value of the high value of the quality control product is 400 +/-15% ng/mL.
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| CN114295567A (en) * | 2021-12-30 | 2022-04-08 | 青岛汉唐生物科技有限公司 | Phospholipase A2 detection kit and preparation method thereof |
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| CN109541201A (en) * | 2018-12-27 | 2019-03-29 | 海格德生物科技（深圳）有限公司 | A method of promoting immunoturbidimetry accuracy |
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| CN102692507B (en) * | 2011-07-29 | 2015-09-16 | 南京诺尔曼生物技术有限公司 | Platelet-activating factor acetylhydro-lase (LPPLA2) measures kit (latex enhancing immune turbidimetry) |
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Application publication date: 20210810 |