CN116068204A - Serum amyloid A detection kit with high sensitivity and good stability - Google Patents

Serum amyloid A detection kit with high sensitivity and good stability Download PDF

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Publication number
CN116068204A
CN116068204A CN202310098385.9A CN202310098385A CN116068204A CN 116068204 A CN116068204 A CN 116068204A CN 202310098385 A CN202310098385 A CN 202310098385A CN 116068204 A CN116068204 A CN 116068204A
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serum amyloid
reagent
detection kit
buffer solution
antibody
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覃肖珍
莫佳陛
苟文来
赵丽珍
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Urit Medical Electronic Co Ltd
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • 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
    • 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
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7095—Inflammation

Abstract

The invention relates to the technical field of medical detection, in particular to a serum amyloid A detection kit with high sensitivity and good stability, which comprises a first reagent, a second reagent, a calibrator and a quality control product; the first reagent comprises: 100-200 mmol/L phosphate buffer solution, 3.0-10.0 g/L sodium chloride, 25-50 g/L polyethylene glycol 6000, 0.5-5.0 g/L Tween-20 and 0.1-0.3 g/LProclin300; the reagent II comprises 0.25 to 0.35 percent of anti-human serum amyloid A antibody sensitization latex microsphere, 100 to 200mmol/L phosphate buffer solution, 1.5 to 4.0g/L sodium chloride, 0.5 to 1g/L fishskin gelatin and 0.1 to 0.3g/LProclin300; the serum amyloid A kit has the advantages of high sensitivity, good repeatability, accurate test result and stable performance, can be widely used in various biochemicals, and has wider universality.

Description

Serum amyloid A detection kit with high sensitivity and good stability
Technical Field
The invention relates to the technical field of medical detection, in particular to a serum amyloid A detection kit with high sensitivity and good stability.
Background
Serum Amyloid A (SAA) is an acute phase response protein with a relative molecular weight of about 12kD, and is mainly produced by hepatocytes, and extrahepatic tissues such as heart and skeletal muscle are also produced.
The clinical value of SAA as an inflammation marker is widely paid attention in recent years, and SAA level change has important clinical value for early diagnosis, risk assessment, curative effect observation and prognosis evaluation of infectious diseases; besides rising in bacterial infection, SAA also rises in viral infection obviously, and according to the rising degree or combined application with other indexes, bacterial or viral infection can be prompted, so that the defect that the current common inflammation markers cannot prompt viral infection is overcome, but the sensitivity of the existing serum amyloid A detection kit is too low, so that the reagent detection result is inaccurate, and the clinical application cannot be satisfied.
Disclosure of Invention
The invention aims to provide a serum amyloid A detection kit with high sensitivity and good stability, and aims to solve the problem that the sensitivity of the existing serum amyloid A detection kit is too low.
In order to achieve the aim, the invention provides a serum amyloid A detection kit with high sensitivity and good stability, which comprises a first reagent, a second reagent, a calibrator and a quality control product;
the first reagent comprises: 100-200 mmol/L phosphate buffer solution, 3.0-10.0 g/L sodium chloride, 25-50 g/L polyethylene glycol 6000, 0.5-5.0 g/L Tween-20 and 0.1-0.3 g/LProclin300;
the reagent II comprises 0.25 to 0.35 percent of anti-human serum amyloid A antibody sensitized latex microsphere, 100 to 200mmol/L phosphate buffer solution, 1.5 to 4.0g/L sodium chloride, 0.5 to 1g/L fishskin gelatin and 0.1 to 0.3g/LProclin300.
Wherein the calibrator and the quality control are diluted to different concentrations for recombinant serum amyloid A antigen with a buffer.
The preparation method of the reagent I comprises the following steps:
and (3) taking purified water, sequentially adding a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative, stirring until the components are completely dissolved, and regulating the pH value to 7.6-8.0 to prepare the reagent I.
The preparation method of the reagent II comprises the following steps:
adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the latex microsphere, and mixing and activating for 30min at room temperature;
adding serum amyloid A antibody into activated latex microspheres, and reacting for 2 hours at room temperature for covalent coupling;
centrifuging the latex microsphere reaction solution obtained after covalent coupling to remove supernatant, and removing free antibody and small molecular impurities to obtain first latex microspheres;
adding ethanolamine and bovine serum albumin into the first latex microsphere, and sealing for 2 hours to prepare a first solution;
and diluting the first solution by 6-10 times by using an R2 preservation solution containing a stabilizer, a preservative and a buffer solution to prepare a reagent II.
The preparation method of the calibrator comprises the following steps:
the recombinant serum amyloid a antigen is diluted with a diluent to a target concentration.
The preparation method of the quality control product comprises the following steps:
the recombinant serum amyloid a antigen is diluted with a diluent to a target concentration.
The serum amyloid A detection kit with high sensitivity and good stability is prepared by using two complementary type anti-human serum amyloid A monoclonal antibodies as the anti-human serum amyloid A antibody sensitization latex microspheres in the reagent II and using a chemical crosslinking method to sensitize the latex microspheres with the average particle size of 60-100 nm; the two serum amyloid monoclonal antibodies are complementary antibodies, can form a space cross-linked structure with the antigen, and play a role in cascade amplification of signal values; the serum amyloid A kit has the advantages of high sensitivity, good repeatability, accurate test result and stable performance, can be widely used in various biochemicals, and has wider universality.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a drawing showing a serum amyloid A kit antibody monoclonal antibody test of the present invention.
FIG. 2 is a graph showing the effect of the serum amyloid A kit antibody two-strain antibody mixing test of the present invention.
FIG. 3 is a table of the sensitivity test of the serum amyloid A kit of the present invention.
FIG. 4 is a table showing the precision of serum amyloid A kit of the present invention.
FIG. 5 is a table of stability tests for serum amyloid A kits of the invention.
FIG. 6 is a flow chart of a method for preparing the reagent one of the present invention.
FIG. 7 is a flow chart of a method for producing the reagent II of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Referring to fig. 1 to 7, the invention provides a serum amyloid a detection kit with high sensitivity and good stability: comprises a first reagent, a second reagent, a calibrator and a quality control product;
the first reagent comprises: 100-200 mmol/L phosphate buffer solution, 3.0-10.0 g/L sodium chloride, 25-50 g/L polyethylene glycol 6000, 0.5-5.0 g/L Tween-20 and 0.1-0.3 g/LProclin300;
the reagent II comprises 0.25 to 0.35 percent of anti-human serum amyloid A antibody sensitized latex microsphere, 100 to 200mmol/L phosphate buffer solution, 1.5 to 4.0g/L sodium chloride, 0.5 to 1g/L fishskin gelatin and 0.1 to 0.3g/LProclin300.
In the embodiment, the anti-human serum amyloid A antibody sensitization latex microsphere in the reagent II is obtained by sensitization of two complementary anti-human serum amyloid A monoclonal antibodies and latex microsphere with average particle size of 60-100 nm by a chemical crosslinking method; the two serum amyloid monoclonal antibodies are complementary antibodies, can form a space cross-linked structure with the antigen, and play a role in cascade amplification of signal values; the serum amyloid A kit has the advantages of high sensitivity, good repeatability, accurate test result and stable performance, can be widely used in various biochemicals, and has wider universality.
Further, the calibrator and the quality control are diluted to different concentrations for recombinant serum amyloid a antigen with a buffer.
Further, the preparation method of the reagent I comprises the following steps:
s101, taking purified water, sequentially adding buffer solution, anti-interference agent, sensitizer, electrolyte and preservative, stirring until the components are completely dissolved, and regulating the pH value to 7.6-8.0 to prepare the reagent I.
Further, the preparation method of the reagent II comprises the following steps:
s201, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into latex microspheres, and mixing and activating for 30min at room temperature;
s202, adding serum amyloid A antibody into activated latex microspheres, and reacting for 2 hours at room temperature for covalent coupling;
s203, centrifuging the latex microsphere reaction solution obtained after covalent coupling to remove supernatant, and removing free antibody and small molecular impurities to obtain first latex microspheres;
s204, adding ethanolamine and bovine serum albumin into the first latex microsphere, and sealing for 2 hours to prepare a first solution;
s205, diluting the first solution by 6-10 times by using an R2 preservation solution containing a stabilizer, a preservative and a buffer solution to prepare a reagent II.
Further, the preparation method of the calibrator comprises the following steps:
the recombinant serum amyloid a antigen is diluted with a diluent to a target concentration.
Further, the preparation method of the quality control product comprises the following steps:
the recombinant serum amyloid a antigen is diluted with a diluent to a target concentration.
The present invention will be described in further detail by way of examples.
1. Preparation of serum amyloid A kit.
The main raw material sources involved in the serum amyloid A kit provided by the invention are as follows:
anti-human serum amyloid a antibodies: anti-human serum amyloid A antibody (accession number: MS01-4C 7), paired anti-human serum amyloid A antibody (accession number: MS 01-Y01) (Gui Linyou Rui Biotech Co., ltd.);
latex microspheres: latex microsphere with carboxyl group and particle size of 80nm (Holms (Beijing) diagnostic technique Co., ltd.)
Recombinant serum amyloid a antigen: SAA-C (Gui Linying Meite Biotechnology Co., ltd.);
EDC: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (Sigma Co.);
NHS: n-hydroxysuccinimide (Sigma Co.);
the main reagents of this example were formulated as follows:
reagent one: 0.1M phosphate buffer solution containing 25g/L polyethylene glycol 6000, 4g/L sodium chloride, 1.0g/L Tween-20 and 0.1g/LProclin300, and having a pH of 7.60+ -0.05;
and (2) a reagent II: the method comprises the following specific steps:
1. 20mL (solid content 10%) of latex microspheres were mixed and dispersed with 40mL25 mmoles of PH6.5 MES buffer.
2. 1.6mL of freshly prepared 50mg/mL 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution and 3.2mL of freshly prepared 50 mg/mLN-hydroxysuccinimide solution were added and reacted at room temperature for 30min;
3. the activated latex microsphere is divided into two parts averagely;
4. 40mg of antibody MS01-4C7 and 40mg of antibody MS01-Y01 were added respectively, and reacted at room temperature for 2 hours.
5. Adding 0.1mL of 10% ethanolamine respectively for blocking for 1h, adding 5mL of 10% BSA solution, and continuing blocking for 1h;
6. after centrifugation for 20min, the supernatant was removed, and 40mL of R2 preservation solution (0.1MPH7.2 phosphate buffer, 2.0g/L sodium chloride, 1g/L fishskin gelatin, 0.1g/LProclin 300) was added and dispersed by sonication.
7. Two latex microspheres were mixed at 1:1, diluting the anti-human serum amyloid A antibody sensitized latex microsphere with R2 preservation solution until the mass fraction is 0.35%, namely the reagent II.
Preparation of recombinant serum amyloid A antigen calibrator and quality control: different recombinant serum amyloid A antigens are added into the buffer solution, and the concentration range of the calibrator is 12.5mg/L, 25.0mg/L, 50.0mg/L, 100.0mg/L and 300.0mg/L. The concentration range of the quality control product is 10.0mg/L and 50.0mg/L.
2. A detection method of serum amyloid A kit.
Detection tool: hitachi 7180 model automatic analyzer.
The analysis method comprises the following steps:
a two-point end point method; a wavelength of 600nm; sample amount 2uL; r1:200uL; r2:50uL; the reaction direction is as follows: ascending; measuring temperature: 37 ℃; after the sample is uniformly mixed with R1, the absorbance A1 is read in 10 seconds; r2 was added at 4 to 5min, and the absorbance A2 was read after 5 min. The absorbance of the reaction was calculated as the difference between A2 and A1.
The calculation method comprises the following steps:
curve fitting calculations were performed using multipoint calibration, using either logic or Spline. Serum amyloid a concentration was determined as Δa.
Figure BDA0004072448670000051
Serum amyloid a kit antibody single-strain antibody test and two-strain antibody mixed test.
As can be seen from fig. 1 and 2, the signal of the single antibody test results is low, and the signal value is high and the linearity is good after mixing the two strains.
4. Serum amyloid a kit sensitivity test.
Purified water is used as a blank sample, which should be free of the test object. Continuously detecting 20 times on a biochemical analyzer, and calculating the average value of 20 test results
Figure BDA0004072448670000061
And Standard Deviation (SD), twice the standard deviation +.>
Figure BDA0004072448670000062
The detection limit of the report range of the kit is used as the report range of the kit. The results are shown in FIG. 3, with a sensitivity of 0.27mg/L.
5. And (5) testing the precision of the serum amyloid A kit.
Samples with the test concentration of (10.0+/-2.0) mg/L and (50.0+/-5.0) mg/L are tested repeatedly for 10 times, and the average value of the measured values is calculated respectively
Figure BDA0004072448670000063
And standard deviation (S) to calculate Coefficient of Variation (CV). The results are shown in FIG. 4, and as can be seen from FIG. 4, the repetitive CV is 3.52% and 2.15%, respectively.
6. Serum amyloid a kit stability test.
Simulating clinical laboratory operation of a hospital, storing the serum amyloid A kit in an environment of 2-8 ℃, keeping the reagent bottle mouth open all the time in the whole process, detecting the internal standard substance (international standard substance NIBSC 92/680) with the concentration of 150.0mg/L in 0 day, 7 day, 14 day, 21 day, 28 day and 30 day respectively, repeatedly testing for 3 times, calculating the relative deviation of the average value and the test average value of 0 day, and lasting for 30 days. The results are shown in fig. 5, and as can be seen from fig. 5, the relative deviation of the test value of the kit for 30 days when the bottle is opened and the test value of the kit for 0 day when the bottle is opened is within 10%.
Through the test, the serum amyloid A kit has the advantages of high sensitivity, good repeatability, accurate test result and stable performance, can be widely used in various biochemicals, and has wider universality.
The above disclosure is only a preferred embodiment of the present invention, and it should be understood that the scope of the invention is not limited thereto, and those skilled in the art will appreciate that all or part of the procedures described above can be performed according to the equivalent changes of the claims, and still fall within the scope of the present invention.

Claims (6)

1. A serum amyloid A detection kit with high sensitivity and good stability is characterized in that,
comprises a first reagent, a second reagent, a calibrator and a quality control product;
the first reagent comprises: 100-200 mmol/L phosphate buffer solution, 3.0-10.0 g/L sodium chloride, 25-50 g/L polyethylene glycol 6000, 0.5-5.0 g/L Tween-20 and 0.1-0.3 g/LProclin300;
the reagent II comprises 0.25 to 0.35 percent of anti-human serum amyloid A antibody sensitized latex microsphere, 100 to 200mmol/L phosphate buffer solution, 1.5 to 4.0g/L sodium chloride, 0.5 to 1g/L fishskin gelatin and 0.1 to 0.3g/LProclin300.
2. A highly sensitive, stable serum amyloid A detection kit as claimed in claim 1 wherein,
the calibrator and the quality control are diluted to different concentrations for recombinant serum amyloid A antigen with a buffer.
3. A highly sensitive, stable serum amyloid A detection kit as claimed in claim 1 wherein,
the preparation method of the reagent I comprises the following steps:
and (3) taking purified water, sequentially adding a buffer solution, an anti-interference agent, a sensitizer, an electrolyte and a preservative, stirring until the components are completely dissolved, and regulating the pH value to 7.6-8.0 to prepare the reagent I.
4. A highly sensitive, stable serum amyloid A detection kit as claimed in claim 1 wherein,
the preparation method of the reagent II comprises the following steps:
adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide into the latex microsphere, and mixing and activating for 30min at room temperature;
adding serum amyloid A antibody into activated latex microspheres, and reacting for 2 hours at room temperature for covalent coupling;
centrifuging the latex microsphere reaction solution obtained after covalent coupling to remove supernatant, and removing free antibody and small molecular impurities to obtain first latex microspheres;
adding ethanolamine and bovine serum albumin into the first latex microsphere, and sealing for 2 hours to prepare a first solution;
and diluting the first solution by 6-10 times by using an R2 preservation solution containing a stabilizer, a preservative and a buffer solution to prepare a reagent II.
5. A highly sensitive, stable serum amyloid A detection kit as claimed in claim 1 wherein,
the preparation method of the calibrator comprises the following steps:
the recombinant serum amyloid a antigen is diluted with a diluent to a target concentration.
6. A highly sensitive, stable serum amyloid A detection kit as claimed in claim 1 wherein,
the preparation method of the quality control product comprises the following steps:
the recombinant serum amyloid a antigen is diluted with a diluent to a target concentration.
CN202310098385.9A 2023-02-10 2023-02-10 Serum amyloid A detection kit with high sensitivity and good stability Pending CN116068204A (en)

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