CN112268868A - Kit for determining calprotectin by latex immunoturbidimetry - Google Patents
Kit for determining calprotectin by latex immunoturbidimetry Download PDFInfo
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- CN112268868A CN112268868A CN202011110762.9A CN202011110762A CN112268868A CN 112268868 A CN112268868 A CN 112268868A CN 202011110762 A CN202011110762 A CN 202011110762A CN 112268868 A CN112268868 A CN 112268868A
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
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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
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Abstract
The invention relates to a kit for determining calprotectin by a latex immunoturbidimetry, which comprises an R1 reagent and an R2 reagent; the R1 reagent comprises an electrolyte, a stabilizer, a surfactant, a preservative, and a buffer; the R2 reagent comprises latex particles coated with anti-human calprotectin polyclonal antibody, electrolyte, stabilizer, surfactant, preservative and buffer solution; the average particle size range of the latex particles is 50-500 nm; the stabilizer of the R1 reagent is selected from sorbitol and/or PEG (polyethylene glycol), and the stabilizer of the R2 reagent is selected from sorbitol and/or PEG (polyethylene glycol). The kit for determining calprotectin by the latex-enhanced immunoturbidimetry has higher stability, and can still maintain higher accuracy under the condition of long-term storage.
Description
Technical Field
The invention relates to the field of medical examination and in-vitro diagnosis, in particular to a kit for determining calprotectin in human blood or excrement by adopting a latex immunoturbidimetry method.
Background
Calprotectin (Calprotectin) is a calcium and zinc binding protein with molecular weight of 36kD, which is composed of heterotrimers formed by covalently linking two heavy chains with molecular weight of 14kD and a light chain with molecular weight of 8kD, each chain can bind two Ca ions, thereby having the characteristics of heat resistance and hydrolysis enhancement, and the protein structure of the Calprotectin is a compound composed of 2 subunits of S-100A8 and S-100A 9. It is distributed in granulocytes, monocytes, keratinocytes and various tissues and body fluids of the human body and is a protective protein.
Calprotectin is an acute inflammatory marker derived from neutrophils and macrophages, is expressed with tissue or cell specificity, is a hybrid calbindin, has protease resistance, and has the ability to chelate zinc ions to provide heat resistance. Calprotectin has multiple biological functions of antimicrobial, immune regulation, antiproliferation, signal transmission, etc., and is elevated in many inflammatory conditions. In an inflammatory reaction, the neutrophil enters into the tissue as an active and positive process, under the action of chemotactic factors, the neutrophil is drilled out of the blood vessel wall to reach an inflammatory area, is differentiated and matured into macrophage, and the macrophage is taken as an effector cell of inflammation to release various inflammatory factors and phagocytize to play the biological function. In neutrophils, calprotectin is predominantly present in the cytoplasm outside of lysozyme, accounting for approximately 5% of the total neutrophil protein. Upon neutrophil activation, phosphorylated calprotectin translocates to the cell membrane and acts as a competitive inhibition substrate for enzymes that inhibit enzymes (casein kinase ii, topoisomerase, etc.) that play an important role in cell proliferation. Calprotectin may also stimulate immunoglobulin production, chemokine activation, and neutrophil fixative activation.
The Latex-enhanced immunoturbidimetric turbidimetric assay (Latex-enhanced immunoturbidimetric turbidimetric assay) is a method for detecting humoral protein homogeneous transmission immunoturbidimetric assay, and is characterized in that polyclonal antibodies are crosslinked on the surfaces of nano-scale polymer Latex microspheres, and after the microspheres crosslinked with the antibodies are combined with antigens, the microspheres can be rapidly gathered together in a short time, so that the light transmittance of a reaction solution is changed; the change of the light transmittance (i.e. absorbance) of the reaction solution has strong correlation with the concentration of the antigen to be detected, and can reflect the concentration of the antigen to be detected in a certain range. The advantages of the latex enhanced immunoturbidimetry are embodied in particular in that: 1. time and labor are saved: antigen and antibody reaction is carried out in a homogeneous reaction system, a full-automatic biochemical analyzer is utilized to directly measure the absorbance value of reaction liquid, the result can be obtained within minutes, and complicated operation steps such as repeated incubation and plate washing of an enzyme-linked immunosorbent assay are omitted; 2. the method is stable and accurate: the simplification of the operation steps correspondingly avoids the interference of a plurality of human operation factors and external factors such as reagents, environment and the like, has better stability and repeatability, and can reflect the content of calprotectin in the blood or excrement of the tested human body more truly; 3. the application is wide: the sensitivity of the latex enhanced immunoturbidimetry is enough to detect the lower limit value of calprotectin, can completely meet the clinical detection requirement, and is obviously superior to the immunochromatography (colloidal gold).
In a prior application CN106771232A of the present application, a kit for determining calprotectin by nano latex enhanced immunoturbidimetry is disclosed, comprising an R1 reagent, an R2 reagent and calprotectin antigen calibrators and quality control solutions, wherein the R1 reagent comprises an electrolyte, a stabilizer, a surfactant, a preservative and a buffer; the R2 reagent comprises latex particles of an anti-human calprotectin polyclonal antibody, electrolyte, a stabilizer, a surfactant, a preservative and a buffer solution; the average particle size range of the latex particles is 50-500 nm. The calprotectin antigen calibrator solution comprises calprotectin and a stabilizing agent. Although the kit has the advantages of stability, accuracy, time saving and labor saving, the kit has the problem of insufficient stability, and particularly has large deviation of detection results after long-term storage.
Disclosure of Invention
The invention aims to provide a kit for determining calprotectin by a latex immunoturbidimetry method, which aims to solve the problem in the background technology, namely how to improve the storage stability of the kit. In order to achieve the purpose of the invention, the following technical scheme is adopted.
The invention relates to a kit for determining calprotectin by a latex immunoturbidimetry method, which comprises an R1 reagent and an R2 reagent; the R1 reagent comprises an electrolyte, a stabilizer, a surfactant, a preservative, and a buffer; the R2 reagent comprises latex particles coated with anti-human calprotectin polyclonal antibody, electrolyte, stabilizer, surfactant, preservative and buffer solution; the average particle size range of the latex particles is 50-500 nm; the stabilizer of the R1 reagent is selected from sorbitol and/or PEG (polyethylene glycol), and the stabilizer of the R2 reagent is selected from sorbitol and/or PEG (polyethylene glycol).
In a preferred embodiment of the invention, the stabilizer of the R1 reagent is selected from sorbitol and the stabilizer of the R2 reagent is selected from PEG (polyethylene glycol).
In a preferred embodiment of the invention, the kit comprises a closable kit body and a reagent fixing piece arranged in the kit body, wherein the reagent fixing piece is a paper fixing piece or a high-molecular fixing piece, a cavity is formed in the reagent fixing piece and comprises a first cavity and a second cavity, the first cavity is provided with a reagent bottle R1, and the second cavity is provided with a reagent bottle R2.
In a preferred embodiment of the present invention, the electrolyte is selected from sodium chloride, potassium chloride, magnesium sulfate, or a combination thereof, and has a concentration of 0.1 to 10%.
In a preferred embodiment of the invention the concentration of the calprotectin antigen in the calibrator and control solutions is in the range 1ng/mL to 100 mg/mL.
In a preferred embodiment of the present invention, the surfactant is selected from tween, fatty alcohol polyglycol ethers, polyoxyethylene phenyl ether or a combination thereof, and the concentration is 0.1-10%.
In a preferred embodiment of the invention, the preservative is selected from sodium azide, phenol, parahydroxybenzoic acid, ethyl parahydroxybenzoate and Proclin series, and the concentration is 0.1-10%.
In a preferred embodiment of the invention, the buffer is selected from MES buffer, boric acid buffer, acetate buffer, phosphate buffer and glycine buffer, the concentration is 10-500 mmol/L, and the pH is 5-9.
In a preferred embodiment of the invention, the anti-human calprotectin polyclonal antibody is coupled to the surface of the latex particles by chemical cross-linking.
In a preferred embodiment of the invention, the method is carried out by chemical cross-linking agents in a cross-linking buffer; the chemical crosslinker is selected from EDC, N-hydroxysuccinimide, N-hydroxythiosuccinimide, carboximides, hydrazides, potassium isocyanate or combinations thereof; the crosslinking buffer solution is selected from MES, MOPSO, MOPS, HEPES and PBS buffer solution, and has the pH value of 6-9.
Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
the kit for determining calprotectin by the latex immunoturbidimetry has higher stability, and can still maintain higher accuracy under the condition of long-term storage.
Drawings
Fig. 1 is a graph showing the test results for sample 1 with different kits as the storage time increases.
Fig. 2 is a graph showing the test results for sample 2 with different kits as the storage time increases.
Fig. 3 is a diagram showing a calibration curve.
Fig. 4 is a graph showing a reaction curve of sample 1.
Fig. 5 is a graph showing a reaction curve of sample 2.
Detailed Description
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Materials and sources:
calprotectin purified human calprotectin was obtained by reference to the genetic recombination technique described in example 1 of CN 106771232A; latex particles coated with anti-human calprotectin polyclonal antibodies were prepared according to CN106771232A examples 2 and 3.
Example 1: preparation of the kit
A kit for determining calprotectin by a latex immunoturbidimetry method comprises an R1 reagent, an R2 reagent and a calprotectin antigen calibrator solution, and comprises a closable box body and a reagent fixing piece arranged in the box body, wherein the reagent fixing piece 2 is a paper fixing piece or a high-molecular material fixing piece, a cavity is formed in the reagent fixing piece and comprises a first cavity and a second cavity, a reagent bottle R1 is arranged in the first cavity, and a reagent bottle R2 is arranged in the second cavity.
R1 reagent: the composition is prepared from the following components in percentage by weight: 3% sodium chloride, 2.5% sorbitol, 5% Tween 80, 0.5% sodium azide, pH7.4, 250mmol/L MES buffer.
R2 reagent: the composition is prepared from the following components in percentage by weight: 4mg/mL of latex particles coated with anti-human calprotectin polyclonal antibody, 3% of sodium chloride, 2.5% of sorbitol, 5% of Tween 80, 0.5% of sodium azide, pH7.4, 250mmol/L of MES buffer.
The prepared kit was stored in a refrigerator at 4 ℃.
Example 2:
sorbitol in the R1 reagent of example 1 was replaced with the same weight of PEG-600, and sorbitol in the R2 reagent was also replaced with the same weight of PEG-600.
Example 3:
only sorbitol in the R2 reagent of example 1 was replaced with the same weight of PEG-600.
Comparative example 1:
sorbitol in the R1 and R2 reagents of example 1 was replaced with the same weight of mannitol.
Comparative example 2
Sorbitol in the reagents R1 and R2 of example 1 was replaced with 1% by weight bovine serum albumin.
Making standard curve examples:
preparing a standard sample by taking human calprotectin purified by a gene recombination technology as a mother solution and taking a bovine serum albumin solution as a dilution mother solution: s0: 0. mu.g/mL, S1: 50. mu.g/mL, S2: 100. mu.g/mL, S3: 250. mu.g/mL, S4: 750 ug/mL, bovine serum albumin concentration 1 mg/mL.
Test examples:
preparation of a standard substance: preparing a sample 1 by using human calprotectin purified by a gene recombination technology as a mother solution and using a bovine serum albumin solution as a dilution mother solution: 200. mu.g/mL and sample 2: 300. mu.g/mL, bovine serum albumin concentration 1 mg/mL.
The kit uses a full-automatic biochemical analyzer to detect the dominant wavelength: 450nm, sub-wavelength: 800 nm.
Reagent: 250. mu.l of the R1 reagent of example 3; 30 μ l of R2 reagent.
Measurement method (two-point end-point method): mu.l of the R1 reagent was added to 5. mu.l of the sample, reacted at 37 ℃ for 3-4 minutes, then 30. mu.l of the R2 reagent was added to start reading the absorbance A1, after 5 minutes the absorbance A2 was read again, the absorbance difference Δ A-A2-A1 was calculated, and the test results were determined from the standard curve (FIG. 3) of calprotectin standard samples (FIGS. 4 and 5).
Examples 1 to 3 and comparative examples 1 and 2 were stored in a refrigerator at 4 ℃ and 3 parts of each were taken out as a detection reagent every 10 days, and the results of the detection were averaged to obtain the results of the detection as shown in FIGS. 1 and 2.
As can be seen from FIGS. 1 and 2, the test results of the kits of comparative examples 1 and 2 were greatly deviated from the calibration values of the standard samples over time, while the test results of examples 1 to 3, particularly example 3, were less deviated from the calibration values of the standard samples, thereby illustrating that the use of the specific stabilizer of the present invention contributes to the improvement of the stability of the kit.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (10)
1. A kit for determining calprotectin by latex immunoturbidimetry, comprising a reagent R1 and a reagent R2; the R1 reagent comprises an electrolyte, a stabilizer, a surfactant, a preservative, and a buffer; the R2 reagent comprises latex particles coated with anti-human calprotectin polyclonal antibody, electrolyte, stabilizer, surfactant, preservative and buffer solution; the average particle size range of the latex particles is 50-500 nm; the stabilizer of the R1 reagent is selected from sorbitol and/or PEG (polyethylene glycol), and the stabilizer of the R2 reagent is selected from sorbitol and/or PEG (polyethylene glycol).
2. The kit of claim 1, wherein the stabilizer of the R1 reagent is selected from sorbitol and the stabilizer of the R2 reagent is selected from PEG (polyethylene glycol).
3. The kit according to claim 1, which comprises a closable box body and a reagent fixing member arranged in the box body, wherein the reagent fixing member is a paper fixing member or a high molecular material fixing member, a cavity is formed in the reagent fixing member and comprises a first cavity and a second cavity, the first cavity is provided with a reagent bottle R1, and the second cavity is provided with a reagent bottle R2.
4. The kit according to claim 1, wherein the electrolyte is selected from sodium chloride, potassium chloride, magnesium sulfate or a combination thereof, and the concentration is 0.1-10%.
5. The kit of claim 1 wherein the concentration of calprotectin antigen in the calibrator and control solutions is from 1ng/mL to 100 mg/mL.
6. The kit according to claim 1, wherein the surfactant is selected from tween, fatty alcohol polyglycol ethers, polyoxyethylene phenyl ether or a combination thereof, and the concentration is 0.1-10%.
7. The kit according to claim 1, wherein the preservative is selected from sodium azide, phenol, parahydroxybenzoic acid, ethyl parahydroxybenzoate, Proclin series, and the concentration is 0.1-10%.
8. The kit according to claim 1, wherein the buffer solution is selected from MES buffer solution, boric acid buffer solution, acetate buffer solution, phosphate buffer solution and glycine buffer solution, the concentration is 10-500 mmol/L, and the pH is 5-9.
9. The kit of claim 1 wherein the anti-human calprotectin polyclonal antibody is coupled to the surface of a latex particle by chemical cross-linking.
10. The kit of claim 1, wherein the method is performed by a chemical cross-linking agent in a cross-linking buffer; the chemical crosslinker is selected from EDC, N-hydroxysuccinimide, N-hydroxythiosuccinimide, carboximides, hydrazides, potassium isocyanate or combinations thereof; the crosslinking buffer solution is selected from MES, MOPSO, MOPS, HEPES and PBS buffer solution, and the pH value is 6-9.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011110762.9A CN112268868A (en) | 2020-10-16 | 2020-10-16 | Kit for determining calprotectin by latex immunoturbidimetry |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011110762.9A CN112268868A (en) | 2020-10-16 | 2020-10-16 | Kit for determining calprotectin by latex immunoturbidimetry |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114062691A (en) * | 2022-01-12 | 2022-02-18 | 苏州和锐生物科技有限公司 | Diluent and calprotectin calibrator |
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| CN102662061A (en) * | 2012-04-17 | 2012-09-12 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
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| CN109613265A (en) * | 2018-12-29 | 2019-04-12 | 中拓生物有限公司 | A kind of kit with latex immunoturbidimetry measurement apoC 3 |
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2020
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114062691A (en) * | 2022-01-12 | 2022-02-18 | 苏州和锐生物科技有限公司 | Diluent and calprotectin calibrator |
| CN114062691B (en) * | 2022-01-12 | 2022-04-01 | 苏州和锐生物科技有限公司 | Diluent and calprotectin calibrator |
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