CN109358009B - Cystatin C determination kit, preparation method and detection method thereof - Google Patents
Cystatin C determination kit, preparation method and detection method thereof Download PDFInfo
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- CN109358009B CN109358009B CN201811260548.4A CN201811260548A CN109358009B CN 109358009 B CN109358009 B CN 109358009B CN 201811260548 A CN201811260548 A CN 201811260548A CN 109358009 B CN109358009 B CN 109358009B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
Abstract
The invention relates to a cystatin C determination kit, a preparation method and a detection method thereof, wherein the cystatin C determination kit comprises a reagent R1 and a reagent R2, and the reagent R1 is an aqueous solution prepared from the following components in concentration: 7.3-7.7 g/L of glycine, 18.0-22.0 g/L of polyethylene glycol, 0.8-1.2 mL/L of biological preservative and sodium hydroxide, wherein the molecular weight range of the polyethylene glycol is 5000-8000; the reagent R2 is an aqueous solution prepared from the following components in concentration: 7.3-7.7 g/L of glycine, 1.8-2.2 g/L of protective agent, 0.8-1.2 mL/L of biological preservative, 147.0-153.0 mL/L of anti-human Cys-C antibody latex particles and sodium hydroxide. According to the cystatin C determination kit, a reagent R1 and a reagent R2 are respectively formed by screening components containing specific proportions, so that the lower limit of detection on Cys-C in serum reaches 0.2mg/L, the sensitivity is high, the overall linear range can reach 10.0mg/L, the precision is high, the interference capability on other components in serum is strong, and the specificity is good.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a cystatin C determination kit, and a preparation method and a detection method thereof.
Background
Cystatin C (cysstatin C, Cys-C), also known as cystatin C, is a low molecular weight protein consisting of 122 amino acids with a positive charge (isoelectric point 9.3) that can pass freely through glomerular filtration membranes. All nucleated cells of the human body produce Cys-C at a constant rate, and its concentration in serum is closely related to the Glomerular Filtration Rate (GFR). In the diagnosis of various diseases such as renal insufficiency, liver cirrhosis, tumor and the like, the serum creatinine and creatinine elimination rate can be well replaced, and the serum creatinine and creatinine elimination rate can be a new ideal marker capable of reflecting GFR.
Cystatin C is a secreted protein that is widely found in various body fluids, such as cerebrospinal fluid, blood, urine, saliva, seminal plasma, and the like. The concentration in cerebrospinal fluid and seminal plasma of human body is higher, and the concentration in urine is lowest. The concentration of Cys-C in normal blood plasma is 1.0mg/L, the concentration of Cys-C in amniotic fluid is similar to that of blood plasma, and the concentration of Cys-C in cerebrospinal fluid is 5.8mg/L, which is 5-6 times higher than that of normal blood plasma; the concentration in seminal plasma is highest, and is 51mg/L on average; the mean urine concentration and plasma concentration were 0.1:0.8 mg/L.
In clinical practice, cystatin C is used more and more widely in renal function and thyroid gland detection. With the clinical application of Cys-C, methods for detecting serum cystatin C are increasing, and the methods are roughly divided into the following two types according to the principle:
(1) heterogeneous methods such as a one-way immunodiffusion method (RID), a Radioimmunoassay (RIA), a Fluorescent Immunoassay (FIA), an enzyme-linked immunoassay (ELISA), etc.;
(2) homogeneous methods, such as particle counting immunoassays, Particle Enhanced Transmission Immunoturbidimetry (PETIA), particle enhanced scattering immunoturbidimetry (PENIT), and the like.
Because the concentration of cystatin C in serum is low, the determination method needs higher analysis sensitivity and specificity. Lofberg et al first determined cystatin C content using one-way immunodiffusion and enzyme immunoassay, however, both methods are not only time consuming, but also have poor detection limits (300 ug/L and 30ug/L, respectively). The subsequent reports of simpler and more sensitive methods for radiation, fluorescence and various enzyme immunoassays have shown that the detection limit is reduced, but the assay time is still long, and cannot be used conventionally or even in emergency treatment. I.e., using heterogeneous assay methods, automation is difficult to achieve. The sensitivity and specificity of the homogeneous particle-enhanced transmission turbidimetric immunoassay (PETIA) and the particle-enhanced scattering turbidimetric immunoassay (PENIT) are relatively high, but are still greatly influenced by components such as bilirubin, hemoglobin and chyle in serum.
Disclosure of Invention
Based on this, it is necessary to provide a cystatin C assay kit with high sensitivity, and a preparation method and a detection method thereof.
A cystatin C determination kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is an aqueous solution prepared from the following components in concentration:
wherein the molecular weight range of the polyethylene glycol is 5000-8000;
the reagent R2 is an aqueous solution prepared from the following components in concentration:
in one embodiment, the reagent R1 is an aqueous solution prepared from the following components in concentration:
the reagent R2 is an aqueous solution prepared from the following components in concentration:
in one embodiment, the biological preservative in both the reagent R1 and the reagent R2 is Proclin 300.
In one embodiment, the protein protectant is bovine serum albumin.
In one embodiment, the polyethylene glycol has a molecular weight of 5500-7500.
In one embodiment, the anti-human Cys-C antibody latex particles are selected from anti-human Cys-C antibody latex particles manufactured by yuans biotechnology (shanghai) ltd.
In one embodiment, the cystatin C assay kit further comprises a calibrator; the calibrator is selected from Cys-C calibrators produced by Beijing century Ward Biotechnology Limited, and the concentrations of the calibrator are respectively 0.5mg/L, 1mg/L, 2mg/L, 4mg/L and 8 mg/L.
A preparation method of the cystatin C determination kit comprises the following steps:
preparing a reagent R1: weighing the components according to the composition of the reagent R1, stirring and dissolving glycine with purified water which is 4/5 times of the total volume of the prepared solution, adding sodium hydroxide to adjust the pH value of the solution to 8.60 +/-0.1, sequentially adding polyethylene glycol and a biological preservative, dissolving, fixing the volume, and storing at 2-8 ℃;
preparing a reagent R2: weighing the components according to the composition of the reagent R2, stirring and dissolving glycine in purified water which is 4/5 times of the total volume of a pre-prepared solution, adding sodium hydroxide to adjust the pH value of the solution to 8.60 +/-0.1, adding a biological preservative to dissolve, then mixing a protein protective agent and anti-human Cys-C antibody latex particles uniformly, fixing the volume, and storing at the temperature of 2-8 ℃;
and subpackaging the prepared reagent R1 and the reagent R2 according to the volume ratio of 4:1, and assembling the subpackaged reagent R1 and the subpackaged reagent R2 in a one-to-one manner to obtain the reagent.
A detection method of the cystatin C determination kit comprises the following steps:
the test conditions selected are as follows:
| dominant wavelength | 530nm～550nm | Sub-wavelength | Is free of |
| Analytical method | Two-point end point method | Reaction time | 10min |
| Correction method | Spline | Reaction direction | Up |
The tests were carried out according to the following table:
and (3) calculating: the standard curve was obtained using 6 points: taking distilled water from the 1 st tube, inputting the value of 0.00g/L, taking the calibrators from the 2 nd tube to the 6 th tube, inputting the absorbance difference value of each calibrator obtained by the test, and drawing a standard curve by taking Delta A as a vertical coordinate and a concentration value as a horizontal coordinate; correcting the standard curve by adopting a nonlinear Spline mode or a Logit-Log5P mode to obtain a corrected standard curve; and calculating the content of cystatin C in the sample to be detected according to the corrected standard curve.
The invention has the beneficial effects that:
the cystatin C (CysC) determination kit (latex enhanced turbidimetry) adopts a latex enhanced immunoturbidimetry, and the reagent R1 and the reagent R2 are respectively formed by screening components containing specific proportions, so that the lower limit of detection on Cys-C in serum can reach 0.2mg/L, the sensitivity is high, the overall linear range can reach 10.0mg/L, the precision is high, the anti-interference capability on other components in serum is strong, and the specificity is good.
Detailed Description
The present invention will now be described more fully hereinafter for purposes of facilitating understanding thereof, but it may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1
The embodiment provides a cystatin C (Cys-C) determination kit (latex enhanced turbidimetry), which comprises a reagent R1, a reagent R2 and a calibrator which are packaged independently, wherein the reagent R1 is an aqueous solution prepared from the following components in concentration:
the reagent R2 is an aqueous solution prepared from the following components in concentration:
the particles are selected from anti-human Cys-C antibody latex particles manufactured by Yuanliter Biotechnology (Shanghai) Inc., having a product number of C0323.
The calibrator is Cys-C calibrator prepared by Beijing century Ward Biotechnology Limited, and the concentrations are 0.5mg/L, 1mg/L, 2mg/L, 4mg/L and 8mg/L respectively.
The preparation method of the cystatin C (Cys-C) assay kit (latex enhanced turbidimetry) provided in this embodiment includes the following steps:
preparation of reagent R1: accurately weighing glycine, stirring with 4/5 times of purified water to dissolve glycine, adding appropriate amount of sodium hydroxide to adjust pH value of the solution to 8.60 + -0.1, sequentially adding accurately weighed polyethylene glycol 6000 and Proclin300, stirring, dissolving, mixing, adding the rest purified water to constant volume to the prepared volume after completely dissolving, slowly mixing, and storing at 2-8 deg.C.
Preparation of reagent R2: accurately weighing glycine, stirring and dissolving the glycine by using purified water with 4/5 times of the pre-prepared volume, adding a proper amount of sodium hydroxide to adjust the pH value of the solution to 8.60 +/-0.1, sequentially adding the accurately weighed Proclin300, stirring, dissolving and uniformly mixing, adding bovine serum albumin and anti-human CysC antibody latex particles after complete dissolution, stirring and uniformly mixing, finally adding the rest of purified water, fixing the volume to the pre-prepared volume, slowly mixing, and storing at 2-8 ℃.
Subpackaging; subpackaging the prepared reagent R1 and reagent R2 according to the volume ratio of 4:1, and assembling the subpackaged reagent R1, the subpackaged reagent R2 and the calibrator in a one-to-one manner to obtain the reagent.
Comparative example 1
The comparative example provides a cystatin C (Cys-C) assay kit (latex enhanced turbidimetry), comprising a reagent R1 and a reagent R2 which are independently packaged, wherein the reagent R1 is an aqueous solution prepared from the following components in concentration:
the reagent R2 is an aqueous solution prepared from the following components in concentration:
comparative example 2
The comparative example provides a cystatin C (Cys-C) assay kit (latex enhanced turbidimetry), comprising a reagent R1 and a reagent R2 which are independently packaged, wherein the reagent R1 is an aqueous solution prepared from the following components in concentration:
the reagent R2 is an aqueous solution prepared from the following components in concentration:
reliability verification
The invention also provides a detection method of the cystatin C (Cys-C) determination kit (latex enhanced turbidimetry), which comprises the following steps:
the test conditions (different computer parameters can be requested according to different detection instruments) are selected as the following table 1:
TABLE 1 test conditions
| Dominant wavelength | 548(530～550)nm | Sub-wavelength | Is free of |
| Analytical method | Two-point end point method | Reaction time | 10min |
| Correction method | Spline | Reaction direction | Up |
The test procedure is shown in table 2 below:
TABLE 2 specific test procedures
Remarking: the reagent and sample amount can be properly increased and decreased according to the requirements of different biochemical analyzers in proportion.
And (3) calculating:
the standard curve was determined using 6 points: taking distilled water from the 1 st tube, taking a calibration product from the 2 nd to 6 th tubes, inputting the absorbance difference value of each calibration product obtained by testing, drawing a standard curve by taking the Delta A as a vertical coordinate and the concentration value as a horizontal coordinate, correcting the standard curve by adopting modes such as a nonlinear method Spline, a Logit-Log5P and the like, and storing the corrected standard curve for calculating a result. Different batches of reagents must be re-plotted against the standard curve.
And (4) advising each laboratory to establish a quality control system and selecting proper quality control products to perform quality control. The quality control measurement value should be within a prescribed range. If the content is out of the specified range, corresponding measures are necessary or a manufacturer is contacted.
According to the detection method, the tested serum samples are all from the hospital clinical laboratory, the linear range test, the precision test and the interference resistance test are respectively carried out on the example 1, the comparative example 2 and the conventional commercial determination kit (latex enhanced turbidimetry) of the nine-strong and Ningbo-sourced cystatin C (CysC), and the statistical results are shown in the following tables 3 and 4:
TABLE 3 statistical table of results
Table 4 results of precision test of example 1 (N ═ 8)
As can be seen from the above table 3 and the above table 4, compared with the comparative example 1 and the comparative example 2, the cystatin C (Cys-C) determination kit (latex enhanced turbidimetry) in example 1 has the advantages that the lower limit of detection of Cys-C in serum reaches 0.2mg/L, the sensitivity is high, the overall linear range reaches 10.0mg/L, the precision is high, and the linear requirement of the cystatin C (Cys-C) determination kit which is commercially available conventionally can be met by forming the reagent R1 and the reagent R2 by using the components in the specific ratio. Compared with the conventional cystatin C (Cys-C) determination kit, the cystatin C (Cys-C) determination kit in the embodiment 1 has strong anti-interference capability on other components in serum, particularly has no cross reaction with beta 2-MG, and has better specificity.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (4)
1. The cystatin C determination kit is characterized in that the detection wavelength of the cystatin C determination kit is 530nm-550nm, the analysis method is a two-point end point method and a nonlinear Spline mode or a Logit-Log5P mode is adopted to correct a standard curve, and the cystatin C determination kit comprises a Cys-C calibrator, a reagent R1 and a reagent R2, the concentrations of the calibrator are 0.5mg/L, 1mg/L, 2mg/L, 4mg/L and 8mg/L respectively, and the reagent R1 is an aqueous solution prepared from the following components:
wherein the molecular weight range of the polyethylene glycol is 5500-7500;
the reagent R2 is an aqueous solution prepared from the following components in concentration:
the biological preservative in the reagent R1 and the reagent R2 is Proclin300, the protein protective agent is bovine serum albumin, and the anti-human Cys-C antibody latex particles are selected from anti-human Cys-C antibody latex particles produced by Yuansheng Biotechnology (Shanghai) Co., Ltd;
when the reagent is used for detection, the volume ratio of the reagent R1 to the reagent R2 is 4: 1.
2. The cystatin C assay kit according to claim 1, characterized in that the reagent R1 is an aqueous solution prepared from the following components in concentration:
the reagent R2 is an aqueous solution prepared from the following components in concentration:
3. a method for preparing a cystatin C assay kit according to claim 1 or 2, characterized by comprising the steps of:
preparing a reagent R1: weighing the components according to the composition of the reagent R1 in claim 1 or 2, stirring and dissolving glycine with purified water which is 4/5 times of the total volume of the pre-prepared solution, adding sodium hydroxide to adjust the pH value of the solution to 8.60 +/-0.1, sequentially adding polyethylene glycol and a biological preservative, dissolving, fixing the volume, and storing at 2-8 ℃;
preparing a reagent R2: weighing the components according to the composition of the reagent R2 in claim 1 or 2, stirring and dissolving glycine in purified water which is 4/5 times of the total volume of the pre-prepared solution, adding sodium hydroxide to adjust the pH value of the solution to 8.60 +/-0.1, adding a biological preservative to dissolve, then adding a protein protective agent and anti-human Cys-C antibody latex particles, uniformly mixing, fixing the volume, and storing at 2-8 ℃;
and subpackaging the prepared reagent R1 and the reagent R2 according to the volume ratio of 4:1, and assembling the subpackaged reagent R1 and the subpackaged reagent R2 in a one-to-one manner to obtain the reagent.
4. A detection method of the cystatin C assay kit according to claim 1 or 2, characterized by comprising the steps of:
the test conditions were chosen as indicated in the following table:
The tests were carried out according to the procedure shown in the following table:
and (3) calculating: the standard curve was obtained using 6 points: taking distilled water from the 1 st tube, and inputting the value of 0.00g/L, respectively taking the calibrators from the 2 nd tube to the 6 th tube, inputting the absorbance difference value of each calibrator obtained by the test, and drawing a standard curve by taking Delta A as a vertical coordinate and concentration as a horizontal coordinate; correcting the standard curve by adopting a nonlinear Spline mode or a Logit-Log5P mode to obtain a corrected standard curve; and calculating the content of cystatin C in the sample to be detected according to the corrected standard curve.
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| WO2016132182A1 (en) * | 2015-02-20 | 2016-08-25 | Cells For Cells | Devices for studying chemotaxis/adherence in cells wherein gradients and channels are formed using hydrogels |
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| EP2806272A1 (en) * | 2009-08-07 | 2014-11-26 | Myriad RBM, Inc. | Methods and devices for detecting kidney transplant rejection |
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