CN113740539A - Kit for determining specific growth factor - Google Patents
Kit for determining specific growth factor Download PDFInfo
- Publication number
- CN113740539A CN113740539A CN202110917705.XA CN202110917705A CN113740539A CN 113740539 A CN113740539 A CN 113740539A CN 202110917705 A CN202110917705 A CN 202110917705A CN 113740539 A CN113740539 A CN 113740539A
- Authority
- CN
- China
- Prior art keywords
- kit
- reagent
- surfactant
- triton
- buffer solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- 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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
-
- 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
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Endocrinology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kit for determining a specific growth factor, which comprises a reagent R1 and a reagent R2, wherein the reagent R1 comprises a buffer solution, a surfactant 1, a surfactant 2, an inorganic salt and an organic solvent, and the reagent R2 comprises a buffer solution, a color developing agent and a preservative. According to the method, a certain amount of organic solvent (DMSO) is added into the detection reagent, so that the positive progress of the ninhydrin color reaction can be effectively promoted, and the color reaction is close to complete reaction. Meanwhile, the problem of sample precipitation after the organic solvent is added is solved by adding the composition of the surfactant and the salt. The improvement measures are matched with the reaction system of the invention, so that the detection performance of the kit is effectively improved, including precision and the quantifiability of the kit to the object to be detected in a linear range.
Description
Technical Field
The invention relates to the field of medical inspection, in particular to a kit for determining a specific growth factor.
Background
Specific Growth Factor (SGF) is a special substance including saccharides, amino acids and metabolites thereof, which has a property that when a tumor is produced, the content of the Specific growth factor in blood is increased, which can stimulate the angiogenesis of malignant tumor, and after the angiogenesis, can provide sufficient nutrition for tumor tissue, thereby promoting the rapid growth of the tumor. The specific growth factor can be obviously increased in the early stage of the tumor, and the content of the specific growth factor in serum is clinically determined to be used as an effective reference basis for early detection of the tumor, change of the disease condition of a patient and postoperative monitoring.
Currently known SGF detection means include enzyme-linked immunosorbent assay (ELISA) and common chemical methods, wherein the enzyme-linked immunosorbent assay is complex in operation, long in reaction time and poor in detection repeatability, so that the SGF detection method is not favorable for being widely applied clinically. The common chemical method utilizes the mixed solution in the reagent kit to carry out superposition coupling reaction with specific growth factors (substances such as glycolipid with specific amino group, glycoprotein metabolite, free alpha-amino acid and the like) in a sample to be detected at 37 ℃, the reaction product and a color developing agent in the reagent kit carry out color development reaction under the same reaction condition to form a blue product, the absorbance value of the color developing product is measured at the position of 570m of wavelength, and the content of the specific growth factors in the sample to be detected is converted through a standard concentration curve and a formula. As the color development reaction of the color development agent ninhydrin in the reagent is a reversible reaction and is incomplete, the detection reagent used clinically at present generally has the problem of poor quantifiability and further poor linearity.
Disclosure of Invention
The inventors have unexpectedly found that the forward progress of the ninhydrin color reaction can be effectively promoted by adding a certain amount of organic solvent (DMSO) to the detection reagent, so that the color reaction approaches a complete reaction. Meanwhile, the problem of sample precipitation after the organic solvent is added is solved by adding the composition of the surfactant and the salt. The improvement measures are matched with the reaction system of the invention, so that the detection performance of the kit is effectively improved, including precision and the quantifiability of the kit to the object to be detected in a linear range.
In order to achieve the purpose, the invention adopts the following technical means: a kit for determining a specific growth factor, comprising a reagent R1 and a reagent R2, wherein: the reagent R1 comprises a buffer solution, a surfactant 1, an inorganic salt and an organic solvent.
Preferably, the reagent R2 comprises a buffer solution, a color developing agent and a preservative.
Preferably, the organic solvent is selected from at least one of DMSO, DMF or methanol, preferably DMSO.
Preferably, the content of the organic solvent is 200-600g/L, preferably 400-600 g/L.
Preferably, the reagent R1 also comprises a surfactant 2; the reagent R2 comprises a buffer solution, a color developing agent and a preservative.
Preferably, the surfactant 1 and the surfactant 2 are selected from two of Triton X-100, Triton X-305, Emulgen LS-114, Emulgen 709, Tween-20 or Tween-80, preferably combinations of Triton X-100 and Emulgen LS-114, Triton X-305 and Emulgen LS-114 or Triton X-305 and Tween-20; the content of the surfactant 1 is 5-20g/L, and the content of the surfactant 2 is 0.1-1 g/L.
Preferably, the buffer solution in the reagent R1 is selected from at least one of 4-hydroxyethyl piperazine ethanesulfonic acid, Tris buffer solution or GOOD' S buffer solution, and the inorganic salt is selected from at least one of sodium chloride or potassium chloride; the buffer solution in the reagent R2 is at least one selected from phosphate buffer solution, GOOD' S buffer solution or succinic acid buffer solution, the color developing agent is ninhydrin, and the preservative is at least one selected from sodium azide or Proclin 300; the content of the buffer solution in the reagent R1 is 50-150mM, and the content of the inorganic salt is 1-5 g/L; the content of the buffer solution in the reagent R2 is 30-50mM, the content of the color developing agent is 10-100mM, and the content of the preservative is 0.1-1 g/L.
A composition effective for enhancing the detection of a biochemical reagent, the composition comprising: organic solvent, surfactant 1, surfactant 2, and inorganic salt.
Preferably, the organic solvent is selected from at least one of DMSO, DMF or methanol, preferably DMSO; the surfactant 1 and the surfactant 2 are selected from two of Triton X-100, Triton X-305, Emulgen LS-114, Emulgen 709, Tween-20 or Tween-80, preferably combinations of Triton X-100 and Emulgen LS-114, Triton X-305 and Emulgen LS-114 or Triton X-305 and Tween-20; the inorganic salt is selected from at least one of sodium chloride or potassium chloride.
Preferably, the reagent is a kit for detecting specific growth factors.
The invention has the beneficial effects that: according to the invention, a certain amount of organic solvent (DMSO) is added into the detection reagent R1, so that the positive progress of the ninhydrin color reaction is effectively promoted, the color reaction is close to complete reaction, and the quantifiability of the detection reagent to the substance to be detected in a linear range is remarkably improved. Meanwhile, the problems of sample precipitation and detection precision reduction are brought along with the addition of the organic solvent, and further, the problems of subsequent sample precipitation and precision reduction caused by the addition of the organic solvent are solved by adding the composition of the surfactant and the salt into the reagent R1, so that the detection performance of the kit is effectively improved.
Drawings
FIG. 1 is a graph showing a linear relationship between theoretical concentration and detection value of group A kit samples in example 3 of the present invention;
FIG. 2 is a graph showing a linear relationship between theoretical concentration and detection value of group B kit samples in example 3 of the present invention;
FIG. 3 is a linear relationship graph of theoretical concentration and detection value of group C kit samples in example 3 of the present invention;
FIG. 4 is a graph showing a linear relationship between theoretical concentrations and detection values of group A kit samples in example 4 of the present invention;
FIG. 5 is a graph showing the linear relationship between the theoretical concentration and the detection value of the group B kit sample in example 4 of the present invention;
FIG. 6 is a graph showing the linear relationship between the theoretical concentration and the detection value of the group C kit sample in example 4 of the present invention;
FIG. 7 is a graph showing the linear relationship between the theoretical concentration and the detection value of the group D kit sample in example 4 of the present invention;
FIG. 8 is a graph showing the linear relationship between the theoretical concentration and the detection value of the group E kit sample in example 4 of the present invention;
FIG. 9 is a graph showing the linear relationship between the theoretical concentration and the detection value of the group F kit sample in example 4 of the present invention;
FIG. 10 is a graph showing the linear relationship between the theoretical concentration and the detection value of the group G kit sample in example 4 of the present invention;
FIG. 11 is a graph showing a linear relationship between theoretical concentrations and detection values of group H kit samples in example 4 of the present invention;
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following examples are included to more clearly and clearly illustrate the technical solutions of the present invention by way of illustration. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. The specific embodiments of the present invention are merely illustrative of the invention and are not intended to limit the invention in any way.
Example 1 preparation of TSGF assay kit
The Specific Growth Factor (TSGF) detection kit comprises a reagent R1 and a reagent R2 double liquid components which are independent of each other.
1. Preparation of reagent R1
The preparation is carried out according to the following formula, fully stirred and uniformly mixed, and stored at 2-8 ℃.
2. Preparation of reagent R2
The preparation is carried out according to the following formula, fully stirred and uniformly mixed, and stored at 2-8 ℃.
Example 2 method of Using the kit
In this example, a fully automatic biochemical analyzer (Hitachi 7180) was used in combination with the kit of the present invention to perform sample detection.
(1) Instrument parameter setting
(2) Assay protocol
(3) Computing method
And (3) using a two-point linear calibration mode, taking a linear function as a calculation mode, and making a dose/response curve according to the value of the calibrator and the absorbance change value, wherein the content of the specific growth factor in the sample can be calculated on the dose/response curve according to the absorbance change value.
The detection principle of the invention is as follows: the mixed solution in the kit is subjected to superposition coupling reaction with specific growth factors (substances such as glycolipids with specific amino groups, glycoprotein metabolites, free alpha-amino acids and the like) in a sample to be detected at 37 ℃, the reaction products are subjected to chromogenic reaction with a chromogenic agent in the kit under the same reaction condition to form blue products, the absorbance value of the chromogenic products is measured at the wavelength of 570nm, and the content of the specific growth factors in the sample to be detected is converted through a standard concentration curve and a formula (the TSGF clinical concentration is 100-0.18TSGF equivalent concentration).
EXAMPLE 3 Performance testing of the kits
In order to verify all performances of the kit, 3 groups of kits are arranged for performance verification:
group A: the kit prepared in the invention example 1;
group B: specific growth factor determination kit (chemical method) (Beijing nine strong);
group C: the kit is obtained according to the method described in patent CN104515768B specification example 1.
The kit of group C was prepared using the reagents available in the laboratory (i.e., the same components were used in group A and group C, e.g., the developer ninhydrin was from the same manufacturer and lot) wherein the kit of group A was tested according to the method of use described in example 2, group B was tested according to the instructions, and group C was tested according to the method of use described in the examples.
(1) Accuracy verification
And (3) respectively carrying out accuracy test on the clinical assigned samples by using three groups of kits, setting 2 times of the tests, reading signals by using a full-automatic biochemical analyzer (Hitachi 7180), and calculating the relative deviation between the measured mean value and the target value to carry out accuracy verification. The results are shown in the following table:
TABLE 1 accuracy verification
From the above experimental results, the relative deviations of the test value 1 and the target value 1 of the three sets of kits were 0.21%, 0.61%, and-2.12%, respectively, and the relative deviations of the test value 2 and the target value 2 were-0.25%, 0.71%, and-1.13%, respectively. The detection accuracy of the kit (group A) prepared in the embodiment 1 of the invention is obviously better than that of the kit (group C).
(2) Precision verification
Selecting low-value samples, medium-value samples and high-value samples of the clinical specific growth factors, testing the samples by using three groups of kits, respectively repeating the measurement for 10 times, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), respectively calculating a measurement mean value and a standard deviation, and calculating a variation coefficient to perform precision investigation. The results are shown in the following table:
TABLE 2 precision verification
From the above experimental results, the variation coefficients of the three sets of kits in the detection of the low value sample are 0.59%, 2.87% and 2.30%, the variation coefficients of the median sample in the detection are 0.49%, 0.53% and 1.91%, and the variation coefficients of the high value sample in the detection are 0.58%, 1.00% and 2.87%, respectively, and the experimental results show that the precision of the kit (group a) prepared in example 1 of the present invention in the detection of the low value sample, the median sample and the high value sample is better than that of the control kit-1 (group B) and the control kit-2 (group C).
(3) Linear range verification
Selecting a clinical ultrahigh-value sample and a clinical low-value sample, then utilizing the high-value sample and the low-value sample to prepare each concentration gradient sample in proportion, using three groups of kits to test the samples respectively, repeatedly measuring each sample for 2 times, reading signals through a full-automatic biochemical analyzer (Hitachi 7180), and calculating a measured mean value respectively to perform linear range investigation. The results are shown in the following table:
TABLE 3 group A kit Linear Range validation
| H-L | L-H | Theoretical value | Mean value | Relative deviation 1 | Estimated value | Relative deviation 2 | |
| H | 283.94 | 282.72 | 283.33 | 283.33 | 0.00％ | 282.81 | 0.18％ |
| 3/4H+1/4L | 241.81 | 237.19 | 241.20 | 239.50 | -0.71％ | 240.58 | -0.45％ |
| 1/2H+1/2L | 198.5 | 201.08 | 199.08 | 199.79 | 0.36％ | 198.35 | 0.72％ |
| 1/4H+3/4L | 157.41 | 152.02 | 156.95 | 154.72 | -1.42％ | 156.12 | -0.90％ |
| 1/8H+7/8L | 133.9 | 134.61 | 135.89 | 134.26 | -1.20％ | 135.01 | -0.56％ |
| 1/16H+15/16L | 125.73 | 123.87 | 125.36 | 124.80 | -0.44％ | 124.45 | 0.28％ |
| L | 114.08 | 115.57 | 114.83 | 114.83 | 0.00％ | 113.89 |
Note: the relative deviation 1 is the relative deviation of the mean from the theoretical value, and the relative deviation 2 is the relative deviation of the mean from the estimated value.
TABLE 4 group B kit Linear Range validation
| H-L | L-H | Theoretical value | Mean value | Relative deviation 1 | Estimated value | Relative deviation 2 | |
| H | 295.25 | 301.8 | 298.53 | 298.53 | 0.00％ | 287.32 | 3.90％ |
| 3/4H+1/4L | 220.15 | 231.96 | 240.60 | 226.06 | -6.04％ | 229.64 | -1.56％ |
| 1/2H+1/2L | 161.7 | 154.23 | 182.67 | 157.97 | -13.52％ | 171.95 | -8.14％ |
| 1/4H+3/4L | 107.54 | 106.52 | 124.74 | 107.03 | -14.20％ | 114.27 | -6.34％ |
| 1/8H+7/8L | 86.67 | 86.77 | 95.77 | 86.72 | -9.45％ | 85.43 | 1.51％ |
| 1/16H+15/16L | 72.61 | 73.60 | 81.29 | 73.11 | -10.07％ | 71.01 | 2.95％ |
| L | 65.87 | 67.75 | 66.81 | 66.81 | 0.00％ | 56.59 |
Note: the relative deviation 1 is the relative deviation of the mean from the theoretical value, and the relative deviation 2 is the relative deviation of the mean from the estimated value.
TABLE 5 group C kit Linear Range validation
| H-L | L-H | Theoretical value | Mean value | Relative deviation 1 | Estimated value | Relative deviation 2 | |
| H | 247.56 | 248.15 | 247.86 | 247.86 | 0.00％ | 248.92 | -0.43％ |
| 3/4H+1/4L | 210.36 | 204.58 | 214.60 | 207.47 | -3.32％ | 216.04 | -3.97％ |
| 1/2H+1/2L | 195.5 | 201.08 | 181.34 | 198.29 | 9.35％ | 183.15 | 8.26％ |
| 1/4H+3/4L | 152.41 | 152.02 | 148.08 | 152.22 | 2.79％ | 150.27 | 1.29％ |
| 1/8H+7/8L | 126.9 | 132.61 | 131.45 | 129.76 | -1.29％ | 133.83 | -3.04％ |
| 1/16H+15/16L | 125.73 | 123.87 | 123.14 | 124.80 | 1.35％ | 125.61 | -0.64％ |
| L | 114.10 | 115.57 | 114.83 | 114.83 | 0.00％ | 117.39 |
Note: the relative deviation 1 is the relative deviation of the mean from the theoretical value, and the relative deviation 2 is the relative deviation of the mean from the estimated value.
From the above experimental results, it can be seen that in the linear range of sample concentration 114-A kit. Meanwhile, the detection results of the three groups of kits are subjected to correlation analysis with the theoretical value of the sample concentration (shown in the attached figures 1-3): the correlation between the detection values of the kit in the group A and the theoretical values is obviously better than that between the detection values of the kit in the group B and the group C, wherein the correlation R between the detection values of the group A and the theoretical values20.9997, group B R20.9893 for group C, 0.9783 for group C. The experimental results show that the kit (group A) prepared in example 1 of the present invention has better linear correlation, i.e., better quantifiability, in the linear range than the kits of group B and group C.
EXAMPLE 4 Effect of organic solvents on the Linear Range of the kits
(1) In order to verify the effect of adding organic solvent to the reagent R1 on the linear range of the kit, a total of 4 sets of kits were set up:
group A: the kit prepared in the embodiment 2 of the invention;
group B: the kit differs from the kit of example 2 only in that no organic solvent is added to the reagent R1;
group C: the kit is different from the kit in example 2 only in that the organic solvent added in the reagent R1 is DMF (N, N-dimethylformamide);
group D: the kit is different from the kit in example 2 only in that the organic solvent added in the reagent R1 is methanol;
selecting a clinical ultrahigh-value sample and a clinical low-value sample, then preparing each concentration gradient sample by using the high-value sample and the low-value sample in proportion, respectively testing the samples by using the four groups of kits, respectively repeating the measurement for 2 times for each sample, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), and respectively calculating a measurement mean value to perform linear range investigation. The results are shown in the following table:
TABLE 6 kit Linear Range verification
Note: the relative deviation 1 is the relative deviation of the mean value from the theoretical value.
From the above experimental results, the relative deviation between the detection value and the theoretical value of the groups a, C and D is smaller than that of the group B in the linear detection range, and the detection result of the group a is optimal. Meanwhile, the detection results of the 4 groups of kits are subjected to correlation analysis with the theoretical value of the sample concentration (shown in the attached figures 4-7): correlation R of A group of test values and theoretical values20.9997, group B R20.9774, group C R20.9992, group D R2Is 0.9976. The experimental result shows that the quantifiability of the reagent kit for detecting the TSGF can be obviously improved by adding an organic solvent (especially DMSO) into the reagent kit R1 for detecting the TSGF.
(2) In order to verify the influence of the addition amount of the organic solvent in the reagent R1 on the linear range of the kit, 4 groups of experiments are set up:
group A: the kit is different from the kit in example 2 only in that the content of an organic solvent DMSO in the reagent R1 is 100 g/L;
group B: the kit is different from the kit in example 2 only in that the addition amount of an organic solvent DMSO in the reagent R1 is 200 g/L;
group C: the kit is different from the kit in example 2 only in that the addition amount of an organic solvent DMSO in the reagent R1 is 600 g/L;
group D: the kit is different from the kit in example 2 only in that the addition amount of the organic solvent DMSO in the reagent R1 is 1000 g/L.
Selecting a clinical ultrahigh-value sample and a clinical low-value sample, then preparing each concentration gradient sample by utilizing the high-value sample and the low-value sample in proportion, respectively testing the samples by using the three groups of kits, respectively repeating the measurement for 2 times for each sample, reading signals by a full-automatic biochemical analyzer (Hitachi 7180), and respectively calculating a measurement mean value to perform linear range investigation. The results are shown in the following table:
TABLE 7 kit Linear Range verification
Note: the relative deviation 1 is the relative deviation of the mean value from the theoretical value.
And (3) carrying out correlation analysis on the detection results of the 4 groups of kits and the theoretical value of the sample concentration (shown in the attached figures 8-11): correlation R of A group of test values and theoretical values20.9887, group B R20.9971, group C R20.9996, group D R20.9960, the experimental result shows that the invention adds 200-600g/L organic solvent in the reagent R1, which can significantly improve the detection quantifiability in the linear range of the kit.
EXAMPLE 5 Effect of surfactant and inorganic salt composition on kit precision
(1) Influence of surfactant and inorganic salt composition on detection precision
In order to verify the effect of adding the surfactant and the inorganic salt composition in the reagent R1 on the detection precision of the kit, 8 sets of experiments are provided in the present example, wherein the kit used in each set of experiments is different from that in example 1 only in whether the surfactant 1Triton X-100, the surfactant 2Tween-20 and the inorganic salt NaCl are added in the reagent R1, and the preparation methods of the rest of the kits are the same as those in example 1. And simultaneously, the 8 groups of kits are adopted to carry out precision detection on the median sample, and the detection results are shown in the following table:
TABLE 8
Note: "√" indicates inclusion of the component; "×" indicates that the component is not contained.
Experimental results show that when the surfactant 1TritonX-100, the surfactant 2Tween-20 and the inorganic salt NaCl are simultaneously added into the reagent R1, the detection precision of the kit is higher.
(2) Influence of surfactant and inorganic salt concentration
In order to verify the effect of adding the surfactant and inorganic salt composition to the reagent R1 on the linear range of the kit, 5 sets of experiments were provided in this example, wherein each set of experiments only differed from example 1 in the concentrations of the surfactant 1Triton X-100, the surfactant 2Tween-20 and the inorganic salt NaCl in the reagent R1, and the preparation methods of the other kits were the same as example 1. The five kits are simultaneously adopted to carry out precision detection on the median sample, and the detection results are shown in the following table:
TABLE 9
The experimental result shows that when the concentration range of the surfactant 1 in the reagent R1 is 5-20g/L, the concentration range of the surfactant 2 is 0.1-1g/L and the concentration range of the inorganic salt is 1-5g/L, the detection precision of the kit is higher.
(3) Influence of surfactant and inorganic salt species
In this example, 5 sets of experiments were set, wherein each set of experiments was performed using a kit different from example 1 only in the kinds of surfactant 1, surfactant 2 and inorganic salt in the reagent R1, and the other kits were prepared in the same manner as in example 1. The five kits are simultaneously adopted to carry out precision detection on the median sample, and the detection results are shown in the following table:
watch 10
The experimental result shows that when the surfactant 1, the surfactant 2 and the inorganic salt in the reagent R1 are combined by the 7 methods, the detection precision of the kit is high, and particularly when the surfactant 1 is Triton X-100 or Triton X-305, the surfactant 2 is LS-114 or Tween-20 and the inorganic salt is NaCl, the detection precision is highest.
In conclusion, the method can effectively promote the positive progress of the ninhydrin color reaction by adding a certain amount of organic solvent (DMSO) into the detection reagent R1, so that the color reaction is close to a complete reaction, the quantifiability of the detection reagent to the object to be detected in a linear range is remarkably improved, and simultaneously, the problems of sample precipitation and detection precision reduction are brought along with the addition of the organic solvent. Furthermore, the composition of the surfactant and the salt is added into the reagent R1, so that the problems of sample precipitation and precision reduction after the addition of the organic solvent are solved, and the detection performance of the kit is effectively improved.
Although embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments without departing from the principles and spirit of the present invention.
Claims (10)
1. A kit for determining a specific growth factor, comprising a reagent R1 and a reagent R2, wherein: the reagent R1 comprises a buffer solution, a surfactant 1, an inorganic salt and an organic solvent.
2. The kit for detecting the specific growth factor according to claim 1, wherein the reagent R2 comprises a buffer solution, a color developing agent and a preservative.
3. The kit for detecting a specific growth factor according to claim 2, wherein: the organic solvent is at least one selected from DMSO, DMF or methanol, and is preferably DMSO.
4. The kit for detecting a specific growth factor according to claim 3, wherein: the content of the organic solvent is 200-600g/L, preferably 400-600 g/L.
5. The kit for detecting a specific growth factor according to claim 4, wherein: the reagent R1 also comprises a surfactant 2; the reagent R2 comprises a buffer solution, a color developing agent and a preservative.
6. The kit for detecting a specific growth factor according to claim 5, wherein: the surfactant 1 and the surfactant 2 are selected from two of Triton X-100, Triton X-305, Emulgen LS-114, Emulgen 709, Tween-20 or Tween-80, preferably combinations of Triton X-100 and Emulgen LS-114, Triton X-305 and Emulgen LS-114 or Triton X-305 and Tween-20; the content of the surfactant 1 is 5-20g/L, and the content of the surfactant 2 is 0.1-1 g/L.
7. The kit for detecting the specific growth factor according to any one of claims 5 to 6, wherein the buffer in the reagent R1 is at least one selected from 4-hydroxyethylpiperazine ethanesulfonic acid, Tris buffer or GOOD' S buffer, and the inorganic salt is at least one selected from sodium chloride or potassium chloride; the buffer solution in the reagent R2 is at least one selected from phosphate buffer solution, GOOD' S buffer solution or succinic acid buffer solution, the color developing agent is ninhydrin, and the preservative is at least one selected from sodium azide or Proclin 300; the content of the buffer solution in the reagent R1 is 50-150mM, and the content of the inorganic salt is 1-5 g/L; the content of the buffer solution in the reagent R2 is 30-50mM, the content of the color developing agent is 10-100mM, and the content of the preservative is 0.1-1 g/L.
8. A composition effective for enhancing the detection of a biochemical reagent, the composition comprising: organic solvent, surfactant 1, surfactant 2, and inorganic salt.
9. The composition for improving the detection performance of a biochemical reagent according to claim 8, wherein the organic solvent is at least one selected from DMSO, DMF or methanol, preferably DMSO; the surfactant 1 and the surfactant 2 are selected from two of Triton X-100, Triton X-305, Emulgen LS-114, Emulgen 709, Tween-20 or Tween-80, preferably combinations of Triton X-100 and Emulgen LS-114, Triton X-305 and Emulgen LS-114 or Triton X-305 and Tween-20; the inorganic salt is selected from at least one of sodium chloride or potassium chloride.
10. The composition for improving the detection performance of biochemical reagents according to claims 8-9, wherein the reagent is a kit for detecting specific growth factors.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110917705.XA CN113740539B (en) | 2021-08-11 | 2021-08-11 | Kit for determining specific growth factors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110917705.XA CN113740539B (en) | 2021-08-11 | 2021-08-11 | Kit for determining specific growth factors |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113740539A true CN113740539A (en) | 2021-12-03 |
| CN113740539B CN113740539B (en) | 2023-07-25 |
Family
ID=78730790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110917705.XA Active CN113740539B (en) | 2021-08-11 | 2021-08-11 | Kit for determining specific growth factors |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113740539B (en) |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4274833A (en) * | 1979-10-18 | 1981-06-23 | Pickering Michael V | Ninhydrin reagent for use in amine and amino acid analyses |
| WO1990008320A1 (en) * | 1989-01-19 | 1990-07-26 | Teijin Limited | Immunoassay method, reagent and kit |
| AU2001251510A1 (en) * | 2000-04-10 | 2001-10-23 | Sugen, Inc. | Human protein kinases and protein kinase-like enzymes |
| WO2003074005A2 (en) * | 2002-03-01 | 2003-09-12 | Dyax Corp. | Kdr and vegf/kdr binding peptides and their use in diagnosis and therapy |
| CN102924458A (en) * | 2004-04-02 | 2013-02-13 | Osi制药有限责任公司 | 6,6-bicyclic ring substituted heterobicyclic protein kinase inhibitors |
| CN102967571A (en) * | 2012-12-24 | 2013-03-13 | 北京利德曼生化股份有限公司 | Liquid single reagent for detecting alpha-L-fucosidase and preparation method thereof |
| CN104515768A (en) * | 2014-08-29 | 2015-04-15 | 湖南新大陆生物技术有限公司 | Tumor specific growth factor detection kit |
| CN106053444A (en) * | 2016-04-28 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | A kit for measuring a specific growth factor and a preparing method thereof |
| CN109425580A (en) * | 2017-08-28 | 2019-03-05 | 江苏福隆医疗器械有限公司 | A kind of tumor specific growth factor detection kit and its application method |
| CN109580504A (en) * | 2018-10-19 | 2019-04-05 | 东软威特曼生物科技(南京)有限公司 | A kind of lipoprotein cholesterol measurement reagent and kit |
| CN109799331A (en) * | 2019-03-18 | 2019-05-24 | 湖南海源医疗科技股份有限公司 | A kind of Tumor specific factor TSGF analysis strip |
| CN111721934A (en) * | 2020-06-24 | 2020-09-29 | 湖南新大陆生物技术有限公司 | Improved specific growth factor detection kit and application thereof |
| CN112858690A (en) * | 2021-01-21 | 2021-05-28 | 宁波职业技术学院 | Urine albumin/urine creatinine composite quality control product and preparation method thereof |
-
2021
- 2021-08-11 CN CN202110917705.XA patent/CN113740539B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4274833A (en) * | 1979-10-18 | 1981-06-23 | Pickering Michael V | Ninhydrin reagent for use in amine and amino acid analyses |
| WO1990008320A1 (en) * | 1989-01-19 | 1990-07-26 | Teijin Limited | Immunoassay method, reagent and kit |
| AU2001251510A1 (en) * | 2000-04-10 | 2001-10-23 | Sugen, Inc. | Human protein kinases and protein kinase-like enzymes |
| WO2003074005A2 (en) * | 2002-03-01 | 2003-09-12 | Dyax Corp. | Kdr and vegf/kdr binding peptides and their use in diagnosis and therapy |
| CN102924458A (en) * | 2004-04-02 | 2013-02-13 | Osi制药有限责任公司 | 6,6-bicyclic ring substituted heterobicyclic protein kinase inhibitors |
| CN102967571A (en) * | 2012-12-24 | 2013-03-13 | 北京利德曼生化股份有限公司 | Liquid single reagent for detecting alpha-L-fucosidase and preparation method thereof |
| CN104515768A (en) * | 2014-08-29 | 2015-04-15 | 湖南新大陆生物技术有限公司 | Tumor specific growth factor detection kit |
| CN106053444A (en) * | 2016-04-28 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | A kit for measuring a specific growth factor and a preparing method thereof |
| CN109425580A (en) * | 2017-08-28 | 2019-03-05 | 江苏福隆医疗器械有限公司 | A kind of tumor specific growth factor detection kit and its application method |
| CN109580504A (en) * | 2018-10-19 | 2019-04-05 | 东软威特曼生物科技(南京)有限公司 | A kind of lipoprotein cholesterol measurement reagent and kit |
| CN109799331A (en) * | 2019-03-18 | 2019-05-24 | 湖南海源医疗科技股份有限公司 | A kind of Tumor specific factor TSGF analysis strip |
| CN111721934A (en) * | 2020-06-24 | 2020-09-29 | 湖南新大陆生物技术有限公司 | Improved specific growth factor detection kit and application thereof |
| CN112858690A (en) * | 2021-01-21 | 2021-05-28 | 宁波职业技术学院 | Urine albumin/urine creatinine composite quality control product and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| STANFORD MOORE: "Amino Acid Analysis: Aqueous Dimethyl Sulfoxide As Solvent for the Ninhydrin Reaction", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| XIUQIN SONG 等: "Clinical value of color Doppler ultrasound combined with serum CA153, CEA and TSGF detection in the diagnosis of breast cancer", 《EXPERIMENTAL AND THERAPEUTIC MEDICINE》 * |
| 林莉 等: "血液标本保存方式对恶性肿瘤特异生长因子检测结果的影响", 《实用医学杂志》 * |
| 邵迎春 等: "肝素和乙二胺四乙酸二钾对TSGF检测的影响", 《国际检验医学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113740539B (en) | 2023-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106198961A (en) | The latex of a kind of detection by quantitative serum amyloid A protein strengthens Immunoturbidimetric kit and preparation method thereof | |
| CN104198473A (en) | Stable uric acid detection kit | |
| CN107727869A (en) | Kit of antinuclear antibodies and preparation method thereof in a kind of measure serum | |
| CN109613280B (en) | Serum iron determination kit and preparation method and application thereof | |
| CN109374902A (en) | A kind of latex enhancing Immunoturbidimetric kit of quantitative detection IgG4 and preparation method thereof | |
| CN111208220A (en) | Preparation and application of quality control substance of catecholamine substances and metabolites of plasma type freeze-dried powder | |
| Ladenson | Evaluation of an instrument (Nova-1) for direct potentiometric analysis of sodium and potassium in blood and their indirect potentiometric determination in urine. | |
| CN112029817B (en) | Creatinine detection kit and application method thereof | |
| CN106093418A (en) | A kind of test kit measuring Troponin I and preparation method thereof | |
| CN111289758B (en) | Kit for quantitative detection of H-FABP and method for quantitative detection of H-FABP | |
| CN108169145A (en) | A kind of kit for measuring serum complement C1q and its preparation application method | |
| CN113092746B (en) | Biochemical calibration material | |
| CN112710853B (en) | Anti-interference and stable serum direct bilirubin (enzyme method) determination kit and preparation method and application thereof | |
| CN106353505B (en) | ApoE kits based on catalyzed signal amplification | |
| CN113740539B (en) | Kit for determining specific growth factors | |
| CN112858690A (en) | Urine albumin/urine creatinine composite quality control product and preparation method thereof | |
| CN111721934A (en) | Improved specific growth factor detection kit and application thereof | |
| CN106324234B (en) | Neu 5 Ac aldolase of modification and its preparation method and application | |
| CN108362892B (en) | Procalcitonin colloidal gold immunoturbidimetry detection reagent | |
| CN113984689B (en) | Kit for determining glutathione reductase | |
| CN105929176A (en) | Kit for determining heart-type fatty acid binding protein and preparation method thereof | |
| CN113655006B (en) | Urinary system knot Dan Chengdan risk factor detection and test system | |
| CN113008820A (en) | Method for measuring content of sulfydryl | |
| DK169415B1 (en) | Process and reagent for quantitative photometric determination of phosphorus in body fluids | |
| CN105548171B (en) | A kind of cerebrospinal fluid and Urine proteins liquid single-reagent and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| CB02 | Change of applicant information |
Address after: 400037 1st-4th floor, building 30, No.6 Taikang Road, Zone C, Jianqiao Industrial Park, Dadukou District, Chongqing Applicant after: Zhongyuan Huiji Biotechnology Co.,Ltd. Address before: 400037 1st-4th floor, building 30, No.6 Taikang Road, Zone C, Jianqiao Industrial Park, Dadukou District, Chongqing Applicant before: CHONGQING ZHONGYUAN HUIJI BIOTECHNOLOGY Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |