CN117607440B - Phosphatidylinositol proteoglycan 3 protein assay kit and preparation method and application thereof - Google Patents
Phosphatidylinositol proteoglycan 3 protein assay kit and preparation method and application thereof Download PDFInfo
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- CN117607440B CN117607440B CN202311601434.2A CN202311601434A CN117607440B CN 117607440 B CN117607440 B CN 117607440B CN 202311601434 A CN202311601434 A CN 202311601434A CN 117607440 B CN117607440 B CN 117607440B
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- 101710127914 Proteoglycan 3 Proteins 0.000 title claims abstract description 6
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- 238000002731 protein assay Methods 0.000 title description 2
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- 239000000243 solution Substances 0.000 claims abstract description 14
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000007853 buffer solution Substances 0.000 claims abstract description 12
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- YDEXUEFDPVHGHE-GGMCWBHBSA-L disodium;(2r)-3-(2-hydroxy-3-methoxyphenyl)-2-[2-methoxy-4-(3-sulfonatopropyl)phenoxy]propane-1-sulfonate Chemical group [Na+].[Na+].COC1=CC=CC(C[C@H](CS([O-])(=O)=O)OC=2C(=CC(CCCS([O-])(=O)=O)=CC=2)OC)=C1O YDEXUEFDPVHGHE-GGMCWBHBSA-L 0.000 claims description 11
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Classifications
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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
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- 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
- G01N33/531—Production of immunochemical test materials
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
The invention provides a kit for measuring glypican 3 by a latex immunoturbidimetry, which comprises a reagent R1 and a reagent R2; reagent R1 comprises the following components: 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid buffer solution, sodium chloride, blocking agent, polyethylene glycol 8000, surfactant, pc300; reagent R2 comprises the following components: 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid buffer solution, phosphatidylinositol proteoglycan 3 antibody coated latex microsphere, stabilizer, dispersant and Pc300. The kit is a liquid kit with strong stability, high accuracy, wide linear range, high sensitivity and interference resistance.
Description
Technical Field
The invention relates to the technical field of biochemical detection, in particular to a kit for measuring glypican 3 and application thereof.
Background
Glypican-3 (glypican, GPC 3) is a member of the heparan sulfate glycoprotein family, which is anchored to the cell outer membrane by binding of the C-terminus to glycosyl phosphatidyl alcohols. In the study of a syndrome of excessive growth of X-chromosome-linked recessive genetic disease Simpson-Golabi-Behmel (SGBS), the university of Yersinia 1996 first discovered the GPC3 gene (also called MXR-7 or OCI-5 gene) which was located on human chromosome Xq26 and had a gene structure of over 900kb, which is one of the largest genes in the human genome, consisting of 8 exons and 7 introns. The relative molecular weight of the polypeptide is 60000, arginine at 358 and serine at 359 are dissociated, the N end enters blood and is called soluble GPC3, the relative molecular weight of the polypeptide is 40000, and the research discovers that the GPC3 plays an important role in the signal transmission of growth factors, thereby playing an important role in cell proliferation, differentiation and migration and playing a role in regulating the activity of tumor cell proliferation. In recent years, a great deal of research has found that GPC3 is expressed at high level in embryonic liver tissue, liver cancer tissue and most liver cancer cell lines, but is hardly expressed in normal liver tissue, which suggests that GPC3 can be used as a valuable liver cancer histological tumor marker.
Primary liver cancer is a common malignant tumor of the digestive tract, wherein hepatocellular carcinoma (hepatocellular carcinoma, HCC) is a major pathological type, accounting for 85% -90% of primary liver cancer. HCC is hidden, and the survival rate of HCC in 5 years is less than 15% because HCC is frequently in middle and late stages and lost the opportunity for radical surgery. Thus, early diagnosis is of great importance for improving HCC clinical outcome. Currently, alpha-fetoprotein (AFP) is the most commonly used diagnostic marker for clinical HCC, but the sensitivity is only 20% -65%, and positive results can be detected in other benign liver diseases, gastrointestinal tumors and the like. Thus, there is a need to find more sensitive diagnostic markers to improve HCC diagnostic efficacy.
Glypican 3 (glypican-3, GPC 3) is a member of the GPC family and is a class of carcinoembryonic proteins anchored to the cell membrane. There is increasing evidence that GPC3 protein is highly expressed in serum from HCC patients and peripheral blood GPC3 mRNA. Furthermore, studies have shown that GPC3 can identify AFP negative HCC and benign nodules of liver. Thus, GPC3 is considered to be a very potential diagnostic marker for HCC.
The current method for clinically detecting the glypican 3 mainly comprises a chemiluminescence method, an enzyme-linked immunosorbent assay (ELISA) method and the like, wherein the chemiluminescence method has high accuracy and sensitivity, but instruments and equipment are expensive, and the cost is high; enzyme-linked immunosorbent assay (ELISA) is widely applied to clinic at present, and the technology is mature, and has advantages in sensitivity, specificity, reagent price and the like, but has disadvantages in detection time, operability and the like. The latex immunoturbidimetry has the advantages of high specificity, simple and quick operation, accuracy and safety, automatic analysis and lower cost, but the method has the problems of insensitivity in reaction, narrow linear range, poor stability, difficult application and the like in the prior art, and the invention aims at the development of the latex enhanced immunoturbidimetry of the phosphatidylinositol proteoglycan 3 so as to meet the requirements of large-scale clinical detection and chemical analysis in hospitals.
Disclosure of Invention
In order to solve the problems, the invention provides a kit for measuring glypican 3 by a latex immunoturbidimetry, which is a liquid kit with strong stability, high accuracy, wide linear range, high sensitivity and interference resistance.
The invention is realized by the following technical scheme:
a kit for measuring glypican 3 comprises the following components;
reagent R1 comprises the following components:
50-200mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Sodium chloride 4.5g/L
Blocking agent 8-15g/L
Polyethylene glycol 8000 g/L
Surfactant 1-4g/L
Pc300 1ml/L
Reagent R2 comprises the following components:
50-100mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Glypican 3 antibody coated latex microsphere 10-200mg/L
Dispersing agent 1-10 g/L
Stabilizer 1-10 g/L
Pc300 1ml/L
Preferably, the reagent R1 has a pH of 6.0-8.0; the pH of the reagent R2 is 6.0-8.0.
Preferably, the surfactant in the reagent R1 is one or more of the flower king Emulgen102, emulgen103, emulgen107, emulgen 109.
Preferably, the reagent R1 blocker is one or more of sheep IgG, mouse IgG and rabbit IgG.
Preferably, the stabilizing agent in the reagent R2 is calcium levorotatory saccharate and dextran, and the mass ratio is 1:1.
Preferably, the dispersant in the reagent R1 is sodium lignin sulfonate.
The preparation method of the glypican 3 antibody coated latex microsphere comprises the following steps: adding a proper amount of polystyrene latex microspheres into 50mmol/L MES buffer solution with pH of 6.0 to make the final concentration of the latex microspheres be 1%, adding 10mg/ml of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDAC solution, reacting for 1 hour at room temperature, centrifuging at 12000rpm for 20min, and washing twice with 50mmol/L MES buffer solution with pH of 6.0; then adding glypican 3 antibody, incubating for 2 hours at 37 ℃, and coupling with latex microspheres to form an antibody latex complex; then, a proper amount of 1% casein and 0.5% N, N-dimethylformamide are added to seal for 1 hour at 37 ℃, and centrifugation is carried out at 12000rpm for 30 minutes, the supernatant is removed, and the obtained precipitate is added with 50mmol/L HEPES buffer solution to wash and disperse, thus obtaining the glypican 3 antibody coated latex microsphere.
Preferably, the glypican 3 antibody in the reagent R2 is a goat anti-human glypican 3 polyclonal antibody and a mouse anti-human glypican 3 monoclonal antibody, and the mass ratio is 3:2.
Preferably, the particle size of the polystyrene latex microspheres in the reagent R2 is 418nm and 122nm respectively, and the volume ratio of the two latex microspheres is 1:4.
The application of the glypican 3 assay kit is used for assaying the concentration of glypican 3 for the purpose of diagnosis and treatment of non-diseases.
The surfactant in the reagent R1 is the pollen Emulgen series, so that the interference of blood fat and chyle can be effectively removed, the reagent R1 system is uniformly dispersed, and the anti-interference capability of the reagent is greatly enhanced.
The reagent R1 of the invention is added with a blocking agent, which can eliminate or reduce endogenous interference caused by rheumatoid factors and amphotropic antibodies in immunoassay, and improve the accuracy and specificity of the reaction.
According to the invention, the stabilizer of calcium levorotatory sulfonate, glucan and dispersant of sodium lignin sulfonate are added into the reagent R2, so that the stability of the latex microsphere coated by the glypican 3 antibody in the reagent is improved, and the stability of the reagent R2 is excellent.
The antibody of the invention is a mixture of monoclonal antibody and polyclonal antibody, and improves the specificity and sensitivity of the reaction.
The glypican 3 determination reagent prepared by the invention has the advantages of good stability, high accuracy, high sensitivity and wide linear range, and is obviously superior to the products of the same type sold in the market.
Drawings
FIG. 1 is a correlation curve of the reagents of example 1 and comparative example 1;
FIG. 2 is a correlation curve of the reagents of comparative example 1 and comparative example 3;
FIG. 3 shows the concentration changes of glypican 3 assay reagent for stability test provided in example 1 and comparative examples 1, 4, 5, 6, and 7.
Detailed Description
The following describes in detail the examples of the present invention, which are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following examples.
The kit provided by the invention is used for measuring glypican 3 in serum, and the test conditions are as follows:
The method comprises the following steps: a terminal method; primary/secondary wavelength: 546nm/700nm; temperature: 37 ℃; correction type: non-linearities; the calibration method comprises the following steps: multipoint scaling; the reaction direction is as follows: upward.
The specific operation is shown in table 1.
TABLE 1 operation procedure of glypican 3 assay reagent
Calculation results:
Sample requirements:
1, serum is not hemolyzed.
Sample stability: the specimen can be stored stably for 3 days at 2-8 ℃ and for 2 weeks at-20 ℃.
The invention is further illustrated by the following examples:
Example 1
A kit for measuring glypican 3 comprises the following components;
reagent R1 comprises the following components:
100 mmol/L4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Sodium chloride 4.5g/L
Mouse IgG 10g/L
Polyethylene glycol 8000 g/L
Flower king Emulgen 102 2g/L
Pc300 1ml/L
pH 7.5
Reagent R2 comprises the following components:
100 mmol/L4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Glypican 3 antibody coated latex microsphere 120mg/L
Sodium lignin sulfonate 2g/L
L-calcium gluconate 3g/L
Dextran 3g/L
Pc300 1ml/L
pH 7.0
The preparation method of the glypican 3 assay kit comprises the following steps:
a) Preparation of reagent R1: the preparation method comprises the steps of weighing purified water with the same volume as the preparation volume, calculating and weighing a proper amount of 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid according to the preparation volume, adjusting the pH value to 7.5, calculating and weighing a proper amount of sodium chloride, polyethylene glycol 8000, peanut Emulgen 102, mouse IgG and the like according to the preparation volume, and uniformly stirring to obtain the reagent R1.
B) Preparation of reagent R2: and (3) measuring the purified water with the same volume as the preparation volume, calculating according to the preparation volume, weighing a proper amount of 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid, regulating the pH value to 7.0, calculating according to the preparation volume, weighing a proper amount of phosphatidylinositol proteoglycan 3 antibody coated latex microsphere, sodium lignin sulfonate, calcium levorotatory gluconate, dextran and the like, and uniformly stirring to obtain the reagent R2.
The preparation method of the glypican 3 antibody coated latex microsphere comprises the following steps: adding a proper amount of polystyrene latex microspheres into 50mmol/L MES buffer solution with pH of 6.0 to make the final concentration of the latex microspheres be 1%, adding 10mg/ml of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDAC solution, reacting for 1 hour at room temperature, centrifuging at 12000rpm for 20min, and washing twice with 50mmol/L MES buffer solution with pH of 6.0; then adding glypican 3 antibody, incubating for 2 hours at 37 ℃, and coupling with latex microspheres to form an antibody latex complex; then, a proper amount of 1% casein and 0.5% N, N-dimethylformamide are added to seal for 1 hour at 37 ℃, and centrifugation is carried out at 12000rpm for 30 minutes, the supernatant is removed, and the obtained precipitate is added with 50mmol/L HEPES buffer solution to wash and disperse, thus obtaining the glypican 3 antibody coated latex microsphere. Wherein the particle diameters of the latex microspheres are 418nm and 122nm respectively, and the mass ratio of the two latex microspheres is 1:4. The glypican 3 antibody is a sheep anti-human glypican 3 polyclonal antibody and a mouse anti-human glypican 3 monoclonal antibody, and the mass ratio is 3:2.
Example 2
A kit for measuring glypican 3 comprises the following components;
reagent R1 comprises the following components:
80 mmol/L4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Sodium chloride 4.5g/L
Mouse IgG 9g/L
Polyethylene glycol 8000 g/L
Flower king Emulgen 107 g/L
Pc300 1ml/L
pH 7.5
Reagent R2 comprises the following components:
50 mmol/L4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Glypican 3 antibody coated latex microsphere 100mg/L
Sodium lignin sulfonate 4g/L
L-calcium gluconate 4g/L
Dextran 4g/L
Pc300 1ml/L
pH 7.0
The preparation method of the glypican 3 assay kit is the same as that of example 1.
Example 3
A kit for measuring glypican 3 comprises the following components;
reagent R1 comprises the following components:
100 mmol/L4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Sodium chloride 4.5g/L
Mouse IgG 8g/L
Polyethylene glycol 8000 g/L
Flower king Emulgen 103 4g/L
Pc300 1ml/L
pH 7.5
Reagent R2 comprises the following components:
100 mmol/L4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Glypican 3 antibody coated latex microsphere 120mg/L
Sodium lignin sulfonate 8 g/L
L-calcium gluconate 3g/L
Dextran 3g/L
Pc300 1ml/L
pH 7.0
The preparation method of the glypican 3 assay kit is the same as that of example 1.
Comparative example 1
Glypican 3 chemiluminescent kit.
Comparative example 2
The kit for glypican 3 assay in example 1 was different from that in example 1 only in that the reagent R1 did not contain the King Emulgen 102, and the other was the same as in example 1.
Comparative example 3
The difference from the glypican 3 assay kit of example 1 is that the reagent R1 does not contain mouse IgG, and the other is the same as in example 1.
Comparative example 4
The kit for measuring glypican 3 in example 1 was different from the kit for measuring glypican 3 in that the reagent R2 did not contain sodium lignin sulfonate, and the other steps were the same as those in example 1.
Comparative example 5
The kit for glypican 3 assay in example 1 was different from that in example 1 only in that the reagent R2 did not contain calcium levorotatory sulfate.
Comparative example 6
The kit for glypican 3 assay in example 1 was different from that in example 1 only in that the reagent R2 did not contain dextran.
Comparative example 7
The kit for glypican 3 assay in example 1 was different from that in example 1 only in that the mass ratio of calcium levorotatory gluconate to dextran in the reagent R2 was 2:1, and otherwise the same as in example 1.
Comparative example 8
The kit for measuring glypican 3 in example 1 was different from that in example 1 only in that the latex microspheres in the reagent R2 had particle diameters of 418nm in all cases, and the other was the same as in example 1.
Comparative example 9
The kit for measuring glypican 3 in example 1 was different from the kit for measuring glypican 3 in example 1 only in that the latex microspheres in the reagent R2 had a particle diameter of 122nm in all cases, and the other was the same as in example 1.
Comparative example 10
The kit for glypican 3 assay was different from that of example 1 only in that glypican 3 antibody-coated latex microsphere was prepared using only a mouse anti-human monoclonal antibody in the reagent R2, and otherwise the same as in example 1.
Comparative example 11
The kit for glypican 3 assay was different from that of example 1 only in that glypican 3 antibody-coated latex microsphere was prepared using only goat anti-human polyclonal antibody in the reagent R2, and otherwise the same as in example 1.
Performance verification
Experiment one, correlation experiment
And carrying out a correlation test, wherein the test scheme is as follows: in example 1, comparative example 1 and comparative example 3, 40 clinical serum samples were simultaneously tested, correlation analysis was performed on two sets of test results, and a correlation coefficient r was calculated; the relative deviation (Bias%) of 40 pairs of data was calculated using the detection result of comparative example 1 as a target value. It is required that r is not less than 0.990 and the relative deviation is not more than + -10%.
TABLE 2 correlation comparison experiment results
Table 3 correlation coefficients of comparative example 1 and comparative example 3, respectively
As can be seen from table 2, table 3 and fig. 1, the maximum deviation of the serum samples measured by the reagents of example 1 and comparative example 1 is 7.5%, the correlation coefficient of the two reagents is 0.9931, and the detection results of example 1 and comparative example 1 are very close, so that the accuracy of the detection reagent of example 1 provided by the invention is higher; as can be seen from table 2, table 3 and fig. 2, the test results of comparative example 3 and comparative example 1 have large deviation, and the correlation coefficient is 0.703, which indicates that the addition of the blocking agent to the reagent R1 of the present invention is beneficial to eliminating endogenous interference and significantly improving the accuracy of the reagent.
Test two stability test
Stability tests were performed on glypican 3 assay reagents provided in example 1 and comparative examples 1, 4, 5, 6, 7, with the following test protocols: the reagents provided in example 1 and comparative examples 1, 4, 5, 6, and 7 were placed together in a 37℃water bath, and a quality control having a target value of 56.+ -. 3.1ng/ml was detected every day, and the quality control was monitored for changes in measurement.
Table 4 results of thermal stability verification (quality control) of reagents
As can be seen from Table 4 and FIG. 3, the reagent of example 1 provided by the invention has better stability in 12 days under the water bath condition of 37 ℃ similar to that of comparative example 1; the control value of the reagents of comparative examples 4, 5,6 and 7 is obviously reduced by 12 natural gift under the water bath condition of 37 ℃, the stability of the kit of example 1 is better than that of the kits of comparative examples 4, 5,6 and 7, the mass ratio of the calcium gluconate to the dextran is 2:1 although sodium lignosulfonate and the stabilizing agent calcium levorotatory sulfonate and the dextran are added in comparative example 7, and the quality control value is also reduced, which indicates that the invention adds the dispersing agent sodium lignosulfonate, the stabilizing agent calcium levorotatory sulfonate and the dextran into the reagent R2, optimizes the proportion of the stabilizing agent and is beneficial to improving the stability of the reagent R2.
Test three anti-interference test
Triglyceride samples, chyle and rheumatoid factor interference samples with different concentration levels are prepared, the samples with different concentrations of interference substances are measured and added by using the reagents provided in the example 1 and the comparative examples 1,2 and 3, the measurement result of the glypican 3 in the level 1 sample is taken as a base value, the deviation between the measurement value of the glypican 3 in the interference sample and the base value is calculated, and the deviation percentage is more than or equal to +/-10 percent, so that the interference exists. The results are shown in tables 5 and 6
TABLE 5 anti-interference experimental determination results
TABLE 6 results of interference experiments with rheumatoid factor
The results show that: example 1 was similar to comparative example 1, and when the concentration of triglyceride in the sample to be tested was 13.5mmol/L and the chyle was 1000mg/dL, there was no interference with the measurement result of glypican 3. Comparative example 2, when the concentration of triglyceride in the sample to be measured is 4.2mmol/L and chyle is 200mg/dL, interference is generated to the measurement result of glypican 3; comparative example 3 interference to glypican 3 assay results was observed when the rheumatoid factor in the test sample was 200 mg/dL. Therefore, the addition of the flower king Emulgen 102 plays an important role in eliminating interference of chyle and blood fat, and the addition of the blocker is beneficial to eliminating endogenous interference, so that the accuracy of the reagent is improved.
Test four-line test
Taking 200ng/mL of glypican 3 high-value sample, carrying out gradient dilution, preparing 6 samples with different concentrations, namely 0ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL and 200ng/mL in sequence, detecting the reagents provided in the comparative examples 1, 4, 8 and 9 by using the reagents of the example 1 and the comparative examples, measuring each sample with each concentration level for 3 times, and taking the average value of each sample. The test results are shown in Table 7:
TABLE 7 results of reagent linear validation
As can be seen from Table 7, similar to comparative example 1, inventive example 1 was linearly varied with dilution concentration, and the linear correlation coefficient reached 0.9995, indicating that example 1 was more linear and comparative example 8 was less linear, but comparative example 9 was a large particle size latex microsphere, and was relatively linear. Comparative example 4 although latex microspheres of different particle sizes were used, sodium lignin sulfonate dispersant was not added and linearity was poor, so that the simultaneous addition of dispersant to latex microspheres of different particle sizes could synergistically increase the linear range of the reagent.
Test five sensitivity test
Samples of known concentrations at 35ng/ml were measured with the reagents provided in example 1, comparative examples 1,4, 8, 9, 10, 11, respectively, and absorbance changes (. DELTA.A) were recorded. The detection results are shown in Table 8:
Table 8 analytical sensitivity vs. experimental results
As can be seen from the detection data, the absorbance change of the assay kit of example 1 is similar to that of the kit of comparative example 1, indicating that the kit of the invention has better analysis sensitivity. Comparative examples 4, 8, 9, 10 and 11 are poor in sensitivity, comparative example 8 adopts latex microspheres with large particle sizes, the sensitivity is high, comparative example 4 does not add sodium lignin sulfonate dispersant although latex microspheres with different particle sizes are adopted, the sensitivity is low, so that the simultaneous addition of the dispersant by adopting latex microspheres with different particle sizes can synergistically improve the analysis sensitivity of the reagent, and comparative example 10 and comparative example 11 add an antibody, the sensitivity is low, so that monoclonal antibodies and polyclonal antibodies are added proportionally, the reaction specificity is strong, recognition sites are many, and the sensitivity is improved.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Various modifications or additions to the described embodiments may be made by those skilled in the art to which the invention pertains or may be substituted in a similar manner without departing from the spirit of the invention or beyond the scope of the appended claims.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Claims (5)
1. A kit for determining glypican 3, which is characterized by comprising a reagent R1 and a reagent R2;
reagent R1 comprises the following components:
50-200mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Sodium chloride 4.5g/L
Blocking agent 8-15g/L
Polyethylene glycol 8000 g/L
Surfactant 1-4g/L
Pc300 1ml/L
Reagent R2 comprises the following components:
50-100mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid buffer
Glypican 3 antibody coated latex microsphere 10-200mg/L
Dispersing agent 1-10 g/L
Stabilizer 1-10 g/L
Pc300 1ml/L
The pH of the reagent R1 is 6.0-8.0; the pH of the reagent R2 is 6.0-8.0;
the surfactant in the reagent R1 is one or more of flower king Emulgen102, emulgen103, emulgen107 and Emulgen 109;
The blocking agent in the reagent R1 is mouse IgG;
The stabilizer in the reagent R2 is calcium levorotatory saccharate and glucan, and the mass ratio is 1:1;
the dispersing agent in the reagent R2 is sodium lignin sulfonate.
2. A method for preparing the glypican 3 assay kit according to claim 1, comprising the steps of:
Preparation of reagent R1: weighing purified water, weighing 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid according to the preparation volume, adjusting pH, weighing sodium chloride, polyethylene glycol 8000, surfactant and mouse IgG according to the preparation volume, and stirring uniformly to obtain a reagent R1; the surfactant is one or more of Emulgen102, emulgen103, emulgen107 and Emulgen 109;
Preparation of reagent R2: measuring purified water, weighing 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid according to the preparation volume, regulating pH, weighing phosphatidylinositol proteoglycan 3 antibody coated latex microsphere, sodium lignin sulfonate, calcium levo-gluconate and dextran according to the preparation volume, and uniformly stirring to obtain the reagent R2.
3. The method for preparing glypican 3 assay kit according to claim 2, wherein the method for preparing glypican 3 antibody-coated latex microspheres comprises the steps of: adding polystyrene latex microspheres into 50mmol/L MES buffer solution with pH of 6.0 to make the final concentration of the polystyrene latex microspheres be 1%, adding 10mg/ml of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride EDAC solution, reacting for 1 hour at room temperature, centrifuging at 12000rpm for 20min, and washing twice with 50mmol/L MES buffer solution with pH of 6.0; then adding glypican 3 antibody, incubating for 2 hours at 37 ℃, and coupling with polystyrene latex microspheres to form an antibody latex compound; then, 1% casein and 0.5% N, N-dimethylformamide were added and blocked at 37℃for 1 hour, and the supernatant was removed by centrifugation at 12000rpm for 30 minutes, and the obtained precipitate was washed with 50mmol/L HEPES buffer and dispersed to obtain glypican 3 antibody-coated latex microspheres.
4. The method for preparing glypican 3 assay kit according to claim 3, wherein the polystyrene latex microspheres are two, the particle sizes are 418nm and 122nm respectively, and the volume ratio of the two polystyrene latex microspheres is 1:4.
5. The method for preparing glypican 3 assay kit according to claim 3, wherein the glypican 3 antibody in the reagent R2 is a goat anti-human glypican 3 polyclonal antibody and a mouse anti-human glypican 3 monoclonal antibody, and the mass ratio is 3:2.
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