CN113866406B - Kit for specifically detecting sugar-deficient transferrin - Google Patents
Kit for specifically detecting sugar-deficient transferrin Download PDFInfo
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- CN113866406B CN113866406B CN202111212203.3A CN202111212203A CN113866406B CN 113866406 B CN113866406 B CN 113866406B CN 202111212203 A CN202111212203 A CN 202111212203A CN 113866406 B CN113866406 B CN 113866406B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
- G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Abstract
The invention is applicable to the technical field of biological detection, and provides a specificity detection kit for sugar-deficient transferrin and a preparation method thereof. The kit comprises a first reagent and a second reagent, wherein the first reagent comprises: a first buffer, a sugar-deficient transferrin complex and a coagulant, and a second reagent comprising: a second buffer, a sugar-deficient transferrin antibody conjugate, and a protective agent. The kit can accurately detect the sugar-deficient transferrin in human serum, blood plasma or cerebrospinal fluid, and has strong specificity; the result can be obtained by only one-step test, and the step of chromatographic separation of the sample is omitted.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a specificity detection kit for sugar-deficient transferrin and a preparation method thereof.
Background
Alcohol addiction is a global problem that can lead to irreversible physiological damage. Alcoholic Liver Disease (ALD) is one of the common liver diseases in China, and seriously harms the health of people. Extensive hepatocellular necrosis, and even liver failure, can be induced by severe alcohol abuse. In 2001, the Food and Drug Administration (FDA) approved sugar-deficient transferrin (CDT) as a marker for assessing drinking status in the clinic. CDT has desirable specificity as a marker for evaluating the drinking level. Particularly, compared with other liver enzyme markers, the CDT has better specificity and anti-interference capability on alcohol intake in the presence of liver diseases, is better than other traditional alcohol markers, and has better specificity. Has better specificity and sensitivity for detecting liver diseases caused by alcohol.
Transferrin (TRF), which is synthesized mainly by liver cells, has a relative molecular mass of 795,000Da, and contains 3 domains, i.e., 1 polypeptide chain consisting of 679 amino acids and 2 sugar chains, and 2 sugar chain binding sites are located at positions 413 and 611 of the peptide chain, respectively, and different sugar chain molecules can be bound. CDT is an isoform of transferrin, mainly the deletion of Sialic Acid (SA) residues on TRF. The proportion of different transferrin subtypes in the total amount of transferrin in normal human serum is different, the transferrin subtype with the highest content is the tetrasialo molecular subtype, and sialic acid molecules contained in transferrin in a human body are reduced after drinking wine, so that the levels of trisialo, disialo, monosialo and asiaialo transferrin subtypes are increased. Studies have shown that asialo transferrin, which is the main part of CDT, is due to the deletion of 2 sugar chains, and asialo transferrin, monosialyl transferrin and disialyl transferrin are clinically classified as CDT. The increased CDT content is usually found in the body of the drinker, and can disappear after drinking for a period of time, and the half-life period is 14 days. Studies have shown that the ratio of CDT to total transferrin is more specific for the diagnosis of alcohol status than is the absolute quantitative level of CDT.
The current CDT detection methods mainly comprise an isoelectric focusing electrophoresis method, a chromatography method, an immunization method and the like. The isoelectric focusing electrophoresis method comprises the following steps: separating the gel with pH gradient according to different isoelectric points of different subtypes, observing bands of various transferrin subtypes after the steps of immune fixation and dyeing, and finally performing density measurement to obtain CDT detection results. The method can calculate the absolute concentration of CDT according to a calibration curve. Anion exchange chromatography is commonly used in chromatography, but the sensitivity is low; high performance liquid chromatography can also detect hereditary TRF in blood samples of healthy people, but column exchange is time consuming and expensive, limiting its application in large-scale CDT detection. In recent years, CDT can be isolated from plasma by lectin affinity chromatography, followed by quantitative detection with the addition of anti-human transferrin serum. The method is relatively simple and convenient to operate and widely applied to laboratories. However, this method does not exclude interference from genetic variation of TRF and congenital glycosylation disorders. Genetic variation of TRF may be common in other races except caucasians, and CDT detection separated from TRF by chromatography may result in higher or lower results, resulting in inaccurate detection results.
Disclosure of Invention
In order to solve the problems, the invention provides a kit for specifically detecting the sugar-deficient transferrin and a preparation method thereof, the kit can be used for quantitatively detecting the sugar-deficient transferrin, and aims to make up for the defects of detection means in the prior art and reduce the detection cost.
The embodiment of the invention is realized by providing a kit for specifically detecting the sugar-deficient transferrin, which comprises a first reagent and a second reagent, wherein the first reagent comprises: a first buffering agent, a carbohydrate-deficient transferrin complex and a coagulant, the second reagent comprising: a second buffer and a saccharide deficient transferrin antibody conjugate.
Preferably, the first buffer and the second buffer are the same in composition and are each one of a phosphate buffer, a Tris-HCl buffer, a glycine sodium hydroxide buffer, a borate buffer, a carbonate buffer, a HEPES buffer, and a MES buffer.
Preferably, the first agent further comprises at least one of an inorganic salt, a surfactant, a protective agent and a preservative.
Preferably, the second agent further comprises a protective agent and/or a preservative.
Preferably, the sugar-deficient transferrin complex comprises sugar-deficient transferrin and a carrier, wherein the sugar-deficient transferrin and the carrier are linked together by physical adsorption or covalent coupling, and 2-4 sugar-deficient transferrins are linked to one carrier.
Preferably, the carrier is a biological macromolecule, a high molecular polymer, an avidin-biotin system or a streptavidin-biotin system, wherein the biological macromolecule is bovine serum albumin, casein or ovalbumin; the high molecular polymer is polyamino acid.
Preferably, the saccharide-deficient transferrin antibody conjugate comprises a saccharide-deficient transferrin antibody and polystyrene microspheres, and the saccharide-deficient transferrin antibody and polystyrene microspheres are bound together by covalent cross-linking, and the saccharide-deficient transferrin antibody is a monoclonal antibody that specifically binds to a non-glycosylation site of TRF.
Preferably, the polystyrene microsphere has a particle size of 50-500nm, and the surface of the polystyrene microsphere has one or more functional groups of carboxyl, amino, aldehyde group and chloromethyl.
Preferably, the sugar-deficient transferrin antibody is covalently crosslinked to polystyrene microspheres by the carbodiimide method, the glutaraldehyde method, the sodium periodate method, the N-hydroxysuccinimide ester method, or the maleimide method.
Preferably, the coagulant is at least one of polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000 and dextran 20000.
Preferably, the protective agent is at least one of BSA, casein and gelatin.
It is another object of embodiments of the present invention to provide a method for preparing a specific detection kit for sugar-deficient transferrin, comprising:
s1 preparing a first reagent comprising: covalently coupling a carrier and saccharide-deficient transferrin to prepare a saccharide-deficient transferrin complex, wherein the carrier is a biological macromolecule, a high molecular polymer, an avidin-biotin system or a streptavidin-biotin system, and the biological macromolecule is bovine serum albumin, casein or ovalbumin; the high molecular polymer is polyamino acid, and the first reagent further comprises a first buffering agent and a coagulant;
s2 preparing a second reagent comprising: covalently crosslinking a sugar-deficient transferrin antibody and a polystyrene microsphere by a carbodiimide method, a glutaraldehyde method, a sodium periodate method, an N-hydroxysuccinimide ester method or a maleimide method to prepare a sugar-deficient transferrin antibody microsphere conjugate, wherein the polystyrene microsphere has a particle size of 50-500nm and one or more of carboxyl, amino, aldehyde and chloromethyl functional groups on the surface, and the second reagent further comprises a second buffering agent;
s3 a sample to be detected and a first reagent are mixed evenly, incubated for 1-5 minutes, a second reagent is added, a transmission or scattering optical signal is detected under the specific wavelength of 340-.
Preferably, in the step S2, the operation of covalently crosslinking the polystyrene microspheres containing carboxyl groups includes: washing polystyrene microspheres with functional groups for 2-3 times by using MES or HEPES pH6.0 buffer solution, then fixing the volume by using the buffer solution, adding carbodiimide and N-hydroxy thiosuccinimide into the buffer solution, stirring at room temperature for activation, washing by using the buffer solution, fixing the volume, adding the washed microsphere suspension into an isometric sugar-deficient transferrin antibody solution, and adding a blocking agent under the stirring condition.
The detection kit provided by the invention has the following beneficial effects:
1. the kit for detecting the specific sugar-deficient transferrin by the immunoturbidimetry can accurately detect the sugar-deficient transferrin in human serum, plasma or cerebrospinal fluid by using the sugar-deficient transferrin antibody which is specifically combined with a TRF non-glycosylation site, does not detect other forms of transferrin such as genetic variant and congenital glycosylation disorder (CDG), has strong specificity, and can accurately evaluate the state of alcoholic liver diseases.
2. The kit for detecting the specific sugar-deficient transferrin by the immunoturbidimetry is applied to a biochemical analyzer or a specific protein analyzer, a result can be obtained by one-step testing, the step of chromatographic separation of a sample is omitted, large-batch and high-throughput testing of the sample can be realized, and clinical requirements are met.
3. The kit for detecting the specific sugar-deficient transferrin by the immunoturbidimetry provided by the invention has the advantages of high detection sensitivity, good repeatability and stable mass production process.
Drawings
FIG. 1 is a calibration curve obtained by fitting a calibrator concentration to a measured reactivity in an example of the present invention;
FIG. 2 is a linear range plot of the mean of the test versus the concentration of each dilution in example 5 of the present invention;
FIG. 3 shows a linear regression analysis curve of the test values of the reference kit in example 5 and the test values of the reagent combination in the example of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The embodiment of the invention provides a kit for specifically detecting sugar-deficient transferrin by using an immunoturbidimetry method, which comprises a first reagent and a second reagent, and preferably further comprises a calibrator and a quality control product, wherein the first reagent comprises: a first buffer, a sugar-deficient transferrin complex, and a coagulant, preferably further comprising inorganic salts, surfactants, protective agents, preservatives, and the like, and a second agent comprising: the second buffer and the sugar-deficient transferrin antibody microsphere conjugate preferably further comprise a protective agent, a preservative and the like. Preferably, the calibration material and the quality control material respectively comprise at the same time: a third buffer, a carbohydrate-deficient transferrin antigen, a protective agent, a preservative, and the like.
The kit provided by the embodiment of the invention utilizes a latex enhanced immunoturbidimetry method, is matched with a full-automatic biochemical analyzer or a specific protein analyzer for detection, can test the content of the sugar-deficient transferrin in samples such as serum, plasma or cerebrospinal fluid and the like, and can carry out high-throughput test. The kit does not need to pretreat a sample, and the content of the sugar-deficient transferrin can be obtained by directly testing on a computer. The detection kit is not influenced by TRF genetic variation, congenital glycosylation disorder (CDG) subtype and different sugar type iron saturation degrees of TRF. The sample is directly used for detection without chromatography separation of CDT, and the result is obtained within 2-10 minutes.
Specifically, the first buffer, the second buffer and the third buffer may be the same or different and may be one of a phosphate buffer, a Tris-HCl buffer, a glycine sodium hydroxide buffer, a borate buffer, a carbonate buffer, a HEPES buffer, a MES buffer, and the like. The inorganic salt may be one or more of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, etc. The surfactant can be nonionic surfactant, such as one or more of Triton-100, Tween-20 and Triton-308, or zwitterionic surfactant, anionic surfactant or cationic surfactant. The protective agent is inert protein, can be one or more of BSA, casein, gelatin and the like, and preferably also comprises polyalcohol or polysaccharide, such as one or more of glycerol, sucrose, sorbitol and the like. The coagulant can be polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, dextran 20000, etc. The preservative can be one or more of sodium azide, thimerosal, proClin300 and the like.
Specifically, the sugar-deficient transferrin complex in the first reagent is a composition formed by physically adsorbing or covalently coupling sugar-deficient transferrin and a carrier, wherein the carrier comprises a biomacromolecule, a high molecular polymer carrier and the like, and the biomacromolecule is an inert protein which is not specifically bound with the sugar-deficient transferrin, such as Bovine Serum Albumin (BSA), casein, ovalbumin and the like; the high molecular polymer includes polyamino acid, such as polylysine, polyaspartic acid, etc. Preferably, the carrier may further comprise an avidin-biotin system or a streptavidin-biotin system. Wherein 2-4 sugar-deficient transferrins are linked to one vector.
Specifically, the sugar-deficient transferrin antibody microsphere conjugate in the second reagent refers to a complex formed by covalently crosslinking a sugar-deficient transferrin antibody and a polystyrene microsphere. Wherein the glycodeficient transferrin antibody is a monoclonal antibody that specifically binds to a non-glycosylation site of TRF, is a murine, or rabbit anti-human monoclonal antibody, and can specifically bind CDT without binding to other forms of TRF (e.g., intact TRF and genetic variants and congenital glycosylation disorder (CDG) TRF); the polystyrene microsphere is a polystyrene microsphere with functional groups on the surface, and the functional groups can be one or more of carboxyl, amino, aldehyde group, chloromethyl and the like.
Preferably, the particle size of the polystyrene microsphere can be 50-500nm, preferably 120-300 nm.
The covalent cross-linking (covalent coupling) method in the preparation process of the sugar-deficient transferrin antibody microsphere conjugate in the second reagent comprises but is not limited to: a carbodiimide method, a glutaraldehyde method, a sodium periodate method, an N-hydroxysuccinimide ester method, a maleimide method, or the like. Taking carboxyl polystyrene microsphere coupling as an example, the concrete preparation steps of the conjugate are as follows: washing carboxyl polystyrene microsphere with 10-100mM MES pH6.0 buffer solution for 2-3 times, then using the buffer solution to keep the volume to 0.5-2% (w/v), adding carbodiimide and N-hydroxy thiosuccinimide, stirring for 15-40 minutes at room temperature to activate, using 10-100mM MES or HEPES buffer solution to wash for 2-3 times, keeping the volume to 0.5-2% (w/v), adding the microsphere suspension after activation and washing into 0.01-0.2% sugar-deficient transferrin antibody solution with the same volume, stirring for 2-4 hours at room temperature, adding blocking agent, stirring at room temperature for 2-4 hr, washing with 10-100mM MES or HEPES buffer solution for 2-3 times, and dispersing in appropriate storage solution to obtain sugar-deficient transferrin antibody microsphere conjugate.
Specifically, the carbohydrate-deficient transferrin antigen in the calibrator or the quality control product refers to natural human carbohydrate-deficient transferrin or recombinantly expressed carbohydrate-deficient transferrin.
In the kit for detecting transferrin deficient in sugar provided by some embodiments of the invention, the pH of the first reagent is 6.0 to 9.0, and table 1 shows the specific formulation in some embodiments:
TABLE 1 formulation of the first reagent
| Components | Content (wt.) |
| Buffering agent | 10-500mM |
| Sugar-deficient transferrin complexes | 0.1-2g/L |
| Inorganic salt | 0.2-5％(w/v) |
| Surface active agent | 0.005-1％(w/v) |
| Protecting agent | 0.1-5％(w/v) |
| Preservative | 0.05-1％(w/v) |
| Setting accelerator | 0.1-3％ |
In the kit for detecting transferrin deficient in sugar provided by some embodiments of the present invention, the pH of the second reagent is 7.0 to 9.0, and table 2 shows the specific formulation of the second reagent:
TABLE 2 formulation of the second reagent
The specific carbohydrate-deficient transferrin immunoturbidimetry detection kit can be applied to the determination of the carbohydrate-deficient transferrin content in serum, plasma or cerebrospinal fluid samples. The determination method comprises the following specific steps: taking a proper amount of serum, plasma or cerebrospinal fluid sample and the first reagent, mixing uniformly, incubating for 1-5 minutes, adding the second reagent, marking the instrument reading point as A1, detecting a transmission or scattered light signal at a specific wavelength of 340-800nm, continuing incubating for 1-5 minutes, marking the instrument reading point as A2, detecting a transmission or scattered light signal at a specific wavelength of 340-800nm, recording the difference value of the optical signals of the reaction endpoint A2 and the reaction initiation point A1 after the second reagent is added, namely the change amplitude of the transmission or scattered light signals of the reaction endpoint A2 and the reaction initiation point A1, and substituting the change amplitude into a standard curve to calculate the content of the sugar-deficient transferrin in the sample. The kit can be applied to the kit for measuring the transferrin with sugar deficiency by immunoturbidimetry, the detection principle can be transmission turbidimetry or scattering turbidimetry, and the detection instrument can be applied to but not limited to biochemical analysis and a specific protein analyzer.
The transferrin monoclonal antibody, the polystyrene microsphere, the coupling blocking agent, the coupling activating agent and the like which are related to the sugar deficiency can be obtained by purchasing.
Example 1 preparation of a kit for detecting sugar-deficient transferrin
The first reagent was prepared according to the composition shown in table 3:
TABLE 3 formulation of the first reagent
The preparation method comprises the following steps: adding part of purified water into a beaker, placing the mixture on a magnetic stirrer, stirring at medium speed, adding the components according to the table 3, stirring all the components to be completely dissolved, adjusting the pH value to 7.5 by using hydrochloric acid, and fixing the volume to 1L by using the purified water.
A second reagent was prepared according to the composition shown in table 4:
TABLE 4 formulation of the second reagent
| Components | Dosage of |
| Tris (hydroxymethyl) aminomethane | 12.1g |
| Sucrose | 10g |
| Tween-20 | 0.05mL |
| Bovine serum albumin | 0.5g |
| Sodium azide | 0.95g |
| Carbohydrate-deficient transferrin antibody conjugates | 1.5g |
Adding part of purified water into a beaker, placing the mixture on a magnetic stirrer, stirring at a medium speed, adding the components according to the table 4, stirring until all the components are dissolved, adjusting the pH value to 8.0 by using hydrochloric acid, and fixing the volume to 1L by using the purified water.
Example 2 preparation of sugar-deficient transferrin-Bovine Serum Albumin (BSA) complexes in this example, a sugar-deficient transferrin-Bovine Serum Albumin (BSA) composition was prepared as follows:
(1) adding 10mg of sugar-deficient transferrin into 50mM Tris-HCl buffer solution (pH7.5), magnetically stirring at room temperature, mixing, adding 10mg of BSA, and stirring at room temperature for 0.5 h;
(2) adding 50 mu L of 5% (w/v) glutaraldehyde, and stirring at room temperature for 2 hours;
(3) dialyzing with 50mM Tris-HCl buffer (pH7.5) at 4 deg.C overnight, changing the solution for 3 times, transferring into 50mM Tris-HCl buffer (pH8.0, containing 0.09% NaN3, 0.9% NaCl), dialyzing at 4 deg.C overnight, and changing the solution for 3 times;
(4) subpackaging the prepared composition of sugar-deficient transferrin-Bovine Serum Albumin (BSA), and storing at-20 deg.C.
Example 3: preparation of sugar-deficient transferrin-biotin-streptavidin complexes in this example, a sugar-deficient transferrin-biotin-streptavidin composition was prepared as follows:
(1) dialyzing the sugar-deficient transferrin protein with 100mM borate buffer (pH 8.6) overnight at 4 ℃ and changing the solution for 3 times;
(2) dissolving 1mg of N-hydroxysuccinimide biotin in 1mL of DMSO;
(3) mixing 1mL of the sugar-deficient transferrin solution (5mg/mL) with 200 mu L N-hydroxysuccinimide biotin solution, and stirring at room temperature for 2 hours;
(4) dialyzing with 50mM phosphate buffer (pH7.4) at 4 deg.C overnight, and changing the solution for 3 times;
(5) adding 0.1mg of streptavidin into 1mg of prepared biotinylated sugar-deficient transferrin, and stirring at room temperature for 0.5 hour;
(6) filtering with Superdex G-200 gel chromatography column (GE, cat # 17104301), separating sugar-deficient transferrin-biotin-streptavidin composition, packaging, and storing at-20 deg.C.
Example 4 preparation of a saccharide deficient transferrin antibody conjugate
This example provides a method for preparing a transferrin antibody conjugate with sugar deficiency, comprising the following steps:
washing carboxyl polystyrene microspheres for 3 times by using 50mM MES pH6.0 buffer solution, then using the MES buffer solution to fix the volume until the concentration of the carboxyl polystyrene microspheres is 1% (w/v), adding excessive carbodiimide and N-hydroxy thiosuccinimide into the mixture, stirring the mixture for 30 minutes at room temperature to activate the mixture, using 50mM MES pH6.0 buffer solution to wash the mixture for 3 times, fixing the volume until the concentration of the carboxyl polystyrene microspheres in the mixture is 1% (w/v), adding the microsphere suspension after activation and washing the mixture into 0.05% of sugar-deficient transferrin antibody solution with the same volume, stirring the mixture for 2 hours at room temperature, adding one tenth volume of blocking agent (the volume of the blocking agent is one tenth of the volume of the mixture before adding the blocking agent), continuing stirring the mixture for 2 hours at room temperature, using 50mM HEPES buffer solution (pH7.5) to wash the mixture for 3 times, finally dispersing the mixture in proper buffer solution, the final concentration of the carboxyl polystyrene microspheres in the obtained dispersion is 0.13% (w/v), and the obtained dispersion is the sugar-deficient transferrin antibody conjugate. The carbohydrate-deficient transferrin antibody of this example is a murine anti-human monoclonal antibody, derived from: biochem system of the medical college of Buddmont university, Berlington, Va.
The CDT antigens used in the calibrators and quality controls in the examples herein can be prepared by expression from prokaryotic or eukaryotic cells, the details of which are well known to those skilled in the art.
Example 5 evaluation of the Performance of the specific sugar-deficient transferrin immunoturbidimetry assay kit
This example selects the components prepared in examples 1-2 and 4 for performance evaluation, specifically for the evaluation of the performance of linear range, methodological alignment and reproducibility.
Testing an instrument: hitachi 7180 biochemical analyzer;
the specific test method comprises the following steps: and (2) uniformly mixing a2 mu L serum sample and 200 mu L of the first reagent prepared in the embodiment 1, incubating for 5 minutes, adding 50 mu L of the second reagent prepared in the embodiment 2, continuing to incubate for 5 minutes, detecting a transmitted light signal at a specific wavelength of 600nm (the signal value is obtained by an Hitachi 7180 biochemical analyzer according to the automatic detection and calculation of turbidity of reaction liquid), recording the difference value of the amplitude of a reaction endpoint and a reaction initiation point after the second reagent is added, and substituting the difference value into the standard curve shown in the figure 1, so that the content of the sugar-deficient transferrin in the sample to be detected can be calculated. Fig. 1 is a calibration curve obtained by fitting the concentrations of the calibrators and the measured reactivities in the present embodiment, in fig. 1, the ordinate represents reactivities, specifically, transmitted light signal values, and the abscissa represents the concentrations of the calibrators.
The operation method is very simple and convenient.
In this embodiment, the buffer composition of the quality control material is the same as the buffer composition of the calibrator.
The calibrator and the quality control product are prepared by dissolving a sugar-deficient transferrin antigen in a buffer solution, wherein the buffer solution comprises the following components: 20mM PBS buffer, 1% BSA, pH 7.4.
The reagent evaluation results were as follows:
1. linear range
The low-concentration sugar-deficient transferrin serum samples and the high-concentration sugar-deficient transferrin serum samples were mixed to 6 dilution concentrations according to table 5 below. The mixed samples were tested separately with the above reagent combinations (kits), 3 times per concentration, and the correlation coefficient and deviation were calculated.
TABLE 5 formulation of two samples
| Dilution gradient | Concentration 1 | Concentration 2 | Concentration 3 | Concentration 4 | Concentration 5 | Concentration 6 |
| Low concentration sample volume | 1mL | 0.8mL | 0.6mL | 0.4mL | 0.2 |
0 |
| High |
0 | 0.2mL | 0.4mL | 0.6mL | 0.8mL | 1mL |
Wherein the concentration of the low-concentration sample is 20mg/L, and the concentration of the high-concentration sample is 660 mg/L.
The test results are shown in table 6 below:
TABLE 6 test results
Wherein the absolute deviation is | mean-regression | value; relative deviation ═ [ (mean-regression)/regression ] × 100%. "/" indicates no statistics, absolute deviations were observed for concentrations less than 100mg/L and relative deviations were observed for concentrations greater than 100 mg/L.
FIG. 2 shows the linear range curve of each dilution concentration and the test mean, the kit has a [20.0, 660.0] mg/L interval, k is 0.9608, b is 12.1379, the linear correlation coefficient R is 0.9996, the absolute deviation of linearity is not more than + -10 mg/L in the [20.0, 100.0] mg/L interval, and the relative deviation of linearity is not more than + -5% in the (100.0, 660.0] mg/L interval, the results show that the kit of the present application has a very good linear relationship in the range of 20-660 mg/L.
2. Methodological comparison
40 samples of fresh serum were taken and tested with a saccharide-deficient transferrin assay kit (which uses latex enhanced nephelometry, the test method being different from that of the present application) purchased from siemens medical diagnostics products limited, germany (siemens short) (reference kit), and the reagent combinations (kits) prepared according to the methods of examples 1 to 4 of the present invention were used to test at the same time, and the correlation coefficients of the two sets of test values were calculated by linear regression, and the test results are shown in table 7.
TABLE 7 test results
FIG. 3 shows the results of a linear regression analysis of the test values of a reference kit with the test values of the reagent combinations of the examples of the present invention, where R 2 0.9935, correlation R0.9967.
3. Repeatability test
The sugar-deficient transferrin assay kit of example 1 was used to repeat the test 10 times on the low, medium and high 3 concentrations of serum samples, respectively, and the coefficient of variation was calculated, and the results are shown in table 8.
TABLE 8 measurement results of coefficient of variation
It can be seen that the coefficients of variation of the low, medium and high concentration sera tested 10 times were 1.99%, 1.54% and 2.63%, respectively.
The results show that the kit for determining the sugar-deficient transferrin by the immunoturbidimetry method prepared by the embodiment of the invention has good performance, and the linear correlation coefficient R is 0.9996; wherein the correlation R of 40 samples and the reference kit is 0.9967; the test samples were repeated 10 times with a coefficient of variation within 5%. Compared with a reference kit, the kit provided by the application is simple and convenient to operate, and the preparation cost is greatly reduced.
Example 6 assay kit Performance evaluation
The combination of reagents of examples 1 and 3-4 (the sugar-deficient transferrin-bovine serum albumin complex in the second reagent was replaced with the sugar-deficient transferrin-biotin-streptavidin complex) was evaluated in the same manner as in example 5, and the results were substantially the same as in example 5.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit of the present invention are intended to be included therein.
Claims (3)
1. A kit for specifically detecting a carbohydrate-deficient transferrin comprising a first reagent and a second reagent, wherein the first reagent is formulated as follows:
The second reagent formula is as follows:
The sugar-deficient transferrin complex is a sugar-deficient transferrin-biotin-streptavidin complex and is prepared by the following steps:
(1) dialyzing the sugar-deficient transferrin protein overnight at 4 ℃ with 100mM borate buffer solution, pH 8.6, and changing the solution for 3 times;
(2) dissolving 1mg of N-hydroxysuccinimide biotin in 1mL of DMSO;
(3) 1mL of the sugar-deficient transferrin solution with the concentration of 5mg/mL is mixed with 200 mu L N-hydroxysuccinimide biotin solution and stirred for 2 hours at room temperature;
(4) dialyzing with 50mM phosphate buffer solution, pH7.4, at 4 deg.C overnight, and changing the solution for 3 times;
(5) adding 0.1mg of streptavidin into 1mg of prepared biotinylated sugar-deficient transferrin, and stirring at room temperature for 0.5 hour;
(6) filtering with Superdex G-200 gel chromatographic column, separating sugar-deficient transferrin-biotin-streptavidin composition,
the conjugate of the sugar-deficient transferrin antibody comprises the sugar-deficient transferrin antibody and polystyrene microspheres, the sugar-deficient transferrin antibody and the polystyrene microspheres are covalently crosslinked, and the sugar-deficient transferrin antibody is a monoclonal antibody specifically binding to a non-glycosylation site of transferrin.
2. The kit of claim 1, wherein the polystyrene microspheres have a particle size of 50-500nm and one or more of carboxyl, amino, aldehyde and chloromethyl groups on the surface.
3. The kit of claim 1, wherein the sugar-deficient transferrin antibody is covalently crosslinked to polystyrene microspheres by the carbodiimide method, the glutaraldehyde method, the sodium periodate method, the N-hydroxysuccinimide ester method, or the maleimide method.
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