CN112129947A - Method for detecting abnormal sugar chain glycoprotein - Google Patents
Method for detecting abnormal sugar chain glycoprotein Download PDFInfo
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- CN112129947A CN112129947A CN202010822167.1A CN202010822167A CN112129947A CN 112129947 A CN112129947 A CN 112129947A CN 202010822167 A CN202010822167 A CN 202010822167A CN 112129947 A CN112129947 A CN 112129947A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- 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
- G01N21/76—Chemiluminescence; Bioluminescence
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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
Abstract
The present invention relates to a method for detecting an abnormal sugar chain glycoprotein, comprising: the method comprises the steps of firstly, preparing and detecting a sample, namely processing an S1 sample, incubating the S2 sample, separating S3, sealing S4, identifying carbohydrate chain abnormal protein by lectin labeled by S5 biotin, carrying out signal amplification by streptavidin labeled by horseradish peroxidase or alkaline phosphatase by S6, preparing a standard curve, preparing 4 or more abnormal carbohydrate chain glycoprotein samples with different mass concentrations as standard products in the range of 0-20000ng/ml, processing the standard products according to the steps S2-S6, obtaining a luminous value based on a chemiluminescence immunoassay instrument or a semi-automatic luminometer, converting the mass concentration except the 0 value and the corresponding luminous value into logarithm with the base of 2, and substituting the logarithm with the base of 2 into a four-parameter logistic curve y = (A-D)/[1+ (x/C) ^ B ] + D to obtain the standard curve; and thirdly, calculating the concentration of the sample. The method of the invention can be used for independently analyzing different types of abnormal sugar chain glycoprotein, realizes quantitative detection and has high accuracy.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a method for detecting abnormal sugar chain glycoprotein.
Background
Protein glycosylation plays an important role in protein structure and function as an important post-translational modification, and it has been found that glycosylation occurs in about 50% of proteins in mammalian cells, and this modification plays a key role in many cellular functions such as cell recognition, adhesion, cell-cell interaction, and growth and development.
The abnormal sugar chain glycoprotein is mainly caused by incomplete glycosylation or new glycosylation caused by the activation of new glycosyl transferase, and a great deal of research shows that the generation of the abnormal sugar chain glycoprotein is closely related to tumors, such as alpha fetoprotein, transferrin, alkaline phosphatase, r-glutamyltransferase, human chorionic gonadotropin, T antigen, a1 antitrypsin, prostatic acid phosphatase and the like of malignant tumor patients, and after the sugar chain structure is changed to a certain degree, the substances are discharged into blood and are mostly present in peripheral blood species.
Lectin is a glycoprotein or sugar-binding protein purified from various plants, invertebrates and higher animals. It has the characteristic of being capable of being combined with glycoprotein in a specific and non-covalent reversible way. The strength of lectin binding to glycoproteins may increase with the number of molecular interactions, and the dissociation constant of lectin binding to glycoproteins is about Kd10-5～10-7。
In the prior art, the use of the affinity of abnormal sugar chain glycoproteins with different phytolectins for the diagnosis of various types of tumors has been suggested. However, the detection method in the prior art has the following defects:
firstly, the agglutinin is not effectively pretreated, and the stability of the agglutinin is insufficient, so that the reliability of detection is influenced; the anti-proteolytic ability of lectins also needs to be further improved by means of a pretreatment.
Thirdly, the operation steps of the detection method have an optimizable space, and the prior art cannot realize quantitative detection.
The present invention aims to provide a method for detecting an abnormal sugar chain glycoprotein based on a novel abnormal sugar chain glycoprotein detection reagent.
Disclosure of Invention
It is an object of the present invention to solve the problems of the prior art and to provide a method for detecting an abnormal sugar chain glycoprotein by using a novel reagent for detecting an abnormal sugar chain glycoprotein. The method can effectively give consideration to both broad spectrum and accuracy, and in addition, the lectin is pretreated, so that the stability is obviously improved, the reliability of the detection method is good, and the proteolysis resistance of the pretreated lectin is also obviously improved. In addition, the detection method has more reasonable operation steps and high reliability.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for detecting an abnormal sugar chain glycoprotein, comprising the steps of:
first, sample preparation and detection
S1 sample processing
Diluting the sample to be detected to 1-20000 times by using a sample diluent, and fully and uniformly mixing the sample to be detected to obtain a sample solution;
s2 sample incubation
Placing the sample solution in a hole of a solid phase A, wherein the solid phase A is a polystyrene and/or polypropylene luminescent plate, controlling the pH of the sample solution at 9.40-9.60, incubating at room temperature for 2-3h or incubating at 37 ℃ for 30-60min, and binding the diluted sample on the solid phase A through incubation; or placing the sample liquid in a reaction cup, adding a solid phase B, wherein the solid phase B is a magnetic microsphere, controlling the pH of the sample liquid to be 7.00-7.20, incubating at room temperature for 2-3h or incubating at 37 ℃ for 30-60min, and binding the diluted sample on the solid phase B through incubation;
s3 separation
Pouring out residual sample diluent combined with the solid phase A, adding 300ul of washing solution into each hole, standing for 30-60 s, pouring out the washing solution, and repeating the steps for 2-3 times; standing the reaction cups on a magnetic separator for 1-2 min for the solid phase B, adding 300ul of washing solution into each reaction cup, standing for 30-60 s, pouring the washing solution, and repeating the steps for 2-3 times;
s4 sealing
Adopting one or more of bovine serum albumin, whey protein and casein with the mass percentage of 0.5-1.5% as a sealant, sealing at pH7.00-7.20, overnight at 4 ℃ or constant temperature at 37 ℃ for 1.5h, and using the sealant in each hole or reaction cup: 300ul, after sealing, separating different solid phases according to the separation operation of S3;
s5 biotin-labeled lectin recognizing sugar chain-abnormal protein
Concentration and amount of biotin-labeled lectin used per well or cuvette: 0.1-10ug/ml, 50-200ul, pH: 7.00-7.20, reaction time: incubating at room temperature for 2-3h or incubating at 37 ℃ for 1h, and separating different solid phases according to the separation operation of S3;
s6 Signal amplification Using horseradish peroxidase or alkaline phosphatase labeled streptavidin
Labeling ratio of horseradish peroxidase or alkaline phosphatase and SA: 1: (1-10), the concentration and dosage used in each hole or reaction cup are as follows: 0.05-0.2ug/ml, 50-200ul, pH: 7.00-7.20, reaction time: incubating at room temperature for 30-60min or incubating at 37 ℃ for 15min, and separating different solid phases according to the separation operation of S3;
second, making a standard curve
Preparing 4 or more abnormal glycophorin protein samples with different mass concentrations as standard substances within the range of 0-20000ng/ml, processing the standard substances according to the steps S2-S6, obtaining luminescence values based on a chemiluminescence immunoassay analyzer or a semi-automatic luminometer, converting the mass concentrations except the 0 value and the corresponding luminescence values into logarithm taking 2 as a base, substituting the logarithm into a four-parameter logistic curve y = (A-D)/[1+ (x/C) ^ B ] + D, and performing linear fitting to obtain a standard curve;
third step, calculation of sample concentration
And (5) detecting the sample obtained in the step (S6) based on a chemiluminescence immunoassay analyzer or a semi-automatic luminometer to obtain a luminescence value, substituting the luminescence value into the standard curve, and calculating to obtain the concentration of the sugar chain abnormal protein in the sample to be detected.
Preferably, in step S1, the sample is any one of serum, plasma and body fluid, and the ability of the solid phase A to bind to abnormal sugar chain glycoproteins is not less than 1000ng/cm2The capacity of the solid phase B for combining the abnormal sugar chain glycoprotein is not less than 80ng of abnormal sugar chain glycoprotein/10 ug of magnetic microspheres.
Preferably, the solid phase A adopts a 96-hole luminescent plate, the sample amount of each hole is 50-200 mu l, the concentration of the solid phase B is 0.1-0.5mg/ml, the sample amount in each reaction cup is 10-200 mu l, and the addition amount of the solid phase B is 50-200 mu l.
Preferably, in step S5, the mass ratio of the lectin to the biotin is 1: (1-5).
Preferably, the sample diluent for solid phase a has the following composition: 0.5-1.0g of sodium carbonate, 10-15g of sodium bicarbonate and purified water with constant volume of 1L; sample dilutions for solid phase B, consisting of: NaH2PO4.2H2O：0.5-1g，Na2HPO4.12H2O: 2-3g, and the volume of purified water is up to 1L.
Preferably, the diluent for biotin-labeled lectin and horseradish peroxidase-or alkaline phosphatase-labeled streptavidin is composed of:
NaH2PO4.2H2O：0.5-1g
Na2HPO4.12H2O：2-3g
Zn2+：0.01-0.02g
Mg2+：0.2-0.4g
the purified water was made to 1L.
Preferably, the lectin is one or more selected from the group consisting of concanavalin, stramonium lectin, lentil lectin, wheat germ lectin, E-type red kidney bean lectin, L-type red kidney bean lectin, pinocembrus aurantiaca lectin, peanut lectin, castor agglutinin I, Maackia amurensis lectin, Leptospira aurantiaca lectin, Agaricus bisporus lectin, Sambucus nigra lectin and Huai agglutinin І І, and is subjected to PEG pre-modification treatment.
Preferably, the PEG pre-modification treatment of the lectin is performed as follows:
s1: mixing carbodiimide, methoxy polyethylene glycol active ester and lectin, and reacting at room temperature for 2-6 h;
s2: and (3) separating the target product in the reaction system in the step S1 by using sephadex SwphadexG-50 to obtain a substance of a first section peak, and obtaining the target product, namely the lectin after the PEG pre-modification.
Preferably, the mass ratio of the carbodiimide to the methoxypolyethylene glycol active ester to the lectin is 1:100: 1.
In the invention, before the lectin is used, PEG is used for modification so as to enhance the stability of the lectin, improve the protease hydrolysis resistance of the lectin, improve the solubility of the lectin in the reaction process after drying, and facilitate the formation of a uniform and specific reaction system.
The invention has the following beneficial effects: the method of the invention can be used for carrying out independent analysis on different types of abnormal sugar chain glycoprotein, and in addition, the quantitative detection is realized, the accuracy is high, and the reliability is good.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The reagents and raw materials required in the present invention are commercially available.
Example 1 method for detecting abnormal sugar chain glycoprotein
A method for detecting an abnormal sugar chain glycoprotein, comprising the steps of:
first, sample preparation and detection
S1 sample preparation
Diluting a sample to be detected to 1000 times by using a sample diluent, and fully and uniformly mixing the sample to be detected to obtain a sample solution;
s2 sample incubation
Placing the sample solution in a hole of a solid phase A, wherein the solid phase A is a polystyrene luminescent plate, controlling the pH value of the sample solution at 9.40, incubating at the constant temperature of 37 ℃ for 30min, and combining the diluted sample on the solid phase A through incubation;
s3 separation
Pouring out the residual sample diluent, adding 300ul of washing solution into each hole, standing for 30s, pouring out the washing solution, and repeating the steps for 2-3 times;
s4 sealing
Adopting bovine serum albumin with the mass percentage of 0.5% as a blocking agent, and blocking the mixture at pH7.00 and 4 ℃ overnight, wherein the dosage of the blocking agent in each hole is as follows: 300ul, after the sealing is finished, separating according to the separation operation of S3;
s5 biotin-labeled lectin recognizing sugar chain-abnormal protein
Concentration and amount of biotin-labeled lectin used per well: 0.1ug/ml, 50ul, pH: 7.00, reaction time: incubating for 3h at room temperature, and separating according to the separation operation of S3;
s6 Signal amplification Using horseradish peroxidase labeled streptavidin
Labeling ratio of horseradish peroxidase and SA: 1:1, using concentration and using amount of each hole: 0.05ug/ml, 50ul, pH: 7.00, reaction time: incubating at room temperature for 60min, and separating according to the separation operation of S3;
second, prepare the standard curve
Preparing 6 abnormal glycoglycoprotein samples with different mass concentrations as standard substances, processing the standard substances according to the steps S2-S6, obtaining luminescence values based on a chemiluminescence immunoassay analyzer, converting the mass concentrations except for 0 value and the corresponding luminescence values into logarithm with base 2, and substituting into a four-parameter logistic curve y = (A-D)/[1+ (x/C) ^ B]+ D, linear fitting to obtain a standard curve;
| sample numbering | Concentration of | Luminous value | Sample type |
| 1 | 10000.00 | 5231270 | Standard article |
| 2 | 5000.00 | 3195169 | Standard article |
| 3 | 2500.00 | 1510488 | Standard article |
| 4 | 1250.00 | 711602 | Standard article |
| 5 | 625.00 | 256759 | Standard article |
| 6 | 0.00 | 31620 | Standard article |
The data fitting method comprises the following steps: a four parameter logistic equation.
The two-point calibration method comprises the following steps: and a second two-point calibration method.
The standard curve obtained by fitting is: y = (26.33117-5.33984)/[ 1+ (x/8.04767) ^ 2.88728] + 5.33984, correlation coefficient R = 0.99931.
And thirdly, detecting and obtaining a luminous value, wherein the luminous value is 361228, substituting the luminous value into a standard curve, calculating to obtain the concentration of the abnormal sugar chain glycoprotein in the sample to be detected to be 780ng/ml, and multiplying the concentration by the dilution factor 1000 to finally obtain the concentration of the abnormal sugar chain glycoprotein in the sample to be detected to be 780 ug/ml.
In example 1, the sample is serum, wherein the serum sample is diluted at a ratio of 1:1000, and the ability of solid phase A to bind to an abnormal sugar chain glycoprotein is 1000ng/cm2The solid phase A adopts a 96-hole luminescent plate, the sample volume of each hole is 50 mu l, and in the step S5, the mass ratio of the agglutinin to the biotin is 1:1, sample dilution for solid phase a, consisting of: 0.5g of sodium carbonate, 10g of sodium bicarbonate and purified water with the constant volume of 1L, wherein the diluent for biotin-labeled lectin and horseradish peroxidase-labeled streptavidin comprises the following components:
NaH2PO4.2H2O：0.5g
Na2HPO4.12H2O：2g
Zn2+：0.01g
Mg2+：0.2g
the purified water was made to 1L.
In an embodiment, the lectin is reticulocyte aurantiamarin lectin, the lectin is subjected to PEG pre-modification treatment, and the PEG pre-modification treatment of the lectin comprises the following steps:
s1: mixing carbodiimide, methoxy polyethylene glycol active ester and lectin, and reacting for 2 hours at room temperature; the mass ratio of the carbodiimide to the methoxypolyethylene glycol active ester to the lectin is 1: 1;
s2: and (3) separating the target product in the reaction system in the step S1 by using sephadex SwphadexG-50 to obtain a substance of a first section peak, and obtaining the target product, namely the lectin after the PEG pre-modification.
Example 2
A method for detecting an abnormal sugar chain glycoprotein, comprising the steps of:
s1 sample processing
Diluting a sample to be detected to 1000 times by using a sample diluent, and fully and uniformly mixing the sample to be detected to obtain a sample solution;
s2 sample incubation
Placing the sample liquid in a reaction cup, adding a solid phase B, wherein the solid phase B is a magnetic microsphere, controlling the pH value of the sample liquid at 7.00, incubating at the constant temperature of 37 ℃ for 30min, and binding the diluted sample on the solid phase B through incubation;
s3 separation
Standing the reaction cups on a magnetic separator for 1min, adding 300ul of washing solution into each reaction cup, standing for 30s, pouring the washing solution, and repeating the steps for 2-3 times;
s4 sealing
Adopting 1.5 percent of whey protein as a sealant by mass percent, sealing at the constant temperature of 37 ℃ for 1.5h at the pH of 7.20, and using the sealant in each hole: 300ul, after the sealing is finished, separating according to the separation operation of S3;
s5 biotin-labeled lectin recognizing sugar chain-abnormal protein
Concentration and amount of biotin-labeled lectin used per well: 10ug/ml, 200ul, pH: 7.20, reaction time: incubating at the constant temperature of 37 ℃ for 1h, and separating according to the separation operation of S3;
s6 Signal amplification Using alkaline phosphatase-labeled streptavidin
Labeling ratio of alkaline phosphatase and SA: 1:10, concentration and dosage used per well: 0.2ug/ml, 200ul, pH: 7.20, reaction time: incubating at 37 ℃ for 15min, and separating according to the separation operation of S3;
second, prepare the standard curve
Preparing 6 abnormal glycophorin samples with different mass concentrations as standard substances, processing the standard substances according to the steps S2-S6, obtaining luminescence values based on a semi-automatic luminometer, converting the mass concentrations except 0 value and the corresponding luminescence values into logarithm with base 2, and substituting the logarithm with base 2 into a four-parameter logistic curve y = (A-D)/[1+ (x/C) ^ B]+ D, linear fitting to obtain a standard curve;
| sample numbering | Concentration of | Luminous value | Sample type |
| 1 | 10000.00 | 18695750 | Standard article |
| 2 | 5000.00 | 11426035 | Standard article |
| 3 | 2500.00 | 5305425 | Standard article |
| 4 | 1250.00 | 2358954 | Standard article |
| 5 | 625.00 | 856230 | Standard article |
| 6 | 0.00 | 111848 | Standard article |
The data fitting method comprises the following steps: a four parameter logistic equation.
The two-point calibration method comprises the following steps: and a second two-point calibration method.
The standard curve obtained by fitting is: y = (26.43402-14.85330)/[ 1+ (x/9.92860) ^ 4.85743] + 14.85330, correlation coefficient R = 0.99953.
And thirdly, detecting and obtaining a luminous value 1509306, substituting the luminous value into a standard curve, calculating to obtain the concentration of the abnormal sugar chain glycoprotein in the sample to be detected as 920ng/ml, and multiplying the concentration by the dilution factor 1000 to finally obtain the concentration of the abnormal sugar chain glycoprotein in the sample as 920 ug/ml.
In this example, the sample was plasma, the ability of the solid phase B to bind to abnormal glycoglycoproteins was 80ng of abnormal glycoglycoproteins/10 ug of magnetic microspheres, the concentration of the solid phase B was 0.5mg/ml, the amount of the sample in each reaction cup was 200 μ l, the amount of the solid phase B added was 200 μ l, and the mass ratio of lectin to biotin in step S5 was 1: 5; sample dilutions for solid phase B, consisting of: NaH2PO4.2H2O： 1g，Na2HPO4.12H2O: 3g, the volume of purified water is fixed to 1L, and the diluted solution used by biotin-labeled lectin and alkaline phosphatase-labeled streptavidin comprises the following components:
NaH2PO4.2H2O： 1g
Na2HPO4.12H2O： 3g
Zn2+： 0.02g
Mg2+： 0.4g
the purified water is up to 1L, the agglutinin is alexandrium aurantiatum agglutinin, the agglutinin is processed by PEG pre-modification, and the steps of the PEG pre-modification of the agglutinin are as follows:
s1: mixing carbodiimide, methoxy polyethylene glycol active ester and lectin, and reacting at room temperature for 6 h; the mass ratio of the carbodiimide to the methoxypolyethylene glycol active ester to the lectin is 1:100: 1;
s2: and (3) separating the target product in the reaction system in the step S1 by using sephadex SwphadexG-50 to obtain a substance of a first section peak, and obtaining the target product, namely the lectin after the PEG pre-modification.
Example 3
A method for detecting an abnormal sugar chain glycoprotein, comprising the steps of:
s1 sample processing
Diluting the sample to be detected to 1000 times by using a sample diluent, fully and uniformly mixing the sample to be detected to obtain a sample solution, incubating the S2 sample
Placing a sample solution in a hole of a solid phase A, wherein the solid phase A is a luminescent plate made of polypropylene, controlling the pH value of the sample solution at 9.60, incubating at room temperature for 2h, and binding the diluted sample on the solid phase A through incubation;
s3 separation
Pouring out the residual sample diluent, adding 300ul of washing solution into each hole, standing for 60s, pouring out the washing solution, and repeating the steps for 2-3 times;
s4 sealing
Adopting casein with the mass percent of 1.0 percent as a blocking agent, carrying out overnight blocking at the pH value of 7.20 and the temperature of 4 ℃, wherein the dosage of the blocking agent in each hole is as follows: 300ul, after the sealing is finished, separating according to the separation operation of S3;
s5 biotin-labeled lectin recognizing sugar chain-abnormal protein
Concentration and amount of biotin-labeled lectin used per well: 10ug/ml, 100ul, pH: 7.10, reaction time: incubating for 3h at room temperature, and separating according to the separation operation of S3;
s6 Signal amplification Using horseradish peroxidase labeled streptavidin
Labeling ratio of horseradish peroxidase and SA: 1: 5, using concentration and using amount of each hole: 0.2ug/ml, 200ul, pH: 7.20, reaction time: incubating at room temperature for 60min, and separating according to the separation operation of S3;
second, prepare the standard curve
Preparation of 6The abnormal glycophorin samples with different mass concentrations are used as standard substances, the standard substances are processed according to the steps S2-S6, the luminescence values are obtained based on a chemiluminescence immune analyzer, the mass concentrations except the 0 value and the corresponding luminescence values are converted into logarithm with the base 2, and the logarithm with the base 2 is substituted into a four-parameter logistic curve y = (A-D)/[1+ (x/C) ^ B]+ D, linear fitting to obtain a standard curve;
| sample numbering | Concentration of | Luminous value | Sample type |
| 1 | 10000.00 | 8830184 | Standard article |
| 2 | 5000.00 | 6958103 | Standard article |
| 3 | 2500.00 | 3241230 | Standard article |
| 4 | 1250.00 | 1038689 | Standard article |
| 5 | 625.00 | 512948 | Standard article |
| 6 | 0.00 | 55413 | Standard article |
The data fitting method comprises the following steps: a four parameter logistic equation.
The two-point calibration method comprises the following steps: and a second two-point calibration method.
The standard curve obtained by fitting is: y = (23.24926-18.64688)/[ 1+ (x/10.86724) ^ 16.41368] + 18.64688, correlation coefficient R = 0.99993.
And thirdly, detecting and obtaining a luminous value 881878, substituting the luminous value into a standard curve, calculating to obtain the concentration of the abnormal sugar chain glycoprotein in the sample to be detected as 1120ng/ml, and multiplying the concentration by the dilution factor 1000 to finally obtain the concentration of the abnormal sugar chain glycoprotein in the sample as 1120 ug/ml.
Step S1 this point, the sample is a body fluid, and the ability of the solid phase A to bind an abnormal sugar chain glycoprotein is 1200ng/cm2The solid phase A adopts a 96-hole luminescent plate, the sample volume of each hole is 200 mu l, and in the step S5, the mass ratio of the agglutinin to the biotin is 1: sample dilutions for solid phase a, consisting of: 1.0g of sodium carbonate, 15g of sodium bicarbonate and purified water with the constant volume of 1L, wherein the diluent for biotin-labeled lectin and horseradish peroxidase-labeled streptavidin comprises the following components:
NaH2PO4.2H2O： 1g
Na2HPO4.12H2O： 3g
Zn2+： 0.02g
Mg2+： 0.4g
the purified water is added to a constant volume of 1L, the agglutinin is alexandrium aurantiacum agglutinin, the agglutinin is subjected to PEG pre-modification treatment, and the steps of the PEG pre-modification treatment of the agglutinin are as follows:
s1: mixing carbodiimide, methoxy polyethylene glycol active ester and lectin, and reacting for 4 hours at room temperature; the mass ratio of the carbodiimide to the methoxypolyethylene glycol active ester to the lectin is 1:100: 1;
s2: and (3) separating the target product in the reaction system in the step S1 by using sephadex SwphadexG-50 to obtain a substance of a first section peak, and obtaining the target product, namely the lectin after the PEG pre-modification.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (9)
1. A method for detecting an abnormal sugar chain glycoprotein, comprising the steps of:
first step, sample preparation
S1 sample processing
Diluting the sample to be detected to 1-20000 times by using a sample diluent, and fully and uniformly mixing the sample to be detected to obtain a sample solution;
s2 sample incubation
Placing the sample solution in a hole of a solid phase A, wherein the solid phase A is a polystyrene and/or polypropylene luminescent plate, controlling the pH of the sample solution at 9.40-9.60, incubating at room temperature for 2-3h or incubating at 37 ℃ for 30-60min, and binding the diluted sample on the solid phase A through incubation; or placing the sample liquid in a reaction cup, adding a solid phase B, wherein the solid phase B is a magnetic microsphere, controlling the pH of the sample liquid to be 7.00-7.20, incubating at room temperature for 2-3h or incubating at 37 ℃ for 30-60min, and binding the diluted sample on the solid phase B through incubation;
s3 separation
Pouring out residual sample diluent combined with the solid phase A, adding 300ul of washing solution into each hole, standing for 30-60 s, pouring out the washing solution, and repeating the steps for 2-3 times; standing the reaction cups on a magnetic separator for 1-2 min for the solid phase B, adding 300ul of washing solution into each reaction cup, standing for 30-60 s, pouring the washing solution, and repeating the steps for 2-3 times;
s4 sealing
Adopting one or more of bovine serum albumin, whey protein and casein with the mass percentage of 0.5-1.5% as a sealant, sealing at pH7.00-7.20, overnight at 4 ℃ or constant temperature at 37 ℃ for 1.5h, and using the sealant in each hole or reaction cup: 300ul, after sealing, separating different solid phases according to the separation operation of S3;
s5 biotin-labeled lectin recognizing sugar chain-abnormal protein
Concentration and amount of biotin-labeled lectin used per well or cuvette: 0.1-10ug/ml, 50-200ul, pH: 7.00-7.20, reaction time: incubating at room temperature for 2-3h or incubating at 37 ℃ for 1h, and separating different solid phases according to the separation operation of S3;
s6 Signal amplification Using horseradish peroxidase or alkaline phosphatase labeled streptavidin
Labeling ratio of horseradish peroxidase or alkaline phosphatase and SA: 1: (1-10), the concentration and dosage used in each hole or reaction cup are as follows: 0.05-0.2ug/ml, 50-200ul, pH: 7.00-7.20, reaction time: incubating at room temperature for 30-60min or incubating at 37 ℃ for 15min, and separating different solid phases according to the separation operation of S3;
second, making a standard curve
Preparing 4 or more abnormal glycophorin protein samples with different mass concentrations as standard substances within the range of 0-20000ng/ml, processing the standard substances according to the steps S2-S6, obtaining luminescence values based on a chemiluminescence immunoassay analyzer or a semi-automatic luminometer, converting the mass concentrations except the 0 value and the corresponding luminescence values into logarithm taking 2 as a base, substituting the logarithm into a four-parameter logistic curve y = (A-D)/[1+ (x/C) ^ B ] + D, and performing linear fitting to obtain a standard curve;
third step, calculation of sample concentration
And (5) detecting the sample obtained in the step (S6) based on a chemiluminescence immunoassay analyzer or a semi-automatic luminometer to obtain a luminescence value, substituting the luminescence value into the standard curve, and calculating to obtain the concentration of the sugar chain abnormal protein in the sample to be detected.
2. The method for detecting an abnormal sugar chain glycoprotein according to claim 1, wherein the sample is any one of serum, plasma and body fluid in step S1, and the ability of the solid phase A to bind to the abnormal sugar chain glycoprotein is not less than 1000ng/cm in step S22The capacity of the solid phase B for combining the abnormal sugar chain glycoprotein is not less than 80ng of abnormal sugar chain glycoprotein/10 ug of magnetic microspheres.
3. The method for detecting an abnormal sugar chain glycoprotein according to claim 2, wherein said solid phase A is a 96-well luminescent plate, the amount of sample per well is 50 to 200. mu.l, the concentration of said solid phase B is 0.1 to 0.5mg/ml, the amount of sample per reaction cup is 10 to 200. mu.l, and the amount of addition of said solid phase B is 50 to 200. mu.l.
4. The method of detecting an abnormal sugar chain glycoprotein according to claim 1, wherein in step S5, the mass ratio of lectin to biotin is 1: (1-5).
5. The method for detecting an abnormal sugar chain glycoprotein according to claim 1, wherein the sample diluent for the solid phase A is composed of: 0.5-1.0g of sodium carbonate, 10-15g of sodium bicarbonate and purified water with constant volume of 1L; sample dilutions for solid phase B, consisting of: NaH2PO4.2H2O：0.5-1g，Na2HPO4.12H2O: 2-3g, and the volume of purified water is up to 1L.
6. The method for detecting an abnormal sugar chain glycoprotein according to claim 1, wherein the diluent for biotin-labeled lectin and horseradish peroxidase-or alkaline phosphatase-labeled streptavidin is composed of:
NaH2PO4.2H2O：0.5-1g
Na2HPO4.12H2O：2-3g
Zn2+ ：0.01-0.02g
Mg2+：0.2-0.4g
the purified water was made to 1L.
7. The method for detecting an abnormal sugar chain glycoprotein according to claim 1, wherein the lectin is one or a mixture of several selected from the group consisting of concanavalin, stramonium lectin, lentil lectin, wheat germ lectin, type E red kidney bean lectin, type L red kidney bean lectin, pinobacillus citriodorus lectin, peanut lectin, ricinus communis lectin I, Maackia amurensis lectin, Dictyocaulus aurantiaca lectin, Agaricus bisporus lectin, Sambucus nigra lectin, and Huaackia lectin І І, and the lectin is subjected to PEG pre-modification treatment.
8. The method for detecting an abnormal sugar chain glycoprotein according to claim 1, wherein the step of the PEG pre-modification treatment of the mixed lectin is as follows:
s1: mixing carbodiimide, methoxy polyethylene glycol active ester and lectin, and reacting at room temperature for 2-6 h;
s2: and (3) separating the target product in the reaction system in the step S1 by using sephadex SwphadexG-50 to obtain a substance of a first section peak, and obtaining the target product, namely the lectin after the PEG pre-modification.
9. The reagent for detecting an abnormal sugar chain glycoprotein according to claim 8, wherein the mass ratio of the carbodiimide, the methoxypolyethylene glycol active ester and the lectin is 1:100: 1.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101308141A (en) * | 2007-05-16 | 2008-11-19 | 陕西北美基因股份有限公司 | Method for analyzing glucoprotein |
| CN103674918A (en) * | 2013-12-12 | 2014-03-26 | 复旦大学 | Method for detecting glycoprotein carbohydrate chain structure based on lectin liquid suspension chip |
| CN104502611A (en) * | 2014-12-25 | 2015-04-08 | 西安交通大学医学院第一附属医院 | Kit for detecting protein glycosylation subtype and detection method thereof |
| CN105652002A (en) * | 2016-01-07 | 2016-06-08 | 西北大学 | Lectin microarray for detecting carbohydrate chain marker based on sialoprotein and detection method of carbohydrate chain marker using same |
| CN110763849A (en) * | 2019-11-28 | 2020-02-07 | 陕西医药控股医药研究院有限公司 | Method for detecting content of SA α 2-3Gal sugar chains in saliva sample by ELISA (enzyme-Linked immuno sorbent assay) |
| CN110763848A (en) * | 2019-11-28 | 2020-02-07 | 陕西医药控股医药研究院有限公司 | Method for detecting content of SA α 2-6Gal sugar chains in saliva sample by ELISA (enzyme-Linked immuno sorbent assay) |
| CN111381043A (en) * | 2018-12-30 | 2020-07-07 | 糖谱(北京)科技有限公司 | Sample pretreatment composition suitable for mass spectrometry detection, sample pretreatment method and application |
-
2020
- 2020-08-16 CN CN202010822167.1A patent/CN112129947A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101308141A (en) * | 2007-05-16 | 2008-11-19 | 陕西北美基因股份有限公司 | Method for analyzing glucoprotein |
| CN103674918A (en) * | 2013-12-12 | 2014-03-26 | 复旦大学 | Method for detecting glycoprotein carbohydrate chain structure based on lectin liquid suspension chip |
| CN104502611A (en) * | 2014-12-25 | 2015-04-08 | 西安交通大学医学院第一附属医院 | Kit for detecting protein glycosylation subtype and detection method thereof |
| CN105652002A (en) * | 2016-01-07 | 2016-06-08 | 西北大学 | Lectin microarray for detecting carbohydrate chain marker based on sialoprotein and detection method of carbohydrate chain marker using same |
| CN111381043A (en) * | 2018-12-30 | 2020-07-07 | 糖谱(北京)科技有限公司 | Sample pretreatment composition suitable for mass spectrometry detection, sample pretreatment method and application |
| CN110763849A (en) * | 2019-11-28 | 2020-02-07 | 陕西医药控股医药研究院有限公司 | Method for detecting content of SA α 2-3Gal sugar chains in saliva sample by ELISA (enzyme-Linked immuno sorbent assay) |
| CN110763848A (en) * | 2019-11-28 | 2020-02-07 | 陕西医药控股医药研究院有限公司 | Method for detecting content of SA α 2-6Gal sugar chains in saliva sample by ELISA (enzyme-Linked immuno sorbent assay) |
Non-Patent Citations (3)
| Title |
|---|
| 上海炎怡生物科技有限公司: "聚乙二醇修饰化学和PEG修饰剂选择指南", 《HTTP://WWW.CNREAGENT.COM/SHOW1037510/TECHNICAL_2767.HTML》 * |
| 余海忠 等: "《食品营养学概论》", 31 December 2018 * |
| 王晓波: "《药物运释系统》", 31 August 2007 * |
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