CN104502611A - Kit for detecting protein glycosylation subtype and detection method thereof - Google Patents
Kit for detecting protein glycosylation subtype and detection method thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting protein glycosylation subtype and a detection method thereof. The detection kit comprises agglutinin, confining liquid, a biotin labeled antibody, horse radish peroxidase labeled streptavidin, a 3',3',5',5'-tetramethyl benzidine substrate developing solution, a stop solution and a cleaning buffer solution. The detection method comprises the following steps: fixing the agglutinin onto a polystyrene solid phase carrier to be used for recognizing and binding glycoprotein in the sample based on specific recognition of agglutinin and an oligosaccharide chain, recognizing the glycoprotein needing to be detected by virtue of specific binding of the protein and the antibody thereof, and realizing semi-quantitative analysis of the glycoprotein. According to the detection kit and the detection method disclosed by the invention, semi-quantitative detection of single protein glycosylation subtype can be realized, and the kit is easy and convenient to operate and low in cost.
Description
Technical field
The invention belongs to protein and posttranslational modification detection technique field thereof, be specifically related to a kind of detection kit and detection method thereof of protein glycosylation hypotype.
Background technology
Glycosylation (Glycosylation) is one of the most general protein post-translational modification, and mammalian proteins matter about over half all there occurs glycosylation.By affecting the maintenance of the solubleness of protein, folding, location and conformation, it can the function of Function protein matter and degraded, thus the interaction between reporter molecule and molecule, between molecule and cell, between cell and cell.In recent years, the research of protein glycosylation in tumour is risen day by day.Large quantity research shows, protein glycosylation can change and have corresponding function affect in the process of tumour cell vicious transformation, migration, invasion and attack.In clinical diagnosis field, protein glycosylation hypotype is a kind of important malignant tumour mark.In kinds of tumors, as lung cancer, breast cancer, liver cancer, the carcinoma of the rectum, cancer of pancreas, cancer of the stomach etc., tumor tissues or patients serum in, some glycoprotein glycosylation hypotypes are found to there is significant difference between normal or optimum control group.Therefore, set up the protein glycosylation detection method that high sensitivity, selectivity are strong, easy and simple to handle, the early diagnosis of cancer and treatment tool are of great significance.
At present, the glycosylation for protein detects mainly by the specific binding of agglutinin (Lectin) with sugar chain.The lectin chip technology of the foundation such as letter is strong by being fixed on epoxidation sheet base by the agglutinin of multiple specific recognition different sugar chain structure, then is reacted with sample to be tested, achieves quick to glycoprotein, high-throughout analysis.Jung-Hyun Rho etc. utilizes high-efficient liquid phase chromatogram technology (High performance liquidchromatography, HPLC) and tandem mass spectrum technology (MS/MS) coupling, can realize the separation andpreconcentration of glycopeptide.In addition, the method such as agglutinin trace, Lectin histochemistry also can be used for the detection of protein sugar chain in tissue or body fluid sample.But these method complicated operations, reactions steps is many, length consuming time, needs to be equipped with expensive experimental facilities; In addition, these technology all can not realize the detection of certain glycosylation modified hypotype of specified protein in biological specimen, be not suitable for clinical tumor mark real-time, detect fast.Some having been found that be can be used as to the protein glycosylation hypotype of diagnosing tumor mark, as the alpha-fetoprotein etc. of fucosylation, exploitation is applicable to clinical detection method that is real-time, quick, higher sensitivity and is necessary.
Summary of the invention
The object of the invention is the test problems in order to solve protein glycosylation hypotype in biological sample, provide kit and the detection method thereof of the detection of a kind of protein glycosylation hypotype, it not only can realize the half-quantitative detection of single protein glycosylation hypotype, simultaneously easy and simple to handle, cost is lower.
For achieving the above object, the present invention is achieved through the following technical solutions:
A detection kit for protein glycosylation hypotype, comprising:
Agglutinin: be fixed on after on polystyrene solid phase carrier, utilizes its carbohydrate recognition domain specific recognition and in conjunction with the oligonucleotide chain on glycoprotein, agglutinin bag is 3 ~ 30 μ g/mL by concentration;
Confining liquid: containing 0.025 ~ 0.05g/mL bovine serum albumin(BSA) (Bovine serum albumin, BSA) phosphate buffer (Phosphate Buffered Saline, PBS, pH=6 ~ 8), for closing the solid phase carrier site be not combined with agglutinin, reduce background value;
Biotin labeled antibody: for the target protein in the glycoprotein that specific binding agglutinin is caught, and biotin-avidin system has Cascaded amplification effect, during use, be diluted to 100 ~ 2000 times with the PBST (pH=6 ~ 8) containing volume fraction being 0.02 ~ 0.1%Tween-20;
Horseradish peroxidase (Horseradish peroxidase, HRP) streptavidin marked: the Avidin molecule combining enzyme and the biotin molecule being combined with specific antibody react, serve Cascaded amplification effect, and horseradish peroxidase develops the color after running into catalytic substrate, reach the object detecting target glycoprotein glycosylation hypotype, during use, be diluted to 2000 ~ 10000 times with the PBST (pH=6 ~ 8) containing volume fraction being 0.02 ~ 0.1%Tween-20;
3', 3', 5', 5'-tetramethyl benzidine (3', 3', 5', 5'-Tetramethylbenzidine, TMB) substrate nitrite ion: can form solubility blue product under horseradish peroxidase enzyme catalytic, for half-quantitative detection target glycoprotein;
Stop buffer: 1 ~ 2mol/L sulfuric acid, appeals catalytic reaction for stopping;
Cleaning buffer solution: be the PBST of 0.1% ~ 0.2%Tween-20 containing volume fraction, pH=6 ~ 8.
A detection method for protein glycosylation hypotype, comprises the following steps:
1) after the carbonate buffer solution of agglutinin pH=9 ~ 9.6 being diluted to 3 ~ 30 μ g/mL, be coated in each polystyrene reactant hole of 96 orifice plates, add agglutinin 50 ~ 200 μ L in every hole, 2 ~ 6 DEG C leave standstill 12 ~ 18h;
2) add in reacting hole by confining liquid, every hole adds 200 ~ 300 μ L, closes the position be not combined with agglutinin, incubated at room 1 ~ 3h;
3) 96 orifice plate is cleaned 4 times with cleaning buffer solution;
4) biological sample (Tissue lysates, cell pyrolysis liquid or serum etc.) is added 96 orifice plates with containing after PBS (pH=6 ~ 8) dilution of 0.005 ~ 0.015g/mLBSA, adding 50 ~ 200 μ L has wrapped in each reacting hole of quilt in above-mentioned, incubated at room 1 ~ 3h; After question response terminates, clean 96 orifice plate 4 times with cleaning buffer solution;
5) dilute biotin labeled specific antibody 100 ~ 2000 times with the PBST containing volume fraction being 0.02 ~ 0.1%Tween-20, add in 50 ~ 200 μ L to each reacting hole, incubated at room 1 ~ 3h; After reaction terminates, wash plank 4 times with cleaning buffer solution; Wherein, containing pH=6 ~ 8 of the PBST of Tween-20;
6) dilute the streptavidin 2000 ~ 10000 times of HRP mark with the PBST containing volume fraction being 0.02 ~ 0.1%Tween-20, add 50 ~ 200 μ L to 96 orifice plates, incubated at room 0.5 ~ 1.5h, washes plank 4-5 time with cleaning buffer solution;
7) 50 ~ 200 μ LTMB substrate nitrite ions are added in each reacting hole, incubated at room 15 ~ 20min;
8) 50 ~ 100 μ L stop buffer cessation reactions are added in each hole;
9), after question response completes, utilize microplate reader to detect each reacting hole at the absorbance of wavelength for 450nm place, half-quantitative detection is carried out to the target protein glycosylation hypotype in sample to be tested.
Compared with prior art, the present invention has following useful technique effect:
1, the detection kit of protein glycosylation hypotype provided by the invention and detection method thereof, be that first can realization carries out for a certain specific glycosylation modified hypotype of single protein the method that detects, semiquantitative determination can be carried out to the content of target glycoprotein glycosylation hypotype in sample; To the detection of protein glycosylation hypotype in various biological specimen, there is broad applicability.
2, the detection kit of protein glycosylation hypotype provided by the invention and detection method thereof is a kind of enzyme-linked immuno sorbent assay kit based on agglutinin and antibody and detection method thereof.React Cascaded amplification by agglutinin and oligonucleotide chain, antigen and antibody, biotin and streptavidin etc., be then translated into the convenient signal detected, thus realize the half-quantitative detection of glycoprotein glycosylation hypotype.
3, the detection kit of protein glycosylation hypotype provided by the invention and detection method thereof, agglutinin and antibody is utilized to identify sugar chain structure and the proteantigen itself of glycoprotein respectively, before detection without the need to by target glycoprotein extraction purification from biological specimen, the biological specimen directly can applying collection detects.The method not only increases detection efficiency, does not need complicated purifying instrument and equipment simultaneously; Moreover, avoid the adverse effect of protein purification to target glycoprotein activity, structure etc.
4, the detection kit of protein glycosylation hypotype provided by the invention and detection method thereof, the interference that the sugar chain carried to overcome antibody itself detects for target glycoprotein candy chain, agglutinin bag is adopted to be gathered the third ethene hole, then sample to be tested is added, finally add the experimental technique of antibody, make first to add glycoprotein excessive in the sample to be tested in reacting hole and be enough to closed all agglutinin binding sites, and form space steric effect, thus reach and avoid the sugar chain of antibody to combine with agglutinin, reduce the object of background value.
5, the detection kit of protein glycosylation hypotype provided by the invention and detection method thereof, compared with operating with the method that protein glycosylation detected in the past, operate easier, step is few, and does not need expensive equipment.
Accompanying drawing explanation
Fig. 1 is the absorbance detection result figure of different extension rate blood serum sample galactosylation alpha1-antitrypsin hypotype in embodiment 1;
Fig. 2 is the absorbance detection result figure of different extension rate blood serum sample fucosylation alpha1-antitrypsin hypotype in embodiment 2;
Fig. 3 is the absorbance detection result figure of the aminated alpha1-antitrypsin hypotype of different dilutability blood serum sample poly lactoses in embodiment 3.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Embodiment 1:
1) by BSLi (Bandeiraea simplicifolia, BS-I) dissolve with the carbonate buffer solution of pH=9.6 and be diluted to 3 μ g/mL, add in each polystyrene reactant hole of 96 orifice plates, it is made to cover at the bottom of whole hole, add this agglutinin solution 100 μ L in every hole, 4 DEG C are spent the night.
2) next day, discard agglutinin solution in hole, every hole adds 200 μ L confining liquids, closes the position be not combined with agglutinin, incubated at room 1h.
3) clean 96 orifice plate 4 times with cleaning buffer solution, during each cleaning, cleaning buffer solution should fill it up with whole reacting hole, softly shakes 3min, discards liquid in hole and pat dry on thieving paper.
4) PBS (pH=7.4) of serum sample containing 0.01g/mL BSA is added 96 orifice plates respectively by after 1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560 doubling dilution, every hole adds 100 μ L, incubated at room 1h; After question response terminates, clean 96 orifice plate 4 times with cleaning buffer solution.
5) with containing volume fraction be the PBST (pH=7.4) of 0.05%Tween-20 by biotin labeled alpha1-antitrypsin antibody dilution 100 times, every hole adds 100 μ L, incubated at room 1h; After reaction terminates, wash plank 4 times with cleaning buffer solution.
6) with the PBST (pH=7.4) containing volume fraction being 0.05%Tween-20, the streptavidin that HRP marks is diluted 2000 times, every hole adds 100 μ L, incubated at room 30min.Plank is washed 5 times with cleaning buffer solution.
7) in each reacting hole, 100 μ LTMB substrate nitrite ions are added, incubated at room 15 ~ 20min.
8) in each hole, add 50 μ L stop buffer cessation reactions.
9), after question response completes, utilize microplate reader to detect each reacting hole at the absorbance of wavelength for 450nm place, half-quantitative detection is carried out to galactosylation alpha1-antitrypsin hypotype in different extension rate blood serum sample.
As shown in Figure 1, horizontal ordinate is serum diluting multiple to concrete testing result, and ordinate is absorbance.Agglutinin BS-I specific recognition galactose and N-acetyl galactose in embodiment 1, alpha1-antitrypsin antibody can with alpha1-antitrypsin specific binding, the serum levels of joint-detection galactosylation alpha1-antitrypsin hypotype.
Embodiment 2:
1) by Aleuria agglutinin (Aleuria Aurantia Lectin, AAL) dissolve with the carbonate buffer solution of pH=9.6 and be diluted to 3 μ g/mL, add in each polystyrene reactant hole of 96 orifice plates, it is made to cover at the bottom of whole hole, add this agglutinin solution 100 μ L in every hole, 4 DEG C are spent the night.
2) next day, discard agglutinin solution in hole, every hole adds 200 μ L confining liquids, closes the position be not combined with agglutinin, incubated at room 1h.
3) clean 96 orifice plate 4 times with cleaning buffer solution, during each cleaning, cleaning buffer solution should fill it up with whole reacting hole, softly shakes 3min, discards liquid in hole and pat dry on thieving paper.
4) PBS (pH=7.4) of serum sample containing 0.01g/mL BSA is added 96 orifice plates respectively by after 1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560 doubling dilution, every hole adds 100 μ L, incubated at room 1h; After question response terminates, clean 96 orifice plate 4 times with cleaning buffer solution.
5) with containing volume fraction be the PBST (pH=7.4) of 0.05%Tween-20 by biotin labeled alpha1-antitrypsin antibody dilution 100 times, every hole adds 100 μ L, incubated at room 1h; After reaction terminates, wash plank 4 times with cleaning buffer solution.
6) with the PBST (pH=7.4) containing volume fraction being 0.05%Tween-20, the streptavidin that HRP marks is diluted 2000 times, every hole adds 100 μ L, incubated at room 30min.Plank is washed 5 times with cleaning buffer solution.
7) in each reacting hole, 100 μ LTMB substrate nitrite ions are added, incubated at room 15 ~ 20min.
8) in each hole, add 50 μ L stop buffer cessation reactions.
9), after question response completes, utilize microplate reader to detect each reacting hole at the absorbance of wavelength for 450nm place, half-quantitative detection is carried out to fucosylation alpha1-antitrypsin hypotype in different extension rate blood serum sample.
As shown in Figure 2, horizontal ordinate is serum diluting multiple to concrete testing result, and ordinate is absorbance.Agglutinin AAL specific recognition fucose in embodiment 2, alpha1-antitrypsin antibody can with alpha1-antitrypsin specific binding, the serum levels of joint-detection fucosylation alpha1-antitrypsin hypotype.
Embodiment 3:
1) by trade route, America agglutinin (Phytolacca americana, PWM) dissolve with the carbonate buffer solution of pH=9.6 and be diluted to 3 μ g/mL, add in each polystyrene reactant hole of 96 orifice plates, it is made to cover at the bottom of whole hole, add this agglutinin solution 100 μ L in every hole, 4 DEG C are spent the night.
2) next day, discard agglutinin solution in hole, every hole adds 200 μ L confining liquids, closes the position be not combined with agglutinin, incubated at room 1h.
3) clean 96 orifice plate 4 times with cleaning buffer solution, during each cleaning, cleaning buffer solution should fill it up with whole reacting hole, softly shakes 3min, discards liquid in hole and pat dry on thieving paper.
4) PBS (pH=7.4) of serum sample containing 0.01g/mL BSA is added 96 orifice plates respectively by after 1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560 doubling dilution, every hole adds 100 μ L, incubated at room 1h; After question response terminates, clean 96 orifice plate 4 times with cleaning buffer solution.
5) with containing volume fraction be the PBST (pH=7.4) of 0.05%Tween-20 by biotin labeled alpha1-antitrypsin antibody dilution 100 times, every hole adds 100 μ L, incubated at room 1h; After reaction terminates, wash plank 4 times with cleaning buffer solution.
6) with the PBST (pH=7.4) containing volume fraction being 0.05%Tween-20, the streptavidin that HRP marks is diluted 2000 times, every hole adds 100 μ L, incubated at room 30min.Plank is washed 5 times with cleaning buffer solution.
7) in each reacting hole, 100 μ L tmb substrate nitrite ions are added, incubated at room 15 ~ 20min.
8) in each hole, add 50 μ L stop buffer cessation reactions.
9) after question response completes, utilize microplate reader to detect each reacting hole at the absorbance of wavelength for 450nm place, half-quantitative detection is carried out to the aminated alpha1-antitrypsin hypotype of poly lactose in different dilutability blood serum sample.
As shown in Figure 3, horizontal ordinate is serum diluting multiple to concrete testing result, and ordinate is absorbance.Agglutinin PWM specific recognition poly lactose amine in embodiment 3, alpha1-antitrypsin antibody can with alpha1-antitrypsin specific binding, the serum levels of the aminated alpha1-antitrypsin hypotype of joint-detection poly lactose.
The biotin labeled alpha1-antitrypsin antibody used in above-described embodiment 1,2,3 is purchased from Abcam company, the streptavidin that the HRP used in embodiment 1,2,3 marks, tmb substrate nitrite ion are all purchased from Sigma-Aldrich company, agglutinin BS-I, AAL of using in embodiment 1,2 are purchased from Vector company, and the agglutinin PWM used in embodiment 3 is purchased from Sigma-Aldrich company.
Claims (6)
1. a detection kit for protein glycosylation hypotype, is characterized in that, comprising:
Agglutinin: be fixed on after on polystyrene solid phase carrier, utilizes its carbohydrate recognition domain specific recognition and in conjunction with the oligonucleotide chain on glycoprotein, agglutinin bag is 3 ~ 30 μ g/mL by concentration;
Confining liquid: containing the phosphate buffer of 0.025 ~ 0.05g/mL bovine serum albumin(BSA), pH=6 ~ 8, for closing the solid phase carrier site be not combined with agglutinin, reduce background value;
Biotin labeled antibody: for the target protein in the glycoprotein that specific binding agglutinin is caught, and biotin-avidin system has Cascaded amplification effect;
The streptavidin of horseradish peroxidase-labeled: the Avidin molecule combining enzyme and the biotin molecule being combined with specific antibody react, serve Cascaded amplification effect, and horseradish peroxidase develops the color after running into catalytic substrate, reach the object detecting target glycoprotein glycosylation hypotype;
3', 3', 5', 5'-tetramethyl benzidine substrate nitrite ion: can form solubility blue product under horseradish peroxidase enzyme catalytic, for half-quantitative detection target glycoprotein;
Stop buffer: 1 ~ 2mol/L sulfuric acid, appeals catalytic reaction for stopping;
Cleaning buffer solution: be the PBST of 0.1 ~ 0.2%Tween-20 containing volume fraction, pH=6 ~ 8.
2. the detection kit of a kind of protein glycosylation hypotype according to claim 1, is characterized in that, is diluted to 100 ~ 2000 times when biotin labeled antibody uses with the PBST containing volume fraction being 0.02 ~ 0.1%Tween-20.
3. the detection kit of a kind of protein glycosylation hypotype according to claim 1, it is characterized in that, during use, the streptavidin of horseradish peroxidase-labeled is diluted to 2000 ~ 10000 times with the PBST containing volume fraction being 0.02 ~ 0.1%Tween-20.
4. a detection method for protein glycosylation hypotype, is characterized in that, comprises the following steps:
1) after the carbonate buffer solution of agglutinin pH=9 ~ 9.6 being diluted to 3 ~ 30 μ g/mL, be coated in each polystyrene reactant hole of orifice plate, add agglutinin 50 ~ 200 μ L in each hole, 2 ~ 6 DEG C leave standstill 12 ~ 18h;
2) add in reacting hole by confining liquid, every hole adds 200 ~ 300 μ L, closes the position be not combined with agglutinin, incubated at room 1 ~ 3h;
3) cleaning buffer solution cleaning orifice is used;
4) biological sample is added orifice plate with containing after the PBS dilution of 0.005 ~ 0.015g/mL BSA, add 50 ~ 200 μ L and wrapped in each reacting hole of quilt in above-mentioned, incubated at room 1 ~ 3h; After question response terminates, use cleaning buffer solution cleaning orifice; Wherein, containing pH=6 ~ 8 of the PBS of BSA;
5) dilute biotin labeled specific antibody 100 ~ 2000 times with the PBST containing volume fraction being 0.02 ~ 0.1%Tween-20, add in 50 ~ 200 μ L to each reacting hole, incubated at room 1 ~ 3h; After reaction terminates, wash plank 4 times with cleaning buffer solution; Wherein, containing pH=6 ~ 8 of the PBST of Tween-20;
6) dilute the streptavidin 2000 ~ 10000 times of HRP mark with the PBST containing volume fraction being 0.02 ~ 0.1%Tween-20, add 50 ~ 200 μ L to orifice plate, incubated at room 0.5 ~ 1.5h, then use cleaning buffer solution hole flushing plate;
7) 50 ~ 200 μ LTMB substrate nitrite ions are added in each reacting hole, incubated at room 15 ~ 20min;
8) 50 ~ 100 μ L stop buffer cessation reactions are added in each hole;
9), after question response completes, utilize microplate reader to detect each reacting hole at the absorbance of wavelength for 450nm place, half-quantitative detection is carried out to the target protein glycosylation hypotype in sample to be tested.
5. the detection method of a kind of protein glycosylation hypotype according to claim 4, it is characterized in that, orifice plate selects 96 orifice plates.
6. the detection method of a kind of protein glycosylation hypotype according to claim 4, is characterized in that, biological sample is Tissue lysates, cell pyrolysis liquid or serum.
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| CN109655622A (en) * | 2019-01-25 | 2019-04-19 | 西北大学 | Based on early-stage breast cancer correlation screening/assessment product of the special glycoprotein candy chain structure of saliva and application |
| CN110045126A (en) * | 2019-04-03 | 2019-07-23 | 中国医学科学院北京协和医院 | A kind of biomarker and application thereof for diagnosis of autoimmune pancreatitis |
| CN112129948A (en) * | 2020-08-16 | 2020-12-25 | 浙江瑞生医疗科技有限公司 | Reagent for detecting abnormal sugar chain glycoprotein and process for producing the same |
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| CN105954518A (en) * | 2016-05-23 | 2016-09-21 | 中国人民解放军总医院 | Application of lectin chip in analysis of urine protein and sugar chain spectrum |
| CN106248959A (en) * | 2016-07-21 | 2016-12-21 | 首都医科大学附属北京佑安医院 | A kind of immunity percolation method detecting serum fucose albumen and immunity percolation device |
| CN109655622A (en) * | 2019-01-25 | 2019-04-19 | 西北大学 | Based on early-stage breast cancer correlation screening/assessment product of the special glycoprotein candy chain structure of saliva and application |
| CN109655622B (en) * | 2019-01-25 | 2021-10-22 | 深圳格道糖生物技术有限公司 | Early-stage breast cancer related screening/evaluation product based on saliva specific glycoprotein carbohydrate chain structure and application |
| CN110045126A (en) * | 2019-04-03 | 2019-07-23 | 中国医学科学院北京协和医院 | A kind of biomarker and application thereof for diagnosis of autoimmune pancreatitis |
| CN110045126B (en) * | 2019-04-03 | 2022-08-09 | 中国医学科学院北京协和医院 | Biomarker for diagnosing autoimmune pancreatitis and application thereof |
| CN112129948A (en) * | 2020-08-16 | 2020-12-25 | 浙江瑞生医疗科技有限公司 | Reagent for detecting abnormal sugar chain glycoprotein and process for producing the same |
| CN112129947A (en) * | 2020-08-16 | 2020-12-25 | 浙江紫荆生物技术有限公司 | Method for detecting abnormal sugar chain glycoprotein |
| CN112129946A (en) * | 2020-08-16 | 2020-12-25 | 陆修委 | Preparation method and application of sugar-free chain type inert protein sealant |
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Application publication date: 20150408 |