CN103645325A - Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip - Google Patents
Method for multiply detecting carbohydrate chain structure of glycoprotein through antibody-assisted lectin liquid-phase suspension chip Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to the technical field of biology and relates to a method for multiply detecting a carbohydrate chain structure of glycoprotein through an antibody-assisted lectin liquid-phase suspension chip. According to the method, lectins are coupled to magnetic beads marked with different dyes, the magnetic beads which are coupled with different lectins are identified through a Bioplex detection system, and different glycoforms on the same protein can be simultaneously detected by adding the magnetic beads which are coupled with different lectins and have different serial numbers into a reaction, so that the detection flux of the carbohydrate chain structure is remarkably improved. According to the method, the carbohydrate chain structure on the glycoprotein can be quickly and specifically detected with high flux. The method can be further applied to detection of a clinical biological marker and provides an efficient way for research on disease glycomics.
Description
Technical field
The invention belongs to biological technical field, relate to the new method of the auxiliary agglutinin liquid phase suspending chip Multiple detection glycoprotein candy chain structure of a kind of antibody; The auxiliary agglutinin liquid-phase chip of preparation, antibody that is specifically related to the auxiliary agglutinin liquid phase suspending chip of antibody carries out Multiple detection to the sugared type on haptoglobin (Hp); The method can be carried out Multiple detection to the sugar chain structure on glycoprotein, improves the flux of lectin chip technology.
Background technology
The glycosylation of eukaryotic protein is a kind of general, most important but complicated posttranslational modification; The variation of glycosylation structure is a kind of sign widely in cancer.The sugar that some are positioned sugar-chain end, comprises Slex (sLe
x), sialylated Tn (sTn), Globo H, Lewis y (Le
y) and polysialic acids can be in many malignant tissues cross and express.Although sugar chain has important function, for glycosylated research, be but hampered by and because it is synthetic, lack structure heterogeneity and the diversity that template causes.In order to resolve sugar chain, many analysis means that arisen at the historic moment are as liquid chromatography, mass spectrum, Capillary Electrophoresis etc.Yet, mass spectrum cannot be differentiated isomers and also have difficulties in resolution data, and the traditional sugar chain structure analytic method such as chromatogram, mass spectrum is because sample pre-treatments step is many, repeatability is bad, and flux is not low, be difficult to be applied in the analysis of sugar chain structure in large-scale clinical sample.
It is a kind of sugar chain structure analytic technique having a great attraction that prior art discloses lectin chip technology, and it can be fast, high flux, resolve sugar chain structure and screen glycosylation difference on a large scale; Yet, the interaction between agglutinin and sugar (dissociation constant, Kd=10
-7– 10
-3m) very weak, to cause lectin chip to detect muting sensitivity.And lectin chip technology and liquid phase suspending chip technology are combined, owing to having realized the reaction in three-dimensional environment of sugar chain and agglutinin, can greatly improve the sensitivity of lectin chip technology.While carrying out sugar chain structural analysis for the target glycoprotein of purifying from biological specimen, can adopt antibody assisted reaction to improve the specificity of detection.Described Bioplex suspension chip system utilizes 100 kinds of different microballoons to detect the concern that 100 kinds of different antigens are more and more subject to people because it has simultaneously; The detection method of this system is that antibody is fixed on different microballoons, utilizes two bundle laser of flow cytometer simultaneously to being combined in the fluorescence on magnetic ball surface and the fluorescence of magnetic ball itself detects; It is quick that described suspending chip detection technique has the data of acquisition, remarkable sensitivity and the advantage of specificity and multiple analysis.In order to make lectin chip technology can better be applied to clinical sample glycosylation detection and glycosylation differential screening, the detection flux of lectin chip technology is in urgent need to be improved.
Summary of the invention
The object of the present invention is to provide the method for the auxiliary agglutinin liquid phase suspending chip Multiple detection glycoprotein candy chain structure of a kind of antibody, improve the flux of lectin chip technology.Chip system of the present invention can be rapidly, in high sensitivity, the glycosylation of high flux ground detection molecules mark, be the powerful measure of analyzing glycosylation difference, there is good potential applicability in clinical practice.
For achieving the above object, the technical solution used in the present invention is as follows:
(1) agglutinin carries out covalent coupling with the magnetic bead that is marked with different dyes, prepares agglutinin liquid-phase chip
Adopt cleaning buffer solution (coupling reagent kit carries) to clean after magnetic bead, add the resuspended magnetic bead of activation damping fluid (coupling reagent kit carries); To adding under 50 μ g/ μ L EDC and 50 μ g/ μ L sulfo-NHS oscillating conditions lucifuge room temperature reaction in magnetic bead 20 minutes; With PBS, clean excessive EDC and sulfo-NHS, and repeat cleaning process once; Finally, use the magnetic bead of the resuspended activation of PBS; 4 ℃ of reaction overnight of magnetic bead after configuring a series of agglutinin and activate with PBS; Centrifugally go, after supernatant, to add " Stabil Guard Choice " resuspended without protein blocking solution, incubated at room 30 minutes.After centrifugal, with the resuspended magnetic bead of storage buffer (coupling reagent kit carries);
(2) with amino reaction biotin labeling kit labelled antibody
Antibody is joined to the ultrafiltration of 50kDa MWCO centrifugal ultrafiltration pipe, and clean (labelling kit carries) with cleaning solution; 10 μ L DMSO are joined to amino reaction biotin (labelling kit carries), and piping and druming makes its dissolving; In super filter tube, add reaction buffer (labelling kit carries) and amino reaction biotin solution, in 37 ℃ of reactions 10 minutes; Reactant liquor is removed in ultrafiltration, the antibody of collection of biological element mark;
(3) the auxiliary agglutinin liquid phase suspending chip reaction of antibody
In incubation buffer (1%Triton X-100PBS), add haptoglobin (Hp) and a kind of (substance detection) or multiple magnetic bead (Multiple detection), react at 4 ℃ and spend the night; After reaction finishes, add 100 μ L cleaning buffer solutions (1%Triton X-100PBS) to clean magnetic bead; Add 20 μ g/ holes not by biotin labeled Ig G, further sealing not with the agglutinin binding site of haptoglobin (Hp) combination; Under room temperature, hatch 30 minutes; After incubation reaction finishes, add 100 μ L cleaning buffer solutions (1%Triton X-100PBS) to clean magnetic bead, repeat 3 times; Add and be dissolved in the 100 μ L incubation buffer biotin labeled haptoglobin of 0.3 μ g/mL (Hp) antibody, under room temperature, react 1 hour; Repeated washing step; Add again under 1 μ g/mL streptomysin-phycoerythrin room temperature of 100 μ L incubation buffer dilutions oscillating reactions 30 minutes; After reaction finishes, repeated washing step; Finally, with the resuspended magnetic bead of cleaning buffer solution;
(4) utilize Bio-Plex liquid phase suspending chip detection system to gather signal
50 magnetic beads are detected in each hole, and record fluorescence median (MFI); For Multiple detection, during image data, select multiple magnetic bead numbering.
In the present invention, the auxiliary agglutinin liquid-phase chip method of described antibody has shown high sensitivity, good reappearance and the very wide range of linearity.In the present invention, different types of agglutinin is coupled on the magnetic bead of different dyes mark, and classification laser identifiable marker in Bioplex detection system has the magnetic bead of different dyes, therefore can be by adding coupling to have the magnetic bead of the difference numbering of different agglutinins in a reaction, can to the different sugar type on same albumen, detect simultaneously, and then significantly improved the detection flux of agglutinin liquid-phase chip technology, and save time, save human and material resources.The described agglutinin suspension liquid-phase chip multiple detection method based on antibody assisted reaction, can be rapidly, high flux ground, the glycosylation of detection molecules mark efficiently, is the powerful measure of analyzing glycosylation difference, has good potential applicability in clinical practice.
Accompanying drawing explanation
Fig. 1 is the experiment flow of the method for the invention.
Fig. 2 has shown that the auxiliary agglutinin liquid-phase chip method of described antibody detects detectability and the range of linearity of haptoglobin (Hp), wherein,
A RCA120 detectability;
The B RCA120 range of linearity;
C SNA-I detectability.
Fig. 3 has shown the agglutinin liquid-phase chip multiple detection method experimental result that antibody is auxiliary, wherein,
Multiple detection method and the substance detection method of the auxiliary agglutinin liquid phase suspending chip of A contrast antibody;
The dose dependent curve of the auxiliary agglutinin liquid phase suspending chip multiple detection method of B antibody.
Embodiment
Example is below further illustrating of sugar chain structure being detected based on the auxiliary agglutinin liquid phase suspension liquid-phase chip multiple detection method of antibody that the present invention is proposed.
The lowest detectable limit of the auxiliary agglutinin liquid phase suspending chip of embodiment 1 antibody and range of linearity experiment
With haptoglobin (Hp) solution of a series of concentration, with 4 ℃ of night incubation of 2.5 μ L magnetic beads, after cleaning, add the sealing of IgG room temperature; After cleaning, add biotinylated antibody-solutions, incubated at room is after 1 hour again, cleans and adds two anti-reactant liquors; After incubated at room 30 minutes, by Bioplex suspending chip detection system, signal is gathered; With having similar sugar chain structure to haptoglobin (Hp), but the transferrins (Tf) that can not identify for haptoglobin (Hp) antibody is as negative control; When net signal is greater than 3 times of standard deviation, and the minimum glycoprotein concentration of the ratio of signal and noise while being greater than 1.5 times is detectability.Result is as shown in Fig. 2 A, Fig. 2 B, Fig. 2 C, and RCA120 is limited to 500pg/mL to the detection of haptoglobin (Hp), and the range of linearity is 500～25,000pg/mL; SNA-I is limited to 60pg/mL to the detection of haptoglobin (Hp).
The specificity experiment of the auxiliary agglutinin liquid phase suspending chip multiple detection method of embodiment 2 antibody
In haptoglobin (Hp) solution of 3ng/mL, add the magnetic bead of each 2.5 μ L RCA120 and SNA-I coupling, 4 ℃ of night incubation, add the sealing of IgG room temperature after cleaning; After cleaning, add biotinylated haptoglobin (Hp) antibody-solutions, incubated at room is after 1 hour again, cleans and adds two anti-reactant liquors; After incubated at room 30 minutes, by Bioplex suspending chip detection system, signal is gathered; With having similar sugar chain structure to haptoglobin (Hp), but the transferrins (Tf) that can not identify for haptoglobin (Hp) antibody is as negative control; As shown in Figure 3A, the signal intensity of multiple detection method and separately detection method is consistent for result, shows the different sugar chain structure of multiple detection method on can the same albumen of specific identification.
Increasing property of the dosage experiment of the auxiliary agglutinin liquid phase suspending chip multiple detection method of embodiment 3 antibody
In haptoglobin (Hp) solution of configuration variable concentrations, to the magnetic bead that adds each 2.5 μ L RCA120 and SNA-I coupling in each sample, 4 ℃ of night incubation, add the sealing of IgG room temperature after cleaning; After cleaning, add biotinylated haptoglobin (Hp) antibody-solutions, incubated at room is after 1 hour again, cleans and adds two anti-reactant liquors; After incubated at room 30 minutes, by Bioplex suspending chip detection system, signal is gathered; With having similar sugar chain structure to haptoglobin (Hp), but the transferrins (Tf) that can not identify for haptoglobin (Hp) antibody is as negative control; As shown in Figure 3 B, in Multiple detection, agglutinin signal increases with the increase of glycoprotein concentration result.
Claims (6)
1. a method for the auxiliary agglutinin liquid phase suspending chip Multiple detection glycoprotein candy chain structure of antibody, is characterized in that, step is as follows:
(1) agglutinin and the magnetic bead covalent coupling that is marked with different dyes, prepare agglutinin liquid-phase chip,
Adopt cleaning buffer solution to clean after magnetic bead, add the resuspended magnetic bead of activation damping fluid; To adding under 50 μ g/ μ L EDC and 50 μ g/ μ L sulfo-NHS oscillating conditions lucifuge room temperature reaction in magnetic bead 20 minutes; With PBS, clean excessive EDC and sulfo-NHS, and repeat cleaning process once; Finally, use the magnetic bead of the resuspended activation of PBS; 4 ℃ of reaction overnight of magnetic bead after configuring a series of agglutinin and activate with PBS; Centrifugally go, after supernatant, to add " Stabil Guard Choice " resuspended without protein blocking solution, incubated at room 30 minutes; After centrifugal, with the resuspended magnetic bead of storage buffer;
(2) with amino reaction biotin labeling kit labelled antibody
Antibody is joined to the ultrafiltration of 50kDa MWCO centrifugal ultrafiltration pipe, and clean with cleaning solution; 10 μ LDMSO are joined to amino reaction biotin, and piping and druming makes its dissolving; In super filter tube, add reaction buffer and amino reaction biotin solution, in 37 ℃ of reactions 10 minutes; Reactant liquor is removed in ultrafiltration, the antibody of collection of biological element mark;
(3) the auxiliary agglutinin liquid phase suspending chip reaction of antibody
In incubation buffer, add glycoprotein and one or more magnetic beads, 4 ℃ of reactions are spent the night; After reaction finishes, add 100 μ L cleaning buffer solutions to clean magnetic bead; Add 20 μ g/ holes not by biotin labeled Ig G, the agglutinin binding site that further sealing is not combined with haptoglobin; Under room temperature, hatch 30 minutes; After incubation reaction finishes, add 100 μ L cleaning buffer solutions to clean magnetic bead, repeat 3 times; Add and be dissolved in the biotin labeled protein antibodies of 100 μ L incubation buffer 0.3 μ g/mL, under room temperature, react 1 hour; Repeated washing step; Add again under 1 μ g/mL streptomysin-phycoerythrin room temperature of 100 μ L incubation buffer dilutions oscillating reactions 30 minutes; After reaction finishes, repeated washing step; Finally, with the resuspended magnetic bead of cleaning buffer solution;
(4) utilize Bio-Plex liquid phase suspending chip detection system to gather signal
50 magnetic beads are detected in each hole, and record fluorescence signal median MFI; For Multiple detection, during image data, select multiple magnetic bead numbering.
2. the auxiliary agglutinin liquid phase suspending chip Multiple detection new method of antibody according to claim 1, is characterized in that, in described step (2), adopts amino reaction biotin labeling kit antagonist to carry out biotin labeling.
3. the method for the auxiliary agglutinin liquid phase suspending chip Multiple detection glycoprotein candy chain structure of antibody according to claim 1, it is characterized in that, in described step (3), to the coupling that adds different dyes mark in the glycoprotein solution of reaction buffer dilution, there is the magnetic bead of different agglutinins, hatch.
4. the method for the auxiliary agglutinin liquid phase suspending chip Multiple detection glycoprotein candy chain structure of antibody according to claim 1, is characterized in that, the incubation buffer in described step (3) is 1%Triton X-100PBS.
5. the method for the auxiliary agglutinin liquid phase suspending chip Multiple detection glycoprotein candy chain structure of antibody according to claim 1, is characterized in that, the cleaning buffer solution in described step (3) is 1%Triton X-100PBS.
6. the method for the auxiliary agglutinin liquid phase suspending chip Multiple detection glycoprotein candy chain structure of antibody according to claim 1, it is characterized in that, in described step (4), utilize Bioplex detection system to have the multiple magnetic bead of different agglutinins to detect to the coupling in agglutinin liquid-phase chip simultaneously.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104020299A (en) * | 2014-06-20 | 2014-09-03 | 复旦大学 | Method for quantitatively detecting glycosylation level of peptide fragment |
| CN105259006A (en) * | 2014-07-16 | 2016-01-20 | 温州医科大学 | Applications of 4H-[1]-benzopyran[4,3-b]thiophene-2-carboxylic acid hydrazide and derivative thereof in specific fluorescent pre-dyeing detection method of glycoproteins |
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
| 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 |
| CN108761088A (en) * | 2018-06-28 | 2018-11-06 | 北京热景生物技术股份有限公司 | Composition, kit and method and purposes for the detection of sugar chain abnormal Protein Separation |
| CN113214403A (en) * | 2021-04-25 | 2021-08-06 | 重庆威斯腾前沿生物研究院有限责任公司 | Efficient streptavidin magnetic bead and preparation method thereof |
| CN114324557A (en) * | 2021-12-03 | 2022-04-12 | 融智生物科技（青岛）有限公司 | Zeta-globin detection method based on MALDI-TOF MS |
| CN116840487A (en) * | 2023-05-30 | 2023-10-03 | 中拓生物有限公司 | Method for detecting sugar-deficient transferrin |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104020299A (en) * | 2014-06-20 | 2014-09-03 | 复旦大学 | Method for quantitatively detecting glycosylation level of peptide fragment |
| CN105259006A (en) * | 2014-07-16 | 2016-01-20 | 温州医科大学 | Applications of 4H-[1]-benzopyran[4,3-b]thiophene-2-carboxylic acid hydrazide and derivative thereof in specific fluorescent pre-dyeing detection method of glycoproteins |
| CN105259006B (en) * | 2014-07-16 | 2018-05-08 | 温州医科大学 | 4H- [1]-application of chromene [4,3-b] the thiophene -2-carboxylic acid acyl hydrazine and its derivative in the pre- dyeing detection method of glycoprotein specificity fluorescent |
| CN105779398A (en) * | 2015-01-12 | 2016-07-20 | 北京亿森宝生物科技有限公司 | Immunomagnetic bead purification kit and method for 12 kinds of swine common viruses and germs |
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| CN108761088A (en) * | 2018-06-28 | 2018-11-06 | 北京热景生物技术股份有限公司 | Composition, kit and method and purposes for the detection of sugar chain abnormal Protein Separation |
| CN113214403A (en) * | 2021-04-25 | 2021-08-06 | 重庆威斯腾前沿生物研究院有限责任公司 | Efficient streptavidin magnetic bead and preparation method thereof |
| CN114324557A (en) * | 2021-12-03 | 2022-04-12 | 融智生物科技（青岛）有限公司 | Zeta-globin detection method based on MALDI-TOF MS |
| CN114324557B (en) * | 2021-12-03 | 2024-05-10 | 融智生物科技（青岛）有限公司 | Zeta-globin detection method based on MALDI-TOF MS |
| CN116840487A (en) * | 2023-05-30 | 2023-10-03 | 中拓生物有限公司 | Method for detecting sugar-deficient transferrin |
| CN116840487B (en) * | 2023-05-30 | 2024-03-19 | 中拓生物有限公司 | Method for detecting sugar-deficient transferrin |
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