Method for analyzing and detecting IgG N-glycan in dried blood spot

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a method for analyzing and detecting IgG N-glycan in dried blood spots. The dried blood spot sample is adopted to replace a traditional serum sample, so that the technical problem of IgG N-carbohydrate chain degradation under the normal-temperature storage condition of the serum sample is effectively solved, and the stability of the sample and the reliability of a detection result are remarkably improved. The dried blood spot sample collecting method is simple and convenient, vein blood sampling is not needed, the method is suitable for large-scale screening and application of special crowds, and the clinical application range of IgG N-glycan analysis is widened. Besides, the dried blood spot sample can be stored for a long time at room temperature, the transportation and storage cost is reduced, meanwhile, analysis can be completed only through a trace amount of blood, the method is suitable for repeated sampling and long-term monitoring, and a technical support is provided for research and application of IgG glycosylation as a biomarker.

Description

Technical Field

[0001] This application belongs to the field of biological detection technology, specifically relating to a method for the analysis and detection of IgG N-glycans in dried blood spots. Background Technology

[0002] Immunoglobulin G (IgG), as a core functional component of the immune response, directly influences antibody effector function and immunomodulatory activity through the modification pattern of its conserved N-glycosylation sites in the Fc region. Glycans in IgG molecules have been proven to be highly promising biomarkers related to aging; the dynamic changes in their glycosylation modification patterns are significantly statistically correlated with an individual's chronological age and can reflect immune remodeling during the inflammatory aging process, providing crucial theoretical support for the development of personalized medicine.

[0003] Currently, traditional IgG N-glycan detection primarily uses serum as the sample matrix. This requires first separating and purifying IgG molecules from serum using a standardized process, and then performing targeted analysis on the N-glycans they bind to. However, among the N-glycans carried by IgG molecules, those modified with sialic acid are not very stable and are prone to hydrolysis and shedding under suboptimal storage conditions, which affects the accuracy and reproducibility of the detection results.

[0004] The degradation of IgG N-glycans is particularly prominent in serum samples stored at room temperature. Long-term storage or temperature fluctuations can lead to damage to the structural integrity of N-glycans and abnormal fluctuations in their content, severely affecting the accuracy and reproducibility of test results and limiting the widespread application of IgG glycosylation analysis in clinical diagnosis and biomarker research. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a method for analyzing and detecting IgG N-glycans in dried blood spots.

[0006] This application also provides a method for analyzing and detecting IgG N-glycans in dried blood spots, comprising the following steps: S1. Provide dried blood spot samples; S2. Soak the dried blood spot sample in buffer solution and shake. S3. Filter the soaked liquid; S4. Use immunoaffinity purification media to purify and separate IgG from the filtered sample; S5. The purified IgG is denatured, digested with enzymes, labeled and purified to prepare oligosaccharide chains; S6. The oligosaccharide chains are separated and detected by electrophoresis.

[0007] In some embodiments, the dried blood spot sample comprises dried blood spot on filter paper.

[0008] In some embodiments, the buffer solution comprises a phosphate buffer.

[0009] In some embodiments, the soaking and shaking time in step S2 is 1 hour to 3 hours.

[0010] In some embodiments, the filtration process in step S3 includes filtration using a vacuum manifold.

[0011] In some embodiments, the immunoaffinity purification medium includes Protein G plates.

[0012] In some embodiments, the electrophoretic separation detection in step S6 includes capillary gel electrophoresis separation detection.

[0013] In some embodiments, the dried blood spot sample is prepared from fingertip blood.

[0014] In some embodiments, the enzymatic digestion in step S5 includes digestion using a glycosidase.

[0015] In some embodiments, the marking in step S5 includes a fluorescent marking.

[0016] This application has the following beneficial effects: 1. This application effectively solves the technical problem of IgG N-glycan degradation under room temperature storage conditions by using dried blood spot samples instead of traditional serum samples, and significantly improves the stability of the samples and the reliability of the test results.

[0017] 2. The dried blood spot sample collection method of this application is simple and does not require venous blood collection. It is suitable for large-scale screening and special populations (such as newborns and children), and broadens the clinical application scope of IgG N-glycan analysis.

[0018] 3. The dried blood spot samples of this application can be stored for a long time at room temperature, which reduces transportation and storage costs. At the same time, only a small amount of blood is needed to complete the analysis, making it suitable for repeated sampling and long-term monitoring, and providing technical support for the research and application of IgG glycosylation as a biomarker. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of a sugar chain structure; Figure 2 Image of IgG-N glycans on the first day of plasma storage at room temperature; Figure 3 Image of IgG-N glycans in plasma stored at room temperature on day 3; Figure 4 Image of IgG-N glycans in plasma stored at room temperature for seven days; Figure 5 Image of IgG-N glycans on day 1 of DBS blood stored at room temperature; Figure 6 Image of IgG-N glycans on the third day after DBS blood card was stored at room temperature; Figure 7 Image of IgG-N glycans on DBS blood sample stored at room temperature for seven days. Detailed Implementation

[0021] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0022] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] the term Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings: The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0024] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0025] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0026] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0027] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0028] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0029] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0030] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0031] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0032] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0033] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0034] The first aspect of this application provides a method for the analysis and detection of IgG N-glycans in dried blood spots, comprising the following steps: S1 provides dried blood spot samples; S2 immerses the dried blood spot samples in buffer solution and shakes; S3 filters the soaked liquid; S4 uses an immunoaffinity purification medium to purify and separate IgG from the filtered sample; S5 denatures, digests, labels, and purifies the purified IgG to prepare oligosaccharide chains; S6 performs electrophoretic separation and detection of the oligosaccharide chains. This method effectively solves the technical problem of N-glycan degradation under room temperature storage conditions by using dried blood spot samples instead of traditional serum samples. Dried blood spot samples can significantly improve the stability of IgG N-glycan chains, allowing for long-term storage at room temperature without degradation, ensuring the accuracy and reproducibility of the detection results. The buffer solution can be selected from physiological buffers such as phosphate buffer, Tris buffer, and HEPES buffer, for example, phosphate buffer; the immunoaffinity purification medium can be selected from Protein G, Protein A, antibody affinity columns, for example, Protein G plates; the electrophoretic separation and detection can be selected from capillary gel electrophoresis, polyacrylamide gel electrophoresis, for example, capillary gel electrophoresis.

[0035] In some embodiments, the dried blood spot sample is a filter paper dried blood spot. Filter paper dried blood spots have good blood adsorption properties and stability, effectively fixing blood components and preventing the degradation of IgG N-glycan chains. Specifically, the filter paper can be selected from commonly used filter paper materials such as Ahlstrom BioSample TFN card, Whatman WB100014, Whatman DMPK-C, Whatman 903 filter paper, FTA filter paper, and standard filter paper, for example, Whatman 903 filter paper.

[0036] In some embodiments, the buffer solution is a phosphate buffer. Phosphate buffers have good pH buffering capacity and biocompatibility, providing a suitable physiological environment for IgG extraction. Specifically, the concentration of the phosphate buffer can be selected from any value among 0.01M, 0.05M, 0.1M, and 0.15M, and the pH value can be selected from any value among 7.0, 7.2, 7.4, and 7.6, for example, a 0.1M phosphate buffer with pH 7.4.

[0037] In some embodiments, the soaking and shaking time in step S2 is 1-3 hours. An appropriate soaking time ensures that IgG is fully released from the dried blood spot, improving extraction efficiency. Specifically, the soaking time can be any value from 1 hour, 1.5 hours, 2 hours, 2.5 hours, and 3 hours, for example, 2 hours.

[0038] In some embodiments, the filtration process in step S3 is performed using a vacuum manifold. Vacuum manifold filtration can effectively remove impurities and particulate matter from the sample, providing a clean sample for subsequent purification. Specifically, the filter plate can be selected from filter plates with pore sizes of 0.22 μm, 0.45 μm, and 1.0 μm, such as a filter plate with a pore size of 0.45 μm.

[0039] In some embodiments, the immunoaffinity purification medium is a Protein G plate. Protein G can specifically bind to the Fc fragment of IgG, achieving efficient purification of IgG. Specifically, the Protein G plate can be selected from commercially available Protein G affinity plates, homemade Protein G affinity plates, etc., for example, commercially available Protein G affinity plates.

[0040] In some embodiments, the electrophoretic separation detection in step S6 is capillary gel electrophoresis. Capillary gel electrophoresis has the advantages of high resolution, high sensitivity, and high automation, making it suitable for the precise analysis of oligosaccharide chains. Specifically, capillary gel electrophoresis can be selected from capillary electrophoresis systems based on laser-induced fluorescence detection, capillary electrophoresis systems based on ultraviolet detection, etc., such as capillary electrophoresis systems based on laser-induced fluorescence detection.

[0041] In some embodiments, the dried blood spot sample is prepared using fingertip blood. Fingert-prick blood collection has the advantages of being minimally invasive and easy to operate, making it suitable for large-scale population screening and sampling of special populations. Specifically, the amount of blood collected from the fingertip can be any value among 10 μL, 20 μL, 30 μL, 40 μL, and 50 μL, for example, 30 μL.

[0042] In some embodiments, the enzymatic digestion in step S5 is performed using a glycosidase. Glycosidases specifically cleave the glycosidic bond between IgG and N-glycan, releasing the intact N-glycan. Specifically, the glycosidase can be selected from PNGase F, Endo H, Endo F, etc., for example, PNGase F.

[0043] In some embodiments, the labeling in step S5 is fluorescent labeling. Fluorescent labeling can significantly improve the sensitivity of oligosaccharide chain detection and is suitable for the analysis of trace samples. Specifically, the fluorescent labeling reagent can be selected from fluorescent dyes such as 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), and trisodium 8-aminopyrene-1,3,6-trisulfonic acid (APTS), for example, trisodium 8-aminopyrene-1,3,6-trisulfonic acid (APTS).

[0044] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0045] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0046] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0047] Example 1 The raw materials for the preparation of the method for analyzing and detecting IgG N-glycans in dried blood spots include: dried blood spots on filter paper (Whatman 903 filter paper, 30 μL of fingertip blood collection), phosphate buffer (0.1 M, pH 7.4), filter plate (0.45 μm pore size), Protein G plate (commercial Protein G affinity plate), PNGase F glycosidase, and 8-aminopyrene-1,3,6-trisulfonate trisodium salt (APTS) fluorescent labeling reagent.

[0048] The analytical detection method for IgG N-glycans in dried blood spots includes the following steps: Step 1: Prepare dried blood spots on filter paper using fingertip blood. Collect 30 μL of fingertip blood and drop it onto Whatman 903 filter paper. Let it dry at room temperature for 2 hours. Step 2: Punch holes in the dried blood spots from Step 1 to obtain dried blood spot discs with a diameter of 6 mm, and soak them in 200 μL of phosphate buffer (0.1 M, pH 7.4) and shake for 2 hours; Step 3: Place the soaking liquid from Step 2 into a 0.45μm pore size filter plate and filter the DBS sample into a clean collection plate using a vacuum manifold. Step 4: Transfer the filtered DBS sample to a Protein G plate using a pipette tip for purification and separation to obtain purified blood spot IgG; Step 5: Denature the purified blood spot IgG at 95℃ for 5 minutes, add PNGase F glycosidase and digest at 37℃ for 2 hours, add 8-aminopyrene-1,3,6-trisulfonate trisodium salt (APTS) fluorescent labeling reagent and label at 65℃ for 2 hours to purify and prepare oligosaccharide chains; Step 6: Separate and detect the oligosaccharide chains obtained in Step 5 by capillary gel electrophoresis, and then detect them using a laser-induced fluorescence detection system.

[0049] Example 2: This example provides a method for the analysis and detection of IgG N-glycans in dried blood spots.

[0050] Unlike Example 1, the soaking time in step 2 is 1 hour, while the other steps and raw materials are the same as in Example 1.

[0051] Example 3: This example provides a method for analyzing and detecting IgG N-glycans in dried blood spots.

[0052] Unlike Example 1, the soaking time in step 2 is 3 hours, while the other steps and raw materials are the same as in Example 1.

[0053] Example 4: This example provides a method for the analysis and detection of IgG N-glycans in dried blood spots.

[0054] Unlike Example 1, venous blood was used to prepare the dried blood spots on the filter paper, while the other steps and raw materials were the same as in Example 1.

[0055] Example 5: This example provides a method for the analysis and detection of IgG N-glycans in dried blood spots.

[0056] Unlike Example 1, Tris buffer (0.1M, pH 7.4) was used instead of phosphate buffer, while the other steps and ingredients were the same as in Example 1.

[0057] Example 6: This example provides a method for the analysis and detection of IgG N-glycans in dried blood spots.

[0058] Unlike Example 1, Protein A plate was used instead of Protein G plate, but the other steps and raw materials were the same as in Example 1.

[0059] Comparative Example 1: This comparative example did not use dried blood spot samples, but instead used serum samples.

[0060] In this comparative example, serum samples were used instead of dried blood spot samples, and the other steps and raw materials were the same as in Example 1. The specific steps were as follows: venous blood was collected to prepare serum samples, and the serum samples were directly used for subsequent IgG purification, enzyme digestion, labeling, and detection steps.

[0061] Comparative Example 2: This comparative example did not use immunoaffinity purification media.

[0062] This comparative example did not use Protein G plates for IgG purification and separation. Instead, the filtered samples were directly denatured, digested with enzymes, labeled, and detected. Other steps and raw materials were the same as in Example 1.

[0063] Comparative Example 3: This comparative example did not use the buffer soaking step.

[0064] In this comparative example, the dried blood spots were not soaked in phosphate buffer, but were directly perforated and filtered. Other steps and raw materials were the same as in Example 1.

[0065] Performance testing methods Sample stability test: Plasma samples and dried blood spot samples were stored at room temperature (25℃) for 1 day, 3 days, and 7 days, respectively, and then IgG N-glycan analysis was performed to compare the changes in glycan peak shapes at different storage times. The specific glycan structures of peaks 1 to 6 are shown in the figure below. Figure 1 As shown.

[0066] Glycan peak area ratio determination: The peaks were separated and detected by capillary gel electrophoresis. The area ratio of different glycan peaks was calculated to assess sample stability. The results are as follows: Figures 2-7 And as shown in Tables 1 and 2.

[0067] Table 1: Ratio of plasma IgG glucose peak area Table 2: Ratio of peak area of ​​IgG in blood glucose As shown in Table 1, the stability of IgG N-glycans in plasma samples was poor under room temperature storage conditions. By day 3, the area ratios of the sialic acid-containing glycan peaks (peaks 1, 2, and 3) had significantly decreased, from 0.0182, 0.050, and 0.045 on day 1 to 0.0015, 0.0075, and 0.0021, respectively. By day 7, these glycan peaks were almost completely degraded, with area ratios approaching 0. In contrast, after 7 days of storage at room temperature, the area ratios of the glycan peaks in dried blood spot samples remained relatively stable, with peaks 1, 2, and 3 at 0.023, 0.041, and 0.049, respectively. These ratios showed no significant change compared to 0.026, 0.044, and 0.051 on day 1, indicating that dried blood spot samples could effectively maintain the structural integrity of IgG N-glycans.

[0068] As can be seen from the repeatability test results in Table 2, the detection repeatability of Examples 1-6 is good, with relative standard deviations (RSD) between 3.2% and 4.1%, indicating that the method of this application has good repeatability and reliability. However, the RSD values ​​of Comparative Examples 1-3 are significantly higher, at 15.7%, 22.3%, and 18.9% respectively, indicating that not adopting the key technical features of this application would lead to a significant decrease in the repeatability of the detection results.

[0069] Comparative Example 1 used serum samples instead of dried blood spot samples. Because IgG N-glycans in serum samples are easily degraded under room temperature storage conditions, the test results were unstable and reproducible. Comparative Example 2 did not use immunoaffinity purification media, resulting in insufficient IgG purity and severe interference from impurities, affecting detection accuracy. Comparative Example 3 did not use a buffer soaking step, leading to low IgG extraction efficiency, insufficient sample volume, and reduced detection sensitivity.

[0070] Example 4 used venous blood to prepare dried blood spots. The test results were similar to those of the fingertip blood used in Example 1, indicating that different blood collection methods have little impact on this method. Example 5 used Tris buffer instead of phosphate buffer. The test results were similar to those of Example 1, indicating that different types of physiological buffers are suitable for this method. Example 6 used Protein A plates instead of Protein G plates. The test results were similar to those of Example 1, indicating that different types of immunoaffinity purification media can achieve effective purification of IgG.

[0071] from Figure 1 It is evident that after three days of storage at room temperature, the peak shape of plasma showed significant changes, particularly the sialic acid-containing sugar peaks; while after seven days of storage at room temperature, the peak shape of blood glucose remained unchanged. The percentage of IgG sugar peak area showed that after seven days of storage, the sialic acid-containing sugar peaks in plasma were largely degraded, while the sugar peak area in blood glucose remained unchanged during the same period. In conclusion, blood glucose can maintain the integrity of the sugar chains at room temperature.

[0072] In summary, this application effectively solves the technical challenge of IgG N-glycan degradation under room temperature storage conditions by using dried blood spot samples instead of traditional serum samples and combining them with an optimized pretreatment process, thus significantly improving the stability and reliability of the detection results.

[0073] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting IgG N-glycan analysis in a dried blood spot, characterized by, The method comprises the following steps: S1, providing a dried blood spot sample; S2, soaking and shaking the dried blood spot sample in a buffer solution; S3, filtering the soaked liquid; S4, using an immunoaffinity purification medium to purify and separate IgG from the filtered sample; S5, denaturing, enzyme cutting, labeling and purifying the purified IgG to prepare oligosaccharide chains; S6, performing electrophoretic separation and detection on the oligosaccharide chains.

2. The method for detection of IgG N-glycan analysis in dried blood spot according to claim 1, characterized in that, The dried blood spot sample comprises a filter paper dried blood spot.

3. The method for detection of IgG N-glycan analysis in dried blood spot according to claim 1, characterized in that, The buffer solution comprises a phosphate buffer solution.

4. The method for detection of IgG N-glycan analysis in dried blood spot according to claim 1, characterized in that, The soaking and shaking in step S2 lasts for 1-3 hours.

5. The method for detection of IgG N-glycan analysis in dried blood spot according to claim 1, characterized in that, The filtering in step S3 comprises filtering using a vacuum manifold.

6. The method for dried blood spot IgG N-glycan analysis and detection according to any one of claims 1 to 5, characterized in that, The immunoaffinity purification medium comprises a Protein G plate.

7. The method for the detection of IgG N-glycan analysis in dried blood spot according to any one of claims 1 to 5, characterized in that, The electrophoretic separation and detection in step S6 comprises capillary gel electrophoretic separation and detection.

8. The method for detection of IgG N-glycan analysis in dried blood spot according to any one of claims 1 to 5, characterized in that, The dried blood spot sample is prepared from fingertip blood.

9. The method for detection of IgG N-glycan analysis in dried blood spot according to any one of claims 1 to 5, characterized in that, The enzyme cutting in step S5 comprises using a glycosidase for enzyme cutting.

10. The method for detection of IgG N-glycan analysis in dried blood spot according to any one of claims 1 to 5, characterized in that, The labeling in step S5 comprises fluorescent labeling.

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