Now that defense and immune response to pathogens have become the object of intense research, antibodies are integral to host defense mechanisms. The circulation’s most common antibody, IgG, is thought to be involved in the defense against viral and bacterial infections, and especially the Glycosylation of its fragment crystallizable (Fc) soluble part plays a major role in the function of immune effectors. But until recently, research on antibody glycosylation has only looked at the broad picture, without taking into account individual antigens, and thus was not able to provide us with the exact pattern of glycosylation and its role in various disease states. Modulations of antibody glycosylation are tightly associated with many disease conditions, allogeneic immunity and autoimmune response. However, existing methods often fail to provide high-resolution glycosylation information for specific antigens, resulting in limited understanding of changes in antibody glycosylation in diseases. For more fully and correctly characterising antibodies in infection, allogeneic immunity and autoimmune diseases, researchers desperately need a method that can do specific Antibody Glycosylation Analysis.
To solve this issue, David Falck and Manfred Wuhrer of Leiden University Medical Center in The Netherlands have published a paper in Nature Protocols titled "GlYcoLISA: antigen-specific and subclass-specific IgG Fc glycosylation analysis based on an immunosorbent assay with an LC–MS readout". They proposed the GlYcoLISA method, an analytical scheme that combines immunosorbent assay and liquid chromatography mass spectrometry (LC-MS) technology. This method uses a 96-well plate-based immunosorbent assay to purify antibodies with high throughput and high sensitivity, and then uses LC-MS to perform detailed analysis of antigen-specific IgG Fc glycosylation. This method enables scientists to comprehensively evaluate the glycosylation of low-abundance specific IgG in large-scale clinical samples, providing more comprehensive and specific information.
Figure 1 shows the Structure of IgG and its expected glycosylation structure, with a particular focus on glycosylation in the CH2 region. First, the figure clearly states that the glycosylation of the CH2 region of IgG is conserved in all subclasses. This indicates that in the molecular structure of IgG, the glycosylation of the CH2 region is highly consistent, and similar glycosylation patterns are maintained regardless of the subclass of IgG. This consistency provides researchers with a key observation point, allowing them to gain a deeper understanding of the common characteristics of IgG molecules. Further observation of the specific results of each part of the figure shows that the researchers have classified and displayed the glycosylation structure of IgG. Each part is clearly indicated by a label, such as "a", "b", etc. The presentation of these structures provides researchers with a systematic framework to understand the possible differences between different subclasses and variants. By classifying these structures, researchers can identify and compare the glycosylation differences in the CH2 region in each subclass. The significance of this figure is that it provides a comprehensive understanding of the structure and glycosylation pattern of IgG, especially in the CH2 region. This is crucial for further studying the function and regulatory mechanism of antibodies in disease processes such as infection, alloimmunity and autoimmunity.
Fig. 1 IgG structure and expected glycosylation structures. (Falck, et al., 2024)
Figure 2 shows the workflow of GlYcoLISA, which is designed to achieve efficient and accurate glycosylation analysis of antigen-specific antibodies. By combining immunosorbent assay with LC-MS technology, this method enables comprehensive and detailed analysis of antibody glycosylation in high throughput. In the figure, the GlYcoLISA process first involves the classic immunosorbent assay for purification of antigen-specific antibodies from clinical samples. The result of this step is high-throughput affinity purification of antibodies, which lays the foundation for subsequent analysis. Next, LC-MS technology is applied to perform high-resolution analysis of IgG Fc N-glycosylation. The detailed steps of LC-MS are not directly shown in the figure, but the role of this key technology in glycosylation analysis is emphasized. The significance of GlYcoLISA is that it overcomes the limitations of traditional methods and makes glycosylation analysis of antigen-specific antibodies more feasible. By combining high-throughput sample processing in 96-well plates and high-resolution analysis by LC-MS, this method provides a comprehensive understanding of antibody glycosylation while maintaining efficiency. The application of automated data processing strategies further accelerates the process of obtaining and interpreting results.
Fig. 2 GlYcoLISA workflow. (Falck, et al., 2024)
This article introduces a new method, GlYcoLISA, for high-resolution and specific analysis of antibody glycosylation. This method combines traditional immunosorbent assays with LC-MS to enable analysis of antigen-specific antibody glycosylation in large-scale samples. In addition, through GlYcoLISA, researchers are able to address the problem of insufficient analysis of specific antibody glycosylation in traditional methods. This innovation is expected to advance our understanding of the functional impact of antibody glycosylation in immunity and disease. Finally, the introduction of GlYcoLISA also emphasizes the importance of subclass- and site-specific analysis of antibody glycosylation, providing a new means for more refined studies.
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