GlycoStation™, LecChip™, Introduction of Glycan Profiling Analysis Technology using Lectin Microarrays
The principle of glycan structure profiling analysis is based on the principle of lectin to glycan binding affinity. Lectins are glycan binding proteins and certain lectins will only bind with particular glycans. A GlycoTechnica’s LecChip™ (Lectin Microarray/Lectin Array) contains 45 different lectins immobilized on a slide glass in a X-Y array. Cy3 labeled glycoproteins are applied to the LecChip surface and their glycans then bind to the lectin with which it has affinity. The LecChip is then loaded into the GlycoStation™ Reader 1200 where an evanescent-field is generated above the LecChip array. The Cy3 marker attached to glycans that have successfully bound with lectins will fluoresce. The total LecChip™ fluorescence pattern generated by the glycan to lectin binding is then captured by the scanner (profiler) and analyzed by ToolsPro software differentially.
For a detailed explanation, please refer to “Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profling,” A. Kuno et al., Nature Methods 2, p. 851 (2005)
Shown below is an actual glycan profiling pattern generated from a LecChip™ scanned on a GlycoStation™ Reader 1200
The evanescent-field fluorescent excitation method used to excite the Cy3 marker on the glycans is an essential enabling technology as it allows for detection of very weak molecular interactions. Lectin-glycan interactions are well known to be relatively weak compared to antigen-antibody and biotin-avidin interactions (see Table 1). Given this fact, if a washing process is applied to the LecChip™ to remove non-lectin binding redundant glycoproteins, much of the affinity information will be lost. Fortunately, the GlycoTechnica’s glycan profiling analysis technology allows for generation of lectin-glycan affinity data from unwashed samples thereby taking advantage of the higher signal strength.
The evanescent-field is formed on the surface of the LecChip™ when light enters into the slide glass from the sidewall and propagates through the glass. This is known as the principle of internal reflection mode. The evanescent field that is formed has a depth equal to the wavelength of the light and the field strength decreases exponentially with the distance away from the slide surface. Therefore, the further away from the slide glass, the weaker the evanescent-field becomes. Therefore Cy3 tagged glycans floating above the slide glass in a liquid phase exhibit a relatively low level of excitation as they are in the weakest portion of the evanescent-field. Whereas Cy3 tagged glycans that interact with the lectins located on the LecChip™, are contained in the stronger portion of the evanescent-field and are effectively excited. This technology, jointly developed by Moritex and the National Institute of Advanced Industrial Science and Technology (AIST), allows monitoring of very weak molecular interactions directly from a liquid phase without washing of the sample.
A significant piece of the overall technology used in this glycan profiling is the comprehensive database developed by AIST which covers more than 10,000 lectin-glycan interactions and developed utilizing Frontal Affinity Chromatography (FAC). The AIST database is an indispensable tool for understanding and calculating any given sample’s most probable glycan structure taken from its complex lectin-glycan affinity pattern. The database has already been open to the public.
You can take a look of outline of GlycoStation operation on video.
Technical Reports (Notes)
A number of technical notes are ready for your refrence. Please download pdf files from the folliwing list.
- Technical Note No.1: Differential glycan profiling of cell lysates between CHO and Lec1.
- Technical Note No.2: Glycan profiling of secreted proteins from cancer cells into culture media
- Technical Note No.3: Glycan profiling of living cells
- Technical Note No.4: Differential glycan profiling of commercial EPOs