With NIS’s rapid Epitope Mapping Service, you can expect to receive a fully refined, 3D reconstruction within 2 weeks of sample receipt, for well behaved samples. What happens once your sample is in NIS’s lab?
Each antibody-antigen complex begins with an initial sample optimization and screening. NIS’s experienced lab technicians will vitrify grids by taking an aliquot of sample in the buffer and apply it to a grid before plunging it into liquid ethane. The goal is to obtain a thin, uniform layer of ice, with particles embedded in a variety of orientations in order to capture a variety of different views of the sample. Grids will be screened on one of our four ThermoFisher Scientific Glacios microscopes for suitability. If issues such as preferred orientation are observed, NIS’s lab techs will try a variety of optimization techniques including use of detergents, changing grid types, or using a different vitrification method.
After the grid is optimized, an overnight data collection is carried out on the Glacios microscope with cryoSPARC Live running in parallel. By the next morning, an initial set of 2D classifications for the sample will be available. The 2D classes are extremely informative with regard to sample suitability, and we are able to determine if the sample is a candidate for moving forward based on characteristics such as the visibility of secondary structure elements and whether a variety of orientations are available.
If the sample is suitable, NIS’s scientists will then spend a few days working on further data processing in order to generate optimized 2D class averages, and an initial map from them. The initial map is a low resolution map with no refinement, however, it allows the scientist to further see and address sample suitability issues such as preferred orientation visible in the 2D classes, but confirmed in the initial map.
Once the 2D classes and initial map are collected, if no further sample optimization is required, the sample will be further imaged in order to collect the data necessary to get a high resolution three dimensional structure, which may require another overnight data collection on the microscope. If further sample optimization is required, or the sample is found to be unsuitable for further data collection, we will work with you to collaborate on next steps for your project.
Initial sample optimization, 2D class averages and initial map, and 3D structure reconstruction at 3.5 Å or resolution at the epitope/paratope interface suitable for tracing of the chains and assigning 75% of the side chains will be available in 2 weeks or less.
What is a well behaved sample?
In order for your sample to be eligible for the Epitope Mapping service, the antibody-antigen complex or fab complex has to have an ordered mass of at least 80-100 kDa, with no upper size limit. If the sample is smaller than 80 kDa, we’re often unable to distinguish individual particle orientation in the micrographs, which makes aligning particles for 2D classification and subsequent 3D structure reconstruction very difficult.
The epitope should not be on a loop or other flexible parts of the antigen, and the complex should not show extreme behaviors such as severe preferred orientation. Severe preferred orientation makes the process of aligning particles for 2D class averaging challenging, and therefore takes time and effort to correct. While severe preferred orientation can be corrected with methods including detergents, changing grid substrates, or tilted collections, this means the experiment is no longer a routine Epitope Mapping service. To read more about methods for overcoming preferred orientation during vitrification, you can read our blog here.
If the epitope is on a loop, or another flexible part of the antigen, the particles can be characterized by a large conformational flexibility, which could make it very challenging to align particles in two and three dimensions. This conformational flexibility can be addressed, but it will require further experiments outside of the routine Epitope Mapping service.
What kind of antibody-antigen complex can you map?
NIS can work with both full length antibodies and Fab fragments attached to antigens. We are also able to map antigens with multiple antibodies, as long as each antibody binds in a unique site, and they are not competitive. In fact, having multiple antibodies may even help reduce the amount of preferred orientation a sample exhibits, and increase the mass of the particle, making classification and reconstruction easier.
When it comes to using a Fab or a full length antibody, there are benefits and drawbacks to both. NIS commonly works with Fabs.
When working with a full length antibody, we need to keep in mind that often the antibody is so flexible that we are only able to focus on and visualize the Fab and antigen in the micrographs. The other Fab and the FC are generally completely washed out due to their flexibility and disordered nature. You can see an example of this flexibility in the short movie below, depicting negative stain imaging of Immunoglobulin M (IgM). Having a full length antibody may reduce the amount of preferred orientation, however, the full length antibody can negatively impact vitrification due to the formation of aggregates or extended chains that are the focus of visualization.
Visualization of flexibility in Immunoglobulin M (IgM) antibody-antigen complex with negative stain transmission electron microscopy.
Fabs are preferred for visualization of antibody-antigen complexes, because they minimize the complexity of the sample. However, Fabs do tend to exhibit preferred orientation more strongly than a full length antibody, and often tend to vitrify in very specific positions. The issue of preferred orientation happens in approximately half of the cases we see. While preferred orientation can slow down sample optimization and data collection, we have several practical ways to address it, which you can read more about here. In cryo-EM, Fab fragments from a digested Mab can be used, eliminating the need for recombinant Fab production and simplifying the sample production process.
What other sample requirements are there for Epitope Mapping with cryo-EM?
Cryo-EM is fairly tolerant of many buffer compositions, as long as the components do not interfere with the vitrification process or the signal to noise ratio. However, compounds with high carbon contents, like glycerol or sucrose, can increase the background signal, resulting in lower resolution maps. Organic solvents, like DMSO, can interfere with the vitrification process. To learn more about the effects of glycerol on the resolution of cryo-EM imaging and resulting map quality, you can read our whitepaper here.
When it comes to concentration, cryo-EM imaging works with samples at a micromolar concentration; 50-100 microliters of sample at 0.5-5 mgs/mL is appropriate for imaging. If the KD for the complex is worse than micromolar, the complex will tend to dissociate at the concentrations used for cryo-EM, making imaging difficult. However, our lab techs are able to assist with samples that do not have an ideal concentration by changing vitrification methods, performing sample dilution immediately before vitrification, or using appropriate additives.
Get started mapping your antibody-antigen complexes today.
To hear more from NIS CSO Dr. Giovanna Scapin about epitope mapping with cryo-EM, how cryo-EM epitope mapping compares to other techniques, and see a case study, watch the webinar and interactive discussion here.
With NIS’s rapid cryo-EM Epitope Mapping Service, you receive a 3D reconstruction within 2 weeks of sample receipt, for well behaved antibody-antigen complexes.