Frequently Asked Questions

1. What is the size of the printed array and each ROI “spot”?

LSA biosensor chips consist of a gold surface mounted on a trapezoidal glass prism, both of which are encased in a plastic cartridge. The printed array is approximately 16 mm wide by 5 mm high. Each printed ROI on the chip surface measures approximately 24 µm wide by 90 µm high.

For more information on chip surfaces: https://carterra-bio.com/lsa/#chip-chemistry

3. Which binding-curve analysis models that are available in the Kinetics™ analysis software?

Models available in Kinetics include:

• k_d = Off-rate only model
• k_dk_a = 1:1 Langmuir binding model
• k_dk_ak_m = Mass transport model

Use of more complex models to describe interactions beyond a simple 1:1 model requires a detailed knowledge of binding mechanisms. Instead, assay optimization is recommended to achieve 1:1 binding for example through use of monovalent analytes and minimal ligand densities.

In instances where advanced models are absolutely required, data can be exported for analysis into third party software.

5. What is the sensitivity of the LSA for smaller MW analytes? Is there a minimum MW cutoff for analytes?

Sensitivity limitations for lower MW analytes will depend on the interaction being measured. SPR is a mass-based detection technology and the analyte binding signal is proportional to the stoichiometry of the interaction and can be theoretically calculated. As a general rule, in order to maintain a specific RU signal, ligand density will need be increased as analyte MW decreases, with attention paid to the binding stoichiometry of the ligand and analyte.

Here is an example of a lower MW analyte binding to captured antibodies:

• Ligand: Panel of mouse mAbs (MW 150 kDa) captured as an array on an anti-mouse surface
• Analyte: 7 amino acid peptide (MW 1 kDa)
• Using the R_max equation, to achieve a 40 RU R_max signal of analyte binding to a 150 kDa ligand with a valency of 2, the total RU of mAb captured is 3000 RU:

40 RU R_max = 3000 RU x (1 kDa / 150 kDa ) x 2

7. What is the maximum number of analyte cycles with regeneration that can be performed on the LSA?

Deck capacity of the LSA single channel needle sampler enables approximately 400 cycles of analytes in a single experiment, each of which can contain a regeneration pulse. The ability of the immobilized ligand to tolerate a given regeneration condition will dictate the number of cycles with regeneration that still allow for adequate analyte binding. Additionally, the acceptable loss of ligand activity due to regeneration will be somewhat dependent on assay objectives. Therefore, the maximum number of regenerations will be ligand and assay specific.

For a kinetics experiment, a non-regenerative approach is a good option to ensure there is minimal loss of ligand activity throughout an experiment and avoids the need to identify optimal regeneration conditions. An additional benefit is that this assay format uses minimal quantities of ligands. For kinetic experiments, most capture surfaces (anti-Fc, Protein A/G) are unaffected by the typical cycle numbers required if regeneration is chosen.

In epitope binning, where hundreds of regeneration events are required, regeneration scouting prior to the experiment is highly recommended. It is difficult to select a regeneration strategy that is optimal for an entire array of antibodies, however selecting the least harsh regeneration condition that thoroughly regenerates the majority of the panel is the most practical approach. Even if some ligands lose activity during the experiment, both the antigen signal normalization feature in Epitope™ and the bidirectionally competitive design of epitope binning can still facilitate meaningful data interpretation.

9. What is meant by floating R_max?

Floating R_max is an option in the Kinetics™ analysis software allowing the binding curves in a kinetic titration to be fit independent of one another with regard to R_max. Floating R_max should be used only in specific circumstances. Here are 2 scenarios where it is appropriate to use the floating R_max option:

1. Performing a multi-cycle kinetics experiment with regeneration and the chip surface is losing activity from cycle-to-cycle
2. Performing a multi-cycle capture kinetics experiment with regeneration and the ligand capture levels vary significantly from cycle to cycle.

Since R_max is changing across the assay in these 2 scenarios, differences between the data and the fitting model are increased when applying a global R_max value and therefore local R_max fitting is more appropriate.