How to analyse bispecific antibodies?

Welcome to our instructional guide on using the Fida instrument for assaying bispecific antibodies. This guide complements our video demonstration, providing step-by-step details for both the experimental setup and data analysis. Let's dive into the terminologies and methodologies for executing these assays effectively.

Terminologies:

• Indicator: The fluorescent molecule whose hydrodynamic radius is being measured.
• Analyte: The compound that is titrated in the experiment.

Methodologies for Fida Experiments:

1. Premix Method:
2. Involves pre-mixing equal amounts of indicator and analyte in vials before injection.
3. Capillary Mix (Cap Mix) Method:
4. Utilises a single indicator vial and multiple analyte vials. Mixing occurs in the capillary by the Fida instrument.
5. Capillary Dissociation (CapDisc) Method:
6. Consists of a titration of mixed indicator and analyte vials and a single buffer vial. The complex is injected into a buffer.
7. Capillary Flow (CapFlex) Method:
8. Primarily used for liquid phase separation and condensate analysis (optional in most cases).

Ternary Complex Assay Setup for Bispecific Antibodies:

1. Rapid Confirmation Approach:
2. Ideal for quick verification of binary and ternary complex formation. Uses CapDisc method.
3. Plate setup includes various indicator vials mixed with analyte and a single buffer vial.
4. Measure sizes of labeled antigen mixed with unlabeled antigen, labeled antigen with bispecific antibody, and labeled antigen in presence of both bispecific antibody and excess unlabeled antigen.
5. Complete Biophysical Characterization:
6. Suitable for thorough analysis of interesting bispecific candidates. Can be executed as either a premix or a Cap Mix experiment.
7. Essential to label one of the two antigens fluorescently.
8. The experiment measures various parameters, including the size of labeled antigen, binary and ternary complexes, affinities, and cooperativity.

Data Analysis Using Fida Software:

1. Loading Data:
2. Access the data analysis tab and load data files for analysis.
3. Evaluating and Fitting Data:
4. Select appropriate time intervals and fit data for single or multiple species.
5. Utilise auto mode for fitting multiple data points efficiently.
6. Manually evaluate and correct each fit, if necessary.
7. Finalising the Analysis:
8. Compensate for viscosity and observe fluorescence areas.
9. Select the ternary complex model and input initial parameters.
10. Apply the model and fit the binding curve for detailed results.

Conclusion:This guide provides a detailed walkthrough for conducting and analyzing bispecific antibody assays using the Fida instrument. By following these steps, you can efficiently confirm the formation of binary and ternary complexes and perform a complete biophysical characterisation of your samples.

We hope this guide enhances your understanding and aids in your research. For visual assistance, refer to our accompanying video. Happy experimenting with your Fida assays!