Biacore X100 reference portal

Screening assays are evaluated by comparison of the response levels obtained from the sample injection between different samples.

Classification of binding strength

With the same concentration of different analytes, the response value gives a direct indication of the binding strength. The simplest approach to evaluating screening is to compare response levels from one report point.

A plot of cycle number against report point values on the y-axis.

Classification of kinetic properties

Comparison of response levels measured at different times during sample injection and dissociation can also give an approximate indication of kinetic parameters for the binding that complements the classification of samples in terms of binding strength.

A plot of report point binding_late against stability_late can provide a rough visualization of kinetic properties for all samples in a single plot. The binding_late report point reflects the level of binding reached during the association phase, while stability_late reflects the extent of dissociation during a fixed period. Non-binders among the samples cluster close to the origin. Points with a significant binding_late response but low stability_late represent samples that bind but dissociate rapidly, while points with high stability_late values bind more strongly.

In a plot of binding_late against stability_late, high values on the y-axis indicate rapid association while high values on the x-axis indicate high stability (slow dissociation).

Screening provides two main types of data:

• “Yes/no” answers for binding of individual compounds
• Ranking of a set of compounds in terms of relative binding levels

An additional goal of the screening is to obtain preliminary kinetic or affinity information on the antibody clones, since these parameters can be important deciding factors in selection of antibodies for both biopharmaceutics and biotechnology applications.

The essential difference from screening for small molecules apart from analyte size is that the antibody concentration is not known.

Assay format

Screening for antibody production from hybridoma cultures can in principle be set up in a format analogous to small molecule screens, with antigen immobilized on the surface and cell culture supernatant injected as sample. A more convenient and informative approach is however to immobilize anti-mouse immunoglobulin antibody on the surface as a capturing molecule, inject the cell culture supernatant over this surface to capture the MAbs, and then inject antigen in solution to confirm the MAb identity and to provide information on antibody-antigen characteristics. Although this is a somewhat more complicated approach and the sample throughput is lower compared with immobilized antigen, there are a number of advantages:

• Immobilization and regeneration of anti-mouse immunoglobulin antibody is well characterized, so the assay development work involved in finding immobilization and regeneration conditions for antigen is eliminated.
• Anti-mouse immunoglobulin antibody interacts with the constant region of MAbs, so that the response obtained from injection of the supernatant over anti-mouse immunoglobulin antibody gives a direct indication of the concentration of MAb produced by the hybridoma clone, independently of the antibody-antigen binding characteristics.
• The antigen response can be normalized to the level of captured MAb, so that an indication of the kinetic and/or affinity properties of the MAb can be obtained directly from the first screening. This can help to eliminate cell clones early in the development procedure that produce MAbs unsuitable for the intended application.
• Suitable antibodies for use as capturing molecules are available from Biacore.