Often, antibody-containing hybridoma supernatants have to be purified and, in some cases, labeled before off-rate determination; this is time-consuming, labor-intensive, and introduces complications such as loss of yield or aggregation. To save time and labor, we have screened unpurified hybridoma supernatants containing serum directly against an antigen on the sensor surface (referred to as a direct approach) with good results using the Attana A200 system.
Hybridoma supernatants are often screened using a capture approach, where an antibody is first captured before the antigen is injected and the antibody-antigen interaction studied. However, this approach consumes a large amount of sample and is time-consuming due to the extra antibody-capturing step. The direct approach saves both time and sample, thereby decreasing the cost of expensive antigens while increasing throughput.
A key requirement of the direct approach is that it has to be performed on surface chemistries that have low, nonspecific binding. To address this need, we have developed a low, nonspecific binding surface. A tagged antigen was immobilized on one surface, and the tag without the antigen was immobilized on the reference surface.
Hybridoma supernatants were then injected and screened directly against the antigen. The system’s response over the antigen-coupled surface is shown in Figure 3A, and the reference surface response in Figure 3B. Reference subtraction is then made to correct for bulk effects and drift (Figure 3C). Off-rate constants were subsequently determined directly from the resulting curves and are displayed either in a comprehensive list (Figure 3D) that can be printed or exported or as a report also containing graphical data of the interaction.
Screening E.coli Lysates
As with hybridoma supernatants, lysates often have to be diluted or purified before off-rate screening. To save time and labor, and to increase sensitivity, we have screened unpurified, undiluted E. coli lysates containing scaffold proteins with good results using the Attana A200 system.
Due to debris, nonspecific binding, and poor referencing, lysates are frequently diluted and then screened using a capture approach. Diluting the samples, however, decreases the sensitivity of the assay and introduces errors, directly affecting the results. Capturing surfaces could be an alternative, but for a variety of reasons, including the fact that they cannot be made generic since proteins, apart from antibodies, are very diverse. Again, the direct approach saves both time and sample consumption, thereby decreasing the cost of expensive antigens while increasing throughput.