(1314-C) Homogeneous Antibody-Binding Assay Using High-Throughput Microscopy
Tuesday, February 6, 2024
12:00 PM – 1:00 PM EST
Location: Exhibit Halls AB
Abstract: For various applications in research, diagnosis, and therapy, the production of high-affinity, antigen-specific antibodies is essential. Typically, the antibody-producing cells are generated via hybridoma technology and subsequent single cell cloning (SCC). This requires a reliable method for selecting high-producing clones. Various screening methods for antibodies in hybridoma supernatants exist, each with advantages and disadvantages. For example, enzyme-linked immunosorbent assay (ELISA) is common but requires extensive washing and can compromise antigen conformation. Flow cytometry is highly sensitive, but entails large sample volumes and complex data analysis, while fluorometric microvolume assay technology (FMAT) employs only a single laser. Moreover, these assays do not offer images of the cells for optical control and documentation. Here, we present a homogeneous antibody-binding assay using our automated microscope NYONE Scientific. This assay eliminates the need for washing steps and utilizes minimal sample volumes. We used an anti-HER2 antibody as a model antibody. The HER2-expressing breast cancer cell line SK-BR-3 served as target cells, while HER2-negative MDA-MB-468 cells were used as a negative control. We seeded the cells into 384 well plates. Subsequently, we first added the primary anti-Her antibody and then a fluorescently labeled secondary antibody to the wells and imaged the plate. The images were automatically analyzed using the newly developed Suspension Cell AB Binding (1F) application of our YT-SOFTWARE. This application detects the cells in a brightfield image and analyzes the average fluorescence intensity of the cells using a fluorescence image. The image processing application is specialized in the detection of weak signals with a high background. By optimizing the assay and the image settings, we achieved a detection limit of primary antibody up to 1 ng/mL. The scanning time was less than 6 min for a whole 384-well plate making the assay suitable for high-throughput screening. Moreover, the NYONE Scientific offers the versatility of different fluorescence channels and the ability to measure additional characteristics through the brightfield channel. Combined with our SCC application, both the proof of monoclonality and the screening of hybridoma supernatant can be analyzed with the same device and software reducing time and cost for additional equipment, training, and support.