Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody.

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.

Determining the Ratio of Two Types of Prostate Specific Antigens with Biochips and Gold Nanoparticles for Accurate Prostate Cancer Diagnosis.

Prostate-specific antigen (PSA) is a well-known biomarker for prostate-cancer diagnosis. However, the serum PSA measurement alone is insufficient for accurate diagnoses because the correlation with cancer is weak within the gray zone-the biomarker level range wherein a clear-cut diagnosis is impossible. As such, accurate prostate cancer diagnosis has been supplemented by measurements of the ratio of two types of PSA: free PSA (fPSA) and complexed PSA (cPSA; α-1-antichymotrypsin-bound PSA). Herein, we describe a new method for measuring the ratio of these two types of PSA by using gold nanoparticles (AuNPs) and biochips. Both types of PSA in a sample are captured by the antibody immobilized on a biochip based on self-assembled monolayers on gold. fPSA and cPSA on the biochip are then distinguished by AuNPs that present antibodies against fPSA and cPSA, respectively. The presence of PSAs in a sample is detected with laser desorption/ionization time-of-flight mass spectrometry by observing reporter molecules, called amplification tags (Am-tags), on the AuNPs. One of the reporter molecules is an Am-tag without isotope labeling, and the other is a deuterium-labeled Am-tag (dAm-tag). These tags amplify mass signals so as to enhance the sensitivity of the method. A comparison of the mass intensities between the Am-tag and dAm-tag signals allows the determination of the ratio between fPSA and cPSA. We validated the selective measurement of fPSA and cPSA at different ratios in 50, 75, and 100 pM of total PSA (fPSA + cPSA) solutions corresponding to the gray zone in prostate-cancer diagnosis (4 - 10 ng/mL). Finally, the two types of PSA were spiked in fetal bovine serum at various ratios, and our strategy greatly afforded their accurate ratios as spiked based on a constructed calibration curve. These results clearly indicate that the strategy is applicable to human serum as a diagnostic and prognostic assay for prostate cancer.