Volume 9 Supplement 9
PAIA assays: A new bead-based assay system for high throughput protein quantification
© Wosnitza et al. 2015
Published: 14 December 2015
Key words: protein quantification / cell line development / IgG assay kit / high producer screening / high throughput
Material and methods
The PAIA platform is based on 384-well plates and all assays were performed in a volume of 60 µL. If not stated otherwise, a sample volume of 6 µL was used. The sample as well as fluorescence marker in reaction buffer were added to the microplate which contained dried capture beads. After addition of buffer and sample, the microplate was incubated for 30minutesat 2000 rpm on an orbital shaker at room temperature and subsequently allowed to stand for 10-15 minutes. The read-out is obtained with either a plate reader or a fluorescence microscope (SynenTec, Elmshorn, Germany) in less than five minutes per plate. The data depicted in Figure 1D was obtained by incubating the assay on the BioShakeXP (Q.Instruments, Jena, Germany) at 1800 rpm for 40 minutes, followed by 5 minutes at 1000 rpm and 10 minutes without agitation. The protrusion on the microplate bottom ensures that the beads are settling outside the detection window. Hence, only the unbound fluorescent marker was detected when measuring on a fluorescence plate reader in bottom read modus at 485/520 nm (Safire, Tecan, Männedorf, Switzerland).Calibration curves were calculated in the PAIA evaluation software with a 4-parameter fit.
Results and discussion
Performance values of the Human IgG Fc/Fab assay
5 - 200 μg/mL
Limit of detection
~ 5 μg/mL
We believe that the high throughput, the small sample volume, and the low overall costs represent substantial advantages over existing methods. In future, we will expand the range of assays to other proteins to fully exploit the potential of this versatile technique.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.