Abstract POSTER-TECH-1131: Next generation protein multiplexing
Authors: Sloan D , Nelson J , Ure D , Votaw G , Stevens E 
Published: August 2015

While a variety of technologies exist to measure proteins, such as ELISA’s, Mass Spec, and 2D Gel’s, the most promising for screening multiple proteins are biochip or microarray based technologies. However, even with biochip devices, measuring specific interactions between multiple protein combinations remains problematic. Proteins are complex and fragile bio-molecules and often interact in complex and unpredictable ways with other proteins and/or protein analysis equipment, causing non-specific (false) signals. Furthermore, many proteins of interest to the life science industry are present in samples at very different concentrations, limiting which proteins can be screened in a single multiplex test and sample dilution.

To address these problems, Inanovate has developed a new multiplexed protein quantification technology called Longitudinal Assay Screening (LAS), combining high sensitivity confocal imaging and microfluidics alongside protein based microarrays. Inanovate has completed testing and benchmarking of the first platform integrating LAS technology across a range of demonstration assays. Instead of depending on a 96-well micro-titer plate, the new LAS platform utilizes a glass slide based protein microarray and microfluidics for dispensing and binding samples and detection antibodies. In its most basic form, the protein microarray is composed of capture antibodies for the proteins being measured, as well as positive and negative quality control features for ensuring sample to sample, run to run, lot to lot, and user to user consistency. Conceptually very similar to a real-time PCR reaction, the LAS platform iteratively flows small volumes of sample and labeled detection antibodies across the protein microarray (housed on Inanovate’s fluidic cartridges) and fluorescently measures the formation of the sandwich between capture antibody, analyte of interest, and detection antibody in real-time.

Due to the time-resolved nature of the assay, the resulting data is a rate of reaction, instead of a simple final fluorescent reading. This ‘rate of reaction’ based analysis helps deliver the following core advantages of LAS technology, each of which has been demonstrated through the development and validation of a five-plex assay consisting of CRP, IL-6, IL-1a, IL-8 and IL-1b.

1. Large detection range: LAS enables the accurate quantitation of protein concentrations across a 7 log range in a single multiplex test. This eliminates the need for serial dilutions, making multiplexing faster, cheaper and helping preserve precious samples.

2. Multiplexing flexibility: Due to its large detection range, LAS allows users to run virtually any assay of interest in one test, enabling the development of biologically relevant multiplexes.

3. Improved accuracy: LAS produces and analyses real-time kinetic data on protein interactions (rate of reaction data), improving identification and discrimination of background and nonspecific signals, delivering more accurate quantitation at low analyte concentrations.

LAS technology holds the potential to become to proteomics what PCR was to genomics. It offers a new approach to multiplexed protein screening that helps address many of the problems presently affecting the utility of biochips in protein biomarker discovery, validation and clinical screening applications. The Poster will both summarize the technical components of the new LAS based platform and present data from the five-plex demonstration work plus recent work on serology assays.

Citation Format: Sloan D, Nelson J, Ure D, Votaw G, Stevens E. Next generation protein multiplexing [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1131.