Mark J. Cameron

The choice of an appropriate immunoassay technology platform can mean a difference measured in days instead of months for immunoassay development time. In today's life-science environment, when time and cost efficiencies are crucial for meeting condensed development timelines, many biopharmaceutical companies are relying more on contract research organizations (CROs) with highly automated technologies to improve laboratory productivity, shorten immunoassay development and validation timelines, and speed new drugs to market.

The development of immunoassays for sample analysis is important to support drug safety and toxicology studies and the critical decisions that lead to advancing drug candidates. Ideally, immunoassays are developed and validated prior to initiating in-life safety assessment studies, and those assays are validated in a good laboratory practice (GLP) or a fit-for-purpose compliant manner. Placement of toxicology and drug-safety studies often hinge on the ability of the ligand-binding assay laboratory to rapidly develop and validate pharmacokinetic and/or biomarker immunoassays.

SANDWICH ELISA

The sandwich enzyme-linked immunosorbent assay (ELISA) is the traditional method used to measure analytes and requires two layers of antibodies. The antibody captures the analyte from a complex liquid matrix such as serum or plasma. The ELISA can be custom-built on a variety of platforms, maintaining its original form; that is, a capture antibody on a solid support with the analyte sandwiched between the capture and detection antibody and a reporter indicating that the sandwich has completed.

Widely available platforms for the sandwich ELISA format include the following 96-well plate colorimetric ELISAs:

• Luminex's bead-based flow cytometry platform
• Meso Scale Discovery's (MSD's) platform, based on electrochemiluminescence
• The Gyros Gyrolab, the newest, automated platform that uses a novel compact disk (CD) format.

In addition to traditional ELISA, now decades old, these three technology platforms have proliferated in the past 10 years and are commonly found at life-science companies and CROs.

COLORIMETRIC SANDWICH ELISA

The traditional ELISAs, still commonly used, are labor intensive. The assays typically have a narrow dynamic range resulting in frequent repeat analyses when the unknown sample concentration is above the limit of quantification, and are less sensitive than sandwich assays performed on other platforms. Although readily available for many analytes and species, this platform is not capable of multiplexing. The development of multiplexing formats originated from customer demand to obtain more information from smaller sample volumes. Rodent species, especially mouse, have very limited blood volumes, which places limits on the number of samples that can be run.

LUMINEX

The widely available Luminex platform is a bead-based flow cytometry system capable of multiplexing. When the assay is complete, a laser passes over the individual 5.5 µm beads. The laser excites the fluorochrome-conjugated detection antibody, and the light energy emitted is captured by a photomultiplier tube. In theory, 100 different ELISAs could be analyzed simultaneously from a single 100-µL sample.

MESO SCALE DISCOVERY