Fluorescence Polarization based assay for rapid, precise, high-throughput measurement of IgG & Fc containing derivatives

The accurate, rapid, and high-throughput measurement of IgG is essential in the development and manufacture of most therapeutic antibodies. Monoclonal antibodies are becoming increasingly dominant in biopharmaceuticals, where a vast number of samples must be screened for the development of each potential therapeutic. Here we present the Valita™TITER assay, a novel assay for the quantification of IgG in cell culture supernatant.

Current workflows, which include HPLC protein A, ELISA, protein A surface interferometry, and HTRF (homogenous time resolved fluorescence) have major drawbacks, which include costly, labour-intensive methods and long analysis times. 

The Valita^TMTITER assay provides a very accurate and cost-effective solution to measure IgG in a range from 2.5-80mg/L. The Valita^TMTITER assay relies on the detection of IgG Fc interactions with Protein G by measurement of fluorescence polarization (Fig. 1). The assay comes in a 96-well format and is simple, high-throughput, rapid, and fully automatable. Analysis can be carried out in cell culture media with a low sample volume and no complex preparation steps.

In this application note, we show the optimal protocol for performing IgG quantification using Valita^TMTITER assay kits obtained by the PHERAstar^® and the CLARIOstar^® plate readers.

FP effectively analyzes changes in the size of molecules. “Fixed” fluorophores that are excited by polarized light preferentially emit light in the same plane of polarization. However, rotation of the molecules between absorption and emission of the photon has the effect of “twisting” the polarization of the light. Small molecules tumble faster in solution than larger molecules. Hence, the change of size of molecules, with an associated fluorophore, can be measured using the degree of light depolarization. Consequently, when fluorescently labelled Protein G is unbound, it tumbles rapidly and depolarizes the light more than when it is bound to an IgG (which is 5 times larger) (Fig. 2). This change in polarization is used to measure the amount of IgG in the solution. FP is measured by exciting the solution with plane polarized light and measuring the intensity of light emitted in the plane parallel to the exciting light (polarized proportion) and perpendicular to the exciting light (depolarized portion). The FP is expressed as a normalized difference of these two intensities, which is typically in millipolarization units (mP).

Experimental Procedure
Samples were prepared according to the respective product instruction for use. A standard curve was prepared using an IgG standard as per IFU. 60 μl of ValitaMab reconstitution buffer was pipetted into each well of the Valita^TMTITER plate, along with 60 μl of prepared standards and samples. Contents were mixed and incubated in the dark for 30 minutes before being read on the PHERAstar, and CLARIOstar plate readers using preconfigured protocols in the BMG LABTECH software (detailed settings below). Data was then analysed using exported .csv files from the raw data, using the ValitaAPP software. 

Instrument settings

A standard curve (2.5-80 mg/l) was quantified with the Valita^TMTITER assay on three FP capable multi-mode readers by BMG LABTECH (CLARIOstar, and PHERAstar). All readers showed low variation of replicate measurements (Fig. 3).