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 and Valita Titer Plus assays, for the quantification of IgG in cell culture supernatants for both low and high sensitivity applications.

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

The Valita Titer assays provide a very accurate and cost-effective solution to measure IgG from 10 – 100 mg/L or 100 – 2000 mg/L, respectively. The Valita Titer assays rely on the detection of IgG Fc interactions with Protein G by measurement of fluorescence polarization. The assays come in 96-well or 384-well formats and are 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 quantifi cation using Valita Titer assay kits obtained by the PHERAstar^®, CLARIOstar^® and VANTAstar^® plate readers.

Assay principle

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). 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. All liquid handling steps were completed using fresh culture media. A 7-point standard curve was prepared using an IgG1 kappa standard as per IFU. 60 μL of CD-CHO cell culture medium was pipetted into each well of the Valita Titer plate, along with 60 μL of prepared standards and samples. Contents were mixed and incubated in the dark for 5 min in case of the Valita Titer assay or 15 min in case of the Valita Titer Plus assay before being read on the PHERAstar, CLARIOstar, and VANTAstar plate readers using preconfigured protocols in the BMG LABTECH software (detailed settings below). Data was then analysed using the MARS software. 

Instrument settings

A 7-point standard curve (0 100 mg/L or 0-2000 mg/L, respectively) was quantifi ed with both the Valita Titer and Valita Titer Plus assays on three FP capable multi-mode readers by BMG LABTECH (VANTAstar, CLARIOstar and PHERAstar). All readers showed a high correlation (R2>0.95 with a 4-parameter fit) and low variation of replicate measurements for both assays (figure 3). The high signal range of the Valita Titer Plus assay was addressed with a special Valita Titer optic module on the PHERAstar that is also suitable for performing the standard range Valita Titer assay.