IgG Quantification

Valita®Titer provides an easy, rapid and high-throughput solution for IgG quantification – results are available in minutes, not hours. Valita®Titer kits consist of a 96- or 384-well plate precoated with lyophilised probes specific to Fc-containing IgG-based antibodies. The simple workflow follows an add, mix and read procedure, which means it can be easily integrated into any automation system. The assay is based on fluorescence polarisation (FP), so measurements can be taken on any microplate reader that includes an FP module. Valita®Titer is a homogeneous assay, so no washing steps are required and cells do not need to be removed – crude sample can be used.

Fluorescence Polarisation assay

With Valita®Titer, IgG concentration is determined using Fluorescence Polarisation (FP). The principle of FP is based around changes in molecule size. Small molecules rotate rapidly in solution, while larger molecules rotate slower in solution. When a rapidly rotating small molecule is excited with polarised light, the emission light will be depolarised. Larger molecules, under the same conditions, will retain polarisation. In Valita®Titer, this principle applies to the proportion of bound probe to IgG – unbound probe will tumble quickly and emit depolarised light, while bound probe will rotate slowly and retain polarisation. The polarisation (mP) values are then plotted as a standard curve, which can be used to quantify IgG in unknown samples.

Applications

Valita®Titer can be used at any stage of the drug development process to determine IgG concentration, from discovery to manufacturing. This includes cell line development, clone ranking, productivity ranking, stable pool characterisation and clone expression optimisation. Valita®Titer is optimised for human, rabbit and dog samples, and work is underway to validate for other mammalian samples.

Technical Information

The detection range of 2.5 – 100 mg/L and 100 – 2000 mg/L are available. Assay precision has been determined to be 2 – 4 mg/L or 20 – 40 mg/L respectively, depending on instrument performance.

The fluorescent label is a fluorescein (FITC) molecule, which has an excitation maximum of 490 nm, and an emission maximum of 525 nm. The fluorescent label is bound to GB1 (a subunit of Protein G) which selectively binds the Fc region of Fc-containing IgG-based antibodies.