Fast, accurate and high-throughput detection of IgG and Fc-containing derivatives by fluorescence polarization

Fast, accurate and high-throughput detection of IgG and Fc-containing derivatives by fluorescence polarization

Dr. Carolanne Doherty
Valitacell, NIBRT, Fosters Avenue, Blackrock, Dublin, Ireland

1. BMG multi-function microplate reader can be applied to the new technology of quantitative IgG ValitaTMTITER

2. Detection of IgG directly in cell culture supernatants by fluorescence polarization

3. Microplate reader preset detection program and data analysis template to improve research speed

Introduction

Accurate, rapid and high-throughput detection of IgG is essential for the development and production of most therapeutic antibodies. Monoclonal antibodies are gaining momentum in the biopharmaceutical field, and a large number of samples are awaiting screening for further development. Here we describe a new screening technique for the direct quantification of IgG in cell culture supernatants: ValitaTM TITER.

Existing technologies, including protein A, ELISA, protein A surface interferometry, and HTRF (homogeneous time-resolved fluorescence) have significant drawbacks, including cost, labor, and time consuming. .

The ValitaTM TITER method is a new, very accurate, low-cost solution for detecting IgG with a detection range of 2.5-80 mg/L. ValitaTM TITER uses fluorescence polarization to detect binding of the Fc portion of IgG to protein G (Figure 1). The analysis is performed in a 96-well plate that is simple to operate, high throughput, fast and automated. Analysis can be performed in very small amounts of cell culture medium without cumbersome preparation procedures.

In this application note, we introduced the use of the ValitaTM TITER kit for quantitative detection of IgG on PHERAstar®, POLARstar® Omega and CLARIOstar® multi-plate readers.

Figure 1: Schematic diagram of the ValitaTM TITER analysis principle for quantitative determination of IgG by fluorescence polarization. Each well of a 96-well ValitaTM TITER microplate was pre-coated with a fluorescently labeled IgG-binding polypeptide-protein G (A). The IgG sample binds to the polypeptide and is determined by fluorescence polarization and dispersion (depolarization) (B).

Figure 2: Analytical principle: Analysis of IgG quantification using fluorescence polarization. The concentration of the sample is determined by measuring the fluorescence polarization due to molecular rotation. Small, unbound molecules rotate faster in solution, while large, bound molecules rotate slowly.

Fluorescence polarization can effectively analyze changes in molecular size. A "immobilized" fluorophore emits fluorescence in the same polarization direction as the excitation light under excitation of polarized light. However, the fluorescence polarization direction of the rotating fluorophore will have a certain change compared to the polarization direction of the excitation light. Small molecules rotate faster in the solution than macromolecules, so the change in the size of the molecule to which the fluorophore is attached can be determined by the degree of fluorescence depolarization. Therefore, when the protein G labeled with fluorescence does not bind to other molecules, it rotates faster and depolarizes more, whereas when combined with IgG, it is reversed (5 times larger) (Fig. 2). This change in polarization is applied to detect IgG in solution. Fluorescence polarization (FP) illuminates the solution by irradiating the solution with excitation light in a parallel polarization direction and measuring the emission in parallel and in the vertical direction. FP is the normalized difference between these two polarized fluorescences, usually expressed in mP.

ValitaTM TITER Analysis Kit
ValitaTMTITER APP software (available in the kit)
IgG standard (IgG obtained from human serum)
PHERAstar, POLARstar Omega and CLARIOstar Multi-Function Microplate Reader (BMG LABTECH)

Prepare the sample in accordance with the appropriate requirements of the kit instructions. The standard curve was drawn using a method in which each relative fluorescence value corresponds to an IgG standard. 60 μl of reconstitution buffer and 60 μl of standard or sample were added to each well of a ValtaTM TITER microplate. Incubate and incubate for 30 minutes in the dark. Then, in the POLARstar Omega, PHERAstar or CLARIOstar multi-function microplate reader (BMG LABTECH), the readings are performed using a preset test procedure (see the table below for parameter settings). The Raw Data obtained in the MARS analysis software was converted into a .csv format file and analyzed by ValitaTM APP software.

Results and discussion

The standard curve (2.5-80 mg/L) of Figure 3 was drawn on a ValitaTM TITER kit on BMG LABTECH's multi-function microplate readers (POLARstar Omega, CLARIOstar and PHERAstar) with fluorescence polarization detection. The results obtained by all microplate readers are highly stable.

Figure 3: IgG quantitative standard curve constructed with ValitaTM TITER on a CLARIOstar multi-function microplate reader. Error bars indicate the standard deviation of the three duplicate holes

Finally, we used samples cultured under different conditions, and the results of quantifying IgG with ValitaTM TITER were compared with those obtained by conventional methods. Figure 4 shows the correlation between the two methods, showing that both methods have good accuracy, while ValitaTM TITER is faster because it does not require a cumbersome sample preparation process.

Figure 4: ValitaTM TITER quantified IgG results compared to conventional HPLC results

in conclusion

It is important to perform rapid IgG concentration determinations on mixed samples in the development and production of such biopharmaceuticals. Here we present a fast and simple new technique for the determination of high-throughput IgG and Fc-segment derivatives based on the fluorescence polarization method, the ValitaTM TITER assay. The ValitaTM TITER assay allows direct determination of IgG in a test sample without cumbersome sample preparation or purification. CLARIOstar, PHERAstar and POLARstar Omega deliver outstanding performance when applied to ValitaTM TITER analysis. Compared to the convenience of other IgGs, ValitaTM TITER is high-throughput, simple, accurate and fast.

video:
QUICK-Titer: A new technology for the quantitation of recombinant IgG in high-throughput cell supernatants by BMG with a fluorescence polarimetric plate reader

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