Quick, accurate and high-throughput detection of IgG and Fc segment-containing derivatives using fluorescence polarization method

Dr. Carolanne Doherty

Valitacell, NIBRT, Fosters Avenue, Blackrock, Dublin, Ireland

BMG multifunctional microplate reader can be applied to the new technology Valita?TITER for quantification of IgG. It uses fluorescence polarization method to directly detect IgG in cell culture supernatant.

Microplate reader preset detection programs and data analysis templates increase research speed

Introduction

Accurate, rapid and high-throughput detection of IgG for most therapeutics development and production of antibodies. Monoclonal antibodies are making their mark in the field of biopharmaceuticals, and large numbers of samples are waiting to be screened for further development. Here we introduce a new screening technology for direct quantification of IgG in cell culture supernatant: Valita?TITER.

Existing technologies, including protein A high-performance liquid chromatography (HPLC protein A), ELISA technology, protein A surface interference method, and HTRF (homogeneous time-resolved fluorescence) all have obvious shortcomings, including price Expensive, labor intensive and time consuming.

The Valita?TITER method is a new, very accurate and low-cost method for detecting IgG, with a detection range of 2.5-80mg/L. Valita?TITER uses fluorescence polarization technology to detect the binding of the Fc segment of IgG to protein G (Figure 1). The analysis is performed in a 96-well plate, which is simple, high-throughput, fast, and can be automated. Assays can be performed on very small amounts of cell culture medium and without tedious preparation.

In this application note, we introduce the protocol for quantitative detection of IgG using Valita? TITER kit on PHERAstar?, POLARstar? Omega and CLARIOstar? multi-function microplate readers.

Fluorescence polarization can effectively analyze changes in molecular size. An "immobile" fluorophore will emit fluorescence in the same polarization direction as the excitation light when excited by polarized light. However, the fluorescence polarization direction of the rotating fluorophore will change to a certain extent compared to the polarization direction of the excitation light. Small molecules rotate faster than large molecules in solution. Therefore, changes in the size of molecules connected to fluorescent groups can be measured by the degree of fluorescence depolarization. Therefore, when fluorescently labeled protein G is not bound to other molecules, it rotates faster and depolarizes more, while the opposite occurs (5 times larger) when bound to IgG (Figure 2). This change in polarization is used to detect IgG in solution. Fluorescence polarization (FP) is performed by illuminating a solution with excitation light of parallel polarization direction and measuring the emitted fluorescence in parallel and perpendicular directions. FP is the normalized difference between these two polarized fluorescences, usually expressed in mP.

Materials and methods

Valita?TITER analysis kit

Valita?TITER APP software (provided in the kit)

IgG standard Products (IgG obtained from human serum)

PHERAstar, POLARstar Omega and CLARIOstar multi-function microplate readers (BMG LABTECH)

Experimental process

Sample preparation Follow the appropriate requirements in the kit instructions. The standard curve is drawn using the method that each relative fluorescence value corresponds to an IgG standard. Add 60 μl of reconstitution buffer and 60 μl of standard or sample to each well of the Valta?TITER microplate. Mix well and incubate in the dark for 30 minutes.

Then use the preset detection program for reading in the POLARstar Omega, PHERAstar or CLARIOstar multifunctional microplate reader (BMG LABTECH Company) (see the table below for parameter settings). Convert the obtained Raw Data into a .csv format file in MARS analysis software, and then use Valita? APP software for analysis.

Instrument Settings

Results and Discussion

The standard curve (2.5-80mg/L) in Figure 3 is obtained from BMG LABTECH Company with fluorescence polarization detection function Plotted using Valita?TITER kit on multifunctional microplate readers (POLARstar Omega, CLARIOstar and PHERAstar). The results obtained by all microplate readers are highly stable.

Finally, we compared the results of IgG quantification using Valita?TITER with the results of conventional HPLC using samples cultured under different conditions. Figure 4 shows the correlation of the measurement results of the two methods, showing that both quantitative methods have good accuracy, and Valita?TITER analysis is faster because it does not require tedious sample preparation processes.

Conclusion

Rapid IgG concentration determination of mixed samples is very important in the development and production of such biopharmaceuticals. Here we introduce a new technology for rapid and simple high-throughput determination of IgG and Fc segment derivatives based on fluorescence polarization method - Valita?TITER analysis method. The Valita?TITER assay can directly quantify IgG in the sample to be tested without the need for cumbersome sample preparation or purification processes. CLARIOstar, PHERAstar and POLARstar Omega achieve excellent performance when used in Valita?TITER analysis. Compared with other IgG quantitative methods, Valita?TITER has the characteristics of high-throughput, simple, accurate and fast.