Quantitation of IgG and His-tagged proteins using the Carterra LSA

Key Highlights

• Quantitate 1,152 samples in a single run using ready-to-use capture surfaces

• Broad dynamic range across three orders of magnitude

• Single digit ng/ml limit of detection

• Requires as little as a few microliters of supernatant

Introduction

High-throughput quantitation of protein samples is desirable, particularly in early-stage antibody discovery workflows and cell line development. The Carterra LSA offers an HT-SPR protein quantitation workflow which can be performed as a standalone experiment or in conjunction with capture kinetics to obtain both detailed quantitation and kinetics. This workflow has an uninterrupted throughput of 1,152 samples per experiment, owing the sample plate capacity of up to three 384-well plates. As a capture-based approach, it works equally well for purified and crude protein samples, such as cell culture supernatants.

The Quant application requires use of a capture surface, and several ready-to-use sensor surfaces are available from Carterra, including Protein A, Proteins AG, and NiNTA. For these surfaces, capture sensitivities range from 10s ng/mL to 10’s of μg/mL depending on molecule type and construct size. Custom capture surfaces can also be created for characterization of protein concentration based on other affinity tags or interactions (anti-V5, anti-Fab, etc.). The Quant feature in the Kinetics software utilizes a set of standard dilutions to be included for each set of 96-sample array captures. The choice of standard is important, as the similarity and behavior of the standard relative to the unknowns determine the accuracy of the measurement. A good standard should meet several criteria, including being as identical as possible in terms of sequence of the affinity tag, species, isotype (Fc capture) and molecular weight as the unknown proteins. Additionally, standard dilutions should extend above and below the anticipated unknown concentrations.

Experimental parameters, such as capture contact time, also depend on the expected unknown concentration range. If unknown samples are expected to be in the sub- to mid-μg/mL range, shorter contact times (5 min) will be sufficient, and quantitation can be performed using capture levels or initial capture slopes. For low-to-mid ng/mL samples, longer contact times of 20-40 minutes are suggested to allow more time for association and enhance detection sensitivity. The unique bidirectional injection fluidics of the LSA allow for extended injection times without additional consumption of sample in these cases where enhanced sensitivity is required.

Posted by Maria McGresham