Proteomics: Resource Efficiency

In the era of personalized medicine, the proteomic analysis of an individual patient provides critical information in treating a disease. One of the popular methods in proteomic analysis is based on the availability of antibodies for target proteins. This article describes a method of monoclonal antibody screening that can lead to the expedited expansion of an antibody library that is crucial for protein analysis.

In a typical process of antibody screening, hybridoma cells and their supernatants are screened by a range of assays such as Enzyme- Linked Immunosorbent Assay (ELISA), cell immunostaining assay, and western blots.

One potential bottleneck in the process is the screening of hybridoma supernatants by ELISA and immunostaining assay in 96 or 384 multiwell plates. These assays are tedious to run and consume significant amounts of supernatants that lead to lower throughput.

Ideally, it is beneficial to perform these assays with less labor and hybridoma supernatant while producing more information content.

In order to overcome the bottleneck, DropArray technology was used. It offers benefits in performing an assay that requires a washing step in between the addition of reagents and samples – such as ELISA and cell-based assays. With this technology, the traditionally-used microtiter plate is replaced by a proprietary “wallless” flat plate, that is patterned with an array of hydrophilic wells surrounded by a hydrophobic coating.

Ease of Separation

The key technical innovation of the technology is the integration of a proprietary immiscible fluid with the hydrophobic coating of the plates. This creates “virtual walls” thereby separating the hydrophilic array into “wells”.

The conventional microtiter plates comprise of wells that are separated by physical walls, in which reagents and samples are added (one at a time) for a reaction to occur. The presence of these walls allows reagents to be added, mixed, incubated and washed without cross-contamination across the different wells.

However, it also creates the main bottleneck for conducting assays – impeding the washing process, and creating accuracy and sensitivity problems for both manual washing and automated washing.

By eliminating the physical walls, DropArray technology allows the washing process to be performed in a single automated whole plate wash, with no pipetting required. The platform thereby overcomes the limitations of microtiter platforms, while offering the same functionality and compatibility with existing microtiter plate readers.

It uses a 384-well plate that follows the same Society for Biomolecular Sciences (SBS) standard specification. The array of hydrophilic wells of 2mm diameter exposes a treated surface that is optimized for cell attachment and growing, and holds 2-3 μL of an assay volume. In the process, the entire plate is washed between incubations using a rinsing station.

The platform has been evaluated by the Experimental Therapeutics Center (ETC), one of the research institutes under A*STAR, for suitability to screen hybridoma supernatants using High Content Screening (HCS) methodologies. Through the “wall-less” miniaturization, the technology is able to reduce the volume of reagents and time that are spent on the assays, as well as the efforts for washing a plate.

Materials and Methods

The workflow that is employed for monoclonal antibody production and screening is outlined in Figure 1. To evaluate monoclonal antibodies that are generated against Apoptosis- Inducing Factor (AIF) and Adenomatous Polyposis Coli (APC), HCT116 wild-type (“negative” control cells) and HCT116-AIF knockdown were added to the 384-well plate wells (500 cells in 2 _l per well), allowed to attach overnight and fixed the next day.

The cells were stained with 2 _l of positive and negative control sera as well as hybridoma supernatants, followed by incubation with a secondary staining solution containing rabbit anti-mouse Alexa 488 antibodies and Hoechst 33342.

For comparison purposes, the same cells were plated out on 96-well plates and stained with the same positive and negative control sera as well as hybridoma supernatants followed by the secondary staining solution (conventional antibody screening). High resolution imaging was performed using the epifluorescence InCell1000 HCS reader and the confocal ImageXpress Ultra.

The plates are available in 48, 96 and 384 “well” formats. They expose flat hydrophilic glass surfaces that are surrounded by a hydrophobic polytetrafluoroethylene (PTFE) coating. The PTFE film in the presence of hydrophobic proprietary immiscible liquid Rinsing Oil, forms a virtual wall to prevent cross-contamination, even in the absence of physical walls.

In addition, the oil, which immerses an array of drops on the plate completely prevents evaporation from the drops. Glass surfaces that are exposed on DropArray plates have different treatments for a range of assays. For example, the glass surfaces expose protein-binding coatings for ELISA. The surfaces expose cell-friendly coatings for cell-based assays such as general tissue culture, collagen or polylysine coating.

Following the SBS standard in the specification and arrangement of exposed glass surfaces, the plates are compatible with existing fluorescent and cell imaging systems, as well as, dispensing systems. The plates require an automated washing station in order to perform the proprietary washing process accurately. The washing station receives a plate and performs a whole-plate wash by introducing a bulk-washing buffer at once. It shakes an entire plate with washing buffer and drains to leave minimal carry-over of washing buffer.

The plate can be washed repeatedly depending on the level of washing that is required by the assay. Often, one washing cycle in the washing station corresponds to one manual washing cycle in a microtiter plate.

Currently, two types of washing stations are available, LT for semi-automated operation by manual dispensing and HT for fully-automated operation to be integrated into an existing highthroughput screening system by simple plug-in (Figure 2).

In the screening, cell ELISA with HCT116 cell line against AIF and APC was performed by employing the wild type and AIF knockdown cells. The wild type cells were employed as negative control.

Table 1 shows the staining pattern of the AIF cell ELISA, where the conventional 96-well microtiter plates and the 384- well plates show consistent images. This shows that the exact same materials and procedures can be applied for the plates in cell seeding and culture as well as staining process, albeit with reduced reagent requirements.

Table 2 shows images of antibody staining in selected wells to illustrate the selection of monoclonal antibodies with desired staining patterns and characteristics. Hybridomas producing antibodies with the desired staining characteristics of selectivity and specificity were successfully identified.

The proof of concept studies with the 384 well plate suggests that it is suitable for carrying out monoclonal antibody screening. The major advantage is a reduction in the amounts of hybridoma supernatants that are required (Table 3). The technology also allows the selection of antibodies with desirable characteristics and staining patterns using HCS technology, which is scalable and automation-friendly.