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Biomedical Optics & Medical Imaging

A new method for therapeutic antibody screening

Fast screening of antibody-secreting cells by extraordinary optical transmission could be a new mechanism for drug discovery.
6 July 2010, SPIE Newsroom. DOI: 10.1117/2.1201006.002995

Therapeutic antibodies are among the most common drug candidates in pipelines, with many in clinical trials.1 Their mechanism of action involves specific binding to infectious agents, disease-related cells, or molecules such as viruses, cancer cells, or cytokines. This binding triggers different responses, depending on the disease. For instance, it might signal the immune system to attack a flagged cell or organism, or it might simply inhibit cellular growth. The concept of antibody therapy thus centers on strong specific interactions between antibodies and particular disease-related targets. The requirement for high specificity, high binding affinity, and consistent quality is best realized by monoclonal antibodies (MAbs), which are discovered by identifying a unique antibody-secreting cell (ASC) from a significantly larger population.2 The drug-discovery process for therapeutic antibodies requires screening thousands of individual ASCs to find a MAb target for a predetermined disease-related cell, molecule, or organism. Once an ASC that produces the antibody with the best binding affinity and specificity is identified, either the cell itself or the genes encoding the antibody must be retrieved and cloned. The overall process is time-consuming. For example, the identification of only 1 to 10 ASCs producing high-affinity MAbs typically takes 4 to 6 weeks.

Our current efforts are focused on developing an integrated microfluidic system for reversible cell trapping that should significantly shorten the ASC screening time. This device is coupled to an immunobiosensor unit based on the phenomenon of extraordinary optical transmission (EOT).

EOT occurs when much more light than expected passes through tiny (nanometric) holes in opaque metal films.3 In other words, the material becomes much more transparent than expected for certain colors. The concept of EOT-based biosensing is shown in Figure 1. White light from a microscope is transmitted through an array of 200nm-diameter nanoholes milled through a 100nm-thick gold film coated with a biological target. We used a peptide derived from an outer surface protein of influenza A called biotinylated hemagluttinin A (Bio-HA). The transmitted light was captured by an optical fiber and analyzed, yielding a spectrum that peaks at a certain wavelength. The transmitted spectrum changes when the 17/9 MAb in solution phase specifically binds to the immobilized target Bio-HA peptide, leading to a peak shift Δλ proportional to the mass of the bounded 17/9 MAb. Among the advantages of this technique are the label-free detection of biological molecules within normal transmission geometry, the small footprint of the arrays, and real-time binding detection.

Figure 1. The concept of extraordinary optical transmission-based biosensing. White light from a microscope is transmitted through an array of nanoholes milled through a gold (Au) film coated with a biological target. The transmitted light is captured by an optical fiber and analyzed to yield a spectrum that peaks at a certain wavelength (λ). A peak shift (Δλ) occurs when the transmitted spectrum changes as a solution binds to an immobilized target. (Figure is not to scale.)

Our antibody discovery platform involved trapping hybridoma ASCs producing 17/9 MAb within an array that is 200μm in diameter and 60–80μm deep and has microwells fabricated in a polymeric matrix (see Figure 2). The cells trapped inside the microwells survived for more than 24 hours when incubated in the appropriate conditions. In a separate gold-coated plate, arrays of nanoholes were milled to match the pattern of square microwells containing the ASCs (see Figure 3A). The Bio-HA peptide was then chemically conjugated to the arrays of nanoholes (see Figures 3B–D).

Figure 2. The antibody discovery platform involves trapping hybridoma antibody-secreting cells within an array of microwells fabricated in a polymeric matrix. The cells survived for more than 24 hours when incubated in the appropriate conditions.

Figure 3. (A) In a gold (Au)-coated plate, nanohole arrays are milled to match the pattern of square microwells containing antibody-secreting cells. (B-D) The biotinylated hemagluttinin A (HA) peptide is then chemically conjugated to the nanohole arrays.

Finally, the slide containing the arrays of nanoholes was aligned over the microwells containing the trapped ASCs. This exposed the gold surface bearing the Bio-HA peptide to the microwells, allowing the 17/9 MAbs secreted by the ASCs to diffuse and bind to the Bio-HA peptide. After a few hours of incubation, the gold plate containing the arrays was separated from the microwells and washed. The gold nanohole arrays were then optically probed, as shown in Figure 1. The affinity of the secreted 17/9 MAb to the Bio-HA peptide was inferred from the observed Δλ. Using our procedure, the ASCs that secreted the best MAbs could be easily identified and retrieved, allowing the genes encoding their antibody to be cloned.

Preliminary results and controls indicate that we are now able to detect antibodies from approximately 200 ASCs trapped within a single microwell. We are now working to optimize the trapping efficiency of the microwells through refinements to the microfluidics design. Attempts to improve the sensitivity of the EOT-based sensor are also underway. This methodology is a promising alternative to conventional ASC-screening techniques, and it could drastically streamline the drug discovery process for therapeutic antibodies.

Alexandre Brolo, Rajan Nirwan
Department of Chemistry
University of Victoria
Victoria, BC, Canada

Alexandre Brolo is an associate professor of chemistry. His main interests are in surface spectroscopy and in finding plasmonic-based solutions to biomedical problems.

Jamie Scott, Naveed Gulzar
Department of Molecular Biology and Biochemistry
Simon Fraser University
Burnaby, BC, Canada
Bonnie Gray, Sean Romanuik, Samantha Grist
School of Engineering Science
Simon Fraser University
Burnaby, BC, Canada
Karen Kavanagh, Donna Hohertz
Department of Physics
Simon Fraser University
Burnaby, BC, Canada
Reuven Gordon
Department of Electrical Engineering
University of Victoria
Victoria, BC, Canada