esm.doi.bio/esm33/out2

Response: Experimental Protocol to Identify Antibodies Binding to IDH-Wildtype Glioblastoma Using Phage Display and Ginkgo Bioworks' Platform

Introduction

The objective of this protocol is to develop a high-throughput experimental workflow to identify monoclonal antibodies that specifically bind to IDH-wildtype glioblastoma (GBM) cells using phage display technology, integrated with Ginkgo Bioworks' synthetic biology and automation platforms. Phage display is a powerful method for discovering peptides or antibody fragments with high affinity and specificity to target molecules or cells. By coupling phage display with Ginkgo Bioworks' capabilities, we aim to accelerate the discovery process through automation, advanced screening methods, and bioinformatic analysis.

Materials and Reagents

1. Cell Lines and Culture:

• IDH-wildtype glioblastoma cell lines (e.g., U87MG, LN-229)
• IDH-mutant glioblastoma cell lines for negative selection (e.g., BT142)
• Normal human astrocyte cell lines for counter-selection
• Cell culture media and supplements appropriate for each cell line
• Cell culture flasks, plates, and related consumables

1. Phage Display Libraries:

• Human scFv (single-chain variable fragment) phage display library
• Phagemid vector system (e.g., pComb3XSS)
• Helper phage (e.g., M13KO7)

1. Reagents and Buffers:

• Phosphate-buffered saline (PBS)
• Blocking buffer (e.g., PBS with 5% BSA)
• Washing buffer (e.g., PBS with 0.1% Tween-20)
• Elution buffer (e.g., 0.2 M glycine-HCl, pH 2.2)
• Neutralization buffer (e.g., 1 M Tris-HCl, pH 9.1)

1. Bacterial Strains and Growth Media:

• Escherichia coli TG1 or ER2738 cells for phage amplification
• LB broth and agar plates with appropriate antibiotics

1. Equipment:

• Ginkgo Bioworks’ automated workstations and liquid handlers
• Incubators and shakers
• Centrifuges
• Spectrophotometer/Nanodrop
• ELISA plate reader
• Flow cytometer
• PCR thermocycler
• Sequencing platform (e.g., Illumina MiSeq)

1. Additional Consumables:

• ELISA plates
• Flow cytometry tubes
• Pipettes and tips
• Sterile filters and tubes

Experimental Procedure

1. Preparation of Target and Control Cells

1.1. Cell Culture and Maintenance

• Thaw and culture IDH-wildtype GBM cells following standard cell culture protocols.
• Similarly, culture IDH-mutant GBM cells and normal human astrocytes for negative selection and counter-selection.
• Ensure cells are healthy and in the logarithmic growth phase before experiments.

1.2. Cell Harvesting

• Detach cells using non-enzymatic cell dissociation buffer to preserve surface antigens.
• Wash cells three times with cold PBS to remove residual media and enzymes.
• Count cells and adjust concentration to 1 × 10^7 cells/mL in cold PBS with 1% BSA.

2. Phage Library Preparation

2.1. Amplification of Phage Display Library

• Infect E. coli cells with the phage display library following the library provider's instructions.
• Amplify the phage library by super-infection with helper phage (e.g., M13KO7).
• Purify phage particles from the culture supernatant using PEG/NaCl precipitation.
• Determine phage titer by plating serial dilutions on LB agar plates with antibiotics.

2.2. Quality Control

• Confirm phage display library diversity via sequencing a subset of clones.
• Ensure that the phage preparation is free from bacterial contamination.

3. Biopanning (Selection Process)

3.1. Positive Selection with IDH-Wildtype GBM Cells

• Incubate the phage library with IDH-wildtype GBM cells (1 × 10^7 cells) in PBS with 1% BSA for 1 hour at 4°C with gentle rotation.
• Phage particles displaying antibodies will bind to cell surface antigens.

3.2. Washing Steps

• Separate unbound phage by centrifugation at 300 × g for 5 minutes at 4°C.
• Wash the cell-phage complex extensively with cold washing buffer to remove non-specifically bound phage (e.g., 10 washes with PBS + 0.1% Tween-20).

3.3. Elution of Bound Phage

• Elute bound phage by resuspending the cells in elution buffer and incubate for 10 minutes at room temperature.
• Neutralize the eluted phage with neutralization buffer.

3.4. Amplification of Eluted Phage

• Infect fresh E. coli cells with the eluted phage.
• Amplify the phage as described in step 2.1.
• Purify amplified phage for the next round of selection.

3.5. Negative Selection with Control Cells

• To increase specificity, perform negative selection by incubating the amplified phage with IDH-mutant GBM cells and normal astrocytes.
• Collect unbound phage, which are enriched for those not binding to control cells.
• Use these unbound phage for the next round of positive selection.

3.6. Repeat Biopanning for Multiple Rounds

• Perform 3–5