(Theoretical Concepts Only — Fully Detailed)
Phage display is a selection system where billions of phages, each displaying a different antibody variant, are exposed to a target (protein or cell). Only the phages whose antibodies bind survive the selection.
Low-abundance, biologically valuable markers (like cancer-specific receptors) may be lost during repeated rounds because they’re rare and weakly represented.
When the goal is biomarker discovery, one-round selection is theoretically superior — if you can control background noise.
Some phages bind not because their antibody recognizes a target, but because:
In a 1-round selection, most recovered phages are these background binders.
Develop helper phage systems that reduce background. (Details skipped in lecture, but the key idea: make phage viability dependent on real antigen binding.)
Keratinocytes are epidermal skin cells with differentiation stages (stem-like → differentiated → dead keratin layer).
The goal was to discover cell-type-specific surface markers on living keratinocytes.
When the one-round selection was done:
This reveals an important theoretical principle:
Even cells of similar lineage or tissue origin can have drastically different surface antigen repertoires.
One antibody (B3) pulled down plectin — a cytoskeletal protein every cell expresses.
Yet it bound specifically to keratinocytes. Why?
Alternative splicing
This illustrates the principle:
Surface diversity arises from:
A second selection (young vs old endothelial cells) revealed something shocking:
Expected location: inside the cell, as an intermediate filament protein.
But the antibody stained the cell surface of old cells.
Theoretical implications:
Proteins canonically described as “intracellular” may:
Extracellular vimentin had:
Extracellular vimentin affects:
The theoretical meaning:
Real tissues are not uniform. Cells vary due to:
A selection performed on bulk cultures misses rare but crucial subpopulations.
Two technologies were developed to isolate phages bound to one specific cell:
📌 Precision-targeted molecular discovery 📌 Identify markers of rare cancers, fetal cells, or specialized brain cells 📌 No need for whole-cell purification
Pericytes wrap capillaries; they support vessel stability and blood-brain-barrier integrity.
A single-cell selection yielded:
Cell-type specificity is shaped by:
C3 recognized fibronectin, which is normally ubiquitous.
But pericytes seem to have:
This reinforces the theme:
Theoretical insight from tube formation + migration assays:
Antibodies are more than markers — they can:
Binding to the same protein does not imply the same biological effect. The epitope targeted determines the downstream signaling outcome.
Even when those antigens are “housekeeping” proteins (due to isoforms or PTMs).
Even within one tissue or cell type.
Vimentin and fibronectin illustrate this paradigm shift.
They don’t just label cells; they modulate signaling, migration, angiogenesis.
Useful for: