Blocking assays
Binding is not blocking. An antibody can bind a receptor with picomolar affinity on a surface it shares with nothing important and leave the ligand interaction untouched. If the mechanism is blockade, the blocking assay is the real assay and binding is a prerequisite.
The biochemical version
Immobilize one partner, add the other in the presence of a titration of antibody, and measure how much complex forms. The readout can be ELISA, a sensor surface, or a homogeneous proximity format.
Two design choices decide whether the result means anything.
Which partner is immobilized. Blocking a soluble ligand from reaching an immobilized receptor is a different experiment from blocking a soluble receptor from reaching an immobilized ligand, and an antibody can look potent in one and weak in the other. Choose the orientation that matches the biology, and state it.
The ligand concentration. Blockade is competition, so the apparent potency of the blocker depends on how much ligand it is competing against. Run the ligand at a concentration near its own binding midpoint, not at saturation, or every blocker will look weak. Changing the ligand concentration between experiments is a reliable way to generate irreproducible potencies.
The cell version
Cells give the interaction its real context: receptor density, membrane environment, co-receptors, and the possibility that the antibody works by driving internalization rather than by steric blockade.
A standard format is labeled ligand binding to cells in the presence of the antibody, read by flow cytometry. A functional version measures the downstream signal instead, which is the more meaningful readout and adds the complication that signaling is amplified, so partial receptor occupancy can produce full signal suppression or none at all depending on receptor reserve.
Controls
- An isotype control at matched concentration and format.
- A known blocker, where one exists, to anchor the scale.
- A non-blocking binder to the same target, which is the control that proves the assay is measuring blockade rather than binding.
- A no-ligand well to define the floor, and a no-antibody well to define the top.
That third control is the one most often missing, and it is the one that distinguishes a blocking assay from an elaborate binding assay.
Reading it
Report the full inhibition curve, the maximum inhibition, and the conditions. Partial blockade that plateaus below complete inhibition is a real and common finding: it can mean two binding modes, a subpopulation of receptor in a different state, or an antibody that slows the interaction rather than preventing it.
Where blockade and binding potencies disagree by a lot, that gap is usually the most interesting number in the dataset, and it is worth chasing before it is averaged away.