Biomarker ELISA: Antibody Binding Kinetics & Detection Limit

Assay Reagents

Capture antibody density 70

Antibody affinity K_d (nM) 1.00

Nonspecific background 4%

Incubation

Incubation time (min) 45

Assay Readout

Top-well signal

Signal:background

Apparent EC50

True K_d

Limit of detection

Low signal (unreacted) High signal (developed)

How it works

Each of the 8 wells holds the same capture antibody but a different biomarker (antigen) concentration [Ag] — a two-fold dilution series, plus a zero-antigen blank. Binding follows pseudo-first-order kinetics (antibody in excess):

k_obs = k_on·[Ag] + k_off,   K_d = k_off / k_on
bound(t)/B_max = [Ag]/([Ag]+K_d) · (1 − e^(−k_obs·t))

At long incubation times this converges to the Langmuir isotherm bound = B_max·[Ag]/([Ag]+K_d) — the classic dose-response curve. The developed signal also carries a fixed nonspecific background that scales with capture density, exactly like real ELISA plate noise.

The panel reports two numbers you would normally only get by running the assay: the apparent EC50 — the concentration giving half-maximal signal, found by bisection on the simulated curve — and the limit of detection, the lowest concentration whose signal clears the blank by 3 standard deviations of assay noise. Because binding hasn't reached equilibrium at short incubation times, the apparent EC50 sits above the true K_d — drag the incubation slider down and watch the gap open, then let it run to equilibrium and watch EC50 converge back onto K_d.

Bound complex Free antibody site

⚠ Couldn't load the 3D engineThree.js failed to load from the CDN. Check your connection and reload.