Question 1

How do you calculate concentration from ELISA absorbance?

Accepted Answer

Subtract the blank mean from every well, fit the standards to a 4PL curve OD = D + (A - D) / (1 + (C/C50)^B), then invert the curve for each sample OD to get its concentration and multiply by the dilution factor. Labreador does this automatically in the browser at labreador.com/elisa.

Question 2

Should I use 4PL or 5PL for an ELISA standard curve?

Accepted Answer

Use 4PL for symmetric sigmoidal standard curves — it is the regulatory default. Switch to 5PL only when residuals show systematic asymmetry near one asymptote; the extra asymmetry parameter costs a degree of freedom.

Question 3

Why can't the zero standard be used as the LLOQ?

Accepted Answer

4PL/5PL fitting works on log concentration and log(0) is undefined, so the zero standard cannot define a quantifiable level. It anchors the lower asymptote instead, and the LLOQ is the lowest non-zero standard that passes recovery and CV% acceptance.

Question 4

How do I know if my ELISA run is valid?

Accepted Answer

Check five things: curve R² and the quality tier, residual pattern, back-calculated standard recovery within 80-120%, replicate CV% below about 15%, and whether samples fall inside the LLOQ-ULOQ range. A small OD span between the top and bottom standard (for example under 0.5 OD) indicates a failed assay regardless of R².

ELISA Analysis & 4PL/5PL Standard Curve Fitting — Labreador

Import Plate Data