ELISA Analysis (4PL + Endpoint Titer)

What this is for: Quantitative ELISA analysis — blank subtraction, duplicate CV flags, 4PL standard curve with sample concentrations by inversion — plus an endpoint-titer sheet.

The formula: 4PL: y = D + (A − D) ÷ (1 + (x/C)^B); endpoint cutoff = mean of negatives + 3 × SD

How to use:

Enter the blank wells, then the standards as one row per standard: concentration, OD rep 1, OD rep 2.
Check the blank mean, the duplicate CV flags, and the linear fit in the first section.
Adjust the 4PL parameters A, D, C and B in the second section until the charted curve tracks the standards.
Enter samples as name, OD rep 1, OD rep 2, dilution factor, and read each concentration from the 4PL (and from the linear fit for comparison).
For endpoint titer, enter the negative control ODs and k, then each sample's dilution rows (dilution, OD 1, OD 2), and read the cutoff, endpoint titer and interpolated titer.

Quantitative ELISA — 4-Parameter Logistic (4PL)

Enter blank wells, standard concentrations with duplicate ODs, and sample ODs. Blank-corrected ODs and duplicate CVs (flagged above 20%) are automatic. Two fits are provided: (i) a linear fit on raw values (only valid in the near-linear range) and (ii) a 4PL whose parameters you adjust until the charted curve tracks the standards; sample concentrations come from inverting the 4PL. 4PL: y = D + (A−D)/(1+(x/C)^B); inverse: x = C × ((A−y)/(y−D))^(1/B). Note: the workbook does not do a true nonlinear least-squares fit — the 4PL parameters below are the adjustable ones from the sheet, exactly as in the workbook.

Blank wells (OD, space-separated)

Standards — one row per standard: concentration, OD rep 1, OD rep 2

Blank mean (OD)

Linear fit — slope

Linear fit — intercept

Linear fit — R-squared

(ii) 4PL parameters — adjust until the curve fits the standards

A — min asymptote (OD)

D — max asymptote (OD)

C — EC50 (conc. units)

B — slope factor

Samples — one row per sample: name, OD rep 1, OD rep 2, dilution factor

Sample 1 — conc. from 4PL

Sample 2 — conc. from 4PL

Sample 3 — conc. from 4PL

Sample 1 — conc. from linear fit

Sample 2 — conc. from linear fit

Sample 3 — conc. from linear fit

ELISA Endpoint Titer (serial dilution)

Cutoff = mean OD of negative controls + k × SD (k editable, default 3). Titer = reciprocal of the highest dilution whose mean OD is still ≥ cutoff. The interpolated titer uses proportional distance (Reed–Muench style) between the dilutions bracketing the cutoff.

Negative control ODs (space-separated)

Cutoff multiplier k

Sample 1 — one row per dilution: dilution, OD 1, OD 2

Sample 2

Sample 3

CUTOFF (OD)

Sample 1 — endpoint titer

Sample 1 — interpolated titer

Sample 2 — endpoint titer

Sample 2 — interpolated titer

Sample 3 — endpoint titer

Sample 3 — interpolated titer

Prefer to work offline or keep a copy? The download link is right below.
Download the ELISA Analysis (4PL + Endpoint Titer) workbook (.xlsx)

Notes & limits (from the workbook): Blank-corrected ODs and duplicate CVs (flagged above 20%) are automatic. The linear fit is only valid in the near-linear range. The 4PL parameters are adjusted by hand until the charted curve tracks the standards — the workbook does not perform a true nonlinear least-squares 4PL fit; sample concentrations come from inverting the 4PL with the parameters you set: x = C × ((A−y)/(y−D))^(1/B), multiplied by the dilution factor. Endpoint titer: cutoff = mean OD of negative controls + k × SD; titer = reciprocal of the highest dilution whose mean OD is still ≥ cutoff; the interpolated titer uses proportional distance (Reed–Muench style) between the dilutions bracketing the cutoff, assuming a constant 2-fold dilution step (factor LOG10(2)), exactly as the sheet's formulas do.