Example data table
| Type | Concentration (ng/mL) | OD | Notes |
|---|---|---|---|
| Standard | 0.5 | 0.12 | Low end |
| Standard | 1 | 0.20 | |
| Standard | 2 | 0.33 | |
| Standard | 4 | 0.55 | Mid range |
| Standard | 8 | 0.86 | Upper end |
| Sample | — | 0.42 | Compute from curve, then adjust DF |
Formula used
- Dilution: C₂ = C₁ × (V₁ / V₂). Dilution factor DF = V₂ / V₁.
- Mass/Volume: C = mass / volume with unit normalization to ng/mL.
- Standard curve (linear): Fit OD = m×C + b, then C = (OD − b)/m.
- Standard curve (log-linear): Fit OD = m×log10(C) + b, then C = 10^((OD − b)/m).
- Fit quality: R² = 1 − SSres/SStot using observed and predicted ODs.
How to use this calculator
- Select a calculation method that matches your data source.
- Enter values and choose units carefully for each field.
- For assay data, enter at least two standards and one sample OD.
- Optionally include blank OD and a sample dilution factor.
- Press Calculate to view results above the form.
- Use the export buttons to download a CSV or PDF summary.
Sample matrices and reporting units
Antigen concentration is commonly reported as mass per volume (ng/mL, µg/mL) so results stay comparable across batches and instruments. When preparing a stock, record weighed mass, final volume, and the conversion used. If molecular weight is known, you may also report nM for binding and stoichiometry work. For serum, plasma, or lysate, document extraction yield and any pre‑dilution so reported values represent the original sample.
Dilution planning for linear range
Most immunoassays have a linear window where signal changes proportionally with concentration. Choose a dilution factor (DF) that places expected levels near the mid‑range of standards, not at detection limits. Single dilution: DF = Vfinal/Valiquot. Serial dilution: multiply step DFs for the overall DF, then back‑calculate. Use pipettes within their optimal range and keep steps simple (1:2, 1:5, 1:10) to reduce cumulative error.
Standard curves and regression checks
Standards map concentration to absorbance (OD). Linear fit: OD = mC + b. Log‑linear: OD = m·log10(C) + b for wider ranges. Apply blank subtraction and average replicates before prediction. Look for high R² (often ≥0.98) and non‑systematic residuals. Confirm a positive slope and check residuals near the LLOQ and ULOQ for visible bias. If sample OD is outside the standard range, re‑dilute and rerun rather than extrapolate.
Recovery, precision, and controls
Run duplicates or triplicates and track CV% = SD/mean × 100; targets are often ≤10–15% for standards and ≤20% for unknowns. Spike‑and‑recovery checks matrix effects; 80–120% recovery is common, but follow your method’s acceptance limits. Include blanks and negative controls to confirm background and specificity. Add a mid‑level control on every plate and trend it over time to spot drift. Define LLOQ/ULOQ so out‑of‑range results are flagged early.
Documentation and reproducibility
Save concentrations with units, curve slope/intercept, dilution factors, and timestamps. Keep the standards table and blank value to support audits and troubleshooting. When reporting, state whether results are dilution‑corrected and whether they reflect native sample concentration. Use consistent sample IDs and file names so CSV/PDF exports trace back to plate records. Store raw ODs separately from calculated outputs to preserve an immutable record. Good documentation reduces repeats and improves reproducibility.
FAQs
1) What does the dilution factor mean?
It is the ratio of final to transferred volume. The calculator divides the stock concentration by this factor, or multiplies curve-derived values by your sample dilution factor to report native concentration.
2) When should I choose a log-linear standard curve?
Use it when standards span multiple orders of magnitude and the response is approximately linear versus log10(concentration). If your assay is truly sigmoidal, restrict the fit to the near-linear region or use a 4PL method externally.
3) Why is my calculated concentration negative?
Negative values usually come from blank over-correction, an intercept larger than the sample OD, or standards entered in the wrong order. Recheck blanks, confirm a positive slope, and ensure the sample OD lies within the standards range.
4) How are concentration units converted?
All calculations are normalized internally to ng/mL, then converted to your selected output unit. This keeps dilution, mass/volume, and curve modes consistent even when you mix units across inputs.
5) How can I improve curve fit quality?
Use fresh standards, mix thoroughly, avoid edge effects, and run replicates. Remove obvious outliers only with documented rationale. Keep ODs within the reader’s linear range, and ensure blank subtraction is applied consistently.
6) Do the CSV and PDF exports include my inputs?
Yes. Exports include the selected method, key inputs, calculated concentration, units, dilution factors, and timestamps. For curve mode, they also include slope, intercept, R², and a standards report when available.