Antibody dilution calculator: working dilutions, serial dilutions and ELISA endpoint titre

What it does

Antibodies are used at tiny amounts, so most of the work is arithmetic: how many microlitres of stock go into a blocking buffer, how to set up a dilution series to find the best concentration, and how to read a titre off an ELISA plate. This page does four of those jobs, and everything is worked out in your browser.

1. Working dilution. From the stock concentration on the vial (mg/mL), make a dilution written as 1:N or aim for a concentration in µg/mL. You get the stock and diluent volumes, the final concentration and the micrograms you use. When the stock volume is under 1 µL, the page suggests a 1:10 or 1:100 intermediate dilution so you never have to pipette a fraction of a microlitre.
2. Serial dilution. Pick the first dilution, the step (2-fold, 3-fold, 4, 5, 10-fold or any custom fold), the number of tubes and the volume each tube should end with. The table lists every tube’s dilution, concentration, diluent and transfer.
3. Titre. Paste dilutions with their OD readings (replicates side by side) and the blank wells. The cutoff is the blank mean plus k standard deviations (3 by default), k times the blank mean, or a fixed OD. The endpoint titre is the highest dilution whose mean OD is above the cutoff, read more precisely by drawing a straight line, on a log scale of dilution, to the next dilution below the cutoff.
4. Antibody for a run. For a number of blots (mL each) or plates (wells × µL), at a dilution and with a spare margin, you get the working volume, the stock volume, the micrograms and how many blots or plates one vial covers.

Here 1:N means 1 part in N parts in total, the way antibody data sheets use it: 1:1000 is 1 µL of antibody in 1 mL of finished dilution. Some labs write 1:N for 1 part plus N parts of diluent; for large N the difference is tiny, but for 1:2 or 1:5 it matters, so check which your protocol means.

How to use it

1. Type the stock concentration from the vial in mg/mL (leave it empty if the data sheet gives only a suggested dilution).
2. Choose A dilution 1:N or A concentration, then the final volume.
3. For a titration, set the first tube, the step and the number of tubes. Put the diluent in every tube first, then add the antibody to tube 1, mix, and carry the transfer volume down the row with a fresh tip each time.
4. After the plate is read, paste the dilutions and ODs, add the blank ODs and pick the cutoff rule.
5. Download the series or the titre table as CSV, or all four parts as an image, PDF or Word file.

Worked example

The example uses made-up numbers: a stock of 0.8 mg/mL diluted 1:2000 in 10 mL of blocking buffer. The page shows 5 µL of stock + 9.995 mL of diluent, a final concentration of 400 ng/mL, and 4 µg of antibody used.

The serial dilution starts at 1:100 and goes 2-fold over 8 tubes of 100 µL: tube 1 is 198 µL of diluent plus 2 µL of stock, and you move 100 µL each step, from 1:100 to 1:12,800.

The made-up ELISA has eight 3-fold dilutions from 1:100 to 1:218,700 in duplicate, and six blank wells with a mean of 0.053 and an SD of 0.0058. The cutoff (blank mean + 3 SD) is OD 0.071. The last dilution above it is 1:24,300 (mean 0.146); the next, 1:72,900, reads 0.066. A straight line between them on log dilution meets the cutoff at an endpoint titre of 1:68,382 (log₁₀ 4.83). Switch to k × blank mean with k = 2 and the cutoff rises to 0.107, giving 1:41,850: always say which rule you used.

For 6 blots of 10 mL each at 1:2000 with 10 % spare, you need 66 mL of working dilution, 33 µL of antibody (26.4 µg), and a 100 µL vial covers 18 blots, leaving 67 µL after this run.

Tips

• Pipette at least 1 to 2 µL. Below that, make an intermediate dilution, as the page suggests.
• Spin the vial briefly before opening so the antibody is not lost in the cap.
• Read the titre from the straight middle part of the curve’s tail: if every dilution is above the cutoff, the page tells you to dilute further; if the OD dips and rises again, check for a pipetting slip or a hook effect.
• The same cutoff rule must be used to compare titres between plates and days.

FAQ

Which cutoff should I use? Blank mean + 3 SD is common for endpoint titres; 2 × or 2.1 × the blank or negative-control mean is common too. A fixed OD (for example 0.1) is simple but depends on the reader and substrate time. Use the rule your lab or protocol states.

Should I use replicate means? Yes. The page averages the replicates on each line and shows their SD, and the titre uses the means.

Is the titre the same as an EC50? No. The endpoint titre is where the signal disappears into the background. For the dilution that gives half of the maximum signal, fit the curve with the dose-response tool.

Does anything get uploaded? No. Everything is worked out on this page and nothing is stored or sent.

Last updated 2026-11-09.