How to Decide ELISA Dilution Ratio

What is a Dilution Ratio?

Dilution ratio describes a simple dilution – a unit volume of solute (or sample) is combined with a desired unit volume of solvent (or diluent), to reach a desired total volume (Vsolute + Vsolvent = Total Vsolution)

Thus, a dilution ratio of 1:4 describes 1 part solute + 4 parts solvent = 5 parts total. The sum of both solute plus solvent equals total, final volume.

General Sample Dilution Procedure

• Review Literature

  Find expected concentration range in sample.

• Dilute Sample to Desired Volume

  Typically ~2x predicted analyte concentration in pg/mL

• Perform Pilot Test

  Serial dilution of sample into a full row on a multi-well plate (8 wells on a 96-well plate)

• Find Concentration that Results in Saturation of Signal (Sigmoidal Response)

  Data is typically plotted as optical density (or fluorescence) vs. concentration

• Quantitative Assay

  Compare experimental signals with a standard curve precisely prepared from known concentrations

Boster Bio offers an in-depth ELISA data analysis guide:

How to Prepare a Serial Dilution

Serial dilutions are a series of dilutions prepared using the same dilution factor – often performed to use as standard concentrations (or quantities) for quantification assays. Using the same dilution factor over a series of dilutions results in a logarithmic decrease in solute concentration across the series. The standard concentrations are used to create a calibration, or standard curve spanning the linearity range of an assay. The target concentration or amount may then be calculated from this curve.

Here’s what a tenfold serial dilution looks like

Dilution Ratio Calculator

Need a little help with your calculations?

Click on the image below for calculators for everything from simple dilutions to complex serial dilutions.

Is there a difference between a dilution ratio and a dilution factor?

Yes, but depending on the application the difference may be negligible.

Dilution factor describes the ratio of the initial volume of solute to the total volume of the diluted solution (Vsolute x Vsolvent = Total Vsolution). Dilution factors are of particular importance when performing serial dilutions.

Thus, a dilution factor of 1:4 describes 1 part solute dissolved in 4 parts solvent = 4 total parts. The product of solute times solvent equals total, final volume.

Put in Context

The ambiguity between the two terms derives from the convention of presenting both as a ratio. In other words, asking different people to perform a 1 to 2 dilution will get varying results over requesting to dilute a sample in half. Both are commonly expressed in writing and textbooks though as 1:2. In strictest terms, a dilution is expressed as parts over parts to differentiate, ½ in this example, or as an exponent.