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Posted on Thu, Jun 03, 2021

ELISA standard curve

Background on ELISA

ELISA stands for enzyme-linked immunosorbant assay. To help you understand the usefulness of this technique we’ll start with a brief description of how it works. The first step is to immobilize a probe molecule to your ELISA plate, these plates are usually purchased through a vendor. A probe is a molecule that binds to a target (analyte) you are hoping to capture from a sample mixture. The probe binds to the bottom of the wells through passive adsorption to the plastic. You next add your sample and allow time for your target of interest to bind to the probe in the ELISA plate wells. Lastly, a secondary antibody is added to visualize where binding has occurred through a colorimetric or fluorescent signal. There are various versions of ELISA that modify the assay for what kind of molecule you are trying to detect in a sample and whether a primary detection antibody is available for your assay for example. You can discover which ELISA is best for you in these articles about direct and indirect ELISA, sandwich ELISA, competitive ELISA. 

Free PDF guide:   "Validation of Magnetic Bead Separation Processes" 

Applications of ELISA technology

ELISA is considered the gold standard for detection of molecules from samples. Patient samples can be urine, blood, or saliva. ELISA is used around the world in clinical settings to detect many types of diseases to help diagnose, reduce spread, control symptoms and cure. For example ELISA is used to detect makers of hepatitis B and C in serum or SARS-CoV-2 antibodies from blood. 

What information can ELISA give you?

ELISA data can be used to gain qualitative information about the binding/presence of target molecules. This can be binary information, meaning you can answer whether or not a molecule is present (yes or no). You can also look at relative binding, how different samples have more or less binding than each other. ELISA can also be used to get quantitative data, meaning you can get the concentration of an analyte in a sample. 

How to get quantitative data using an ELISA standard curve

If you are interested in knowing the concentration of a certain analyte in your patient samples, you can do an ELISA standard curve. Every time you do the ELISA technique for your samples (which you do in triplicate), you also do an ELISA for known concentrations of that analyte (also in triplicate). You use several dilutions of that analyte, and plot the OD for each dilution. A binding curve is made from data points of binding from very low to high concentrations of analyte, and it is fit using a 4 or 5 parameter logistic model which describes 1:1 binding interactions on a surface. You want to use a wide range of analyte concentrations because you ensure you have found the linear part of the binding curve. The linear part of this curve will give you a relationship between OD value and what concentration of analyte you have in an unknown sample. 

Final thoughts on ELISA standard curve

The ELISA standard curve is a powerful tool that can give quantitative answers to better understand disease states. Aside from knowing that an antibody or antigen exists, the concentration of these molecules can give more in depth information about disease progression in clinical labs and help research laboratories understand the concentration changes of molecules for various diseases. The separation process is a crucial part of this technique. Modern ELISA can be done using magnetic beads which can offer a different binding method for your target of interest. Magnetic beads are a simple and efficient method of isolation to investigate the kinds of binding events mentioned in this article. 

biomagnetic separation processes

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