ELISA (Enzyme Linked Immuno-Sorbent Assay) is a highly sensitive plate-based assay for in vitro quantification of soluble analytes in liquid samples. ELISA can be used to detect and/or quantify any analyte that can be bound by an antibody, such as cytokines, CD antigens, apoptosis markers, hormones, metabolites, growth factors, and more.
How Does ELISA Work Again?
You can find an overview of the ELISA principles and the various assay formats available in our first ELISA 101 post, but for now here’s a quick recap on how a typical assay works:
Let’s say you want to detect a certain protein in a blood sample:
- Your target protein or an antibody that recognises it is immobilised directly to an assay plate, or indirectly to a capture antibody that has been attached to the assay plate.
- A primary antibody recognises an epitope on your target protein within the blood sample.
- Depending on the assay format, a secondary antibody enzyme conjugate binds the primary antibody or the target protein itself, and reacts with a substrate to produce a signal that is detectable on a spectrophotometer, fluorometer or luminometer.
Where Do ELISA Standards Come In?
ELISAs may be quantitative or qualitative. A qualitative assay gives a yes/no readout about whether or not the target analyte is present in the sample, within a certain assay-dependent detection range. Qualitative assays don’t require the presence of standards and are typically read by comparing unknown samples to a sample-free well of the assay plate or an unrelated positive control sample that contains a fixed concentration of the target analyte.
Standards come into the picture when the assay goal is quantification. Quantitative ELISAs are much more powerful and commonplace that the yes/no assays, providing information about the amount and concentration of a target analyte present in a given sample, which allows comparative experiments, such as diseased vs. healthy samples, as well as studies of how certain changes affect analyte levels, e.g., dietary changes, pH fluctuations, presence of medications, etc.
Quantitative ELISAs rely on the presence of a standard curve that is included in every ELISA run. A standard curve is usually prepared by assaying a serial dilution of a standard preparation of the target analyte, of which the starting concentration is known (see example in Figure 1).
Then, when the ELISA is run, the detectable signal from every sample is compared to the signals obtained from the standard curve. For example, if a test sample yields an optical density (OD) of 0.5, the concentration of the standard dilution that yielded OD = 0.5 must match the target analyte concentration in the test sample.
Use The Right Standards!
Quantitative ELISAs are a very powerful tool within research and clinical applications, but only when they are based on high-quality reliable standards. Most commercial ELISA kits available today include standard preparations from which a standard curve can be prepared. In these cases, the manufacturer will also provide guidelines for dilution and handling of the standards to help you obtain the best possible results.
However, if you want to assay an analyte for which there is no kit available, e.g., a newly discovered hormone or rare metabolite, you will need to develop your own assay and procure your own standards. The next sections cover the most important consideration to bear in mind when preparing ELISA standards.
Standard Type and Source
A purified analyte, e.g., a pure aliquot of your target protein, should be used to prepare the standard curve if possible. You may be able to purchase this from a vendor who can demonstrate proof of purity. Commercial standards are generally provided as lyophilised aliquots (powder) with lot-specific instructions for reconstitution.
If your target analyte is not available commercially, you can use either try to purify it yourself, or a recombinant version, and use HPLC or another chromatographic method to measure its concentration. Since ‘home-made’ standards are unlikely to be completely pure, it is not advisable to use all-in-one protein quantification methods such as Bradford assay to determine their concentration.
Standard Curve Range
In commercial assays, the standard curve will often range from 0 to 1,000 pg/ml although this is not always the case. The standard curve range should reflect the anticipated concentration range of the target analyte in typical samples. If you are measuring a very abundant analyte, you may need to dilute your sample so that it ‘fits’ into the range of your standard curve.
If you are preparing your own standards and you already have insight into the abundance of your target analyte in your samples, you can use this information to define an appropriate standard curve range. For consistently low-abundance analytes, e.g., certain hormones, the standard curve range may be set lower while it may be higher for high-abundance analytes.
Regardless of whether you use commercial or home-made ELISAs, you should always take certain precautions to help promote accuracy and high quality data. For example:
- Use clean tubes to prepare reconstituted standards and standard dilutions.
- Ensure that pipettes are calibrated regularly.
- Use fresh pipette tips after each dilution.
- Aim to use fresh standards for every assay and avoid freeze-thaw cycles.
- Repeat measurements in duplicate or triplicate for accuracy and to ensure that you can still use your data should something go wrong with one measurement.
- Construct a new standard curve for every plate and each experiment.
Reach Out to Us!
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