Choosing the right ELISA kit
Loading Control Antibody Guide
Knockout-validated primary antibodies
Secondary antibodies resources
Alexa Fluor secondary antibodies
Biotinylated secondary antibodies
Gold-labelled secondary antibodies
How to prepare Phosphate Buffered Saline (PBS)
How to design a flow cytometry panel
Enhancing Detection of Low-Abundance Proteins
9 tips for detecting phosphorylation events using a Western Blot
Western Blotting with Tissue Lysates
Western blot membrane stripping protocol
Immunohistochemistry and Immunocytochemistry
Chromogenic and Fluorescent detection
Preparing paraffin-embedded and frozen samples for Immunohistochemistry
Competitive ELISA assay protocol
Measuring analyte concentration using serial dilutions and standard curve
Understanding ELISA results, controls and optimization:
Quantitative ELISA results:
One way to interpret the data from this assay is to compare it to a standard curve. This curve is essentially a representation of serial dilution of a purified antigen, and placing the two plots side to side allows the precise calculation of antigen concentrations across different samples.
Qualitative ELISA results:
Aside from the more mathematical outcomes of ELISA results, the assay in itself does at least one basic thing, which is determine whether a specific antigen is present in a given sample. This yes or no method is performed by comparing the sample of interest to either a blank sample or an unrelated antigen.
Semi-quantitative ELISA results:
Lastly, this assay can be efficiently used to compare the levels of antigen across several samples by using the varying signal intensity, which is directly related to the antigen concentration.
ELISA standard curve:
Usually the ELISA assay is graphed with log concentration against the optical density, so a sigmoidal curve can be produced. This curve later allows measuring the concentration of unknown samples by comparing the two graphs.
One way to do that is by using a software, that can fit the curve directly on the graph. This curve fitting software is usually found on the assay’s plate readers.
ELISA and sensitivity:
This assay is one of the most sensitive immunoassays, with 0.01 ng 0.1 ng detection range. This range depends on the antibody-antigen reaction and its characteristics alongside using substrates, that can improve results. Such substrates have enhanced fluorescent or chemiluminescent signal.
Using indirect detection can increase the signal levels, which lead to the assay being more sensitive. The drawback to this method is that it also increases the background, leading to less specific signals.
Using controls in this assay is as important as having valid standards. Having both positive and negative controls can ensure that the experiment is accurate and running smoothly.
This type of control can be either a natural or a recombinant sample, that will be detected in the assay. Positive controls help with verifying true negative samples. One form of this control is the standard curve. It allows fast and simple comparison between the obtained results and a known sample.
Opposite to positive controls, negative ones can verify that no false positives or non-specific binding are obtained during the experiment.
If the experiment has sophisticated sample matrices, knowing that the matrix is clear from anything that can possibly interfere with the assay is important. Spiking recombinant or natural protein in the assay and verifying the readout is recommended.
Optimizing the ELISA assay:
Achieving consistent conditions ensures that the results can be easily reproduced with high accuracy. Having a checklist during the first steps of the assay helps to maintain the parameters in range, like standard temperature, humidity and buffers.
Calibrating the pipettors on a regular basis and closely observing the amount of liquid in the clear tips helps with maintaining consistency. The previous practices are quite important when using multi-channel pipets, as the tips at the ends can prove to be more difficult to attach to the pipettor.