General ELISA Procedure
ELISAs are used to measure unknown protein concentrations in samples. In the assay, the protein is fixed to a solid surface, such as a 96-well plate. A primary antibody specific to the protein is added to the wells, where secondary antibodies that recognize the primary antibody can be added in specific models. Bound to the antibodies are reporter enzymes. Added substrates activate these enzymes, resulting in colorimetric changes that are measured on a spectrophotometer.
Because the samples have unknown protein concentrations, standards must be added to each experiment to generate a reference curve. The unknown sample concentrations are determined by plotting their measured values along this curve.
Types of Common ELISAs
There are four major types of ELISAs. These include:
- Direct
- Indirect
- Sandwich
- Competitive
The direct ELISA was the original form of the assay. The protein of interest is directly added to the plate, and enzyme-linked antibodies are subsequently added. Generally, this method is not very sensitive and is best for high molecular weight proteins.
Meanwhile, indirect ELISAs use two types of antibodies. After the proteins attach to the plate, the primary antibody binds to the proteins. Then, enzyme-linked secondary antibodies bind to the primary antibody. This method is more sensitive and is best used for endocrinology.
In sandwich ELISAs, the primary antibody pre-coats the plate, and the protein is added. The enzyme-linked secondary antibody follows this, thus “sandwiching” the protein between two antibodies. As a highly sensitive model, this is the most common type of ELISAs performed.
In competitive ELISAs, antibodies are again pre-attached to the plate. However, in this model, the protein and enzyme-linked antibodies are added simultaneously, competing for binding on the pre-attached antibodies. This is a fairly sensitive model capable of quantitating small molecules.
Benefits of a Fully-Automated ELISA Reader
Manual ELISAs can be time-consuming, especially for the common sandwich models. Thus, many scientists can benefit from using a fully-automated ELISA reader. The automated ELISA workstation intertwines the actions of the SOLO liquid handler, Micro10x reagent dispenser, and RapidWash plate washer to complete all the steps necessary for the ELISA in a 96-well plate. In addition, by being a largely “hands-off” system, the fully-automated ELISA reader is a trustworthy robot giving scientists more time to devote to more demanding tasks.
In this “ELISA machine,” up to 12 samples can be added at a time across a 96 well plate, where the SOLO liquid handler performs serial dilutions across the plate. The SOLO adds reagents as needed for the ELISA, and the RapidWash, acting as an automated ELISA plate washer, completes the repetitive washing steps. These steps are done with precision, ensuring the proteins and antibodies remain attached to the plate. Once the plate is prepared, the fully-automated ELISA reader automatically emails the scientist, stating the plate is ready for the next batch of samples.
Using a fully automated ELISA reader, the scientist is ensured that reagent aliquoting, incubation timing and plate washing are accurate and consistent. There is less room for error, thus producing reliable and reproducible results.
Furthermore, the reagent dispensers and RapidWash stations in this ELISA system are faster than a human technician, allowing more ELISAs to be completed in a given time. This is especially beneficial for high throughput laboratories, such as in pharmaceutical and biotech companies, where quick turnover time is required.
The ELISAs is a critical method to many laboratories. To learn more on how your lab will benefit from a fully automated ELISA reader, request a quote today!
