Background on ELISAs
Enzyme linked immunosorbent assays (ELISAs) are designed to measure a specific target or marker from a biological sample in either a quantitative or qualitative fashion. The quantitative aspect provides advantages over other detection methods such as western blotting, with ELISAs also providing higher sensitivity, consistency, and the potential for high throughput.
There are multiple formats for an ELISA, each with their own pros and cons, including:
- Direct – where an antigen is immobilized to the ELISA well plate, and then detected by a conjugated antibody (e.g. conjugated to horseradish peroxidase or HRP)
- Indirect – again an antigen is immobilized to the ELISA well plate, but detection is a two-step process with an unlabelled primary followed by a conjugated secondary antibody
- Sandwich – using matched “capture and detection” antibody pairs, where the “capture” antibody binds the antigen (with the capture antibody being plate immobilized rather than the antigen), which the capture antibody then “detected” in a direct or indirect method
- Competitive – also known as a blocking ELISA, where a sample antigen/antibody competes for binding to a limited amount of labeled antibody/antigen, against a reference
This post focuses on the sandwich ELISA format and the potential benefits provided by using a kit rather than developing an in-house assay.
Benefits of Sandwich ELISA Kits
One of the main issues with developing a sandwich ELISA is antibody compatibility. The capture and detection antibodies need to be able to fulfil their individual roles without impeding each other. To achieve this, each antibody needs to bind to a different and non-overlapping region, or epitope, of the antigen.
Setting this up in house means you need to validate and optimize the compatibility of the two antibodies which can take both a lot of time and money (with antibodies sold as compatible often not working this way). This can be particularly difficult if working with a native protein, where the two binding epitopes could be potentially near each other and hinder the antibodies from binding.
Using a premade ELISA kit, means the vendor has already verified compatibility of the antibodies as a pair, ready to instantly use. The kit will also provide the small quantities of antibody you need, rather than full vials of two or more antibodies being purchased for optimization or when antibodies have been chosen.
Other advantages include the kit providing all the other reagents needed, often with the capture antibody already coated on the plate for you. With all of this supplied from one vendor shipping costs are also reduced.
Commercially Available Sandwich ELISA Kits
There are a selection of commercially available sandwich ELISA kits, including from:
- Abcam – who supply a wide variety of ELISA kits
- RevMAb – who provide unique human IgG ELISA kits, which allow detection of therapeutic antibodies from samples from experimental animal models to provide accurate PK data
- ThermoFisher – providing a wide kit supply for cytokines, chemokines, and growth factors and across many experimental indications