What is it ELISA?
Enzyme-linked immune absorbent assay or ELISA is the tried and true, quick and dirty way of assessing the immune response of the body to vaccines, infections or other immune challenges. It has other applications as well, however. The food industry typically uses ELISA to detect the presence of allergens in food products. It is used to detect human chorionic gonadotrophin (hCG) in urine in pregnancy screenings. ELISA is the tripwire used in initial cancer screenings. It can even be used to detect the concentrations of illicit drugs, including cannabinoids, amphetamines, opiates, cocaine, benzodiazepines, and methadone in urine samples.
There are three common ELISA variations. Each is based on the binding of antibodies tagged with enzymes generating a measurable biochemical reaction to the target antigen, but the differences in approach can be significant for your research result validity, so choose wisely!
This is the original method pioneered by Perlman and Engval. Simply coat the surface with the antigen containing sample, and then apply the enzyme tagged antibodies. After incubation wash the plate to remove the unattached enzymes. Simple, right? Unfortunately directly labeling primary antibodies takes time, is wasteful/expensive – and may also have unexpected influence of the immunoreactivity of the antibody.
In Indirect ELISA a secondary antibody, usually a polyclonal anti-species antibody is applied to the primary antibody following its incubation with the antigen coated plates. A substrate is used to boost the signal. This is the method most often used to assess infections and immune responses to vaccines. It enjoys greater sensitivity and flexibility than direct ELISA, but non-specific hits and cross-reactivity are common issues..
Double antibody Sandwich ELISA
Two antibodies specific to the antigen bind to the antigens from both sides. This method is very specific – but also very inflexible. Each antibody pair, and usually the component thereof is specific to only one antigen.
Here, antigens compete over limited antibody binding sites. Accordingly, the relationship between antigen concentration and substrate turnover is inverted. This reaction is best suited (indeed, it is the only suitable reaction) for antigens with a few epitopes or antibody-binding sites, generally due to their low length and molecular weight.
Are you wondering which ELISA variation is right for you? Contact Merkel and we will be happy to advise you on the method and kit optimal for your research needs.