Product Survey: ELISA Kits
Pimp Your ELISA
by Harald Zähringer, Labtimes 03/2013
The basic concept of the Enzyme-Linked-Immunosorbent-Assay (ELISA) hasn’t changed for more than forty years. However, ELISAs are anything but outdated and old-fashioned.
The Romans already knew how to colour glass with gold and silver nanoparticles.
Even forty years after its invention by two, independent research groups, the Enzyme-Linked-Immunosorbent Assay (ELISA) is still one of the most popular techniques for the determination of target analytes (antigens).
That’s quite a career for an assay, which started out as a simple dip-and-read pregnancy test project by the two Organon (now Merck) researchers, Anton Schuurs and Bauke van Weemen, back in the late nineteen-sixties. Schuurs’ and van Weemen’s pregnancy strip project failed; however, the resultant idea of using enzyme-linked antibodies to detect immunochemical reactions turned out to be a bull’s-eye hit.
The two immediately realised that their new method to quantify chorionic gonadotropin concentrations in urine, which they called Enzyme Immunoassay (EIA), was perfectly suited to also detect various other analytes.
Virtually at the same time, Peter Perlmann and his PhD student Eva Engvall were tinkering around on a similar assay to quantify IgG levels in rabbit serum at the University of Stockholm, which they called enzyme-linked immunosorbent assay. Perlmann and Engvall published their ELISA assay in 1971 just a few months before Schuurs and van Weemen presented their EIA method.
Though the technical approaches of the two groups were slightly different, their basic concepts were more or less identical and are still at the heart of modern ELISAs. A primary poly- or monoclonal antibody binds to an antigen immobilised on the surface of a microplate. A secondary, enzyme-conjugated detection antibody is added, which recognises a target epitope of the primary antibody. The enzyme finally converts an added substrate into a coloured, fluorescent or luminescent product, which may be easily detected by an ELISA-reader.
Antigens may be directly coupled to the well surface. However, most researchers prefer sandwich ELISAs, where antigens are captured by an immobilised primary antibody and sandwiched between the first and a second primary antibody, targeted to another antigen epitope.
It took almost ten years, from the invention of the ELISA to the first commercial kit, actually an EIA kit, in the early nineteen-eighties. Some thirty years later, myriads of commercial ELISA kits for diagnostic or research purposes targeted to any thinkable antigen are flooding the market. All of them contain more or less the same basic ingredients, i.e., a (96)-microplate, block- and wash buffers, primary and secondary antibodies and a colour substrate.
Finding the right kit for a certain application, however, is not always easy. You may choose between numerous different types of microplate surfaces, buffer compositions, or colour substrates as well as primary and secondary antibodies. ELISA plates, for example, are available in many surface formats ranging from pure polystyrene to surface coatings with protein A, G, and L or Biotin, Ni2+, glutathion and maleic acid to promote the binding of primary antibodies, proteins, streptavidin or other biomolecules used in ELISAs.
The majority of ELISA kits is still based on the traditional assay format, which includes repeated, time-consuming washing steps. However, some manufacturers have created interesting, alternative ELISA concepts in recent years. Typical examples are bead-based ELISAs, which completely omit the annoying washing steps. The colour-signal generation of this so-called no wash ELISA is pretty sophisticated; the underlying concept, however, is borrowed from the good old sandwich ELISA.
Instead of immobilising antibody number one on the surface of a microplate, it is coupled via Streptavidin-Biotin to so called donor beads. Another bead set (acceptor beads), is coated with antibody number two, targeted to a different antigen epitope. Binding of the analyte to both antibodies puts donor and acceptor beads into close proximity. Exciting the donor beads with a laser beam releases singlet oxygen, which in turn leads to the emission of a light signal from the acceptor beads.
The Dissociation-Enhanced Lanthanide Fluorescent Immuno Assay (DELFIA) developed by the Finnish researcher, Ikka Hemmilä, in the early nineteen-nineties is a another example of a tweaked sandwich ELISA. Hemmilä and his team labelled Streptavidin with a fluorogenic europium ion and coupled it via biotin to a detection antibody. In today’s DELFIA assays not only europium but also other lanthanide-labelled antibodies are used for antigen detection.
Very recently, Molly Stevens’ group from the Imperial College London came up with a new signal generation technique, based on nanocrystal formation that boosts the sensitivity of ELISAs to new levels (de La Rica and Stevens, Nature Nanotechnology, 7(12):821-4). Stevens’ ELISA is set up like a traditional sandwich assay, except for the colour reaction design, which is both simple and pretty darn brilliant.
The basic components of the British colour generation system are a catalase-labelled, secondary antibody, gold ions and hydrogen peroxide solution. Without bound analyte, Au3+ ions are reduced by hydrogen peroxide to non-aggregated, spherical gold nanoparticles, which turn the assay solution red. If an analyte binds and is sandwiched between capture and second primary antibody, the catalase disproportionates hydrogen peroxide to water and oxygen. This slows down the reduction of gold ions and leads to aggregated nanoparticles, which give rise to an intensive blue colour.
According to the Stevens’ group, this so-called plasmonic ELISA enables life scientists to detect minute amounts of antigens (10-18 g ml-1) with the naked eye.
Looks like the simple, forty year-old ELISA assay is still going strong and remains a rewarding playground for creative researchers.
First published in Labtimes 03/2013. We give no guarantee and assume no liability for article and PDF-download.
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