ELISA

What is an ELISA?

The well-known term ELISA is an abbreviation of Enzyme-Linked Immunosorbent Assay, yet although ELISA readouts have traditionally relied on the use of an antibody which has been linked to an enzyme, the term is now broadly used to describe any plate-based immunoassay in which a molecule has been adsorbed on to a solid surface. ELISAs are popular for several reasons:

  1. Easy to perform
  2. Quantitative
  3. Sensitive – limit of detection typically µg-pg
  4. Relatively inexpensive
  5. Suitable for screening large numbers of samples
  6. Amenable to automation – suitable for HTS

To find out more about ELISA, including the wide range of products which we offer to help streamline your assay, why not download our free guide?

Assay formats

There are several ways in which an ELISA can be configured:

  • During an antigen-down ELISA, the microplate wells are coated with the antigen, and the plate is then washed prior to the addition of a sample containing an antigen-specific antibody. This type of assay is often used to verify the presence of antibodies in a sample, for example during the diagnosis of allergic conditions.
  • A sandwich ELISA requires the microplate wells to be coated with an antigen-specific antibody. After washing to remove any unbound antibody, a sample which is thought to contain the target antigen is added. Detection relies on the addition of a second, antigen-specific antibody. Sandwich ELISAs are typically more sensitive than antigen-down ELISAs.
    Sandwich ELISA
Sandwich_ELISA process diagram

Schematic representation of a sandwich ELISA using indirect versus direct detection.

  • Antigen-down and sandwich ELISAs can both be adapted to produce a competitive ELISA. This format is often used when the analyte is small and cannot be bound by two different antibodies simultaneously. During a competitive ELISA, an unknown amount of antigen in the sample competes with a known amount of reference antigen for binding to the sites which are available in the microplate well. Samples which contain a high concentration of the antigen will give rise to a low signal in a competitive ELISA.

Indirect versus direct detection

Although ELISAs have traditionally relied on the use of labeled secondary antibodies for detection, directly labeled primary antibodies offer several clear advantages:

  • Non-specific binding is avoided since secondary antibodies are not used
  • Multiplexing is possible with antibodies from the same species
  • Faster since there is no secondary antibody incubation step and therefore fewer wash steps
  • Data quality is improved through assay simplification

Unfortunately, in many cases the directly conjugated reagents required are unavailable, while methods of antibody labeling can require specialist knowledge of chemical modification techniques. To overcome these issues, we offer a range of easy to use bioconjugation kits.

Lightning-Link® for direct antibody labeling

Our Lightning-Link® kits enable direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups to enzymes, biotin, streptavidin, fluorescent dyes or fluorescent proteins. To produce the conjugate, the biomolecule is simply pipetted into a vial of lyophilized mixture containing the label of interest, incubated, and is then ready for use in the downstream application. Since no separation or washing steps are necessary, antibody recovery is 100%. Furthermore, the kits are fully scalable, allowing easy transfer from R&D to manufacturing.

Lightning-Link process diagram

The Lightning-Link® antibody labeling process.

Our Lightning-Link® kits have been widely literature cited, with over 300 references to date. Many of these articles describe the use of Lightning-Link® antibody labeling kits in ELISA.

Europium Conjugation Kits for antibody labeling with Europium particles

Lanthanides such as Europium can provide a further level of assay sensitivity, due to their large Stokes shift and long fluorescent lifetime, and are therefore ideal for assays which utilize a time resolved fluorescence (TRF) readout. Our Europium Conjugation Kit enables direct labeling of antibodies, proteins, peptides or any other biomolecule with free amine groups to 200nm Europium (Eu) chelate microspheres.

The specially treated surface of these particles allows the generation of highly stable, covalent conjugates which are resistant to aggregation and require no extensive pH optimization.
We have demonstrated assay sensitivity of as low as approximately 10pg/ml when using an antibody-Europium particle conjugate in an ELISA.

FlexLISA® – quick and easy ELISA development and optimization

One disadvantage of an ELISA is that, since it relies on the use of antibodies, it can become rather expensive to run. To significantly drive down costs, we have developed FlexLISA®, an assay which reduces the required amount of capture antibody by up to 40-fold, providing a significant cost-saving.

The FlexLISA® product contains a Lightning-Link® Streptavidin kit for conjugating your choice of capture antibody to streptavidin, and either a Lightning-Link® HRP kit or a Lightning-Link® Alkaline Phosphatase kit for conjugation of your chosen detection antibody. Each of these kits is suitable for the conjugation of 3 x 10µg antibody, allowing you to directly label a range of capture and detection antibodies. Also included is a 96-well microplate (in 12 x 8 strip well format) which has been pre-coated with biotin; this can be supplied as clear or black, depending on the enzyme substrate.
FlexLISA® employs a rapid one-step protocol. The assay is run by simply adding the sample and the antibody mix to the pre-blocked microtiter well strips, incubating for one hour, then washing and reading the assay plate. The kit is ideal for quick and reliable ELISA assay development, antibody pair screening and ELISA assay optimization.

ELISA schematic process

Schematic representation of the FlexLISA process. 50µl sample is added to a microplate which has been pre-coated with biotin. 50µl antibody mix, consisting of a capture antibody-streptavidin conjugate and a detection antibody-enzyme conjugate is then added, and the plate is incubated for 1 hour at room temperature with shaking. After washing, an assay readout is produced using an appropriate enzyme substrate.

In addition to our Lightning-Link® antibody labeling kits, Europium Conjugation Kit and FlexLISA®, to further improve the efficiency and utility of your ELISA we offer:

  • LifeXtend™ HRP Conjugate Stabilizer to protect antibody-HRP conjugates from factors which can diminish performance
  • Protein A conjugates – secondary reagents formed by covalent conjugation of Protein A to enzymes or fluorescent proteins
  • Streptavidin conjugates – secondary reagents formed by covalent conjugation of streptavidin to enzymes, fluorescein, RPE or Europium
  • Check&Go! product range for quick and easy confirmation of successful antibody conjugation to colored labels or to biotin
  • Biotinylated BSA – ideally suited as a plate coating reagent, significantly reducing the amount of capture antibody that is required

Further information on all these reagents can be found within our free ELISA guide.

Proteoloc

The addition of protease inhibitors to lysis buffers is an effective means of protecting extracted proteins from enzymatic degradation by endogenous proteases. Expedeon offers protease inhibitor preparations for use with protein extraction lysis buffers. EDTA-free cocktails are available for specific cases where EDTA is not compatible.

Visit our website to find out more about the Expedeon products that can assist you further in ELISA application.

Custom services from Expedeon

Expedeon are experts in bioconjugation, and we offer a range of custom services to assist you in the development and implementation of your ELISA. These include our:

  • Antibody Micro-Optimization Service
  • Custom Antibody Conjugation Service
  • Bulk Ordering
  • Inventory Control
  • Complete ELISA Development Service

For further information on any of our products or services, please contact us.

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