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ELISA (Enzyme-linked Immunosorbent Assay) detects particles with a simple color change by using antibodies. When antibodies are exposed to their corresponding antigen, it will bind to it to instigate an immune response. Researchers can utilize this ‘hook-like’ mechanism to detect particles like peptides, proteins, and hormones. There are also a variety of ELISA types, such as Direct or Indirect, Sandwich, and Competitive ELISA. Each of these have their own applications, allowing ELISA to be used for various tests, especially for medical reasons or toxicology reports.

Expand your understanding in our Knowledge Section. Perfect your experiments with our Troubleshooting Guides. Join in the discussion within our Community. Learn about ZAGENO at How It Works.

Experimental Setup

Simply put, what is required for an ELISA test, in general, are wells with which contain an antigen, detection enzymes, antibodies, and your sample. Although some types of ELISA work in different ways, the underlying principle always remain the same: the immobilization of an antigen. Modifications on this theme, like whether the antigen is caught with an antibody attached to a plate or suspended in solution, is what makes ELISA so useful for detection. Once particles of interest are stuck onto the ELISA plate, then all that is necessary is to wash the plate’s surface, separating attached from unattached particles.

To make researchers lives easier, detection enzymes (or any other tag) can be attached to primary or secondary antibodies, or proteins. Once a detection enzyme encounters its corresponding substrate, light is usually emitted and measured depending on your experimental setup (fluorometer, luminometer, spectrophotometer).

Our ELISA Troubleshoot provides further assistance.

Types of ELISA

Direct ELISA - Refers to direct detection; a primary antibody (labeled with a detection enzyme), reacts directly with an antigen, which is immobilized on the plate.

Indirect ELISA – Indirect detection; a labeled secondary antibody with known specificity for a primary antibody

Sandwich ELISA – one of the most common methods of ELISA, and the steps are as follows:

  1. Capture antibody is placed onto a plate
  2. Sample antigen is added, binding to the capture antibody
  3. Detection antibody is introduced, binding also to the antigen
  4. Secondary antibody with a detection enzyme is added, binding to the detection antibody
  5. Substrate is added, thus triggering all successfully captured antigens within sample

Competitive ELISA

  1. Sample antigen is incubated with complementary unlabeled
  2. The antibody/antigen complexes are inserted into a well coated with competitive antigen.
  3. Washing the plate removes unbound antibodies. (The higher the volume of antigen in the sample, the more Ag-Ab complexes present and therefore less unbound antibodies free to bind to the antigen coating the well)
  4. Secondary antibody coupled to an enzyme is added, which binds the primary antibody.
  5. Adding the substrate for the enzyme produces a chromogenic or fluorescent signal, depending on the reaction
  6. Stopping the reaction prevents the signal from saturating

Enzyme-linked antigen can be used in place of enzyme-linked antibody. In this case labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). A stronger signal is seen if the there is less antigen in the sample, as more labeled antigen is retained in the well.

Compare Clusterin ELISA Kits

With our compare function, you can avoid all the time and energy wasted sifting through multiple web pages from different suppliers. At ZAGENO you can clearly see kits side-by-side, with the relevant attributes for each kit neatly in line for easy selection of the best product for you.

Click on the comparison below for a clearer view!

For example, this comparison shows:

  • that the CLUSTERIN HUMAN ELISA Kit from Invitrogen is the cheapest per reaction.
  • that the HUMAN CLUSTERIN ELISA Kit from Abcam employs sandwich ELISA and takes 90 minutes to complete.
  • that the HUMAN CLU / CLUSTERIN ELISA KIT Kit from Sigma-Aldrich can accept samples from blood and urine.

This comparison clearly exemplifies how every kit has strengths and weaknesses. Depending on what features you require for your experiment, these details should enable you to make an informed decision on the right kit for you.

The ZAGENO comparison does not highlight one kit to be better than the other, as the kit of choice may vary between researchers - depending on each individual's preferred attributes. The best kit is the one that meets your needs - ZAGENO allows you to make an informed decision with minimum effort.

Check out our How It Works page for a guide to using the comparison function.

Video Credits

Video: openmichigan/YouTube


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