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ELISA

Enzyme Vs. Fluorescent Conjugates

Choosing the best detection method for ELISA/IHC

In order for an ELISA to produce useful data, the proteins that become bound in each well must be detected in such a way that the signal strength correlates linearly to the protein concentration. Toward this end, labelled secondary antibodies are used. The choice of which detection system to use is influenced by the equipment available and the needs of the specific application being considered. Use this guide to help decide whether to use enzyme-linked secondary antibodies, or fluorescently labelled secondary antibodies as your detection system, as part of ELISA optimization.

Enzyme Conjugates Fluorescent Conjugates
Advantages
  • Signal amplification gives greater sensitivity
  • Greater shelf-life
  • Able to produce chromogenic, chemiluminescent, or fluorescent signals
  • Can be used with simpler equipment
  • No substrate requirement
  • Signal not affected by endogenous enzymes
  • Less likely to interfere with the binding affinity of antibodies
Disadvantages
  • May interfere with binding affinity
  • Require substrate that is sensitive to light and prone to degradation
  • Endogenous enzymes may produce competing signal
  • Less versatile
  • Require more specialized equipment
  • Less sensitive
  • Shorter shelf-life
  • Sample autofluorescence may produce competing signal

Keywords: ELISA, optimization, enzyme, fluorescent, enzyme, conjugate, secondary antibodies

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