ELISpot: Difference between revisions

The FluoroSpot Assay is a variation of the ELISpot assay. The FluoroSpot Assay uses [[Fluorescence|florescence]] in order to analyze multiple analytes, meaning it can detect the secretion of more than one type of protein.

 

As assay is a scientific technique used to qualitatively and quantitatively describe the presence, concentration, or activity of a substance, such as a drug, protein, or toxin. An assay generally includes the desired substance to be studied, reactants that are added to the assay in order to create a chemical or physical change that can later be observed and described, and a final product that can be quantified or qualitatively analyzed through different methods such as using a standard color spectrum or through simple analysis under a microscope.

 

[[File:Culture multiwell plate.png|thumb|Plate with multiple wells typically used in assays]]

 

 

#'''''Antibody coating''''': Throughout the ELISpot Assay technique, different substances are added to and washed away from wells. Wells are found on a laboratory plate with tiny dishes/bowls that can be filled with a substance to be examined; the amount of wells on a plate varies, but it usually ranges from 16-100. The first substance added to the wells are cytokine specific monoclonal antibodies. These antibodies coat the walls of the wells for future binding to cytokine. The [[Monoclonal antibody|monoclonal antibodies]] means that the antibody is produced from a single cell lineage, and is only able to bind to one protein [[epitope]]. [[Polyclonal antibodies|Polyclonal]] antibodies, on the other hand, are capable of binding to multiple epitopes of the same protein.

#'''''Analysis''''': The spots that are formed can then be read on an automated ELISpot reader, or counted under a dissection microscope, and further used to calculate the frequency of cytokine secretion.

 

<ref name=":0">{{Cite web|url=https://www.mabtech.com/knowledge-center/assay-principles/fluorospot-assay-principle|title=FluoroSpot Assay Principle|last=|first=|date=|website=MABTECH|archive-url=|archive-date=|dead-url=|access-date=6 December 2018}}</ref>

The FluoroSpot assay is very similar to the ELISpot assay. The main difference is that the FluoroSpot assay is able to analyze the presence of multiple analytes on one plate of wells, whereas the ELISpot assay can only analyze one analyte at a time. The FluoroSpot assay accomplishes this by using fluorescence rather than an enzymatic reaction for detection. The steps for a FluoroSpot assay are also similar, with a few differences.