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Competitive vs Sandwich ELISA

Competitive vs Sandwich ELISA - ELISA Assay Forum

Competitive vs Sandwich ELISA - ELISA Forum. Discuss and post questions including optimization, troubleshooting, protocols and theory of ELISA assays using monoclonal, and polyclonal antibodies or peptides.


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Old 09-23-2012, 12:43 PM
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Default Competitive vs Sandwich ELISA



Hi!

Due to which reason(s) is a competitive ELISA more sensitive than a sandwich ELISA? I've often heard this (e.g. my former P.I. often said that, and it is said here as well: ncbi.nlm.nih.gov/books/NBK92434/#immunometh.J_INITIAL_CONCEPT_AND_METHOD), but I never got to know the reasons...

Thank you!
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Old 09-29-2012, 04:40 PM
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Default Re: Competitive vs Sandwich ELISA

Competitive EIAs and sandwich ELISAs are two really different beasts, and it will depend on the analyte to be measured and critical reagents used as to which is really more sensitive.

Typically sandwich formats are not so good for small molecules simply because there is insufficient space around the analyte to allow efficient binding of two different antibodies. So if you set up a competitive format and compare it head to head with a sandwich format for a small molecule, the competitive format may well be more sensitive.

Also, because competitive formats are often more applied to small molecules (5 KDa or less) the ng/mL sensitivity may apper to be higher than sanwich formats which are more often applied to larger molecules but on a molar basis, the sensitivity may be similar.

Interference can be more prevalent with competitive formats because only one ligand binding interaction is involved, whereas as sandwich format has a double safety net of two ligand binding interactions, so if there is some interfering molecule affecting one, it is unlikely to affect both. Interference may have to be diluted out, indirectly affecting assay sensitivity. Conversely, the more critical reagents that there are in an assay, the more complicated things get, increasing the chance of having a weak link!

There is no hard and fast rule. The sensitivity is really down to careful selection of critical reagents, avoiding streric issues, careful assay development to maximize signal to noise ratios, to maximize precision, and to obtain consistent spike recovery profiles in different matrix lots.

Let us know if you have any specific questions relating to sensitivity to address
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