Technique / Immunology / ELISA assay

ELISA protocol

ELISA protocol

1. To coat the ELISA plate with diluted capture antibody and incubate overnight at 4c.
2. Wash the plate wells with ddH2O, wash with PBS-Triton twice.
3. Block non-specific binding using 1% BSA/PBS and incubate for 30-60minutes at Room Temperature.
4. Wash plate. Add standards and 100uL of diluted samples to appropriate wells.
5. Incubate for 1hour at RT. Wash.
6. Add 100ul appropriate dilution of the secondary antibody conjugated with Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP) and incubate for 1 hour. Wash.
7. Add 100uL of substrate to well and incubate at RT for 1 hour. (Add stopping solution)
8. Read plates on an ELISA microplate reader.

Last update 07-Jan-2005, Rating of 10 votes.

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By uya on 22-Mar-2009

hi evrybody...i want analyse serum factors(TNF-a, aCL, sFasL and anti dsDNa) of SLE patients and normal control samples by using ELISA..is the method above suitable for that??or can anybody give me suggestion..thanx
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By uya on 22-Mar-2009

Sir,
I need a protocol for TNF-a, anti dsDNA,aCL and sFasL measurement from serum by ELISA. Thank you.
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By Morteza Izadyar on 18-Aug-2008

If you get no color by mixing conjugate and substrate, one of the conjugate or substrate does not work properly. you con do cross tests by changing source of substrate at one time and source of conjugate another time.
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By eka on 26-Jun-2008

Hi everybody, I've made my first elisa experiment I got no color reaction. I'm using HRP conjugated sec. antibody and OPD as a substrate. When I mixed just conjugate and substrate didn't get the colour as well. What can be the problem?
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By paari on 10-Mar-2008

dear friends ?
can any one tell me how to calculate p/n ratio in elisa after reading the sample
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By Aly Kodio on 27-Feb-2008

je suis à la rcherche du protocole ELISA d'anticorps anti-pLDH
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By herve on 21-May-2007

i performed an Elisa test using Biotinylated alkaline phosphatase as detection enzyme and pNPP as substrate,but i obtained always a very high background signal and no linearity depending on the concentration.could one give me some tip please?
Thanks
herve
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By LISBETHH on 02-May-2007