I am working with multiplex immunoassay. As I am new into multiplexing I am facing some problem.
I have coupled Protein X to carboxylated magnetic beads (BioRad) using EDC-NHS. Now, while evaluating those coupled beads, I am getting same FI with positive control (contains primary antibody against protein X) as well as my negative control (does not contain antibody against protein X).
What can be the possible reason for it?
Unfortunately, there might be many reasons for this. The main areas of troubleshooting may be:
1. Failure of the conjugation of Protein X: Wrong buffer, wrong protocol, protein is denatured or has a wrong structure.
2. Primary antibody is poor:
Article Ten Basic Rules of Antibody Validation
Article Quality Issues of Research Antibodies
In most cases, you need one or several independent assays, for example on a microtitration plate, to verify the functionality of your reagents.
Unfortunately, there might be many reasons for this. The main areas of troubleshooting may be:
1. Failure of the conjugation of Protein X: Wrong buffer, wrong protocol, protein is denatured or has a wrong structure.
2. Primary antibody is poor:
Article Ten Basic Rules of Antibody Validation
Article Quality Issues of Research Antibodies
In most cases, you need one or several independent assays, for example on a microtitration plate, to verify the functionality of your reagents.
Sayak Ghosh ; just a few thoughts: A reason happening very often is old, hydrolysed EDC; It has to be used directly after opening the bottle or it should be withdrawn under nitrogen for re-use of the reagent; otherwise there is no conjugation. It needs only a few humidity to hydrolyse this material.
In order to avoid non-specific binding, end-capping of the NHS groups with ethanolamine or glycine could be helpful.
And the functionality of the X protein and antibody could be tested by a blot.
thank you Roland Fabis , can you please provide me some more information regarding Blocking? why you have chosen Ethanolamine or Glycine over BSA for Blocking the active sites?
Sayak Ghosh : The molecular weight of BSA is much higher than EA or Glycine, so I would prefer them due to their lower sterical demand, where a large molecule could reduce the AB functionality. Also, smaller molecules can react faster and more completely. With EA you have a neutral end group, with Glycine a negatively charged.
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