ELISA problems - sample recovery! - why is this happening? (Oct/26/2006 )
Okay so I am trying to developed an ELISA for use with inactivated virus.
My curve made from virus sample in coating buffer (carbonate-bicarbonate) is great as are my control samples (also virus in coating buffer).
The problem is with recovery of the virus. To test this I am using the same virus sample and spiking it (at set concs similar to the control samples) into the supernatant of drug product containing said virus. The supernatant should be PBS with a negligable amount of sucrose and formalin.
Does anyone know if either sucrose or formalin could somehow be inhibiting the binding of the virus onto the plate wells? I have already tested to see if PBS vs coating buffer makes a significant difference and it doesn't so unsure why this is happening?
Any ideas? please help?
sucrose should have no effect but formalin may be a problem. if the virus has been treated with formalin then the proteins are probably denatured (and crosslinked?). their epitopes may not be available to your antibody.
The virus is the same in all cases - yes it has been inactivated by the formalin but it happily binds to the antibody when in coating buffer so I think it is something to do with the virus binding to the wells in the first place when added to the wells in PBS (+sucrose and formalin)
I don't know anything about formalin effect, but I do know that we used to use a sucrose solution as a stabilizing agent after coating with peptides (before blocking with BSA). I don't know what it could be doing to your virus though...