ELISA

eevyhayes

Hi guys,

Just wondering if any of you can help me understand the concept of a competitive Elisa. I have looked up a few things on the net but still dont fully understand it. In an experiment I am doing, I have my analyte coated onto the plate (solid phase). Then i have in the well my antisera and analyte. after incubation i add my secondary anti-rabbit then my substrate TMB. I know that a high signal means a negative result and a low signal means a positive result. But I'm just having a bit of trouble understanding whats going on! what is actually competing in my Elisa. Is the analyte bound to the plate and the analyte in the well competing for binding with the antisera?

If someone could try explain to me I would be greatful.

GLaDOS

The antigen (for the signal antibody) bound to your plate competes with the free anylyte (also acting as an antigen for the signal antibody). The more free analyte is added, the less signal antibody can bind to the one on the plate. The solution is then washed away so that all that remains is what is attached to your well. (so low signal = more free analyte.)

Conversely, if there is little free analyte, it means more signal antibody can bind to the antigen attached to the well, resulting in a higher signal.

There can be some variation in technique but that's the basic principle as I understand it

eevyhayes

ScienceNerd wrote: »

The antigen (for the signal antibody) bound to your plate competes with the free anylyte (also acting as an antigen for the signal antibody). The more free analyte is added, the less signal antibody can bind to the one on the plate. The solution is then washed away so that all that remains is what is attached to your well. (so low signal = more free analyte.)

Conversely, if there is little free analyte, it means more signal antibody can bind to the antigen attached to the well, resulting in a higher signal.

There can be some variation in technique but that's the basic principle as I understand it

Thanks for that. But what im a little confused about is the antigen bound to the plate in my Elisa is the same as the free analyte. so if the antisera doesnt bind the free analyte it still binds to the antigen on the plate which is the same as my free analyte? Its hard to explain this through words!

GLaDOS

The antigen bound to the plate and th free analyte are both antigens for the secondary antibody (antisera). So the antisera has to bind to one of them.

eevyhayes

yes but if it binds to the antigen on the plate, it must have antibodies against that analyte. so how can that be a negative result if both analytes are the same? Am i making any sense!

GLaDOS

The antibodies that bind to the free analyte are washed away along with he free analyte, so only antibodies that bound to the antigens attached to the well remain.

The antisera will be more likely to bind to the free analyte than the bound antigen. So if having washed it all away there is a strong signal, it means there mustn't have been much free analyte to mop up the antibody so to speak.