High Background help
Posted 28 September 2011 - 07:13 AM
Posted 28 September 2011 - 08:06 PM
Either the OPD is contaminated and producing colour itself, or something is resulting in the conjugated antibody remaining within the wells containing the zero spikes. Detective work will identify where the problem lies, but performing an assay and removing a layer at a time.
Possible causes include: analyte contamination of matrix, reagents or buffers, matrix lot change (have you confirmed that the assay gives consistent blanks among multiple different lots of matrix), or degradation of the matrix (I have seen progressive increase in background with stored diluent/matrix) improper or variable blocking step. Improper storage of the peroxidase conjugate....-20 in 50%glycerol is preferred. Lot change of critical reagent.
These are just a few, but with more specifics of the assay, it may be possible to identify the weak links.
Is this something that you just need to get up and running again, or is there some development work required?
Posted 05 October 2011 - 07:53 AM
What would Improper storage of the peroxidase conjugate do? Is there a way to tell if this is the problem (besides buying some new stock)?
Posted 09 October 2011 - 04:00 AM
The other thing to consider, is reagent contamination. Contamination of either the conjugate or coating antibody with analyte can cause problems. In most labs, this is an unpopular but real subject to get to grips with. Routinely, when spiking standard/QC stocks, we separate the activity from the rest of the assay and use aerosol resistant tips. Nanolitre quantities of mg/mL stocks can have a dramatic effect on the assay. This can be a particular problem if multiple individuals within the lab are working onthe same molecule using the same equipment.
Let us know how you get on!
Posted 10 April 2012 - 07:16 PM
Posted 11 April 2012 - 11:34 PM
Hemoglobin, Iron and metal can work as a peroxidase.