High Background help - (Sep/28/2011 )

I'm running a Sandwich ELISA with OPD as my substrate. All of the sudden I've been getting high background (high blank wells). Doesn't seem to be any particular changes in reagents between the two runs when the problem started. Any suggestions on things to check or possible causes? Thanks.

Best,
Grad

Is this an assay that you have been running routinely or something that is relatively recent?

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?

Thank you for your response. This is an assay that we've had up and running for a while without problems. Suddenly a couple weeks ago the background increased and has stayed elevated. The OPD starts out clear when pipetting it into the wells, so I assume it's fine. We remade all of our buffers and our wash machine is working fine, so I'm at a little bit of a loss.

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)?

If the problem occurred suddenly, it is unlikely to be a reagent going 'off'. In my experience, peroxidase conjugates gradually lose the activity of the peroxidase over a period of months rather than overnight. Unless, it is being stored at 4 degrees and it doesn't contain an appropriate preservative and now has microbial contamination which has now got to such a level that a large proportion of the conjugated molecules are now degraded or partially degraded.

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!

Follow-up on this: Not sure what it was, but something in the batch of deionized water we were using was responsible.

glad you got round the problem

Do you use peroxidase method?

Hemoglobin, Iron and metal can work as a peroxidase.