Hi Preetha, 

is the ELISA a commercial one, or did you develop this assay yourself? It could be that the lowest point of your standard curve is below your lower limit of quantitation; your assay cannot reliably detect that amount of antigen. Usually this point is defined as the amount of antigen that can not be detected with a cv < 15%, at which point your assay simply does not have the analytical sensitivity.

There are several hints if you are working with an in-house assay - such as extending your incubation step - , but you might simply be doing something 'wrong' if you have a protocol to work with.

Hi Preetha,

In my experience as the concentration gets lower the CV% usually gets higher. However, if you curve consists of 7 serial dilutions you should be focused on the middle 3 dilutions - which is where ideally your sample concentrations should fall.  Has this high CV% affected the R2 value of your curve?

I'm presuming each standard is in triplicates- if the OD for one of the technical replicates is off you may want to delete it and that might help tighten the CV%.

Usually the wash step is the most crucial and needs to be consistent for an ELISA to work. Also make sure to remove as much of the wash buffer from the wells before pipetting.

Hope that helps,

Padmaja

Hi Padmaja,

Many thanks for your swift response. My Standard curve contains 8 serial dilutions(with 2 replicates each) and unfortunately I am not allowed to remove/delete any points from the curve.

Also, how many times do you bang your  plate before you add in your sample? Does banging too much affect your results?Sometimes I could see tiny bubbles inside the well before I add in my samples. I have tried to remove it but it was not possible sometimes.

In addition, the OD value of my last Std curve seem to the same or greater than the one above it which makes my ELISA more difficult to pass.

Please pass me your thoughts.

Thanks,

Preetha.

Hi Wesley,

Thanks for your reply.

The ELISA I am doing is developed in house and I am following a protocol specific to that kind of ELISA. The OD value of my lowest is equal or greater than the point above it and I don't know why. I have made my std curve by serial dilution using a rainin multichannel pipette. Any thoughts on why this would happen?

Thanks again,

Preetha.

Hi Preetha,

Wesley raised a good point about whether you are using a commercial kit or if this was an in-house ELISA method- in which case you may want to modify the steps accordingly. Also technical replicates of three are always better.

It sounds like you have hit the detection limit, so try using a 7 point curve or increase your top standard.

If you are seeing tiny bubbles than that will hold some of the buffer and create a dilution effect which will distort your reading. You will need to remove it as much as possible and repeated patting of the plate onto paper towels does not affect your results.

Good luck

Padmaja