Poor replicates help! - (Jan/14/2015 )

Hi,

I am trying to do some home made ELISAs to look at antibody levels in serum. I am doing two ELISAs one looking at IgG1 antibodies the other for IgG2. I am running the two plates in parallel, everything is identical except for on one plate I put anti-IgG1 conjugate and the on the other plate anti-IgG2 conjugate. Each time I run the ELISA I see a high level of variation between duplicates on the IgG2 plate. Wheras the dupliates on IgG1 plate show low variation.

I just can't figure out why this is happening. Any advice?

Thanks!!!

I am assuming you have the plates coated with IgG1 and IgG2. You are adding the serum that is supposed to have anti-IgG1 and anti-IgG2 antibodies.

In any case, when first plate works and the second shows a lot of variation,

1. You either have very low or very high antibody titer (prozone phenomenon-dilute serum-less likely) in the second plate. 

2. Since it is homemade, antigen coating is not uniform in the second plate.

3. You are tired pipeting after the first plate, do second plate first!

Also, if a commercial assay is available, first get the correct results for each sample, so you are not going after a wild-goose chase.

Thanks for your reply. I am coating my plates with an antigen. I am adding the serum to look for antibodies specific for antigen.

The highest OD reading I am getting on my IgG2 plate is about 1 but mmost of my samples are below that. For the IgG1 plate the ODs are consistently lower than the IgG2 plate. I am diluting my serums 1:100.

I am using three different antigens for coating and all 3 antigens have variable IgG2 results.

I have tried doing the IgG2 plate first and still see the same thing.

As an example out of 24 sampes I had 9 with a CV greater than 15% for IgG2 and only 1 sample with a CV greater than 15% for IgG1.

I am becoming paranoid that I am the problem!!