Hello,

In my lab we are using ELISA to determine antibody titres in sheep sera by several different immunisation regimens. Previously this assay was done last year (four times, due to the serum not being diluted enough to get an endpoint calculated, and various other problems) though consistently we were able to show titres from group A were higher than group B (about 700-fold increase in endpoint titre). We calculate endpoint cutoff using the mean + 2 SD of negative samples (from the sheep before they were immunised). I know this is not 'ideal' but this is looking for differences between groups, rather than seroconversion or some other measurement.

We have repeated the assay to look at a different immunisation regimen, but now group B is coming up higher than group A. This is quite confusing.

The sera have been freeze-thawed a few times (frozen, thawed, aliquoted into plates to freeze, and then thawed for assay). We always make sure we compare sera that have had the same number of freeze-thaw cycles.

This time doing the assay, the student noticed that the thawed serum samples of Group B appeared strange, the protein had aggregated into an oily goo at the bottom, and after vortexing and pipetting mixed back together, but sort of 'settled' again when the samples sat on the bench for about 1 minute (I have observed this too but generally once mixed it stays mixed for at least half an hour).

Is it possible that this is a sign of some aggregation between antibodies, or with lipids in serum, that are increasing signal in the assay? Is there something that can be done to resolubilise the proteins, such as incubating for a long time in detergent?

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