Hi Natalie

Are you positive the sheep samples are serum and not plasma? If they are plasma, sounds to me like you have cryoprecipitation, which is a phenomenon that occurs particularly if the samples have been slowly thawed on ice or in the refrigerator. 

Regards

Rosemary

repeat thawing and refeze  affect the protein of the samples so decrease the level  , alliqutte the samples first for ever sample 2 or 3 alliquttes done, use one evey time you repeat the test, sure the refrezing temprature at - 20 , thawing the samples at 37 degree

if your samples are serum , investigate the lipids content, if increase , extract lipid befor assay. 

this paper may answer your quation

Hello,

I am certain these are serum samples, not plasma. They were allowed to fully clot (over 1 hour) before being spun and analysed.

Thank you for the paper. Perhaps some IgG is getting in these aggregates too, but I wonder if anyone has noticed this effect specifically by ELISA for IgG?

Hi Natalie

Thanks for your additional information. I still think it sounds like cryoprecipitation. This can occur in thawed serum samples and is due to the presence of residual fibrin carried over following the clotting process.  Cryoprecipitation can become more apparent with repeat freeze-thaw cycles.

Also I wonder whether part of the problem could be that the clot was not fully retracted when you collected the serum. This could mean that you have some residual clotting proteins present in your 'serum'.  Did you use a standard tube to collect the sheep blood or a serum separator tube with a gel barrier? Did you place the tube in the refrigerator to aid clot retraction. It is best to allow longer than 1 hour for the clot to retract - preferably overnight in the refrigerator.

Regards

Rosemary

Hi Rosemary,

The sera were prepared in clotting tubes with a gel layer, and allowed to clot at room temp for 1 hour before spinning and pipetting serum. Perhaps we could wait later, but we don't really have a problem in any of our other samples, I think this is due to freeze thaw in these specifically. We do aliquot and freeze for single-use, but this has been an unusual case where we have had to repeat assays more times than we could predict.

If it is cold agglutination, could this affect antibody titre to increase it? I am trying to work out why a sample would suddenly have a higher titre, not a lower titre as I would expect with repeated freeze-thaw cycles.

Hi Natalie

I do not have a definite answer to your question.  It is possible that the presence of proteins with the potential to cold agglutinate could interfere with antibody binding in your ELISA and once these proteins are depleted by cryoprecipitation allow for more antibody to bind or higher affinity of binding.  

Hi Rosemary,

Thanks for your input. I hadn't considered that. I am imagining a scenario where cold agglutinins (or other proteins that can agglutinate) form clumps that the Ab gets trapped or non-specifically bound to, and then when one exposed Ag binding site binds to the plate there are lots of antibodies gunked together and so I detect more antibodies and the titre is higher. We'll try a different freeze down and if it looks funny on thawing maybe a high speed spin.