I am attempting to optimize a basic microbial killing assay using previously collected serum samples of my study species. The microbe of interest is E. coli ATCC 8739, the defense against which is complement-dependent. However, I am concerned that my samples have simply been frozen too long for me to see any killing activity of the challenged serum. Is anyone aware of a maximum length for which complement can persist in a frozen (-80) sample? My "most recent" samples are from May 2014, which I fear is still too old. Otherwise, I will have to wait until April 2015 to process within the generally accepted 20-30 day window.
I determined C5a concentration in mouse sera that were stored for more than a year and had no problems with it. Especially in serum, where complement is additionally activated via extrinsic pathways. Of course, I do not know how functionally active are other proteins, but I think complement should be pretty stable
There was a paper recently published in 2015 by the American Journal of Clinical Pathology that demonstrated complement factors are stable in citrated plasma when stored up to 6 years at -80˚C. We have good reproducibility in our assays with NHP serum in storage for 1 year at -80˚C. As long as the serum you collected wasn't sitting at RT for more than 3 hours prior to storage, you should have good hemolytic or microbe killing assay results. The article I mentioned gave a comprehensive investigation in preanalytical effects on complement biomarker assays. It's not the end-all guideline for processing and storing serum/plasma for complement activity preservation, but it clarifies the impact of long-term storage on complement studies and I highly suggest giving it a read for your future experiments.
http://www.ncbi.nlm.nih.gov/pubmed/25780008
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