Analyzing complement activity in the serum and body homogenates of different fish species, using rabbit and sheep red blood cells

Alternative complement activity was determined in whole body homogenates (WBHs) and serum samples of different fish species, by measuring the amount of sample that induces 50% hemolysis of red blood cells using the ACH50 assay (Alternative Complement pathway Hemolytic activity). Values of ACH50 obtained for serum samples were about two-fold higher when using rabbit red blood cells (RRBC), as compared to sheep red blood cells (SRBC). The increase in ACH50 when using RRBCs for WBH samples was 28, 7 and 4 folds for guppy, molly and zebrafish, respectively. Large variability in complement activity was evident between fish species for both serum and WBHs. Evaluating the effect of freeze-thaw cycles on complement revealed significant reduction in complement activity in all tested samples. Loss of activity following three freeze-thaw cycles amounted to 48-59% when serum was tested and over 95% loss in activity for WBH. To our knowledge, this is the first study where fish WBHs were used for assaying complement activity. Our results support the suitability of this method in evaluating complement activity in small fish species or larvae, where blood cannot be obtained, as long as samples can be tested upon first thawing.