[Effect of multiple freeze-thaw cycles on detection of IgA, IgG and IgM antibodies to selected bacterial antigens].

Abstract

Introduction: In presented study we investigated the effect of multiple freeze-thaw cycles of human sera on the determination of IgA, IgG and IgM antibodies to selected bacterial antigens.

Methods: A panel of 15 serum samples with elevated levels of antibodies to Mycoplasma peumoniae, Yersinia enterocolitica and Salmonella spp. were used (5 positive sera for each pathogen). One set of aliquots designed as the baseline, was taken and stored at 4-8o C for the remainder for the study. The remaining seven sets of aliquots were divided into two parts and repeatedly frozen respectively at two different temperatures: -65 degrees C and -25 degrees C. Once a day the aliquot sets were removed from the freezer and allowed to stand at room temperature for approximately 1 h until completely thawed. For the determination of the level of antibodies the sera after: 2, 5, 10, 15, 20, 25 and 30 freeze/cycle were used. The measurement of IgA, IgG and IgM antibodies was done using a home-made ELISA with four different antigens: whole-cell antigen of M. pneumoniae FH strain, LPS and Yop antigens of Y. enterocolitica serotype O:3 and LPS extracted by Westphal method from Salmonella serogroup B +D. The results were presented as the arithmetic mean of the antibody titre in five sera which were treated by the same number of freeze-thaw cycles.

Results: There was no significant statistic difference between levels of antibodies in unfrozen and frozen sera even after 30 freeze-thaw cycles. Depending of the antigen used in ELISA a slight varations in the level of antibodies were observed but the changes were small and not clinically significant. Examination of the ELISA values does not suggest any consistent nonlinear trend in levels of IgA, IgG and IgM antibodies in sera frozen at -65 degrees C as well at -25 degrees C.

Conclusions: Our study demonstrates that the IgA, IgG and IgM antibody activity levels measured for M. pneumoniae, Y enterocolitica and Salmonella antigens are stable even after 30 freeze-thaw cycles.