A Study on the Impact of Repeated Freeze-Thaw Cycles on the Serum Cytokine Enzyme-Linked Immunosorbent Assay (Elisa)

Abstract

Sandwich ELISA is a modified form of the standard ELISA technique used by researchers and in routine laboratories for detecting and quantifying specific analytes in samples. This study is designed to evaluate the impact offreeze-thaw cycles on the results of serum cytokine measurement by sandwich ELISA technique. Serum samples were collected from four healthy donors and stored in 3 aliquots at -80oC for three days per thaw-freeze cycle. The concentrations of IL-4, IL-6, IL-8 and IL-9 in the aliquots of serum samples exposed to one, two and three freeze-thaw cycles respectively were determined. Data shows an increase in the cytokine levels following two and three freeze-thaw cycles compared to the expression levels obtained after one freeze-thaw cycle (baseline). The increases reported are as follows: IL-4; 6.56pg/ml (19%) and 3.52pg/ml (10%), IL-6; 103pg/ml (20.9%) and 24.6pg/ml (5%), IL-8; 105.05pg/ml (14%) and 102.76pg/ml (11%) and IL-9; 117pg/ml (17.77%) and as high as 312pg/ml (47.52%) respectively. These results suggest that the repeated thawing and freezing of serum samples can alter the values of serum cytokines measured by ELISA, in most cases with an increase. The reasons for the increase in the values of these cytokines following repeated thawing and freezing have not been investigated. To minimize the effects of thaw-freeze cycles on serum cytokine ELISA, fresh serum samples should be used for the analysis of serum cytokines. Alternatively, aliquots of serum samples can be stored in the -80°C Freezers for single use or by the application of a methodology that will subject samples to similar treatments, for instance, analysing several samples in parallel as in Multiplex assay. These approaches will minimise variations due to repetitive freeze-thaw cycles.