20-P: BACK TO THE FUTURE: HEAT-TREATING FOR SOLID PHASE ANTIBODY TESTING

Aim In seeking a method of reducing the background on the control beads in order to clarify the results of the Gen-Probe™ Luminex Single Antigen (LSA) assays, we hypothesized that heat- treating sera would be of great benefit. Methods When initially processed in the laboratory, five hundred microliter aliquots of sera are frozen at −80 °C for a minimum of 10 minutes. The sample is then thawed to room temperature (22 °C), heated in a 63 °C water bath for 12 minutes then ultra-centrifuged for 10 minutes at 13,200 rpm. Results We have seen no noticeable change in the negative or positive control beads. Heat treating appeared to intensify our positive results. We see an increase in Mean Fluorescence Intensity (MFI) of the positively-reacting Luminex beads while also noting a decrease of the MFI in the negatively-reacting beads. We are uncovering antibodies that were obscured before. However, there has been an unexpected negative side effect of heat treatment. In approximately 30% of our population we are finding the sera are unfit for testing due to coagulation. We found that after ultracentrifuging for 10 minutes at 13,200 rpm, 75% of the coagulated samples are suitable for testing. Conclusions The heat treating process appears to rid the sample of a heat-labile inhibitor, presumably IgM. We believe that through the heat treating process we are able to better detect antibodies in patient sera. In an effort to streamline our testing algorithm, we now heat treat all sera that will be used for single antigen testing.