Impact of high temperatures on enzyme-linked immunoassay (ELISA) performance for leptin measurements in human milk stored under varied freeze/thaw conditions

Ambient temperature conditions are a common concern during laboratory analysis. Due to unexpected shipping conditions, leptin ELISA kits (Leptin Ultrasensitive, ALPCO USA; Catalog #22-LEPHUU-E01) arrived from the manufacturer at our laboratory at a temperature (76.3°F/24.6°C) well above the 2-8°C conditions recommended by the manufacturer. Since no data are available on the effects of high ambient temperature exposure on the performance of this commercial assay, we opportunistically assessed assay performance using human milk samples. Leptin measurement of recently collected and frozen human milk samples was compared between the warm temperature exposed assay kits and Normal kits that arrived and were stored at recommended temperatures (2-8 °C). We found that assay kit exposure to warm temperature during shipping resulted in sample results that were significantly different from Normal kits despite similar standard curve performance. Measurement variability from human milk samples increased with warmed kits in association with greater freeze/thaw times. This suggests that even under high temperature transportation conditions, this leptin assay performance is robust with kit reagents but compromised with human milk samples. We conclude that kits exposed to high temperature during shipment and/or storage should not be used to run human milk samples and that our concerns may extend to other biological media (i.e., serum, urine, or saliva). This study fills a critical gap in the literature on assay performance validation under non-ideal conditions, such as high temperatures. As global temperatures continue to rise, this question will become more pertinent to research integrity if left unaddressed. In light of our findings, we propose that industry standards for ELISA kit shipping and handling should be evaluated to ensure that all kits are being received in an optimal condition.