Enzyme-linked immunosorbent assay procedure at higher temperature enhances speed of the assay

Abstract

In this protocol, we report substantial decrease in time for an enzyme-linked immunosorbent assay (ELISA) technique by performing it at elevated temperature on an activated surface. The activated plate was able to bind covalently anti-human IgG at 50 degree Celsius over 40 min to form a solid phase. Blocking, human IgG and antibody–enzyme conjugate binding were performed on this solid phase at 40 degree Celsius in 40 min, 50 degree Celsius in 45 min and 50 degree Celsius in 40 min, respectively. The ELISA readings obtained were 1.5-fold higher than those obtained at 37degree Celsius over similar incubation times. Total IgE was also determined by the heat-mediated ELISA (HELISA) technique in less than 3 h without compromising sensitivity and specificity of the assay. As surface activation can be achieved by application of a simple technique, the HELISA procedure could be a powerful alternative to conventional ELISA.