Heat-mediated enzyme-linked immunosorbent assay procedure on a photoactivated surface.
The human burden of infectious and allergic disease has necessitated rapid screening and measurement of antibodies in sera. Here, we report an enhancement of speed and sensitivity of an enzyme-linked immunosorbent assay (ELISA) technique by performing it at elevated temperature on an activated surface. The activated polymer was able to bind covalently anti-human IgG at 50 degrees C over 40 min to form a solid phase. The covalently bound solid phase was stable enough to withstand subsequent ELISA steps at elevated temperature. Thus, when blocking, human IgG and antibody-enzyme conjugate binding were performed on this solid phase at 40 degrees C in 40 min, 50 degrees C in 45 min and 50 degrees C in 40 min, respectively. The ELISA readings obtained were 1.5-fold higher than those obtained at 37 degrees C over similar incubation times. Total IgE was also determined by the heat-mediated ELISA (HELISA) technique in less than 3 h and gave similar ELISA values to those obtained by the conventional procedure carried out for 18 h on an untreated surface. A stable covalently bound solid phase is a prerequisite for the HELISA technique and was further verified when a solid phase prepared through adsorption onto an untreated surface showed less than half the ELISA absorbance values obtained with the activated surface at elevated temperatures. As surface activation can be achieved by application of a simple technique, the HELISA procedure could be a powerful alternative to conventional ELISA.
Bora
Utpal
U
Institute of Genomics and Integrative Biology (CSIR) (formerly Centre for Biochemical Technology), Mall Road, Delhi-110007, India.
Kannan
Krishnamoorthy
K
Nahar
Pradip
P
D000818:Animals; D019411:Clinical Laboratory Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006041:Goats / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D011137:Polystyrenes; D013997:Time Factors
D007074:Immunoglobulin G; D011137:Polystyrenes; D007073:Immunoglobulin E