Notes & Tips Microwave-mediated enzyme-linked immunosorbent assay procedure Pradip Nahar ⇑ , Utpal Bora 1 , Gainda L. Sharma, Dileep Kumar Kannoujia Institute of Genomics and Integrative Biology (CSIR), Delhi 110 007, India article info Article history: Received 24 May 2011 Received in revised form 23 September 2011 Accepted 29 September 2011 Available online 6 October 2011 Keywords: ELISA Microwave-mediated ELISA Microwave Immunoassay abstract Here we demonstrate a novel microwave-mediated enzyme-linked immunosorbent assay (MELISA) method that has dramatically reduced the enzyme-linked immunosorbent assay (ELISA) timing to less than 5 min with a result comparable to that obtained by 18-h conventional ELISA. Efficacy of the MELISA procedure is demonstrated by detecting human immunoglobulin G (IgG), rabbit IgG, human immuno- globulin E (IgE), human interleuken 1b (IL-1b), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. MELISA could be an excellent substitute for time-consuming conventional ELISA for rapid diag- nosis of diseases in cases of medical urgency, outbreak of infectious diseases, and screening of samples in blood banks or emigration counters. Ó 2011 Elsevier Inc. All rights reserved. The enzyme-linked immunosorbent assay (ELISA) 2 technique is an important tool for diagnosis of infectious and allergic diseases [1– 3], detection of biothreat agents [4], and high-throughput screening of drugs and drug targets [5,6]. Conventionally, ELISA is a time- consuming procedure. It has been found that ELISA timing can be re- duced using an activated solid support for covalent binding of an antigen or antibody [3,7]. Several authors have shortened the ELISA procedure to 3h by performing heat-mediated ELISA (HELISA), where ELISA was carried out at 50 °C on an activated polystyrene microtiter plate [8–10]. Kannoujia and Nahar [11] used pressure en- ergy instead of thermal incubation to reduce the ELISA timing to 1 h, whereas Sharma and Nahar [12] further reduced the ELISA timing to 40 min by employing ultrasound energy. In all of these processes, the ELISA procedure was shortened without using any additional or expensive reagent such as monoclonal antibody or sensitive tags. van Dorp and coworkers attempted to shorten the ELISA timing by microwave irradiation; nevertheless, the time gain for such ELISA was not attractive given that they did it in 2 h [13]. In this study, we demonstrate an ultrafast ELISA procedure in which the steps were carried out by microwave irradiation in a domestic microwave oven (BPL–Sanyo, India) operating at a fre- quency of 2450 MHz. Efficacy of the microwave-mediated ELISA (MELISA) procedure was demonstrated by detecting human immu- noglobulin G (IgG), rabbit IgG, human immunoglobulin E (IgE), hu- man interleuken 1b (IL-1b), Entamoeba histolytica antibody, and Aspergillus fumigatus antibody. The amoebic antigen and A. fumiga- tus antigen were obtained from the culture as per the published procedure [14]. E. histolytica antibody was raised in New Zealand white rabbits, and consent from an animal ethics committee was obtained before the experiment. A. fumigatus antibody was ob- tained from the sera of patients (n = 9) suffering from allergic bron- chopulmonary aspergillosis (ABPA) and used as positive test sera, whereas pooled sera samples from healthy volunteers (n = 9) was used as a negative control. Patients were from the V.P. Chest Insti- tute outpatient center in Delhi, India. Informed consent was ob- tained from each patient and normal individual. Human IgG, rabbit IgG, and their antibodies and antibody conjugates were pur- chased from Sigma (USA). ELISA kits of human IgE and human IL-1b were purchased from Bethyl Laboratories (USA) and BD Biosciences (USA), respectively. For coating a well, 1 lg of antigen or antibody (1 ll for anti-human IL-1b) was diluted in 100 ll of sodium phos- phate buffer saline (pH 7.2, 0.01 M). Antibody and conjugates were used with the optimum titer value or according to the supplier’s protocol. MELISA was performed in the wells of a polystyrene mod- ule (Dynatech, USA) or a polystyrene microtiter plate (Greiner Bio One, Germany), each of which was activated by coating the wells with 1-flouro-2-nitro-4-azidobenzene (FNAB) and exposing them to ultraviolet (UV) radiation at 365 nm according to the published procedure [15]. Conventional ELISA was carried out by a procedure involving the steps of (i) antigen or antibody coating into the wells by over- night incubation (12 h) at 4 °C, (ii) blocking the unbound surface with 200 ll of 2% (w/v) bovine serum albumin (BSA) solution in 2 h at 37 °C, (iii) binding of human IgG, rabbit IgG, human IgE, hu- man IL-1b, E. histolytica antibody, and A. fumigatus antibody in 2 h 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.09.029 ⇑ Corresponding author. Fax: +91 11 27667471. E-mail address: pnahar@igib.res.in (P. Nahar). 1 Current address: Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781 039, India. 2 Abbreviations used: ELISA, enzyme-linked immunosorbent assay; MELISA, micro- wave-mediated ELISA; IgG, immunoglobulin G; IgE, immunoglobulin E; IL-1b, interleuken 1b; BSA, bovine serum albumin; HRP, horseradish peroxidase. Analytical Biochemistry 421 (2012) 764–766 Contents lists available at SciVerse ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio