Journal article Open Access

A novel enzyme-linked immunosorbent assay procedure in one hour by pressure incubation


Authors: Pradip Nahar ### Abstract In this protocol, we describe a unique ELISA method for the detection of an antigen or antibody, in which ELISA steps are carried out by pressure incubation instead of conventional thermal incubation. Pressure-mediated ELISA (PELISA), carried out in 1 h shows more than a 2-fold increase in absorbance value than the control experiment carried out at the same time and temperature without applying pressure. Estimation of analyte by the 1-h PELISA method gives similar absorbance value to that obtained by 3-h heat-mediated ELISA (HELISA) on an activated surface. As PELISA is sensitive, specific, and reproducible, it could be an excellent alternative to HELISA or conventional ELISA procedure. ### Introduction The enzyme-linked immunosorbent assay (ELISA) technique is the back-bone of the diagnostic assay for detection of infectious and allergic diseases. Conventional ELISA is a time-consuming 18 h procedure. Earlier, we have shown that photoactivated microtiter plate (1) can reduce ELISA timing to around 7.5 hours by binding an antigen or antibody by covalent bond rather than adsorption (2). We have further shortened the ELISA timings to around 3 h by doing it at higher incubation temperature on an activated polystyrene microtiter plate with results comparable to conventional ELISA carried out in 18 h (3). In this protocol, we reported a novel and rapid method for ELISA technique in which ELISA steps are carried out in one hour under pressure (4). ### Materials 1. 1-fluoro-2-nitro-4-azidobenzene (FNAB, IUPAC nomenclature: 4-azido-1-fluoro-2-nitrobenzene) can be purchased from Apollo scientific ltd, UK (cat no. 248-878-6). Alternatively, it can be made from 4-fluoro-3-nitroaniline by a simple diazotization reaction as described earlier (1,5) CAUTION. FNAB is explosive and should be handled with care, especially when using large quantities. We did not encounter any untoward incident while working with FNAB in last 12 years. - Polystyrene microtiter plates (Greiner Labortechnik, Germany). - Anti-human IgG, (Sigma Aldrich, cat. no. I3382) CAUTION Store all reagents at 2-8ºC. If slight turbidity occurs upon prolonged storage clarify the solution by centrifugation before use. - Human IgG, (Sigma Aldrich, cat. no. I4506). - Anti-human IgG-HRP conjugate, (Sigma Aldrich, cat. no. A8419 ). - Rabbit IgG, (Sigma Aldrich, cat. no. I4508) - Bovine serum albumin (BSA), Sigma (USA). CRITICAL BSA solution should be filtered prior to use to avoid microbial contamination. - o-phenylenediamine dihydrochloride (OPD), (Sigma Aldrich, USA cat. no. P1526). Store in cool place. Recommended storage temperature: -20 °C; Keep container tightly closed. CAUTION Avoid contact with skin and eyes. Avoid formation of dust and aerosols. - Phosphate buffered saline (PBS) was prepared by mixing 0.85% Sodium chloride to 0.01 M phosphate buffer (pH 7.2). To make 0.01 M phosphate buffer add 1.217 g disodium phosphate, 0.379 g monosodium phosphate. Add distilled water to make 1 liter solution. - Washing buffer was made by adding 0.1% Tween 20 to PBS. - Substrate -dye buffer was prepared by mixing 12 ml of citrate buffer (0.025M citric acid and 0.05M sodium phosphate dibasic , pH 5), 5 µl of hydrogen peroxide (30% w/v), and 4 mg of o-phenylenediamine dihydrochloride. - Stop Solution- 5% sulphuric acid ### Equipment 1. Refrigerator (Godrej, India) - UV Stratalinker 2400 (Stratagene, USA) - Polystyrene microtiter plates (Greiner Labortechnik, Germany) - ELISA reader (Biorad iMark™ Microplate Reader, USA). - Pressure cooker (Hawkins Cookers Ltd., Mumbai, India). - 1-m pressure tube made of natural rubber having i.d. 6 mm and o.d. 16 mm (Local supplier, Delhi, India) - Pressure gauge (Laser Gasses India, New Delhi, India) - Laboratory cylinder filled with argon gas (Laser Gasses India, New Delhi, India) ### Procedure 1.Activation of polystyrene surface The wells of a polystyrene plate were activated by coating with FNAB followed by photoactivation by UV light for 15 min at a wavelength of 365 nm as described (1). - CRITICAL We have decreased UV exposure time for activation of microtiter plate from 20 minutes to 15 minutes without any significant difference in results. 2.Designing of a pressure incubator Pressure incubator was designed from a 5-L pressure cooker (Domestic kitchen pressure cooker, Hawkins Cookers Ltd., Mumbai, India). The pressure regulator (vent weight) from the lid was removed and one end of a 1-m pressure tube (or laboratory high vacuum tube) was inserted through the vent pipe of the pressure cooker; the other end of the tube was connected to a three-way connector. One of the outlays of the connector is connected to a pressure gauge through a rubber tube and further connected to a laboratory argon cylinder. Third outlay of the connector was used for release of excess pressure and also release of pressure after each ELISA step. All PELISA steps were carried out inside the pressure incubator. 3.Detection of analyte - PELISA steps in the pressure incubator In the activated and untreated wells of a polystyrene microtiter plate capture molecule (anti-human IgG) was added and the plate was kept inside the pressure incubator. The pressure cooker was closed and a pressure of 1.5 × 105 Pa was applied by opening the gas cylinder. In this pressure the plate was kept for 10 minutes after which the pressure was released. During this time capture molecule was immobilized in the activated and untreated wells of a polystyrene microtiter plate. After wshing, blocking was carried out similarly by applying pressure of 1.5 × 105 Pa for 10 minutes. After the stipulated time period, the pressure was released and the plate was washed by washing buffer. Binding of analyte was carried out by adding the analyte to the wells and incubating the plate inside the pressure incubator 20 minutes by applying pressure of 2 × 105 Pa. Binding of secondary antibody-conjugate was also carried out similarly by applying pressure of 2 × 105 Pa for 20 minutes. After all the four ELISA steps were carried out inside the pressure incubator, the color development was done by adding 100 µl of substrate- dye buffer at room temperature outside pressure cooker. Color development was stopped by adding 20 µl of 5% sulfuric acid and absorbance was recorded at 490 nm. - CRITICAL - CAUTION Pressure must be carefully monitored. Excess pressure may lead to explosion. Applying and release of pressure should be slow/ gradual and should not be abrupt. ### Timing 1. Activation of polystyrene surface –approx. 25 minutes - a) Coating of FNAB to polystyrene surface 5-10 minutes - b) UV light exposure- 15 minutes - c) Washing and drying – 5 minutes - Detection of Analyte – 1 hour + washing time after each step - a.) Immobilization of capture molecule (0.25 μg/100 μL 0.01 M carbonate buffer, pH 9.6/well ) onto the photoactivated surface-10 minutes by applying pressure of 150000 Pa (0.15 megapascals) - b.) Blocking (200 µl/ well of 2% BSA) -10 minutes by applying pressure of 150000 Pa (0.15 megapascals) - c.) Incubation of analyte ( 0.25 μg/100 μL 0.01 M PBS, pH 7.2/well) -20 minutes by applying pressure of 200000 Pa (0.20 megapascals) - d.) Incubation time of secondary antibody – enzyme conjugate (100 μLof 1:5000 v/v dilution /well) – 20 minutes by applying pressure of 200000 Pa (0.20 megapascals). ### Anticipated Results - Pressure-mediated ELISA showed 2-3-fold increase in absorbance value than control experiment carried out in the same time and temperature without applying pressure (fig 1). - PELISA is as good as 18-h conventional ELISA or 3-h HELISA. - PELISA is found to reduce assay time to 1 h without affecting specificity or sensi-tivity and could be a good substitute for immunoassay procedures widely used in clinical and research laboratories. ### References 1. (a) Nahar P. Light-Induced Activation of an Inert Surface for Covalent Immobilization of a Protein Ligand, Community Contributed Protocol Exchange 26/11/2013 doi:10.1038/protex.2013.086. - (b) Nahar, P., Wali, N.M., Gandhi, R.P., 2001. Light-induced activation of an inert surface for covalent immobilization of protein ligand. *Anal. Biochem*. 294, 148. - (a) Nahar P., Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures. Community Contributed Protocol Exchange 05/12/2013 doi:10.1038/protex.2013.090. - (b) Bora, U., Chugh, L., Nahar, P., 2002. Covalent immobilization of proteins onto photoactivated polystyrene microtiter plates for enzyme-linked immunosorbent assay procedures. *J. Immunol. Methods* 268, 171. - (a) Nahar P., Enzyme-linked immunosorbent assay procedure at higher temperature enhances speed of the assay. Community Contributed Protocol Exchange 09/12/2013 doi:10.1038/protex.2013.091. - (b) Bora, U., Kannan, K. and Nahar, P. (2004) Heat-mediated enzyme-linked immunosorbent assay (HELISA) Procedure on a photoactivated surface. *Journal of Immunol Methods*. 293: 43-50. - (c) Nahar P and Bora U. Rapid Heat-Mediated Method for Enzyme Linked Immunosorbent Assay Procedure. European Patent (EP) 1608971. - (d) Kumar S, Ghosh L, Kumar S, Ghosh B, Nahar P (2007) A rapid method for detection of cell adhesion molecules (CAMs) on human umbilical vein endothelial cells (HUVECs). *Talanta* 73: 466–470. - (e) Sharma P, Gupta B, Basir S F, Das H R, Nahar P (2008). Rapid and sensitive detection of autoantibody in rheumatoid arthritis patients by heat-mediated ELISA. *Clinical Biochemistry* 41: 97–102 - (a) Kannoujia DK and Nahar P (2009) Pressure: a novel tool for enzyme-linked immunosorbent assay procedure. *BioTechniques* 46: 468–472. - (b) Nahar Pradip and Kumar Dileep. A Novel Pressure -Induced Immunoassay Procedure. Patent pending. - Naqvi, A., Nahar, P., 2004. Photochemical immobilization of proteins on microwave-synthesized photoreactive polymers. *Anal. Biochem*. 327, 68. ### Figures **figure 1: Comparison of PELISA with conventional ELISA and HELISA procedures**. ![Figure 1]( "Figure 1") Comparison of PELISA with conventional ELISA and HELISA procedures. Conventional ELISA steps involve 11 h at 4 degree Celsius ; 1 h at 37 degree Celsius , 3 h at 37 degree Celsius, and 3 h at 37 degree Celsius. HELISA steps involve 45 min at 50 degree Celsius, 40 min at 40 degree Celsius, 45 min at 50 degree Celsius and 45 min at 50 degree Celsius. PELISA steps are 10 min at 1.5 × 105 Pa, 10 min at 1.5 × 105 Pa, 20 min at 2.0 × 105 Pa, and 20 min at 2.0 × 105 Pa pressure. A control ELISA experiment was carried out at the same time as in PELISA without applying pressure. Human IgG was used as positive test sera whereas rabbit serum was a negative control. *Source: [Protocol Exchange]( (2013) doi:10.1038/protex.2013.092. Originally published online 10 December 2013*. ### Associated Publications **Pressure: a novel tool for enzyme-linked immunosorbent assay procedure**. Dileep Kannoujia and Pradip Nahar. BioTechniques 46 (6) 468 - 472 [doi:10.2144/000113118]( ### Author information Pradip Nahar, Nahar's Lab Correspondence to: Pradip Nahar (
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