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Pressure: a novel tool for enzyme-linked immunosorbent assay procedure
Dileep Kumar Kannoujia Pradip Nahar
Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research, Delhi, India
BioTechniques, Vol. 46, No. 6, May 2009, pp. 468–472
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The enzyme-linked immunosorbent assay (ELISA) technique is the backbone of the diagnostic assay for detection of infectious and allergic diseases. Here, we demonstrate a unique ELISA method for the detection of an antigen or antibody, in which ELISA steps are carried out under pressure 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 total IgE by the 1-h PELISA method gives similar absorbance value (1.081±0.031, 823.12 IU) to that obtained by 3-h heat-mediated ELISA on an activated surface (HELISA) (1.165±0.037, 810.96 IU). Since PELISA is sensitive, specific, and reproducible (intra- and interassay CVs were 6.47% and 9.65%, respectively), it could be an excellent alternative to HELISA or conventional ELISA procedures.

The enzyme-linked immunosorbent assay (ELISA) is a very useful technique for clinical diagnostics, detection of biothreat agents, high-throughput screening of drugs and drug targets, and biomedical research (1,2,3,4,5). ELISA is conventionally carried out by overnight incubation of an antigen or an antibody at low temperature (generally 4–8°C) on a microtiter plate through adsorption. This makes conventional ELISA a time-consuming procedure. Some commercial entrepreneurs have used large excess of the exogenous binding partner, monoclonal antibody, or sensitive tags—such as fluorescent, chemiluminiscent, or radioactive moieties—to shorten ELISA timing (6,7,8). In other approaches, a reduction in ELISA timing was achieved by binding an antigen or antibody to an activated solid support by covalent bond rather than adsorption (9,10,11,12,13). Recently, Bora et al. have further shortened the ELISA timings to 3 h by doing it at higher incubation temperature on an activated polystyrene microtiter plate (HELISA) with results comparable to conventional ELISA carried out in 18 h (14). In this communication, we report a novel and rapid method for ELISA technique in which ELISA steps are carried out under pressure.

Pressure-mediated ELISA (PELISA) was carried out on a microtiter plate that binds a protein molecule by covalent bonding. To enable covalent binding, a polystyrene microtiter plate (Greiner Bio-One, Frickenhausen, Germany) was activated by a photolinker, 1-fluoro-2-nitro-4-azidobenzene (FNAB). FNAB was prepared from 4-fluoro-3-nitroaniline (Sigma-Aldrich, St. Louis, MO, USA) by a simple diazotization reaction as described previously (15). Activation was done by coating the wells of the microtiter plate with FNAB and exposing them to 365-nm wavelength UV light as reported earlier (16). PELISA was carried out in a tightly closed 5-L pressure cooker (Hawkins Cookers Ltd., Mumbai, India). The pressure regulator from the lid was removed and one end of a 1-m pressure tube (natural rubber, i.d. 6mm, o.d. 16 mm) was inserted through the vent pipe of the pressure cooker; the other end of the tube was passed through a pressure gauge (Laser Gasses India, New Delhi, India) and connected to a laboratory argon cylinder.

Pressure-mediated ELISA steps were optimized by doing each step at a fixed time at variable pressure and subsequent steps by conventional ELISA procedure. In optimization experiments, goat anti–human IgG (0.25 μg/100 μL 0.01 M carbonate buffer, pH 9.6/well; Sigma-Aldrich) was poured into the activated and untreated wells of a polystyrene microtiter plate and the plate was put inside the pressure cooker. A pressure of 1.0×105–3.0×105 Pa (measured by the pressure gauge) was applied to the closed pressure cooker by releasingargon gas and was maintained for 10 min. After the stipulated time, pressure was released slowly. Pressure-mediated blocking was optimized by adding 2% BSA (Sigma-Aldrich; 200 μL/well) and subjecting the plate to 1.0×105–3.0×105 Pa of pressure for 10 min. Binding of human IgG (0.25 μg/100 μL 0.01 M PBS, pH 7.4/well; Sigma-Aldrich) and anti–human IgG-peroxidase conjugate (100 μL 1:5000 v/v dilution solution in 0.01 M PBS, pH 7.4/well; Sigma-Aldrich) were optimized by applying variable pressure (1.0×105–3.0×105 Pa) for 10 min. After each step, the plate was washed with washing buffer (0.05% Tween-20 in 0.01 M PBS). Color was developed with the addition of 100 μL substrate-dye solution [2.5 mg o-phenylene-diamine dihydrochloride (Sigma-Aldrich) and 2.5 μL H2O2 in 6 mL 0.2 M citrate buffer, pH 5.0]. The reaction was terminated by adding 20 μL stop solution (5% H2SO4) and absorbance was recorded at a wavelength of 490 nm in an ELISA reader (SpectraMax; Molecular Devices, Sunnyvale, CA, USA) All the experiments were carried out at ambient temperature (25°C±2). Results showed that immobilization of antigen and blocking were best achieved by applying a pressure of 1.5×105 Pa; however, increasing pressure up to 2.0×105 Pa showed a similar absorbance value (Figure 1, A and C). In contrast to other steps, applied pressure had less influence on the blocking step. This was verified by doing the blocking experiments under pressure (absorbance value 0.610±0.019) and without pressure (absorbance value 0.615±0.024). Similar absorbance values were found in experiments carried out with blocking and without blocking. In all these cases, appreciable change in absorbance value in negative control sera (rabbit IgG; Sigma-Aldrich) was not observed. Pressure-mediated primary and secondary antibody binding were found to be more sensitive and both these steps require a pressure of 2.0×105 Pa beyond which a sharp decrease in absorbance value is observed (Figure 1, E and G). Similarly, time was optimized by doing each ELISA step at an optimized pressure at different time. Results showed that 10 min was sufficient for immobilizing antigen onto the solid surface as well as blocking, whereas maximum binding of primary and secondary antibody each occurred in 20 min (Figure 1, B, D, F, and H).

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