Micro-ELISA plate

One of the laboratory tests used in the identification of patients with heparin-induced thrombocytopenia, employs a micro-ELISA plate coated with purified PF4-heparin complex. Upon incubation of patient s plasma, if antibody is present, it binds to the PF4-heparin complex. The bound antibody is detected with goat antihuman antibody coupled to peroxidase, followed by incubation with substrates o-phenylenediamine/H202 (Chapter 8). 

ELISA was performed as described previously Briefly, each well of a 96-well microtiter plate was coated with 100 pi of the sample to be tested in PBS, blocked with 0.5% gelatin, and washed three times with PBS containing 0.05% Tween 20 (washing buffer). The wells were incubated with 0.1 ml of anti-CMA antibody, 3F5, (1 pg/mL) dissolved in washing buffer for 1 hr. The wells were then washed with washing buffer three times and reacted with HRP-conjugated anti-mouse IgG antibody, followed by reaction with 1,2-phenylenediamine dihydrochloride. The reaction was terminated by addition of 0.1 ml of 1 M sulfuric acid, and the absorbance at 492 nm was read by a micro-ELISA plate reader. 

Fig. 20c. 1. ELISA assay, (a) Antibodies to the drug of interest are secured to a solid substratum such as a test tube or micro-well plate. The sample containing the analyte antigen is added to the reaction surface, (b) After the analyte has bound to the antibody, the vessel is rinsed to remove unbound antibody. A second antibody to the analyte is added. This antibody has a bound enzyme which has been chosen because its reaction produces a colored product which can be detected spectrophotometrically. (c) After this second antibody has bound to the first antibody-antigen complex, the surface is again rinsed to remove unbound-antibody enzyme. The enzyme substrate is added in sufficient excess such that the rate of product formed is proportional to the amount of enzyme present. The enzyme-linked assays are very sensitive, since each enzyme can rapidly catalyze thousands of substrate to product reactions.

This author and coworkers at Beckman Coulter first described the use of a low form 96-well plastic microplate for automated micro-ELISA immunoassays (Matson et al., 2001). The polypropylene plate was first modified by a radiofrequency plasma amination process (Matson et al., 1995) followed by conversion to an acyl fluoride surface chemistry for rapid covalent attachment of biomolecules. Proteins (1 to 2 mg/mL) were prepared in 50 mM carbonate buffer, pH 9, containing 4% sodium sulfate (to improve spot uniformity) and printed using a conventional arrayer system. Approximately 200-pL droplets of monoclonal antibodies (anti-cytokine) were deposited into the bottom of the microwells using a Cartesian PS7200 system equipped... 

Read plate on a micro-ELISA reader using a test wavelength of 570 nm and a reference wavelength of 630 nm. 

Coat ELISA plate wells overnight at 4°C with 100 pL of the appropriate dilution ofrlgG in CB (see Note 2). Lise adjustable (micro)pipette(s) to prepare antibody dilutions and to coat microwells. 

The test can be performed in 2-2.5 h and as it is in a micro titre plate it is quite economical and can be automated. The woik flow is very similar to the ELISA test (Sigma-Aldrich, 2014b Taskila, Tuomola, Kronlof, Neubauer, 2010). 

Most of the immunoassay methods for bacterial detection are currently performed in solid-phase ELISA format conducted on micro-titer plates. Flow inununofiltration assay, can be an excellent alternative for detection of bacterial pathogens because it not only overrides the effects of diffusional limitations in conventional ELISA, but also allows the concentration of bacteria on the membrane by filtering a large volume of... 

Voller, A. Bidwell, D. E. Bartlett, A. "The Enz3rme-linked Immunosorbent Assay (ELISA), A Guide with Abstracts of Micro-Plate Applications" Dynatech Publication, Nuffield Laboratories of Comparative Medicine Zoological Soc. of London Reagents Park. Campbell, G. S. Mageau, R. P. Schwab, B. Johnston, R. W. Antimicrob. Agents Chemother. 1984, 25, 205-11. 

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