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High Sensitivity, Wide Dynamic Range, Horseradish Peroxidase (HRP) ELISA Using the LMax Microplate Luminometer (MaxLine Application Note #41)

Evelyn McGown, Ph.D. and Michael Su, M.S.
Molecular Devices Corporation, 8/00


INTRODUCTION
Immunoassays have traditionally employed colorimetric indicators. As such, multiple dilutions of samples were necessary to ensure that they fell within the dynamic range of the assay. Recently, chemiluminescent immunoassays, with greatly improved sensitivity and dynamic range, have become commercially available1. The QuantiGlo TNF-α Immunoassay is a chemiluminescent ELISA designed to measure human TNF-α in cell culture supernate, serum and plasma. The assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-α is pre-coated onto a microplate. Standards and samples are pipetted into the wells, and any TNF-α present is bound by the immobilized antibody. After washing away any unbound substances, horseradish peroxidase (HRP)-linked polyclonal antibody specific for TNF-α is added to the wells. Unbound antibody-enzyme reagent is removed by washing. An enhanced luminol/peroxide substrate solution is added to the wells to produce light. The reaction involves the oxidation of luminol to an excited 3aminophthalate anion (not shown) which decays to the product shown and releases light (Figure 1). Over a wide dynamic range, the amount of light produced is a function of the amount of TNF-α bound in the initial reaction and is easily measured in the LMax microplate luminometer. A brief description of the procedure and results is presented below.


MATERIALS
1. LMax microplate luminometer with SoftMax Pro for LMax (Molecular Devices Corp.)

2. QuantiGlo Human TNF-α Immunoassay kit (R&D Systems1, Cat #QTA50). The kit contains all reagents and standards needed for the assay, including the polystyrene microplate (12 strips of 8 wells@) pre-tre
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