( ally carried out using 125 I-labeled a...)Genetic Reporter Systems

ally carried out using ¹²⁵I-labeled antibodies against the growth hormone. Several manufacturers supply a non-radioactive alternative in the form of ELISA kits, with anti-hGH antibodies bound to the surface of a microtiter plate. First, the hGH from the supernatant of the culture medium binds to the antibody on the plate. Subsequently, the bound hGH is detected in two steps via a digoxigenin- coupled anti-hGH antibody and a peroxidase-coupled anti-digoxigenin antibody. Bound peroxidase is quantified by incubation with a substrate.

Another secreted reporter protein is the SEAP (secreted alkaline phosphatase). This protein is quantified directly by measuring the enzyme activity in the supernatant of the culture medium. Fluorescence and chemiluminescence assays are available for this purpose.

Firefly Luciferase

The enzyme from the North American firefly (Photinus pyralis) catalyzes a bioluminescence reaction. In the luciferase assay, the lysates of transfected cells are incubated with luciferin, molecular oxygen, ATP and Mg²⁺. In the following reaction, the luciferase catalyzes the oxidation of luciferin in oxyluciferin and CO². In this reaction, light with a wavelength of 562 nm is emitted, which then fades rapidly. The light emitted can be measured in a luminometer or in a liquid-scintillation counter. Light emission is proportional to the amount of luciferase in the lysate, thus enabling the indirect quantification of the transfection rate of the reporter gene. The sensitivity of the luciferase assay is further increased by adding co-enzyme A to the reaction preparation, rendering it 1020 times more sensitive than the CAT assay.

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