A General Fluorogenic Assay for Catalysis Using Antibody Sensors

Abstract

A general assay for monitoring catalysis by fluorescence in real time has been developed by use of an antibody sensor. The sensor consists of a product-specific antibody tightly bound to a product analogue covalently labeled with the fluorescent tag acridone. Acridone fluorescence is quenched in the bound state. The reaction is monitored by following the fluorescence increase caused by displacement of the acridonelabeled product from the antibody combining site by the released product. Fluorescence detection of enzymatic hydrolysis of a b-galactoside and butyrate by b-galactosidase and esterase, respectively, are demonstrated. The assay operates by modulation of fluorescence intensity at 445 nm, a signal compatible with currently available instruments measuring in 96-well or 384-well plastic microtiter plates. The method is potentially general and only limited by binding selectivities of the product versus the substrate that can be encountered in antibodies.

Fluorogenic Polypropionate Fragments for Detecting Stereoselective Aldolases

“…From the various methods known for screening catalysis, [18] fluorogenic substrates are the most attractive in terms of simplicity of use and sensitivity, and their use effectively allows the isolation of catalytic antibodies. [19] The fluorogenic polypropionate fragments reported here offer a convenient tool for the isolation of new stereoselective aldolases by screening of catalyst libraries.…”

High Throughput Screening Methods for Asymmetric Synthesis

“…An alternative technique similar to cat-ELISA (catalyst enzyme-linked immuno-sorbent assay) [29] was introduced by Reymond [30] using an "antibody sensor" to follow the reaction progress. This antibody sensor was comprised of a product-specific antibody bound to a fluorescently-labelled product analog.…”

Substrate Arrays for Fluorescence‐Based Enzyme Fingerprinting and High‐Throughput Screening

“…Furthermore, the assay does not reproduce the activity of the enzymes in solution, suggesting that other phenomena than substrate binding and turnover determine the outcome of the assay. (5) and (8) (FIG. 2) with different substitution patterns and enzyme reactive functional groups as shown in the array reference at lower right (cpds numbers from original Ref.…”

“…Initial experiments used acridone‐labeled substrates that were separated and identified at very low concentration by thin‐layer chromatography, however high‐throughput screening was still quite difficult to carry out in this format 7 . We next investigated antibody‐based fluorogenic sensors to selectively reveal product formation 8 . This sensor format was compatible with high‐throughput liquid handling in microtiter plates but required expensive reagents, rendering it unpractical.…”

“…7 We next investigated antibodybased fluorogenic sensors to selectively reveal product formation. 8 This sensor format was compatible with high-throughput liquid handling in microtiter plates but required expensive reagents, rendering it unpractical. We therefore turned our attention to simple fluorogenic substrates and sensors.…”