Robust technology is required to underpin rapid point-of-care and in-field diagnostics to improve timely decision making across broad sectors. An attractive strategy combines target recognition and signal generating elements into an “active” enzyme-switch that directly transduces target-binding into a signal. However, approaches that are broadly applicable to diverse targets remain elusive. Here, an enzyme–inhibitor switch sensor was developed by insertion of non-immunoglobulin Affimer binding proteins, between TEM1-β-lactamase and its inhibitor protein, such that target binding disrupts the enzyme–inhibitor complex. Design principles for a successful switch architecture are illustrated by the rapid (min), simple (wash-free), and sensitive (pM) quantification of multimeric target analytes in biological samples (serum, plasma, leaf extracts), across three application areas. A therapeutic antibody (Herceptin), protein biomarker (human C-reactive protein), and plant virus (cow pea mosaic virus) were targeted, demonstrating assays for therapeutic drug monitoring, health diagnostics, and plant pathogen detection, respectively. Batch-to-batch reproducibility, shelf-life stability, and consistency with validated enzyme-linked immunosorbent assay analysis confirm that the principle of an Affimer–enzyme–inhibitor switch provides a platform for point-of-care and in-field diagnostics.

Therapeutic antibodies are the fastest growing class of drugs in the treatment of cancer, and autoimmune and inflammatory diseases that require the concomitant development of assays to monitor therapeutic antibody levels. Here, we demonstrate that the use of Affimer nonantibody binding proteins provides an advantage over current antibody-based detection systems. For four therapeutic antibodies, we used phage display to isolate highly specific anti-idiotypic Affimer reagents, which selectively bind to the therapeutic antibody idiotype. For each antibody target the calibration curves met US Food and Drug Administration criteria and the dynamic range compared favorably with commercially available reagents. Affimer proteins therefore represent promising anti-idiotypic reagents that are simple to select and manufacture, and that offer the sensitivity, specificity and consistency required for pharmacokinetic assays.

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Diacylamide precursors of amide impurities have been detected in spectral tests of the addition of carboxylic acids to N-tert-butylacylketenimines. Variations in the product distribution with an inefficient acylketenimine in media of differing acidity suggest that diacylamide formation involves a second intermediate adduct that does not convert rapidly to the desired enol ester. A high free acid concentration results in interception of the intermediate to give acid anhydrides. Partial deuterium incorporation a t the vinylic position of the enol esters indicates that intramolecular 0,O-acyl migration is relatively slow for the adducts of N-tert-butyiacylketenimines, and possible substituent influences are discussed. The preparation of p-acyloxy-N-tertbutylcrotonamide enol ester acylating agents from iV-tertbutyl-Gmethylisoxazolium perchlorate succeeds with unprotected hydroxyl groups and the carboxamide function of glutamine. However, amide dehydration was observed in the case of asparagine and competing azlactone formation was detected with benzoylleucine. Crystalline esters were not obtained with Z-Ala-OH, Z-Tyr-OH, and Z-Met-OH. Test couplings have established compatibility of the enol esters with unprotected hydroxyl groups in the amine component but results are not markedly improved relative to iV-ethyl-5-phenylisoxazolium 3'-sulfonate. A new side reaction, condensation of the amine component with the coupling by-product, is shown to be a likely source of impurities in the use of the esters of hindered carboxylic acids. The original zwitterionic isoxazolium salt reagent is much less susceptible to the side reaction.(8) Other workers# have reported that the enol ester of 3,5-diamino-6-chloropyrazine-2-carboxylic aaid similarly gives the Naert-butylamide on treatment with triethylamine, hut a compound assigned the diacylamide structure was obtained with methoxide in polar media. If the latter structure is correct, it remains unclear why fragmentation took place only in the former medium and what special factors account for imide stability in the latter experiment. (9) I<. L. Shepard, W. Halczenko, and A. J. Cragoe, Jr., Tetrahedron Ldt., 4757 (lQ69).ADDITION O F ACIDS TO N-led-BUTYLACYLKETENIMINES J. Ory. Chem., Vol. 38, No. 25, 1073 4289 R1 CMe, + R3C02--la, R1 "Me; % = H b, R1= Ph; & P H c,R1 =Me,C;&-H e, Rl = H &=Me f, Rl 3 Ph; &=Me d, R1= H % H NHCMe, NCMe, %-ON 2 RIYoH I Rz +OCOR3 I NCMe3 J 3 5 6J.

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