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Current diagnosis of acute myocardial infarction involves quantification of circulating cTn levels. This work endeavoured to generate and enhance recombinant antibody fragments targeting various epitopes on the N- and C-terminals of the cTnI molecule, thereby facilitating highly sensitive detection of the troponin molecule. From this approach, two anti-cTnI scFv antibodies were successfully selected using either phage display or structural reformatting of full length anti-cTnI IgG. Their antibody binding affinity was further optimised via chain shuffling and/or site directed mutagenesis, resulting in scFv with heightened sensitivity when compared to the wild-type scFv. If used in conjunction with existing anti-mid fragment cTnI antibodies, these N- and C- terminal-targeting scFvs show high potential for the enhancement of current cTnI detection assays by limiting the effects from cTnI degradation or troponin complex formation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antibody-based diagnostics and therapeutics have huge commercial value. However, applications of antibodies are often limited by instability, particularly for recombinant antibody formats. This paper describes the conversion of a single-chain variable fragment (scFv) antibody to a single-chain antibody fragment (scAb) with notably improved stability characteristics. This scAb retains antigen-binding activity (i) at high temperature (up to 60 °C), (ii) in guanidine hydrochloride (GdnHCl, up to 1 M), and (iii) when stored at 37 °C for 6 months. However, limited improvement was observed when the original scFv was converted to a larger fragment antigen-binding (Fab) format. Certain Cys-to-Ala mutations in the third complementarity determining region of the antibody heavy chain (CDRH3) also led to stability improvements. Our findings indicate that the stability of an antibody derivative depends on its format and on the positions of cysteines in the CDRs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Laccase from two different source organisms, Myceliophthora thermophila and Trametes hirsuta, were subjected to chemical modification in solution by (i) two bifunctional reagents, ethylene-glycol-N-hydroxy succinimide (EGNHS) and glutaraldehyde and (ii) by the monofunctional citraconic anhydride. The untreated and chemically modified forms of both enzymes were then immobilised onto three different types of mesoporous silicate (MPS) particle (MCM, CNS and SBA-15). Thermal stabilities of native, modified-soluble and immobilised laccases were then evaluated. Although the two laccases have similar lysine contents, those of M. thermophila are clearly more amenable to chemical modification. Treatment of the M. thermophila enzyme with EGNHS led to a 8.7-fold increase in thermal stability over the free soluble enzyme while glutaraldehyde gave a 5.7-fold increase. Increased activity of M. thermophila laccase occurred only with citraconic anhydride modification (a 3-fold increase), while the glutaraldehyde modification marginally increased the activity of the T. hirsuta enzyme (by 1.2-fold). Upon immobilisation onto MPS, the greatest increase in stability was for the glutaraldehyde-treated M. thermophila preparation on SBA-15 (24-fold over the soluble enzyme). Chemical modification of laccase from T. hirsuta with both glutaraldehyde and EGNHS gave only a 2-fold increase in stability, increasing >4-fold upon immobilisation onto SBA-15 and MCM-41/98., (Copyright © 2010 Elsevier Inc. All rights reserved.)