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The quantification of L-thyroxine (T4) is crucial for regulating metabolism, diagnosing diseases, and monitoring the efficacy of T4 replacement therapy. However, because T4 is a hapten biomarker with a molecular weight of 777 g/mol, conventional immunoassay approaches, including Western blotting and some types of ELISA, have limited accuracy in the quantification of small molecules, including T4. Furthermore, these methods are time-consuming and involve multiple incubation and reaction steps. Therefore, a novel immunoassay method is required for simple and rapid on-site detection of T4. In this study, we expressed a recombinant anti-T4 single-chain variable fragment (scFv) in soluble form using Escherichia coli. The scFv exhibited high T4-binding efficiency, and T4 concentration-dependent titration curves indicated that the sandwich ELISA could detect T4 in the nanogram range. We labeled the scFv using a fluorescent dye for a Quenchbody (Q-body)-based one-pot immunoassay, which yielded a T4 concentration-dependent fluorescent response in 3 min. A comparison of the Q-body-based T4 detection system with ELISA-based methods demonstrated that the ELISA system was more sensitive but the Q-body assay was more rapid. Therefore, both ELISA and Q-body systems can be used depending on the experimental purpose, with the newly developed anti-T4 Q-body system being applicable for convenient in situ immunoassay of T4., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Recently, we reported a novel immunoassay reagent Quenchbody (Q-body): a single chain antibody variable region (scFv) fragment labeled with fluorescent dye, whose fluorescence intensity increases when it binds to the antigen. Here we analyze its working mechanism by immuno- and fluorescence polarization (FP) assays. In an enzyme-linked immunosorbent assay, we found that in the presence of antigen osteocalcin peptide (BGP-C7), more TAMRA-labeled Q-bodies bound to anti-TAMRA antibody than in its absence. Moreover, we found that anti-BGP Q-body with the shortest linker that exhibits the largest antigen-dependency in fluorescence showed the highest binding signal. Similar results were obtained with anti-bisphenol A (BPA) Q-bodies, with inversed correlation with their linker lengths. In the FP assay, when the ATTO 520 labeled Q-body was added with antigen, the Brownian motion of the dye became more active, which resulted in reduced fluorescence anisotropy r. In other words, in the presence of antigen, 1/r showing that the dye mobility is larger than in the absence of its antigen. In addition, anti-BGP Q-body with the largest antigen-dependency in fluorescence showed the highest mobility. Overall, these results clearly suggest that the antigen-dependent fluorescence quenching and recovery of Q-body is caused by the movement of the dye within and around scFv, which moves out of scFv upon binding with its antigen.

Programmed cell death‐ligand 1 (PDL1) is a transmembrane protein that is characterized as an immune regulatory molecule. We recently developed a recombinant single‐chain fragment of variable domain (scFv) against PDL1, which showed high binding efficiency to purified recombinant PDL1 protein. However, at that time, proof‐of‐concept data for the effect of scFv using PDL1‐expressing cells was lacking. In this study, we conducted two kinds of cell‐based immunoassays, western blotting and enzyme‐linked immunosorbent assay, using anti‐PDL1 scFv. The results indicate that scFv can selectively and sensitively detect PDL1 from PDL1 positive human cancer cell lines. Our findings suggest that scFv could be used as a potential PDL1 inhibitor agent and probe for cell‐based immunoassays to detect PDL1. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major pathogen that causes infections and life-threatening diseases. Although antibiotics, such as methicillin, have been used, methicillin-resistant S. aureus (MRSA) causes high morbidity and mortality rates, and conventional detection methods are difficult to be used because of time-consuming process. To control the spread of S. aureus, a development of a rapid and simple detection method is required. In this study, we generated a fluorescent anti-S. aureus antibody, and established a novel fluorescence-linked immunosorbent assay (FLISA)-based S. aureus detection method. The method showed high sensitivity and low limit of detection toward MRSA detection. The assay time for FLISA was 5 h, which was faster than that of conventional enzyme-linked immunosorbent assay (ELISA) or rapid ELISA. Moreover, the FLISA-based detection method was applied to diagnose clinically isolated MRSA samples that required only 5.3 h of preincubation. The FLISA method developed in this study can be widely applied as a useful tool for convenient S. aureus detection. Key points: • A fluorescence-linked immunosorbent assay-based S. aureus detection method • Simultaneous quantification of a maximum of 96 samples within 5 h • Application of the novel system to diagnosis clinical isolates [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a major pathogen that causes infections and life-threatening diseases. Although antibiotics, such as methicillin, have been used, methicillin-resistant S. aureus (MRSA) causes high morbidity and mortality rates, and conventional detection methods are difficult to be used because of time-consuming process. To control the spread of S. aureus, a development of a rapid and simple detection method is required. In this study, we generated a fluorescent anti-S. aureus antibody, and established a novel fluorescence-linked immunosorbent assay (FLISA)-based S. aureus detection method. The method showed high sensitivity and low limit of detection toward MRSA detection. The assay time for FLISA was 5 h, which was faster than that of conventional enzyme-linked immunosorbent assay (ELISA) or rapid ELISA. Moreover, the FLISA-based detection method was applied to diagnose clinically isolated MRSA samples that required only 5.3 h of preincubation. The FLISA method developed in this study can be widely applied as a useful tool for convenient S. aureus detection. Key points: • A fluorescence-linked immunosorbent assay-based S. aureus detection method • Simultaneous quantification of a maximum of 96 samples within 5 h • Application of the novel system to diagnosis clinical isolates [ABSTRACT FROM AUTHOR]

Aim: This cross‐sectional study aimed to examine the diagnostic ability of salivary matrix metalloproteinase (MMP)‐9 lateral flow test (LFT) point‐of‐care (POC) kit and develop an algorithm for diagnosis of periodontitis. Materials and Methods: Through Seoul National Dental Hospital, 137 participants (46 LFT negatives, 91 LFT positives) were recruited. For salivary diagnostics, 150 μl of the unstimulated saliva was applied to LFT‐POC kit. To make a diagnosis of periodontitis, stage II‐IV in modified new international classification system was used. Covariates encompassing age, sex, smoking and obesity were evaluated through face‐to‐face interview. Enzyme‐linked immunosorbent assay was used for quantification of salivary MMP‐9. To develop a diagnostic algorithm, multivariable logistic regression analysis was used. Receiver operating characteristic curve was applied for evaluating diagnostic ability. Results: Diagnostic ability of salivary MMP‐9 LFT‐POC test was 0.82 (sensitivity of 0.92, specificity of 0.72) in total participants. Diagnostic algorithm using POC test resulted in a response equation, that is algorithm score = −3.675 + 2.877*LFT + 0.034*age + 0.121*sex + 0.372*smoking + 0.192*obesity. Diagnostic ability of the algorithm was 0.88 (sensitivity of 0.92, specificity of 0.85) with cut‐off score of 0.589. Conclusions: Salivary MMP‐9 LFT‐POC kit showed appropriate diagnostic ability for periodontitis and would be an efficient tool for screening of periodontitis. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase 9 (MMP9) contributes to several aspects of inflammation and cancer pathology, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant fragment antigen-binding (Fab)-type anti-MMP9 antibody in Escherichia coli with high purity within five days and confirmed the nanomolar order of antigen-binding efficiency of the recombinant Fab. Moreover, we optimized the experimental time for performing enzyme-linked immunosorbent assay (ELISA), and decreased the reaction time from the conventional 20.5 h to 3.5 h. The rapid and sensitive MMP9 detection system developed in this study can be applied to a range of applications, including the diagnosis of diseases with MMP9 overexpression including inflammatory and cancer-related diseases. • A recombinant Fab against MMP9 was produced in Escherichia coli with high purity within five days. • The nanomolar order of antigen-binding efficiency of anti-MMP9 Fab was confirmed using a rapid ELISA, which required only 3.5 h. • The rapid and sensitive MMP9 detection system can be applied to the diagnosis of diseases with MMP9 overexpression. [ABSTRACT FROM AUTHOR]