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Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we used preparative gel electrophoresis to develop a capture format for WB. Fractions with soluble, size-separated proteins facilitated parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). This pipeline provided the means for large-scale implementation of antibody validation concepts proposed by an international working group on antibody validation (IWGAV).
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http://dx.doi.org/10.1038/s41592-018-0179-8 | DOI Listing |
Elife
September 2025
Chinese Academy of Medical Science Oxford Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
Influenza virus neuraminidase (NA) is a crucial target for protective antibodies, yet the development of recombinant NA protein as a vaccine has been held back by instability and variable expression. We have taken a pragmatic approach to improving expression and stability of NA by grafting antigenic surface loops from low-expressing NA proteins onto the scaffold of high-expressing counterparts. The resulting hybrid proteins retained the antigenic properties of the loop donor while benefiting from the high-yield expression, stability, and tetrameric structure of the loop recipient.
View Article and Find Full Text PDFACS Sens
September 2025
School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, China.
Alpha-2-macroglobulin (A2M) is a critical biomarker implicated in inflammation, immune regulation, coagulation, and various pathological conditions such as liver fibrosis, neurodegenerative diseases, and cancers. However, its precise quantification remains challenging due to complex conformational dynamics, subtle abundance fluctuations, and interference from plasma proteins. Here, we present a label-free dynamic single-molecule sensing (LFDSMS) strategy for the sensitive and specific detection of A2M.
View Article and Find Full Text PDFThyroid Res
September 2025
Department of Translational Medical Sciences, University of Naples "Federico II", Via S. Pansini, Naples, 5 - 80131, +39817462038, Italy.
Objectives: Serum thyroglobulin (Tg) is a key biomarker in the post-surgical monitoring of differentiated thyroid cancer (DTC). However, inter-assay variability among different immunoassay platforms can impact clinical interpretation, particularly at low Tg concentrations. This study aimed to compare the analytical performance and concordance of three widely used Tg immunoassays, Access (Beckman Coulter, Tg-B), Atellica (Siemens, Tg-A), and Liaison (Diasorin, Tg-L), with a focus on their agreement across clinically relevant Tg ranges.
View Article and Find Full Text PDFJ Mol Histol
September 2025
Department of Bioinformatics, School of Life Sciences, Pondicherry University, Kalapet, Puducherry, 605014, India.
Survivin, an inhibitor of apoptosis protein, is minimally expressed in normal adult tissues but overexpressed in multiple cancers. This study investigates survivin expression alongside autophagy markers ATG7 and LC3B in seven solid tumor types in Indian patient samples. Immunohistochemical analysis was performed on 48 cancer tissue samples (breast n = 7, buccal n = 6, cervical n = 5, colon n = 8, renal n = 6, liver n = 10, thyroid n = 6) and adjacent normal tissues (n = 9) using anti-human antibodies against survivin, ATG7, and LC3B.
View Article and Find Full Text PDFMikrochim Acta
September 2025
Department of Surgical Oncology, Shaanxi Provincial People's Hospital, 256 Friendship West Road, Beilin District, Xi'an, 710068, Shaanxi, China.
Mycoplasma pneumonia, a primary aetiological agent of atypical pneumonia, necessitates the implementation of rapid point-of-care diagnostics. Lateral flow immunoassays (LFIAs) hold promise for point-of-care testing (POCT), yet their sensitivity levels are frequently constrained by probe affinity and matrix interference. We introduce an orientational labelling strategy that employs magnetic nanoparticles (MNPs) functionalized with staphylococcal protein A (SPA) to simultaneously enhance antibody orientation and facilitate magnetic enrichment.
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