PubMed · 7764917
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D Medynski. 1994. Synthetic peptide combinatorial libraries.. https://doi.org/10.1038/nbt0794-709
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Aspergillus ochraceus contaminates agricultural products and produces nephrotoxic, carcinogenic ochratoxin A (OTA), posing severe food safety hazards. A dual-signal lateral flow immunochromatographic assay (dLFIA) based on ZrO₂@C nanoprobes was established for quantitative detection of A. ochraceus biomass. A novel monoclonal antibody (mAb 4B4) was prepared as the capture antibody to immobilize A. ochraceus mycelial lysate antigen on the test line, and a rabbit polyclonal antibody (pAb G2801) as the detection antibody to modify ZrO₂@C composites (synthesized via UiO-66 pyrolysis) into 200 nm colorimetric/photothermal nanoprobes. This dLFIA achieved limits of detection of 0.164 μg/mL (colorimetric) and 0.517 μg/mL (photothermal). This efficient and reliable method allows quantitative analysis of A. ochraceus biomass, which is suitable for routine monitoring of fungal contamination in agro-food matrices.
MAbs (monoclonal antibodies) are becoming increasingly important as diagnostic tools for pharmaceutical biotechnology, and hence it is crucial that they are produced under controlled conditions to assure their consistency and reproducibility, not only in terms of protein sequence and bioactivity, but also in terms of post-translational modifications, e.g. for N-glycosylation. Hybridoma CB.Hep-1, which secretes an IgG2b mAb, was cultured in vivo in ascites and in vitro in static-flask, spinner-flask, dialysis-membrane and perfusion systems using protein-free, low-serum-containing medium (1% foetal-calf serum) and high-serum-containing medium (8% foetal-calf serum). These CB.Hep-1 mAbs were fully characterized, and insignificant differences in the affinity constant were observed. Glycosylation profiling was performed by labelling the N-glycans released by peptide N-glycosidase F with either of the fluorophore tags 8-aminonaphthalene-1,3,6-trisulphonic acid and 4-aminobenzoic acid. The mAb produced in vivo showed two major biantennary-complex-type N-glycans: monogalactosylated, core-fucosylated and agalactosylated, core-fucosylated. The mAbs produced in vitro in static flasks and spinner flasks were not significantly influenced by the serum content in the culture media and showed a higher degree of N-glycan galactosylation compared with those produced in mouse-ascites, hollow-fibre and membrane systems. The monogalactosylated, core-fucosylated structure was the most abundant N-glycan except for those produced in ascites and hollow fibres, where the agalactosylated, core-fucosylated glycoform was the major specie. MAbs produced in high-cellular-yield systems displayed greater galactosylation heterogeneity influenced by changes in culture media.
We describe the use of fluorophore-doped nanoparticles as reporters in a recently developed ArcDia TPX bioaffinity assay technique. The ArcDia TPX technique is based on the use of polymer microspheres as solid-phase reaction carrier, fluorescent bioaffinity reagents, and detection of two-photon excited fluorescence. This new assay technique enables multiplexed, separation-free bioaffinity assays from microvolumes with high sensitivity. As a model analyte we chose C-reactive protein (CRP). The assay of CRP was optimized for assessment of CRP baseline levels using a nanoparticulate fluorescent reporter, 75 nm in diameter, and the assay performance was compared to that of CRP assay based on a molecular reporter of the same fluorophore core. The results show that using fluorescent nanoparticles as the reporter provides two orders of magnitude better sensitivity (87 fM) than using the molecular label, while no difference between precision profiles of the different assay types was found. The new assay method was applied for assessment of baseline levels of CRP in sera of apparently healthy individuals.