Effect of double antigen bridging immunoassay format on antigen coating concentration dependence and implications for designing immunogenicity assays for monoclonal antibodies.
ABSTRACT The double antigen bridging immunoassay has been used extensively for detection of immunogenicity responses to therapeutic monoclonal antibodies. We have analyzed parameters affecting performance of this type of immunoassay including microtiter plate antigen coating concentration, enzyme-labeled antigen conjugate dilution and assay format (one-step versus two-step). We present results demonstrating that the format of the assay has a significant impact on the optimal parameters to maximize assay performance. A one-step assay format achieves maximal sensitivity across a broad range of coating concentrations and at a lower concentration of conjugate than that in a two-step format. In contrast, a two-step format requires very low coating concentrations and higher conjugate concentrations to achieve maximal sensitivity and suffers from significantly reduced sensitivity at higher coating concentrations. Together, these findings indicate that a one-step assay format can greatly reduce the effect of coating concentration variation on assay performance.
- SourceAvailable from: Roberto Mallone[Show abstract] [Hide abstract]
ABSTRACT: Type 1 diabetes (T1D) is an autoimmune disease which results from the destruction of pancreatic beta cells. Autoantibodies directed against islet antigens are valuable diagnostic tools. Insulin autoantibodies (IAAs) are usually the first to appear and also the most difficult to detect amongst the four major islet autoantibodies. A non-radioactive IAA bridging ELISA was developed to this end. In this assay, one site of the IAAs from serum samples is bound to a hapten-labeled insulin (GC300-insulin), which is subsequently captured on anti-GC300 antibody-coated 96-well plates. The other site of the IAAs is bound to biotinylated insulin, allowing the complex to be detected by an enzyme-streptavidin conjugate. In the present study, 50 serum samples from patients with newly diagnosed T1D and 100 control sera from non-diabetic individuals were analyzed with our new assay and the results were correlated with an IAA radioimmunoassay (RIA). Using IAA bridging ELISA, IAAs were detected in 32 out of 50 T1D children, whereas with IAA RIA, 41 out of 50 children with newly diagnosed T1D were scored as positive. In conclusion, the IAA bridging ELISA could serve as an attractive approach for rapid and automated detection of IAAs in T1D patients for diagnostic purposes.PLoS ONE 07/2013; 8(7):e69021. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Monoclonal antibodies (mAbs) represent a therapeutic strategy that has been increasingly used in different diseases. mAbs are highly specific for their targets leading to induce specific effector functions. Despite their therapeutic benefits, the presence of immunogenic reactions is of growing concern. The immunogenicity identified as anti-drug antibodies (ADA) production due to the continuous administration of mAbs may affect the pharmacokinetics (PK) and/or the pharmacodynamics (PD) of mAbs administered to patients. Therefore, the immunogenicity and its clinical impact have been studied by several authors using PK modeling approaches. In this review, the authors try to present all those models under a unique theoretical mechanism-based framework incorporating the main considerations related to ADA formation, and how ADA may affect the efficacy or toxicity profile of some therapeutic biomolecules.Journal of Pharmacokinetics and Pharmacodynamics 07/2014; · 1.46 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Cuticular fractions of the important northern berries sea buckthorn (Hippophaë rhamnoides) and black currant (Ribes nigrum), and the suberized membrane of red peel potato (Solanum tuberosum var. Asterix), were investigated for the first time with solid state spectroscopic methods. In addition, the ester-bonded aliphatic monomers were analyzed as TMS derivatives by GC–MS after depolymerization with NaOMe catalyzed methanolysis. The fractions inspected by solid state 13C CP-MAS NMR and FT-IR spectroscopy included isolated cuticular and suberin-enriched membranes, i.e. raw cutin and suberin, the non-depolymerizable fractions and further cellulase- and pectinase-resistant fractions. Spectroscopic results supported the findings of the monomeric composition of ester-bound cutin, confirmed the presence of polysaccharides and an aromatic, possibly lignin-like, fraction and also suggested the presence of a non-degradable cutan-type polymer in the berry cuticular fraction. Black currant peel cutin and receptacle cutin were found to differ from each other in composition, notably in terms of the abundance of monomeric, aromatic and polysaccharide fractions. Some effect of latitudinal growth on the composition of cuticular fractions was observed for black currants grown in northern and southern Finland. The spectra of potato suberin were similar to the spectra of black currant receptacles, but the berry had even higher proportions of aromatic components.Journal of Food Composition and Analysis 05/2011; 24(3). · 2.26 Impact Factor