Complement C1q in plasma induces nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticle antibody conjugates.

Upconverting nanoparticles are attractive reporters for immunoassays, because their high specific activity and lack of autofluorescence background enable their detection at extremely low concentrations. However, the sensitivity achieved with heterogeneous sandwich immunoassays using nanoparticle reporters is generally limited by the nonspecific binding of nanoparticle antibody conjugates to solid supports. In this study, we characterized plasma components associated with elevated nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticles in heterogeneous two-step sandwich immunoassays. Plasma was consecutively fractionated using various chromatographic methods by selecting after each step the fractions producing the highest nonspecific binding of upconverting nanoparticle conjugates in an immunoassay for cardiac troponin I. Finally, the proteins in the fractions associated with highest amount of nonspecific binding were separated by gel electrophoresis and identified with mass spectrometry. The results indicated that complement component C1q was present in the fractions associated with the highest signal from nonspecific binding. The interference was not limited to only poly(acrylic acid)-coated nanoparticles or certain antibody combination, but occurred more generally. The interference was removed by increasing the ionic strength of the assay buffer in the sample incubation step or by adding a negatively charged blocker to bind on positively charged C1q, suggesting that the interaction is mostly electrostatic. Hence, we assume that the interference is likely to affect various negatively charged nanoparticles. The identification of complement component C1q as the major interfering protein allows for more rational design of countermeasures in future immunoassay development utilizing nanoparticle reporters.

Combinatorial mutagenesis with alternative CDR-L1 and -H2 loop lengths contributes to affinity maturation of antibodies.

Loop length variation in the complementary determining regions (CDRs) 1 and 2 encoded in germline variable antibody genes provides structural diversity in naïve antibody libraries. In synthetic single framework libraries the parental CDR-1 and CDR-2 length is typically unchanged and alternative lengths are provided only at CDR-3 sites. Based on an analysis of the germline repertoire and structure-solved anti-hapten and anti-peptide antibodies, we introduced combinatorial diversity with alternative loop lengths into the CDR-L1, CDR-L3 and CDR-H2 loops of anti-digoxigenin and anti-microcystin-LR single chain Fv fragments (scFvs) sharing human IGKV3-20/IGHV3-23 frameworks. The libraries were phage display selected for folding and affinity, and analysed by single clone screening and deep sequencing. Among microcystin-LR binders the most frequently encountered alternative loop lengths were one amino acid shorter (6 aa) and four amino acids longer (11 aa) CDR-L1 loops leading up to 17- and 28-fold improved affinity, respectively. Among digoxigenin binders, 2 amino acids longer (10 aa) CDR-H2 loops were strongly enriched, but affinity improved anti-digoxigenin scFvs were also encountered with 7 aa CDR-H2 and 11 aa CDR-L1 loops. Despite the fact that CDR-L3 loop length variants were not specifically enriched in selections, one clone with 22-fold improved digoxigenin binding affinity was identified containing a 2 residues longer (10 aa) CDR-L3 loop. Based on our results the IGKV3-20/IGHV3-23 scaffold tolerates loop length variation, particularly in CDR-L1 and CDR-H2 loops, without compromising antibody stability, laying the foundation for developing novel synthetic antibody libraries with loop length combinations not existing in the natural human Ig gene repertoire.

Emerging Technologies and Platforms for the Immunodetection of Multiple Biochemical Markers in Osteoarthritis Research and Therapy.

Biomarkers, especially biochemical markers, are important in osteoarthritis (OA) research, clinical trials, and drug development and have potential for more extensive use in therapeutic monitoring. However, they have not yet had any significant impact on disease diagnosis and follow-up in a clinical context. Nevertheless, the development of immunoassays for the detection and measurement of biochemical markers in OA research and therapy is an active area of research and development. The evaluation of biochemical markers representing low-grade inflammation or extracellular matrix turnover may permit OA prognosis and expedite the development of personalized treatment tailored to fit particular disease severities. However, currently detection methods have failed to overcome specific hurdles such as low biochemical marker concentrations, patient-specific variation, and limited utility of single biochemical markers for definitive characterization of disease status. These challenges require new and innovative approaches for development of detection and quantification systems that incorporate clinically relevant biochemical marker panels. Emerging platforms and technologies that are already on the way to implementation in routine diagnostics and monitoring of other diseases could potentially serve as good technological and strategic examples for better assessment of OA. State-of-the-art technologies such as advanced multiplex assays, enhanced immunoassays, and biosensors ensure simultaneous screening of a range of biochemical marker targets, the expansion of detection limits, low costs, and rapid analysis. This paper explores the implementation of such technologies in OA research and therapy. Application of novel immunoassay-based technologies may shed light on poorly understood mechanisms in disease pathogenesis and lead to the development of clinically relevant biochemical marker panels. More sensitive and specific biochemical marker immunodetection will complement imaging biomarkers and ensure evidence-based comparisons of intervention efficacy. We discuss the challenges hindering the development, testing, and implementation of new OA biochemical marker assays utilizing emerging multiplexing technologies and biosensors.