Probing Interactions of Therapeutic Antibodies with Serum via Second Virial Coefficient Measurements

Antibody-based therapeutics are the fastest growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. While the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, in ways that are incompletely understood, thereby altering therapeutic effect and disposition. While excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy (FCS) is able to measure diffusive B2 values directly in undiluted serum. Apparent second virial coefficient (B2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the common approach of isolating specific components and completing independent cross-term virial coefficient measurements is an incomplete representation of nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general, and advance the development of therapeutic antibodies and other protein-based therapeutics.

Multiplexed Detection of Sepsis Markers in Whole Blood using Nanocomposite Coated Electrochemical Sensors

ABSTRACTSepsis is a leading cause of mortality worldwide that is difficult to diagnose and manage because this requires simultaneous analysis of multiple biomarkers. Electrochemical detection methods could potentially provide a way to accurately quantify multiple sepsis biomarkers in a multiplexed manner as they have very low limits of detection and require minimal sensor instrumentation; however, affinity-based electrochemical sensors are usually hampered by biological fouling. Here we describe development of an electrochemical detection platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a recently developed nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling. Using nanocomposite coated planar gold electrodes, we constructed a procalcitonin sensor and demonstrated sensitive PCT detection in undiluted serum and clinical samples, as well as excellent correlation with a conventional ELISA (adjusted r2 = 0.95). Sensors for two additional sepsis biomarkers — C-reactive protein and pathogen-associated molecular patterns — were developed on the same multiplexed platform and tested in whole blood. Due to the excellent antifouling properties of the nanocomposite coating, all three sensors exhibited specific responses within the clinically significant range without any cross-reactivity in the same channel with low sample volume. This platform enables sensitive simultaneous electrochemical detection of multiple analytes in human whole blood, which can be expanded further to any target analyte with an appropriate antibody pair or capturing probe, and thus, may offer a potentially valuable tool for development of clinical point-of-care diagnostics.GRAPHICAL ABSTRACT

Electrochemical ELISA Protein Biosensing in Undiluted Serum Using a Polypyrrole-Based Platform

An electrochemical enzyme-linked immunosorbent assay (ELISA) biosensor platform using electrochemically prepared ~11 nm thick carboxylic functionalized popypyrrole film has been developed for bio-analyte measurement in undiluted serum. Carboxyl polypyrrole (PPy-COOH) film using 3-carboxy-pyrrol monomer onto comb-shaped gold electrode microarray (Au) was prepared via cyclic voltammetry (CV). The prepared Au/PPy-COOH was then utilized for electrochemical ELISA platform development by immobilizing analyte-specific antibodies. Tumor necrosis factor-alpha (TNF-α) was selected as a model analyte and detected in undiluted serum. For enhanced performance, the use of a polymeric alkaline phosphatase tag was investigated for the electrochemical ELISA. The developed platform was characterized at each step of fabrication using CV, electrochemical impedance spectroscopy and atomic force microscopy. The bioelectrodes exhibited linearity for TNF-α in the 100 pg/mL–100 ng/mL range when measured in spiked serum, with limit of detection of 78 pg/mL. The sensor showed insignificant signal disturbance from serum proteins and other biologically important proteins. The developed platform was found to be fast and specific and can be applicable for testing and measuring various biologically important protein markers in real samples.

A case of IgE myeloma transformed into IgE-producing plasma cell leukaemia

This is a case report of a challenging diagnosis of IgE monoclonal gammopathy of undetermined significance, which transformed into myeloma, then transformed into IgE-producing plasma cell leukaemia in a 71-year-old male who was followed in Brest, France, from 2015 to 2019. The IgEproducing variant is the rarest sub-type of multiple myeloma, and plasma cell leukaemia is considered to be the rarest and the most aggressive of human monoclonal gammopathies. In November 2015, hypogammaglobulinemia was detected during a systematic check-up. A kappa light chain monoclonal gammopathy was first diagnosed due to an increase of the free kappa/lambda light chains ratio. No monoclonal immunoglobulin was detected by either serum protein electrophoresis (Capillarys 2, Sebia, Issy-les-Moulineaux, France) or immunofixation (Hydrasys 2, Sebia, Issy-les- Moulineaux, France). In June 2018, a blood smear led to the diagnosis of plasma cell leukaemia. A monoclonal peak was detected and identified as IgE-kappa. Analysis of an archival sample taken three years earlier, revealed the presence of a monoclonal IgE, which had been missed at diagnosis. Chemotherapy with bortezomib and dexamethasone was introduced. The patient survived 10 months after the diagnosis of leukaemia. This case shows that an abnormal free light chain ratio should be considered as a possible marker of IgE monoclonal gammopathy even in the absence of a solitary light chain revealed by immunofixation. In addition, the use of an undiluted serum may increase the sensitivity of the immunofixation for the detection of IgE monoclonal gammopathies compared to the 1:3 dilution recommended by the manufacturer.

μPAD Fluorescence Scattering Immunoagglutination Assay for Cancer Biomarkers from Blood and Serum

A microfluidic paper analytical device (μPAD) was created for the sensitive quantification of cancer antigens, carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), from human whole blood and serum, toward diagnosis and prognosis of colorectal cancer. Anti-CEA and anti–CA 19-9 antibodies were covalently linked to submicron, fluorescent polystyrene particles, loaded, and then dried in the center of the μPAD channel. CEA- or CA 19-9–spiked blood or serum samples were loaded to the inlet of μPAD, and subsequent immunoagglutination changed the fluorescent scatter signals upon ultraviolet (UV) excitation. The total assay time was about 1 min. Detection limits were 1 pg/mL for CEA and 0.1 U/mL for CA 19-9 from both 10% diluted blood and undiluted serum. The use of UV excitation and subsequent fluorescence scattering enabled much higher double-normalized intensities (up to 1.28–3.51, compared with 1.067 with the elastic Mie scatter detection), successful detection in the presence of blood or serum, and distinct multiplex assays with minimum cross-reaction of antibodies. The results with undiluted serum showed the larger dynamic range and smaller standard errors, which can be attributed to the presence of serum proteins, functioning as a stabilizer or a passivating protein for the particles within paper fibers.