Label-free detection of immune complexes with myeloid cells.

The aim of this study was to provide proof-of-concept for quantitative and qualitative label-free detection of immune complexes through myeloid cells with imaging surface plasmon resonance. Surface plasmon resonance imaging was first applied to monitor the binding of human sera from healthy and rheumatoid arthritis (RA) patients to immobilized citrullinated RA-specific peptide antigens, histone citrullinated peptide 2 (HCP2) and viral citrullinated peptide 2 (VCP2). Next, the binding of monocytoid cell line U937 to the resulting immune complexes on the sensor surface was monitored. As control, binding of U937 was monitored to immunoglobulin (Ig)G subclasses simultaneously. Cell response results were compared to results of cyclic citrullinated peptide 2 (CCP2) enzyme-linked immunosorbent assay (ELISA), clinical RA diagnosis and antigen-specific antibody distribution of the samples. Human IgG3 triggered the most pronounced response, followed by IgG1 and IgG4, while IgG2 did not result in U937 cell binding. Serum samples obtained from RA patients resulted in a significantly increased cell response to VCP2 compared to healthy controls. The strength of cell response towards VCP2 immune complexes showed significant correlation with levels of antigen-specific IgA, IgG and IgG3. Cellular responses on VCP2 immune complexes showed significant association with both CCP2-based serological positivity and European League Against Rheumatism (EULAR) criteria-based clinical RA diagnosis. Immunoglobulin-triggered binding of monocytoid cells can be monitored using a label-free multiplex technology. Because these binding events are presumably initiated by Fc receptors, the system provides a tool for biological detection of autoantibodies with diagnostic value, here exemplified by anti-citrullinated antibodies. This provides added information to antibody levels, as interaction with Fc-receptor-expressing cells is also affected by post-translational modification of the immunoglobulins.

Ultrasensitive Impedimetric Biosensor Fabricated by a New Immobilisation Technique for Parathyroid Hormone.

This paper presents a novel ultrasensitive and rapid impedimetric biosensor with new immobilisation materials for parathyroid hormone (PTH) with the aim to determine the PTH level in serum for the diagnosis and monitoring of parathyroid diseases such as hyperparathyroidism, adenoma, and thyroid cancer. The interaction between PTH and the biosensor was investigated with an electrochemical method. The biosensor was based on the gold electrode modified by mercaptohexanol (6-MHL). Anti-parathyroid hormone (anti-PTH) was covalently immobilised onto a self-assembled monolayer (SAM) by using epiclorhidrina (EPI) with ethanolamine (EA). The EPI-EA interaction represents the first use of these for the construction of biosensors in published reports. The immobilisation of the anti-PTH was monitored by electrochemical impedance spectroscopy, cyclic voltammetry and scanning electron microscopy (SEM) techniques. After the optimisation studies of immobilisation materials such as 6-MHL, EPI, EA and glutaraldehyde, linearity, repeatability and sensitivity of biosensor were evaluated as the performance of biosensor. PTH was detected within a linear range of 0.1-0.6 pg/ml, and the detection limit was 0.1 fg/ml. The specificity of the biosensor was also investigated. Finally, the described biosensor was used to detect the PTH levels in artificial serum samples.

High throughput profiling of serum phosphoproteins/peptides using the SELDI-TOF-MS platform.

Protein phosphorylation is a dynamic post-translational modification that plays a critical role in the regulation of a wide spectrum of biological events and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis. Determination of the sites and magnitudes of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. A high throughput analysis of phosphorylation of proteins would provide a simple, logical, and useful tool for a functional dissection and prediction of biological functions and signaling pathways in association with these important molecular events. We have developed a functional proteomics technique using the ProteinChip array-based SELDI-TOF-MS analysis for high throughput profiling of phosphoproteins/phosphopeptides in human serum for the early detection and diagnosis as well as for the molecular staging of human cancer. The methodology and experimental approach consists of five steps: (1) generation of a total peptide pool of serum proteins by a global trypsin digestion; (2) rapid isolation of phosphopeptides from the total serum peptide pool by an affinity selection, purification, and enrichment using a novel automated micro-bioprocessing system with phospho-antibody-conjugated paramagnetic beads and a hybrid magnet plate; (3) high throughput phosphopeptide analysis on ProteinChip arrays by automated SELDI-TOF-MS; and (4) bioinformatics and statistical methods for data analysis. This method with appropriate modifications may be equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins and for selectively isolating, profiling, and identifying phosphopeptides present in a highly complex phosphor-peptide mixture prepared from various human specimens such as cells, tissue samples, and serum and other body fluids.

Adhesion frequency assay for in situ kinetics analysis of cross-junctional molecular interactions at the cell-cell interface.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics to the binding curve returns the 2D affinity and off-rate. The assay has been validated using interactions of Fcγ receptors with IgG Fc, selectins with glycoconjugate ligands, integrins with ligands, homotypical cadherin binding, T cell receptor and coreceptor with peptide-major histocompatibility complexes. The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology, membrane anchor, molecular orientation and length, carrier stiffness, curvature, and impingement force, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules. The method has also been used to study the concurrent binding of dual receptor-ligand species, and trimolecular interactions using a modified model. The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods. To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin α(L)ß(2) on neutrophils with dimeric E-selectin in the solution to activate α(L)ß(2).