The surface plasmon resonance (SPR) wavelength of colloidal gold particles coated with a monoclonal antibody is red-shifted when the antibody interacts with its specific ligand. This shift results from the change in the refractive index of the particles as induced by ligand binding. This property is used to monitor in real-time the association and dissociation kinetics of the interaction in solution. The monitoring is performed in a clinical chemistry automated analyzer during a few minutes of incubation at 37 degrees C. Data treatment allows calculation of the affinity constant of the interaction. The SPR wavelength shift does not necessarily require agglutination or aggregation of the particles to occur since particles coated with one monoclonal antibody specific for a single epitope on the ligand can be used in the procedure. The affinity constants measured by this procedure correlate with those calculated from Scatchard plots or BIAcore data.

Surface plasmon resonance (SPR) is a phenomenon occuring at metal surfaces (typically gold and silver) when an incident light beam strikes the surface at a particular angle. Depending on the thickness of a molecular layer at the metal surface, the SPR phenomenon results in a graded reduction in intensity of the reflected light. Biomedical applications take advantage of the exquisite sensitivity of SPR to the refractive index of the medium next to the metal surface, which makes it possible to measure accurately the adsorption of molecules on the metal surface and their eventual interactions with specific ligands. The last ten years have seen a tremendous development of SPR use in biomedical applications. The technique is applied not only to the measurement in real-time of the kinetics of ligand–receptor interactions and to the screening of lead compounds in the pharmaceutical industry, but also to the measurement of DNA hybridization, enzyme–substrate interactions, in polyclonal antibody characterization, epitope mapping, protein conformation studies and label-free immunoassays. Conventional SPR is applied in specialized biosensing instruments. These instruments use expensive sensor chips of limited reuse capacity and require complex chemistry for ligand or protein immobilization. Our laboratory has successfully applied SPR with colloidal gold particles in buffered solution. This application offers many advantages over conventional SPR. The support is cheap, easily synthesized, and can be coated with various proteins or protein–ligand complexes by charge adsorption. With colloidal gold, the SPR phenomenon can be monitored in any UV-vis spectrophotometer. For high‒throughput applications, we have adapted the technology in an automated clinical chemistry analyzer. This simple technology finds application in label-free quantitative immunoassay techniques for proteins and small analytes, in conformational studies with proteins as well as in the real-time association-dissociation measurements of receptor–ligand interactions, for high-throughput screening and lead optimization.

We propose a high-throughput screening method which involves colloidal gold nanoparticles sensitized with the binding protein. Upon interaction with a specific ligand (a polypeptide or a small organic molecule), the surface plasmon resonance absorbance peak of the colloidal gold reagent shifts toward longer wavelengths due to the change in refractive index at the particle surface caused by changes in mass. The shift is proportional to the dose of ligand involved for a fixed amount of binding protein and occurs according to the kinetics of interaction. We applied this property to the analysis of association and dissociation of ligand-binding protein interactions in a small random access clinical chemistry analyzer. The instrument measures the changes in A 600 nm over a period of 20 min for each sample. Due to the high degree of automation, the instrument throughput amounts to 144 samples an hour and can be run during 24 h a day in a walk-away mode. When connected to a computer for data handling, a single instrument can consequently handle over 3000 samples a day. Higher throughput instruments are available which can handle as much as ten times more samples. We validated the technique by comparing the affinity constants (range 10 3 210 12 mol 21 ) calculated for 30 pairs of ligand-protein interactions at different ligand doses with those obtained from other methods, including the BIAcore (slope 0.84; coefficient of correlation r = 0.82).

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A 37-kDa binding polypeptide accumulates in peripheral blood mononuclear cell (PBMC) extracts from chronic fatigue syndrome (CFS) patients and is being considered as a potential diagnostic marker (De Meirleir, K., Bisbal, C., Campine, I., De Becker, P., Salehzada, T., Demettre, E., and Lebleu, B. (2000) Am. J. Med. 108, 99 -105). We establish here that this low molecular weight 2-5A-binding polypeptide is a truncated form of the native 2-5A-dependent ribonuclease L (RNase L), generated by an increased proteolytic activity in CFS PBMC extracts. RNase L proteolysis in CFS PBMC extracts can be mimicked in a model system in which recombinant RNase L is treated with human leukocyte elastase. RNase L proteolysis leads to the accumulation of two major fragments with molecular masses of 37 and 30 kDa. The 37-kDa fragment includes the 2-5A binding site and the N-terminal end of native RNase L. The 30-kDa fragment includes the catalytic site in the C-terminal part of RNase L. Interestingly, RNase L remains active and 2-5A-dependent when degraded into its 30-and 37-kDa fragments by proteases of CFS PBMC extract or by purified human leukocyte elastase. The 2-5A-dependent nuclease activity of the truncated RNase L could result from the association of these digestion products, as suggested in pull down experiments.Chronic fatigue syndrome is characterized by long-lasting and debilitating fatigue, myalgia, impairment of neurocognitive functions, and flu-like symptoms, which are often severely worsened after physical exercise. A case definition has been proposed by the Center for Disease Control in Atlanta under the name of Holmes (2) and Fukuda (3) criteria. Most of these symptoms are unfortunately common to other diseases, thus complicating a diagnosis that still relies on extensive clinical testing to exclude other pathologies (4). A large proportion of the patients report an infectious episode at the onset of their chronic fatigue. No single agent has been conclusively associated with the disease, although several candidates including human T-cell lymphotrophic virus-1, human herpesvirus-6, Enterovirus, or mycoplasma have been proposed (5-8). Dysregulation of immune functions has also been suggested and natural killer cell cytotoxicity was significantly diminished in patients (when compared with normal controls) (9).These observations have prompted studies of possible dysfunctioning of interferon-induced responses. An up-regulation of the 2-5A/RNase L antiviral pathway in peripheral blood mononuclear cells (PBMC) 1 of CFS patients has been described (10). RNase L is the terminal enzyme in the 2-5A synthetase/ RNase L antiviral pathway and plays an essential role in the elimination of viral mRNAs (for review, see Ref. 11). Activation of RNase L requires the binding of a small 2Ј,5Ј-linked oligoadenylate (2-5A). Intriguingly, a low molecular weight 2-5A-binding polypeptide has been observed in a subset of patients diagnosed for CFS (12). Similar observations have been made in a larger study including CFS patients and control ...

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