Patrick Englebienne

Use of colloidal gold surface plasmon resonance peak shift to infer affinity constants from the interactions between protein antigens and antibodies specific for single or multiple epitopes

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: principles, methods and applications in biomedical sciences

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.

Ribonuclease L proteolysis in peripheral blood mononuclear cells of chronic fatigue syndrome patients

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.

High-throughput screening using the surface plasmon resonance effect of colloidal gold nanoparticles

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 A600 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 103−1012 mol−1) 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).

Advances in High-Throughput Screening: Biomolecular Interaction Monitoring in Real-Time with Colloidal Metal Nanoparticles

The post-genomic era is revolutionizing the drug discovery process. The new challenges in the identification of therapeutic targets require efficient technological tools in order to be properly addressed. Label-free detection systems use proteins or ligands coupled to materials of which the physical properties are measurably modified upon specific interactions. Among the label-free systems currently available, the use of metal nanocolloids offers enhanced throughput and flexibility for real-time biomolecular recognition monitoring at a reasonable cost.

Chronic fatigue syndrome: a risk factor for osteopenia?

No data documenting a possible depletion of bone mineral density in patients with chronic fatigue syndrome (CFS) are currently available. However, recent pathophysiological observations in CFS patients may have deleterious consequences on bone density. Firstly, the deregulation of the 2,5A synthetase RNase L antiviral pathway and its associated channelopathy, implicates increased demands for calcium and consequent increased calcium-re-absorption from the skeletal system. Secondly, Mycoplasma fermentans which has been frequently associated with CFS, produces a lipopeptide, named 2-kDa macrophage-activating lipopeptide (MALP-2), which stimulates macrophages. MALP-2 has been shown to enhance bone resorption in a dose-dependent manner, at least in part by stimulating the formation of prostaglandins. Thirdly, decreased levels of insulin-like growth factor I (IGF-I) have been reported in CFS-patients. IGF-I is critical to the proliferation of osteoblasts. Consequently, depleted levels of IGF-I may shift the balance between osteoclastic and osteoblastic activity towards bone resorption.

37-Kilodalton/83-kilodalton RNase L isoform ratio in peripheral blood mononuclear cells: analytical performance and relevance for chronic fatigue syndrome.

A French group has reported results (5) supporting the use of the RNase L 37-kDa/83-kDa ratio (37/83 R) in peripheral blood mononuclear cells (PBMC) as a diagnostic test for chronic fatigue syndrome (CFS). More recently (6), the same group cautioned about the diagnostic value of the 37/83 R, based on a small patient follow-up study which was likely to indicate analytical variability among duplicate assays, lack of reproducibility over time, and a weak correlation with the multidimensional fatigue inventory (MFI) score. Because of our long-term experience with this assay, we would like to offer some comments. First, we tested the analytical performance of the 37/83 R assay according to CLSI (formerly NCCLS) procedure EP5-A (4), with control samples at three different levels made of extracts of the monocytic U937 cell line spiked with various concentrations of recombinant RNase L. The guideline protocol involves assaying the samples in duplicate twice daily over a total period of 20 days. The results summarized in Table 1 indicate that both within- and between-run variation does not exceed 13%. In another series of experiments, we assayed eight patient samples in duplicate (average 37/83 R ranging from 0.5 to 245). Although in accordance with the NCCLS protocol results, the variation did not exceed 12% for samples with 37/83 R levels up to 20, and it rose significantly to 30% and more for samples with 37/83 R levels above 20. This should be expected, because beyond this level, more than 70% of the 83-kDa isoform is cleaved, and consequently, the faint 83-kDa band is difficult to scan with accuracy. Thus, in our opinion, the lower level of correlation between the duplicate assay results observed with the CFS group versus those with the controls (6) reflects the prevalence of high 37/83 R levels in the CFS group rather than a low test reproducibility as claimed by these authors. This is further supported by the good correlation found for the control group (r 0.95). During validation, the lowest detectable ratio measured with a sample containing the 83kDa isoform only was estimated (3 independent experiments with 26 replicates each) to be 0.13 0.06 (average three standard deviations). Thus, the clinical cutoff ratio of 0.4 found by the authors (5, 6) to best discriminate CFS patients from controls falls within the measurable range. Second, although long-term unexplained fatigue is a hallmark symptom, CFS is a complex clinical condition, and other important symptoms reflect an exacerbated inflammatory response. Because the inflammatory protease elastase has been shown to be responsible for the cleavage of 83-kDa RNase L into the 37-kDa isoform (2), one would expect the 37/83 R assay to reflect inflammatory activity in the immune system. In a recent study, we compared the 37/83 R with human leukocyte elastase activity (Molecular Probes kit) with 52 CFS PBMC samples. The correlation was highly significant (r 2 0.76; P 0.001), supporting this proposal. In our opinion, both fatigue and RNase L cleavage result from similar dysfunctions in the interferon-activated pathways that occur upstream of RNase L activation and exert their effects on different targets (3). Consequently, we do not feel particularly concerned by the weak correlations observed between the MFI and 37/83 R (6). The poor reproducibility of the test results over time (6) might reflect changes in the immune inflammatory status rather than in fatigue. In particular, the authors do not provide any information concerning the drugs CFS patient might have been using during the longitudinal study, which is critical as some drugs may act on the inflammatory response. Finally, by combining the results of both studies by this group (5, 6) with ours (1), which included 57 CFS patients and 28 healthy controls, we note that both diagnostic sensitivity (76%) and specificity (65%) remain attractive for such a poorly defined pathological condition.