Preparation of a portable point-of-care in vitro diagnostic system, for quantification of canine C-reactive protein, based on a magnetic two-site immunoassay.

In this study, characterization of the binding kinetics and optimization of a magnetic permeability based point-of-care (POC) immunoassay system for quantification of canine C-reactive protein (cCRP) is described. The reagent is based on a two-site heterogeneous immunoassay system utilizing conjugated superparamagnetic nanoparticles (SPION) and silica particles, both particles carrying covalently linked antibodies directed to the cCRP analyte. Detection is carried out using a magnetic permeability-based small instrument, adjusted in order to apply it in a POC setting near the patients. The kinetic parameters are characterized and applied in the final design of the assay system. In the cCRP system studied, 90% of the binding between immobilized solid-phase silica antibody and cCRP is complete after only 15 s, and 30 s for the binding between the antibody on the SPION and the bound cCRP on the silica particle. Additionally, the binding rate constants are determined to be 149 and 30 M(-1)s(-1), respectively. The analytical sensitivity, clinical sensitivity, and imprecision verifies the clinical usefulness of the system. Also, quantification of cCRP, using the system described, in dog clinical samples from mixed breeds shows a high correlation to a commercially available comparative cCRP ELISA system (y = 0.98 × +3.2, R(2) = 0.98, n = 47). The immunoassay system described can thus provide the veterinarian a valuable tool for rapid diagnosis and monitoring of inflammatory diseases in dogs in a setting near the patients.

C1-TEN is a negative regulator of the Akt/PKB signal transduction pathway and inhibits cell survival, proliferation, and migration.

We have previously identified C1 domain-containing phosphatase and TENsin homologue (C1-TEN) as being an intracellular binding partner for Axl receptor tyrosine kinase (RTK). C1-TEN is a tensin-related protein that houses an N-terminal region with predicted structural similarity to PTEN. Here, we report our observations on the effects of ectopic expression of C1-TEN in HEK293 cells, which resulted in profound molecular and phenotypic changes. Stable expression of C1-TEN altered cellular morphology, with less cell spreading and weaker filamentous actin staining. Cells overexpressing C1-TEN were inhibited greatly in their proliferation and migration rates as compared with mock-transfected cells. Furthermore, serum starvation-induced apoptosis caused a twofold increase in caspase 3 activity in C1-TEN-overexpressing cells vs. mock cells. In addition, C1-TEN-overexpressing cells showed a markedly reduced phosphorylation of Akt/PKB kinase and its substrate GSK3, as well as reduced Akt enzymatic activity. No such effects on JNK were observed. Also, serum-stimulated activation of Akt was delayed in C1-TEN-overexpressing cells, while no difference in profile of ERK activation was observed. Furthermore, cells expressing a C1-TEN mutant where the putative phosphatase active site cysteine at position 231 was substituted for a serine displayed full restoration of both cell proliferation and Akt activation. In conclusion, C1-TEN appears to be a novel intracellular phosphatase that negatively regulates the Akt/PKB signaling cascade, and is similar to its relative PTEN in this respect. However, the particular domain organization of C1-TEN may enable it to regulate RTK and other signaling complexes that are linked to Akt/PKB signaling in a unique manner.

A combination of magnetic permeability detection with nanometer-scaled superparamagnetic tracer and its application for one-step detection of human urinary albumin in undiluted urine.

A rapid (6.5 min) and simple one-step magnetic immunoassay (MIA) has been developed for analysis of human urinary albumin in near patient settings. Polyclonal rabbit anti-human albumin was used as a capture antibody and monoclonal mouse anti-human albumin as a detection antibody in a two-site immunometric assay requiring no additional washing procedures. The polyclonal anti-human albumin was conjugated to silica microparticles (solid phase) and the monoclonal antibody to dextran-coated nanoscaled superparamagnetic particles (tracer). Quantification of human albumin in undiluted urine was performed by adding 2 microL urine to a measuring vial containing solid-phase, superparamagnetic tracer and reaction buffer and then inverting the vial by hand for 20 s. The measuring vial was allowed to stand for 6 min prior to detection, in order for the solid-phase sediment to form at the bottom of the vial. Lastly, the measuring vial was placed into a magnetic permeability detector, which measured the enrichment of superparamagnetic tracer in the sediment due to complex formation with human albumin. Total analysis time was 6.5 min. A linear response was obtained for 0-400 mg/L albumin with a detection limit of 5 mg/L. The total coefficient of variation (CV) was 11% calculated from four consecutive runs on a urine sample containing 11.1 mg/L human albumin during 3 consecutive days. Human urinary albumin analysis was performed on 149 patient samples using the MIA technique and the obtained results showed good correlation with the hospital immunoturbidimetric reference method (y = 1.004x + 4.01, R2 = 0.978, N = 149) and a commercially available point of care albumin analysis provided by HemoCue Inc. (y = 0.98x + 5.8, R2 = 0.833, N = 90).

Rapid one-step whole blood C-reactive protein magnetic permeability immunoassay with monoclonal antibody conjugated nanoparticles as superparamagnetic labels and enhanced sedimentation.

A rapid (5.5 min) one-step whole blood C-reactive protein (CRP) magnetic permeability immunoassay utilizing monoclonal antibody conjugated dextran iron oxide nanoparticles (70 nm) as superparamagnetic labels and mixed fractions (1:1 ratio of 15-40 and 60 microm) of polyclonal anti-CRP conjugated silica microparticles for enhanced sedimentation is described. In this one-step assay procedure, a whole blood sample (4 microl) is applied to an assay glass vial, containing both antibody conjugates, and mixed for 30 s. The target analyte, CRP, forms a sandwich complex between the conjugated nanoparticles and microparticles, and, subsequently, the complex sediments under normal gravitation within 5 min to the bottom of the vial. The magnetic permeability increase of the sediment due to the presence of the complexed superparamagnetic nanoparticles is determined using an inductance-based transducer. Assayed patient whole blood samples were compared with the Abbott Diagnostics Architect reference method. A strong linear correlation was observed for the CRP concentration range 0-260 mg/l in whole blood (y=1.001x+0.42, R2=0.982, n=50). The CRP assay presented showed a limit of detection of 3 mg/l and a total imprecision (coefficient of variation) of 10.5%. On the basis of our observations, we propose a rapid, one-step, CRP assay for near-patient testing.

Detection of C-reactive protein utilizing magnetic permeability detection based immunoassays.

A new sensing technology platform integrating magnetic permeability detection and a two-site heterogeneous immunoassay in a one-step analysis is described. As a platform model, measurements of C-reactive protein (CRP), a cardiac and inflammation marker, were performed in a rapid (11.5 min) high-sensitivity (hs) procedure with a low detection limit (0.2 mg/L) and accuracy (CV = 11%). The two-site heterogeneous immunoassay was performed in 1.2-mL disposable reagents vials containing solid phase (polyclonal anti-CRP conjugated silica microparticles), labeling agent (monoclonal anti-CRP conjugated superparamagnetic nanoparticles), and reaction buffer. Whole blood (20 muL) was assayed by introducing the sample into a reagent vial using a glass capillary and mixing its contents by hand for 30 s. After a 11-min sedimentation step, the vial was placed into the coil of the magnetic permeability detector, which measured the enrichment of superparamagnetic nanoparticles in the solid-phase sediment. Magnetic permeability detection and quantification is based on the principle that when paramagnetic materials are placed inside a coil, the inductance of the coil is influenced. Screening of CRP on whole blood patient samples showed good correlation with central hospital measurements for hsCRP (y = 1.018x - 0.021, R(2) = 0.980, n = 103) and normal range CRP (y = 1.02x + 2.53, R(2)= 0.991, n = 33) analyses. The mean differences of the two methods according to the Bland and Altman plots were -0.03 +/- 1.12 mg/L for hsCRP analysis and -3.4 +/- 8.64 mg/L for normal range CRP analysis.