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

Abstract

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.

Current Status and Future Prospects for Nanoparticle‐Based Technology in Human Medicine

“…In this work, the C-reactive protein forms a sandwich complex between the Fe 2 O 3 nanoparticles and silica microparticles, which is sedimented under normal gravitation. A magnetic permeability increase in the sediment due to the presence of the complexed superparamagnetic nanoparticles was determined using an inductance-based transducer [67]. A recent publication reports the development of conjugated nanoparticle-based magnetic relaxation switch biosensors and their application to protein analysis.…”

Magnetomotive Molecular Nanoprobes

“…Efficient ways to direct these nanoparticles to organs, tissues, tumors, and malignant cells have been reported by conjugating the nanoparticles with enzymes, proteins, antibodies, nucleotides, or peptide ligands. Various biomarkers and oncoproteins like HER2 [46, 60, 61, 67, 68], or MMPs [69-71] that are known to be over-expressed at the sites of malignancy have been targeted using different conjugation techniques. The conjugating procedures depend on the nature of the nanoparticle coating and the conjugating ligand or proteins.…”

Magnetic permeability based diagnostic test for the determination of the canine C-reactive protein concentration in undiluted whole blood

“…The use of superparamagnetic nanoparticles as labels to proteins, including human CRP [13,14] and albumin [15], in a magnetic permeability based immunoassay has previously been shown [16]. This new assay, as well as, eliminating the problem of measuring interferences, is inexpensive, nonhazardous, rapid, and can be used with small sample volumes.…”

“…The following activation, coupling and blocking procedure was also performed according to the previously described procedure [14] where the activated silica microparticles were resuspended in 9.75 mL 0.1 M NaH 2 PO 4 , pH 7.0 containing 0.25 mL polyclonal anti-canine CRP (Innovative Research, Novi, Michigan, USA). After the finalized preparation procedure, the silica microparticles were stored at 4 1C.…”