CRP, short for C-Reactive Protein is an acute phase protein that is naturally occurring in the human body. A person with a chronically lightly increased CRP concentration between 3 mg/l and 10 mg/l has an increased risk of cardiovascular disease in the future. The goal of this thesis is to investigate the biological part of a biosensor for the detection of CRP, with superparamagnetic beads as label. Therefore first a working immunoassay has to be designed. To obtain a sensitive assay the ratio between specific bonds (CRP-antibody) and non-specific bonds should be as high as possible. To determine if a difference can be made between possible non-specific bonds and specific bonds occurring in the assay, insight in the CRPantibody bond present in the assay is required. To optimize the biological part different immunoassays are tested with Horseradish peroxidase (HRP) as label, since it is a well established way for detection. If an enzymatic substrate is added HRP will catalyze a chemical reaction producing light that can be detected. This type of assay is called an ELISA. For the quantification of CRP bound in the immunoassays, first a dose response curve of HRP is performed. The optimized dose response curve for HRP is linear and reproducible for a HRP quantity of 0.2 fmol to 12 fmol, with a sensitivity of 22±0.24 RLU/fmol. The demands for a linear and reproducible dose response curve for HRP are: Block the material in which HRP is measured, always use the same materials for storage, never change the concentration of the stock solution HRP and always preserve it in the same way, the experiments have to be performed the same way always. Two immunoassays are tested, a sandwich assay and a competition assay, both immunoassays are capable to detect CRP. For a CRP sandwich assay a quantity of 40 amol to 8 fmol CRP could be detected with a sensitivity of 19.9±1.83 RLU/fmol CRP. For a CRP competition assay a quantity of 10 to 100 fmol CRP could be detected with a sensitivity of 11±0.5 RLU/fmol CRP. When using HRP as label a sandwich assay for detection of CRP is best, however in a biosensor as less steps as possible are preferred, so a competition assay might be better especially since it is easier to shift the sensitive area of the assay, so no dilution steps of the blood have to be performed. For optimizing the detection of CRP with superparamagnetic beads Streptavidinbeads with a diameter of 2.8 ?m are used. Furthermore different blocking agents are tested: BSA and Casein. Casein appears to be a better blocking agent in a CRP assay with beads, resulting in less non-specific binding, an explanation is given with help of the DLVO theory, which discusses the interaction energies occurring between a sphere and a flat surface. It appeared that the second energy minimum of the sphere and flat surface is further away for Casein than BSA, also the interaction energy is lower for a Casein coated surface at this distance. This difference can be enough to reduce the non-specific interactions between the beads and surface. To get more insight in the antibody-CRP complex the dissociation rate constant of this complex when applying a force is studied. A setup has been used in which forces up to 100 pN can be exerted on the beads. Optical detection is used to observe the decrease in the number of beads that is bound to the surface as a function of time. A kinetic model that describes the dissociation of beads with one or two identical parallel bonds is used to fit the number of beads attached to the surface as a function of time. The obtained force induced dissociation rate constant of the single bound beads is 0.6±0.4 s-1 and for beads bound with two bonds 4·10-3±2·10-3 s-1. With the obtained force induced dissociation rate constants it is possible to determine the effective bond length at the transition state, x? and the dissociation rate constant. The x? calculated has a value of (7.5±1)·10-10 m and the corresponding dissociation rate constant is (5.2±4)·10-5 s-1, which is in the same order of magnitude as values reported in literature. The accompanying activation energy is 0.83±0.02 eV. The obtained activation energy can be compared with other antibody-antigen complexes which values range from 0.8 eV to 1.1 eV. Taking into account all the results it is concluded that this model describes the dissociation of the antibody-antigen complex correctly.
|Date of Award||31 Dec 2008|
|Supervisor||Arthur M. de Jong (Supervisor 1)|