Nonspecific analyte binding

Nonspecific protein binding to the solid phase complicates the method and is a selective pressure driving its evolution. The adaptive response has been the development of intrinsically comparative methods in which specific binding to an immobilized ligand is blocked in one out of two otherwise identical samples. When the respective protein components of the samples are compared, specifically bound proteins are present in one but severely depleted in the other. To allow relative quantitation, the two samples can be made isotopically distinct by a chemical or metabolic process and then mixed for an analytical step that avoids intersample variability [15]. 

A major disadvantage is that the direct sensor detection cannot distinguish between the sensor response to the specific analyte binding from the response to a possible nonspecific adsorption of other compounds. The nonspecific fouling from blood or blood serum seems to be one of the main barriers for practical application of immunosensors in medical diagnostics. 

For analytes that cannot interact with the incorporated fluorophore strongly enough to cause a significant change in the fluorescence intensity and/or wavelength, one can use a nonspecific external quencher or modifier to affect the fluorescence change upon analyte binding (Fig. 1, Scenario 3b). 

The potential issues with bead-based immunocapture methods are saturation, nonspecific adsorption, and nonspecific protein binding, which through different mechanisms lead to nonlinear response of analyte at high and low analyte concentrations. Bead saturation at high protein concentrations can be solved by narrowing the curve range. Bead pretreatment with addition of a... 

Sandwich-type sensors are applicable for measuring large antigens that are capable of binding two different antibodies. Such sensors utilize an antibody that binds the analyte-antigen, which then binds an enzyme-labeled second antibody. After removal of the nonspecifically adsorbed label, the probe is placed into the substrate-containing solution, and the extent of the enzymatic reaction is monitored... 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

A special nonspecific sensor response might be due to the cross-reactivity of immobilized antibodies. Besides the analyte, an antibody can bind also other entities bearing a similar antigenic epitope, e.g. the detection of some pathogenic bacteria can be interfered by the binding of non-pathogenic bacteria with the same surface antigen. 

Similar to micellar assemblies in water, reverse micelles have also been utilized to bring about nonspecific binding interactions in organic solvents. Akiyoshi et al. (2002) have synthesized an amphiphilic block copolymer containing PEO and an amylase chain as receptor for methyl orange (MO Chart 2.2). Amylases are insoluble and methoxy-PEO (MPEO) is soluble in chloroform. Hence, an MPEO-amylase block copolymer forms reverse micelles in chloroform. Akiyoshi et al. established the capability of the buried receptors to extract the complementary analyte by studying the ultraviolet visible (UV-vis) spectra. A solution of polymer was shaken... 

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