Flow injection binding assays

Nandakumar, M. P., and Mattiasson, B. (1999). Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column. Bioseparation 8, 237-245. 

Flow Injection Antigen-Antibody Binding Assay... 

The binding assays that are performed in this flow injection system may be either competitive or sandwich type. An assay for the serum protein transferrin using a competitive ELISA (129) illustrates the principle (Fig. 5). An immunoafiinity purified polyclonal antiserum raised in rabbits against human transferrin is the binder. The immobilized antibody is packed in a small column (100-200 pL), which is placed in a continuous flow of buffer. The experimental setup is shown in Fig. 6. 

We have developed an LC-based sensor which relies upon the displacement of a drug-dye conjugate from a CAP-imprinted polymer upon application of samples containing the original template molecule (Fig. 20.15). This is essentially a competitive binding assay in a flow injection format. The increased number of theoretical plates available during the separation serves to greatly enhance the selectivity of the assay. 

Fig. 15.2 Flow injection set-up for optical sensing and screening library members and competitive binding assay for fructose developed on-bead using fluorescent labeled glucose...

Brandes W, Maschke HE, Scheper T (1993) Specific flow injection sandwich binding assay for IgG using protein A and a fusion protein. Anal Chem 65 3368 - 3371 Cannings LM, Carr PW (1975) Rapid thermochemical analysis via immobilized enzyme reactors. Anal Lett 8(5) 359-367... 

Figure 10 Surface plasmon resonance analysis. A surface displaying ligand is examined under conditions of flow. Injection of lectin results in an increase in signal observed in the association phase of the curve. At equilibrium the surface achieves maximal binding. Measurement of the dissociation can yield off-rates. Competing ligands in solution can also be assayed.

CHROMATOGRAPHY-LIGAND-BINDING ASSAY COUPLED METHODS, IMMUNOAFFINITY SYSTEMS, AND ONLINE (FLOW-INJECTION) LIGAND-BINDING ASSAY METHODS... 

Flow injection analysis offers many attractive features to biosensor analysis. The reproducibility and the speed are two dominant characteristics when combining FIA with proper sampling and sample handling. It can be used for both enzyme-based assays and immunochemical binding assays. 

Another development in HPLC analysis is the interfacing of on-line sample clean-up technologies at the front end of the HPLC analytical column. Using a switching valve, complex samples can be injected directly onto a sample preparation column, the stationary phase of which is designed to bind the analyte(s) of interest to the exclusion of the rest of the matrix, which runs to waste. Then the valve is switched and the mobile phase flows through the sample preparation column where it picks up the analyte(s) and carries them onto the analytical column and through to the detector in the normal way. This automation of sample preparation saves time and effort and reduces errors. There are many applications of HPLC assays in conjunction with on-line sample clean-up methods in the literature ... 

Serum-Adsorption Experiments Protein-resistance measurements were carried out by following the same procedure as above to form a PLL-g-PEG/PEG-NTA monolayer on the waveguide (or after the standard assay experiments of binding 6xHis-tagged proteins), and then the surface was fully regenerated by exposing it to EDTA (200 mM). After a flat baseline was reached, the control human serum was injected into the OWLS flow cell. After 15 min of equilibration, the flow cell was rinsed with HEPES-2 buffer and allowed to equilibrate for 30 additional minutes. The difference to the first baseline corresponds to the amount of non-specific adsorption of proteins. 

Methods very similar to classical immunoassays in the sandwich format are easily implemented in flow systems (Fig. 2d). In this type of noncompetitive assays, again, antigen is captured and concentrated from an appropriate volume of sample on an immunosorbent (-Abi) column while nonantigenic components are eluted. Subsequent to the capture step, labeled second antibody (Abj-label) is introduced into the mobile phase and swept into the column, where it binds to the -Ab]-Ag complex to form -Ab -Ag-Ab2-label. Unbound Ab2-label is swept from the column, and when the label is an enzyme, antigen is quantitated indirectly by conducting an enzyme assay in the column. After substrate incubation, the reaction product is transported to a detector at the column terminus. Ag and Ab2-label can be introduced in the column sequentially or simultaneously. In some instances both modes led to similar sensitivity [55], and in other cases simultaneous injection produced a greater response than sequential injection [56]. The term sandwich has also been applied to the procedure carried out to quantitate Ab by capturing a complex Ab-Ag-label onto a protein G capillary column [57]. In this case detection is performed after elution. 

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