Use of Monoclonal Anti‐enzyme Antibodies for Analytical Purposes

Gunaratna, P C; Wilson, George S.

doi:10.1021/bp00016a002

Cited by 8 publications

(6 citation statements)

References 42 publications

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“…This amount is not unusual when compared to the literature values. 31,32 As already explained, the decrease in assay time is a significant reason for developing microchannel-based immunochemical systems. Therefore, the investigation of the equilibration kinetics 33 is of major interest.…”

Section: Resultsmentioning

confidence: 99%

Characterization of Protein Adsorption and Immunosorption Kinetics in Photoablated Polymer Microchannels

et al. 2000

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A preliminary characterization of protein adsorption and immunosorption kinetics carried out in polymer microchannels is reported. A photoablated poly(ethylene terephthalate) (PET) surface and a PET/polyethylene sealing laminate were used for the channel microfabrication. The surface state of the PET channel substrate and PET/polyethylene lamination were analyzed by using SEM and ATR-FTIR spectroscopy techniques. Protein adsorption and immunosorption studies were carried out using staphylococcal enterotoxin B (SEB) and polyclonal anti-SEB antibody (Ab) samples. Affinity purified polyclonal rabbit (Rb) anti-SEB Ab was radioiodinated and adsorbed in the microchannel. It was determined that the maximum amount of adsorbed antibody was about 13.0 pmol·cm-2 (about 2 μg·cm-2), which corresponds to 0.81 pmol per microchannel. The distribution of the adsorbed protein on the walls of the microchannel depended on the surface state of the polymer exposed to the solution. The amount of the radiolabeled antibody adsorbed on the photoablated PET was about 19.4 pmol·cm-2, whereas it was only 5.5 pmol·cm-2 on the PET/polyethylene lamination. About 30% of the anti-SEB Ab adsorbed on the microchannel surface was found to be biologically active. A study of the kinetics of the SEB−anti-SEB Ab immunochemical reaction was also carried out. It could be substantiated that the forward reaction is diffusion controlled and that the equilibrium for such a reaction could be achieved within about 1 min in the microchannels. This is in good agreement with the calculation of a diffusion-controlled reaction in such a microchannel, according to Fick's second law.

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Section: Resultsmentioning

confidence: 99%

Characterization of Protein Adsorption and Immunosorption Kinetics in Photoablated Polymer Microchannels

et al. 2000

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“…To date, biological recognition elements such as antibodies, − enzymes, − , and DNA 19,21b,23 have been successfully incorporated into thin films. Few studies, however, have been directed at achieving oriented bioassemblies. ,,21a, Of these studies, the majority have centered on exploiting natural affinity ligands as a means of orienting functional proteins in lipid monolayers 22 or into bilayers .…”

Section: Introductionmentioning

confidence: 99%

“…To date, biological recognition elements such as antibodies, 9-12 enzymes, [13][14][15][16][17][18]24 and DNA 19,21b,23 have been successfully incorporated into thin films. Few studies, however, have been directed at achieving oriented bioassemblies.…”

Section: Introductionmentioning

confidence: 99%

Film Architecture in Biomolecular Assemblies. Effect of Linker on the Orientation of Genetically Engineered Surface-Bound Proteins

et al. 1996

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This contribution presents strategies for the optimization of supramolecular architecture aimed at controlling the organization of biomolecules at solid surfaces. Myoglobin, modified by site-directed mutagenesis to include a unique cysteine residue, is selectively chemisorbed to self-assembled haloalkylsilylated silica surfaces of varying n-alkyl chain length (n = 2, 3, 8, 11, 15) to yield a series of surface-immobilized recombinant protein assemblies. These supramolecular assemblies are probed using tapping mode atomic force microscopy, wettability measurements, Fourier transform infrared spectroscopy, and linear dichroism spectroscopy to determine how the individual components comprising these structures (substrate, silane coupling layer, and protein) influence macromolecular protein ordering and stability. Surface roughness is found to be a minor contributor in the determination of macromolecular ordering in these assemblies. In contrast, the nature of the underlying silane self-assembled coupling layer is shown to strongly influence both the spatial and functional properties of the chemisorbed protein. Silane coupling layers with short aliphatic chain lengths (n = 2, 3) produce highly trans-conformationally ordered structures upon which differential heme prosthetic group orientation can be achieved. Long alkyl chain (n ≥ 11) silane-derivatized surfaces also form ordered structures. The stability of myoglobin appended to long chain aliphatic silylated surfaces is poor, however. The apparent protein instability arises due to the increased hydrophobic character of these films. At intermediate alkyl chain length (n = 8), a conformationally disordered coupling layer with a high concentration of gauche defects is produced, regardless of the method of silane deposition or postdeposition processing. Chemisorption of myoglobin to the highly disorganized assembly yields a random orientation of the protein.

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“…Moreover the method is easy to apply and could also be used for protein patterning when based on an underlying DNA template pattern. An alternative approach could be to use an antibody instead of an aptamer and this has indeed been described for other enzymes [41]. However there are compelling cost reasons to choose aptamers over antibodies.…”

Section: Resultsmentioning

confidence: 99%

High Efficiency Acetylcholinesterase Immobilization on DNA Aptamer Modified Surfaces

Chumphukam

Cass

2014

Molecules

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Abstract:We report here the in vitro selection of DNA aptamers for electric eel acetylcholinesterase (AChE). One selected aptamer sequence (R15/19) has a high affinity towards the enzyme (K d = 157 ± 42 pM). Characterization of the aptamer showed its binding is not affected by low ionic strength (20 mM), however significant reduction in affinity occurred at high ionic strength (1.2 M). In addition, this aptamer does not inhibit the catalytic activity of AChE that we exploit through immobilization of the DNA on a streptavidin-coated surface. Subsequent immobilization of AChE by the aptamer results in a 4-fold higher catalytic activity when compared to adsorption directly on to plastic.

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Use of Monoclonal Anti‐enzyme Antibodies for Analytical Purposes

Cited by 8 publications

References 42 publications

Characterization of Protein Adsorption and Immunosorption Kinetics in Photoablated Polymer Microchannels

Characterization of Protein Adsorption and Immunosorption Kinetics in Photoablated Polymer Microchannels

Film Architecture in Biomolecular Assemblies. Effect of Linker on the Orientation of Genetically Engineered Surface-Bound Proteins

High Efficiency Acetylcholinesterase Immobilization on DNA Aptamer Modified Surfaces

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