Binding immobilization

Figure 6.2 A Likely purification procedure for tPA produced in recombinant E. coLi cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltra-tion step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed, as tPA is known to bind immobilized Lysine molecules. Adapted with permission from Bio/Technology (1993), 11, 351... 

In the hybridization step, the ability of labeled targets to bind immobilized probes are tested. Referring again to the cases where DNA microarrays are... 

The carbonyl compounds formed due to autoxidation of lipids and in catabolic processes postmortem in muscles, or introduced with wood smoke, can bring about undesirable effects by interacting with a.a. residues. On the other hand, some carbonyl compounds are used intentionally to modify the proteins. Formaldehyde can harden the collagen dope in the manufacturing of sausage casings, protect fodder meals against deamination by the rumen microflora, and bind immobilized enzymes on supports. 

A primary example of biological binding immobilization is the avidin-biotin complex. Biotin (vitamin H) is a low-molecular-weight cofactor distributed ubiquitously in cells. Biotin specifically binds to avidin, an egg white protein, or to strepavidin, a similar protein which occurs in Streptomyces sp. 

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Eig. 5. Determination of the for the binding of DAS to its polyclonal antibody raised in mice. A fixed amount of immobilized antibody in a microtiter plate is reacted with increasing amounts of DAS and the amount of DAS bound at each concentration is deterrnined using an EIA based on alkaline... 

This same experimental approach can be used to determine the appHcabiUty of the aDAS—AP to a competitive assay for DAS. As shown in Eigure 6, increasing amounts of free DAS were used to define the 50% inhibition level (ID q) of DAS for binding of two aDAS—AP conjugates to immobilized DAS. This approach was also used to determine the sensitivity of an EIA, as well as the specificity of the assay, as shown in Table 2. Increasing amounts of trichothecene mycotoxins closely related to DAS were added to microtiter plate wells containing a constant amount of prereacted DAS—aDAS—AP. After 30 min, excess toxin and any free toxin—aDAS—AP were washed out, and substrate was added. Quantification of the color produced was directly related to the abihty of the added toxin to displace aDAS—AP from the immobilized DAS, which is an indication that the aDAS also has an avidity for that toxin. 

Fig. 6. Determination of the amount of free DAS required to cause 50% binding inhibition, ID q, of aDAS—AP to immobilized DAS, as a means to...

In a second example, a cell—gelatin mixture is cross-linked with glutaraldehyde (43). When soluble enzyme is used for binding, the enzyme is first released from the cell, then recovered and concentrated. Examples of this type of immobilization include binding enzyme to a DEAE-ceUulose—titanium dioxide—polystyrene carrier (44) or absorbing enzyme onto alumina followed by cross-linking with glutaraldehyde (45,46). 

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

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