Antibodies flexibility

Smith, T. J., Olson, N. H., Cheng, R. H., Chase, E. S., and Baker, T. S. (1993). Structure of a human rhinovirus-bivalently bound antibody complex Implications for virus neutralization and antibody flexibility. Proc. Natl. Acad. Sci. USA 90, 7015-7018. 

IgG antibody molecules are composed of two light chains and two heavy chains joined together by disulfide bonds. Each light chain has one variable domain and one constant domain, while each heavy chain has one variable and three constant domains. All of the domains have a similar three-dimensional structure known as the immunoglobulin fold. The Fc stem of the molecule is formed by constant domains from each of the heavy chains, while two Fab arms are formed by constant and variable domains from both heavy and light chains. The hinge region between the stem and the arms is flexible and allows the arms to move relative to each other and to the stem. 

The important advantage of the immunosorbents based on WPG-PA is the fast rate of biospecific interaction between the oligosaccharides and antibodies. For B-trisaccharide-WPG-PA the average sorption time of monoclonal B8 antibodies was 20 times shorter than with B-trisaccharide-Sepharose 4B. The role of the flexible polymeric spacer, therefore, is in this case very pronounced. 

For systems that do not offer online HIAR, these procedures must be performed manually or off-line prior to loading slides on the instrument. Online HIAR methods are usually found as part of the closed-type automated staining systems, and are therefore less flexible in terms of what a technician can do to change the HIAR portion of a staining protocol to help optimize staining for particularly difficult antibodies. In spite of this, the ability to perform online HIAR is advantageous for many antibodies because it is extremely consistent and frees up technician time to complete other laboratory tasks. 

In general, online versus off-line HIAR offers similar pros and cons as does the open versus the closed automated staining systems. Closed systems and online HIAR not only produce extreme consistency and require less technical knowledge, but it can also limit the number of antibodies that can be used successfully. Open systems and off-line HIAR allow the flexibility to use many more types of antibodies, but technicians need to be well trained and diligent in their record keeping in order to insure consistent staining. 

Using the same versatile modular synthetic strategy, the same group developed biotinylated bi- (438) and tetra-antennary (439) mannosylated glycoconjugates to capture and detect E. coli cells, and compared the relative capturing ability of these molecules to commercial polyclonal antibodies (Fig. 47).318 Instead of aliphatic spacers, tetraethylene glycol linkers were used to diminish nonspecific binding and to impart flexibility for a better fit in the active sites. 

The following protocol should be compared to the method described for SATA thiolation in Chapter 1, Section 4.1. Although the procedures are slightly dissimilar, the differences indicate the flexibility inherent in the chemistry. For convenience, the buffer composition indicated here was chosen to be consistent throughout this section on enzyme-antibody conjugation using SMCC. Other buffers and alternate protocols can be found in the literature. 

Brown JK, Pemberton AD, Wright SH, Miller HRP (2004) Primary antibody Fab fragment complexes a flexible alternative to traditional direct and indirect immunolabeling techniques. J Histochem Cytochem 52 1219 1230... 

Spectrophotometric plate readers Perkin-Elmer s lambda reader, an automated microprocessor-controlled, microplate reader, offers the flexibility of configuring a reliable, user-friendly, versatile system, capable of accommodating a wide variety of assays requiring calorimetric measurement on microscale (<300pl) samples. These assays include ELISA, protein determination, cytotoxicity, cytoproliferation and antibody sensitivity testing. 

As described above, the dendrimer-coupled antibody conjugates show uniquely enhanced properties compared to the classical double antibody systems. Important characteristics such as complete solubility in aqueous buffers, flexibility in immunoassay format, ability to improve assay sensitivity, consistent, reproducible manufacturing and favorable stability has driven the utilization of these dendrimer-based reagents in Stratus CS, the latest member of the Stratus family of immunochemistry analyzers. In this new analyzer system, the primary... 

The general method utilized to prepare E5-Ab solutions obviates the need for stocking large numbers of reagents which would be necessary if different activation methods were used for each antibody. A number of specific antibodies immobilized by this process have shown response similar to that of the same antibodies when adsorbed as immune complexes in the Stratus system. In addition, the dendrimer-coupled antibodies have shown dramatic improvements in sensitivity, flexibility and precision for the enzyme immunoassay system. Feasibility demonstration of an assay for DNA probes is a prelude to what can possibly be achieved with these dendrimer-based reagents. 

The basic structure of an immunoglobulin molecule, such as the major serum antibody IgG, consists of four polypeptide chains two identical light chains (molecular weight around 25 000 daltons) and two identical heavy chains (with a molecular weight around 50 000 daltons), cross-linked by disulfide bonds to form Y-shaped molecules with two flexible arms (Fig. 11.2). The binding sites are located on the arms and vary from one molecule to another (variable region) [22b]. 

Limited proteolysis. Flexible regions of proteins can sometimes be removed by digestion of the protein with different proteases. This technique is based on the techniques that were used to determine the core folded regions of proteins, most notably antibodies (Porter, 1973). Limited proteolysis can be used to remove flexible loops of proteins, or separate multidomain proteins into separate domains and has been used successfully in a number of instances (Noel et al., 1993 Sondek et al., 1996 Mazza et al., 2002). 

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