Monoclonal antibodies probes

Acceptable bridging molecule systems have been developed which have also simplified the utilization of different detection systems. To illustrate this point, a researcher who has developed a unique monoclonal antibody (a primary antibody) in the mouse may select from a variety of commercially available products consisting of different detection systems (e.g. fluorescein, alkaline phosphatase, colloidal gold) attached to an immunoglobulin that will specifically bind to mouse antibodies (a secondary antibody). In this way the researcher may readily obtain and test a number of detection methods for visualizing target-probe interactions without having to directly label the monoclonal antibody probe. For nucleic acid probes, which in themselves are not readily immunodetectable, it is useful to incorporate or attach detectable moieties to the nucleotides. 

The development of monoclonal antibody probes specific to human albumin is the subject of this chapter. These antibody probes and the test methods were developed and evaluated for use in forensic science situations. Although these methods work well on soluble extracts of dried blood stains that are several years old, they have not been applied to archaeological material. The successful development of monoclonal antibodies specific to the human albumin molecule suggests that an approach similar to that used for identifying human tissues and blood on forensic evidence could be applied to any species of interest to the archaeologist. 

The investigation and characterization of cell surfaces by probing with monoclonal antibodies is one of the most vital areas of application. In this context they have been used in the following ways ... 

Blount, S., Griffiths, H.R., Staines, N.A. and Lunec, J. (1992). Probing molecular changes induced in DNA by reactive oxygen species with monoclonal antibodies. Immunol. Lett. 34, 115-126. 

Bonfante et al. (73) used monoclonal antibodies and enzyme-gold complexes to reveal pectins and cellulose at the interface between the fungal wall and the host plasma membrane in AM roots (Fig. 6), and additional wall components have been investigated with other molecular probes (74-76). These studies indicate that the interface is an apoplastic space of high molecular complexity where the boundaries of the partners are defined. The examination of other endomycorrhizal systems has demonstrated that their interface is morphologically similar but different in composition. Cellulose and pectins are present at the interface... 

K.L. Egodage, B.S. de Silva, and G.S. Wilson, Probing the conformation and orientation of adsorbed protein using monoclonal antibodies cytochrome c3 films on a mercury electrode. J. Am. Chem. Soc. 119, 5295-5301 (1997). 

There are literally hundreds of markers that are currently available for the mouse and human than can be used to characterize lymphoid and myeloid cells and subsets in primary and secondary lymphoid organs. Many of the markers expressed in mammals are highly conserved across species and have been designated as genetic clusters of differentiation (CD). CDs can be identified with fluorescently labeled monoclonal antibodies. As presented previously, when combined with other fluorescent probes, important information on intracellular biochemistry and cell function can be obtained. Many of the biochemical markers used by immunotoxicologists are common to other... 

However, recombinant antibodies may be less stable and have lower binding affinities than monoclonal antibodies (Valle and Jendoubi, 2003). Therefore, in order to fully implement the microarray format, a host of diverse capture agents could be required in addition to antibodies. These include peptides, small molecules, aptamers, ribozymes, or other molecular recognition probes yet to be discovered. However, it is also xmderstandable because of the diverse nature of proteins that additional technologies besides microarrays will be used in proteomics research (Hanash, 2003). 

Anti-Aurora-A (see Note 1) monoclonal antibody (610939, BD) and Alexa Fluor 488 Goat Anti-Mouse (A-11001, Molecular Probes). 

Egodage etal. [103] have described a novel application of monoclonal antibodies for the probing of conformation and orientation of the adsorbed protein using cytochrome c(3) films on mercury electrode. Antibodies were utilized to confirm the presence of three confor-mationally distinct electrochemical forms of cytochrome dependent on the applied potential. 

Zhang, T.-H. Luo, J. Zhou, J.-M. Conformational changes at the active site of adenylate kinase detected using a fluorescent probe and monoclonal antibody binding. Biochimie, 84, 335-339 (2002)... 

Using Antibodies to Probe Structure-Function Relationships in Proteins A monoclonal antibody binds to G-actin but not to F-actin. What does this tell you about the epitope recognized by the antibody ... 

Another possible participant in hemozoin nucleation is the histidine-rich protein (HRP). Using monoclonal antibodies to probe the proteins of the digestive food vacuole of P. falciparum, Sullivan et al. identified two histidine-rich proteins, HRP II and HRP III, and demonstrated that these proteins could mediate the formation of hemozoin [37]. HRP II (Mr 35 kD) contains 51 repeats of the sequence Ala-His-His (76% of the mature protein is histidine and alanine) [38], while HRP III (Mr 27 kD) contains 28 Ala-His-His sequences (56% of the mature protein is histidine and alanine) [39]. While a clone lacking both HRP II and III has been shown to produce hemozoin, a third protein which cross reacted with anti-HRP monoclonal antibodies has also been found in the digestive vacuole of this strain. Although this protein has not been isolated or shown to mediate hemozoin formation, a candidate is HRP IV (Mr 10 kD) which is 31% histidine [40]. 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

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