Selecting an antibody

An alternative aspect of specificity to consider is that of the antigen, for the same epitope may be presort on more than one antigen species. This is an important concept which is often overlooked in fervour of the belief that since monodonals are specific reagents, an assay employing them will be equally specific ft)r the expected antigen. 

For many proteins, a purified protein standard is ideal and straightforward to incorporate since the concentration is known. However, in reality such standard/ sample accord is often not possible to obtain because the antigen contained in the sample may be a mixture of species. For example, it may come in a variety of molecular sizes (e.g. monomeric versus oligomeric forms of immimoglobulin A), or it may exist in complexes with other molecules (e.g. a protease bound to a macromolecular inhibitor, or a ligand bound to its soluble receptor), or as a 

In some cases it miy be possible to use a single sample to provide the standard antigen, providing it contains a similar mix of molecular species. This can also be useful if a purified protein standard is not available or scarce. Samples may then be designated as standard sample , or in other units if the standard sample can be alternatively quantitated by some means (e.g. its functional activity). 

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Selecting an antibody 297 Selecting an assay standard 297 Sample matrix 299 Sample preparation and dilution 299 Assay turnaround time 300... 

Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

Pollack, 5.J., Jacobs, J.W., Schultz, RG. Selective chemical catalysis by an antibody. Science 234 1570-1573,... 

Wallraff, E., Schleicher, M., Modersitzki. M Reiger, D., Isenberg, G., Gerish, G. (1986). Selection of Dictyostelium mutants defective in cytoskeletal protein Use of an antibody that binds to the ends of alpha-actinin rods. EMBO J. 5,61-67. 

The remarkable selectivity that is inherent in the reaction of an antibody with the antigen or hapten against which it was raised is the basis for the extensive use of immunoassay for the rapid analysis of samples in clinical chemistry. Immunochemical reactions offer a means by which the applicability of potentiometric techniques can be broadened. A number of strategies for incorporating immunoassay into the methodology of potentiometry have been explored... 

An electrode in which an antibody or an antigen/hapten is incorporated in the sensing element is termed an immunoelectrode . The potential response of the immuno-electrode is based on an immunochemical reaction between the sensing element of the electrode and antibody or antigen/hapten in the sample solution. One example of such an electrode is the polymer membrane electrode shown in Fig. 7. The selective response of this electrode to specific immunoglobulins is based on the interaction between antibody in solution and an antigen-ionophore complex in the membrane ... 

Another subset of SPE is immunoaffinity extraction, in which an antibody specific to the analyte is incorporated into the SPE sorbent. This technique is very selective to the analyte and would be very effective in separating the marker residue from tissue-related matrix components. Disadvantages of immunoaffinity extraction are the need to develop a specific antibody-based SPE for each analyte. This approach holds promise for the future as the development of antibody-based methods becomes more commonplace. 

Immunosorbents have also found applicability in on-line SPE analysis. An antibody is immobilized on to a silica support and used as an affinity ligand to retain targeted analytes. Components not recognized by the antibody are not retained and some degree of selectivity is attained. Recoveries of 87-103% were obtained for atrazine, simazine, DEA, propazine, and terbuthylazine at the 0.2 xgL concentration level when using immunosorbent SPE (80 mg silica and 2 mg anti-atrazine and anti-chlortoluron antibodies) on-line with LC/APcI-MS however, this method is not applicable to DIA (0% recovery). This compound may be better retained when using an... 

Laden JC, Philibert P, Torreilles F, Pugniere M, Martineau P (2002) Expression and folding of an antibody fragment selected in vivo for high expression levels in Escherichia coli cytoplasm. Res Microbiol 153 469-474... 

Thorpe, P.E., Mason, D.W., Brown, A.N.F., Simmonds, S.J., Ross, W.C.J., Cumber, A.J., and Forrester, J.A. (1982) Selective killing of malignant cells in a leukaemic rat bone marrow using an antibody-ricin conjugate. Nature (London) 297, 594. 

Many precolumns and trap cartridges for sample clean-up are commercially available. In our experience, a 2 to 3 cm short column with twice the analytical column inner diameter and packed with the same particles performs satisfactorily. An antibody affinity column for selective removal of highly abundant proteins from human serum samples provides better sensitivity for the discovery of low abundance protein markers that may represent revolutionary therapeutic diagnosis and monitoring. 

In 1975, Kohler and Milstein observed that if an antibody-producing cell was fused with a myeloma tumor cell, a rapidly dividing hybrid was produced that synthesized a monospecific antibody. Each hybridoma formed then became a factory, producing antibodies monospecific to a particular sensitizing antigenic epitope. Cell cloning allows selection of hybrids producing antibodies with the desired characteristics. 

Colloidal gold A suspension (or colloid) of submicrometer-sized particles of gold in a fluid, usually water. A colloidal gold conjugate consists of gold particles coated with a selected protein or macromolecule, such as an antibody, protein A or protein G. Because of their high electron density, the gold particles are visible in the electron microscope without further treatment. 

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