Antibodies specificity determination

The preparation of antibodies specific for the individual plasma proteins has greatly facilitated their smdy, allowing the precipitation and isolation of pure proteins from the complex mixmre present in tissues or plasma. In addition, the use of isotopes has made possible the determination of their pathways of biosynthesis and of their turnover rates in plasma. 

When an antigen is injected into an animal, the resulting antibodies are polyclonal, being synthesized by a mixture of B cells. Polyclonal antibodies are directed against a number of different sites (epitopes or determinants) on the antigen and thus are not monospecific. However, by means of a method developed by Kohler and Milstein, large amounts of a single monoclonal antibody specific for one epitope can be obtained. 

Figure 7.1. Protein expression mapping using an antibody array. The antibody array consists of monoclonal antibodies specific for a set of proteins in the organism of interest gridded onto a filter. To determine if a protein is expressed under the conditions being tested, a crude lysate is obtained and the proteins within the lysate are labeled with a fluorescent tag. The lysate is applied to the filter and the proteins are allowed to bind to the relevant antibody. Bound proteins are visualized via the fluorescent tag.

Immunohistochemistry is a powerful method for identification of proteins in cells and tissues, but control procedures are necessary. The specificity of the results depends on two independent criteria the specificity of the antibody and of the method used (Burry 2000). The antibody specificity is best determined by immu-noblot and/or immunoprecipitation. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG (must be the same species as primary antibody at the same concentration), and a positive control, testing the primary antibody with tissues known to contain the antigen. 

Alternative methods to MS for the analysis of protein phosphorylation would involve immunoaffinity techniques using antibodies specific for phosphorylated residues. Surface plasmon resonance technology has been recently used for the determination of active concentration of phosphotyrosine-containing proteins at picomolar levels.117... 

In general, four factors help to determine the sensitivity of the sandwich ELISA. These factors are (1) the number of molecules of the first antibody that are bound to tbe solid phase (2) the avidity of the first antibody for the antigen (3) the avidity of the second antibody for the antigen (4) the specific activity of the second antibody. By diluting or concentrating the antibody solution, the amount of capture antibody that is bound to the solid phase can be adjusted. In contrast, tbe avidity of the antibodies for the antigen can be altered only by substituting other antibodies. The specific activity of the second antibody is determined by the number and type of labeled moieties it contains. 

Libraries can also be made from naturally occurring peptides (e.g. melanocortin [30] and somatostatin [31]) and their variants, or be derived from protein-fragments. The former have been employed mainly for the identification of peptide variants with unproved characteristics, and the latter for the identification of antigenic determinants present on targets, for example by selecting for binders to monoclonal antibodies specific for an antigen-fragment peptide library. 

The issue of which antibody to select for an assay is not a new problem. Certainly anyone involved in the development of an immunoassay has been faced with this choice. Consider attempting to create a multianalyte, microarray-based micro-ELISA of modest density (10 to 100 analytes) and determining which capture antibodies to use based upon their affinities, stabilities, and cross-reactivities. For a sandwich assay, add in the 10 to 100 analyte-specific secondary (reporter) antibodies and determine their levels of cross-reactivity with each other and with the specified antigens and capture antibodies. In other words, achieving high performance for all analytes with a microarray immunoassay is indeed a formidable challenge. 

The IgD profile in a normal Nigerian population was similar to that of the British and American populations (T7). Interestingly, in more than half the patients with African trypanosomes, IgD was absent from their sera (M35). Three out of six diabetic patients had insulin antibodies of the IgD class (D3), and IgD levels were five to six times higher in Ethiopian children than in Swedish children of the same age (J4). Patients with penicillin-induced hemolytic anemia had IgD receptors on their erythrocyte surfaces and sera from some subjects allergic to penicillin G were shown to have IgD antibodies specific for the benzyl-penicilloyl-antigenic determinant of this antibiotic (C19). 

While invertebrate tropomyosins are likely pan-allergens, vertebrate tropomyosins appear to be nonallergenic (Reese et ah, 1999). Using bioinformatics approaches to compare the sequences of tropomyosins from various species, Goodman et ah (2002) determined that tropomyosins from vertebrate species — rabbit, pig, chicken, and human — share 53-57% amino acid sequence identity to the known shrimp tropomyosin allergen. Met e 1. This comparison likely explains why vertebrate tropomyosins are not allergenic and do not cross-react with IgE antibodies specific to invertebrate tropomyosins. 

For these reasons, microbial sensors are less suitable for the determination of individual analytes. However, some practical apphcations for biosensors based on enzymes or antibodies for the specific determination of environmentally relevant compounds can be expected soon [11]. Furthermore, in some cases defined specific metabolic pathways in microorganisms are used, leading to microbial sensors for more selective analysis for those environmental pollutants which cannot be measured by the use of simple enzyme reactions, e.g., aromatic compounds and heavy metals. In this context it is also important to mention the aspect of bio availability, a parameter which is included by the measuring procedure of microbial sensors as an integral effect. 

Capromab pendetide is a murine monoclonal antibody specific for prostate specific membrane antigen. It is coupled to isotopic indium (111In) and is used in immunoscintigraphy for patients with biopsy-confirmed prostate cancer and post-prostatectomy in patients with rising prostate specific antibody level to determine extent of disease. 

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