Sandwich immunoassays antibody selection

Figure 4.12 describes two popular antibody microarrays formats that are constructed for antigen capture in small sample volumes with detection by either sandwich immunoassay or antigen labeling. In sandwich immunoassay, capture antibodies are arrayed and immobilized to select specific proteins which are then found by a second labeled detection antibody. In protein target labeling, all proteins in the sample are prelabeled (i.e., fluorescent dyes) prior to capture by immobilized antibody arrays. In direct assay systems, sample proteins are directly immobilized onto... 

Replicates Repeated independent determinations on the same sample by the same analyst at essentially the same time under the same conditions Response Measured characteristics when a sample is analysed Sandwich immunoassay Immunoassay variant in which the analjde is detected by binding to two different antibodies Selectivity Characteristic of a method to distinguish between true/specific and inaccurate/non-specific results... 

For triazine herbicides a very selective and sensitive immunological technique is recommended. The triazine herbicides are covalently bound to soil humic acids. A sandwich-immunoassay based on both polyclonal humic acid-antibodies and monoclonal triazine-antibodies111 was used for triazine detection. The schematic view of sandwich immunoassay is presented in Figure 4.3. The technique is very selective and sensitive. It can assure the best reliability of the analytical information because it does not require a prior separation. 

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. 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

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