Noncompetitive immunoassay ELISA

More recently, noncompetitive immunoassays (IRMA, ELISA, and ICMA) have been reported for calcitonin. Com-... 

FIGURE 12.2 Noncompetitive immunoassay (A) direct ELISA (B) enhanced assay using a biotin-streptavidin system (C) indirect ELISA. 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

More recently, noncompetitive methods have been developed for measuring human OC. These immunoassays use monoclonal antibodies and affinity-purified polyclonal antisera against synthetic peptides of human OC or intact human or bovine OC. Many antibody pairs have been reported including capture [and signal] antibodies, respectively, against amino acid sequences 12-33 [and 34-49], 43-49 [and 5-13], 25-37 [and 5-13], 20-43 [and 7-19], 1-19 [and 20-49], and 1-12 [and 15-30 or 38-49], These noncompetitive methods have used signal antibodies labeled with radioactivity (IRMA), enzymes (EIA/ELISA), or chemiluminescent compounds (ICMA). 

Table 1 The plethora of formats of immunoassay. Ab = antibody, Ag = antigen. Noncompetitive and competitive heterogenous binding assays where the probe is labelled with an enzyme are known as enzyme-linked immunosorbent assay (ELISA)...

Figure 5 Schematic representation of the most widely used noncompetitive biologieal immunoassay, the enzyme-linked immunosorbent assay (ELISA), (a) Antibody, selective for analyte, is immobilized on microtiter plate well surface (b) sample added (c) analyte in sample binds to antibody while other compounds in matrix remain in solution (d) sample solution containing nonbound molecules discarded and wells washed (e) analyte remains bound to antibody (f) second antibody nzyme conjugate added (g) conjugate binds to bound analyte (h) solution containing nonbound conjugate discarded and wells washed (i) conjugate remains bound 0 enzyme substrate added (k) substrate S converted to colored or fluorescent product P at a rate which is proportional to the amount of bound enzyme and hence to the concentration of analyte in the sample.

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