Tracer antibody

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

An evanescent wave fiber optic immunosensor has been used for the detection of ricin in river water.(131) A tapered fiber optic waveguide with covalently bound anti-ricin IgG is used in a sandwich format, with tetramethylrhodamine-labeled antibody as tracer. In a two-step format, ricin in the sample is bound to the fiber first, and then the fiber is exposed to the tracer antibody. Sensitivity is 1 ng/ml for the two-step assay. The one-step assay, in which the fiber optic probe contacts the sample and labeled antibody simultaneously is less sensitive, but more convenient. 

This principle requires two different antibodies (capture and tracer antibody). 

For example, several strategies have been used for immunoassay techniques with fiber-optic biosensors. In the sandwich format, the receptor is immobilized on the stu"face of the fiber waveguide and a secondary or tracer antibody (which is labelled with a fluorescent dye) is added to the solution. In the absence of the analyte, the tracer remains in solution and little fluorescence is observed. However, after addition of the analyte, a molecular sandwich is formed on the sensor smface within the evanescent excitation volume. The sandwich assay is usually more sensitive than a competitive-binding assay because the fluorescence intensity increases with analyte concentration. 

The assay is carried out on a test strip and based on a sandwich format with two antibodies. One antibody, the capture antibody, is immobilised, i.e. covalently attached to the device surface. The second antibody, the tracer antibody, is labelled, usually with a dye. This tracer antibody is impregnated onto the surface of the device, but is not permanently attached. The strip component is composed of an adsorbent material. Once the urine sample is applied, the liquid moves along the strip by capillary action and the assay-reactions are carried out in flow. 

A schematic of a test strip is shown in Fig. 5.12. The adsorbent material is usually enclosed within a plastic casing featuring a sample input window, a test result window and a control window. A drop of urine is applied at the sample input and the liquid first moves over the zone, which contains the labelled tracer antibody (Fig. 5.13). If hCG is present in the sample, it forms a complex with the tracer antibody. This complex continues to move along the adsorbent material and passes over the area with the immobilised capture antibody. A sandwich is formed between the immobilised capture antibody and the tracer antibody with the hCG in between. Thus, the initially mobile antibody with the label becomes immobilised. The amount of sandwich complexes formed is directly proportional to the amount of hCG present in the sample. If the hCG concentration exceeds a minimum concentration, the dye colour becomes visible to the eye. 

MPO assay kit (Hycult Biotech) containing anti-MPO antibody-coated 96 weU plates, dUution buffer, Streptavidin-peroxidase, biotinylated tracer antibody, TMB substrate, and stop solution. 

FIGURE 51 Scheme of the flow-through channel-resolved immunosensing system for the determination of AFP and CEA (a) flow cell (b) flow-through CL immunosens-ing system and process of immunoassay (c) transect of flow cell for immunoassay and (d) optical shutter. WB wash buffer RB regeneration buffer CS CL substrate S mixture of sample and tracer antibody M I anti-AFP immobilized membrane M II anti-CEA immobilized membrane. (Adapted from Fu, Z. et al. 2008. Biosens. Bioelectron. 23 1063-1069. With permission.)... 

A typical sequence of responses of the heterogeneous amperometric immunosensor for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is shown in Pig. 13.4. The mixture of sample and tracer (antibody-peroxidase conjugate) is added to the sensor with immobilised 2,4-D and allowed to incubate. After a washing step, the surface-bound peroxidase label becomes quantified amperome-trically, and the signal is inversely proportional to the amount of herbicide in the sample (as in Pig. 13.1b, plots at the top right comer). The highest response is obtained in the absence of the analyte, when maximum binding of the tracer is achieved, and the blank obtained in the absence of the tracer allows one to correct for nonenzymatic reactions of the substrate mixture. 

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