Secondary antibodies

A sensor array where different haptens are immobilized at well-defined areas on a plain glass surface has been developed [66], Using an automated flow injection system it was possible to incubate all areas on the chip with analytes, specific antibodies, secondary HRP-labeled antibodies, and CL substrate. Measurement of the light output via imaging performed with a CCD device allowed determination of the analytes present in the sample on the basis of the spatial localization of the CL signal. 

Autl-F roxidasq Antibody Fazoxidasa Enzyme Anti-Peroxidase Antibody Secondary LinJclng Antibody Frimazy Antibody... 

Immunolocalization is a technique for identifying the presence of a protein within the cell, its relative abundance and its subcellular localization. After suitable preparation of the cells, they are treated with an antibody (the primary antibody) that binds to the protein of interest. An antibody that binds to the primary antibody (the secondary antibody) is then allowed to bind and form an antigen—primary antibody—secondary antibody complex. The detection system generally consists of the formation of a colored insoluble product of an enzymatic reaction, the enzyme, such as alkaline phosphatase or horseradish peroxidase, being covalently linked to the secondary antibody. 

Horseradish peroxidase is one of several enzymes that are commonly used for the detection of antigens in the ELISA. Like HRP-conjugated antibodies, secondary antibodies conjugated with these other enzymes are commercially available. A list of ELISA detection enzymes, the substrates that they use, and the wavelengths of detection for the soluble colored products that they produce is presented in Table 17-1. 

Preparations are incubated with secondary antibody. The secondary antibody will recognize and bind to the primary antibodies. This tertiary (protein-primary antibody-secondary antibody) complex can now be subjected to detection methods. 

Enzyme-linked secondary antibody is added to the well. The solution is incubated for a sufficient time to allow for the tertiary complexes (antigen-primary antibody-secondary antibody) to form. 

Key words Immunohistochemistry, Fluorescence microscopy, IgG, Primary antibody, Secondary antibody, Monoclonal antibody, Polyclonal antibody, Fluorochrome... 

Key words Enzyme, Peroxidase, Alkaline phosphatase, Primary antibody, Secondary antibody, Avidin, Biotin, Substrate, Immunepolymer, Immune complex... 

Indirect or secondary assay antigen attaches to primary antibody, secondary antibody with multiple reporter labelling then attaches to same antigen... 

Fig. 7. Two-dimensional Western blot analysis of anti-3-nitrotyrosine proteins in a human pituitary (70 p,g protein per 2D gel). (A) Silver-stained image on a 2D gel before transfer of proteins to a PVDF membrane. (B) Silver-stained image on a 2D gel after transfer of proteins to a PVDF membrane. (C) Western blot image of anti-3-nitrotyrosine proteins (anti-3-nitrot5TOsine antibodies + secondary antibody). (D) Negative control of Western blot to show the cross-reaction of the secondary antibody (only the secondary antibody no anti-3-nitrotyrosine antibody). Reproduced from Zhan and Desiderio [19], with permission from Elsevier Science (USA), copyright 2004.

Secondary antibodies. Secondary antibodies are diluted in PBS containing 2% normal serum, which is obtained from the same species as the secondary antibody. Depending on the immimostaining method used, the following antibodies are used ... 

---