Alkaline phosphatase avidin-labeled

Reaction with avidin-labeled alkaline phosphatase (AP)-biotin conjugate... 

As an example of the use of antibodies labeled with alkaline phosphatase for detection of in situ hybridization, an infection with BNYVV virus in sugar beet is shown in Fig. 3C. Lectins labeled with an avidin-biotin fluorescein conjugate was used to visualize a-galactosyl groups on the surface of S. pombe in Fig. 3D. 

Enzyme labels are usually coupled to secondary antibodies or to (strept)avidin. The latter is used for detection of biotinylated primary or secondary antibodies in ABC methods (see Sect. 6.2.1). Enzyme labels routinely used in immunohisto-chemistry are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E. coli (3-galactosidase are only rarely applied. 

Fig. 1. Dual nucleic acid detection. The target DNAs (A and B) are denatured and hybridized with complementary DNA probes labeled with biotin (Bio ), or digoxigenm (Dig ). Biotin and digoxigenm residues are detected, respectively, with avidin peroxidase (red) and antibody to digoxigenin labeled with alkaline phosphatase (blue).

Fig. 2. Diagrammatic representation of three-step digoxigemn detection. The target nucleic acid (1) is labeled with digoxigemn (Dig.). Monoclonal antidigoxigenm (2) is linked via biotinylated (Bio) rabbit antimouse (3) to avidin alkaline phosphatase (4)...

Simultaneous, double immunostaining of two antigens in single cells in sections of formalin-fixed and paraffin-embedded archival tissues can be carried out. This is accomplished by using microwave heating to detect otherwise undetectable nuclear antigens, followed by the labeled avidin-biotin (LSAB) procedure and the alkaline phosphatase (APAAP) protocol to detect cytoplasmic or membranous antigens (Bohle et al 1997). 

A variety of techniques (Fig. 1), which use a variable combination of antibody steps, enzyme labels, and chromogens, are employed in immunohistochemistry. The most common immunohistochemical methods are the PAP (peroxidase-antiperoxidase), ABC (avidin-biotin) peroxidase or alkaline phosphatase, and the APAAP (alkaline phosphatase-anti-alkaline phosophatase) techniques however,... 

Immunohistochemical staining can be direct or indirect. Direct immunohis-tochemical staining methods utilize only a primary antibody, which may be conjugated to horseradish peroxidase, biotin, alkaline phosphatase, or other chromogens. In the case of biotin-labeled primary antibodies, avidin or strep-tavidin linked to peroxidase binds to the biotin allowing detection of reactivity of the test antibody with the tissue. Indirect immunohistochemical staining methods utilize secondary, tertiary, or even quaternary antibodies, any of which may be linked either to biotin or enzyme (e.g., peroxidase). 

Incubate for 2 hr at 37° with avidin labeled with alkaline phosphatase (diluted 1/4000 in 1% BSA-PBS-T). 

However, the same basic principles can be applied (with appropriate modifications) to the labeling of antigens with other enzymes (peroxidase, alkaline phosphatase, -galactosidase). This is particularly true for those labeling methods involving the use of biotinylated antigens, since biotinylated enzymes as well as avidin-enzyme conjugates are available from numerous commercial sources. 

The three amplification procedures used with this functionahzed Au-quartz crystal interface consisted of detection first using avidin and biotin-labelled liposomes, secondly using avidin-Au-nanoparticle conjugate and the catalysed electroless deposition of gold, and thirdly avidin-alkaline phosphatase interaction with 5-bromo-4-chloro-3-indolyl phosphate causing the biocatalysed precipitation of the insoluble product on the piezoelectric crystal. The separation of surface treatment outside the QCM cell coupled with... 

Fig. 4.1. Amplified detection paths of a single-base mismatch in nucleic acids (a) using avidin and hiotin-labelled liposomes (h) using an avidin-Au-nanoparticle conjugate and the catalysed deposition of gold (c) using an avidin-alkaline phosphatase bioconjugate and the biocatalysed precipitation of the insoluble product 4. (Reproduced from Ref. [63] with permission from Elsevier.)...

---