Avidin alkaline phosphatase conjugate

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.)...

PAP, peroxidase-antiperoxidase ABC, avidin-biotin conjugate APAAP, alkaline phosphatase antialkaline phosphatase B-SA, biotin-streptavidin PCNA, proiiferating cell nuclear antigen EPOS, enhanced polymer one-step staining (Dako) CARD, catalyzed reporter deposition HRP, horseradish peroxidase. 

Figure 1 Immune complexes formed in typical PCR immunoassay. The two general configurations for DNA immunoassay that are shown differ in the means of capture. Analyte DNA is assumed to have been amplified with a 5 -biotinylated primer. In the immunocapture approach, biotin provides the means for capture onto an avidin-coated multiwell plate. In the hybridization approach, the analyte is captured by hybridization to an immobilized probe followed by immunoenzymatic detection with an avidin-alkaline phosphatase conjugate.

Figure 26 Faradaic impedance spectra corresponding to (a) The (33)-modified electrode, (b) After hybridization with M13< DNA, 2.3 X 10 M. (c) After the polymerase-induced replication and formation of the double-stranded assembly for 45 minutes, (d) After the binding of the avidin-alkaline phosphatase conjugate to the surface, (e) After the biocatalyzed precipitation of (12) for 20 minutes in the presence of (11), 2 X 10 M, in 0.1 M Tris-buffer, pH = 7.2.

Figure 1. Immunoblots and avidin blots of recombinant E, coli DH5ocF cells expressing C. cryptica ACCase. Lane A - Control cells containing pBluescript KS+. Lane B - pACC20M9-containing ceUs. Lane C - purified C. cryptica ACCase. Secondary antibodies and avidin were conjugated to alkaline phosphatase. The gels used for these blots were loaded with equal amounts of protein.

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

Perhaps the most common conjugates of (strept)avidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 20), by far the most commonly used enzymes for this purpose are HRP and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 26). 

If P-galactosidase is used to conjugate with an SMCC-activated (strept)avidin, then there is no need to thiolate the enzyme, since it contains sulfhydryls in its native state (Fujiwara et al., 1988 Sivakoff and Janes, 1988). For conjugations using HRP, alkaline phosphatase, or glucose oxidase, however, thiolation is necessary to add the requisite sulfhydryls. 

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. 

Incubate sections for 30 min with (strept)avidin conjugated with a fluorophore or with any enzyme (peroxidase or alkaline phosphatase). For localization of low-density antigens (<10K molecules/cell), VECTASTAIN ABC reagent (http //www.vectorlabs.com/) is preferable. 

Incubate the slides m a mixture of avidin—peroxidase conjugate diluted 1.100 and alkaline phosphatase-conjugated antidigoxigenin diluted 1 600 m TBT, in practice, 1 pL of antibody and 6 pL of avidin are added to 600 pL of TBT. 

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). 

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. 

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