Alkaline phosphatase antibody conjugates

Jeanson, A., Cloes, J.M., Bouchet, M., and Rentier, B. (1988) Preparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent. Anal. Biochem. 172, 392. 

Incubate with 100 pL of an antihuman Fab (IgG-specific) alkaline phosphatase antibody conjugate (Sigma) diluted to 1.1000 in PBS containing 1% BSA for 1 h. 

Jeanson, A., et al. (1988). Preparation of Reproducible Alkaline Phosphatase-antibody Conjugates for Enzyme Immunoassay Using a Heterobifunctional Linking Agent, Anal. Biochem. 172 392-396. 

Ford, D.J., Radin, R., and Pesce, A.J. (1978) Characterization of glutaraldehyde coupled alkaline phosphatase-antibody and lactoperoxidase-antibody conjugates. Immunochemistry 15, 237. 

Fig. 4. Testing of a conjugate prepared from purified antibodies to mouse immunoglobulin labeled with alkaline phosphatase. Ordinate conjugate dilution abscissa absorbance at 405 nm of samples diluted 1 4 after 15 min of incubation with substrate. — , Dose response in microtiter wells coated with mouse serum proteins O—O, dose response in uncoated wells.

Fig. 4. Schematic diagram of the steps in the automated PCR/OLA procedure performed with a robotic workstation. The assay contains three steps (1) DNA target amplification (2) analysis of target nucleotide sequences with biotin (B)-labeled and digoxigenin (D)-labeled oligonucleotide probes and T4 DNA ligase (L) and (3) capture of the biotin-labeled probes on streptavidin (SA)-coated microtiter wells and analysis for covalently linked digoxigenin by using an ELISA procedure with alkaline phosphatase (AP)-conjugated antidigoxigenin (aD) antibodies and a substrate (S). Reprinted with the permission of Nickerson el al. (N2) and the Proc. Natl. Acad. Sci. (U.SA.).

Figure 3 Immuno localization of acetyl esterase. Sections were incubated with antibodies raised against the acetyl esterase, followed by visualization with alkaline phosphatase conjugated secondary antibodies and staining with Fast Red.

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

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). 

Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate. Proc. Natl. Acad. Sci. USA 85, 4842. 

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. 

Note Do not use PBS for alkaline phosphatase-conjugated antibodies, since phosphate is an inhibitor of alkaline phosphatase. 

The RPIA technology has been enhanced in the Stratus CS system by utilization of a dendrimer-antibody complex in which the analyte-specific capture antibody is covalenty coupled onto a dendrimer. The test packs in the Stratus CS system include dendrimer-capture antibody complex reagent, the alkaline phosphatase labeled antibody conjugate reagent, the substrate-wash reagent and a piece of glass fiber filter paper as the solid phase. Preparation and unique properties associated with these dendrimer-coupled antibody complexes are described below. 

The Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

---