Avidin HRP conjugate

The K138C mutants (-1.5 mg in 0.5 mL of PBS) were treated with 50 mM DTT (removed by gel filtration) and then biotinylated using biotin-maleimide (Sigma) (2 1 molar ratio). The biotinylated product was purified by gel filtration on Superdex 75. Evidence of biotinylation was routinely obtained by western blots probed with an avidin-HRP conjugate (Pierce) and by binding of the proteins to streptavidin-coated BIAcore chips (Pharmacia SA5). 

Figure 9 Stepwise assembly of a DNA-sensing electrode by the functionalization of the conductive support with the sensing oligonucleotide (13), interaction with the target DNA (14) pre-hybridized with the biotinylated oligonucleotide (15), and the association of the avidin-HRP conjugate (16). The biocatalyzed precipitation of the insoluble product (8) is used as an amplification route for the electrochemical transduction of the biorecognition event.

Figure 11 Time-dependent frequency changes resulting upon the biocatalyzed precipitation of (8) onto an Au-quartz crystal modified with the (13)-oligonucleotide sensing monolayer, which was interacted with the target DNA, (14), 5.8 x 10- mg-mL that was pre-hybridized with the biotinylated oligonucleotide, (15), 2.6 x IQ- mg-mL . The resulting tri-component ds-assembly on the Au-quartz crystal was then reacted with the avidin-HRP conjugate, (16). Curve (a) shows the frequency changes upon the precipitation of (8) in the presence of (7), 5 x lO 5 x IQ- M. Curve (b) shows the frequency in...

Results from a Western blot. A SDS-PAGE gels, 12%, were run and transferred to nitrocellulose. Lane 1, MW standards lane 2, biotinylated standards lane 3, human transferrin lane 4, . coli lysate lane 5, total human serum lane 6, biotinylated standards. Gel A was stained with a protein dye. Blot B was assayed using rabbit anti-human transferrin as the first antibody. The second antibody solution contained anti-rabbit HRP conjugates. Only the transferrin bands and the prestained biotinylated standards were detected by the antibodies and the avidin-HRP treatment. 

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. 

Originally described several years ago for use in immunoassay systems (36), biotinylated tyramine amplification has recently been adapted for immuno-cytochemical use (37). The system is based on the ABC method. H2O2 is catalysed to H2O and oxygen-free radicals by HRP as usual. This then reacts with biotinylated tyramine to produce activated biotinylated tyramine. which binds to nearby proteins in a highly localized manner. The high concentrations of biotin thus produced can then be detected by the application of one additional layer of avidin-reporter conjugate. The system has been used to demonstrate antigens previously only detectable in frozen sections, and to increase primaiy antibody dilution. Several manufacturers produce kits that are based on the system (CSA from Dako Ltd. and TSA from NEN life Science Products), or home-made biotinylated tyramine can be produced. 

Figure 13 Illustration of the principle of the competitive assay using an avidin-horse-radish peroxidase conjugate. (A) Incubation of the components. (B) After washing, only bound avidin-HRP is detected. The more free biotin in the incubation mixture, the less bound avidin-HRP after the washing step. B, biotin Av, avidin HRP, horseradish peroxidase.

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. 

Conjugation of HRP by reductive amination can be done by oxidizing the carbohydrate on the enzyme and subsequently coupling to the amines on (strept)avidin (Figure 23.5). 

Figure 23.5 Oxidation of the polysaccharide components of HRP produces reactive aldehyde groups. Conjugation to avidin then may be done by reductive amination.

Conjugation of Periodate-Oxidized HRP with (Strept)avidin... 

Figure 8 Chemiluminescent (A and B) and bioluminescent (C) detections for immobilized hybridizations of PCR product. Dg, digoxigenin Bt, biotin Ad, avidin. Procedure A [30] Biotin moiety is incorporated into PCR products during the amplification reaction, using one 5 -biotinylated primer. The product is hybridized with a Dg-labeled probe and is immobilized on streptavidin-coated magnetic beads. This capture reaction is carried out for 30 min at 37°C. A permanent magnet is used to sediment the beads during washing to remove unbound DNA. By incubation with the washed beads for 45 min at 37°C, anti-Dg antibody conjugated to HRP enzyme is bound to the Dg-labeled probe, and luminol reaction is performed for CL detection. Procedure B [31] Wells of apolystyrene microtiter plate are activated with l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and then coated with a labeled cDNA probe complementary to an internal region of the target DNA.

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