Streptavidin coated microtiter plates

PCR-ELISA is a hybrid assay combining PCR as a first step and using ELISA as a detection system for the amplicons (Figure 9.1). The PCR step produces biotinylated amplicons that are bound further to a streptavidin-coated microtiter plate. The amplicons are denatured to produce single-stranded PCR products, and only the biotinylated strand stays in the well of the microtiter plate. A FITC-Iabeled probe is bound to the PCR strand, a step that adds an extra level of specificity to the assay. An enzyme-labeled anti-FITC antibody is used to produce a colorimetric signal that can be measured by an ELISA reader. This method has been applied successfully to the detection of <10 ppm hazelnut in processed foods and food ingredients, which eliminated the potential for cross-reactivity observed in ELISA (Holzhauser et al., 2002). 

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.

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

Figure 5 A penicillin-binding activity-based high-throughput SPA assay for the identification of potential inhibitors of the transpeptidase activity of PBPs. A particular PBP of interest is first labeled with biotin. The biotinylated PBP is then mixed with a 3H-labeled P-lactam in 96-well microtiter plates and the reaction mixtures are incubated at room temperature for a period of time. After incubation, the biotionylated PBP-3H-P-lactam complexes are captured by mixing with streptavidin coated SPA beads. The P-lactam-binding activity of the PBP is measured by counting the microtiter plates after mixing with the SPA beads.

Pipet 0.2 mL of streptavidin-AP solution into all antibody-coated wells. Cover the microtiter plate with aluminum foil and incubate for 1 h at 37°C. 

The wells of the microtiter plate were coated with rabbit anti-fluorescein IgG which were 5 pg/mL in 50 mmol/L sodium bicarbonate buffer (pH 9.5). The solution was added 100 pL to each well. The wells were then post-coated by adding 100 pL of a 1% water-soluble gelatin solution containing 0.05% NaN3. The plates were stored at 4°C prior to use. After washing the plate, 100 pL of a PCR product solution were added to each well. The plate was incubated for 1 h at room temperature. After washing, 150 pL of b-Luc/streptavidin complex (1 xl0"9 mol/L), Aq-labeled anti-Dig Fab fragment (495 ng/mL) and HRP-labeled anti-DNP (500-fold dilution) mixed solution was added and allowed to stand for 1 h at room temperature. The microtiter plate was re-washed and Aq, b-Luc and HRP activities were assayed by the simultaneous bio- and chemiluminescent method described above. 

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