9. Pipet 1 mL of PBSTB into the syringe reservoir and start the pump at a 1 mL/min flow rate. After approx 1 min, allow each channel to fill with air and stop the pump (see Note 16).
10. Pipet 0.4 mL of the “tracer” antibody into each reservoir, start the pump at a 0.1 mL/min flow rate. After the “tracer” has flowed through each channel and reser- voir is empty, allow each channel to fill with air and stop the pump.
11. Pipet 1 mL of PBSTB into the syringe reservoir and start the pump at a 1 mL/min flow rate. After approx 1 min, allow each channel to fill with air and stop the pump.
12. Remove all fluidics connections from the assay template and disassemble assay template.
13. Rinse the entire slide with water and dry under a stream of nitrogen.
14. Image slide immediately or store in the dark for up to 24 h for later imaging.
Glassware is then incubated overnight in a base bath and finally rinsed exhaus- tively with water.
2. NeutrAvidin-coated slides stored at 4⬚C are stable for as long as 5 mo (11).
3. As the reaction chemistry links biotin (or Cy5) to the antibody via amine groups, the antibody must be in a buffer that does not contain amine moieties. Amine- based buffers (e.g., Tris, glycine) must be removed from the antibody prep prior to the labeling reaction. Any method suitable for desalting may be used for buffer exchange.
4. The Bio-Gel P-10 column is prepared by first suspending Bio-Gel P-10 gel (medium, 90–180 µm) into PBS to make a slurry. To completely hydrate the Bio-Gel, allow the slurry to sit overnight at room temperature or alternatively, autoclave or boil for 20 min. The hydrated slurry is stable at room temperature for months. Immediately before loading the column, the slurry is degassed by application of a vacuum. The slurry is then loaded into a 25-mL column prefilled with approx 3 mL of PBS.
Once the column has been filled, it is then flushed with at least three volumes of Fig. 5. Charge-coupled device image of a sandwich assay for Salmonella, Campylobacter, and staphylococcal enterotoxin B (SEB) at a 1.5-s exposure time. The slide was patterned with 20 µg/mL of biotinylated rabbit antibodies against Salmo- nella (Rb-Sal), Campylobacter (Rb-Campy), and SEB (Rb-SEB); 10 µg/mL biotinylated monoclonal antibodies against SEB (mAb-SEB); and Biotin-SP-conju- gated AffiniPure rabbit anti-chicken IgY (Chic) in phosphate-buffered saline. Samples were spiked with SEB, Campylobacter (Campy), or Salmonella (Sal), indicated to the right of the image. “Tracer” antibodies consisted of 20 µg/mL Cy5-labeled rabbit anti- Salmonella, 20 µg/mL Cy5-labeled rabbit anti-Campylobacter, and 10 µg/mL Cy5- labeled sheep anti-SEB, each containing 100 ng/mL Cy5-conjugated ChromPure chicken IgY.
PBS. After the elution of the conjugate products, the column must be flushed exhaustively with PBS and stored wet (PBS) at room temperature for future use.
5. The absorbance at 280 nm should be below a value of 1.0 absorbance units for accurate determination of concentration. The concentration of biotinylated anti- body is determined by the Beer Lambert Law (A = εcl), with ε280 nm,1mg/ml, 1cm = 1.4.
6. Each component of the silicon elastomer is very sticky. It is recommended that a paper towel be used to cover the balance as well as the bench top on which the beaker and stock containers are placed.
7. Once the vacuum is applied, the mix must be observed until the rising contents start to drop down (if overflow occurs, the vacuum must be stopped). It is recommended that a paper towel be placed inside the vacuum chamber prior to placing the beaker.
8. It is recommended that positive controls, such as anti-chicken IgY, be patterned at the outmost channels and with the buffer blank (negative control) channels immediately adjacent. This format prevents any interference of the analyte sig- nals in the event of leakage due to faulty contact with the PDMS patterning tem- plate. The positive control channels should be the last to be patterned.
9. The patterning protocol is similar to that described in Subheading 3.2.3., except that the biotinylated analog of the analyte is used as the capture molecule. The samples are prepared accordingly and a constant concentration of the Cy5-labeled antibodies against the analyte (and Cy5 labeled positive control) is added to each sample (13). These then are assayed for 15 min. Initially, a checkerboard-type of assay, whereby different concentrations of capture molecules are exposed to vari- ous concentrations of Cy5-labeled “tracer” antibody in PBSTB, is performed to determine the reasonable working concentrations for both capture molecules and
“tracer” antibody.
10. The Cy5-dye in DMSO is stable for several hours, provided that anhydrous DMSO is used. Otherwise, it is stable for approx 30 min. As only 15 µL of the dissolved Cy5 is used to label 1 mg of antibody, each vial may be used to label multiple batches of antibody, as long as all labeling reactions are performed within a period of several hours. Cy5-dye is light-sensitive and all Cy5 solutions must be protected from light.
11. Incubation time may be extended to increase the dye to protein ratio. However, for optimal labeling efficiency the molar ratio should be maintained at 2:1 to 4:1 because higher ratios have been shown to exhibit quenching characteristics (28).
12. The absorbance at 280 and 650 nm (A280 and A650, respectively) must be less than 1 absorbance unit for accurate determination of concentrations. The concentration of the Cy5-labeled antibody (moles/liter) is given by: [A280 – (0.05 ⫻ A650)] / 170,000.
The dye to protein ratio is calculated by: (0.68 ⫻ A650) / [A280 – (0.05 ⫻ A650)].
13. The carcass wash can be stored frozen at –20⬚C for later analysis. Frozen carcass wash must be thawed before spiking with analyte.
14. As a result of bubbles that occasionally form at the ends of the patterning chan- nels, it is recommended that one not use the outermost assay channels or not to run analyte samples on them. These outermost channels often produce incom- plete “spots” (see Fig. 5 uppermost spots).
15. Sensitivity may be improved by extending the duration in which the sample is exposed to the slide, which could be performed without increasing the volume by recirculating the sample over the slide.
16. The “tracer” species of the positive control is added to all “tracer” antibody solu- tions. If the analysis is geared toward a simultaneous analysis of multiple samples for multiple analytes, then the “tracer” antibody is a cocktail of all tracer antibod- ies plus the positive control.
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From: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O‘Connor © Humana Press Inc., Totowa, NJ