Materials

1. Antibodies: antibody "pairs" can often be bought commercially (see Note 1), comprising a capture antibody and a detection antibody that has been directly biotinylated. Aliquot and freeze the capture antibody at -20°C or lower in small, usable quantities. Most biotinylated antibodies can also be stored frozen for long-term use, although a very slight reduction in the sensitivity of the ELISA may result and it is worth testing a "test freeze" aliquot before aliquoting and freezing an entire batch.

2. Blocking buffer: phosphate-buffered saline (PBS), pH 7.4, supplemented with 1% fatty acid-free BSA.

3. Carbonate coating buffer: For a carbonate coating buffer, use 8.41 g of Na2HCO3 in 1 L of water. Dissolve and adjust to desired pH (see Note 2) with HCl or NaOH. If the buffer has not been recently used check the pH before use. Store at room temperature for no longer than 1 mo or 4°C for up to 3 mo.

4. High-capacity protein binding 96-well microtiter plates: there are a large number of suitable makes including Maxisorp (Nunc), Immunoware (Pierce), Immunlon II (Dynatech), and Costar (see Note 3).

5. Plate sealers or cling film wrap: used to prevent evaporation from the plate during incubations.

6. Plate-washing apparatus: adequate washing is a vital element of achieving a successful ELISA. Although a number of automatic plate washers are available, they are expensive and the use of a wash bottle with good pressure is perfectly suitable, though a little more time consuming.

7. Samples/standards: standards of known amounts are required for positive controls, and for estimation of levels within samples (through comparison of OD values from the sample to those obtained from the 'standard curve' titration of known amounts). All standards should be diluted in a matrix as near to that of the sample solution as is possible, for example culture medium. Standards are best obtained from a reliable commercial source having been mass calibrated and should be frozen in small, concentrated aliquots at -20°C or lower. Repeated freeze-thaw cycles must be avoided.

8. Spectrophotometer: any suitable microplate reader able to measure absorbance at the appropriate wavelength. For TMB, this is 450 nm, having stopped the reaction with 0.5 M H2SO4.

9. Stop solution: 0.5 M H2SO4.

10. Streptavidin HRP: use in accordance with the manufacturers instructions. Numerous companies sell Streptavidin-HRP, including Sigma, Becton Dickinson, and Biosource.

11. Substrate: one step TMB (Zymed) is a common choice (see Note 4). Although many other substrates are available for HRP, TMB has high sensitivity with a quick development time. OD can be monitored at 650 nm as the color develops, then at 450 nm, when the reaction is stopped with H2SO4. TMB may also be obtained in a lyophilized state and made up fresh with hydrogen peroxidase or as a preprepared one-step solution. TMB can vary considerably between different manufacturers, and this can affect the sensitivity and specificity of the ELISA. With one-step TMB, there is often large batch-to-batch variation as well. Each batch therefore needs to be tested before use.

12. Washing buffer: add 0.5 mL of Tween-20 to 1 L of PBS, pH 7.4. Make up fresh as required.

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