Materials

1. Antibodies: antibodies can often be bought commercially (see Note 1). The choice of monoclonal or polyclonal antibody can have a significant effect on the success of the technique. In general, if the antibody is to be coated onto the plate, it is preferable to use a monoclonal antibody because the assay should only require optimiz ing once (polyclonals can vary tremendously between batches). The sensitivity is also often higher using a monoclonal antibody because of high-level affinity for the antigen (this is of course, antibody-dependent, but in general, binding avidity of a monoclonal is greater than a polyclonal because every antibody will bind to the antigen being measured).

If the technique is to be used for the detection of antibodies in serum for example (which will be polyclonal by nature) a polyclonal antibody may be preferable in order to compete more effectively with the antibody in the serum. Batch to batch variation in polyclonal antibodies, does, however, make the establishment of the ELISA more difficult and each the assay must be optimized every time a new batch is prepared. Antibodies may be aliquoted and frozen at -20°C or lower in small, usable quantities.

2. Blocking buffer: PBS, pH 7.4, supplemented with 1% fatty acid-free bovine serum albumin

3. Coating buffer: for 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 microtitre plates: there are a large number of suitable makes of plate for adsorbing hydrophilic molecules such as antibodies (see Note 3). These include Maxisorp (Nunc), Immunoware (Pierce), Immunlon II (Dynatech), and Costar. If coating antigen onto the plate, however, a different plate type may be necessary. For example, hydrophobic molecules such as lipopro-teins may require an alternative such as Polysorp (Nunc)

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 (although a little more time consuming).

7. Samples/standards: standards (enzyme conjugated) are best obtained from a reliable commercial source having been mass calibrated and should be stored in small, concentrated aliquots. It is usually recommended that samples are stored frozen at -20°C or lower and to avoid repeated freeze-thaw cycles.

8. Spectrophotometer: any suitable microplate reader able to measure absorbance at the appropriate wavelength. For example 450 nm for the substrate tetramethylbenzidine (TMB).

9. Stop solution: 0.5 M H2SO4.

10. Streptavidin HRP: use in accordance to 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 lyo-philized state and made up fresh with hydrogen peroxidase or as a preprepared solution one-step solution. TMB can vary considerably between different manufacturer's, 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 (see Note 5).

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