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Diluent
A standard antibody diluent for primary antibodies is 0.01M phosphate-buffered normal saline (0.87% NaCl), pH 7.2 to 7.4 (PBS) containing 0.1% bovine serum albumin (BSA) and 0.1% sodium azide as a preservative. Tris/HCl-buffered saline, pH 7.6 (TBS), may also be used. The BSA acts as an inert protein that can bind to any sticky sites on the walls of the containing vial and thus keep the precious antibody molecules free in the solution. Many antibodies can be stored at their optimal dilution in this solution at 4°C which ensures uniformity in tests, but it is advisable to check their capability for long-term storage.
Optimisation of dilution
Primary antibodies may be supplied ready to use or as a concentrate. It is always advisable to try a series of dilutions to find the optimal dilution for the detection system you are using, as you may be using a more or less sensitive method than the one used by the supplier. Monoclonal antibodies usually react well within a range of 1 to 20 µg/ml. Polyclonal antibodies can vary greatly, from a dilution of 1/100 to 1/10,000 or more.
Testing is done on preparations of tissue known to contain the antigen of interest, using the method that will be used for the diagnostic procedures. For economy, the highest dilution giving a strong immunostain against the lowest non-specific background should be chosen. This should ensure an excess of antibody over tissue antigen and will then be standard for all preparations to be stained. If known positive control cytological preparations are not available, the tests can be done on paraffin sections of suitable tissue, and the chosen optimal dilution will be suitable for cytological preparations.
Secondary antibodies (if not supplied ready to use) should also be tested and are usually found to react well in the range of 1/100 to 1/500. Secondary antibodies carrying labels should not be stored diluted as there is a (remote) danger that the label may become detached. Peroxidase-labelled antibodies must not be diluted or stored in solutions containing sodium azide, as this inhibits the enzyme reaction. Use PBS alone for diluting the antibody.
Time of reaction
The primary antibody is usually left to react with the preparations for one hour at room temperature and the secondary antibody for 30 minutes. However, with all the blocking steps and rinsing periods the procedure from start to finished preparation may take several hours. If the procedure is being done by hand, it may be convenient to leave the primary antibody overnight (at 4°C in a damp chamber) and finish the reaction next day. The dilution chosen for a monoclonal antibody should not need adjusting under these conditions because there is only one kind of antibody in the solution. A polyclonal antibody might give better results at a higher dilution if the longer incubation period is used because the solution will contain a mixture of antibodies to different areas of the same antigen molecule, some of which may be slower to bind and may not attach during the shorter incubation period. In addition, in a polyclonal antiserum, any unwanted immunoglobulins that might bind non-specifically to the tissue will be diluted and thus of less importance. The dilution series should be re-run if the incubation period is changed. After the primary antibody has been applied, preparations should be left in this rather than buffer which might cause lightly bound antibodies to detach.