Division Note:

Always use a polyclonal antibody, when the IF test is used as the principal screening test. In case of a positive IF reading with a polyclonal antibody further screening of the sample with a monoclonal antibody may provide more specificity but can be less sensitive.

Use antibodies to a reference strain of C. m. subsp. sepedonicus. It is recommended that the titre is determined for each new batch of antibodies. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension containing 10⁵ to 10⁶ cells per ml of the homologous strain of C. m. subsp. sepedonicus and using an appropriate fluorescein isothiocyanate (FITC) conjugate according to the manufacturer's recommendations. The crude polyclonal or monoclonal antibodies should have an IF titre of at least 1:2000. During testing, the antibodies should be used at a working dilution(s) (WD) close to or at the titre. Use validated antibodies (see web site http://forum.europa.eu.int/Public/irc/sanco/Home/main).

The test should be performed on freshly-prepared sample extracts. If necessary, it can be successfully performed on extracts stored at -68 to -86 °C under glycerol. Glycerol can be removed from the sample by addition of 1 ml pellet buffer (Appendix 4), re-centrifugation for 15 minutes at 7 000 g and resuspension in an equal volume of pellet buffer. This is often not necessary, especially if slides samples are fixed to the slides by flaming (see 2.2).