The effect of different immunisation schedules on the specificity of rabbit polyclonal antibodies to potato cyst nematode

Nematode surface molecules are of interest because they are potential triggers of plant defence responses. An examination of the nematode surface began with the production of polyclonal antibodies by schedule 1. A rabbit immunised with whole juveniles of Globodera pallida Pa2-3 plus Freund’s incomplete adjuvant (IFC) produced antiserum which bound to the surface of the juveniles of both G. pallida Pa2-3 and G. rostochiensis Ro1. When the antiserum was partially purified by ammonium sulphate precipitation and affinity isolation on a column of Protein A or G, it was separated into immunoglobulin G (lgG) and ‘non lgG’ fractions. Each of the fractions still bound strongly to the whole juveniles of both species when examined by immunofluorescence microscopy. Another rabbit was immunised following a protocol devised by Barclay & Smith (PNAS 83, 4336–4340, 1986) for producing stage specific antibodies for the slime mould Dictyostelium. Schedule 2 began first of all with an immunisation with whole juveniles of G.pallida Pa2-3 plus IFC. Subsequent immunisations were with whole juveniles plus IFC plus antiserum to G. rostochiensis Ro1. Antiserum from the second rabbit also bound to the whole body surface of both species of juveniles. However, after fractionation as previously, the lgG component bound to the amphids and surface of the head, and more faintly to the body, whereas the ‘non 1gG’ fraction bound only to the amphids, small sensory organs on the surface of the head. Reduced binding to the surface of the nematode may be explained in two ways.
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