3.3. Juvenile hormone quantification by radioimmunoassay
Functionally, radioimmunoassays are competition assays, whereby a radiolabelled ligand competes with equivalent nonradioactive moieties from a sample for antigen binding sites of a highly specific antibody. The limiting factor in such assays is always the concentration of the antibody for which the ligands compete. Thus, to be efficient and sensitive, antibody concentrations must be chosen so as to allow a maximal binding ratio of less than 50 % for the radioactive ligand, in a standard solution that is free of unlabelled ligand. Once this competition for antibody binding sites has reached an equilibrium, antibody-complexed antigen is separated from the remaining unbound antigen, either chemically, generally by ammonium sulphate precipitation, or immunologically, by means of a secondary antibody or Protein A. Radioactivity in the resultant precipitate is then counted by scintillation spectrometry. The isotopes most frequently used for labelling antigens are 3H or 125J. Since the latter is suitable only for labelling proteins or peptides, all juvenile hormone and ecdysteroid radioimmunoassays use tritiated (3H) compounds.
An in-depth discussion on the radioimmunoassay for juvenile hormones has been provided by Granger and Goodman (1988), including a very detailed description on antiserum production, which will not be addressed here, as this is a rather complicated process and, once a suitable antibody has been generated, it is usually shared within the community. Sharing suitable antibodies has the further advantage that assays are easily comparable among laboratories that run such assays.
Hence, the protocol described here uses a specific antiserum produced by conjugation of JH III to thyroglobulin (Goodman, 1990) and injection into rabbits. The general radioimmunoassay protocol for the detection of JH by means of this antiserum has originally been given by Goodman et al. (1990) and subsequently, in a slightly modified version, by Goodman et al. (1995). We have used this protocol and adapted it for use with honey bees (Guidugli et al., 2005; Amdam et al., 2007; Marco Antonio et al., 2008). As JH III is the only juvenile hormone homolog present in honey bees (Trautmann et al., 1974), just as in most other insect orders, this greatly facilitates data analysis.
The method described here is certainly not the first serum and RIA protocol used for the quantification of JH titres in honey bees. A highly sensitive but slightly more laborious protocol using a different antiserum and an assay based on equilibrium dialysis for detection of insect JHs has been developed by Strambi et al. (1981) and applied to honey bee larvae (Rachinsky et al. 1990) and adults (Robinson et al., 1987), and a direct comparison using the two radioimmunoassay on honey bee samples has validated both assays as equally sensitive (Goodman et al., 1993). A third RIA, with an enantiomere-specific antiserum developed by Hunnicut et al. (1989) has also been used on honey bee samples, primarily in the context of division of labour in workers (Huang et al. 1994; Jassim et al., 2000). Finally, it is worthy of note that there are no commercially available JH antibodies.
The following protocol, which is the currently most frequently run, uses an antibody developed by Walter G Goodman (Univ. Wisconsin, Madison). It is divided into four parts: sample preparation and JH extraction, preparation of radioimmunoassay solutions and assay standardization, running the assay, and data analysis.