3.4. Quantification of ecdysteroids by radioimmunoassay
As far as ecdysteroid quantification is concerned, the same general principles described for the juvenile hormone RIA also apply to the detection of ecdysteroids, except that not a single but several ecdysteroids may naturally be present in haemolymph or tissues. For this reason, when ecdysteroids are not fractionated and separated by specific chromatography methods prior to radioimmunoassaying, results are given as immunoreactive ecdysteroids in relation to the compound used to set up the standard curve, which is usually 20-hydroxyecdysone.
Basically two ecdysteroid RIAs have been used in honey bees, one developed by de Reggi et al. (1975), in a protocol using equilibrium dialysis to quantify larval and pupal and adult ecdysteroids (Rachinsky et al., 1990; Robinson et al., 1991), and the other based on an antiserum and protocol developed by Bollenbacher et al. (1983). The latter was used more frequently for studies on honey bee ecdysteroids (Feldlaufer and Hartfelder, 1997; Feldlaufer et al., 1985, 1986; Hartfelder et al., 2002; Pinto et al., 2002; Amdam et al., 2004; Nascimento et al., 2004). It is worthy of note that there are no commercially available ecdysone antibodies.
The following protocol uses an antibody developed in the Gilbert laboratory (Univ. North Carolina at Chapel Hill) against a hemisuccinate derivative of ecdysone (Bollenbacher et al., 1983; Warren and Gilbert, 1986). Tritiated ecdysone serves as the labelled ligand and standard curves are established with 20-hydroxyecdysone (20E) as the non-radioactive ligand. Results are, therefore, expressed as 20E equivalents. The protocol is essentially divided into four parts: sample preparation, preparation of radioimmunoassay solutions and assay standardization, running the assay, and data analysis.