A radioimmunoassay for arthropod moulting hormones, introducing a novel method of immunogen coupling

Inokosteron-26-oic acid was coupled to thyroglobulin in aqueous pyridine by a water-soluble carbodiimide. After exhaustive dialysis and gel filtration on Sephadex G-25 in the presence of sodium dodecylsulfate, a coupling ratio of 164 haptens per molecule of thyroglobulin was determined. In all three animals injected with the conjugate, ecdysone-binding antibodies were detected. After one booster injection the antiserum could be diluted 1 : 5000 (1 : 4000, or 1 : 2000) in order to get a 50% binding of [3H]ecdysone. The dissociation constant was calculated as 5.8 X 10(-10) MOL/L. The antiserum has a greater affinity for ecdysone and 22-isoecdysone than for all other ecdysteroids and steroids tested.