A rapid radioimmunoassay, using a nylon support, for amatoxins from Amanita mushrooms

Abstract

A fetuin derivative of alpha-amanitin was prepared and used as an antigen in rabbits. The antigen was superior to previous bovine serum albumin derivatives of beta-amanitin by its lower toxicity and high immunogenicity. On the other hand, the antibodies raised with the alpha-amanitin derivative did not show full crossreactivity with the other amatoxins, as did the immunoglobulins induced by protein derivatives of beta-amanitin. The procedure for activating nylon surfaces and coupling proteins onto them was improved with respect to surface charge and homogeneity. A partially purified IgG-fraction derived from the sera of rabbits immunized against amatoxins was covalently attached to the activated nylon surfaces. The covalently coupled immunoglobulins were complexed with a tritiated amatoxin. Then small pieces of the nylon sheet were punched out and incubated with the amatoxin solution to be analyzed. This procedure represents a method for dosing, in one step and without pipetting, the immunoglobulins and the labelled hapten. Determination of amatoxin concentrations was achieved by counting the radioactivity in the incubation fluid. The limit of detection was about 3 ng of amatoxins per ml. The radioimmunoassay was used to measure amatoxin concentrations in serum, urine, duodenal fluid, and gastric juice of patients with Amanita poisoning. Since such assays can be performed in 2-3 hr, the results can be used to determine the therapeutic protocol. The assay was likewise used to determine the concentration of amatoxins in mushroom tissue. For Amanita phalloides, for example, we found that the amatoxin concentration (mg/g dry weight) is 4.5 times higher in the gills than in the bulb.