Electrophoretic determination of ricin using immunogold silver staining--comparison with simple "protein dot" method.

Studies were performed to establish a sensitive electrophoretic immunodetection system for the highly toxic plant protein, ricin. This has potential criminal application as an agent for causing a delayed death following parenteral administration. The immunodetection system could be used to demonstrate residual traces of the toxin left in certain tissues of the victim's body. Following polyacrylamide gel electrophoresis of ricin added to rat muscle tissue extracts, the gels were electro-blotted onto nitrocellulose paper and ricin bands probed for visualisation by immunostaining. Several immunostaining procedures were investigated in order to select the most sensitive. These included indirect immunoperoxidase, peroxidase-anti-peroxidase (PAP), avidin-biotin complex (ABC) and the immunogold silver staining (IGSS) procedures. The sensitivity of PAP and indirect immunoperoxidase methods were similar at around 50 ng while the ABC technique gave visible staining of 10 ng of electro-blotted ricin. The method with greatest sensitivity was undoubtedly IGSS, which resulted in unequivocal demonstration of 4 ng of ricin. The IGSS-immunoblotting system was considered to readily demonstrate the presence of ricin in muscle tissue from the injection site of dead victims. We compared this system with the very simple method of sample dot staining. Here, samples of ricin were spotted directly onto nitrocellulose. The dots were stained using the IGSS method which was found able to demonstrate less than 10 pg of ricin.

Demonstration of ricin within the mammalian para-aortic lymph node. I. Comparison of the localization, after intramuscular injection, with three immunocytochemical methods.

Following a supralethal injection of ricin into thigh muscle of the adult rat, the toxin was demonstrated post-mortem in the para-aortic lymph node, ipsilateral to the side of injection. The relative merits of two immunoenzyme methods, peroxidase anti-peroxidase (PAP) and avidin-biotin-peroxidase complex (ABC) and a silver-enhanced immunogold method (IGSS) were assessed in the detection of ricin in the lymph node tissue. The toxin was clearly seen to be located in association with histiocytes found both within and lining the sinuses of the nodes and also, in some cases, in the subcapsular sinus of the node; the toxin was not demonstrable within lymphoid follicles by light microscopy. However, using electron microscopy and the IGSS technique, cells carrying discrete particles of gold could be visualized within follicular areas. The IGSS and ABC-peroxidase methods were both found to give excellent results without background staining at the light microscopy level. However, when these techniques were used prior to embedding and viewing by electron microscopy, the IGSS technique proved to be far superior.

Distribution of ricin within the mammalian para-aortic lymph node. II. Comparison of the localization, after intramuscular dosage of colloidal gold-labelled ricin in vivo, with in vitro binding characteristics of the native toxin.

Previous work has shown that, following an intramuscular injection of ricin, the toxin becomes localized within histiocytes in the sinuses of lymph nodes draining the 'wound' site. When ricin labelled with colloidal gold was similarly injected, it was found within the same lymphoid cells as seen with native ricin. Biologically inert Indian ink apparently follows a similar fate, as demonstrated by the appearance of carbon particles within sinus histiocytes, as soon as 1 h after intramuscular injection. When the binding in vitro of Indian ink or ricin toxin to sections of lymph node was examined, ricin was seen to bind to the surfaces of the same sinusoidal cells and also, with a much lower frequency, to follicular lymphocytes, whereas Indian ink failed to bind. This indicated an interaction between ricin and cell membrane components. Moreover, this binding was inhibited markedly by the galactose-containing disaccharide, lactose, a target sugar specified by the lectin binding site of ricin and to a much lesser extent by the monosaccharide mannose.