Ricin activation with

Earlier studies have also shown that ricin induces oxidative stress in mice, resulting in increased urinary excretion of MDA and formaldehyde (FA) (Muldoon et al, 1994). Other toxicants have been shown to induce oxidative stress by macrophage activation with subsequent release of reactive oxygen species and tumor necrosis factor alpha (TNF-a). 

In the absence of an unambiguous history of ricin exposure, the preferred diagnostic method is specific immunoassay of ricin in serum, respiratory secretions, or other clinical samples associated with poisoning. Most of the methods described for ricin detection are experimental or are under development. The CDC and the Federal Laboratory Response Network have the capability to detect ricin in environmental specimens using validated polymerase chain reaction (PCR) tests and time-resolved immunofluorescence assays, with cell-based bioassays to confirm ricin activity. The U.S. Department of Defense has produced experimental field immunoassays, but commercial distribution of field test kits currently is limited. 

A combined phase I/II trial for ricin conjugates with antibodies against the lymphocyte activation markers CD25 (the IL-2 receptor a-chain) or CD30 (a member of the TNF receptor superfamily, possibly involved among others in memory T cell development) for patients with Hodgkin s lymphoma showed some promise [146]. 

A. The enzyme Ricin A with a bound oligonucleotide (CG AG AG) modeled at the active site. B. Interactions involving Arg-180 and Glu-177 are noted. Monzingo, A. F., and Robertus , J. D. X-ray Analysis of Substrate Analogs in the Ricin A-chain Active Site." /. Mol. Biol., 1T7,1136-1145 (1992). 

The following method calls for mixing activated antibody with ricin A chain at a ratio of 2 mg antibody per mg of A chain. Adjustments to the amount of antibody and A chain initially dissolved in the reaction buffers should be done to anticipate this ratio. 

The following protocol is adapted from Myers et al. (1989). It involves activation of ricin with MBS and conjugation with a partially reduced antibody (Figure 21.14). 

Conjugation of MBS-Activated Ricin with Partially Reduced Antibody... 

Mix the MBS-activated ricin with the partially reduced antibody in a molar ratio of 15 1 (or 6.24mg activated ricin per mg of reduced antibody). This represents a volume ratio (at lOmg/ml for both proteins) of 1ml ricin solution mixed with 160pi antibody solution. 

Antibody-toxin conjugates made with ricin A chain, abrin A chain, gelomn, and momordin can be stored for at least 4 yr at -70°C without detectable loss of activity. The bond between the antibody and the RIP breaks down very slowly at 4°C in PBSE but, provided that care is taken to ensure the sterility of the solution, conjugates can be stored under these conditions for up to one year with little deterioration in quality. 

RIPs are plant protein toxins that are able to inhibit enzymatically ribosomal activity and are therefore highly cytotoxic [98]. RIPs are taken up in the cells by means of endocytosis, and only a small fraction (5% or less) are translocated to the cytosol where the toxins inhibit the protein synthesis and eventually kill the cell. PCI may be used to increase both the efficacy and specificity of these toxins. RIPs are divided into two groups, type I and type II. Type II RIPs, like ricin, consists of two polypeptide chains, one cytotoxic A-chain with /V-glycosidase activity and one B-chain which binds to the cell surface. Type I RIPs, like gelonin, agrostin, and saporin, lack the B chain, which make them poorly transported over the cell- and intracellular membranes to the cell cytosol. Hence, the cytotoxic effect of these protein toxins is usually absent or very low. A considerable cytotoxic effect of type I RIPs has been shown in combination with PCI, both in vitro and in vivo [25, 99]. 

Multiple purification-schemes have been applied to the separation of the toxin and the hemagglutinin.144,146,147,150,194,64,-651 Fractionation using salt and ethanol precipitation led to crystallization844,645 of the toxin known as ricin or ricin D. The hemagglutinin was isolated, free from toxic activity, by ion-exchange chromatography and gel filtration.642,646-648 With the introduction of affinity chromatography on Sepharose 4B, to which both proteins bind, purification of the two R. 

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