Ricin A-chain

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. 

Colombatti, M., Nabholz, M., Gros, O., and Brown, C. (1983) Selective killing of target cells by anti-body-ricin A-chain or antibody-gelonin hybrid molecules Comparison of cytotoxic potency and use in immunoselection procedures./. Immunol. 131, 3091. 

Ghetie, V., Ghetie, M.-A., Uhr, J.W., and Vitetta, E.S. (1988) Large scale preparation of immunotoxins constructed with the Fab fragment of IgGl murine monoclonal antibodies and chemically deglyco-sylated ricin A chain./. Immunol. Meth. 112, 267-277. 

Hashimoto, N., Takatsu, K., Masuho, Y., Kishida, K., Hara, T., and Hamaoka, T. (1984) Selective elimination of a B cell subset having acceptor site(s) for T cell-replacing factor (TRF) with biotinylated antibody to the acceptor site(s) and avidin-ricin A chain conjugate./. Immunol. 132, 129-135. 

Masuho, Y., Kishida, K., Saito, M., Umemoto, N., and Hara, T. (1982) Importance of the antigen-binding valency and the nature of the cross-linking bond in ricin A-chain conjugates with antibody. J. Biochem. (Tokyo) 91, 1583. 

Oeltmann, T.N., and Forbes, J.T. (1981) Inhibition of mouse spleen cell function by diphtheria toxin fragment A coupled to anti-mouse Thy-1.2 and by ricin A chain coupled to anti-mouse IgM. Arch. Biochem. Biophys. 209, 362. 

Raso, V., and Basala, M. (1984) A highly cytotoxic human transferrin - Ricin A chain conjugate used to select receptor-modified cells./. Biol. Chem. 259, 1143. 

Cytotoxic drugs including toxins such as saporin, ricin A chain, vinca alkaloids, and radioisotopes have been delivered to tumour cells with BsMAbs that bind to the drug/toxin with one arm and to a surface molecule on the targeted cell with the other arm. This approach has proven successful in animals as e.g. shown by Schmidt et al. [78]. 

Laurent, G., D. Maraninchi, E. Gluckman, J.P Vernant, J.M. Derocq, M.H. Gaspard, B. Rio, M. Michalet, J. Reiffers, F. Dreyfus, et al.. Donor bone marrow treatment with TlOl Fab fragment-ricin A-chain immunotoxin prevents graft-versus-host disease. Bone Marrow Transplant, 1989. 4(4) 367-71. 

Commercially obtained preparations of ricin A chain and abrin A chain may require further purification to eliminate traces of contaminating toxin B chains. The simplest procedure is to pass the A chain preparation over a column of Sepharose-linked asialofetuin, to which the B chains bind avidly (4,11)... 

The single-chain RIPs are generally more stable to a wider range of experimental conditions than ricin A chain However, loss of material can occur in the course of procedures based on selective passage through membranes. Ultrafiltration may be performed using a membrane with a lower Mr cut-off. 

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. 

Ricin A chain in solution is prone to precipitate with even mild agitation It is recommended that the gel-coupling procedure be performed without constant inversion. 

Knowles, P. P. and Thorpe, P. E. (1987) Purification of immunotoxins containing ricin A chain and abrin A chain using Blue Sepharose CL-6B. Anal Biochem. 160,440-443. 

Spooner, R A and Lord, J. M. (1991) Expression of antibody-ricin A chain fusions in mammalian cells, in Monoclonal Antibodies Applications in Clinical Oncology (Epenetos, A A., ed), Chapman and Hall Medical, New York, pp 65-77... 

When using ricin A chains, it has been found that chemical deglycosylation of the subunit prevents its nonspecific binding to receptors for mannose on certain cells of the reticuloendothelial system (Vitetta and Thorpe, 1985 Ghetie et al., 1988, 1991). 

Dissolve deglycosylated ricin A chain (dgA) in 0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.5, at a concentration of 10 mg/ml. The buffer should be degassed under vacuum and nitrogen bubbled through it to remove oxygen. Prepare half the amount of A-chain solution as the amount of antibody prepared... 

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