Activation toxins

Protocol for Thiolation of Antibody with SPDP and Conjugation to an SPDP-Activated Toxin... 

Purify the SPDP-activated toxin from excess reagents and reaction by-products by gel filtration using a desalting resin. For the chromatography use 0.1 M sodium phosphate,... 

Conjugation of SPDP-Activated Toxin with Thiolated Antibody... 

Immediately mix the concentrated, thiolated antibody solution from part B with the SPDP-activated toxin from part A. 

Figure 21.12 SIAB can be used to activate toxin molecules for coupling with sulfhydryl-containing antibodies. In this case, the antibody molecule is thiolated using SATA and deprotected to reveal the free sulfhydryl. Reaction with the SIAB-activated toxin forms the final conjugate by thioether bond formation.

Concentrate the purified, SIAB-activated toxin to lOmg/ml using centrifugal concentrators with a MW cutoff of 10,000. Protect the activated toxin from light to prevent degradation of the iodoacetyl-reactive group. 

Mix activated toxin from part A with thiolated antibody from part B at a ratio of 2.25 mg of antibody per mg of toxin. Protect the solution from light. 

The toxoid is then prepared by treating the active toxin produced with formaldehyde. The product is normally sold as a sterile aqueous preparation. Tetanus vaccine production follows a similar approach. Clostridium tetani is cultured in appropriate media. The toxin is recovered and inactivated by formaldehyde treatment. Again, it is usually marketed as a sterile aqueous-based product. 

We first met nettle stings on p. 253, where methanoic ( formic ) acid was identified as the active toxin causing the pain. Like its... 

Primary hepatocyte cultures have been used in vitro to metabolically activate toxins for evaluation with target tissues. Cocultures of rat embryos with hepatocytes have been used to study the role of metabolism in teratogenesis (Oglesby et al., 1986). Lindahl-Kiessling et al., (1989), in an attempt to bring test conditions closer to in vivo conditions, developed an assay utilizing primary rat hepatocytes and human peripheral lymphocytes to detect metabolism-mediated mutagenesis. 

Binary toxins are unique concerning their structure because they are comprised of two individual, nonlinked proteins represented by an enzyme component and a binding/translocation component. The two components are secreted by the bacterium and assemble upon the surface of targeted eukaryotic cells to form an active toxin complex. For this to occur, both protein components of binary toxins act in a precisely concerted manner. The binding component first engages the cell-surface receptor and then mediates translocation of enzyme compo-nent(s) from the outside of a cell, through acidified endosomes, and into the host cell cytosol where it modifies the substrate (for review see Barth ). 

The outgrowth of C. botulinum requires a suitable medium, temperature, atmosphere, pH, Eh potential, and water activity. Toxin is usually only produced in optimal or close-to-optimal conditions. Nutrient demands of C. botulinum are complex, and include amino acids, B vitamins, and minerals. In broth, non-proteolytic strains of type B and F grow and produce toxin at 4°C, but in crab meat the outgrowth and toxin production occurs solely at 26°C (Alberto et al., 2003). 

The toxin may undergo microbial degradation either while it is free in soil solution or while it is adsorbed. This could destroy all or part of the toxin, and there is evidence that most of the natural organic chemical groups that contain allelopathic compounds can be metabolized by some microorganism. The possibility always exists, however, that the microbial degradation product from the metabolism of an active toxin will itself be an allelopathic chemical. 

Immediately purify the MBS-activated toxin by gel filtration using a column of Sephadex G-25. Apply no more sample than represents 5—8 % of the gel volume. Isolate the protein peak by its absorbance at 280 nm and concentrate to 10 mg/ml using centrifugal concentrators with a molecular weight cut-off of 10,000. 

Aga IVA and ro-conotoxin GVIA are standard tools in elucidating the roles of P/Q-type and N-type calcium channels in synaptic transmission. In many types of synapses, application of either toxin may mediate moderate inhibition of neurotransmitter release, whereas co-application of both blockers may almost abolish synaptic transmission due to the nonlinear dependence of synaptic release on intracellular calcium concentration. On a final note, we should add that there are many other species of cone snails and spiders that produce active toxins which selectivity inhibit specific calcium channel subtypes (for example, co-conotoxins GVIB, GVIC, GVIIA, SVIA, SVIB), and it is likely that many more remain to be discovered (Olivera et al. 1994). 

Fig. 9. Chemical structure of the synthetic neurotoxin l-methyl-4-phenyl-tetrahydro-pyridine (MPTP) and its metabolism, with monoamine oxidase B as substrate, via MPDP+ to the methyl-phenyl-pyridinium ion (MPP+), which is the active toxin. (For further details see Feldman (1997).)...

B. thuringiensis crystals are first solubilized in the midgut of susceptible insects, followed by activation of the protoxins to active toxins by midgut proteases. The activated toxins then bind to midgut membrane receptors, insert into the apical membrane and form pores. Formation of the pores causes loss of osmotic regulation, and eventually leads to cell lyses, which is thought to be responsible for insect death [4,5]. 

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