2-Iminothiolane toxins

Another way of utilizing SPDP is to again activate the antibody to create the pyridyl disulfide derivative, but this time thiolate the toxin component using 2-iminothiolane (Chapter 1,... 

Figure 21.7 An intact A-B subunit toxin molecule may be activated with 2-iminothiolane with good retention of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the immunotoxin conjugate through a disulfide bond.

Protocol for the Conjugation of SPDP-activated Antibodies with 2-Iminothiolane-Modified Toxins... 

Thiolation of Gelonin (or Other Single-Polypeptide Toxins) with 2-Iminothiolane (Traut s Reagent)... 

Concentrate the thiolated toxin to lOmg/ml using centrifugal concentrators. Immediately use the modified protein in the conjugation reaction to prevent inactivation of 2-iminothiolane-modified molecules by recyclization (Chapter 1, Section 4.1). 

Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin components. An intact A-B toxin molecule can be modified to contain sulfhydryls by treatment with 2-iminothiolane. Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conjugation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through thioether bond formation.

Dissolve 2-iminothiolane in degassed, nitrogen-bubbled deionized water at a concentration of 20mg/ml (makes a 0.14M stock solution). The solution should be used immediately. Add 70 pi of the 2-iminothiolane solution to each ml of the toxin solution (final concentration is about lOmM). 

---