Iminothiolane-Modified Antibodies

The number of sulfhydryls created on the immunoglobulin using thiolation procedures such as this one is more critical to the yield of conjugated enzyme molecules than the molar excess of maleimide-activated enzyme used in the conjugation reaction. Therefore, it is important to use a sufficient excess of Traut s reagent to obtain a sufficient number of available sulfhydryls. 

Dissolve the antibody to be modified at a concentration of l-10mg/ml in 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, containing 10 mM EDTA. High levels of EDTA often are required to stop completely metal-catalyzed oxidation of sulfhydryl groups when working with serum proteins—especially polyclonal antibodies purified from antisera. Presumably, carry-over of iron from partially hemolyzed blood is the contaminating culprit. 

Add 2-iminothiolane (Thermo Fisher) to this solution to give a molar excess of 20-40 X over the amount of antibody present (MW of Traut s reagent is 137.63). Addition of solid 2-iminothiolane may be done despite the fact that the compound is relatively insoluble in aqueous solution. As the reagent reacts, it will be completely drawn into solution. Alternatively, a stock solution of Traut s may be made in DMF and an aliquot added to the antibody solution (not to exceed 10 percent DMF in the final solution). 

Antibody-enzyme conjugate formation through thioether bond 

Purify the thiolated antibody by gel filtration using a desalting resin. Perform the chromatography using 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, containing lOmM EDTA as the buffer. To obtain efficient separation between the reduced antibody and excess reductant, the sample size applied to the column should be at a ratio of no more 

---

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

Figure 20.5 Antibodies may be modified with 2-iminothiolane at their amine groups to create sulfhydryls for conjugation with SMCC-activated enzymes. The maleimide groups on the derivatized enzyme react with the thiols on the antibody to form thioether bonds.

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.

---