Maleimide sulfo-SMCC

A common choice of crosslinker for this type of reaction is sulfo-SMCC, which has been used extensively for antibody conjugation (Chapter 20, Section 1.1). A better option for dendrimer conjugation is to use a similar crosslinker design, but one that contains a hydrophilic PEG spacer arm to promote dendrimer hydrophilicity after modification. Derivatization of an amine-dendrimer with a NHS-PEG-maleimide can create an intermediate that is coated with water-soluble PEG spacers. This modification helps to mask any potential for nonspecific interactions that the PAMAM surface may have, while providing terminal thiol-reactive maleimides for coupling ligands (Figure 7.10). 

Figure 19.16 A common way of conjugating sulfhydryl-containing haptens to carrier proteins is to activate the carrier with sulfo-SMCC to create an intermediate maleimide derivative. The maleimide groups then can be coupled to thiols to form thioether bonds.

After a carrier protein has been activated with sulfo-SMCC, it is often useful to measure the degree of maleimide incorporation prior to coupling an expensive hapten. Ellman s reagent may be used in an indirect method to assess the level of maleimide activity of sulfo-SMCC-activated proteins and other carriers. First, a sulfhydryl-containing compound such as 2-mercaptoethanol or cysteine is reacted in excess with the activated protein. The amount of unreacted sulfhydryls remaining in solution is then determined using the Ellman s reaction (Chapter 1, Section 4.1). Comparison of the response of the sample to a blank reaction using... 

Figure 19.18 Carrier proteins may be activated with sulfo-SMCC to produce maleimide derivatives reactive with sulfhydryl-containing molecules. The graphs show the gel filtration separation on Sephadex G-25 of male-imide-activated BSA (A) and OVA (B) after reaction with sulfo-SMCC. The first peak is the protein and the second peak is excess crosslinker. The maleimide groups create increased absorbance at 280 nm in the activated proteins.

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.

The following protocol describes the activation of HRP with sulfo-SMCC. Other enzymes may be activated in a similar manner. The activated enzyme possesses maleimide groups that are relatively unstable in aqueous solution. Therefore, the thiolation reaction should be coordinated with the activation process so that the final conjugation can be done immediately. Note If preactivated enzymes are obtained (Thermo Fisher), this step may be eliminated. 

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