Antibodies conjugation with amine

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

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 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.

Conjugation of Periodate-Oxidized Antibodies with Amine orHydrazide Derivatives... 

In a somewhat similar scheme, Noguchi et al. (1992) prepared a carboxylate spacer arm by reacting 6-bromohexanoic acid with a dextran polymer. The carboxylate then was aminated with ethylene diamine to form an amine-terminal spacer (Figure 25.15). This dextran derivative finally was reacted with N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Chapter 5, Section 1.1) to create the desired sulfhydryl-reactive polymer (Section 2.4, this chapter). The SPDP-activated polymer then could be used to prepare an immunoconjugate composed of an antibody against human colon cancer conjugated with the drug mitomycin-C. 

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