Antibodies sulfo-SMCC

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

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 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.

Dialyze an antibody solution against Soln. C and concentrate to 20-30 mg/ml. Add 3 mg of sulfo-SMCC (3-sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-caboxylate, Mr 436.7) or of sulfo-GMBS (N-(y-maleimidobutyryloxy)-3-sulfo-N-hydroxysuc-cinimide ester, Mr 382.3), dissolved in ddH20 to 60 mg/ml, to 10 mg of antibody, rock at RT for 15 min and add further 3 mg of coupling reagent. Desalt on a Sephadex G-25 column, equilibrated with Soln. A, after total incubation time of 30 min and use the activated antibody (protein) for conjugation immediately (see above). 

One note should be mentioned before proceeding when conjugating antibody molecules with (B-galactosidase, the antibody usually is activated with sulfo-SMCC first to take advantage of the indigenous sulfhydryl groups on the enzyme. Therefore, if (3-gal is being used, substitute the antibody for the enzyme mentioned in this protocol,... 

The two-stage reaction of the immobilization of immunoglobulin on the surface of FeaOzt/DMSA nanocomposites by means of sulfo-SMCC spacer leads to the formation of specific complexes with antibody ... 

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