Thiolation antibodies

The following protocol represents a generalized method for protein thiolation using SATA. For comparison purposes, contrast the variation of this SATA modification method as outlined in Chapter 20, Section 1.1 for use in the preparation of antibody-enzyme conjugates. 

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. 

Add 1-10 mg of a protein or antibody containing an available thiol group to the particle suspension. Alternatively, add the protein to be coupled to the particle suspension in an amount equal to 1-10 X molar excess over the calculated monolayer for the protein type to be coupled. The optimal amount of protein to be added should be determined experimentally. Creating thiol groups on proteins or peptides may be done from disulfides by reduction. Alternatively, a thiolation reagent may be used to add thiols to the protein surface for coupling (see the protocols in Chapter 1, Section 4.1). 

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... 

Although amine-reactive protocols, such as SATA thiolation, result in nearly random attachment over the surface of the antibody structure, it has been shown that modification with up to 6 SATAs per antibody molecule typically results in no decrease in antigen binding activity (Duncan et al., 1983). Even higher ratios of SATA to antibody are possible with excellent retention of activity. 

The following protocol should be compared to the method described for SATA thiolation in Chapter 1, Section 4.1. Although the procedures are slightly dissimilar, the differences indicate the flexibility inherent in the chemistry. For convenience, the buffer composition indicated here was chosen to be consistent throughout this section on enzyme-antibody conjugation using SMCC. Other buffers and alternate protocols can be found in the literature. 

Figure 20.6 Available amine groups on an antibody molecule may be modified with the NHS ester end of SATA to produce amide bond derivatives containing terminal protected sulfhydryls. The acetylated thiols may be deprotected by treatment with hydroxylamine at alkaline pH. Reaction of the thiolated antibody with a maleimide-activated enzyme results in thioether crosslinks.

Add 10—40 ul of the SATA stock solution per ml of lmg/ml antibody solution. This will result in a molar excess of approximately 12- to 50-fold of SATA over the antibody concentration (for an initial antibody concentration of lmg/ml). A 12-fold molar excess works well, but higher levels of SATA incorporation will potentially result in more maleimide-activated enzyme molecules able to couple to each thiolated antibody molecule. For higher concentrations of antibody in the reaction medium, proportionally increase the amount of SATA addition however do not exceed 10 percent DMF in the aqueous reaction medium. 

Immediately mix the thiolated antibody with an amount of maleimide-activated enzyme to obtain the desired molar ratio of antibody-to-enzyme in the conjugate. Use of a 4 1 (enzyme antibody) to 15 1 molar ratio in the conjugation reaction usually results in high-activity conjugates suitable for use in many enzyme-linked immunoassay procedures. 

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

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 Thiolation of Antibody with SPDP and Conjugation to an SPDP-Activated Toxin... 

Conjugation of SPDP-Activated Toxin with Thiolated Antibody... 

Immediately mix the concentrated, thiolated antibody solution from part B with the SPDP-activated toxin from part A. 

Conjugation of SPDP-Activated Antibody with Thiolated Gelonin... 

Mix SPDP-activated antibody with thiolated gelonin in equal mass quantities (or equal volumes if they are at the same concentration). This ratio results in about a 5-fold molar excess of toxin over the amount of antibody. 

Figure 21.12 SIAB can be used to activate toxin molecules for coupling with sulfhydryl-containing antibodies. In this case, the antibody molecule is thiolated using SATA and deprotected to reveal the free sulfhydryl. Reaction with the SIAB-activated toxin forms the final conjugate by thioether bond formation.

Thiolation of Specific Antibody Molecule with SPDP... 

Mix activated toxin from part A with thiolated antibody from part B at a ratio of 2.25 mg of antibody per mg of toxin. Protect the solution from light. 

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.

Collect the peak containing the activated antibody (eluting first) and concentrate to 10 mg/ml using centrifugal concentrators. Use immediately for conjugating to a thiolated toxin. 

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