Thiolation of antibodies

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

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. 

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. 

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. 

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. 

Park, I. S., and Kim, N. (1998). Thiolated Salmonella antibody immobilization onto the gold surface of piezoelectric quartz crystal. Biosens. Bioelectron. 13,1091-1097. 

For instance, if toxin A chain—antibody conjugates are to be prepared, the antibody can be similarly activated with SPDP, but in this case not treated with reductant. After removal of excess cross-linker, the activated antibody can be directly mixed with isolated A chain to create the conjugate (Fig. 320). This procedure makes use of the indigenous sulfhydryl residues produced during reductive separation of the A and B chains and therefore does not require cross-linker thiolation of one of the proteins. 

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