Modified proteins can also be lyophilized and stored for later conjugation to a sulfhydryl-containing molecule. These derivatives can be stored for up to 64 hours in 0.1 M sodium phosphate buffer, pH 7 at 4☌. The stability of the maleimide-derived thioether is enhanced by the cyclohexane group. The maleimide part of the reagent reacts with sulfhydryl groups from another molecule at pH 6.5–7.5 to form stable thioether bonds NHS moiety with primary amines takes place at pH 7–9 to form amide bonds. SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate)Ĭontains both N-hydroxysuccinimide (NHS) ester and maleimide groups. Forms cleavable disulfide bonds with other sulfhydryl-containing molecules Forms highly stable amide bonds with primary amines of lysine residues Allows the long-term storage of the sulfhydryl-modified antibody. Hence protein structure/function is not affected. SATA (N-succinimidyl S-acetylthioacetate)Ĭovalently modifies primary amines to generate a protected sulfhydryl group.Īdds a short-chain (2.8-angstrom spacer arm) reagent Deacylation is done with the addition of a mild reagent such as hydroxylamine- HCl to generate a free sulfhydryl. SPDP comes in a variety of PEGylated forms, allowing for a longer spacer arm length.īeing water-insoluble, a high concentration of the reagent in dimethylformamide (DMF) or dimethylsulfoxide (DMSO) should be used to keep the organic solvent in the reaction mix to a minimum. The resultant disulfide bonds with cysteine sulfhydryls can be cleaved easily by 10-50 mM DTT or even SDS-PAGE sample loading buffer at pH 8.5. The conjugation reaction can be assessed by following the release of pyridine-2-thione at 343 nm. The sulfhydryl moiety introduced can be linked to a cysteine through a disulphide bond. Modifies free amine groups through a short-chain crosslinker. SPDP (N-succinimidyl 3-(2-pyridyldithio) propionate) Use limited to research and diagnostic purposes Modifies amines to sulfhydryls to form a thioether linkage with other molecules MBS (3-maleimidobenzoic acid N-hydroxysuccinimide ester) Maintains positive charge Retains protein solubility Sufficient length of spacer arm reduces steric hindrance To enhance the efficacy of conjugation the antibody may be altered by the introduction of unusual amino acids or chemical modification.Ĭonverts primary amines to sulfhydryls at pH 7.0 Inter-chain cysteine is usually targeted for conjugation due to its solvent accessibility. About 16 pairs of cysteine residues exist as 12 intra-chain and four inter-chain disulfide bonds. Cysteine residues are more uniformly distributed and are fewer in number. On an average, IgG molecules have about 80 lysine residues of which 20 are present in solvent accessible sites. Presence of the lysine and cysteine residues in the Fab which determine Ag binding and therefore should not be conjugated.Larger molecules like enzymes may be restricted to just two molecules per antibody. Size of the conjugate: Small molecules like haptens or drugs can be conjugated in a minimum ratio of antibody: conjugate::1:4 or even 1:10.This ensures that the antibody does not precipitate during the conjugation reaction. This is why most commercial conjugation kits are designed to complete the conjugation reaction within 15- 30 minutes and usually target the primary amine group of lysine (see Table 5).Īnother option is to engineer antibodies to alter the pI from 8-9 to 5-6. Conjugated cysteine residues are prone to fragmentation at alkaline pH. It is crucial to limit the reaction time under these conditions to avoid deterioration of the antibody. There is also a problem of overall antibody stability at alkaline pH. E.g., at an alkaline pH, the reactivity of certain aliphatic amine groups is much higher than other groups. pH dependence of the reaction: Reaction conditions that allow only specific groups to react can be standardized to obtain an optimum Ab: reporter molecule ratio.While the conjugation chemistry treats all accessible residues equally, the number of molecules that can be conjugated is limited by the following: The preferred site for chemical conjugation on the antibody is the –NH 2 (amine) group of a lysine or the free –SH (sulfhydryl) group of cysteine. Antibody conjugation is usually achieved through chemical reaction, although specific enzymatic conjugation, such as sortase-based protein terminal conjugation, or even through protein engineering, is gaining popularity.
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