Hydrazide periodate-oxidized antibodies

For conjugation to hydrazide-containing proteins, dissolve the periodate-oxidized antibody at a concentration of lOmg/ml in 0.1M sodium phosphate, 0.15M NaCl, pH 6.0-7.2. For conjugation to amine-containing molecules and proteins, dissolve the oxidized antibody at lOmg/ml in 0.2M sodium carbonate, pH 9.6. 

Conjugation of Periodate-Oxidized Antibodies with Amine or Hydrazide Derivatives... 

A synthesis of MTX y-hydrazide (VIII.224) was described in 1981 by Rosowsky et al. [299], Condensation of MeAPA with y-methyl a-/-butyl L-glu-tamate with the aid of diethyl phosphorocyanidate afforded the diester (VIII.225) (76% yield), which was then converted sequentially to (VIII.226) (60% yield) on treatment with hydrazine in MeOH solution at 4 °C for 3 days and to (VIII.224) (45% yield) on hydrolysis with 1 M HCl at 50 °C for 1 h. Compound (VIII.224) has gained attention recently because of the opportunity it affords to couple MTX to monoclonal antitumour antibodies through the formation of semicarbazide bonds to periodate-oxidized sugars [331]. The y-hydrazide derivative (VIII.227) of AMT has also been cited briefly [332], although details of its synthesis have not yet been disclosed. 

G. Fleminger, E. Hadas, T. Wolf, and B. Soloman, Oriented immobilization of periodate-oxidized monoclonal antibodies on amino and hydrazide derivatives of Eupergit C, Appl. Biochem. Biotech., 23 (1990) 123-137. 

Figure 20.9 Polysaccharide groups on antibody molecules may be oxidized with periodate to create aldehydes. Modification with biotin-hydrazide results in hydrazone linkages. The sites of modification using this technique often are away from the antibody-antigen binding regions, thus preserving antibody activity.

Analogously to the O Shanessy method, the glycoproteins are detected with digoxigenin-3-0-succinyl-e-aminocaproic acid hydrazide (Bohringer, now Roche Diagnostics). The glycoproteins are oxidized first with periodate and then transformed with the hydrazide. After SDS gel electrophoresis and blot, they are detected with anti-digoxigenin antibodies coupled to alkaline phosphatase. 

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