Larson R S, Menard V, Jacobs H, Kim S W
Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 South 2000 East Room 201, Salt Lake City, Utah 84112, USA.
Bioconjug Chem. 2001 Nov-Dec;12(6):861-9. doi: 10.1021/bc000137e.
Monoclonal antibodies against glutamic acid decarboxylase (anti-GAD) were modified with poly(ethylene glycol) (PEG), and the resulting conjugates were characterized. Monoclonal anti-GAD antibodies were purified from ATCC HB184 hybridoma cells by either cell culture supernatant or ascites fluid from BALB/c mice. Polyclonal rabbit IgG antibodies were also used as a model protein. Polyclonal rabbit IgG or purified anti-GAD was modified by PEG (MW = 5000 or 20000 Da) through either the lysine residues or through the carbohydrate moiety. Lysine modification was performed in PBS (pH 7.4) or 0.1 M borate (pH 9.2) by adding a molar excess (5-80) of a succinimidyl activated propionic acid terminated mPEG (SPA-PEG) while stirring at room temperature. Carbohydrate modifications were performed in PBS (pH 6.2) by first oxidizing the antibody with sodium periodate followed by incubation with hydrazide-terminated PEG followed by reduction with sodium cyanoborohydride. The degree of modification was assessed by 1H NMR or TNBS (trinitrobenzenesulfonic acid). Circular dichroism (CD) spectra were obtained for lysine-modified rabbit IgG at various degrees of modification ranging from 5 to 60 PEG per antibody. Binding was assessed using an ELISA method with GAD or rabbit anti-mouse-IgG (H+L) coated plates. The TNBS and 1H NMR analysis of the modified antibody showed reasonably similar results from 5 to 60 PEG per antibody. The 1H NMR method showed greater sensitivity at low modifications (below 20:1) and was fairly linear up to about 60 PEG per antibody. The CD spectra of the polyclonal rabbit IgG showed only small differences at variously modified antibody. The binding affinity of anti-GAD is lower for all PEG modifications with respect to unmodified anti-GAD. Modifications at pH 7.4 show lower binding to GAD than modifications at pH 9.2. Binding to GAD or anti-mouse-IgG is decreased as the degree of modification is increased. Lysine modifications showed lower binding to GAD or anti-mouse-IgG than carbohydrate modifications. Binding to GAD or anti-mouse-IgG is lower for PEG20000-modified anti-GAD with respect to PEG5000-modified anti-GAD.
用聚乙二醇（PEG）修饰抗谷氨酸脱羧酶单克隆抗体（抗GAD），并对所得共轭物进行表征。通过细胞培养上清液或来自BALB/c小鼠的腹水从ATCC HB184杂交瘤细胞中纯化单克隆抗GAD抗体。多克隆兔IgG抗体也用作模型蛋白。多克隆兔IgG或纯化的抗GAD通过赖氨酸残基或通过碳水化合物部分用PEG（分子量 = 5000或20000 Da）进行修饰。赖氨酸修饰在PBS（pH 7.4）或0.1 M硼酸盐（pH 9.2）中进行，通过加入摩尔过量（5 - 80）的琥珀酰亚胺活化的丙酸封端的甲氧基聚乙二醇（SPA-PEG），同时在室温下搅拌。碳水化合物修饰在PBS（pH 6.2）中进行，首先用过碘酸钠氧化抗体，然后与酰肼封端的PEG孵育，接着用氰基硼氢化钠还原。修饰程度通过1H NMR或TNBS（三硝基苯磺酸）评估。获得了赖氨酸修饰的兔IgG在每抗体5至60个PEG的不同修饰程度下的圆二色性（CD）光谱。使用包被有GAD或兔抗小鼠IgG（H + L）的酶联免疫吸附测定（ELISA）方法评估结合情况。修饰抗体的TNBS和1H NMR分析在每抗体5至60个PEG时显示出相当相似的结果。1H NMR方法在低修饰（低于20:1）时显示出更高的灵敏度，并且在每抗体约60个PEG之前相当呈线性。多克隆兔IgG的CD光谱在不同修饰的抗体上仅显示出微小差异。与未修饰的抗GAD相比，所有PEG修饰的抗GAD与GAD的结合亲和力都较低。在pH 7.4下的修饰比在pH 9.2下的修饰显示出与GAD的结合更低。随着修饰程度的增加，与GAD或抗小鼠IgG的结合减少。赖氨酸修饰比碳水化合物修饰显示出与GAD或抗小鼠IgG的结合更低。与PEG5000修饰的抗GAD相比，PEG20000修饰的抗GAD与GAD或抗小鼠IgG的结合更低。
