Hemminki A, Hoffrén A M, Takkinen K, Vehniäinen M, Mäkinen M L, Pettersson K, Teleman O, Söderlund H, Teeri T T
VTT Biotechnology and Food Research, Espoo, Finland.
Protein Eng. 1995 Feb;8(2):185-91. doi: 10.1093/protein/8.2.185.
Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.
铕螯合物为诊断免疫测定中抗体的灵敏标记提供了一种非放射性替代方法。抗体表面的赖氨酸残基是铕螯合物（Eu3+）异硫氰酸酯衍生物标记的理想靶点。在此，通过蛋白质工程增加重组抗人甲胎蛋白（hAFP）Fab片段的赖氨酸含量，提高了其标记效率。利用分子建模鉴定出三个轻链恒定区表面精氨酸残基R154、R187和R210，并将其突变为赖氨酸残基。这些突变不影响富含赖氨酸的Fab片段的亲和力，且发现其标记效率比野生型Fab片段高约40%。在低标记程度下，两种Fab片段的亲和力相同，且与原始单克隆抗hAFP IgG相当。在较高标记程度下，两种Fab片段的亲和力下降幅度均大于完整IgG，因为在较大分子的额外重链恒定区中有更多赖氨酸残基可供标记。通过BIAcore对Fab片段的静电吸附和共价固定进行了表征，发现富含赖氨酸的Fab片段能更有效地固定在活化的羧甲基表面。
