作者:Nicole E. Pollok、Charlie Rabin、Leilani Smith、Richard M. Crooks
DOI:10.1021/acs.bioconjchem.9b00737
日期:2019.12.18
Here we report on the use of heterobifunctional cross-linkers (HBCLs) to control the number, orientation, and activity of immunoglobulin G antibodies (Abs) conjugated to silver nanoparticles (AgNPs). A hydrazone conjugation method resulted in exclusive modification of the polysaccharide chains present on the fragment crystallizable region of the Abs, leaving the antigen-binding regions accessible. Two HBCLs, each having a hydrazide terminal group, were synthesized and tested for effectiveness. The two HBCLs differed in two respects, however: (1) either a thiol or a dithiolane group was used for attachment to the AgNP; and (2) the spacer arm was either a PEG chain or an alkyl chain. Both cross-linkers immobilized 5 ± 1 Abs on the surface of each 20-nm-diameter AgNP. Electrochemical results, obtained using a half-metalloimmunoassay, proved that Abs conjugated to AgNPs via either of the two HBCLs were 4 times more active than those conjugated by the more common physisorption technique. This finding confirmed that the HBCLs exerted orientational control over the Abs. We also demonstrated that the AgNP-HBCL-Ab conjugates were stable and active for at least 2 weeks. Finally, we found that the stability of the HBCLs themselves was related to the nature of their spacer arms. Specifically, the results showed that the HBCL having the alkyl chain is chemically stable for at least 90 days, making it the preferred cross-linker for bioassays.
在此,我们报告了使用杂多功能交联剂(HBCL)来控制与银纳米粒子(AgNPs)共轭的免疫球蛋白 G 抗体(Abs)的数量、取向和活性。采用腙共轭方法对抗体片段可结晶区的多糖链进行了独家修饰,使抗原结合区成为可接触区。我们合成了两种 HBCL,每种都有一个酰肼末端基团,并对其有效性进行了测试。不过,这两种 HBCL 在两个方面有所不同:(1) 与 AgNP 连接时使用的是硫醇基团或二硫环基团;(2) 间距臂是 PEG 链或烷基链。这两种交联剂都在每个 20 纳米直径的 AgNP 表面固定了 5 ± 1 个 Abs。使用半金属免疫分析法得出的电化学结果证明,通过这两种 HBCL 与 AgNPs 连接的抗体的活性是通过更常见的物理吸附技术连接的抗体活性的 4 倍。这一发现证实了 HBCL 对抗原的定向控制。我们还证明,AgNP-HBCL-Ab 共轭物至少在两周内具有稳定性和活性。最后,我们发现 HBCL 本身的稳定性与其间隔臂的性质有关。具体来说,研究结果表明,具有烷基链的 HBCL 化学稳定性至少可达 90 天,因此是生物测定的首选交联剂。