OOO DNA-Technology TS, Moscow, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
PLoS One. 2019 Jan 4;14(1):e0209860. doi: 10.1371/journal.pone.0209860. eCollection 2019.
Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter.
免疫聚合酶链反应（iPCR）是用于检测广泛分析物的方法之一，具有聚合酶链反应（PCR）方法的高灵敏度。iPCR 使用与 DNA 偶联的抗体，然后使用 RT-PCR 扩增附着的 DNA。目前使用两种主要类型的抗体-DNA 缀合物，它们是通过非共价（生物素-链霉亲和素）或共价相互作用获得的。使用应变促进叠氮-炔环加成（SPAAC），我们合成了共价 DNA-抗体缀合物，优化了反应条件，并开发了一种有效的缀合物纯化方案，通过该方案分离了所有未反应的抗体和寡核苷酸。使用之前开发的用于检测 IgE 和 IgM 抗体的超分子 5'-和 3'-生物素化 DNA 与链霉亲和素的复合物的 iPCR 测定法测试了共价 DNA-抗体缀合物。结果表明，用氨基修饰抗体不允许我们获得单标记抗体或严格定义数量的 DNA-标记的抗体。通过引入叠氮基团的染料确定的标记程度从统计学上反映了实际的标记程度。如果叠氮基团的平均标记程度为 1.1，则缀合物含有 25%的单标记抗体、50%的双标记抗体和 25%的未标记抗体。与寡核苷酸偶联后，针对人 IgE（BE5）的单克隆抗体的特异性发生了变化。使用共价缀合物检测针对 LeC 二糖产生的 IgM 抗体的 iPCR 灵敏度与 5'-和 3'-生物素化 DNA 与链霉亲和素的超分子复合物相似，但新程序短了两步。
