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首页> 外文期刊>BMC research notes >Effect of lyophilization on HRP–antibody conjugation: an enhanced antibody labeling technology
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Effect of lyophilization on HRP–antibody conjugation: an enhanced antibody labeling technology

机译：冻干对HRP-抗体结合的影响：增强的抗体标记技术

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Abstract ObjectiveImmunoassay usually deal with the antibody labeling with various reporter molecules, one such useful reporter molecule is horseradish peroxidase (HRPO). Conjugating enzyme with antibody without losing its enzymatic activity is a challenging task. Our aim is to modify existing classical method of conjugating antibodies with HRP to enhance immunoassay techniques with better sensitivity. We used chemicals such as sodium meta periodate to generate aldehyde group by oxidation of carbohydrate moieties on HRPO. The activated form of HRPO is lyophilized and then mixed with 1?mg/ml concentration of antibodies to be conjugate.ResultsAfter confirming chemical modification of conjugates via UV-Spec and SDS-PAGE independent molecules were used for conjugation and HRP–antibody conjugate. Finally, enzymatic activity of HRP–antibody conjugate was confirmed by performing direct ELISA. Functional properties were analyzed using ELISA with dilution of 1:5000, whereas the conjugate prepared by existing method of conjugation worked with as low dilution of 1:25 with a p value highly significant (?0.001) for classical verses modified method of conjugation preparation. Collectively, this study showed the enhanced ability of antibody to bind more number of HRPO with an additional step of lyophilization in the regular conjugation protocol. Future exploration are necessary on wide range of IgG antibodies.
机译：摘要目的免疫分析通常用各种报道分子标记抗体，辣根过氧化物酶（HRPO）是其中一种有用的报道分子。在不损失酶活性的情况下将酶与抗体结合是一项艰巨的任务。我们的目标是修改现有的经典抗体与HRP结合方法，以增强免疫测定技术的灵敏度。我们使用诸如高碘酸钠之类的化学物质通过HRPO上碳水化合物部分的氧化来生成醛基。将激活的HRPO冻干，然后与浓度为1？mg / ml的待结合抗体混合。结果在通过UV-Spec和SDS-PAGE确认结合物的化学修饰后，将独立分子用于结合和HRP-抗体结合物。最后，通过直接ELISA证实了HRP-抗体结合物的酶活性。使用ELISA以1：5000的稀释度分析功能特性，而对于经典的经修饰的缀合制备方法，通过现有的缀合方法制备的缀合物以1:25的低稀释度进行操作，p值高度显着（<0.001）。总体而言，这项研究表明，在常规偶联方案中，冻干的附加步骤增强了抗体结合更多数量的HRPO的能力。未来有必要对各种IgG抗体进行探索。

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