Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay.
Isolation of alpaca anti-hapten heavy chain single domain antibodies for development of sensitive immunoassay.
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DOI：
10.1021/ac2030255
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发表时间：
2012-01-17
影响因子：
7.4
通讯作者：
Hammock, Bruce D.
中科院分区：
文献类型：
--
作者：
Kim, Hee-Joo;McCoy, Mark R.;Majkova, Zuzana;Dechant, Julie E.;Gee, Shirley J.;Tabares-da Rosa, Sofia;Gonzalez-Sapienza, Gualberto G.;Hammock, Bruce D.
骆驼科抗体的一些独特亚类缺乏轻链，并且抗原结合位点仅由重链可变域 (VHH) 组成。尽管传统抗体在当前检测开发中占主导地位，但重组 VHH 具有作为下一代免疫检测替代试剂的巨大潜力。我们从免疫羊驼中表达 VHH，并使用 3-苯氧基苯甲酸 (3-PBA)（拟除虫菊酯杀虫剂的主要代谢物）作为模型系统开发了一种基于 VHH 的免疫测定法。构建了噬菌体VHH文库，并通过与3-PBA竞争性结合选择了7个VHH克隆。使用这些 VHH 之一开发的最佳免疫测定显示 IC50 为 1.4 ng/mL（检测限 (LOD) = 0.1 ng/mL）。通过使用噬菌体携带的 VHH，这些参数得到进一步改善，IC50 = 0.1 ng/mL，LOD = 0.01 ng/mL。两种测定均显示出对测定缓冲液中高达 50% 的甲醇和二甲亚砜的相似耐受性。该测定对 3-PBA 及其 4-羟基化衍生物 4-羟基 3-PBA（150% 交叉反应性）具有高度特异性，与其他测试的结构类似物的交叉反应性可忽略不计，并且加标尿样的回收率为 80% 至 112%。总之，利用免疫羊驼的序列和用于抗体选择的噬菌体展示技术，开发了针对 3-PBA 的高度特异性和敏感性的 VHH。
Some unique subclasses of Camelidae antibodies are devoid of light chain and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). Although conventional antibodies dominate current assay development, recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. We expressed VHHs from an immunized alpaca and developed a VHH-based immunoassay using 3-phenoxybenzoic acid (3-PBA), a major metabolite of pyrethroid insecticides as a model system. A phage VHH library was constructed and seven VHH clones were selected by competitive binding with 3-PBA. The best immunoassay developed with one of these VHHs showed an IC50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameters were further improved by using the phage borne VHH, IC50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similar tolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA (150% cross reactivity) with negligible cross reactivity with other tested structural analogs and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitive VHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.
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噬菌体显示介导的免疫PCR。
影响因子：
14.9
作者：
Guo, Yong-Chao;Zhou, Ya-Feng;Zhang, Xian-En;Zhang, Zhi-Ping;Qiao, Yan-Mei;Bi, Li-Jun;Wen, Ji-Kai;Liang, Mi-Fang;Zhang, Ji-Bin
通讯作者：
Zhang, Ji-Bin
影响因子：
2.2
作者：
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通讯作者：
Shoemaker, Charles B.
影响因子：
16.6
作者：
Mao, Chuanbin;Liu, Aihua;Cao, Binrui
通讯作者：
Cao, Binrui
DOI：
10.3390/s90100542
发表时间：
2009
期刊：
Sensors (Basel, Switzerland)
影响因子：
--
作者：
Goldman ER;Liu JL;Bernstein RD;Swain MD;Mitchell SQ;Anderson GP
通讯作者：
Anderson GP
影响因子：
3.5
作者：
Ghahroudi, MA;Desmyter, A;Muyldermans, S
通讯作者：
Muyldermans, S