Tianjiao Zhang,Chang Liu,Hongwei Zheng,Xiangning Han,Hong Lin,Limin Cao,Jianxin Sui
Phage display technology is commonly applied for high-throughput screening of single-domain antibodies (sdAbs), and the problem of non-specific adsorption caused by carrier proteins seriously affects the biopanning of single-domain antibodies specific to haptens. In this paper, enrofloxacin (ENR)-functionalized cryogels were prepared by the ethylenediamine (EDA) and carbodiimide methods for application in the biopanning of ENR-specific phages. To improve the efficiency of biopanning, double blocking, a wash solution flow rate of 1 mL/min, and phage pre-incubation were applied to the biopanning process through single-factor experiments. Results of flat colony counting showed that the phage output of AG-ENR cryogels was 15 times higher than that of AG cryogels for the same input amount. And seven complete sequences of ENR-specific shark sdAbs were obtained by monoclonal phage ELISA and sequence alignment. All these results indicate that functionalized cryogels could be used as a novel and efficient method for phage biopanning for single-domain antibodies to haptens.
Zhiwei Qie,Qiqi Liu,Wenliang Yan,Zichen Gao,Wu Meng,Rui Xiao,Shengqi Wang
A universal and ultrasensitive immunochromatographic assay (ICA) was established using antigen as a bifunctional element and antialbumin antibody in a test line. Preincubation was introduced for competitive recognition. After optimization, the linear detection of aflatoxin M1 (AFM1) with quantum dot bead (QB)-based ICA (QB-ICA) sensor ranged from 10 to 52 pg mL–1, with a 50% inhibitory concentration (IC50) of 23 pg mL–1, which was nearly 49.6-fold lower than those of ICA on a traditional structure with traditional pretreatment (IC50 = 1.10 ng mL–1) and 10-fold lower than those of ICA on a traditional structure with acid aid pretreatment (IC50 = 0.25 ng mL–1). The limit of detection (LOD) for AFM1 was 16 pg mL–1 in milk, which was approximately 16.3-fold times higher than those of ICA on a traditional structure with traditional pretreatment and 6.3-fold higher than those of ICA on a traditional structure with acid aid pretreatment. The LOD improved by 20-fold by using the proposed structure compared to that of conventional enzyme-linked immunosorbent assay (ELISA) for AFM1-spiked milk samples (IC10 = 0.12 ng mL–1). The performance and practicability of the established QB-ICA sensor were validated with a commercial ELISA kit. To evaluate universality, we successfully detected chloramphenicol, with IC50 of 0.42 ng mL–1. Given its high sensitivity and universality, the proposed QB-ICA can be used as an alternative for rapid, sensitive, and universal quantitative detection of all small-molecule analytes.
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