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Effects of Conjugating Conditions on Goat Anti-rabbit Antibody Immobilization onto Bacterial Magnetic Particles
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| CHEN Ji-feng;LI Ying;JIANG Wei;WANG Zheng-fang;SUN Jian-bo;Li Jian;LI Ji-lun;LI Shao-hua |
| Department of Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100094, China |
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Abstract Goat anti-rabbit immunoglobulin G (antibody) was applied as a sample for studying antibody conjugation onto bacterial magnetic particles (BMPs). The conditions for this study were different pre-treatment BMPs (-196 ℃, -70 ℃ and -20 ℃ for 5 h) for freeze-dry, temperatures with varied conjugation durations, amount of BMPs and antibody, conjugation buffers, and buffer pH and concentrations. The results showed that different pre-treatments for freeze-dry BMPs had different linkage rate of antibody (LRA). Liquid nitrogen(-196 ℃) as a pre-treatment for 5 h, the LRA was significantly higher than that of -70 ℃ and -20 ℃ pre-treatments. Any kinds of pre-treatments and freeze-dry BMPs, the LRAs were decreased 36%~55% compared with no freeze-dry BMPs. Room temperature (about 15 ℃), and incubation for 18 h resulted in the highest LRA (64.06 μg/mg) in the applied temperatures and conjugation durations. LRAs depended upon the amount of antibody and BMPs, when BMPs decreased from 5.0 mg to 0.1 mg, and antibody increased from 10 μg to 300 μg, the LRAs increased gradually. When BMPs at 0.1 mg /mL and antibody at 300 μg/mL were used, the LRA reached to 762.37 μg/mg. Many buffers could be used for antibody conjugation onto BMPs, and under the normal buffer pH and concentration, the LRAs of 8 buffers were 46.28±0.58 ~ 90.83±1.64 μg/mg. The different buffer had different pH and concentration to reach the peak LRA. When pH 3.5 and 20 mmol / L for HEPES, pH 5.6 and 5 mmol / L for Na3PO4, the LRAs were 108.01 and 76.78 μg/mg respectively. SDS-PAGE showed that antibody was conjugated onto BMPs.
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Received: 30 April 2005
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Corresponding Authors:
LI Shao-hua
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ZHAI Da-Cai, FANG Zhen, LV Cai-Yun, ZHANG Ming-Liang, LIU Shan, WU Meng, FANG Ru-Yue, ZHOU Xun, FANG Jian-Xin, BAI Xiao-Hui. Research on Antibacterial Activity of the Endophytic Bacillus subtilis HJ-2 from Polygonatum cyrtonema[J]. 农业生物技术学报, 2019, 27(9): 1664-1672. |
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