Preparation, characterization, and related optimization of dPEG biotinylation

dPEG biotinylation plays a vital role in our production and life. So what is the preparation, characterization, and related optimization of dPEG biotinylation? Next, let us take a look.

Here is that the content:

l Preparation of 3-PBA Nanobody and Characterization of Activity after dPEG biotinylation

l Optimization of the combination of antigen and antibody concentration

Preparation of 3-PBA Nanobody and Characterization of Activity after dPEG biotinylation

The 3-PBA nano-dPEG biotinylation antibody was purified by Ni-NAT and controlled by SDS-PAGE. When the loading volume is the same, the band of 200 mmol/L eluents is deeper than 50 mmol and 100 mmol/L, indicating that 200 mmol is used /L imidazole has the best elution effect of Nanobody; at the same time, since the isoelectric point of anti-3-PBA Nanobody is around 8.1, to avoid the precipitation of Nano dPEG biotinylation antibody during the purification process, the pH value of the eluate should be adjusted to Keep away from the isoelectric point. Therefore, 200 mmol imidazole solution with pH 7.4 was selected as the eluent. Nanobodies after dPEG biotinylation are detected by ELISA using poly hip-SA as an enzyme label. When the coating mass concentration is 125 ng/mL, and the working mass concentration of dPEG biotinylation Nanobodies is 500 ng/mL, it is at 1 ug/mL 3-the inhibition rate in the PBA standard solution was 92.2%, indicating that the 3-PBA Nanobody dPEG biotinylation was successful.

Optimization of the combination of antigen and antibody concentration

Within the selected dPEG biotinylation coating source and antibody concentration range, the higher the concentration of the coating source, the smaller the antibody dilution factor is when it reaches 1.0 to 1.5, and vice versa. On the one hand, reducing the concentration of the coating source is conducive to the competition between 3-PBA and the coating source for antibody binding sites; on the other hand, reducing the concentration of the antibody is conducive to the full binding of the dPEG biotinylation antibody to the relatively low concentration of 3-PBA, thereby improving the detection sensitivity. However, due to the dPEG biotinylation between 1.0 and 1.5, the antigen and antibody concentrations are in a trade-off relationship, so it is necessary to further select the optimal conditions through the 3-PBA inhibition curve. Select the original mass concentration of coated dPEG biotinylation (ng/mL)-antibody dilution factor of 500-1: 4 000, 250-1: 3 000, 125-1: 2 000, 62.5-1: 500 conditions to determine 3-PBA The inhibition curve. In the process of reducing the original mass concentration of coated dPEG biotinylation from 500 ng/mL to 125 ng/mL, the IC50 value showed a decreasing trend, indicating that the sensitivity increased; and when the original mass concentration of the coating decreased to 62.5 ng/mL, IC50 value suddenly surged. It can also be seen from the Amax/IC50 value that when the original mass concentration of the coating is 125 ng/mL and the antibody is diluted 2,000 times, the ratio is the largest.

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