I am trying to connect the antibody using DSPE-PEG-COOH, but it seems to be unsuccessful. The experimental steps are as follows：Take 1mL liposomes into a 1.5mL centrifuge tube, wash once with 1mL PBS, and remove the supernatant after centrifugation at 16000rpm at 4℃ for 10min; add 400μL EDC (5 mg/mL) and 400μL NHS (5 mg/mL) respectively The solution was added to the centrifuge tube, mixed thoroughly, and then placed in a 37°C water bath to activate the carboxyl group for 30 minutes. Then 1M NaOH was added to adjust the pH to 7.4. Add 25 μL（100μg/mL） of PCT antibody and react at room temperature for 6h. After washing twice with PBS, resuspended in PBS (add 1% BSA) to obtain liposome@Ab2 and store it at 4℃ in the dark for use. But in the follow-up, the ELISA experiment was not successful. Is the concentration of the antibody I added too low? or other reasons？
In which buffer and pH your antibody is supplied? Did you perform a successful ELISA experiment without the liposomes? What is the purpose of the liposomes in your assay?
First of all, thank you for paying attention to my question. The antibody is supplied by PBS with pH 7.4. And At present, the ELISA experiment has not been tested without liposomes, is it necessary? The role of liposomes is to wrap signal molecules instead of enzyme-labeled antibodies to achieve detection. I am mainly worried that the concentration of the antibody I added is too low, which affects the efficiency of liposome coupling and affects the experimental results. @Michael G. Weller
Liposome-based assays might be more complicated and less sensitive than ELISA. Therefore, I prefer to set up an immunoassay in a traditional format and then transfer it to the more sophisticated one. You might be right, however. Your antibody concentration might be ok, if you washed your liposomes, but it seems to be not adequate if the total volume is nearly 1 mL. Did you remove the surplus of EDC?
Thank you again for guiding me. Yes, I am indeed aware of this problem now, but the experiment has been carried out to this point, then it can only be continued. I will design the next system with reference to your suggestions. I removed the excess EDC and NHS after the coupling but did not remove them before adding the antibody. Now it seems that I need to add a larger concentration of antibody to ensure that my experiment can proceed smoothly. @Michael G. Weller
If you want to keep your liposomes, you may wash them with PBS and repeat the protocol with improved conditions. A final concentration of 0.1 mg/ml of antibodies should be fine, pH 8 would be good. Please avoid using NaOH. The correct pH should be achieved by buffer exchange.
Thank you so much, I will use your suggestions to adjust my experimental steps as soon as possible, which is very helpful to me. At the same time, the traditional ELISA experiment will be tested without liposomes to make sure that there are no problems with my other experimental procedures. If the experiment is successful, I will be the first to tell you. @Michael G. Weller
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