The evaluations of immunogenicity were performed in vivo using New Zealand white rabbits. The rabbits were randomly divided into four groups (n = 5) for the evaluation of immunogenicity. The treatment of the rabbits in group 1 consisted of an intraperitoneal injection of 500 μl of RhD-positive RBCs and a second intraperitoneal injection of 500 μl of RhD-positive RBCs after 3 weeks. The treatment of the rabbits in group 2 consisted of an intraperitoneal injection of 500 μl of RhD-positive RBCs and a second intraperitoneal injection of 500 μl of engineered RBCs after 3 weeks. The treatment of the rabbits in group 3 consisted of an intraperitoneal injection of 500 μl of engineered RBCs and a second intraperitoneal injection of 500 μl of engineered RBCs after 3 weeks. The treatment of the rabbits in group 4 consisted of an intraperitoneal injection of 500 μl of RhD-negative RBCs and a second intraperitoneal injection of 500 μl of RhD-negative RBCs after 3 weeks. Sera samples were collected every week. ELISAs were performed to analyze both framework and nonframework binding to RhD-positive RBCs and assess the RhD antibody titers in vivo. Briefly, recombinant human RhD proteins (ab80087, Abcam) were used to coat ELISA plates (Corning, NY) at 2 μg ml−1 in 100 μl of coating solution at pH 9.6 (0.05 M bicarbonate buffer). After overnight incubation at 4°C, the plates were washed three times with PBS containing 0.2% (v/v) Tween (Tween 20, Sigma) [PBS + 0.2% Tween (PBST)] and blocked with blocking buffer (Beyotime, China) for 2 hours at 37°C. The plates were then washed three times, and 100 μl of the sera samples was added. After 1 hour of incubation at 37°C, the plates were washed three times and 100 μl of HRP-conjugated secondary antibody (ab6721, Abcam; dilution, 1:100,000) was added. After 45 min of incubation at 37°C, the plates were washed three times, and 100 μl of TMB chromogen substrate solution (Beyotime, China) was added. Regarding the end-point titers, 50 μl of 1 M H2SO4 was added after 10 min to stop the reaction. The absorbance was measured at 450 nm using a microplate reader (BioTek, USA).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.