Rabbit immune sera raised against DHG from E. faecalis type 2 (α-DHG), SagA (α-SagA), and PpiC (α-PpiC) have been previously described [8 (link), 11 (link), 12 (link)]. For DHG-PpiC and DHG-SagA, New Zealand white rabbits were vaccinated with 2 subcutaneous injections of 10 µg of conjugate in incomplete Freund’s adjuvant given 2 weeks apart. An intravenous injection of 5 µg of the conjugate was given 7 days later, followed by 2 more intravenous injections of 5 µg delivered 2 days apart from each other. On day 35, a test serum was collected, and 7 and 14 days later intravenous boosts with 5 µg of antigen were administered. On day 56, a terminal immune serum was collected from each rabbit. All sera were heat inactivated at 56°C for 30 minutes. Terminal immune sera raised against the conjugates were designated α-DHG-PpiC and α-DHG-SagA. The antibodies were purified using an rProtein A GraviTrap column (GE Healthcare) in accordance with the manufacturers’ instructions. Immunoglobulin G (IgG) contents were measured by enzyme-linked immunosorbent assays (ELISAs) in purified and unpurified sera (Supplementary Materials ).
Rprotein a gravitrap column
Manufactured by GE Healthcare
The RProtein A GraviTrap column is a lab equipment product designed for the purification of monoclonal antibodies. The column utilizes Protein A affinity chromatography to capture and separate antibodies from complex samples. The product's core function is to facilitate the efficient and effective purification of antibodies for various research and analytical applications.
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2 protocols using rprotein a gravitrap column
1
Rabbit Antibodies Production and Purification
2
Monoclonal Antibody Purification and Labeling
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Hybridoma cell lines were adapted to CD Hybridoma AGT medium (Gibco); a serum-free media (SFM), supplemented with 40 mL/L GlutaMAX (ThermoFisher). Supernatants were harvested; mAbs were purified on an rProtein A GraviTrap column (GE Healthcare). mAbs were concentrated and buffer-exchanged (Millipore) to PBS. Glycerol was added to 50% and the purified mAbs were stored at -20°C. Each mAb was quantified by densitometry using ImageJ (27 (link)) and/or by a BCA protein assay. mAb light and heavy chains were verified using a non-reducing SDS-polyacrylamide gel (Bio-Rad). mAbs were either used unlabeled or fluorescently-labelled with an Alexa Fluor 647 labeling kit (Invitrogen).
The anti-CD30 mAb clone AC10, which is the mAb component of brentuximab, was obtained from AdipoGen (unlabeled or APC-labeled). Anti-CD30 clone BerH2 (PE-labelled) was acquired from Invitrogen. Anti-CD20-PE and anti-CXCR3-Alexa Fluor 700 were purchased from BioLegend as were all isotype control antibodies. Goat anti-mouse IgG (H+L) secondary antibody labeled with Alexa Fluor 647 was purchased from ThermoFisher.
The anti-CD30 mAb clone AC10, which is the mAb component of brentuximab, was obtained from AdipoGen (unlabeled or APC-labeled). Anti-CD30 clone BerH2 (PE-labelled) was acquired from Invitrogen. Anti-CD20-PE and anti-CXCR3-Alexa Fluor 700 were purchased from BioLegend as were all isotype control antibodies. Goat anti-mouse IgG (H+L) secondary antibody labeled with Alexa Fluor 647 was purchased from ThermoFisher.
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