Briefly, the VH and VL sequences were cloned into human Igγ1, Igκ and Igλ expression vectors (kindly provided by Michel Nussenzweig, Rockefeller University, New York, NY, USA), essentially as described (47). Recombinant mAbs were produced by transient transfection of EXPI293 cells (Invitrogen), purified by Protein A chromatography (GE Healthcare) and desalted against PBS.
Protein a chromatography
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom
Protein A chromatography is a technique used for the purification of antibodies and other proteins that bind to Protein A, a bacterial protein derived from Staphylococcus aureus. The process involves passing a sample containing the target protein through a column packed with a Protein A-based resin, which selectively binds the protein of interest. The bound protein can then be eluted from the column using a suitable buffer, allowing for its isolation and purification.
Automatically generated - may contain errors
Lab products found in correlation
Expi293f cells, by Thermo Fisher Scientific (6 mentions)
Cd22 microbeads, by Miltenyi Biotec (2 mentions)
Anti mouse igg alkaline phosphatase conjugated goat igg, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Prism 7, by GraphPad (2 mentions)
Freund s adjuvant, by Merck Group (2 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Mab936, by R&D Systems (1 mentions)
Pcdna3.1 expression vector, by Thermo Fisher Scientific (1 mentions)
293f cells, by Thermo Fisher Scientific (1 mentions)
Expicho cells, by Thermo Fisher Scientific (1 mentions)
Anti fitc, by Miltenyi Biotec (1 mentions)
Microbead, by Miltenyi Biotec (1 mentions)
Amicon ultra 15 ml, by Merck Group (1 mentions)
Goat anti human igg, by Southern Biotech (1 mentions)
Erms da470, by Merck Group (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Nucleobond xtra midi plus kit, by Macherey-Nagel (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
27 protocols using protein a chromatography
1
Recombinant Monoclonal Antibody Production
2
Immunization and Antibody Characterization for MMP9
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbits were immunized with the peptide (NH2)-FQTFEGDC conjugated to both ovalbumin and keyhole limpet hemocyanin. Based on serum titers toward the immunizing peptide, animals were selected for hybridoma library generation (Abcam, Burlingame, CA, USA). In total, 40,000 individual hybridomas were screened for immunoreactivity of supernatants with the immunizing peptide. Positive supernatants were screened by Western blot with full-length pro- and active-MMP9 recombinant standards [28 (link)]. Positive hybridoma supernatants were screened by IHC against a series of formalin-fixed HEK293 cell pellets containing active MMP9 (see Appendix A for details). Immunoglobulin G complementary DNA (cDNA) was sequenced from the positive hybridomas by 5′RACE (Abcam), cloned into pcDNA3.1 expression vector (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), expressed in HEK293 cells, and purified by protein-A chromatography (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK). Sandwich immunoassays were conducted by coating the plate in 1 μg/mL recombinant antibody, adding recombinant MMP9 standard ADX, detecting with total MMP9 antibody (MAB936; R&D Systems, Minneapolis, MN, USA), and quantifying with a ruthenium-conjugated anti-mouse antibody (Meso Scale Discovery, Rockville, MD, USA).
3
Isolation and Immortalization of COVID-19 Memory B Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cell (PBMC) isolation was performed via density gradient centrifugation over Ficoll-Paque, then IgG+ memory B cells were isolated from a cryopreserved COVID-19 patient’s PBMC by using CD22 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and immortalized with Epstein–Barr virus (EBV) as previously described (16 (link)). Culture supernatants were tested for their ability to bind SARS-CoV-2 proteins using enzyme-linked immunosorbent assay (ELISA). Positive cultures were collected and expanded. The VH and VL sequences from positive cultures were retrieved by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into human IgG1 and Ig kappa or Ig lambda expression vectors as previously described (17 (link)). Monoclonal antibodies were produced by transient transfection of 293F cells (Invitrogen-Life technologies, Grand Island, USA). Supernatants from transfected cells were collected after 4 days, and IgG was affinity purified by protein A chromatography (GE Healthcare, Chicago, USA) and desalted against PBS.
4
Production of Recombinant Antibodies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UA sequences were determined with reference to the IMGT database27 and produced by gene synthesis (Genscript). VH and VK sequences of RVC20 antibody and derived variants were cloned into human IgG1 and IgK expression vectors and recombinant mAbs were produced by transient transfection of ExpiCHO cells (Thermo Fisher Scientific, Cat# A29127), purified by Protein A chromatography (GE Healthcare) and desalted against PBS.
5
Monoclonal Antibody Production and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain mAbs, 2×106 hybridoma cells, producing anti-G9 antibodies, were resuspended in 0.5 mL of sterile 0.9% NaCl and administered intraperitoneally into 20-week-old BALB/c mice. Selected mAbs 3G11 and 5H5 were purified by ammonium sulfate precipitation from ascitic fluids and then purified using protein A chromatography (GE Healthcare, IL). The purity and size of the purified IgG antibodies were examined by SDS-PAGE and Western blot analyses. Purified mAbs were resolved by 12.5% SDS-PAGE under reducing conditions and transferred onto a nitrocellulose membrane (Bio-Rad, CA). After blocking with 5% casein (skim milk powder) in PBS, the membrane was incubated with anti-mouse IgG alkaline phosphatase-conjugated goat IgG (Sigma Aldrich, MO). Immune complexes were visualized by a mixture of nitro blue tetrazolium (WVR, PA) and 5-bromo-4-chloro-3-indolyl-phosphate (Roche, Germany) for 20 min. The selected mAbs 3G11 and 5H5 were conjugated with horse-radish peroxidase (WVR, PA) using the optimized NaIO4 method as described previously [22 (link)].
6
Immortalization of Memory B Cells
7
Antibody Expression in Expi293F Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody heavy and light chains were cloned into human IgG1, Igκ and Igλ expression vectors and expressed by transient transfection of Expi293F Cells (ThermoFisher Scientific) using polyethylenimine. Cell lines were routinely tested for mycoplasma contamination. The antibodies were affinity purified by protein A chromatography (GE Healthcare).
8
Sortase-Mediated Antibody-Drug Conjugation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
vNAR antibody-drug conjugates were generated upon enzymatic ligation of the antimitotic agent Monomethyl Auristatin E (MMAE) to the Fc fragment. Therefore, a genetically engineered C-terminal LPETGG extension was modified by an activity-optimized sortase variant (eSrtA) (39 (link)) from the Gram-positive bacterial strain Staphylococcus aureus as described elsewhere (40 (link)). Briefly, vNAR-Fc molecules were incubated in the presence of 0.1 equivalents sortase A and 10 equivalents GGG-vc-PABA-PEG3-MMAE in reaction buffer (150 mM NaCl, 50 mM TRIS, 10 mM CaCl2, pH 7.5 adjusted with HCl) for 1 h at 22°C. After protein modification, antibody-drug conjugates were purified via protein A chromatography (GE Healthcare), buffer-exchanged and concentrated to required concentrations (Amicon® Ultra 15 mL, Millipore).
9
Generating Anti-RSV G Protein Hybridomas
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate mouse hybridomas against RSV-G protein, two sets of 5 female C57BL/6 mice were immunized and boosted with recombinant non-glycosylated RSV G 67-298 from RSV-A2 strain (REG A2) or from RSV-B1 strains (REG B2) at 28 days apart. After the fusion of post-immunization splenocytes with mouse myeloma cell line, an initial screen of mouse hybridoma supernatants was performed against glycosylated RMG A2 and RMG B1 by ELISA. After the screening, the RSV G-binding positive hybridomas were single cell purified, and hybridomas screened again for clonality and screen for glycosylated RMG A2 or RMG B1 positive binders. All positive hybridoma clones were subjected for antibody production in serum-free media and MAbs were purified using protein A chromatography (GE Healthcare, Uppsala, Sweden).
10
Recombinant Monoclonal Antibody Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Heavy chain variability, diversity, and joining and light chain variability and joining regions were cloned into human IgG1, Igκ, and Igλ expression vectors. To amplify the resulting plasmids, TOP10 chemically competent Escherichia coli (Thermo Fisher Scientific, Waltham, MA) were transformed and expanded in antibiotics containing LB broth, and amplified plasmids were isolated using the NucleoBond Xtra Midi Plus kit (Macherey-Nagel, Düren, Germany). Amplified plasmids were sequenced to assure identity to original sequence. Expi293F cells (Thermo Fisher Scientific) were transiently transfected with plasmids of corresponding light and heavy chains using polyethylenimine (PEI). Expi293F cells were tested routinely for mycoplasma contamination. mAbs were quantified by ELISA. In short, IgG was quantified using 96-well MaxiSorp plates (Nunc) coated with goat anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470 (Sigma-Aldrich, ERMs-DA470) as standard. Produced mAbs were affinity purified by protein A chromatography (GE Healthcare, Chicago, IL), and concentration of purified recombinant mAbs was quantified by measuring absorption at 280 nm.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!