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Hitrap mabselectsure columns

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HiTrap MabSelectSure columns are affinity chromatography columns designed for the purification of monoclonal antibodies (mAbs). The columns contain MabSelect SuRe ligand, which is a recombinant protein A-derived ligand, immobilized on a cross-linked agarose matrix. HiTrap MabSelectSure columns provide a robust and reliable solution for the capture and purification of mAbs from cell culture supernatants or other sources.

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Lab products found in correlation

Hybridoma sfm, by Thermo Fisher Scientific (6 mentions) Pierce fab preparation kit, by Thermo Fisher Scientific (4 mentions) Hitrap mabselect sure column, by GE Healthcare (4 mentions) Hitrap protein g, by GE Healthcare (3 mentions) Clonacell hy medium e, by STEMCELL (3 mentions) Anti ch1 column, by GE Healthcare (2 mentions) Media e, by STEMCELL (2 mentions) Ptwist cmv betaglobin wpre neo mammalian expression vector, by Twist Bioscience (1 mentions) Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions) Protein g resin, by GE Healthcare (1 mentions) Hitrap excel column, by GE Healthcare (1 mentions) Plasmid maxiprep kit, by Qiagen (1 mentions) Hek293f cell, by Thermo Fisher Scientific (1 mentions) Ouabain, by Merck Group (1 mentions) Hat media supplement, by Merck Group (1 mentions) 225 cm2 flasks, by Corning (1 mentions) 75 cm2 flask, by Corning (1 mentions) Zeba spin column, by Thermo Fisher Scientific (1 mentions) Pierce bca protein assay kit, by Merck Group (1 mentions) Dulbecco s modified eagle medium, by Harvard Bioscience (1 mentions)

15 protocols using hitrap mabselectsure columns

1

Antibody Expression and Purification

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For the expression of recombinant forms of antibody clones, nucleotide sequences of antibody heavy- and light-chain antibody variable genes were codon-optimized for mammalian expression and synthesized at Twist Biosciences. Resulting gene fragments were directly cloned at Twist Biosciences into the pTwist CMV BetaGlobin WPRE NEO mammalian expression vector (Twist Biosciences) that had already been fused with the heavy chain Fc domain of human IgG1 or Fc LALA variant. Recombinant antibodies were expressed by transient transfection of 293F cells with polyethyleneimine transfection reagent and was grown in expression medium (Freestyle 293 Expression Medium; Thermo Fisher Scientific). The supernatants were harvested after 7 days, filter-sterilized with a 0.4-μm filter, and purified by affinity chromatography using HiTrap MabSelect Sure columns (GE Healthcare Life Sciences). Purified recombinant IgG were used for in vivo studies.
Gilchuk I.M., Bangaru S., Gilchuk P., Irving R.P., Kose N., Bombardi R.G., Thornburg N.J., Creech C.B., Edwards K.M., Li S., Turner H.L., Yu W., Zhu X., Wilson I.A., Ward A.B, & Crowe JE J.r. (2019). Influenza H7N9 Virus Neuraminidase-Specific Human Monoclonal Antibodies Inhibit Viral Egress and Protect from Lethal Influenza Infection in Mice. Cell host & microbe, 26(6), 715-728.e8.
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2

Monoclonal Antibody Production and Purification

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After fusion with HMMA2.5 myeloma cells, hybridomas producing MARV-specific antibodies were cloned biologically by two rounds of limiting dilution and by single-cell fluorescence-activated cell sorting. After cloning, hybridomas were expanded in post-fusion medium (ClonaCell-HY Medium E, STEMCELL Technologies #03805) until 50% confluent in 75-cm2 flasks (Corning #430641). For antibody production, cells from one 75-cm2 flask were collected with a cell scraper and expanded to four 225-cm2 flasks (Corning #431082) in serum-free medium (Hybridoma-SFM, Gibco #12045-076). After 21 days, supernates were clarified by centrifugation and sterile filtered using 0.2-μm pore size filter devices. HiTrap Protein G or HiTrap MabSelectSure columns (GE Healthcare Life Sciences #17040501 and #11003494 respectively) were used to purify antibodies from filtered supernates. Fab fragments were generated by papain digestion (Pierce Fab Preparation Kit, Thermo Scientific #44985) and purified by chromatography using a two-column system where the first column contained protein G resin (GE Healthcare Life Sciences #29048581) and the second column contained either anti-kappa or anti-lambda antibody light chain resins (GE Healthcare Life Sciences #17545811 and #17548211 respectively).
Flyak A.I., Ilinykh P.A., Murin C.D., Garron T., Shen X., Fusco M.L., Hashiguchi T., Bornholdt Z.A., Slaughter J.C., Sapparapu G., Klages C., Ksiazek T.G., Ward A.B., Saphire E.O., Bukreyev A, & Crowe JE J.r. (2015). Mechanism of Human Antibody-Mediated Neutralization of Marburg Virus. Cell, 160(5), 893-903.
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3

Generation of Monoclonal Tau Antibodies

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DMR7 and SKT82 hybridoma clones were generated as previously described for other tau mAbs [48 (link)]. Briefly, mice were injected subcutaneously with AD-tau in Freund’s complete adjuvant, and spleens were dissociated and fused with SP2 myeloma cells with PEG treatment. Hybridoma clones were diluted with limiting dilutions and screened for tau antibody binding to T40 tau, AD-P1 PFFs, and AD-tau. Clones with selective binding to AD-P1 PFFs and AD-tau were prioritized and sub-cloned two times to ensure monoclonal populations. Control murine IgG1 antibody, CHL34, is directed against the herpes simplex virus 2 envelope glycoprotein L [49 (link)] and murine IgG2b antibody, 52S, is directed against herpes simplex virus 1 glycoproteins H/L [50 (link)]. Antibodies were purified from hybridoma cell culture media using HiTrap MabSelect SuRe columns (GE Healthcare). All procedures were approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) and performed according to the NIH Guide for the Care and Use of Experimental Animals.
Gibbons G.S., Kim S.J., Wu Q., Riddle D.M., Leight S.N., Changolkar L., Xu H., Meymand E.S., O’Reilly M., Zhang B., Brunden K.R., Trojanowski J.Q, & Lee V.M. (2020). Conformation-selective tau monoclonal antibodies inhibit tau pathology in primary neurons and a mouse model of Alzheimer’s disease. Molecular Neurodegeneration, 15, 64.
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4

Antibody Purification and Fab Preparation

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For antibody purification from hybridoma supernates, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer's protocol. To obtain fragment antigen-binding (Fab) fragments, papain digestion was used (Pierce Fab Preparation Kit, Thermo Scientific). Fab fragments were purified by removing IgG and Fc contaminants using a HiTrap MabSelectSure column followed by purification with an anti-CH1 column (GE Healthcare Life Sciences).
Mousa J.J., Kose N., Matta P., Gilchuk P, & Crowe JE J.r. (2017). A novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein. Nature microbiology, 2, 16271.
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5

Isolation and Characterization of Anti-RSV mAb

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25P13 was isolated from the PBMCs of a Nashville Red Cross donor. PBMCs were isolated from human donor blood samples using Ficoll-Histopaque density gradient centrifugation. PBMCs were transformed with Epstein-Barr virus as described previously34 (link). Cells were screened by ELISA for binding to post-fusion RSV F, and positive wells were fused with HMMA2.5 myeloma cells using the previously published protocol34 (link). The 25P13 hybridoma was biologically cloned by single-cell fluorescence-activated sorting. The 25P13 hybridoma was expanded step-wise into 48-well and 12-well plates followed by 75-cm2 flask in Media E (StemCell Technologies). Antibody production was accomplished by expanding the hybridoma to four 225-cm2 cell culture flasks in serum-free medium (Hybridoma-SFM, GIBCO). After 21 days, supernatants were sterile filtered using 0.45 µm pore size filter devices. For antibody purification, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer’s protocol.
Wen X., Mousa J.J., Bates J.T., Lamb R.A., Crowe JE J.r, & Jardetzky T.S. (2017). Structural basis for antibody cross-neutralization of respiratory syncytial virus and human metapneumovirus. Nature microbiology, 2, 16272.
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6

RSV Subgroup Protein Expression and Purification

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Plasmids encoding cDNAs for pre-fusion (SC-TM) or post-fusion RSV subgroup A strain A2, and subgroup B strain 18537 pre-fusion (DsCav1, a gift from Barney Graham) were expanded in E. coli DH5α cells, and plasmids were purified using Qiagen Plasmid Maxiprep kits (Qiagen). Plasmids encoding cDNAs for the protein sequences of mAb 101F, mAb MPE8, and mAb D25, and motavizumab heavy and light chain sequences were cloned into vectors encoding human IgG1 and lambda or kappa light chain constant regions, respectively. Vectors encoding the heavy and light chain sequences of 54G10 were a gift from Dennis Burton. MAb 131-2a protein was obtained from Sigma. Commercial preparations of palivizumab (Medimmune) were obtained from the pharmacy at Vanderbilt University Medical Center. For each liter of protein expression, 1.3 mg of plasmid DNA was mixed with 2 mg of polyethylenimine in Opti-MEM I cell culture medium (Fisher). After 10 min, the DNA mixture was added to HEK293F cells (ThermoFisher R79007) at 1 x 106 cells/mL. The culture supernatant was harvested after 5 days, and the protein was purified by HiTrap Excel column (GE Healthcare Life Sciences) for RSV F protein variants or HiTrap MabSelectSure columns for mAbs.
Mousa J.J., Binshtein E., Human S., Fong R.H., Alvarado G., Doranz B.J., Moore M.L., Ohi M.D, & Crowe JE J.r. (2018). Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins. PLoS Pathogens, 14(2), e1006837.
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7

Hybridoma Generation for ZIKV E Antibody

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Cells from wells with transformed B cells containing supernatants that exhibited reactivity to ZIKV E protein were fused with HMMA2.5 myeloma cells (gift from L. Cavacini) using an established electrofusion technique26 (link). After fusion, hybridomas were suspended in a selection medium containing 100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT Media Supplement, Sigma), and 7 μg ml−1 ouabain (Sigma) and cultured in 384-well plates for 18 days before screening hybridomas for antibody production by ELISA. After fusion with HMMA2.5 myeloma cells, hybridomas producing ZIKV E-specific antibodies were cloned biologically by single-cell fluorescence-activated cell sorting. Hybridomas were expanded in post-fusion medium (ClonaCell-HY Medium E, STEMCELL Technologies) until 50% confluent in 75-cm2 flasks (Corning).
For antibody production, cells from one 75-cm2 flask were collected with a cell scraper and expanded to four 225-cm2 flasks (Corning) in serum-free medium (Hybridoma-SFM, Life Technologies). After 21 days, supernatants were clarified by centrifugation and filtered using 0.45-μm pore size filter devices. HiTrap Protein G or HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies from filtered supernatants.
Sapparapu G., Fernandez E., Kose N., Cao B., Fox J.M., Bombardi R.G., Zhao H., Nelson C.A., Bryan A.L., Barnes T., Davidson E., Mysorekar I.U., Fremont D.H., Doranz B.J., Diamond M.S, & Crowe JE J.r. (2016). Neutralizing human antibodies prevent Zika virus replication and fetal disease in mice. Nature, 540(7633), 443-447.
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8

COVID-19 Convalescent Plasma IgG Isolation

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Plasma samples collected from COVID-19 convalescent and healthy donors are described in (18 (link)). Human IgGs were isolated from heat-inactivated plasma samples using 5-mL HiTrap MabSelect SuRe columns (GE Healthcare Life Sciences) as described (24 ).
Cohen A.A., Gnanapragasam P.N., Lee Y.E., Hoffman P.R., Ou S., Kakutani L.M., Keeffe J.R., Wu H.J., Howarth M., West A.P., Barnes C.O., Nussenzweig M.C, & Bjorkman P.J. (2021). Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice. bioRxiv.
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9

Antibody Purification and Fab Preparation

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For antibody purification from hybridoma supernates, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer's protocol. To obtain fragment antigen-binding (Fab) fragments, papain digestion was used (Pierce Fab Preparation Kit, Thermo Scientific). Fab fragments were purified by removing IgG and Fc contaminants using a HiTrap MabSelectSure column followed by purification with an anti-CH1 column (GE Healthcare Life Sciences).
Mousa J.J., Kose N., Matta P., Gilchuk P, & Crowe JE J.r. (2017). A novel pre-fusion conformation-specific neutralizing epitope on the respiratory syncytial virus fusion protein. Nature microbiology, 2, 16271.
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10

Recombinant Antibody Production in CHO Cells

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Variable light and heavy chain genes encoded by selected anti-ultravariegin scFv(s) were sub-cloned in pcDNA3.4 based pVCLC102 (Kappa Light Chain; KLC) or pVCLC202 (Lambda Light Chain; LLC) and pVCHC302 (Heavy Chain; HC) vectors for expression of antibodies in IgG format. Purified LC and HC plasmid DNA were mixed 1:1 and transfected in 50/100 ml ExpiCHO cells using the Max Titer expression protocol as per manufacturer’s instructions (Thermo Fisher Scientific, MA, USA). Culture supernatant was harvested 8 days after transfection, purified using 1 ml HiTrap MabSelect SuRe columns (GE Healthcare Life Sciences, IL, USA) and eluted with a linear gradient comprising 0.1 M citrate, pH 3.0 followed by neutralisation of pH using 2 M Tris-HCl. Concentrations of each protein were estimated by absorbance and extinction coefficient values at 280 nm, assuming molar extinction coefficient of human IgG to be 210,000 M−1 cm−1.
Koh C.Y., Shih N., Yip C.Y., Li A.W., Chen W., Amran F.S., Leong E.J., Iyer J.K., Croft G., Mazlan M.I., Chee Y.L., Yap E.S., Monroe D.M., Hoffman M., Becker R.C., de Kleijn D.P., Verma V., Gupta A., Chaudhary V.K., Richards A.M., Kini R.M, & Chan M.Y. (2021). Efficacy and safety of next-generation tick transcriptome-derived direct thrombin inhibitors. Nature Communications, 12, 6912.
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