Captureselect iga affinity matrix

Manufactured by Thermo Fisher Scientific

Sourced in United States

CaptureSelect IgA Affinity Matrix is a laboratory tool designed for the purification of immunoglobulin A (IgA) from various biological samples. It is a resin-based affinity chromatography matrix that selectively binds to IgA, enabling its efficient separation and concentration.

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22 protocols using captureselect iga affinity matrix

Capture and Purification of IgA from Plasma

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IgA1 and IgA2 were captured from five
μL of plasma (containing approximately 350 μg of total
protein and 13 μg of IgA) with camelid antibody domains immobilized
on agarose beads (CaptureSelect IgA Affinity Matrix, Thermo Fisher
Scientific, Waltham, MA) as previously described.^{13 (link)} Ten μL of bead slurry were pipetted into each well
of a 96-well Orochem filter plate (AcroPrep, Pall Corporation, Ann
Arbor, MI, USA). The beads were washed three times with 200 μL
ultrapure water (prepared at 18.2 MΩ with a Purelab Ultra, Veolia
Water Technologies Netherlands B.V., Ede, The Netherlands). and three
times with PBS 1× solution on vacuum manifold. Five μL
of sample were added to 100 μL of PBS in each well and incubated
with shaking for 1 h at room temperature (RT). The beads were washed
on vacuum manifold with 3 × 200 μL PBS 1× and three
times 200 μL water. The captured IgA was released from the beads
with 100 μL of 100 mM formic acid, eluted in PP V-bottom plates
by centrifugation (1 min, 100g), and dried at 50
°C in a vacuum centrifuge.

Clerc F., Reiding K.R., de Haan N., Koeleman C.A., Hipgrave Ederveen A.L., Manetti N., Dotz V., Annese V, & Wuhrer M. (2023). Immunoglobulin A Glycosylation Differs between Crohn’s Disease and Ulcerative Colitis. Journal of Proteome Research, 22(10), 3213-3224.

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Antibody expression and purification

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Antibody heavy and light chains were PCR-amplified and sequenced as previously described (61 (link), 62 (link)) Sequence analysis, including the determination of the VH and VL genes and percentage of somatic mutations, was performed using the International Immunogenetics Information System (IMGT) database (Lefranc, 2014). Antibody V_H or V_L sequences were cloned into plasmids containing an IgG1 or relevant light chain backbone (Genscript) and used to transfect Expi293 cells (Thermo Fisher Scientific). Recombinant IgG was purified using HiTrap Protein A columns (GE Healthcare Life Sciences). For IgA1 or IgA2 antibodies, V_H sequences were also cloned into plasmids containing the IgA1 or IgA2 constant region (Genscript). Recombinant IgA was expressed without a J chain (to express only monomeric IgA) and purified using columns containing the CaptureSelect IgA Affinity Matrix (Thermo Fisher Scientific). To produce antibody Fabs, heavy chain plasmids encoding only the V_H and C_H1 were synthesized and used to transfect Expi293 cells along with light chain plasmids. Fab purification (including for the CV503 crystal structure) was performed with the CaptureSelect KappaXP Affinity Matrix or CaptureSelect LC-lambda (Hu) Affinity Matrix (Thermo Fisher Scientific).

Cho H., Gonzales-Wartz K.K., Huang D., Yuan M., Peterson M., Liang J., Beutler N., Torres J.L., Cong Y., Postnikova E., Bangaru S., Talana C.A., Shi W., Yang E.S., Zhang Y., Leung K., Wang L., Peng L., Skinner J., Li S., Wu N.C., Liu H., Dacon C., Moyer T., Cohen M., Zhao M., Lee F.E., Weinberg R.S., Douagi I., Gross R., Schmaljohn C., Pegu A., Mascola J.R., Holbrook M., Nemazee D., Rogers T.F., Ward A.B., Wilson I.A., Crompton P.D, & Tan J. (2021). Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2 variants of concern. Science Translational Medicine, 13(616), eabj5413.

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Affinity-based Isolation of Human IgA

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Isolation of IgA was performed as described previously [28 ]. In short, the steps included capturing human polyclonal IgA from either 40 μL of human plasma (children's samples) or human serum (adults' samples). This was achieved through affinity chromatography using the CaptureSelect™ IgA affinity matrix (Thermo Fischer Scientific, Massachusetts, USA). The IgA affinity matrix bead slurry (25 μL) was added to each well of a 96-well 0.7 μm Orochem filter plate (Orochem Technologies Inc., Illinois, USA), which was mounted on a vacuum manifold. Equilibration of the beads was achieved by washing the beads with water and four times with PBS. The samples (40 μL) were diluted sevenfold with PBS and added to the Orochem plate containing the equilibrated beads and incubated for 15 min on a plate shaker. After washing three times with PBS, the flowthrough was collected using a vacuum manifold and reapplied to the beads for another 15-min incubation to increase the isolation efficacy. Finally, the IgA was eluted using 200 μL of 0.1 M formic acid and neutralized with 34 μL of 1 M ammonium bicarbonate. Hundred μL of IgA eluate was aliquoted and dried using vacuum centrifugation for further analytical procedures.

Nemčić M., Shkunnikova S., Kifer D., Plavša B., Vučić Lovrenčić M., Morahan G., Duvnjak L., Pociot F, & Gornik O. (2024). N-glycosylation of immunoglobulin A in children and adults with type 1 diabetes mellitus. Heliyon, 10(9), e30529.

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Antibody Sequence Analysis and Production

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Antibody heavy and light chains were PCR-amplified and sequenced as previously described (61 (link), 62 (link)) Sequence analysis, including the determination of the VH and VL genes and percentage of somatic mutations, was performed using the International Immunogenetics Information System database (63 (link)). Antibody V_H or V_L sequences were cloned into plasmids containing an IgG1 or relevant light chain backbone (GenScript) and used to transfect Expi293 cells (Thermo Fisher Scientific). Recombinant IgG was purified using HiTrap Protein A columns (GE Healthcare Life Sciences). For IgA1 or IgA2 antibodies, V_H sequences were also cloned into plasmids containing the IgA1 or IgA2 constant region (GenScript). Recombinant IgA was expressed without a J chain (to express only monomeric IgA) and purified using columns containing the CaptureSelect IgA Affinity Matrix (Thermo Fisher Scientific). To produce antibody Fabs, heavy chain plasmids encoding only the V_H and C_H1 (domain 1 of constant region of the immunoglobulin heavy chain) were synthesized and used to transfect Expi293 cells along with light chain plasmids. Fab purification (including for the CV503 crystal structure) was performed with the CaptureSelect KappaXP Affinity Matrix or CaptureSelect LC-lambda (Hu) Affinity Matrix (Thermo Fisher Scientific).

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Selective Depletion of Antibody Isotypes

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The antibody isotypes present in nasal wash samples were selectively depleted using resins targeting IgG (CaptureSelect IgG CH1 affinity matrix, Thermo, 194320005), IgA (CaptureSelect IgA affinity matrix, Thermo, 194288005), or IgM (POROS CaptureSelect IgM affinity matrix, Thermo, 195289005). Microcentrifuge spin columns (Thermo, 69705) were loaded with 50 µl of suspended resin, followed by equilibration of the resin by passing PBS through the column. Nasal wash samples were diluted 1:10 in PBS and 150 µl were added to the column and incubated with end-over-end mixing for 5 min at room temperature. The remaining volume of the diluted sample was saved for use as the mock-depleted control. The isotype-depleted flow through was collected in a clean microcentrifuge tube and the columns were regenerated with 100 mM glycine (pH 3.0) and equilibrated before reuse. The extent of on- and off-target depletion of IgG, IgA, and IgM was measured using the Fc array assay described previously. The depleted samples were then evaluated in neutralization and phagocytosis assays as described above.

Butler S.E., Crowley A.R., Natarajan H., Xu S., Weiner J.A., Bobak C.A., Mattox D.E., Lee J., Wieland-Alter W., Connor R.I., Wright P.F, & Ackerman M.E. (2021). Distinct Features and Functions of Systemic and Mucosal Humoral Immunity Among SARS-CoV-2 Convalescent Individuals. Frontiers in Immunology, 11, 618685.

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Affinity Purification of IgG and IgA

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CaptureSelect
FcXL Affinity Matrix
beads (binding capacity IgG 25–35 g/L; Thermo Fisher Scientific,
Leiden, Netherlands) and CaptureSelect IgA Affinity Matrix (binding
capacity IgA 8 g/L, Thermo Fisher Scientific) were used for affinity
purification. Ammonium bicarbonate, formic acid, tris(hydroxymethyl)amino-methane,
tris(2-carboxyethyl)phosphine (TCEP) hydrochloride, sodium deoxycholate
(SDC), and 2-chloracetamide (CAA) were from Sigma-Aldrich (Zwijndrecht,
The Netherlands). Water was purified via a Purelab Ultra, maintained
at 18.2 MΩ (Veolia Water Technologies Netherlands B.V., Ede,
Netherlands). Acetic acid was from Honeywell (Seelze, Germany), acetonitrile
from Biosolve (Valkenswaard, The Netherlands), trifluoroAcetic acid
(TFA) from Merck (Darmstadt, Germany), and sequencing grade modified
trypsin was from Promega (Madison, WI).

Momčilović A., de Haan N., Hipgrave Ederveen A.L., Bondt A., Koeleman C.A., Falck D., de Neef L.A., Mesker W.E., Tollenaar R., de Ru A., van Veelen P., Wuhrer M, & Dotz V. (2020). Simultaneous Immunoglobulin A and G Glycopeptide Profiling for High-Throughput Applications. Analytical Chemistry, 92(6), 4518-4526.

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Antibody Fab Preparation for Native MS

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Fab molecules were generated by overnight digestion
at 37 °C of the IgAs bound to CaptureSelect IgA affinity matrix
(Thermo Fisher Scientific) using 1 U/μg of the SialEXO sialidase
cocktail and the OpeRATOR O-glycopeptidase from Akkermansia muciniphila (Genovis AB, Llund, Sweden)
for IgA1s and on-bead digestion of the IgGs bound to CaptureSelect
FcXL affinity matrix using 1 U/μg FabALACTICA IgdE (Genovis
AB, Llund, Sweden) as described previously.^{14 (link)}Prior to native top-down analysis, buffers were exchanged
to aqueous 150 mM ammonium acetate (pH 7.5) through six consecutive
dilution and concentration steps at 4 °C using Amicon Ultra centrifugal
filters with a 10 kDa molecular weight cutoff (Merck KGaA, Darmstadt,
Germany). IgA and IgG concentrations used for native electrospray
ionization mass spectrometry were around 4 μM.

Greisch J.F., den Boer M.A., Beurskens F., Schuurman J., Tamara S., Bondt A, & Heck A.J. (2021). Generating Informative Sequence Tags from Antigen-Binding Regions of Heavily Glycosylated IgA1 Antibodies by Native Top-Down Electron Capture Dissociation. Journal of the American Society for Mass Spectrometry, 32(6), 1326-1335.

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Recombinant Antibody Expression and Purification

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Antibody heavy and light chains were PCR-amplified and sequenced as previously described^{57 (link),58 (link)}. Sequence analysis, including the determination of the VH and VL genes and percentage of somatic mutations, was performed using the International Immunogenetics Information System (IMGT) database^{59 (link)}. Antibody V_H or V_L sequences were cloned into plasmids containing an IgG1 or relevant light chain backbone (Genscript) and used to transfect Expi293 cells (ThermoFisher Scientific). Recombinant IgG was purified using HiTrap Protein A columns (GE Healthcare Life Sciences). For IgA1 or IgA2 antibodies, V_H sequences were also cloned into plasmids containing the IgA1 or IgA2 constant region (Genscript). Recombinant IgA was expressed without a J chain (to express only monomeric IgA) and purified using columns containing the CaptureSelect IgA Affinity Matrix (ThermoFisher Scientific). To produce antibody Fabs, heavy chain plasmids encoding only the V_H and C_H1 were synthesized and used to transfect Expi293 cells along with light chain plasmids. Fab purification (including for the CV503 crystal structure) was performed with the CaptureSelect^™ KappaXP Affinity Matrix or CaptureSelect^™ LC-lambda (Hu) Affinity Matrix (ThermoFisher Scientific).

Cho H., Gonzales-Wartz K.K., Huang D., Yuan M., Peterson M., Liang J., Beutler N., Torres J.L., Cong Y., Postnikova E., Bangaru S., Talana C.A., Shi W., Yang E.S., Zhang Y., Leung K., Wang L., Peng L., Skinner J., Li S., Wu N.C., Liu H., Dacon C., Moyer T., Cohen M., Zhao M., Lee F.E., Weinberg R.S., Douagi I., Gross R., Schmaljohn C., Pegu A., Mascola J.R., Holbrook M., Nemazee D., Rogers T.F., Ward A.B., Wilson I.A., Crompton P.D, & Tan J. (2021). Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants. bioRxiv.

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Recombinant Antibody Expression and Purification

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Selected clonally related heavy and light chain sequences were ordered from GenScript. All heavy chain clonally related sequences were paired with the mature CAP88-CH06 light chain. The LCA heavy chain was paired with the LCA light chain (LCA). These heavy and light chain pairs were sub-cloned into IgG1, IgG3, IgA1 or IgA2 backbones, co-transfected and expressed in HEK293F cells (NIH AIDS Reagent Program), confirmed to be Mycoplasma free, grown in FreeStyle 293 Expression Medium (GIBCO) at 37°C, 5% CO2, 70% humidity and 125 rpm. Cultures were harvested after seven days by centrifugation at 4000 x g, and supernatants were filtered through a 0.22 μm filter. Filtered supernatant of IgG1, IgG3, IgA1 and IgA2 antibodies were purified by chromatography using protein A-Agarose (BioVision), immobilized protein G (ThermoFischer Scientific) and CaptureSelect™ IgA affinity matrix (ThermoFischer Scientific), respectively.

Scheepers C., Bekker V., Anthony C., Richardson S.I., Oosthuysen B., Moyo T., Kgagudi P., Kitchin D., Nonyane M., York T., Mielke D., Mabvakure B.M., Sheng Z., Lambson B.E., Ismail A., Garrett N.J., Karim S.S., Shapiro L., Williamson C., Morris L, & Moore P.L. (2020). Antibody Isotype Switching as a Mechanism to Counter HIV Neutralization Escape. Cell reports, 33(8), 108430.

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Purification of IgA from Plasma

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Bulk IgA was purified from IgG depleted plasma using CaptureSelect IgA Affinity Matrix (Thermo Fisher; catalog # 194288010). IgA was concentrated using Amicon^® filters (Milipore catalog #UFC805024), and purity was confirmed by SDS gel.

Giron L.B., Dweep H., Yin X., Wang H., Damra M., Goldman A.R., Gorman N., Palmer C.S., Tang H.Y., Shaikh M.W., Forsyth C.B., Balk R.A., Zilberstein N.F., Liu Q., Kossenkov A., Keshavarzian A., Landay A, & Abdel-Mohsen M. (2021). Plasma Markers of Disrupted Gut Permeability in Severe COVID-19 Patients. Frontiers in Immunology, 12, 686240.

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