TY - JOUR
T1 - Simplifying the synthesis of SIgA
T2 - Combination of dIgA and rhSC using affinity chromatography
AU - Moldt, Brian
AU - Saye-Francisco, Karen
AU - Schultz, Niccole
AU - Burton, Dennis R.
AU - Hessell, Ann J.
N1 - Funding Information:
We thank Diane Kubitz and staff at the Antibody Production Core at The Scripps Research Institute for purification of dIgA2 b12. We thank Christina Corbaci for graphic design and artwork. This work was supported by the National Institutes of Allergy and Infectious Diseases Grant R01 AI33292 (DRB), Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery Grant UM1AI100663 (DRB), and the Lundbeck Foundation (BM).
PY - 2014/1/1
Y1 - 2014/1/1
N2 - The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiple roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1. M citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6. mg of SIgA2 b12 was achieved from the combination of 20. mg of purified dIgA2 b12 and 2. L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a simplified means of generating a variety of pathogen-specific SIgA antibodies for research and clinical applications.
AB - The mucosal epithelia together with adaptive immune responses, such as local production and secretion of dimeric and polymeric immunoglobulin A (IgA), are a crucial part of the first line of defense against invading pathogens. IgA is primarily secreted as SIgA and plays multiple roles in mucosal defense. The study of SIgA-mediated protection is an important area of research in mucosal immunity but an easy, fast and reproducible method to generate pathogen-specific SIgA in vitro has not been available. We report here a new method to produce SIgA by co-purification of dimeric IgA, containing J chain, and recombinant human SC expressed in CHO cells. We previously reported the generation, production and characterization of the human recombinant monoclonal antibody IgA2 b12. This antibody, derived from the variable regions of the neutralizing anti-HIV-1 mAb IgG1 b12, blocked viral attachment and uptake by epithelial cells in vitro. We used a cloned CHO cell line that expresses monomeric, dimeric and polymeric species of IgA2 b12 for large-scale production of dIgA2 b12. Subsequently, we generated a CHO cell line to express recombinant human secretory component (rhSC). Here, we combined dIgA2 b12 and CHO-expressed rhSC via column chromatography to produce SIgA2 b12 that remains fully intact upon elution with 0.1. M citric acid, pH 3.0. We have performed biochemical analysis of the synthesized SIgA to confirm the species is of the expected size and retains the functional properties previously described for IgA2 b12. We show that SIgA2 b12 binds to the HIV-1 gp120 glycoprotein with similar apparent affinity to that of monomeric and dimeric forms of IgA2 b12 and neutralizes HIV-1 isolates with similar potency. An average yield of 6. mg of SIgA2 b12 was achieved from the combination of 20. mg of purified dIgA2 b12 and 2. L of rhSC-containing CHO cell supernatant. We conclude that synthesized production of stable SIgA can be generated by co-purification. This process introduces a simplified means of generating a variety of pathogen-specific SIgA antibodies for research and clinical applications.
KW - Affinity chromatography
KW - Antibody
KW - IgA
KW - Mucosal IgA
KW - Secretory IgA
UR - http://www.scopus.com/inward/record.url?scp=84891624025&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84891624025&partnerID=8YFLogxK
U2 - 10.1016/j.ymeth.2013.06.022
DO - 10.1016/j.ymeth.2013.06.022
M3 - Article
C2 - 23811333
AN - SCOPUS:84891624025
SN - 1046-2023
VL - 65
SP - 127
EP - 132
JO - Methods
JF - Methods
IS - 1
ER -