Simplifying the synthesis of SIgA: Combination of dIgA and rhSC using affinity chromatography

Brian Moldt, Karen Saye-Francisco, Niccole Schultz, Dennis R. Burton, Ann Hessell

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)127-132
Number of pages6
JournalMethods
Volume65
Issue number1
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

Fingerprint

Secretory Component
Affinity chromatography
Secretory Immunoglobulin A
Affinity Chromatography
Immunoglobulin A
CHO Cells
Pathogens
HIV-1
Purification
Cell Line
Mucosal Immunity
Column chromatography
Antibodies
Adaptive Immunity
Research
Citric Acid
Chromatography
Glycoproteins
Epithelium
Immunoglobulin G

Keywords

  • Affinity chromatography
  • Antibody
  • IgA
  • Mucosal IgA
  • Secretory IgA

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Simplifying the synthesis of SIgA : Combination of dIgA and rhSC using affinity chromatography. / Moldt, Brian; Saye-Francisco, Karen; Schultz, Niccole; Burton, Dennis R.; Hessell, Ann.

In: Methods, Vol. 65, No. 1, 01.01.2014, p. 127-132.

Research output: Contribution to journalArticle

Moldt, Brian ; Saye-Francisco, Karen ; Schultz, Niccole ; Burton, Dennis R. ; Hessell, Ann. / Simplifying the synthesis of SIgA : Combination of dIgA and rhSC using affinity chromatography. In: Methods. 2014 ; Vol. 65, No. 1. pp. 127-132.
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