Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells

H. C. Morton, J. D. Atkin, R. J. Owens, J. M. Woof (Lead / Corresponding author)

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fcα receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.

Original languageEnglish
Pages (from-to)4743-4752
Number of pages10
JournalJournal of Immunology
Volume151
Issue number9
Publication statusPublished - 1993

Fingerprint

Cricetulus
Immunoglobulin A
Ovary
IgA-specific serine endopeptidase
Antibodies
Streptococcus oralis
Streptococcus sanguis
Antibody Affinity
Fc Receptors
Calcitriol
HL-60 Cells
Affinity Chromatography
Lectins
Gel Chromatography
Polyacrylamide Gel Electrophoresis
Digestion
High Pressure Liquid Chromatography

Cite this

@article{906fa74f06b742e48dcdd65ba7eaa534,
title = "Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells",
abstract = "Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fcα receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.",
author = "Morton, {H. C.} and Atkin, {J. D.} and Owens, {R. J.} and Woof, {J. M.}",
year = "1993",
language = "English",
volume = "151",
pages = "4743--4752",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "9",

}

Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells. / Morton, H. C.; Atkin, J. D.; Owens, R. J.; Woof, J. M. (Lead / Corresponding author).

In: Journal of Immunology, Vol. 151, No. 9, 1993, p. 4743-4752.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Purification and characterization of chimeric human IgA1 and IgA2 expressed in COS and Chinese hamster ovary cells

AU - Morton, H. C.

AU - Atkin, J. D.

AU - Owens, R. J.

AU - Woof, J. M.

PY - 1993

Y1 - 1993

N2 - Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fcα receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.

AB - Ag-specific chimeric human IgA molecules, of the two human subclasses, IgA1 and IgA2, have been expressed in two mammalian cell systems. Analysis of the secreted IgA molecules, purified in milligram quantities from stable Chinese hamster ovary transfectants by Ag affinity chromatography, has allowed a direct comparison of the biologic properties of the two subclasses. HPLC gel filtration analysis revealed that in both subclasses, the IgA molecules associate predominantly into dimers. The monomer units are presumed to interact noncovalently, inasmuch as no dimers are evident when the antibodies are subjected to SDS-PAGE. The recombinant antibodies are glycosylated, inasmuch as a lectin blotting procedure revealed that the H chains of both subclasses are recognized by Con A. When subjected to digestion by preparations of IgA1-specific proteases secreted by two pathogenic streptococcal strains, Streptococcus sanguis and Streptococcus oralis, the recombinant IgA molecules behave just as their natural equivalents. Thus, only the chimeric IgA1 molecule is cleaved, with the IgA2 remaining intact. In terms of interaction with natural effector molecules, both recombinant IgA isotypes were shown to interact with Fcα receptors on calcitriol-stimulated HL-60 cells with similar affinity, but neither antibody was found to interact with human C1q. The expression system described readily permits manipulation of the human IgA genes, which should lead to a fuller molecular understanding of how this important antibody mediates its function.

UR - http://www.scopus.com/inward/record.url?scp=0027485053&partnerID=8YFLogxK

M3 - Article

C2 - 8409433

AN - SCOPUS:0027485053

VL - 151

SP - 4743

EP - 4752

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 9

ER -