Immunochemical analysis of a recombinant, genetically engineered, secreted HLA-A2/Q10b fusion protein

Lynn D. DeVito, Beth Mason, Jonathan Schneck, David H. Margulies, Hans W. Sollinger, William J. Burlingham

Research output: Contribution to journalArticlepeer-review

Abstract

We engineered a fusion gene which encodes the α1 and α2 domains of HLA-A2 with the α3 and truncated transmembrane domains of the murine class I-like protein Q10b, and transferred it into mouse L cells along with the gene for human β2-microglobulin (β2m). The secreted rA2/Q10b gene product consisted of a single heavy chain of molecular weight 42 kd that was noncovalently associated with the human β2m light chain. Native detergent-solubilized HLA-A2 and secreted rA2/Q10b proteins were found to be similar by: (a) the binding to mouse monoclonal anti-HLA antibodies in an ELISA; (b) the blocking of lysis of HLA-A2+ cells by human anti-HLA-A2,-B17, anti-HLA-A2,9,28, and anti-HLA-A2,28 cross-reactive group (CREG) antisera in a complement-dependent cytotoxicity assay; and (c) the ability when coupled to Sepharose to selectively purify HLA-A2,9,28 and HLA-A2,28 CREG-specific antibodies. Mouse L cells expressing rA2/Q10b produced as much as 2.5 μg protein per 106 cells/day, or 50- to 100- fold more antigen on a per cell basis than the level of HLA-A2 expressed by B-lymphoblastoid cell line or spleen cells. Thus rA2/Q10b represents a viable alternative to detergent-solubilized HLA-A2 for purification of anti-HLA-A2 antibodies and analysis of anti-HLA-A2 immune responses.

Original languageEnglish (US)
Pages (from-to)125-133
Number of pages9
JournalHuman Immunology
Volume32
Issue number2
DOIs
StatePublished - Oct 1991

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Fingerprint Dive into the research topics of 'Immunochemical analysis of a recombinant, genetically engineered, secreted HLA-A2/Q10<sup>b</sup> fusion protein'. Together they form a unique fingerprint.

Cite this