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Development of a sandwich enzyme linked immunosorbent assay (ELISA) for the quantification of iduronate-2-sulfate sulfatase.

Posted by on in 2011
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Sosa ACEspejo AJRodriguez EALizaraso LMRojas AGuevara JEcheverri OYBarrera LA. 2011. J Immunol Methods. 2011:368(1-2):64-70. Epub 2011 Apr 5.

Instituto de Errores Innatos del Metabolismo, Pontificia Universidad Javeriana, Bogotá, Colombia.

Abstract

Iduronate-2-sulfate sulfatase (IDS; EC 3.1.6.13) is an enzyme that belongs to human sulfatases. IDS deficiency causes the Hunter syndrome or mucopolysaccharidosis type II (MPS II; OMIM 309900). We have been developing an expression system for human recombinant IDS (hrIDS) in Pichia pastoris, therefore a method was required for its detection during production and purification processes, which could be used also to measure the enzyme in human fluids. In this study, an immunoquantification assay for human and recombinant IDS was developed with the combination of two antibodies. Rabbit IgG and chicken IgY were used as IDS capture and detection antibodies, respectively. Chicken IgY antibodies were developed against specific amino acid sequences present in IDS but absent in other human sulfatases. hrIDS produced in P. pastoris, commercial hrIDS, and normal human plasma samples were used as antigens and immunoquantification results were compared to enzyme activity. The technique was linear over the range 8 to 500 ng mL(-1) using commercial hrIDS. The concentration range detected for IDS in normal human plasma was 14.43 to 287.88 ng mL(-1). The hrIDS was detected in P. pastoris cultures even when the enzyme was inactive, which is convenient for monitoring the production of recombinant proteins. These results show that chicken site-specific antibodies provide a good alternative, as a substitute of monoclonal antibodies, for the detection of human proteins. This is the first report on the development of an ELISA system to detect and quantify IDS with IgY antibodies.

Copyright © 2011 Elsevier B.V. All rights reserved.

PMID:
21447345
[PubMed - indexed for MEDLINE]
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