Monoclonal anti-acid-labile subunit oligopeptide antibodies and their use in a two-site immunoassay for ALS measurement in humans

Simone Stadler, Zida Wu, Regina A. Dressendörfer, Katherine M. Morrison, Aruna Khare, Phillip Lee, Christian J. Strasburger

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Quantification of the acid-labile subunit (ALS) has to date been restricted to immunoassays utilizing polyclonal antibodies. By immunization with N-terminal and C-terminal specific ALS oligopeptides, we generated monoclonal antibodies (mAbs) that target ALS-specific sequences outside the nonspecific leucine-rich repeats in the ALS molecule. For mAb selection, a special screening method was developed. Monoclonal antibody 5C9, which targets the N-terminus of ALS, is immobilized and the anti-ALS mAb 7H3, directed against the C-terminus, is biotinylated and used as tracer Ab. Due to the extreme pH-lability of ALS, changes in immunorecognition of ALS were investigated after acidification for protein unfolding in different pH ranges and in a time-dependent manner. It was determined that acidification of the serum samples to pH 2.7 for 30 min, followed by neutralization and dilution to 1:100 was the optimal acid-neutralization method. For standardization purposes, a serum pool derived from healthy volunteers was assigned the value 1 U/ml ALS. The sandwich assay has a working range with a linear dose-response curve in a log/log system between 0.005 and 10 U/ml. ALS levels in seven acromegalic patients ranged from 2.0 to 4.2 U/ml, and in 12 untreated growth hormone deficient patients from 0.036 to 0.986 U/ml (mean = 0.45 U/ml). After 12 months of growth hormone therapy, ALS levels increased significantly to 1.18 ± 0.45 U/ml (mean ± SD; p< 0.0006). The increase ranged from 0.48 to 1.4 U/ml. The change in ALS with growth hormone (GH) therapy correlated closer with the change in IGF-I (r = 0.798, p = 0.0057; Spearman rank correlation) than with the change in insulin-like growth factor binding protein (IGFBP3; r = 0.549, p = 0.057). This specific sandwich assay for the measurement of ALS provides a potentially valuable indicator of growth hormone secretory status. With this mAb-based immunofluorometric assay, the nonspecific detection of other proteins containing leucine-rich repeat sequences can be excluded.

Original languageEnglish (US)
Pages (from-to)73-82
Number of pages10
JournalJournal of Immunological Methods
Volume252
Issue number1-2
DOIs
StatePublished - Jun 1 2001
Externally publishedYes

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Keywords

  • ALS
  • Fluorescence assay
  • IGF-I
  • IGFBP3
  • Monoclonal antibodies

ASJC Scopus subject areas

  • Biotechnology
  • Immunology

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