Monoclonal anti-acid-labile subunit oligopeptide antibodies and their use in a two-site immunoassay for ALS measurement in humans.
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.
Stadler
S
S
Department of Medicine, Innenstadt University Hospital, Ziemssenstrasse, Munich, Germany.
Wu
Z
Z
Dressendörfer
R A
RA
Morrison
K M
KM
Khare
A
A
Lee
P D
PD
Strasburger
C J
CJ
D000172:Acromegaly / Q000097:blood* / Q000188:drug therapy; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western / Q000379:methods; D002352:Carrier Proteins / Q000097:blood* / Q000276:immunology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006023:Glycoproteins / Q000097:blood* / Q000276:immunology; D020249:Hormone Replacement Therapy; D019382:Human Growth Hormone / Q000172:deficiency / Q000627:therapeutic use; D006801:Humans; D016014:Linear Models; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009842:Oligopeptides / Q000097:blood* / Q000276:immunology; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D006023:Glycoproteins; D009842:Oligopeptides; C075944:insulin-like growth factor binding protein, acid labile subunit; D019382:Human Growth Hormone