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

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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.

Introduction

The acid-labile subunit (ALS), a 578 amino acid protein with a calculated molecular weight of 63.3 kDa, forms a 150-kDa ternary complex in conjunction with insulin-like growth factor binding protein (IGFBP3) (and to a lesser extent IGFBP5; Twigg and Baxter, 1998) and insulin-like growth factor I or II (IGF-I or IGF-II) (Baxter, 1990). To form this ternary complex, IGFBP3 and IGF usually first form a binary complex, to which ALS then associates. Recently, some evidences suggest that there might also be complex formation of ALS and IGFBP3 without IGF (Lee et al., 1997).

In serum, the ALS molecule is present as an 82–88-kDa glycoprotein showing a double band on western immunoblot (Baxter et al., 1989). In contrast to the IGF binding protein 3, the synthesis of ALS, which is regulated directly by growth hormone (GH), occurs in the hepatocytes and in the epithelium of the proximal tubule of the renal cortex (Chin et al., 1994).

ALS contains 18–20 leucine-rich repeats of 24 amino acids (Leong et al., 1992) that are found in other human proteins as well, and are thought to be important in protein–protein interaction. For this reason, monoclonal antibodies (mAbs) were generated against the specific amino(N)-terminal and carboxyl(C)-terminal sequences of ALS, which contain no leucine-rich repeats and represent the unique sequences of ALS (Leong et al., 1992).

Under acidic conditions, ALS dissociates from IGF and IGFBP3, and neutralization is unable to restore the ternary complex (Hintz and Liu, 1977). Although binary complex formation between IGFs and IGFBP3 persists, ALS loses almost all binding activities below a pH of 4.5 (Holman and Baxter, 1996). This characteristic of ALS was used when pretreating the sera with an acidic glycine-buffer with the intention of “unfolding” the ALS protein so that it could be recognized by the mAbs that had been generated against the synthetic terminal oligopeptide sequences of ALS.

As ALS is produced mainly in hepatocytes under the direct control of GH, it represents a potential marker of GH secretory status and/or GH effects. The goal of this work was to establish a specific immunoassay based on mAb for quantitative ALS determination in biological samples.

Section snippets

Generation and purification of monoclonal antibodies

The synthetic human ALS oligopeptide sequences of the N-terminal (aa: 1–34) and C-terminal (aa: 551–578) of the ALS molecule were prepared by solid phase synthesis, isolated from the IGF–IGFBP3 column by acidification, and coupled to ovalbumin in a ratio of 2.3–3:1 by the glutardialdehyde method (Avrameas et al., 1978). The resulting immunogen was used to immunize BALB-C mice. The spleen cells were fused with NS0 myeloma cells following the hybridoma technique of Köhler and Milstein (1975) and

Anti-C- and N-terminal monoclonal antibodies of ALS

After the first screening, nine anti-N-terminal ALS mAbs and 10 anti-C-terminal ALS mAbs were identified. A clone was considered to be positive when its supernatant gave a signal in the assay that was more than 10-fold greater than the positive control (diluted blood from the immunized mouse collected at the time of fusion). The immunoglobulin subclasses of the anti-N-terminal ALS mAbs are mainly of subclass G2b, whereas the anti-C-terminal ALS mAbs belong, predominantly, to the subclass G1.

ALS levels in hGH-deficient patients

ALS levels were measured in 12 hGH-deficient individuals before and after 1 year of hGH replacement therapy. Prior to therapy, the ALS levels ranged from 36 to 986 mU/ml. The smallest increase in ALS level after therapy was 241 mU/ml and the maximal increase was 1340 mU/ml (Fig. 6). After 1 year of hGH replacement therapy, the mean ALS concentration had increased from 514 (±347) to 1223 (±458) mU/ml (p<0.0006).

Correlation between the changes in ALS levels with changes in IGF-I and IGFBP3 levels

Conclusion/discussion

The monoclonal N-terminal 5C9 and C-terminal 7H3 antibodies (Ab) against ALS represent an antibody pair that enables the measurement of ALS in human serum in a specific and reliable manner. The method of Ab selection prevents cross-reactions of the mAb with other known serum proteins. By covalently linking the N-terminal and C-terminal ALS oligopeptides to the surface of the microtiter plates in an orientation allowing binding of potential monoclonal antibodies, it was assured that the Abs

Acknowledgements

The authors would like to acknowledge the support of this work by Mr. Gopal Savjani, DSL, Webster, TX.

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