Monoclonal antibodies reveal circulating growth hormone of high molecular weight not detectable by conventional assays

Abstract

A radioimmunoassay based on a monoclonal antibody, Mc-ab 1, which was raised against growth hormone but cross-reacted with human placental lactogen yielded higher GH immunoreactivity levels in serum than one based on a polyclonal antiserum. This discrepancy was noted in subjects with normal GH secretion as well as in patients with GH insufficiency. To characterize this GH immunoreactivity detected by Mc-ab 1, affinity purification and molecular sieve chromatography of serum were performed. High molecular weight proteins with GH immunoreactivity were found with both techniques. These proteins were associated with carbohydrates. Affinity cross-linking showed specific binding of radiolabelled GH to high molecular weight proteins in the serum. After fractionation of serum, the GH immunoreactivity became detectable by the polyclonal antiserum assay as well as by an immunoradiometric assay. GH immunoreactive material with an approximate mass of 80 kD was subjected to isoelectric focusing. When GH immunoreactive fractions at pH 5 were re-chromatographed, GH immunoreactivity was recovered in the elution volume corresponding to monomeric GH. Our results show that sera from normal subjects as well as from patients with deficient GH secretion contain notable amounts of high molecular weight GH which is undetectable by antibodies generally used for GH measurements, but which can be revealed after fractionation of serum.The aim of this work was to elucidate the discre¬ pancies found when growth hormone was mea¬ sured in human serum using a polyclonal antiserum (1) and a monoclonal antibody, Mc-ab 1, raised against GH but cross-reacting with human placental lactogen (2): Patients with GH insufficiency and non-detectable GH levels according to the polyclo¬ nal antiserum showed consistently detectable GH immunoreactivity with Mc-ab 1.Several monoclonal antibodies have been pro¬ duced against the closely related hormones human GH and human placental lactogen. These hor¬ mones consist of a single polypeptide chain, crosslinked by two disulfide bridges and have a mole¬ cular mass of approximately 22 kD (3). Pituitary GH does not, however, occur as only one molecular species, but shows both size and charge variations (4-6). In spite of the heterogeneity of GH, the radioimmunoassays based on monoclonal antibodies have correlated well with RIAs using polyclonal antisera (7-9). Subjects and MethodsSerum samples were obtained from 3 healthy volunteers, 2 patients with panhypopituitarism, 3 with acromegaly, and 2 with prolactinomas. The panhypopituitarism of the 2 was due to a non-secreting pituitary adenoma and a craniopharyngioma, respectively; these 2 patients had non-detectable GH levels in 24-h diurnal profiles, after oral administration of 500 mg L-dopa and during insulininduced hypoglycemia determined with the polyclonal antiserum.From the patients with acromegaly and newly diag¬ nosed disease, pieces of growth hormone producing adenomatous tissue were obtained during pituitary micro-

Serum Levels and Molecular Sizes of Growth Hormone during Pregnancy in Relation to Levels of Lactogens, Insulin-Like Growth Factor I and Insulin-Like Growth Factor Binding Protein-1

“…This is in accordance with our previous findings in patients with GH insufficiency. Despite nondetectable GH levels in these patients, GH was found in high molecular weight complexes after molecular sieve chromatography of their sera [8], It has recently been suggested that the decreasing level of serum GH throughout pregnancy is due to a suppression of pituitary GH [17]. Our results give rise to another explanation for the low levels of pitu itary GH in serum at the end of gestation, namely the presence of GH complexes of large molecular mass, which are detectable in unfractionated serum only by a few antibodies raised against GH such as our here reported Mab 1.…”

“…An initial character ization of a high molecular weight GH was described [8], The aim of this work was to further characterize the GH immunoreactivity detected by Mab 1 by studying the immunoreactivity in serum during pregnancy concomi tantly with determinations of GH, PL, prolactin (PRL), insulin-like growth factor I (IGF-I) and IGF binding pro tein-1 (IGFBP-1). Furthermore, pooled serum from late pregnancy was fractionated according to molecular size and analyzed with regard to GH and PL immunoreactiv ity.…”

Growth Hormone-Binding Protein in Plasma Is Inversely Correlated to Placental Lactogen and Augmented with Increasing Body Mass Index in Healthy Pregnant Women and Women with Gestational Diabetes mellitus

“…Plasma GH was determined in a RIA based on a monoclonal antibody (Mab) against GH, Mab 3, previously described [24], The detection limit for the assay was <1.6 pg/1. Mab 3 does not crossreact with PL, PRL and the gene variant of GH and is accordingly specific for pituitary GH (GH-N) [4], Plasma levels of PL and PRL were measured by RIA kits from Pharmacia Diagnostics AB (Upp sala, Sweden) and Farmos Diagnostica (Turku, Finland), respective ly.…”

Women with Prolactin-Producing Pituitary Adenoma Show Decreased Serum Placental Lactogen during Pregnancy

“…Scrum levels of GH and PL were determined by RIA kits from Wellcome Foundation Ltd. (Dartford, UK) and Pharmacia AB (Uppsala, Sweden), respectively. PL was also measured by an RIA with a monoclonal antibody, Mab 1, against GH which cross-reacts with PL [8], This assay also recognizes high molecular weight GH [9], Human CG, OE, and P4 in serum were analyzed by RIA kits from Diagnostic Products Corporation (Los Angeles, Calif., USA) and serum SHBG with an RIA kit from Farmos Diagnostica. …”