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Supplemental data text 1
Characterization of antibodies
The epitopes of the mAbs were identified by three different types of competitive binding
assays (displacement experiments): The degree of cross-reaction was estimated from the
amount of competitors (several variants and fragments of hGH) required to evoke a 50%
inhibition in binding of biotinylated rhGH by the respective mAb. Apart from the hGH
preparations obtained through NIBSC, preparations obtained through various sources were
used (see supplemental table 1). In addition, the interaction of the mAbs with several other
anti-hGH mAbs already characterized was investigated (23). Finally, the relationship of the
respective epitopes and the hormone/receptor interaction site 1 of hGH was studied by
saturating anti-hGH-mAbs immobilized on microtiter plates with recombinant hGH.
Subsequently, biotinylated recombinant hGH receptor ectodomain (hGH binding protein,
rhGHBP) was added. The amount of the formation of rhGH/rhGHBP complexes was
quantified after addition of Europium-labeled Streptavidin and measurement by a time
resolved fluorometer. Integration of the results from the different experimental approaches
and projection on a structural model of the 22,000 hGH molecule derived from x-ray
crystallography allowed deduction of the respective epitopes.
Sources of hGH variants, fragments and other peptide hormones
Recombinant 22kD hGH (IRP88/624) and prolactin (IRP98/582) as well as pituitary derived
hGH (IRP80/505) were obtained from NIBSC, Hartfordshire, UK.
Recombinant 20kD hGH was kindly provided by Eli Lilly & Co., Indianapolis, Indiana, USA.
A second preparation of hGH purified from pituitary extracts (NHPP hGH, Lot AFP-4793))
was obtained from Dr. A. F. Parlow, NIDDK, Harbour-UCLA Medical Center, Torrence,
California, USA.
Recombinant 17kD hGH was provided by Prof. Dr. Ken Ho, Sydney, Australia.
Recombinant mutations of hGH lacking the first 1, 7 and 13 amino acids were a gift from Dr.
Tikva Vogel, Biotechnology General, Nesziona, Israel.
Human placental lactogen and hGH1-43 were purchased from Firma Sigma, St Louis, Mo,
USA.
Several hGH fragments were generated by digestion, purified by Dr. Jack Kostyo, Ann
Arbour, Michigan, USA and characterized by amino acid analysis (details see Mills JB,
Kostyo JL, Moseley MH, Reagan CR, Wilhelmi AE. Isolation and characterization of
fragments of reduced and S-carbamidomethylated human growth hormone produced by
plasmin digestion. I. Chemistry. Endocrinology 1978; 102:1366-76. and Mills JB, Kostyo JL,
Reagan CR, Wagner SA, Moseley MH, Wilhelmi AE. Fragments of human growth hormone
produced by digestion with thrombin: chemistry and biological properties. Endocrinology
1980; 107:391-9). They were kindly donated to our group.
Mutants of hGH G120K and B2036 were kindly provided by Sensus, Austin, Texas, USA.
E. coli derived recombinant hGHBP was a kind donation by Prof. Dr. A. Gertler, Rehovot,
Israel.
Recombinant placental growth hormone (hGH-V) was obtained from Dr. Susan Kirk,
University of Virginia, Charlottesville, Virginia, USA.
Recombinant hGH-CV, lacking 9 of the 13 amino acids at the C-terminal end of hGH-V was
provided by Dr. Pär Gellerfors, Kabi Pharmacia, Stockholm, Sweden.