Physical mapping of human insulin-like growth factor-I using specific monoclonal antibodies

Abstract The primary structure of recombinant human (h) insulin-like growth factor-I (IGF-I) epitopes recognized by a panel of 28 monoclonal antibodies (mAbs) is characterized. Pairwise mAb epitope mapping defines eight 'epitopic clusters' (I–VIII) which cover nearly the entire solvent-exposed IGF-I surface. Monoclonal antibody reactivity with 32 overlapping synthetic peptides and with IGF-I mutants is used to associate these epitopic clusters with the probable primary IGF-I sequences recognized. Epitopic cluster I involves residues in the C-domain and the first α-helix of the A-domain; clusters II, V and VII involve principally the B-domain; clusters III and IV map to amino acid sequences (55–70) and (1–13) respectively; cluster VI includes the A- and B-domains; and cluster VIII involves mainly the C-terminal part of the B-domain. Data indicate that this mAb panel defines 14 distinct IGF-I epitopes. The specific inhibition of HEL 92.1.7 IGF-I-promoted proliferation by these mAbs was explored. Direct correlation between mAb affinity and inhibitory activity was observed except in the case of clusters III- and VII-specific mAbs. Finally, the combination of epitopic cluster I and II mAbs detect 0·5–10 ng/ml hIGF-I in a sandwich immunoassay, with no IGF-II crossreactivity. These anti-IGF-I mAbs are, therefore, useful for both the inhibition of IGF-I mitogenic activity and for the quantification of this growth factor. The potential use of this mAb panel in tumor cell growth control is discussed. Journal of Endocrinology (1997) 154, 293–302

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Functional Epitope Mapping of Insulin-Like Growth Factor I (IGF-I) by Anti-IGF-I Monoclonal Antibodies*

Endocrinology ◽

10.1210/endo.138.3.4965 ◽

1997 ◽

Vol 138 (3) ◽

pp. 905-915 ◽

Cited By ~ 9

Author(s):

Santos Mañes ◽

Leonor Kremer ◽

Juan Pablo Albar ◽

Catherine Mark ◽

Rafael Llopis ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Epitope Mapping ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I

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Receptors for insulin and insulin-like growth factor-I can form hybrid dimers. Characterisation of hybrid receptors in transfected cells

Biochemical Journal ◽

10.1042/bj2700383 ◽

1990 ◽

Vol 270 (2) ◽

pp. 383-390 ◽

Cited By ~ 133

Author(s):

M A Soos ◽

J Whittaker ◽

R Lammers ◽

A Ullrich ◽

K Siddle

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Insulin Receptors ◽

Insulin Like Growth Factor ◽

Transfected Cells ◽

Factor I ◽

High Affinity ◽

Igf I ◽

Growth Factor I ◽

Nih 3T3

We have demonstrated the formation of hybrid insulin/insulin-like growth factor-I(IGF-I) receptors in transfected rodent fibroblasts, which overexpress human receptors, by examining reactivity with species- and receptor-specific monoclonal antibodies. In NIH 3T3 and Rat 1 fibroblasts, endogenous IGF-I receptors were unreactive with anti-(human insulin receptor)monoclonal antibodies (47-9, 25-49, 83-14, 83-7, 18-44). However, in transfected cells expressing high levels of insulin receptors, 60-80% of high-affinity IGF-I receptors reacted with these antibodies, as assessed either by inhibition of ligand binding in intact cells or by precipitation of solubilized receptors. Conversely, endogenous insulin receptors in NIH 3T3 cells were unreactive with anti-(IGF-I receptor) antibodies alpha IR-3 and 16-13. However, approx. 50% of high-affinity insulin receptors reacted with these antibodies in cells expressing high levels of human IGF-I receptors. The hybrid receptors in transfected cells bound insulin or IGF-I with high affinity. However, responses to these ligands were asymmetrical, in that binding of IGF-I inhibited subsequent binding of insulin, but prior binding of insulin did not affect the affinity for IGF-I. The existence of hybrid receptors in normal tissues could have important implications for metabolic regulation by insulin and IGF-I.

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Purification, amino acid sequences and assay cross-reactivities of porcine insulin-like growth factor-I and -II

Journal of Endocrinology ◽

10.1677/joe.0.1220681 ◽

1989 ◽

Vol 122 (3) ◽

pp. 681-687 ◽

Cited By ~ 28

Author(s):

G. L. Francis ◽

P. C. Owens ◽

K. A. McNeil ◽

J. C. Wallace ◽

F. J. Ballard

Keyword(s):

Amino Acid ◽

Growth Factor ◽

Mammalian Species ◽

Cross Reactivity ◽

Porcine Insulin ◽

Amino Acid Sequences ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I

ABSTRACT Porcine insulin-like growth factor-I (IGF-I) and IGF-II have been characterized to help define the roles of these peptides in the growth process. The amino acid sequence of porcine IGF-I was found to be identical to the human and bovine peptides. Porcine IGF-II was more similar to human IGF-II than to forms of this growth factor in other mammalian species, differing only in the replacement of asparagine for serine at residue 36. In a biological assay that measures the stimulation of protein synthesis in rat L6 myoblasts, porcine IGF-I was approximately ninefold more potent than porcine IGF-II or bovine IGF-II, while recombinant human IGF-I and IGF-II had half the potency of the respective natural peptides. Porcine and recombinant human IGF-I showed essentially equal competition for binding in a human IGF-I radioimmunoassay while between 0·6 and 1·5% cross-reactivity was observed with human, bovine or porcine IGF-II. A receptor assay for IGF-II demonstrated similar potencies for the three IGF-II peptides, while the cross-reactivity of recombinant human IGF-I was only 0·05%. Porcine IGF-I exhibited a higher cross-reactivity, presumably due to very slight contamination with IGF-II. Journal of Endocrinology (1989) 122, 681–687

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Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies.

Clinical Chemistry ◽

10.1093/clinchem/33.11.2019 ◽

1987 ◽

Vol 33 (11) ◽

pp. 2019-2023 ◽

Cited By ~ 3

Author(s):

M G Scott ◽

G C Cuca ◽

J R Petersen ◽

L R Lyle ◽

B D Burleigh ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Growth Factor ◽

Cross Reactivity ◽

Immunoradiometric Assay ◽

Insulin Like Growth Factor ◽

Factor I ◽

Igf I ◽

Growth Factor I ◽

Substantial Bias ◽

Growth Abnormalities

Abstract We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h.

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