Monoclonal antibodies (mAbs) were generated against pituitary porcine growth hormone (pGH). Ten mAbs were selected for their specificity and affinity for pGH. These mAbs were of the immunoglobulin G (IgG)1 κ subclass, with dissociation constants (Kd) between 7.42 and 0.26 nM, and recognised seven non-overlapping epitopes. We measured whether the mAbs detected alterations of the pGH three-dimensional structure by comparing the antibody reactivity to native pGH and to pGH experimentally unfolded by heating at 50°C, 75°C and 100°C or by reduction and S-carboxymethylation. The antibody-antigen interactions were studied with two enzyme-linked immunosorbent assays (ELISA), based either on a direct binding or inhibition format. The results show that: 1) one mAb, mAb D12, is a conformation-sensitive antibody that recognises an epitope present only in the native pGH. Because the intact three-dimensional structure is essential for the expression of biological activity, mAb D12 could be used to detect altered pGH molecules in biological samples (blood, pituitary extracts or material produced with recombinant technology), and for the one-step purification of biologically active pGH by immunoaffinity chromatography; 2) one mAb, mAb I4, binds to a linear epitope that is not significantly modified in the denatured hormone. This mAb was able to detect the hormone in assays where protein conformation is usually strongly altered, i.e. immunoblotting and immunohistochemistry; 3) the performances of the other eight mAbs differed significantly in the competitive and non-competitive ELISA.
|Titolo:||Monoclonal antibodies as a probe for the unfolding of porcine growth hormone|
|Autori interni:||BORROMEO, VITALIANO (Primo)|
GAGGIOLI, DANIELA (Secondo)
BERRINI, ANNA (Penultimo)
SECCHI, CAMILLO LUIGI (Ultimo)
|Parole Chiave:||Conformation; ELISA; Monoclonal antibodies; Porcine growth hormone|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2003|
|Digital Object Identifier (DOI):||10.1016/S0022-1759(02)00433-7|
|Appare nelle tipologie:||01 - Articolo su periodico|
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