Monoclonal antibodies as a probe for the unfolding of porcine growth hormone.
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) kappa subclass, with dissociation constants (K(d)) 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 degrees C, 75 degrees C and 100 degrees 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.
Borromeo
Vitaliano
V
Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Sezione di Biochimica e Fisiologia Veterinaria, University of Milan, Via Celoria 10, Italy.
Gaggioli
Daniela
D
Berrini
Anna
A
Secchi
Camillo
C
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000737:chemistry; D013006:Growth Hormone / Q000737:chemistry* / Q000276:immunology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D011487:Protein Conformation; D011489:Protein Denaturation; D013552:Swine
D000911:Antibodies, Monoclonal; D000939:Epitopes; D013006:Growth Hormone