RESUMEN
For many practical applications, monoclonal antibodies must be chemically modified without any significant loss in their immunoreactivity. In some situations, however, the amino acid residue crucial for antibody activity may be highly reactive toward the modifying agent, which results in antibody inactivation. The method to prevent inactivation of a modification-sensitive antinuclear monoclonal antibody by acylating agents was developed. The method is based on the hypothesis that a highly reactive amino group exists within, or in the vicinity of, the binding site of the antibody, providing crucial interaction with negatively charged moieties of DNA. It has been shown that negatively charged polymers, such as dextran sulfate or heparin, may provide temporary protection, presumably interacting noncovalently with this amino group and thus masking it. The protecting molecule can be removed later by chromatography on a protein A column, thus regenerating modified but not inactivated antibody in the free form for use in subsequent applications. In particular, we have modified antibody 2C5 with a chelating agent, diethylenetriaminepentaacetic acid (DTPA) without the loss of activity. Modified antibody was labeled with radioactive isotope, (111)In, via chelation by antibody-attached DTPA. The labeled antibody was shown to demonstrate the same specificity of binding to nucleosomes as the nonmodified antibody, so it may be used in immunoscintigraphy or biodistribution studies. The method might be useful for the modification of other modification-sensitive antibodies with other acylating chemicals, such as crosslinking agents or biotin derivatives.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Anticuerpos Antinucleares/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Polímeros/química , Polímeros/metabolismo , Acilación , Animales , Anticuerpos Antinucleares/química , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Quelantes/química , Quelantes/metabolismo , Sulfato de Dextran/química , Sulfato de Dextran/metabolismo , Heparina/química , Heparina/metabolismo , Radioisótopos de Indio , Ratones , Ácido Pentético/química , Ácido Pentético/metabolismo , Desnaturalización Proteica , Electricidad Estática , Succinimidas/química , Succinimidas/metabolismoRESUMEN
1. The effects of removal of extracellular divalent cations (experimental calcium paradox conditions) were studied on the whole-cell current in freshly isolated smooth muscle cells (SMCs), and on contraction in rabbit aortic rings. 2. Aortic rings treated for 30-60 min with extracellular Ca2+- and Mg2+-free solution contracted following readmission of extracellular Ca2+, even in the presence of nifedipine. 3. In isolated SMCs, the removal of extracellular Ca2+ and Mg2+ induced a non-inactivating whole-cell inward current and membrane depolarization. This current was a monovalent cation (MC) current which reversed at around 0 mV and conducted K+ >= Cs+ > Na+ > Li+. Extracellular divalent cations (Ca2+, Mg2+, Ba2+, Mn2+ and Ni2+) inhibited MC current. 4. Using noise analysis of the whole-cell MC current, the single MC channel conductance was estimated to be < 450 fS. 5. MC current was insensitive to nifedipine, TEA, 4-aminopyridine, SK&F 96365 and S-nitroso-N-acetyl-penicillamine (SNAP), but was decreased by amiloride and low pH. 6. When EGTA was present in Ca2+- and Mg2+-free solution, a significant nifedipine-sensitive Na+ current through L-type Ca2+ channels developed in addition to MC current. 7. It is concluded that upon the removal of extracellular Ca2+ and Mg2+ from resting SMCs, an inward MC current develops allowing Na+ influx and causing SMC depolarization which could be the important steps leading to vessel contraction upon Ca2+ readmission. Addition of EGTA to Ca2+- and Mg2+-free solution greatly potentiates Na+ influx and vessel contraction by allowing additional Na+ influx through L-type Ca2+ channels which are activated presumably by MC current-induced depolarization.