THESIS. I. Fernandes submitted this thesis for the degree of Doctor of Science in Immunology publicly examined at the Department of Immunology of Institute of Biomedical Sciences of the University of São Paulo, São Paulo, Brazil in 1995.
Advisor: Professor Ivan Mota
ABSTRACT. Methods were developed for isolating horse IgG(T) and IgGa in order to study their role in the protective effect of anti-snake venom sera. Initially, the interaction of horse Igs with protein A-Sepharose was studied. Using a starting buffer at pH 8.5 and at low temperature (4ºC), all IgG isotypes present in horse serum were adsorbed to protein A from which they were eluted with buffers of lower pH. In addition, a procedure was developed for the isolation of IgG(T) using a combination of two chromatographic procedures, salt-mediated hydrophobic chromatography using phenyl-Sepharose followed by affinity chromatography with protein A-Sepharose. The immunoelectrophoretically pure IgG(T) isolated by this technique was used to immunize rats whose cells were fused with non-secreting myeloma cells to obtain a hybridoma secreting monoclonal anti-IgG(T) antibodies. A hybridoma able to produce a high affinity anti-IgG(T) antibody was obtained and named LO-HoGT-1 (rat LOU anti-Ho (horse) IgG(T)). This mAb was coupled to Sepharose to prepare an immunoabsorbent column. Application of normal or hyperimmune horse serum to this column followed by elution at a low pH buffer (pH 5.0) allowed to obtain pure IgG(T) with a high yield. In order to isolate IgGa, a fraction was used containing IgG(T) plus IgGa obtained by passing horse hyperimmune serum in protein A-Sepharose column and eluting the bound protein with citrate buffer, pH 6.0. The IgG(T) and IgGa were then separated using the anti-IgG(T) mAb coupled to Sepharose. The ability of the different Ig isotypes present in anti-bothropic or anti-crotalic serum to neutralize the toxic activities of the homologous venoms was studied. The neutralizing activity of the IgG-free serum (containing all isotypes but IgG) and the isolated IgG(T) and IgGa was assayed against the venoms of Bothrops jararaca or Crotalus durissus terrificus. It was concluded that: 1) Most of the anti-lethal activity of anti-bothropic horse serum was due to antibodies of IgG class; 2) The IgG(T) was the most efficient of the IgG subclasses; 3) The IgGa subclass was also able to neutralize the lethality, but it was less efficient than IgG(T); 4) The hemorrhagic lesion induced by Bothrops jararaca venom was mainly neutralized by IgG(T) and to a lesser degree by IgGa; 5) The blood incoagulability induced by bothropic venom was equally neutralized by IgG(T) and IgGa; 6) Other Igs present in the IgG-free serum had little or no neutralizing ability; 7) Most of the anti-lethal activity of anti-crotalic horse serum was mainly due to antibodies of IgG(T) subclass; 8) The IgGa subclass was also able to neutralize the lethality, but it was less efficient than IgG(T); 9) IgG-free serum did not neutralize the crotalic venom lethality.
CORRESPONDENCE TO:
I. FERNANDES - Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, CEP 05503-900, São Paulo, SP, Brasil.
Publication Dates
-
Publication in this collection
08 Jan 1999 -
Date of issue
1997
