Antigenic cross-reactivity among components of Brazilian Elapidae snake venoms

Snake venoms from M. corallinus (LD5=7.1 + or - 0.83 µg), M.frontalis (LD50=19.3 + or - 3.13 µg), M. ibiboboca (LD50=19.8 + or - 2.07 µg) and M. spiixi (LD50=6.7 + or - 1.25 µg) (family Elapidae, genus Micrurus) injected into horses alone or in combination (M. corallinus with M. frontalis) elicit antibody production, as indicated in vivo by neutralization of venom lethality and in vitro by enzyme-linked immunosorbent assay (ELISA), immunoelectrophoresis (IE) and Western blotting (WB). Venom lethality was efficiently neutralized by the antisera, with the monovalent antivenoms being more efficient than the bivalent antivenom. Antibodies against venom components were detected by all artisera at different titers by ELISA. Upon IE, antisera against M. spiixi and M. frontalis venoms cross-reacted with the four types of venoms studied and recognized several molecular components, the precipitin lines obtained had distinct intensities and electrophoretic motilities, whereas the antivenom against M. corallinus only recognized components of its venom but not of the others. All antivenoms cross-reacted with all the elapid venoms in WB revealing several blands with distinct MWs in M. corallinus and M. spiixi venoms, two very sharp and separate bands in M. corallinus venom and a very sharp band of high MW together with several other smaller and faint bands in M. frontalis venom. The data indicate that snake venoms of the genus Micrurus are good immunogens that contain many cross-reactive molecules, and that their toxic components are neutralized more effectively by monovalent rather than by bivalent antivenom

A rapid and efficient purification method for horse IgG(T) usin a rat monoclonal antibody

1. Louvain rats (IgK-1a) were immunized with horse IgG(T). To generate mAb to IgG(T), popliteal lymph node cells taken from the immunized animals were fused to a non-secreting LOU/C immunocytoma (IR983F). The hybridomas were cultured in HAT -containing medium and cloned under limiting dilution conditions. Supernatants from the growing hybrids were screened by ELISE using plates coated with horse IgG(T) or IgGa+b+c. 2. The anti-IgG(T) mAb obtained was named LO-HoGT-1 (LOU anti-horse IgG(T)). It is an IgG2a rat antibody whose light chain allotype is IgK-1a, and with an affinity constant of 2.9 x 1010 M-1. 3. Ascites was isnduced in LOU (IgK-1b) rats by injecting the hybridoma cells and incomplete Freund's adjuvant ip. To obtain purified mAb, ascitic fluid was applied to a Sepharose anti-rat LOU IgK-1 a chain column. 4. The purified mAb was then coupled to Sepharose. Immunoelectrophoretically pure IgG(T) was obtained by passage of horse serum through this column. The entire procedure took less than 30 min and resulted in a highly purified IgG(T)

Detection and neutralization of B. jararaca venom in mice

1. Bothrops jararaca venom was detected by ELISA at different times in the skin, muscle, blood, liver, lung, heart, kidney and spleen of mice injected with venom im or id. 2. The results showed that even 10 min after im injection the venom is detected mostly in skin rather than in the muscle of the venom injection site. A small amount of venom was detected in the kidney up to 12 h after im venom injection, and none was detected in tissues of lung, heart, liver or spleen. 3. However, in mice injected id, the venom could be detected in the skin up to 24 h after injection. Local necrosis and haemorrhage could be neutralized by antivenom injected by the id or iv routes only if the antivenom was given a short time after venom injection, even when antivenom is adminsitered in high concentration. 4. In contrast, experiments performed in mice receiving venom id and treated by id or iv routes with antivenom injected at different times after envenoming showed that the effect of venom on blood coagulation could be counteracted by antivenom administered by either route up to 2 h after venom injection 5. We suggest that a feasible amount of antivenom administered id could be given as a first aid measure after a snake bite accident. However, further experimental studies using the id route for antivenom administration are essential to confirm this possibility

Isolation of horse IgG with protein A

Horse immunoglobulins were obtained from normal defatted with dextran sulfate and precipitated with ammonium sulfate. Eight mg of this preparation was submitted to affinity chromatography with protein A-Sepharose CL-4B. Low temperature (4-C) and a starting buffer at pH 8.0 were conditions required for all IgG subclasses to bind to protein A, even those with low affinity. The IgGs bound to protein A were eluted with glycine buffer at pH 2.8. The yield was about 90%. Its suggested that isolated IgG, instead of whole Igs, be used in serum therapy, reducing the amount of Igs and diminishing serum-related reactions

The possible mechanism of action of IgG antibodies and platelets protecting against Trypanosoma cruzi infection

1. The role of IgG antibody and platelets in the mechanism of defense against Trypanosoma cruzi infection is reviewed. 2. Experimental data showing the participation of the different IgG subclasses in the immune lysis and immune clearance of the parasites are discussed. 3. The involvement of the platelets in the removal of the parasites from the circulation is considered. 4. It is suggested that IgG anti-T. cruzi antibodies interact with circulating parasites leading to formation of microaggregates, activation of C3 and deposition of C3 and deposition of C3b on the immune aggregates followed by adherence of platelets through C3b receptors. The immune aggregates would then be taken up by cells of the mononuclear phagocytic system

Comparison between the antigenic composition of bloodstream and cell culture-derived trypomastigotes of Trypanosoma cruzi

Antigens of bloodstream and cell culture-derived trypomastigotes of T. cruzi were compared by western blotting using sera of chronic chagasic patient as a source of antiobodies. The immunoblots demonstrated that the two forms display extensive homology except for the 85 - and 52 - kDa bands. These antigens were more strongly stained in culture - derived trypomastigotes. Although the reported differences are not related to major antigens, these results might offer an explanation for previous studies showing that culture - derived trypomastigotes are more antigenic and infective in vitro than bloodstream trypomastigotes