Production of a murine mAb against Bothrops alternatus and B. neuwiedi snake venoms and its use to isolate a thrombin-like serine protease fraction.

Accidents with snakes from the genus Bothrops represent ~90 % of all snakebites in Brazil. Monoclonal antibodies (mAbs) targeting venom components can be important assets for treating envenoming syndromes, for developing diagnostic tests and for research purposes. Therefore, in this study, we aimed to generate murine mAbs against the antigenic mixture of Bothropic venoms traditionally used as immunogen to produce Bothropic antivenoms in Brazil. ELISA showed that one of the produced mAbs recognizes B. alternatus and B. neuwiedi venoms (mAb anti-Ba/Bn) specifically and Western Blot revealed that this mAb binds to a single protein band of molecular mass of ≈50 kDa. MAb anti-Ba/Bn inhibited the coagulant activity but was unable to neutralize hemorrhagic and phospholipase A2 activities caused by the B. neuwiedi venom. MAb anti-Ba/Bn was immobilized to Sepharose beads and used for immunoaffinity chromatography of B. neuwiedi venom. Proteolytic activity assays indicated that the immunoaffinity-purified fraction (BnF-Bothrops neuwiedi fraction) has a serine protease thrombin-like profile, which was supported by coagulability assays in mice. Bottom-up proteomic analysis confirmed the prevalence of serine proteases in BnF using label-free quantification. In conclusion, this work characterized a mAb with neutralizing properties against B. neuwiedi coagulant activity and demonstrates that immunoaffinity chromatography using mAbs can be a useful technique for purification of bioactive toxic proteins from Bothrops spp. snake venoms.

On human tissue kallikrein activity in urine of Brazilian White and Black primary hypertensive patients.

OBJECTIVE: To evaluate the possible relationship between the human tissue kallikrein amidase activity with sex and ethnicity in Brazilian primary hypertensive patients. DESIGN: Population-based study. PARTICIPANTS: One hundred men and women, Black and White primary hypertensive patients aged 20 years and older were selected. Eighty nine healthy individuals, paired according to age, sex, and ethnics were used as controls. METHODS: Early-morning midstream urine was used. Human tissue kallikrein amidase activity was estimated with D-Val-Leu-Arg 4-nitroanilide substrate. Creatinine was determined by a method based on Jaffe's reaction. hK1 amidase activity is expressed in microM/(min x mg creatinine) to correct for differences in urine flow rate. Data are expressed as medians. RESULTS: Human tissue kallikrein amidase activity was significantly lower in the urine of hypertensive patients (0.210 microM/(min x mg creatinine) than in the urine of control subjects (0.260 microM/(min x mg creatinine) (P = .010). This result supports data from the literature. Contrasting to what was already reported, namely that human tissue kallikrein excretion is higher in females than in males, and especially higher in Caucasians than in African Americans, our results show that, in the urine of Brazilian hypertensive patients and control subjects, no significant effect of sex and ethnicity on human tissue kallikrein amidase activity was observed. CONCLUSIONS: The lack of ethnicity effect supports what was already asserted, namely, that, in Brazil, at an individual level, color, as determined by physical evaluation, is a poor predictor of genomic African ancestry, estimated by molecular markers.