Nguyen Thi Nguyet Thu P, Dang Hong Phong V, Nguyen Thi Ke V, Nguyen Le Trang P.
P Pasteur Institute
in
V National
Institute for Manufacturing vaccines and biological products, 9 Pasteur – Nha
Trang.
Serums against bacterial toxins (Diphtheria and Tetanus)
were recently investigated in
In a report (1907), Calmette had mentioned that serotherapy allowed to reduce mortality due to snake bites. Neutralizing lethality effect of the venom while preventing complications are objectives in manufacturing antiserums.
The higest incidence with the highest mortality in
Every serums obtained individually
from immunized horses exhibited protection against the entire venom lethal
toxicity. The average neutralization potency of crude serums was
117 LD50/mL (/i.v./20g mouse). For
determination of antibidies specific to the cobratoxin, an immuno-enzymatic
method was adjusted, consisting of purified toxin attached on solid phase for
capturing the antibodies and biotinylated toxin as detecting reagent with
comercial streptavidin-peroxidase. The neutralization potency data obtained by in vivo method correlated well with the
titers of specific antibodies determined by in
vitro measurements, demonstrating the role of antibodies specific to
cobratoxin in the neutralization against the lethal effect of the entire venom.
The annual production capacity from six horses used in the present experiment was estimated to 25.106 LD50. After processing, 12,000 doses of 1000 LD50 should be available annually for the needs, able for facing an incidence of 5 – 6/100,000 population in a total population of 70 millions.
AFP is a major fetal blood protein, carrier of estrogen, unsaturated fatty acids, billirubin, etc... It is involved in carcinogenic and immuno-regulatory processes which are under investigation. AFP is a serum marker for the diagnosis of hepatocellular carcinoma and fetal malformations.
From fetal sera, we have isolated AFP in mg amounts. Rabbit antiserum and AFP-Sepharose were then prepared for obtaining immuno-affinity purified Fab and F(ab’). Fab was attached covalently to polystyrene microplates and used for capturing AFP. Fab’-s-biotin was prepared by site directed labeling and used as detecting antibodies (with streptavidin-peroxidase conjugated). The enzyme-immuno-assay (EIA) system obtained can be used with serum samples directly without dilution. The sensitivity observed was 0.2ng/mL.
AFP concentrations of 380 serum samples from normal individuals and 240 maternal serum samples were determined at present, for establishing reference values before further epidemiological studies.
A project, conducted since 1992, has been completed with the production of an antivenenum for treatment of Naja naja kaouthia and Naja naja atra venom poisoning.
Using the horse antisera produced, an enzyme-immuno-assay has been carried out as following: Naja naja kaouthia and Naja naja atra venom were immobilized on CNBr-activated Sepharose 4B. IgG fraction was submitted for specific antibody isolation. Fab and Fab’-s-biotin were then obtained from the immuno-affinity purified antibodies.
Polystyrene microplates were coated covalently by a titrated concentration of Fab. Fab’-s-biotin was used as detecting antibody in association with an streptavidin-peroxidase conjugated.
Calibration curve obtained by using separately Naja naja kaouthia and Naja naja atra venoms manifested a same
slope from 1ng/mL to 1mg/mL
of venom concentrations. Probably the high proportions of neurotoxins and
cardiotoxins and their homology in both of these venom
could explain the results observed. The sensitivity of the system was 0.1ng/mL,
suggesting a high affinity for the antibodies used. This EIA system should
satisfy the need of poisoning evaluation for adequate treatment. Up to now, 36
victims of cobra snake bites were rescued at
TNF-a plays a pivotal function in the regulation of immune responses. Clinical data concerning cytokines may give information on the state of immuno-regulation and pathogenesis.
Sera from 73 new non treated patients were adequately (2) collected and submitted for TNF-a determination, using an enzyme-immuno-assay system in which other cytokines and soluble TNF receptors did not interfere (3).
Data obtained showed that 100% of TT patients (11 individuals) presented normal TNF-a concentrations (1,1 to 3.99pg/mL). The proportion of patients presenting normal plasma TNF-a concentrations (<4pg/mL) decreased in the progression toward the LL pole: BT patients, 12/14 (85.7%); BB patients, 15/18 (83.3%); BL patients, 10/18 (55%); LL patients 6/12 (50%). However, the highest values were observed in the unstable leprosy groups: BT patients (9, 8 pg/mL); BB patients (4.4, 14.5, >32pg/mL); BL patients (4.2, 4.7, 4.8, 10, 12, 16, 23, >32pg/mL) compared to the LL group (4.0, 4.6, 5.3, 7, 7.6, 28). High values of TNF-a probably reflect the activities of cellular mediated immune responses. The data may reflect the immuno-stimularory activities of TNF-a in human. Relatively low values found in TT and LL patients may be due to an immuno-suppressive property of Mycobacterium leprae.