A nitrocellulose membrane strip dotted with a specific 50 kDa outer membrane protein of Salmonella typhi was applied for the serodiagnosis of typhoid fever. Using horseradish peroxidase conjugated IgM and IgG antibodies with 4-chloronaphthol as substrate, antibodies in typhoid patients were clearly visualised as bluish purple dots while sera from patients with non-typhoid fevers gave negative results. The detection of specific IgM and IgG antibodies in typhoid patients suggest either recent or current infection. Combined with the high specificity, reliability and rapidity of the test, the dot EIA technique provides a simple and useful method for the serodiagnosis of typhoid using a single serum specimen.
Existing protocols for antivenom treatment of snake envenomations are generally not well optimized due partly to inadequate knowledge of the toxicokinetics of venoms. The toxicokinetics of Naja sputatrix (Javan spitting cobra) venom was investigated following intravenous and intramuscular injections of the venom into rabbits using double-sandwich ELISA. The toxicokinetics of the venom injected intravenously fitted a two-compartment model. When the venom was injected intramuscularly, the serum concentration-time profile exhibited a more complex absorption and/or distribution pattern. Nevertheless, the terminal half-life, volume of distribution by area and systemic clearance of the venom injected intramuscularly were not significantly different (p > 0.05) from that of the venom injected intravenously. The systemic bioavailability of the venom antigens injected by intramuscular route was 41.7%. Our toxicokinetic finding is consistent with other reports, and may indicate that some cobra venom toxins have high affinity for the tissues at the site of injection. Our results suggest that the intramuscular route of administration doesn't significantly alter the toxicokinetics of N. sputatrix venom although it significantly reduces the systemic bioavailability of the venom.
Envenomation by hump-nosed pit viper (Hypnale hypnale, Hh) in Sri Lanka has caused significant morbidity and mortality, attributed to 35% of total venomous snakebites. In Southwestern India (Kerala), H. hypnale was increasingly identified as a dangerous and common source of envenomation, second to the Russell's viper but ahead of the cobra bites. Unfortunately, there is still no specific antivenom to date. This study aims to investigate the immunological properties of the venom and to assess the feasibility of specific Hh antivenom production as well as the development of a diagnostic assay. Hh venom elicited satisfactory titers of anti-Hh IgG in rabbits after 3rd immunization. The anti-Hh IgG, isolated with caprylic acid precipitation method, was effective in neutralizing the venom lethality (potency=48 LD(50) per ml IgG) as well as its procoagulant, hemorrhagic and necrotic effects, indicating the possibility to produce the specific antivenom using the common immunization regime. Cross-reactivity studies using indirect ELISA showed that anti-Hh IgG cross-reacted extensively with several Asiatic crotalid venoms, particularly that of Calloselasma rhodostoma (73.6%), presumably due to the presence of venom antigens common to both snakes. Levels of immunological cross-reactivity were vastly reduced with double-sandwich ELISA. Further work demonstrated that the assay was able to distinguish and quantify venoms of H. hypnale, Daboia russelii and Echis carinatus sinhaleyus (three common local viperid) used to spike human sera at various concentrations. The assay hence may be a useful investigating tool for diagnosing biting species and studying the time course profile of venom concentrations in blood.
In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.