LAB REPORT: ELISA PRACTICAL
Snakebites are clinically challenging because of the diversity of species and varying concentrations which impact the severity of envenoming on the victim. Other clinical problems can arise due to (a) unpredictable dose quality, (b) determination of appropriate dose, (c) local site of the snakebite and (d) route of injection (Warrell, 1996). The time that has elapsed from the snakebite event until presentation at hospital is the most important mortality-determining factor. The health status of the human victim is important for how local effects, systemic coagulopathy, and/or neurotoxicity exhibit (Warrell, 1996).
Intravenous doses are prepared from IgG of a single or multi- venom inoculated into a horse or sheep over several months to produce a monospecific or polyspecific antivenom. A progressive rise in neutralizing antibodies (IgG) does not harm the animal (Lalloo and Theakston, 2003). This antivenom is then suitable for the treatment of systemic pathologies induced by envenoming events.
The effectiveness of antivenom prior to human therapeutic use needs to be evaluated by animal experimentation is prohibitively expensive. Immune assays measuring the IgG reactivity of antivenom to venom proteins offer a potentially cheap alternative to measure antivenom efficacy. The ELISA practical runs a series of end-point-titration ELISA assays to determine the IgG titre of antivenoms (AV1, EchiTABG and AV11, Costa Rica) against venoms from various African vipers and elapids. The objective of the practical is to examine the extent to which an ELISA assay can be reliably used to predict preclinical efficacy of antivenom.
Table 1 contains the abbreviations, the species name, the common name and the country from which the venoms came from for the assays AV1 and AV11.
Table 1. Legend for African species.
Table 2 below contains the results obtained from the EPT ELISA assays to determine the IgG titre of Anti-Venoms 1 (AV1) categorized by the venoms from various African vipers and African elapids. Column 1 in Table 2 contains the control from which the mean average of the background level was computed to be 0.28 nm. Comparing the data between each African Viper species shows that the End Point Titre demonstrate similar IgG titre results - a plateau is reached which then declines as the IgG dilutions increase. This is visually evident in
Table 2. The Optical Density (OD) of the reaction between AV1 and Venoms at 450 nm (nanometres).
Figures 2 and 3. Figures 2 and 3 demonstrate that there is extensive-cross specific immunological reactivity between the Echis venoms and the Echis species-specific IgG antisera from the End Point Titration. ELISA Notice that in Figure 2 each AV concentration dilution for each of the types of African Viper shows a gradual decreasing response. This is because although the venoms differ in intra-generic protein size they must still be members of the same protein families. For example in the highest AV concentrations which were in the dilutions, of 1:1100, 1:1400, and 1:16000, all four of the Viper venoms (Eo, Ec, Epl,and Cc) show an interactivity between 1.4 to 1.5 resulting in the plateua. After that the decline in reactivity increases with the successive IgG dilutions. For example, at the AV concentration dilution of 1:64000 the interactivity decreases to 1.4 or to slightly less than 1.4 and when the AV concentration dilution of 1:1,024,000 is reached the range of interactivity is from Eo, approximately 1.04 (highest) to Cc, approximately 0.85 (lowest). The results compared well to results published by Casewell (et al., 2010) for Eo, Epl, and Ec with the IgG type AV1 (e851). Echis ocellatus and Echis coloratus demonstrate a more immunogenic characteristic in this Assay. In particular the Echis ocellautus shows the least decline with respect to each of the venoms.
Figure 1. Immunological reactivity between the IgG titre of AV1 African Viper venoms determined by EPT ELISA.
Figure 3 depicts the reactivity between the IgG titre of AVI venoms which resulted from the EPT ELISA for the African Elapid specie. Notice that the AVI shows a similar pattern between the African Viper and African Elapid demonstrating a very gradual decrease in interactivity (a similar plateau) at each of the AV concentration dilutions between the Nh, Nnig and Nniv species until a larger amount of decrease occurs for each AV concentration dilution between the species NNiv and Dp and then next between the species Dp to Da. The interactivity of Nnig reaches the highest level at approximately 1.42 and AV concentration dilution factor 1:1,000. The interactivity of Nnig reaches the lowest level at approximately 0.42 and AV concentration dilution of 1:1,024,000. . At the AV concentration dilution of 1:1,024,000 the AV1 of the Nh for the first time in the series demonstrates a higher reactivity (0.6) than Nnig. In this ELISA Assay the Naja Nigricollus shows the most consistent and retarded decline when compared to the other venoms which indicates a more immunogenic characteristic than the others.
Figure 2. Immunological reactivity between the IgG titre of AV1
African Elapid venoms determined by EPT ELISA.
Table 3 below contains the results obtained from the EPT ELISA assays to determine the IgG titre of Anti-Venoms 11 (AV11) categorized by the venoms from the same African vipers and elapids used in AV1. Column 1 in Table 2 contains the control from which the mean average of the background level was computed to be 0.16 nm. Comparing the data between each species shows that the End Point Titre demonstrate similar IgG titre results to each other but very different from those in AV1. Instead of a plateau the decrease in interactivity is great from 1:1000 and 1:400 and then the plateau that can be identified is across the rest of the concentrations and include the OD of the control. This is visually evident in
Table 3. The Optical Density (OD) of the reaction between AVII and venoms at 450
nm (nanometres).
Notice that in Figure 3 each dilution for each of the types of African Viper shows a severe decreasing drop in reactivity between the AV concentration dilutions for 1:1100 where the activity for the five species ranges from 1.7 to 2.3. The drop in reactivity to the AV concentration dilution of 1:1400 is such that the reactivity range for the five species is 0.6 to 1. As to the AV concentration dilution of 1:16,000 the reactivity range for the five species is from approximately 0.4 to 0.48. From the AV concentration dilution of 1:64000 until the control the decrease is very small, in fact the range remains between approximately 0. 2 and 0.4. For the AV concentration dilution of 1:1,000 Ec shows the highest interactivity at about .2, followed by Cc at about 2.1, Eo at about 1.98, Epl at about 1.8 and Ba at about 1.7.
Figure 3. Immunological reactivity between the IgG titre of AV11 African Viper venoms determined by EPT ELISA.
Figure 4 depicts the amount of reactivity for the African Elapids when AV11 was used and the same pattern as seen in Figure 3 with the African Vipers is clearly evident. The decrease in reactivity from the AV concentration dilution of 1:1,000 to 1:1,400 is quite severe. At the AV concentration at dilution 1:1,000 the Da species shows the most reactivity which measures about 0.85 or maybe 0.9., next in terms of reactivity are Nh and Nnig which both show a measure of reactivity of a small increment higher than 0.8, then Dp and Nniv show a similar reactivity of about 0.7. For the next AV concentration dilution of 1:1,400 the drop in reactivity is large with the range for all five species being from about 0.3 to 0.4. For the rest of the diluted concentrations and for the control group the reactivity stays consistently between the 0.2 and 0.3 range for all five species.
Figure 4. Immunological reactivity between the IgG titre of AV11 African Elapids venoms demonstrated by EPT ELISA.
The result of the comparison of the End Point Titre ELISA Assay demonstrates that AV1 is more effective than AV11. Table 4 lists the data available from the published findings of Abubakar (et al., 2010) on antivenom for African viper species. The results of this ELISA assay compare favourably with Abubakar (et al., 2010) for the African viper species. In the Abubakar (et al., 2010) study the most high AV concentration to the least showed gradual decreases in the reactivity which corresponded to this laboratory results.
Table 4. Compared assay results to the published findings of Abubakar et al.
Table 5 Shows an efficacy comparison based upon the laboratory results. One (+) represents the dilutions from strongest AV concentration to least. So for Ec with the AV1 assay showed an efficacy based on reactivity for the four highest dilutions (1:1000, 1:4000, 1:16,000 and 64,000). Compared to the AV11 assay results which demonstrated an efficacy based on reactivity only in at the highest AV concentration (1:1000).
Table 6 demonstrates the reliance on cost on the factors of number of vials needed for efficacy and number of vials in demand. The cost of the AV must be at a level that can be paid by the people who most need it. That is why is important to determine the reliability of End Point Titre ELISA Assays. The amount of concentration of AV in the dilution has a big effect on cost which means the cost is prohibitive for the poorest people in Africa who need it the most. Because The ELISA assay is reliable, simple to use and inexpensive antivenom dosage can be prescribed more efficiently so that the costs can be lessened for the antivenom needed.
Conclusions
The main objective of this laboratory was to determine if the results demonstrate that an ELISA assay can be used to reliably predict the clinical efficacy of antivenom. For the assay of AV1and AV11for the African vipers it was found that the antivenoms were appropriate for the Echis species series from the results and with comparison to two other studies by Abubakar (et al., 2010) and Casewwell (et al., 2010). This highly suggests that they would be effective in the Echis species of Africa. For the African Epalids amount of dose is very sensitive to dilution. Only the first dilution of 1:1000 with the highest concentration of the AV shows that efficacy is good as determined by the reactivity.
The AVI Assay was demonstrated to be more effective more effective than AV11 Assay. It does require more dilutions to reach the best End Point Titre (EPT). Interestingly the Echis pyramidum leakeyi and Echis coloratus are very lethal yet the efficacy of the IgG titre was very similar as that for Echis ocellatus. Knowing that the EchiTAbG can neutralize the lethal venoms is very important.
References
Abubakar, S.B., Abubakar, I.S., Habib, A.G., Nasadi, Durfa, N., Ysuf, P.O., Larnyang, s., Garnvwa, J., Sokomba, E., Salako, L. Laing, G.D., Theakston, R.D.G., Juszczak, E., Alder, N., Warrell, D.A. for the Nigeria-UK EchiTab Study Group. 2010. Pre-clinical and preliminary dose-finding and safety studies to identify candidate antivenoms for treatment of envenoming by saw-scaled or carpet vipers (Echis ocellatus) in northern Nigeria. Toxicon. [e-journal] 55 Available through: Elsevier.com database [Accessed 18 April 2012].
Casewell, N.R., Cook, Darren A.N., Wagstaff, S.C., Nasidi, A., Durfa, N., Wüster, W., Harrison, R.A. 2010. Pre-clinical assays predict Pan-African Echis Viper efficacy for a species-specific antivenom. PloS Negi Tro Dis 4 (10): e851. doi:10.1371/journal.pntd.0000851.
Lalloo, D.G. and Theakston, R.D.G., Warrell, D.A. 2003. J. Toxicol. Clin. Toxicol. 41, pp. 541-557.
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