*3.5.1 Enzyme-linked immunosorbent assay-based (ELISA) methods for detection of snake venoms*

The application of ELISAs or enzyme immunoassays (EIAs) in detecting specific venoms as well as the indirect detection of specific venom antibodies (including antivenom) was first reported in 1977 using the double sandwich method carried out in a microliter plate with 96 wells [15]. According to [27], the ELISA was able to detect 1–5 ng of venom/mL in 3 hours and has since then been applied in detecting various venoms in some parts of the world. ELISAs have been widely used and have become handy for studying the kinetics of snake venoms in blood, magnitude of envenomation, and the adequacy or otherwise of antivenom serotherapy. ELISA continues to be the appropriate method for detecting snake venoms, toxins, and venom antibodies in body fluids and is considered to offer more practical value than any other immunoassay [28, 39, 40].

In principle, soluble antigens are linked to the wells contained in the ELISA plate, allowing the individual components to retain their reactivity. In a double sandwich method, a given venom antibody binds to the plate, which is then washed to remove any unbound material followed by adding a test substance, which contains a venomspecific antigen. The complex, thus, formed between the venom and antibody is detected after further washing by using an enzyme-conjugated specific antibody such as alkaline phosphatase or horseradish peroxidase. After a further washing step, an enzyme-specific substrate is added with the resulting hydrolysis or color change measured spectrophotometrically or visually considered proportionate to the quantity of venom or antigen available in the test sample [14]. In respect of sensitivity of ELISAs, significant improvements have been made to enable the detection of venom concentrations at picogram levels. In addition to using avidin-biotin amplification, further improvement in the sensitivity of ELISA can be achieved by an increase in the affinity of antibodies, attainable by increasing the length of immunization and the rate at which booster injections are given. Also, monoclonal antibodies and venom-specific antibodies purified *via* affinity chromatography are regularly used to accomplish species specificity of an ELISA, and the latter appears to be the ideal for detecting venoms [27].

In a study by Liu and colleagues [41], a sandwich ELISA test and lateral flow assay were developed as a way of improving the clinical management of snakebites in Taiwan. These tests were meant to detect both hemorrhagic and neurotoxic venom proteins of four main snake species responsible for over 90% of all clinical

## *Perspective Chapter: Diagnostic and Antivenom Immunotherapeutic Approaches… DOI: http://dx.doi.org/10.5772/intechopen.112147*

envenomation cases. In the study, species-specific antibodies were generated from antivenoms using a two-step affinity purification procedure. The neurotoxic and hemorrhagic species-specific antibodies were biotinylated and subsequently used to develop sandwich and lateral flow assays. The sandwich ELISA assay was developed based on an interaction between the biotinylated antibodies and alkaline phosphataseconjugated streptavidin with 4-methyl umbelliferyl phosphate as substrate. A SpectraMax M5 microplate reader was then used to measure the fluorescence. Findings from the study showed that both diagnostic assays were able to successfully discriminate between hemorrhagic and neurotoxic venoms. For the ELISA, the limits of quantification for hemorrhagic and neurotoxic venoms were determined as 0.78 and 0.39 ng/mL, respectively, whereas the lateral flow assay detected hemorrhagic and neurotoxic venoms at concentrations lower than 50 and 5 ng/mL, respectively within 10 to 15 minutes.

In another study by Shaikh *et al*. [42], a rapid and sensitive assay and sandwich ELISA assay were developed to detect snake envenoming [notably the Indian Cobra (*N. naja*), Russell's viper (*Daboia russelii*)*,* common Krait (*Bungarus caeruleus*), and saw-scaled viper (*Echis carinatus*)] prior to the administration of antivenoms in India. Here, monovalent antisera were prepared by immunizing rabbits with specific venoms in order to obtain venom-specific antibodies. Immunoaffinity chromatography was then used to remove cross-reacting antibodies so as to obtain venom-specific antibodies. Thus, the venom detection ELISA test was developed using two different species of antibodies that provided increased sensitivity and also ensured that venoms of offending snakes were selectively identified. From the results obtained, the sensitivity of the sandwich ELISA was observed to detect venom up to 0.01 ng/mL. It also revealed that the venom detection ELISA test developed was rapid, specific, and sensitive, detecting venoms of culprit snakes at a venom concentration of up to 1 ng/mL. When experimentally envenomed samples were used, the venom detection ELISA test was able to quantitatively detect venom concentration in the range of <1 ng/mL within 20–25 minutes. At between 1.0–0.1 ng/mL, the device demonstrated the lowest venom detection limit with a 100% agreement recorded between the sandwich ELISA and the venom detection ELISA test device.

In another study by Dong *et al.* [40], an avidin-biotin ELISA (AB-ELISA) assay was developed for detection of venoms of four common snake species (*Calloselasma rhodostoma,Trimeresurus popeorum, O. hannah,* and *N. naja*) in the South of Vietnam. In the study, species-specific antivenom antibodies were generated using a three-step affinity purification protocol and antibodies subsequently used to develop an ELISA test kit for venom detection. From the results obtained, the ELISA kit was able to discriminate between the venoms from the four snakes in different sample types tested. The sensitivity of the AB-ELISA reported in the study was high, with a venom detection limit lower than 1.6 ng/mL in most of the samples tested. Notably, the detection limit was as low as 0.2 ng/mL of the serum or sample buffer in the case of *N. naja* venom. Thus, the sensitivity of the AB-ELISA assay was attributed to the selection of high titer serum samples containing high affinity, avidity antibodies, and antibody purification. In addition, the efficacy of the ELISA kit in detecting snakebite envenoming was successfully illustrated in experimentally envenomed rats. The findings also revealed that of the 140 human samples (blood, urine, wound exudates, and blister fluids) tested for venom detection by the kit, venom was detected in all the body fluids.

In a recent study [43], described an inhibition ELISA prototype for detecting cytotoxic three-finger toxins present in the venoms of African spitting cobra species. Such a diagnostic technique capable of detecting specific venom toxins is crucial for the administration of toxin-specific therapy for snakebite envenoming. Different ELISA parameters were optimized employing an indirect ELISA method. The specificity of the assay was assessed by the observed percent inhibition at various sample antigen concentrations (6–0.008 μg/mL) in three homologous (*N. ashei, N. nigricollis, and N. haje*) and two heterologous (*Bitis arietans* and *D. polylepsis*) venom samples. Similarly, to determine the sensitivity of the inhibition ELISA, crude *N. ashei* venom was analyzed from an initial concentration of 27 μg/mL to a final concentration of 0.04 μg/mL, following which the limit of detection (LOD) of the assay was determined. ELISA plates were coated with purified 3FTx antigen overnight, while sample antigen (crude *N. ashei* venom) was constituted in blocking buffer and diluted 3-fold serially. Following the incubation and washing steps, the plate was analyzed and percent inhibition calculated (Eq. (1)) as:

$$\text{Percent inhibition} = \frac{\text{NACOD} - \text{Test sample}}{\text{NACOD}} \times 100\tag{1}$$

The prototype was further tested for its ability to discriminate between venoms containing 3FTxs and those without using crude venoms from two other Naja sp. *(N. nigricollis* and *N. haje*) and two non-Naja sp. (*B. arietans* and *D. polylepsis*). These sample antigens were prepared as previously described, ELISA implemented, plate analyzed, and percent inhibition determined. At all the inhibitor concentrations tested, a percent inhibition of greater than 18% was observed across all homologous venoms. On the other hand, a% inhibition less than 16% was observed among the heterologous samples across all the inhibitor concentrations tested (**Table 1**).

With a mean and standard deviation (of the negative control) of 0.835 and 0.207, respectively, the limit of detection (LOD) of the assay was determined to be approximately 0.01 μg/mL. From the six-point three-fold dilution of the sample antigen, the percent inhibition was observed to be highest (77.70%) at 27.00 μg/mL and lowest (36.56%) at 0.04 μg/mL of the antigen, respectively (**Figure 1**).

It was also observed that at high inhibitor concentrations, the ODs of all three Naja species were low. The ODs, however, increased with decreasing inhibitor concentrations. As low as 0.008 μg/mL inhibitor concentration, the sample antigens induced varying levels of inhibition (18.80, 21.00, and 40.28% for *N. ashei*,

#### **Figure 1.**

*Inhibition ELISA curve showing % inhibition of the coated antigen by the sample-containing antigen at various concentrations of crude* N. ashei *venom (inhibitor). Sample antigen was diluted 3-fold from an initial concentration of 27 μg/mL to a final concentration of 0.04 μg/mL and run in duplicates [43].*


 *Percent inhibition of 3FTxs induced by* N. ashei*,* N. nigricollis*,* N. haje*,* B. arietans*, and* D. polylepsis *venoms.*
