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Toxins 2014, 6(5), 1667-1695; doi:10.3390/toxins6051667
Published: 23 May 2014
Abstract: In many cases of envenoming following snake bite, the snake responsible for the accident remains unidentified; this frequently results in difficulty deciding which antivenom to administer to the systemically-envenomed victim, especially when only monospecific antivenoms are available. Normally the specific diagnosis of snake bite can be conveniently made using clinical and laboratory methods. Where clinical diagnosis depends upon the recognition of specific signs of envenoming in the patient, laboratory diagnosis is based on the changes which occur in envenomed victims including the detection of abnormalities in blood parameters, presence/absence of myoglobinuria, changes in certain enzyme levels, presence/absence of neurotoxic signs and the detection in the blood of specific venom antigens using immunologically-based techniques, such as enzyme immunoassay. It is the latter which is the main subject of this review, together with the application of techniques currently used to objectively assess the effectiveness of new and existing antivenoms, to assess first aid measures, to investigate the possible use of such methods in epidemiological studies, and to detect individual venom components. With this in mind, we have discussed in some detail how such techniques were developed and how they have helped in the treatment of envenoming particularly and in venom research in general.
“Slash, suck out the venom and apply a tourniquet”—It was partly to challenge this dangerous historical advice that many scientists throughout the world, interested in the treatment of snakebite and other venomous bites and stings, united in a common aim of improving diagnosis and treatment. In snake bite, it is often difficult for clinicians treating patients to determine the species responsible for envenoming, thus making treatment with the correct antivenom more difficult, especially in regions where only monospecific antivenoms are available. This was one of the major reasons which inspired the development of sensitive assay techniques using immunodiagnostic and other laboratory-based methods. Early in investigative studies, it was shown that immunodiagnosis using enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) was useful for the identification of the species responsible for envenoming and also for the detection of specific venom antibody ; this followed the detection of venom using radioimmunoassay (RIA) developed by Sutherland’s group in Australia [2,3]. Later, this group also used EIA, which proved to be much cheaper than RIA and obviously did not require the use of radioisotopes . The method enabled the recognition of accurate diagnostic patterns of envenoming by different, sometimes closely related, snake species. Initially, however, a result could only be obtained within a matter of hours rendering an urgent requirement for a more rapid test which would need to provide a reliable diagnostic result within a few minutes of taking a blood sample from the envenomed victim. Only then could the assay system become appropriate for actual early treatment of the patient with antivenom. Such a rapid test has been developed in Australia but, unfortunately, this is considered too expensive and has problems relating to sensitivity . The value of EIA in the study of new and existing antivenoms is that it provides an important objective assessment of antivenom efficacy; as studies mentioned in this review demonstrate, it has proved a useful tool in supplementing clinical observations following antivenom administration after snake bite. Recent advances in the use and development of EIA have added enormously to its use in the field of venom research . The value of EIA in evaluating currently available and novel first aid measures may also prove invaluable both now and in the future, as well as its application in other aspects of venom research.
The diagnosis of snake bite or determination of which snake is responsible for envenoming of a victim can be conveniently divided into clinical diagnosis and laboratory diagnosis. Clinical diagnosis depends upon recognising specific signs of envenoming in the patient. This includes local signs such as swelling (Figure 1a,b), blistering (Figure 2d), and local necrosis (Figure 1c,d). More importantly for accurate diagnosis, systemic signs, such as haemorrhage (Figure 2b,c,d), incoagulable blood, and hypovolaemic shock (Figure 2d), are common mainly in viper bite, whereas neurotoxic signs (Figure 3a) occur primarily in elapid bite, and rhabdomyolyis (muscle damage) in sea snake bite (Figure 3b). Indeed, the late Alistair Reid, founder of the Venom Research Unit, Liverpool School of Tropical Medicine, UK, made many of the original observations pertaining to this, although it should be noted that there are exceptions to this rule. For example, some Australian elapid venoms can cause haemorrhage and incoagulable blood in addition to neurotoxicity and the venoms of some vipers, such as the tropical rattlesnake, Crotalus durissus terrificus, and the berg adder, Bitis atropos, can cause neurotoxic signs in systemically envenomed victims. Local effects, such as necrosis, may occur, especially following viper bites, but this tends to be a slightly later manifestation and is not necessarily diagnostic (Figure 1c,d). Likewise, some cobras are capable of spitting venom; if this enters the eyes it causes a severe local painful conjunctivitis with accompanied swelling (Figure 1b). It should also be noted that the presence or absence of fang marks are not diagnostic although the distance between the fang marks does provide an indication as to the size of the biting snake; however, the detection of fang marks does not necessarily indicate that venom has actually been introduced (Figure 2a). Indeed, in about 50% of bites no venom is injected. More detailed information on clinical diagnosis of snake bite is provided in a recent review .
Laboratory diagnosis of snake bite is based on the changes which occur in envenomed victims. These include the detection of abnormal changes in blood parameters (e.g., incoagulable blood (Figure 4a) as examined using the simple bedside 20 min whole blood clotting test, WBCT20 (Figure 4b) [8,9,10], dramatic fall in the platelet count, changes in red and white blood cell counts), presence/absence of myoglobinuria, changes in certain enzyme levels (such as creatine phosphokinase) and the detection in the blood of the victims of specific venom antigens (biodetection methods using immunologically-based techniques). It is the latter point which is one of the main subjects of this review together with the application of other immunological techniques for use in venom research such as the objective assessment of the effectiveness of new and existing antivenoms, the assessment of first aid measures, the possible use of such methods in epidemiological studies and the immunodetection of individual venom components.
3. Biodetection Methods Considered for Use in Venom Research
Over the years a number of immunologically-based assay systems have been applied to the detection of specific venom and also to the detection of specific venom antibodies. These include immunodiffusion, immunofluorescence, haemagglutination, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA/EIA), and optical immunoassay, as well as the possible future applications of PCR and antibody microarrays in this respect.
An agar-stabilised precipitation test was first used  to detect king cobra (Ophiophagus hannah) venom in excised bite site tissue and, later, gel immunodiffusion was used to detect the venoms from four common Nigerian snakes in wound aspirates, blister fluid, sera, and urine samples from envenomed patients . Although generally successful in detecting specific venom, the system was not sensitive enough to detect venom in sera and was, therefore, of limited use.
Immunofluorescence has been used to detect specific venom in tissue samples but not in body fluids . Passive haemagglutination of sheep red cells sensitised to venom by the bis-diazo benzidine coupling procedure was used to demonstrate both venom and antivenom at high dilutions in a test system but problems included instability of the coupling agent and imprecise end-point determination . More recently a single-bead-based immunofluorescence assay has been developed for the detection of venom with a detection sensitivity of 5–10 ng/mL within a 3 h assay time .
Immunoelectrophoresis was also used but was found to be unlikely to be of practical use in the routine assay of venom and venom antibodies owing to the high levels of common precipitating bands between venoms and antibodies of closely related species [16,17].
Radioimmunoassay [2,18,19] was used to detect venom in the serum of envenomed animals and patients but, although highly sensitive, the method proved to be impractical in patients as well as being very expensive, requiring specialised and elaborate reading equipment for measuring isotope levels in addition to the problems related to the short half-life of 125I. It was stressed that its use was primarily as a research tool .
Theakston and colleagues  first reported the use of enzyme-linked immunosorbent assay (ELISA) or EIA (enzyme immunoassay), using the double sandwich technique performed in 96 well Microtitre plates  for the detection of specific venom and the indirect method for detection of specific antibody (including antivenom) in the blood of envenomed victims. The principle of the technique is based on the linkage of soluble antigens to an insoluble solid phase (the wells of the plate) in such a way that the reactivity of the immunological components is retained. The double sandwich technique consists of binding specific venom antibody to the solid phase followed by a washing step to remove unbound material and subsequent addition of test material containing specific venom antigen. The detection of the venom-antibody complex thus formed is carried out, after further washing, by using specific antibody conjugated to an enzyme (such as horseradish peroxidase or alkaline phosphatase) (Figure 5a). Following a further washing stage, substrate specific for the enzyme is added, the amount of hydrolysis (colour change measured either visually or spectrophotometrically) being proportional to the amount of antigen (venom) present in the test sample. The test can also be used for detection and quantification of venom antigen in other body fluids such as urine and blister and wound aspirates. The indirect method for venom antibody (or antivenom) detection consists of binding antigen (immunologically active venom components) to the solid phase followed by incubation with the test sample. If the sample contains antibody against the specific antigen, the combination can be detected using anti-species immunoglobulin (e.g., horse, sheep, human, etc.) conjugated to the enzyme marker (Figure 5b). In this case the amount of hydrolysis is proportional to the amount of antibody present. In order to estimate the amounts of venom or venom antibody in the test sample the results (colour intensity) are compared with a standard curve set up on the same plate as the test samples. The basic principle of the method is shown in Figure 5. The sensitivity of the venom assay is in the region of 1 ng/mL serum, but using a modification of the EIA using a biotin/avidin combination the sensitivity can be even further increased . For ease of sample collection in the field, whole blood samples can be placed on filter paper and dried; the blood is then eluted off with phosphate buffered saline in the laboratory and the EIA carried out on the eluate [22,23]. Pre-coated plates can be stored for later use and incubation times can be further reduced, permitting a total assay time of less than 3 hours although this is still not rapid enough for the clinician to decide on whether or not to treat the patient with antivenom.
Ho and colleagues  modified EIA by using a specific blocking system thus decreasing the problem of non-specific false positive reactions. Later, modifications of the EIA were developed  which were capable of detecting venom, antivenom, and venom-antivenom complexes; these assays investigated the problems associated with poor sensitivity and the occurrence of false positive results arising from high background absorbance .
More recently a specific and sensitive optical immunoassay (OIA) for venom detection was developed; this was similar to EIA but based on the principle of the detection of physical changes in the thickness of a molecular film resulting from specific binding events on an optical silicon chip . The reflection of white light through the thin film results in destructive interference of the light from gold to purple-blue depending on the thickness of the thin film formed or the amount of venom in the test sample. A prototype test kit for the simultaneous identification of the snake species causing envenoming and the semi-quantitative detection of venoms from four medically-important snakes from Vietnam was developed and was found to be capable of detecting venom in blood, plasma, urine, wound and blister aspirates, and tissue homogenates. The sensitivity of the test was claimed to be double that of EIA and the time taken to perform the assay was 33 min.
Suntrarachun and colleagues  were the first group to investigate the use of the polymerase chain reaction (PCR) to distinguish the venom of the Thai cobra (Naja kaouthia) from the venoms of other Thai species using an experimental mouse model. In this early study, the sequences of nucleotide primers for the cobrotoxin-encoding gene from the Chinese cobra (Naja atra) were chosen because, at that time, the sequences of N.kaouthia were still unknown. In 2005, mitochondrial DNA (mtDNA) sequences from dried snake venom were used  and a DNA barcoding system for the precise identification of venoms was also developed. The group proposed the use of mtDNA for PCR to identify venoms which could overcome some problems encountered with methods such as EIA, although a sizeable venom sample would be required to extract a sufficient quantity of mtDNA; also one would need to decide exactly what to PCR . It may not be a practical system at present because of the very small amounts of venom (nanogram quantities) present in the blood of snake bite victims.
The use of antibody microarrays has also been proposed for detecting specific venoms but, to our knowledge, these have not yet been investigated in this respect. To be able to succeed, two key elements are necessary, namely a unique specific antigen in the venom and a unique antibody (monoclonal antibody) representing the unique protein in the venom of interest. From a proteomics point of view, it is known that there are unique peptide spectra that represent a sequence found only in a certain protein, which could then be only in a specific venom. The key is to select the correct protein and then the right peptide and hope that a monoclonal antibody can be made against it and that it would then bind an epitope in a native protein. This would be possible in principle but has not yet been achieved as far as we are aware .
In many cases of envenoming the snake responsible for the accident is not identified, a fact pointed out by Alistair Reid during the course of his research in Malaysia and West Africa, and this was one of his many reasons for encouraging the development of immunodiagnosis in Liverpool. Enzyme immunoassay was developed by his group at the Liverpool School of Tropical Medicine  and has proved to be of major use in the biodetection of venom and venom antibodies and in venom research generally. The main application of the EIA is in retrospective identification of the biting species. Although the assay can be shortened to produce a result within three hours, this is still not normally rapid enough to enable the clinician to act on its results. However, together with clinical observations, it enables patterns of envenoming to be established within and between the venomous species present in defined geographical areas.
A rapid, sensitive, simple and affordable test is still required to enable the clinician to treat the patient with the correct monospecific (or polyspecific) antivenom at the bedside as soon as possible after admission to hospital. Such a kit has been developed in Australia but this is far too expensive for use in most developing countries where snake bite is a major problem of social, medical and economic importance . The kit also has problems relating to decreased sensitivity.
Other potentially useful and tested applications of enzyme immunoassay are described above. These include the ability to examine the pharmacokinetics of envenoming and therapy enabling the objective assessment of both new and existing antivenoms by looking at the rate of elimination of venom from the circulation in systemically envenomed patients.
The method is appropriate for studying the efficacy, or lack of efficacy, of currently available and new first aid approaches. A great deal of damage occurs following the use of many such methods and, importantly, a visit to a traditional healer who applies such techniques drastically delays the arrival of the patient at hospital where approved conventional treatment is available. On the other hand, there should also be awareness that perhaps a traditional pre-hospital treatment method may eventually be discovered which is appropriate and which is assessable using an immunoassay, although this seems unlikely at the present time.
The use of enzyme immunoassay in epidemiological studies of snake bite is potentially extremely useful although the requirement for a powerful control (non-bitten) group of the same socio-economic background is stressed. There is a real need to determine the problem associated with snake bite and other bites and stings throughout the world. Funding bodies require reliable epidemiological information before they will consider supporting a project and in the field of venomous bites and stings this is sorely lacking.
Other methods have been used and suggested to attempt to develop improved methods for studying the pharmacokinetics of envenoming and therapy in envenomed humans; these have been referred to earlier in this review. To date EIA has proved to be the method of preference for the reasons given previously (Section 2).
It is now 50 years since Alistair Reid established the unit in Liverpool and in his honour it was renamed the Alistair Reid Venom Research Unit after his death in 1983. His major contributions to advances in the clinical treatment of envenoming by medically-important snakes and other animals , his astute observations on the mode of actions of a wide range of venoms including his role in the development of Arvin from the venom of the Malayan pit viper (Calloselasma rhodostoma), continues within the unit named after him.
We would like to thank our many collaborators throughout the world who have been part of the studies mentioned here. We acknowledge with gratitude the guidance and expertise of the late Hugh Alistair Reid in providing the inspiration for many of the studies reported in this review. Indeed, it was written as a memorial to the man and to the major contributions he made over many years to the field of venom research. We are indebted to David Alan Warrell, Nuffield Department of Clinical Medicine, University of Oxford without whose help, collaboration and advice it would have been impossible to carry out many of the studies described here. Thanks also to Jay William Fox, University of Virginia School of Medicine, who commented on the possible use of alternative methods for immunodiagnosis such as antibody microarrays and PCR. We also acknowledge the encouragement of Robert A. Harrison head of the Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine and the invaluable assistance Paul Douglas Rowley who, with others, provided the venom from taxonomically identified species for use in these and other studies. We are also grateful to Aura Shisuko Kamiguti for helpful comments and suggestions on the manuscript and for invaluable help with the figures.
David Theakston headed up the research on immunodiagnosis and wrote the paper. Gavin Laing was responsible for developing and carrying out many of the immunoassays described in the review and assisted with revisions of the paper.
Conflict of Interest
The authors declare no conflict of interest.
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