WO2021033196A1 - Methods, compositions and kits for snake venom detection - Google Patents

Abstract

The invention discloses venom detection kits, compositions, methods and devices. The kits may be based on any known technique in the art, such as enzyme-linked immunosorbent assay (ELISA) or lateral flow assay (LFA) or a micro capillary method, using combinations of antibodies to detect different venom proteins from various venomous snakes. The kit is suitable for detecting venom proteins present in clinical samples from snakebite victims as well as substances expected to be (or contain) venoms. The samples may include any biological matter including blood, urine, plasma, saliva, secretions and swabs from victims or lyophilised powders, crystals or liquids thought to contain or suspected to possess venom. The claimed kit may be used to corroborate the envenomation caused by at least common krait, saw-scaled viper, Indian cobra, and Russell's viper venoms and assist in management of snakebite victims in order to guide the treatment strategy.

Description

METHODS. COMPOSITIONS AND KITS FOR SNAKE VENOM DETECTION

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority to the Indian Provisional Patent Application No. 201941017253 entitled “METHODS, COMPOSITIONS AND KITS FOR VENOM DETECTION” dated August 16, 2019.

FIELD OF THE INVENTION

[0002] The disclosure relates generally to venom detection and in particular to method, composition, devices and kits for detection of venoms and offending species, diagnosis and treatment of envenomation and associated pathologies.

BACKGROUND OF THE INVENTION

[0003] Snakebite is a major neglected tropical disease that affects a huge number of people who live predominantly in the remote, rural and frequently agricultural areas of developing countries such as India. The two largest families of venomous snakes are the vipers (typically producing haemo- and cytotoxic venoms) and elapids (typically producing neurotoxic venoms), and differentiating between the bites of these two snake families would be of great benefit to clinicians and treatment regimens the world over. Approximate global death as a result of snakebites are reported to be from 80,000-150,000 per year. However, in reality, this number may be much higher when we consider the instances of snakebites which go largely unreported such as snakebite victims without any access to hospitals or medical professionals, those seeking treatment from traditional healers, and those who die on the way to hospitals. Snakebites mainly affect people in the rural tropics due to the increased number of snakes in these warmer climes and the amount of time spent conducting agricultural activities without appropriate protective clothing. In the cases where people do have access to hospitals or a treatment facility, there are limited ways of ascertaining what effective treatment will comprise of. As well as snakebite, scorpion and jellyfish stings and spider and centipede bites (as well as venomous bites from other vertebrate/invertebrate species) provide an additional risk to human health which is frequently complex to rapidly and correctly diagnose. [0004] One such factor is determining whether envenomation has truly occurred. Frequently bites/stings may happen in the dark or in thick grass/bushes/crop stacks/burrows. If a snake or another bite from a venomous species did take place, another factor is whether it actually injected any venom. Because non-venomous (bites from non- venomous species) and dry bites (where a venomous species injected no venom) are both common. Snakebite victims rarely remember the details of the snake’s appearance well enough to describe the exact species of offending snake and fang marks may or may not be present after a bite, and are frequently of limited use in correctly diagnosing the offending species. This is also difficult if the bite occurred in the dark; if no fang markings were present or if fang markings were masked by the developing wound. Moreover, it is also very difficult to ascertain the quantity of venom injected in the victims.

[0005] Currently, there is a diagnostic kit which is commercially available for certain venomous snakes in Australia but of no practical use to the rest of the world. The vast majority of snakebites happen in South and South East Asia, sub-Saharan Africa and South America. However, in these regions, snakebites are currently being treated only based on the clinical symptoms, sometimes alongside the rudimentary 20-minute whole blood clotting test, which poses several challenges for clinicians treating snakebites, particularly in rural areas. Moreover, some species such as Kraits ( Bungarus spp.) are nocturnal (only active at night) and their fang marks are invisible, their bites are painless and victims can quickly enter a state of paralysis. Therefore, it is hard to ascertain if it was a snakebite in these cases and difficult to differentiate from sleep paralysis. Moreover, in some cases, based on the amount of venom injected, the symptoms may develop after several hours following the bite, and this will delay essential treatment.

[0006] Hence, easy-to-use, state-of-the-art, simple and effective/reliable diagnostic devices and/or methods are urgently needed to allow envenomation to be corroborated and effectively treated in accordance with the offending species, and not solely based on assumptions or frequently ambiguous, clinical symptoms. In addition, only polyvalent antivenom (produced against a number of regionally specific venomous snakes) is usually administered to all the victims presenting symptoms of snakebite envenomation. For example, polyvalent antivenom against the ‘Big Four’ snakes is typically administered to all snakebite victims in India. Antivenom is commonly administered before the symptoms develop, including in cases of dry and non-venomous bites where antivenom administration is not only wasteful but frequently dangerous to the patient due to their ability to develop anaphylactic reactions. Correctly diagnosing snakebite envenomation (SBE) would not only reduce wasteful antivenom administration but also pave the way for the development of monovalent (against the venom of a specific species) antivenom, which is more effective if administered correctly and is associated with lesser adverse reactions (such as allergic reactions). The future of snakebite therapy promises more taxon/toxin-specific approaches, but these will only be of use if the identity of the offending snake is unambiguous. Hence, there is an urgent need for the development of a diagnostic kit and/or method to easily and accurately differentiate bites from Viperidae (mostly haemotoxic venoms) and Elapidae (mostly neurotoxic venoms) as well as diagnosing spider bites and scorpion and jellyfish stings in addition to envenomation from other vertebrate/invertebrate venomous species.

DESCRIPTION OF RELATED ART

[0007] The PCT publication W02017109660A1 describes a very basic venom/no venom device for administering antivenom. However, this invention does not give any indication of which species/genus/family of snake was involved in the envenomation and any specific details about the method of detection. Therefore, it does not offer any improvement to current therapy for example to the development of monovalent antivenom. Rather, such a device will prevent unnecessary use of polyvalent antivenom in dry or non-venomous bites. The Indian Patent Application No. 201841028197 describes a LFA device for snake venom detection. However, the claimed device and antibodies are limited in their application. The Chinese publication CN201331528Y describes a colloidal gold containing test strip for identifying specific snake venoms in China. The Chinese publication CN204925132U describes a lateral flow assay (LFA)-based line detection of snake venom using gold nanoparticle labelled antibodies. The publication, Pawade et al. 2016 (Toxicon, 119, pp.299-306) describes one such method for discriminating Indian Cobra and Russell’s viper using LFA. The publication, Liu et al. 2018 (PLoS Negl. Trop. Dis. 12(12): e0007014) describes the development of neurotoxic species-specific antibodies (NSS-Abs) and haemorrhagic species-specific antibodies (HSS-Abs) from antivenoms of top 4 snakes in Taiwan and use thereof in a sandwich or lateral flow assay to differentiate between the two groups. The publication, Dong et al., 2003 (J. Immunol. Methods 260: 125-136) describes a method of preparation of species-specific antivenom for common snakes in South Vietnam. The publication, Liu et al, 2018 (Journal of Proteomics 187, 59-68) describes the proteomic characterization of Taiwanese snakes to identify relative abundance of major components and species-specific venom proteins. Overall, it is apparent that there are no commercial kits/methods/devices currently available to corroborate the envenomation or quantify the venoms of Indian ‘Big Four’ snakes [Russell’s viper ( Daboia russelii), Indian cobra (Naja naja), Common krait (Bungarus caeruleus) and Saw-scaled viper (Echis carinatus )] using clinical samples obtained from snakebite victims.

SUMMARY OF THE INVENTION

[0008] The present invention in its various embodiments provides methods, compositions, devices and kits for detecting the type of snake/taxon and/or concentration of snake venom in various embodiments. Included herein is an immunoassay method and a device using combinations of antibodies to detect different venom proteins/polypeptides from various venomous snakes.

[0009] In various embodiments, an immunoassay method of determining the type of snake/taxon and concentration of snake venom polypeptides present in a biological sample is disclosed. In one embodiment, the method comprises loading a predetermined amount (but this can be variable based on the nature of sample or bite) of a biological sample to at least one sample well of an assay device. The biological sample contacts with at least one capture antigen binding molecule followed by at least one detector antigen binding molecule and thereby captures snake venom polypeptide(s) present in the biological sample. The capture antigen binding molecules help to bind to a unique region of a snake venom polypeptide specific for an individual snake species or taxon. The detector antigen binding molecules are configured to bind to at least Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra (Naja naja), and Russell’s viper ( Daboia russelii) venoms or polypeptides. The detector antigen binding molecules are characterized in that they bind to a polypeptide of SEQ ID NO. 1, 2, 3, 4, 5, 6, and 7. The binding intensity of a label conjugated to the detector antigen binding molecule is detected using an optical reader.

[0010] In another embodiment of said method, the biological sample is selected from the group consisting of blood, plasma, serum, saliva, sweat, urine, secretions, swab and biopsy sample obtained from a victim (i.e. patient or subject) suspected to have a snake bite. Some of these samples are expected to have the venom proteins. The biological sample can also be spiked with a snake venom sample. The detection range for the snake venom polypeptide present in the sample is as low as 160 pg/mL and up to 1 mg/mL in some embodiments.

[0011] In yet another embodiment of said method, the capture antigen binding molecules are used to detect Russell’s viper ( Daboia russelii) venom or its polypeptides. The capture antigen binding molecules bind to the polypeptides of SEQ ID NO. 3 and 4. [0012] In yet another embodiment of said method, the capture antigen binding molecules are used to detect Common krait ( Bungarus caeruleus) venom or its polypeptide. The capture antigen binding molecules bind to the polypeptides of SEQ ID NO. 5 and 6.

[0013] In yet another embodiment of said method, the capture antigen binding molecules are used to detect Indian cobra ( Naja naja) venom or its polypeptide. The capture antigen binding molecules bind to the polypeptide of SEQ ID NO. 7.

[0014] In yet another embodiment of said method, the assay device is used for simultaneous detection of a plurality (multiple) of biological samples from same or different origin and from one or more patients. The biological sample contacts with at least one capture antigen binding molecule followed by at least one detector antigen binding molecule. The biological sample in the first well contact with capture antigen binding molecules attached to the first well, which then detect Common krait ( Bungarus caeruleus) venom or its polypeptide. The biological sample in the second well contact with capture antigen binding molecules attached to the second well, which then detects Saw-scaled viper ( Echis carinatus) venom or its polypeptide. The biological sample in the third well contact with capture antigen binding molecules attached to the third well, which then detect Indian cobra ( Naja naja) venom or its polypeptide. The biological sample in the fourth well contact with capture antigen binding molecules attached to the fourth well, which then detect Russell’s viper ( Daboia russelii) venom or its polypeptide. The biological sample in the first, second, third and fourth well contact with detector antigen binding molecules which then detect Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra ( Naja naja), and Russell’s viper ( Daboia russelii) venom or its polypeptides.

[0015] In one embodiment, the label used in assay device is selected from a group consisting of gold nanoparticles, enzyme, fluorophore, and chemiluminescent markers. [0016] In another aspect, the present invention provides a lateral flow immunoassay device used to detect the type and concentration of a snake venom polypeptide present in a biological sample. The device comprising of one or more lateral flow test strips in a housing, and an optical reader unit for measuring the intensity of indicator.

[0017] In another aspect, the said test strip comprising of one or more sample loading pads for loading one or more biological samples in one end; a conjugate pad with at least one detector antigen binding molecules, a nitrocellulose membrane and an absorbent pad. [0018] In another aspect, the said nitrocellulose membrane comprises indicator lines. The individual indicator lines are immobilized with at least one capture antigen binding molecules. The capture antigen binding molecules binds to unique region of the snake venom polypeptide specific for an individual snake species or taxon. The detector antigen binding molecules are attached with gold nanoparticles.

[0019] In another aspect, the said detector antigen binding molecules binds to at least Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra (Naja naja), and Russell’s viper ( Daboia russelii) venoms or polypeptides. The detector antigen binding molecules bind to the polypeptides of SEQ ID NO. 1, 2, 3, 4, 5, 6, and 7. [0020] In one embodiment, the type of snake venom polypeptide is determined from one or more visible lines in the assay device. The detector antigen binding molecules are present at a concentration of 0.05 mg/mL (i.e. 0.04 pg per strip) to 1 mg/mL (i.e. 1 pg per strip).

[0021] This and other aspects are disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The invention has other advantages and features which will be more readily apparent from the following detailed description of the invention and the appended claims, when taken in conjunction with the accompanying drawings, in which:

[0023] FIG. 1 shows a two-site ELISA-based snake venom detection kit.

[0024] FIG. 2 shows a two-site ELISA-based snake venom detection kit for the determination of offending species.

[0025] FIG. 3A-3D shows LFA-based venom detection kit embodiments.

[0026] FIG. 4A-4B shows LFA-based venom detection kits to detect Indian big four snake venoms either on the same (A) or parallel strip(s) (B).

[0027] FIG. 5 shows a digital venom detection kit to quantify the amount of venom present in clinical samples.

[0028] FIG. 6A-6B illustrate one-site ELISA for antibodies (IgG) raised against URP-A and URP-B using a range of viper and elapid venoms.

[0029] FIG. 7A-7B illustrate one-site ELISA for antibodies (IgY) raised against URP-A and URP-B using a range of viper and elapid venoms.

[0030] FIG. 8A-8B illustrate one-site ELISA to determine the affinity of antibodies (IgG and IgY) raised against URP-A and URP-B using C. atrox venom. [0031] FIG. 9A-9B illustrate one-site ELISA for antibodies (IgG) raised against purified toxins (A: CA1; B: CA2) using a range of viper and elapid venoms.

[0032] FIG. 10 illustrates one-site ELISA to determine the affinity of antibodies (IgG) raised against purified toxins using C. atrox venom.

[0033] FIG. 1 lA-1 IB illustrate two-site ELISA for the detection of viper venoms using toxin (CA1 and CA2)-specific antibodies (IgG).

[0034] FIG. 12A-12B illustrate one-site ELISA for antibodies (IgG) raised against 3FTX-specific URP-C and URP-D using a range of elapid and viper venoms.

[0035] FIG. 13 illustrates two-site ELISA for the detection of krait ( Bungarus species) venoms using 3FTX (URP-C and URP-D)-specific antibodies.

[0036] FIG. 14 shows two-site ELISA for the detection of cobra venoms using 3FTX (HH)-specific antibodies (IgG).

[0037] FIG. 15 illustrates one-site ELISA to determine the affinity of antibody (IgG) raised against a purified 3FTX toxin using HH venom. [0038] FIG. 16 illustrates two-site ELISA for the detection of Indian ‘big four’ venoms using ABF antibodies.

[0039] FIG. 17A-17B illustrate two-site ELISA for the detection of Indian ‘big four’ venoms in spiked plasma (A) and urine (B) samples using ABF antibodies.

[0040] FIG. 18A-18B illustrate use of two-site ELISA-based snake venom detection kit for the detection of Indian ‘big four’ venoms in plasma (A) and urine (B) samples obtained from snakebite victims/patients.

[0041] FIG. 19A-19C illustrate use of two-site ELISA-based snake venom detection kit to determine the offending species of ‘big four’ snakes in buffer (A) and spiked plasma (B), and snakebite victim plasma (C) samples.

[0042] FIG. 20A-20I show LFA-based rapid test for Indian ‘big four’ venoms, A Negative control (buffer), B-Russell’s viper venom, C-cobra venom, D-krait venom, E-saw-scaled viper venom and F-I -venom spiked plasma samples in the same order as in B-E.

[0043] FIG. 21A-21J show LFA-based rapid test for individual species of big four snakes.

[0044] Referring to the drawings, like numbers refer to like parts throughout the views.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] While the invention has been disclosed with reference to certain embodiments it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt to a particular situation or material to the teachings of the invention without departing from its scope.

[0046] Definitions: Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0047] Throughout the specification and claims, the following terms take the meanings explicitly associated herein unless the context clearly dictates otherwise. The meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on." Referring to the drawings, like numbers indicate like parts throughout the views. Additionally, a reference to the singular includes a reference to the plural unless otherwise stated or inconsistent with the disclosure herein.

[0048] In the context of the invention, a “sample”, “biological sample”, “test sample”, “control sample” preferably consists of a component, tissue, human/animal body fluid, or biological fluid, such as (but not limited to) blood, plasma, serum, urine, cerebrospinal fluid, saliva, secretions and swabs from bite victims or lyophilised powders, crystals or liquids thought to contain or suspected to possess venom, cell culture supernatants; fixed tissue specimens and fixed cell specimens.

[0049] The term “label” refers both to direct labeling (via enzymes, radioisotopes, fluorochromes, luminescent compounds, etc.) and to indirect labeling (for example via antibodies which are themselves labeled directly or using reagents consisting of a labeled “affinity pair”, such as, but not exclusively, the pair labeled avidin-biotin, etc.).

[0050] As used herein, the term “antigen binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. It can refer to any whole antibody or functional fragment of an antibody comprising or consisting of at least one antigenic combination site making it possible for said antibody to bind to at least one antigenic determinant of an antigenic compound. It can refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. This term encompasses polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as molecules engineered from immunoglobulin gene sequences. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Examples of antigen binding molecules or antibodies are immunoglobulins and derivatives, e.g. fragments, thereof such as scFv (single chain variable fragment) chains, etc. These functional fragments can in particular be obtained by genetic engineering.

[0051] The antibodies used in the invention may be derived from immunising the whole venoms, purified or recombinantly expressed toxins or synthesised peptides against specific venom toxins through hyperimmunisation of animals or use of monoclonal cell lines, although alternative binding compounds (e.g. such as (but not limited) to small chemical molecules such as toxin-specific substrates and/or inhibitors, small proteins/peptides such as affirmers from various vertebrate and invertebrate species or small nucleotides such as aptamers amongst others) with a high affinity for venom components may also be used in the place of antibodies.

[0052] As used herein, the term “antigen” and “epitope” are used synonymously and refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding molecule binds, forming an antigen-antibody complex.

[0053] The term “capture antibody” is intended to mean an antibody or a part of an antibody, preferably attached to a solid phase, which is capable of retaining an antigen, for example one or more snake venom polypeptides, present in a biological sample, by affinity binding.

[0054] The presence of the snake venom in the biological sample is revealed by detection method. As regards the detection of the antigen, the invention in particular provides detection using at least one “detection antibody”. Such a detection antibody, which is labeled, is capable of binding to the captured antigen through affinity binding, by recognizing an epitope site which is different from that recognised by the capture antibody or identical due to the presence of a repeat motif in the capsid. As regards the detection of the antibodies, use may in particular be made of labelled anti immunoglobulin or anti-isotype antibodies, for example anti-immunoglobulins G, Y, etc. [0055] The invention in its various embodiments discloses toxin/taxon-specific antibodies, and device compositions, methods, and kits for venom detection using the antibodies. The kits may be based on any known technique in the art, such as but not limited to enzyme-linked immunosorbent assay (ELISA) or lateral flow assay (LFA) or a micro capillary method, using combinations of antibodies to detect different venom proteins from various venomous snakes. The kit is suitable for detecting venom proteins present in clinical samples from snakebite victims as well as substances expected to be or containing venoms, for example, in identifying venoms seized and intended for use as recreational drugs.

[0056] In one embodiment, a two-site ELISA-based assay device 100 and detection method using the device 100 are disclosed for identification and quantification of venom in a sample. The device 100, as shown in FIG. 1 detects species based on toxin-specific antibodies for the corresponding species. The toxin-specific antibodies may be coated to detection wells 109-1, 109-2,.... 109-n that are used in the device 100 to determine the identity of the species. The device may further include a negative control 107-1 and a positive control 107-2.

[0057] In one embodiment, an ELISA based assay device 150 is disclosed as shown in FIG. 2, that is configured to detect the presence of the Indian ‘Big Four’ snake venoms (or big four). Device 150 may include a microtitre plate having one or more wells with capturing antibodies to identify krait 109-1, saw-scaled viper 109-2, cobra 109-3, or Russell’s viper 109-4or. In various embodiments, the sample may be obtained from a snakebite victim or materials that are suspected to contain specific venoms. In some embodiments, a big four detector well 107-3, may be present along with negative control 107-1, positive control 107-2 and a blank 107-4. In some embodiments, the assay method includes one well specific for a species/taxon and one well for detecting all of the ‘big four’ snake venoms.

[0058] In various embodiments, the method of detecting venomous species in a sample is disclosed herein. The method may involve applying a clinical sample to a device 100 or 150 to allow binding of antibodies with the venom (if any) present in the sample. The next step involves washing of unbound samples and subsequent incubation of bound materials with the antibodies used for detection. Following incubation, development of colour in appropriate wells upon addition of a suitable chromogenic substrate confirms the identity of the offending species e.g. either the bite/venom was from Russell’s viper, cobra, krait or saw-scaled viper, as shown in FIG. 2. As illustrated in the figure, any of these species may cause colour to be developed in the big four detector well 107-3 and additionally in either of the wells 109-1 to 109-4. [0059] To facilitate detection, the antibodies may be conjugated with a fluorophore or an enzyme such as horse radish peroxidase, alkaline phosphatase or biotin. Following incubation, a substrate or streptavidin-enzyme conjugate may be added prior to incubation and addition of substrate for the appropriate enzyme as necessary. The method may in some embodiments involve detecting the presence of a particular type of venom in a sample by the change in colour of an appropriate sample well. The amount of venom present may be determined by the level of fluorescence or absorbance of the colour developed. In some embodiments the amount of venom in a sample may be quantified by spectrofluorimetry or other suitable techniques known in the art. In some embodiments the method may involve comparison of colour or fluorescence of a well containing an antibody compared to the controls to confirm the presence of specific toxins or venom of the offending species in the clinical sample.

[0060] In some embodiments, the assay device used may be a multi-well assay device. The multi -well assay device may support simultaneous detection of samples from different subjects. The subjects may be suspected of having been bitten by snakes, spiders, jellyfish or any other venomous animal. In one embodiment, the subject is a snake bite victim. In some embodiments, the multi-well assay device may support simultaneous detection of samples of different origin. An example of this is simultaneous detection of blood and urine samples obtained from the same subject or different subjects. In some embodiments, the assay device may support simultaneous detection of samples from different snake venoms.

[0061] In one embodiment, a venom detection kit 200 is disclosed that uses the principle of LFA. Here, the first site is coated with gold-conjugated detection antibodies that bind to the venom proteins present in the sample and their visible conjugates allow the diagnosis to be made. The further site is immobilized with one line of high-affinity taxon- specific antibodies (i.e. capture antibodies) as shown in FIG. 3A-D and 4A-4B. The development of a visible line (309) indicates the presence of venom from a specific species in the taxon in the tested sample.

[0062] The LFA device 200 includes a test strip 300 secured in a housing 203 that has a sample loading pad 201, as shown in FIG. 3A at one end for loading a biological sample. The sample loading pad 201 is configured to receive a sample that flows up the strip by capillary action. If the antigen is present in the sample, it will be bound by gold- conjugated detection antibodies that are unbound on the strip at the first site. In various embodiments the device 200 or strip 300 may have single, or multiple sites immobilized with capture antibodies, each line denoting a particular taxon or toxin/species for identifying the venom present in the sample. The unbound detection antibodies coupled to the venom will then bind to the capturing antibodies and form a visible line 309 by concentrating the gold-conjugated antibodies at a defined region. A negative control 307- 1, and a positive control 307-2 etc. may be included for venoms. In some embodiments other assay controls may be included for quantification, as necessary.

[0063] In some embodiments, the LFA assay device includes a single indicator line as shown in FIG. 3A and 3B. In some embodiments, the LFA assay device includes a single strip with multiple indicator lines as shown in FIG. 3C and 4A. In some embodiments, the LFA assay device includes multiple test strips arranged together as shown in FIG. 3D and 4B.

[0064] The LFA based venom detection strip 300, as shown in FIG. 3B-3C, includes a sample loading pad 301 for loading biological samples in one end; a conjugate pad 303 with at least one detector antigen binding molecules used to bind a snake venom polypeptide in the biological sample, a nitrocellulose membrane 305 having one or more indicator lines 309 and an absorbent pad 311.

[0065] In one embodiment, the individual indicator line in each test strip is immobilized with one or more capture antigen-binding molecules, wherein the individual indicator lines are immobilized with one or more capture antigen-binding molecules. The capture antigen binding molecules are configured to bind to unique region of the snake venom polypeptide specific for an individual snake species or taxon.

[0066] In some embodiments, the device is configured with additional lines 500, such as shown in FIG. 4A, or with parallel strips 600, as shown in FIG. 4B, by coating with a specific line(s) of toxin/species-specific antibodies for the detection of individual offending species in samples. Here, each line denotes a particular venom to determine the identity or nature of the venom (or offending species) present in the sample. The strip(s) includes immobilized capture antibodies for the ‘big four’ 307-2, i.e. the most common venomous snakes found in the Indian subcontinent: Common krait ( Bungarus caeruleus), 309-1 Indian cobra (Naja naja) 309-3, Russell’s viper ( Daboia russelii ) 309-4 and saw- scaled viper ( Echis carinatus) 309-2.

[0067] In another embodiment, the kit may focus on detecting the most common venomous snakes in Africa: Puff adder (Bitis arietans), Gaboon viper (Bitis gabonica), Rinkhals ( Haemachatus hemachatus), Green mamba ( Dendroaspis angusticeps), Jameson's mamba (Dendroaspis jamesoni), Black mamba ( Dendroaspis polylepis), Cape cobra ( Naja nivea), Forest cobra ( Naja melanoleuca), Snouted Cobra ( Naja annulifera ) or the Mozambique spitting cobra ( Naja Mossambica) amongst others. The invention in general may include kits and devices for detecting envenomation where identification could assist in the treatment procedure, as from snakes, spiders, jellyfish or any other venomous animal.

[0068] In another embodiment, the kit 700 may include a built-in or separate electronic device for measuring the intensity of the detection line and thereby quantifying the amount of venom present in the sample based on a standard curve recorded at inception. In this embodiment, the built-in electronic device 700, as shown in FIG. 5, may be a simple spectrophotometer to measure the intensity of the colour which develops in the test band using an optical reader unit 205 within the housing 203, and compare it with the initial standard curve included in the device, by which the concentration of venom present in the blood circulation of victims may be analysed. Based on the analysis, the clinicians may determine the number of vials of antivenom or other medicine required to treat the victim. In some embodiments, the kit 700 is suitable for deciding the treatment strategy for the snake bite victims based on the detection of the species and concentration of venom present in circulation. By ascertaining the amount of venom in the circulation using the device, a clinician will be able to administer the required dose of anti-venom or other medication to treat the victim based on scientific quantification as opposed to conjecture. This will have the added benefit of saving the victim from treatment costs and potential hypersensitivity reactions induced by the use of non-human animal derived antivenom (particularly when used in excess). Notably, this will prevent the unnecessary usage of antivenom in non-venomous and dry bite victims.

[0069] In one embodiment, the monovalent/toxin-specific antibodies may be used as capture antibodies, which are suitable for two-site ELISA or LFA. The capture or detection antibody could be monovalent or polyvalent antivenom, and/or toxin-specific monoclonal or polyclonal antibodies as guided by the use of appropriate capture antibodies. This may be used to detect a specific species or a collection of species (taxon). The antibodies (both capture and detection) are raised in horses, sheep, goats, chickens or any other suitable host animals. In some embodiments, the detection and capture antibodies are highly specific and have little to no cross-reactivity, detecting only single species or even only specific sub-species, while in others the extent of cross-reactivity is such that entire families or even orders may be detected with the kit. Antibodies used in the invention may include but are not limited to IgG, IgY, IgA, IgD, IgE and IgM, and/or recombinantly expressed antibodies that may be single chain antibodies or other. In various embodiments, the antibodies are high-affinity purified antibodies capable of detecting as low as 100, 50, 10, 5 or 1 ng of antigen or any range therebetween.

[0070] In some embodiments, the detection range for the assay kit is in the range will be as low as 160 pg/mL and up to 1 mg/mL. In some embodiments, the lower detection limit is 200, 500, 1000, 10000 pg/mL or any value therebetween. In some embodiments, the upper detection limit is 1, 10, 50, 100, 1000 pg/mL, or any value therebetween.

[0071] In some embodiments, the capture antigen binding molecules, the detector antigen binding molecules, or both are present in an LFA device at a concentration range of 0.05 mg/mL to 5 mg/mL. In some embodiments, the upper concentration limit for the antigen binding molecule is 0.1, 1, 10, 100, 1000, 10000 pg/mL or any number therebetween. In some embodiments, the lower concentration limit for the antigen binding molecule is 0.01, 0.1, 1, 10, 50, 100, 1000, 10000 pg/mL or any number there between. By way of example, the capture antigen binding molecules may be present at a concentration of 0.25 mg/mL (i.e. 0.2 pg per strip) to 5 mg/mL (i.e. 5 pg per strip). The detector antigen binding molecules may be present at a concentration of 0.05 mg/mL (i.e. 0.04 pg per strip) to 1 mg/mL (i.e. 1 pg per strip).

[0072] In one embodiment, the detector antigen binding molecules binds specifically to one or more of Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra {Naja naja), and Russell’s viper ( Daboia russelii ) venoms or polypeptides. In some embodiments, the capture antigen binding molecules binds to one or more of Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra (Naja naja), and Russell’s viper ( Daboia russelii) venoms or polypeptides.

[0073] The antigen binding molecules may be purified antibodies obtained by immunising one or more antigens or a mixture thereof. The antigens may be derived from one or more polypeptides of SEQ ID NO. 1-7. In some embodiments, the antigens are at least 70%, 75%, 80%, 85%, 90%, 95%, 99% identical to SEQ ID NO. 1-7. By way of example, various toxin-specific antibodies may be mixed together for the detection of Indian ‘big four’ venoms. In one embodiment, antibodies derived from each of the antigenic peptides of SEQ ID NO. 1-7 are mixed at a ratio of 2: 1:2:2: 1:1: 1 or any appropriate ratios for the detection of venom polypeptides from different snakes.

[0074] The detector antigen binding molecules or capture antigen binding molecules or both may bind to one or more polypeptides of SEQ ID NO. 1, 2, 3, 4, 5, 6, and 7. [0075] In one embodiment, the capture antigen binding molecules is configured to bind to Russell’s viper ( Daboia russelii) venom or a polypeptide thereof. By way of example, the capture antigen binding molecules bind to a polypeptide of SEQ ID NO. 3 and 4.

[0076] In one embodiment, the capture antigen binding molecules is configured to bind to Saw-scaled viper ( Echis carinatus) venom or a polypeptide thereof. By way of example, the capture antigen binding molecules binds to a polypeptide of SEQ ID NO. 2 and 4.

[0077] In one embodiment, the capture antigen binding molecules is configured to bind to Common krait ( Bungarus caeruleus) venom or a polypeptide thereof. By way of example, the capture antigen binding molecules bind to a polypeptide of SEQ ID NO. 5 and 6.

[0078] In one embodiment, the capture antigen binding molecules is configured to bind to Indian cobra ( Naja naja) venom or a polypeptide thereof. By way of example, the capture antigen binding molecules bind to a polypeptide of SEQ ID NO. 7.

[0079] According to another aspect of the invention there is provided an isolated polynucleotide encoding the capture or detection antibody of the invention or a fragment thereof. The invention further provides a pharmaceutical composition comprising the antigen binding molecule of the invention and a pharmaceutically acceptable carrier. [0080] Compared to other existing kits, this invention has many unique and advanced features to solve the practical barriers on diagnosing and treating victims of envenoming. One embodiment will test for the three main families of venomous snakes; Elapids, Vipers and Colubrids by specifically/innovatively using a sequence-structure-function and phylogenetic analysis to identify venom proteins that are found exclusively in a specific species or taxon of snake(s). An advanced version of this invention with an electronic device embedded in order to quantify the amount of venom present in the blood circulation of snake bite victims is also included.

[0081] The invention in various embodiments discloses methods of identifying and preparing antibodies for venoms of specific members of family/species/genera/order [hence forth ‘taxa/taxon’ - represents a group of related species. For example, in India, we refer to Russell’s and Saw-scaled vipers (both belong to Viperidae family) under one taxon, and similarly, Indian cobra and krait (both belong to elapidae family) under another taxon due to their haemotoxic and neurotoxic nature of the venoms, respectively] -specific venom proteins. The antibodies may be prepared based on the comprehensive sequence- structure-function and phylogenetic analysis to identify taxon-specific proteins. In some embodiments the methods may be used to produce antibodies for at least two families of snakes i.e. vipers and elapids. Distinct regions on the toxic proteins making up the venoms from these families may be utilized to develop antibodies from, for example (but not limited to) purified proteins from venoms, recombinant expressed proteins, digested peptides/proteins, region-specific peptides, or other fundamental differences in venoms to allow this kit to make the basic distinction between venomous taxa anywhere in the world. The method for example (but not limited to) includes identifying at least two specific regions (distantly spaced from each other to allow uninterrupted antibody binding) on a toxin that are unique [unique region peptides (URPs)] to a particular species or conserved among a broader taxon. The stability of the URPs is improved by modifying (adding or removing) specific amino acids (such as adding lysine residues). In some embodiments, the improvement in stability of peptides increases their immunogenicity and coupling properties. In various embodiments, the method further includes synthesizing and conjugating one or more URPs to carrier proteins such as keyhole limpet haemocyanin (KUH) and producing, high affinity toxin-specific antibodies by immunisation of synthesized URPs in animals. Antibodies are also produced for purified (from whole venoms) and/or recombinant expressed family-specific proteins through hyperimmunisation of suitable animals.

[0082] In more detail, the above method to identify URPs includes performing a sequence-structure-function and phylogenetic analysis to identify proteins that are found exclusively in a specific species or taxon. Two specific regions (at a relative distance) within that protein that are either unique to a particular species or conserved among a family or taxon are identified. The amino acids within these regions are mapped. These regions are then modified in order to improve their immunogenicity and coupling properties, and synthesized into smaller peptides, and antibodies are then raised against these peptides. This allows production of highly specific antibodies for a species or taxon, which are able to bind to two distinct regions of the protein of interest without any interference in their binding. The antibodies give high specificity, prevent cross reactivity, and allow the possibility for a larger range of snakes to be detected where appropriate. As well as use in species-specific test kits, these antibodies/approaches may be used to detect more broad ranges of snakes, by identifying these URPs on a protein found within an entire family or across different families. [0083] In some embodiments, the antibodies are raised against specific proteins found in venoms (preferably smaller molecular weight proteins) from various species. In previous detection methods for venoms, the development time has been slow which has been postulated as being due in part to the larger size of many venom components. These larger proteins may take longer to flow up the LFA strip, and consequently slow down the development of lines at the test sites. The smaller proteins are frequently less immunogenic as well, meaning that antibodies raised against the whole venom may contain very few antibodies against the smaller, faster moving proteins. Hence, by developing antibodies specifically to the smallest components, the development time in our kits/methods will be reduced, as is cross reactivity with other snake venom proteins. [0084] In various embodiments, the invention includes unique family-specific antibodies, isolated proteins and synthesized (poly)peptides with varying lengths, amino acids, or recombinant vectors for snake venom proteins. This may facilitate the construction of a universal diagnostic platform to corroborate SBE and characterize the nature of bites to guide the treatment strategy. The method includes obtaining the selective immunogenic peptides using sequence-structure-function and phylogenetic analysis for taxon/species-specific venom proteins or isolating these proteins from whole venoms or recombinantly expressing them, and using them to generate high affinity antibodies. Two- site immunoassays may be developed with these antibodies for detection of venoms. Using this approach, it is possible to clinically validate the two-site immunoassays using a range of distinct snake venoms and biological fluids (e.g. plasma, blood, secretions and urine) and swabs of bite sites obtained from snakebite victims for identification and quantification of venoms. Finally, a design of a prototype diagnostic platform using LFA or a micro capillary-based immune detection method to characterize snakebites is possible using this approach and materials developed herein.

[0085] In addition to snakebite envenomation and snake venoms, the invention in various embodiments includes the detection of all manner of other venoms from diverse range of species. This includes but not limited to: reptile venoms [from the four families of venomous snakes - Viperidae, Elapidae (to include Hydrophiinae), Colubridae and Lamprophiidae (venomous genera e.g. Atractaspis), as well as lizard venoms (notably Heloderma and Varanus species amongst others)]; invertebrate venoms (those from Arachnids, cone snails, jellyfish, insects such as wasps and bees, etc); fish venoms (scorpion fish, stonefish, crocodile fish, etc.) and mammal venoms (shrews, platypuses, vampire bats, lorises, etc) amongst others. The invention will detect the venoms of individual taxa/taxon or species-specific proteins/peptides and other toxic molecules [henceforth toxin(s)] .

[0086] The kit analyses and/or detects different venom proteins present in clinical samples of a victim of envenomation or other substances that are expected/suspected to contain venoms. The clinical samples may include blood, plasma, secretions, other bodily fluids and swabs from the envenomed victims. The venom detection kit typically uses but is not limited to a two-site immunoassay (ELISA) or LFA or a micro capillary approach. The kit contains substances for colorimetric detection (ELISA) or a test strip with gold- conjugated antibodies (LFA) and high-affinity taxon/toxin-specific antibodies where the nature of the venom is identified by colour development or a visible line, respectively. The kit aims to solve practical barriers in correctly diagnosing the nature of a bite or sting and treating victims of envenomation. One embodiment will test for the three main families of venomous snakes; elapids, vipers and colubrids by unique sequence-structure-function and phylogenetic analysis approach to identify venom proteins or unique peptides on the surface of proteins exclusively in a specific taxon. Although the device targets the medical implications of envenomation, the device/kit may also be used for the identification of unknown venoms or substances thought to contain venom.

[0087] In one embodiment, a specific two-site immunoassay (ELISA)-based kit was developed to identify the venoms of Indian ‘Big Four’ snake species (Russell’s viper, Cobra, Krait and Saw-scaled viper) using above-mentioned taxon/toxin-specific antibodies. This kit determines the presence of venoms in clinical samples such as blood, plasma, urine, secretions and swabs obtained from snakebite victims. Moreover, this kit may be used to determine the venoms in substances or materials that are believed to contain them.

[0088] Examples

[0089] Example 1: One-site immunoassay (ELISA) using toxin-specific antibodies (IgG) produced against URPs in snake venom serine proteases [0090] Serine proteases are one of the major constituents of viper venoms, although the venoms of other families are reported to possess a small amount of these proteins. Using sequence-structure-function and phylogenetic analysis, specific URPs were designed based on analysis of over 200 snake venom serine proteases from different venoms. Illustrative snake venom serine proteases include those described in Vaiyapuri et. al, 2012 (Sequence and phylogenetic analysis of viper venom serine proteases, Bioinformation, 8 (16). pp. 763-772, ISSN 0973-2063). URP-A was designed based on the C-terminus region of these proteins: SEQ ID NO. 1 (KGNTDATCPP) and URP-B was designed based on the N-terminus region of these proteins: SEQ ID NO. 2 (VIGGDECNINEHR). These peptides were chemically synthesised (Source: Sigma Aldrich, UK) and conjugated with KLH before immunisation in sheep. The antibodies (IgG) from serum of immunised sheep against these peptides were purified using affinity column chromatography (using URPs). These antibodies were then used in one-site ELISA to screen a range of snake venoms for their ability to be detect. Antibodies raised against URP-A were able to detect all viper venoms screened to a significant level (FIG. 6A). Antibodies raised against the URP-B showed increased performance in detecting all viper venoms screened (FIG. 6B). Notably, both of these antibodies were unable to bind any elapid venom to a significant level.

[0091] FIG. 6 shows One-site ELISA for antibodies (IgG) raised against URPs A and B using a range of viper and elapid venoms. Different venoms (100 pL at 1 pg/mL) or the URPs (100 ng/mL) were coated in separate wells of a microtiter plate and incubated for one hour. Following washing of unbound materials, 100 pL of antibodies (1 pg/mL) (A: URP-A; B: URP-B) were added. Following incubation and washing, 100 pL of HRP- conjugated anti-sheep IgG (500 ng/mL) were added prior to the addition of a HRP-specific substrate and measurement of colour developed by spectrometry. The green bar indicates the relevant URP as a positive control, red colour bars indicate viper venoms and blue bars represent elapid venoms. Data represent mean ± S.D (n=3). The p values (**p<0.01, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism. [0092] Example 2: One-site immunoassay (ELISA) using toxin-specific antibodies (IgY) produced against URPs in snake venom serine proteases.

[0093] Using the above mentioned URPs, IgY antibodies were raised in chicken egg yolks, and used to screen against a similar array of snake venoms in order to gauge any changes to their specificity resulting from change in antibody type. FIG. 7A shows that the IgY raised against the URP-A displayed minimal ability at detecting a range of venoms, with only Crotalus atrox (C. atrox) reaching a significant detection level. However, FIG. 7B shows an improved level of detection from IgY raised against the URP-B against a range of viper venoms. Here, six of the eight viper venoms screened appear to get a significant level of detection when compared to the elapid venoms.

[0094] FIG. 7 illustrates one-site ELISA for antibodies (IgY) raised against URPs A and B using a range of viper and elapid venoms. Different venoms (100 pL at 1 pg/mL) or the URPs (100 ng/mL) were coated in separate wells of a microtiter plate and incubated for different time points. Following washing of unbound materials, 100 pL of antibodies (1 pg/mL) (A: URP-A; B: URP-B) were added. Following incubation and washing, 100 pL of HRP-conjugated anti-chicken IgY (500 ng/mL) was added prior to the addition of HRP-specific substrate and measurement of colour developed by spectrometry. Green colour bar indicates the relevant URPs as positive control, red colour bars indicate viper venoms and blue represents elapid venoms. Data represent mean ± S.D (n=3). The p values (***p<0.001, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism.

Example 3: Determination of the affinity of URP-specific antibodies using standard curves.

[0095] In order to determine the concentrations at which the URP-A/B-specific antibodies (IgG and IgY) are able to detect venoms, Crotalus atrox venom (as an example) at concentrations from 10 ng/mL to 20 pg/mL were coated onto a high binding microtiter plate, before following the one-site ELISA protocol as detailed above. The data demonstrate the differences in sensitivity between the four antibodies tested. Both IgG antibodies against URPs A and B (FIG. 8A) are more sensitive than the IgY antibodies (FIG. 8B) in detecting the C. atrox venom. This could be due to fundamental differences in the types of antibodies, with IgY being slightly larger and less flexible molecule than the IgG.

[0096] FIG. 8 shows one-site ELISA to determine the affinity of antibodies (IgG and IgY) raised against URPs A and B using C. atrox venom. 100 pL of C. atrox (10 ng/mL to 20 pg/mL) was coated in separate wells of a microtiter plate and incubated for one hour. Following washing of unbound materials, 100 pL antibodies (1 pg/mL) (A: IgG A and B; B: IgY A and B) were added. Following incubation and washing, 100 pL of HRP- conjugated anti-sheep IgG or anti-chicken IgY (500 ng/mL) were added prior to the addition of HRP-specific substrate and measurement of colour developed by spectrometry. Data represent mean ± S.D (n=3).

[0097] Example 4: One-site ELISA for the detection of viper venoms using toxin- specific antibodies raised against purified metalloproteases from the venom of C. atrox

[0098] Following the production and analysis of antibodies raised against the URPs of snake (primarily viper) venom serine proteases, toxin-specific antibodies against snake venom metalloproteases (SVMPs) were produced. Metalloproteases are the major constituent in viper venoms, and they exert numerous local and systemic envenomation effects following viper bites. In order to broadly identify viper venoms, two different metalloproteases were purified. Specifically, two metalloproteases [with molecular weights of 23 kDa (named as CA1, a group I SVMP, NCBI sequence ID: Q91401) and 55 kDa (named as CA2, a group III SVMP, NCBI sequence ID: AB257084.1)] were obtained from the venom of C. atrox (Source: Sigma Aldrich, UK) using a combination of ion- exchange and gel filtration chromatography techniques, and these proteins were used to immunise sheep, shown as SEQ ID NO. 3

(EDQQNLSQRYIELVVVADHRVFMKYN SDLNIIRKRVHELVNTINGFYRSLNIDV S LTDLEIWSDQDFITVDSSAKNTLNSFGEWREADLLRRKSHDHAQLLTAINFEGKII GRAYTSSMCNPRKSVGIXKDHSPINFFVGVTMAHEFGHNFGMNHDGEKCFRGAS FCIMRPGFTPGRSYEFSDDSMGYYQSFFKQYNPQIXNK) and SEQ ID NO. 4 (MIQVFFVTICFAAFPYQGSSIIFESGNVNDYEIVYPRKVTAFPKGAVQPKYEDAM QYEFKVNGEPVVFHFEKNKQFFSKDY SETHY SPDGREITTYPFVEDHCYYHGRIE NDADSTASISACNGLKGHFKLQGEMYLIEPLKLSDSEAHAVYKYENVEKEDEAPK MCGVTQNWKSYEPIKKASQLVVTAEHQKYNPFRFVELVLVVDKAMVTKNNGDL DKIKTRMYELANTVNDIYRYMYIHVALVGLEIWSNEDKITVKPEADYTLNAFGE WRKTDLLTRKKHDNAQLLTAIDLDRVIGLAYVGSMCHPKRSTGIIQDYSPINLVV AVIMAHEMGHNLGINHDRGYCSCGDYACIMRPEISPEPSTFFSNCSYFDCWDFITN HNPECIVNEPLGTDIISPPVCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGS QCGHGDCCEQCKFSKSGTECRASMSECDPAEHCTGQSSECPADVFHKNGQPCLD NY GYNGN CPIMYHQCYDLF GAD VYEAED S CFERN QKGNYY GY CRKENGNKIPC APEDVKCGRLY CKDN SPGQNNSCKMFY SNEDEHKGMVLPGTKCADGKVCSNGH CVDVATAY) respectively. The purified antibodies (IgG) were then tested using one-site ELISA with a range of elapid and viper venoms along with the purified proteins (CA1 and CA2). While these antibodies cross reacted with some of the elapid venoms, they largely detected the viper venoms with high levels of detection (FIG. 9).

[0099] FIG. 9 illustrates One-site ELISA for antibodies (IgG) raised against purified toxins using a range of viper and elapid venoms. Different venoms (100 pL at 1 pg/mL) or the purified proteins (CA1 or CA2) (100 ng/mL) were coated in separate wells of a microtiter plate and incubated for different time points. Following washing of unbound materials, 100 pL of antibodies (1 pg/mL) (A: CA1; B: CA2) were added. Following incubation and washing, 100 pL of HRP-conjugated anti-sheep IgG (500 ng/mL) was added prior to the addition of HRP (or another related detection enzyme)-specific substrate and measurement of colour developed by spectrometry. The green bars indicate the purified proteins (CA1 and CA2) as positive controls, red colour bars indicate viper venoms and blue represents elapid venoms. Data represent mean ± S.D (n=3). The p values (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism.

[00100] Example 5: Determination of the affinity of toxin-specific antibodies (CA1 and CA2) using standard curves.

[00101] In order to determine the concentrations at which the toxin-specific (IgG) antibodies (CA1 and CA2) are able to detect venoms, C. atrox venom at concentrations from 10 ng/mL to 20 pg/mL were coated onto a high binding microtiter plate, before following the one-site ELISA protocol (as detailed above). The data demonstrate the differences in sensitivity between the two antibodies tested. Both antibodies against CA1 and CA2 are sensitive in detecting C. atrox venom (FIG. 10) although CA1 showed superior efficiency in binding to this venom.

[00102] FIG. 10 shows one-site ELISA to determine the affinity of antibodies (IgG) raised against purified toxins using C. atrox venom. C. atrox venom (100 pL of 10 ng/mL to 20 pg/mL) was coated in separate wells of a microtiter plate and incubated for one hour. Following washing of unbound materials, 100 pL antibodies (1 pg/mL) (CA1 and CA2) were added. Following incubation and washing, 100 pL of HRP-conjugated anti-sheep IgG (500 ng/mL) was added prior to the addition of HRP-specific substrate and measurement of colour developed by spectrometry. Data represent ± S.D (n=3).

[00103] Example 6: Two-site ELISA for the detection of viper venoms using toxin (CA1 and CA2) specific antibodies.

[00104] The toxin-specific antibodies (CA1 and CA2) were then used to develop a two-site ELISA for the detection of a range of viper and elapid venoms. These antibodies detected a large number of vipers, both in buffer (FIG. 11A) and whole blood (FIG. 1 IB). Here the combination of CA1 (for capture)-CA2 (for detection) antibodies showed no cross reactivity against any of the elapid venoms tested. Notably, the two viper species [Russell’s ( Daboia russelii ) and saw-scaled ( Echis carinatus) vipers] from India were detected by these antibodies to a significant level.

[00105] FIG. 11 illustrates Two-site ELISA for the detection of viper venoms using toxin (CA1 and CA2)-specific antibodies (IgG). CA1 antibodies (100 pL of 5 pg/mL) (for capture) were coated in separate wells of a microtiter plate and incubated overnight. Following washing of unbound antibodies, 100 pL of different venoms (2 pg/mL) in buffer (A) or spiked blood (B) were added and incubated for one hour. After washing unbound materials, 100 pL of biotin -conjugated CA2 antibodies (1 pg/mL) (for detection) were added and incubated for one hour. Following washing, 100 pL of streptavidin- conjugated HRP (500 ng/mL) was added and incubated for 30 minutes prior to the addition of HRP-specific substrate and measurement of colour developed using spectrophotometry. Data represent mean ± S.D (n=3). The p values (*p<0.05, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism. [00106] Example 7: One-site ELISA for the detection of elapid venoms using toxin-specific antibodies raised against three finger toxin (3FTX)-specific URPs. [00107] Following the detection of viper venoms (specifically for Russell’s and saw- scaled vipers), using the aforementioned sequence -structure -function and phylogenetic analysis, we identified three-finger toxins (3FTXs) that are predominantly present in a range of elapid venoms. Illustrative examples of 3FTXs are described in Roly ZY et. al., 2014 (A comparative in silico characterization of functional and physicochemical properties of 3FTx (three finger toxin) proteins from four venomous snakes. Bioinformation. 2014;10(5):281-287).

[00108] After identifying two URPs in the sequences of a number of 3FTXs common to the Bungarus genus, URP-C (SEQUENCE ID NO. 5; AVSCPKAKPNET) and URP- D (SEQUENCE ID NO. 6; TSLICPEKDCQK) were designed and chemically synthesised (Source: Sigma Aldrich, UK) to raise antibodies against them in sheep. The purified antibodies (IgG) were then tested using one-site ELISA with a range of elapid and viper venoms along with the URPs (C and D). These antibodies detected elapid venoms to a higher level compared to the viper venoms used (FIG. 12). Notably, these antibodies significantly detected the venom of Indian krait ( Bungarus caeruleus).

[00109] FIG. 12 shows One-site ELISA for antibodies (IgG) raised against 3FTX- specific URPs (C and D) using a range of elapid and viper venoms. Different venoms (100 pL at 1 pg/mL) or the relevant URPs (C or D) (100 ng/mL) were coated in separate wells of a microtiter plate and incubated for different time points. Following washing of unbound materials, 100 pL antibodies (1 pg/mL) (A: URP-C; B: URP-D) were added. Following incubation and washing, 100 pL of HRP -conjugated anti-sheep IgG (500 ng/mL) was added prior to the addition of HRP-specific substrate and measurement of colour developed by spectrometry. Green bars indicate the relevant URPs as positive control, red bars indicate viper venoms and blue bars represent elapid venoms. Data represent mean ± S.D (n=3). The p values (*p<0.05, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism.

[00110] Example 8: Two-site ELISA for the detection of krait ( Bungarus species) venoms using 3FTX (URP-C/D)-specific antibodies.

[00111] The 3FTX (URP-C/D)-specific antibodies were then used to develop a two- site ELISA for the detection of krait ( Bungarus species) venoms (FIG. 13). The combinations of these antibodies (URP-C/D and UPR-D/C) effectively detected two krait species [Bungarus caeruleus (Indian krait) and Bungarus fasciatus] with high specificity compared to any other elapid or viper venoms tested.

[00112] FIG. 13 illustrates two-site ELISA for the detection of krait ( Bungarus species ) venoms using 3FTX (URP-C and D)-specific antibodies. 100 pL of URP-C or D antibodies (5 pg/mL) (for capture) were coated in separate wells of a microtiter plate and incubated overnight. Following washing of unbound antibodies, 100 pL of different venoms (2 pg/mL) in buffer were added and incubated for one hour. After washing unbound venoms, 100 pL of biotin-conjugated URP C or D antibodies (based on the capture antibodies used) (1 pg/mL) (for detection) were added and incubated for one hour. Following washing, 100 pL of streptavidin-conjugated HRP (500 ng/mL) was added and incubated for 30 minutes prior to the addition of HRP-specific substrate and measurement of colour developed using spectrophotometry. Data represent mean ± S.D (n=3). The p values (*p<0.05, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism.

[00113] Example 9: Two-site ELISA for the detection of cobra venoms using antibodies (IgG) raised against a purified 3FTX.

[00114] Following the development of antibodies against Indian vipers (Russell’s and saw-scaled vipers) and krait, we then sought to develop antibodies specifically for cobra venoms. In order to achieve this, a 3FTX polypeptide (NCBI Sequence ID: P24778, SEQ ID NO. 7;

HHLICHNRPLPFLHKTCPEGQNICYKMTLKKTPMKLSVKRGCAATCPSERPLVQV ECCKTDKCNW) purified from the venom of Rinkhals [Haemachatus hemachatus (HH)] (Source: Sigma Aldrich, UK) and used to immunise sheep to produce 3FTX (HH)-specific antibodies. After raising antibodies against this protein, the antibodies were used in a two- site ELISA for cobra (Naja and Haemachatus) venoms. This method successfully detects a range of cobra species (specifically Indian cobra), and could help to differentiate between Naja/Haemachatus bites and those from others (FIG. 14).

[00115] FIG. 14 shows two-site ELISA for the detection of cobra venoms using 3FTX (HH)-specific antibodies (IgG). 100 pL of 3FTX (HH)-specific antibody (5 pg/mL) (for capture) was coated in separate wells of a microtiter plate and incubated overnight. Following washing of unbound antibodies, 100 pL of different elapid or viper venoms (2 pg/mL) in buffer were added and incubated for one hour. After washing unbound materials, 100 pL of biotin-conjugated 3FTX (HH)-specific antibody (1 pg/mL) (for detection) was added and incubated for one hour. Following washing, 100 pL of streptavidin-conjugated HRP (500 ng/mL) was added and incubated for 30 minutes prior to the addition of HRP-specific substrate and measurement of colour developed using spectrophotometry. Data represent mean ± S.D (n=3). The p values (**p<0.01, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism. [00116] Example 10: Determination of the affinity of 3FTX (HH)-specific antibody using standard curves.

[00117] Affinity of 3FTX (HH)-specific antibody (IgG) was determined using HH venom. HH venom at concentrations from 10 ng/mL to 20 pg/mL were coated onto a high binding microtiter plate, before following the one-site ELISA protocol (as detailed above). The data demonstrate that this antibody is highly sensitive towards HH venom (FIG. 15), and it can detect as low as 20 ng/mL.

[00118] FIG. 15 illustrates one-site ELISA to determine the affinity of antibody (IgG) raised against a purified 3FTX toxin using HH venom. 100 pL of HH venom (10 ng/mL to 20 pg/mL) was coated in separate wells of a microtiter plate and incubated for one hour. Following washing of unbound materials, 100 pL 3FTX (HH) antibody (1 pg/mL) was added. Following incubation and washing, 100 pL of HRP -conjugated anti-sheep IgG (500 ng/mL) was added prior to the addition of HRP-specific substrate and measurement of colour developed by spectrometry. Data represent ± S.D. (n=3).

[00119] Example 11: Two-site ELISA for the determination of the affinity of toxin-specific antibodies against the Indian ‘big four’ venoms.

[00120] Following the successful detection of Indian ‘big four’ venoms using various toxin-specific antibodies [URP-A, URP-B, CA1, CA2, URP-C, URP-D and 3FTX (HH)] in separate assays, a specific mixture (at a ratio of 2: 1:2:2: 1:1:1) of these antibodies was prepared (named anti-big four [ABF] antibodies) based on their affinity towards the Indian ‘big four’ venoms. The affinity of ABF antibodies was determined using ‘big four ‘venoms [prepared in buffer (phosphate-buffered saline)] in two-site ELISA. This specific antibody mixture displayed strong binding affinity towards the Indian ‘big four’ venoms with a detection sensitivity of as low as 8 pg of each venom (FIG. 16).

[00121] FIG. 16 shows Two-site ELISA for the detection of Indian ‘big four’ venoms using ABF antibodies. ABF antibodies (100 pL of 10 pg/mL) (for capture) were coated in separate wells of a microtiter plate and incubated overnight. Following washing of unbound antibodies, 50 pL of ‘big four’ venoms (6 pg/mL to 100 ng/mL) in buffer were added and incubated for 30 minutes. After washing unbound venoms, 100 pL of biotin- conjugated ABF antibodies (10 pg/mL) (for detection) were added and incubated for 30 minutes. Following washing, 100 pL of streptavidin-conjugated HRP (500 ng/mL) was added and incubated for 30 minutes prior to the addition of HRP-specific substrate and measurement of colour developed using spectrophotometry. Data represent ± S.D. (n=3). [00122] Example 12: Two-site ELISA for the detection of ‘big four’ venoms in spiked plasma and urine samples using ABF antibodies.

[00123] Following the determination of their sensitivity, the ability of ABF antibodies to detect the Indian ‘big four’ venoms in spiked plasma and urine samples was determined using two-site ELISA. The results demonstrated the strong ability of these antibodies to detect the venoms in spiked biological samples (FIG. 17).

[00124] FIG. 17 illustrates Two-site ELISA for the detection of Indian ‘big four’ venoms in spiked plasma and urine samples using ABF antibodies. 100 pL of ABF antibodies (10 pg/mL) (for capture) were coated in separate wells of a microtiter plate and incubated overnight. Following washing of unbound antibodies, ‘big four’ venoms spiked in plasma (20 pL, final concentration of 40 pg/mL to 100 ng/mL) and urine (50 pL, final concentration of 970 pg/mL to 250 ng/mL) were added and incubated for 30 minutes. After washing unbound materials, 100 pL of biotin-conjugated ABF antibodies (10 pg/mL) (for detection) were added and incubated for 30 minutes. Following washing, 100 pL of streptavidin-conjugated HRP (500 ng/mL) was added and incubated for 30 minutes prior to the addition of HRP-specific substrate and measurement of colour developed using spectrophotometry. Data represent mean ± S.D. (n=3).

[00125] Example 13: Two-site ELISA-based snake venom detection kit for the detection of Indian ‘big four’ venoms in clinical samples obtained from snakebite victims. [00126] Followed by spiked biological samples, the ABF antibodies were used to develop a two-site ELISA-based snake venom detection kit for the detection of big four venoms in clinical samples obtained from snakebite victims. This kit was tested with 61 plasma and urine samples obtained from different snake bite victims who were admitted to a snakebite referral hospital. The offending species of snake in each case was predicted by clinical experts based on the descriptions provided by the victim or their relatives and relevant clinical symptoms developed. As shown in FIG. 18A, this kit has significantly detected the venoms of Russell’s viper and Indian cobra in most of the plasma samples in line with the predictions made by clinicians who treated these patients. Interestingly, our kit has detected venoms in some samples that were classified as ‘unknown bites’ due to insufficient information provided by the victims about the offending snake and/or the lack of unambiguous clinical symptoms to ascertain the offending species. Notably, the presence of venom was not detected in some cases where although the offending species was predicted, they were considered as dry bite due to lack of obvious clinical symptoms as ascertained by the clinicians. Moreover, in several of Russell’s viper bite victims, the presence of venom in their urine samples was detected (FIG. 18B). This may emphasise the impact of venom on the kidneys, and/or subsequent elimination of venoms via urine. The kit did not detect the presence of any venom in samples obtained from victims who were bitten by non-venomous snakes or a red centipede. This highlights the lack of cross reactivity of antibodies against non-specific proteins present in the plasma or urine. Overall, this kit is able to unambiguously detect the presence of venoms in clinical samples (plasma and urine) from snake bite victims. Hence, this can be used in clinical settings to ascertain SBE in victims in India.

[00127] FIG. 18 shows Two-site ELISA-based snake venom detection kit for the detection of Indian ‘big four’ venoms in plasma and urine samples obtained from snake bite victims. ABF antibodies (100 pL of 10 pg/mL) (for capture) were coated in separate wells of a microtiter plate and incubated overnight. Following washing of unbound antibodies, plasma (25 pL - A) or urine (50 pL - B) samples from snakebite victims were added and incubated for 30 minutes. After washing unbound materials, 100 pL of biotin- conjugated ABF antibodies (10 pg/mL) (for detection) were added and incubated for 30 minutes. Following washing, 100 pL of streptavidin-conjugated HRP (500 ng/mL) was added and incubated for 30 minutes prior to the addition of HRP-specific substrate and measurement of colour developed using spectrophotometry. Plasma (25 pL) and urine (50 pL) samples obtained from healthy volunteers were used as negative controls (NC) in this assay. Similarly, plasma and urine samples obtained from victims who were bitten by non- venomous snakes or a red centipede were used as controls. Data represent mean ± S.D (n=3). The p values (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism.

[00128] A comprehensive analysis of all 61 samples tested with our snake venom detection kit is provided below in Table 1. Out of 61 samples, 43 samples were found to be positive for the presence of venom. The non-venomous snake (2) and red centipede (1) bite samples provided negative results as expected. Furthermore, 6 unknown species samples were provided negative results as possibly they could be associated with some insect bites or non-venomous or dry snakebites. These patients did not display any obvious clinical symptoms for SBE. In some cases where the offending snake was predicted as one of the big four, the presence of venom was not detected as they were dry bites as confirmed by the clinicians based on the clinical symptoms. Hence, this kit is able to unambiguously corroborate the presence of venom in clinical samples obtained from any genuine envenomed victims without any cross-reactivity to plasma or urine proteins.

Table 1: Comprehensive analysis of clinical samples from snakebite victims.

[00129] Example 14: Two-site ELISA-based snake venom detection kit to determine the offending species of ‘big four’ snakes. [00130] Following the collective identification of big four snake venoms, selective toxin-specific antibodies were chosen for species identification based on their affinity with ‘big four’ venoms. Specific antibody pairs (CA1-CA2 for Russell’s viper, 3FTX (HH) for Indian cobra, URP-B-CA2 for saw-scaled viper and URP-C/D for krait) were used in two- site ELISA. The kit has selectively detected the individual species of ‘big four’ venoms in buffer (FIG. 19A) and spiked plasma (FIG. 19B) samples. Although there was some cross reactivity between the species observed, the dominance in the absorbance level for respective species was confirmed to ascertain the offending species. Due to similarities between venom components within the same family (viper or elapid), this cross reactivity is inevitable. Moreover, this kit was tested with plasma samples obtained from snakebite victims who were bitten by Russell’s viper and cobra, and it successfully detected the offending species (FIG. 19C). The samples from victims who are bitten by krait and saw- scaled viper were not tested yet due to the unavailability of these specific samples.

[00131] FIG. 19 Two-site ELISA-based snake venom detection kit to determine the offending species of big four snakes. 100 pL of toxin-specific antibodies (as mentioned above) (10 pg/mL) (for capture) were coated in separate wells of a microtiter plate and incubated overnight. Following washing of unbound antibodies, ‘big four’ venoms in buffer (50 pL - A) and spiked plasma (25 pL - B), and snakebite victim plasma (25 pL - C) samples were added in respective wells and incubated for 30 minutes. After washing unbound samples, 100 pL of biotin-conjugated ABF antibodies (10 pg/mL) (for detection) were added and incubated for 30 minutes. Following washing, 100 pL of streptavidin- conjugated HRP (500 ng/mL) was added and incubated for 30 minutes prior to the addition of HRP-specific substrate and measurement of colour developed using spectrophotometry. Data represent mean ± S.D (n=3 for A and B, and n=2 for C). The p values (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001) shown are as calculated by one-way ANOVA using GraphPad Prism.

[00132] Example 15: LFA-based rapid detection kit for ‘big four’ venoms.

[00133] Following the successful development of an ELISA-based venom detection kit, we sought to use these antibodies to develop an LFA-based rapid test kit for Indian big four venoms. Here, ABF antibodies were used to develop LFA strips for the detection of venoms. The ‘big four’ venoms in buffer and spiked plasma samples (FIG. 20) were used and 5 pg of venom was used in these assays.

[00134] FIG. 20 LFA-based rapid test for Indian ‘big four’ venoms. A-Negative control (buffer), B - Russell’s viper venom in buffer, C - cobra venom in buffer, D - krait venom in buffer, E - saw-scaled viper venom in buffer, and F-I are venom spiked plasma samples in the same order as in B-E. (Concentrations/test: antigen - 5 pg (whole venom & spiked samples) and capture antibody - 2 pg).

[00135] Example 16: LFA-based rapid test for the identification of offending species of big four snakes.

[00136] Selective toxin-specific antibodies were chosen for species identification based on their affinity in ELISA. Specific (CA1-CA2 for Russell’s viper, 3FTX (HH) for Indian cobra, URP-B-CA2 for saw-scaled viper and URP-C/D for krait) antibody pairs were used to develop LFA tests for the detection of venoms from individual species. The individual kits were then tested with ‘big four’ venom spiked plasma samples (FIG. 21). Notably, the Russell’s viper kit was tested with corresponding snakebite victim plasma and it provided positive result. 1 pg venom was used in this assay.

[00137] FIG. 21 LFA-based rapid test for individual species of big four snakes. Plasma samples obtained from healthy volunteers (A-D) and a victim who was bitten by centipede (E) were used as negative controls. Then the kit was used to selectively detect individual venoms in spiked plasma samples; Russell’s viper (F), cobra (G), krait (H) and saw-scaled viper (I). J, plasma obtained from a victim who was bitten by Russell’s viper. (Concentrations/test: antigen - 1 pg (whole venom & spiked samples) and capture antibody - 2 pg).

Claims

WE CLAIM:

1. An immunoassay method of determining the type and concentration of a snake venom polypeptide present in a biological sample, comprising the steps of:

(a) loading a predetermined amount of a biological sample in one or more sample wells of an assay device;

(b) contacting the biological sample with one or more capture antigen binding molecules followed by one or more detector antigen binding molecules thereby capturing one or more snake venom polypeptides present in the biological sample, wherein the capture antigen binding molecules are configured to bind to a unique region of the one or more snake venom polypeptides specific for an individual snake species or taxon, wherein the detector antigen binding molecules are configured to bind to at least Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra ( Naja naja), and Russell’s viper ( Daboia russelii) venoms or polypeptides thereof, characterized in that the detector antigen binding molecules bind to a polypeptide of SEQ ID NO. 1, 2, 3, 4, 5, 6, and 7; and

(c) detecting, by an optical reader, the binding intensity of a label conjugated to the detector antigen binding molecules to determine the type and concentration of the snake venom polypeptide in the biological sample.

2. The method as claimed in claim 1, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, sweat, urine, secretion, swab and biopsy sample.

3. The method as claimed in claim 1, wherein the lower detection limit for the one or more snake venom polypeptides present in the sample is 160 pg/mL).

4. The method as claimed in claim 1, wherein the capture antigen binding molecules is configured to bind to Russell’s viper ( Daboia russelii) venom or a polypeptide thereof, characterized in that the capture antigen binding molecules binds to a polypeptide of SEQ ID NO. 3 and 4.

5. The method as claimed in claim 1, wherein the capture antigen binding molecules are configured to detect Saw-scaled viper ( Echis carinatus) venom or a polypeptide thereof, characterized in that the capture antigen binding molecules binds to a polypeptide of SEQ ID NO. 2 and 4.

6. The method as claimed in claim 1, wherein the capture antigen binding molecules are configured to detect Common krait ( Bungarus caeruleus) venom or a polypeptide thereof, characterized in that the capture antigen binding molecules bind to a polypeptide of SEQ ID NO. 5 and 6.

7. The method as claimed in claim 1, wherein the capture antigen binding molecules is configured to detect Indian cobra ( Naja naja) venom or a polypeptide thereof, characterized in that the capture antigen binding molecules bind to a polypeptide of SEQ ID NO. 7.

8. The method as claimed in claim 1, wherein the detector antigen binding molecules, the capture antigen binding molecules, or both are present at a concentration range of 1 pg/mL to 5 mg/mL.

9. The method as claimed in claim 1, wherein the assay device is configured for simultaneous detection of a plurality of biological samples.

10. The method as claimed in claim 1, wherein said contacting the biological sample with one or more capture antigen binding molecules followed by one or more detector antigen binding molecules, comprises: contacting the biological sample in a first well (109-1) with capture antigen binding molecules conjugated to the first well and configured to detect Common krait ( Bungarus caeruleus) venom or a polypeptide thereof; contacting the biological sample in a second well (109-2) with capture antigen binding molecules conjugated to the second well and configured to detect Saw-scaled viper ( Echis carinatus) venom or a polypeptide thereof; contacting the biological sample in a third well (109-3) with capture antigen binding molecules conjugated to the third well and configured to detect Indian cobra (Naja naja) venom or a polypeptide thereof; contacting the biological sample in a fourth well (109-4) with capture antigen binding molecules conjugated to the fourth well and configured to detect Russell’s viper ( Daboia russelii) venom or a polypeptide thereof; contacting the biological sample in the first, second, third and fourth well with detector antigen binding molecules configured to detect Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra ( Naja naja), and Russell’s viper ( Daboia russelii) venoms or polypeptides thereof.

11. The method as claimed in claim 1, wherein the label is selected from the group consisting of gold nanoparticles, enzyme, fluorophore, and chemiluminescence markers.

12. A lateral flow immunoassay device comprising: a lateral flow test strip (300) secured in a housing (203), the test strip comprising: a sample loading pad (301) for loading a biological sample in one end; a conjugate pad (303) comprising one or more detector antigen binding molecules configured to bind to a snake venom polypeptide in the biological sample, wherein the detector antigen binding molecules are configured to bind to at least Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra ( Naja naja), and Russell’s viper ( Daboia russelii) venoms or polypeptides thereof, characterized in that the detector antigen binding molecules bind to a polypeptide of SEQ ID NO. 1, 2, 3, 4, 5, 6, and 7; a nitrocellulose membrane (305) having a plurality of indicator lines (309), wherein the individual indicator lines are immobilized with one or more capture antigen-binding molecules, wherein the capture antigen binding molecules are configured to bind to unique region of the snake venom polypeptide specific for an individual snake species or taxon; and an absorbent pad (107) an optical reader unit (205) for measuring the intensity of the plurality of indicator lines to display the type and concentration of the snake venom polypeptide in the biological sample.

13. A lateral flow immunoassay device comprising: a plurality of lateral flow test strips (300) secured in a housing (203), each test strip comprising: a sample loading pad (301) for loading a biological sample in one end; a conjugate pad (303) comprising a one or more detector antigen binding molecules configured to bind to a snake venom polypeptide in the biological sample, wherein the detector antigen binding molecules is configured to bind to at least Common krait ( Bungarus caeruleus), Saw-scaled viper ( Echis carinatus), Indian cobra ( Naja naja), and Russell’s viper ( Daboia russelii) venoms or polypeptides thereof, characterized in that the detector antigen binding molecules bind to a polypeptide of SEQ ID NO. 1, 2, 3, 4, 5, 6, and 7; a nitrocellulose membrane (105) having one or more indicator lines (309), wherein the individual indicator line in each test strip is immobilized with one or more capture antigen-binding molecules, wherein the individual indicator lines are immobilized with one or more capture antigen-binding molecules, wherein the capture antigen binding molecules are configured to bind to unique region of the snake venom polypeptide specific for an individual snake species or taxon; and an absorbent pad (311) an optical reader unit (205) for measuring the intensity of the plurality of indicator lines to display the type and concentration of the snake venom polypeptide in the biological sample.

14. The device as claimed in claim 12 or claim 13, wherein the detector antigen binding molecules are conjugated with gold nanoparticles.

15. The device as claimed in claim 12 or claim 13, wherein the type of snake venom polypeptide is determined from one or more visible lines in the assay device.

16. The device as claimed in claim 12 or claim 13, wherein the capture antigen binding molecules, the detector antigen binding molecules, or both are present at a concentration between 0.05 mg/mL to 5 mg/mL.