FIELD OF INVENTION
The present invention relates to methods for development of antibodies and devices for
detection of snake venom.
BACKGROUND
5 Snakebite envenoming is a neglected tropical disease that affects 1.8-2.7 million
people worldwide per year and causes 81,000-1,38,000 deaths per year with women, children and farmers in poor rural communities being the most vulnerable groups. In contrast to many other serious health conditions, snakebite envenoming can be treated using anti-venoms.
However, there are several challenges in treatment of snakebite envenoming. Firstly,
10 after snakebite occurs, there is huge uncertainty regarding the species of snake involved, which is a hinderance for successful management of snakebites. Due to the complexity of snake venoms, no method or device has been developed which can clearly identify the specific snake species.
Secondly, the conventional clinical practice in low and middle-income countries, such
15 as India is to administer polyvalent anti-snake venom formulation comprising antibodies of different species which are common to the specific region. In India, the formulation comprises snake venom antibodies from four different species accounting for most of the bite cases (commonly termed as Big Four), namely the spectacled (Indian) cobra (Naja naja), the common krait (Bungarus caeruleus), saw-scaled viper (Echis carinatus) and russell's viper
20 (Daboia russelii). Heterogeneous and non-abundant antibodies present in the formulation leads to chronic side effects of existing antivenin therapy. The polyvalent formulations often cause severe side effects in the victim, (reported up to 30 percent of the victims worldwide) demanding secondary treatment. Treatment using polyvalent formulations results in collateral damage, affecting internal organs and lead to death.
25 Current approaches in production of antibodies against antigens involves production of
monoclonal antibodies using technologies such as hybridoma technology, phage display technologies etc. against specific epitopes. However, the approaches using monoclonal antibodies fail to address the above issues as snake venoms are extremely complex due to diversity of antigenic targets and presence of several toxins in snake venom.
30 Approaches utilizing polyclonal antibodies have also failed to address the issues as the
polyclonal antibodies have cross-reactivity to several antigenic targets and therefore, unsustainable. Further, no snake venom detection kit or enzyme assay technique is available which specifically addresses the complexity of the venom of the Big Four snake species.

To overcome this difficulty, the inventor has invented processes for development of affordable monospecific and bispecific antibodies against the Big Four snake species. These antibodies have been used to address the challenge of identification of species of snake involved in envenoming by development of a snake venom detection kit (SVDK). The invention makes diagnosis and treatment of snake envenomation more rapid, affordable and effective to a huge proportion of population living in low and middle-income nations. SUMMARY OF THE INVENTION Technical Problem
The technical problem to be solved in this invention is to provide inexpensive monospecific and bi-specific anti-venom antibodies and kits for determination of snake species involved in envenomation. Solution to the problem
The problem has been solved by inventing a process for selection of monospecific and bispecific anti-venom antibodies. Further, the antibodies have been used to develop devices which are can detect the snake species responsible for envenomation. The devices are highly species specific, sensitive, cost effective, rapid, simple, stable and portable. Advantages of the invention
The developed process can be used for generation of monospecific and bispecific anti-venom antibodies, can be used for effective treatment of snake envenomation. Further, the developed devices provide for rapid and on-site determination of the snake species involved in envenomation. The SVDK is highly species specific, sensitive, cost effective, rapid, simple, stable and portable for field use. Overview of the invention
In one aspect, the invention provides a process for selection of monospecific antibodies from a pool of antibodies having binding affinity to a snake venom (cobra venom, russel's viper venom, krait venom and saw scale viper venom) comprising the steps of:
a. incubating the pool of antibodies with first venom-coupled beads;
b. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first venom;
c. incubating the supernatant obtained in step (b) with second venom-coupled
beads;
d. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first or second venom;
e. incubating the supernatant obtained in step (d) with third venom-coupled beads;

f. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first, second or third venom; and
g. purifying the supernatant to obtain monospecific antibodies.
In another aspect, the invention provides for a process for selection of bispecific antibodies from a pool of antibodies having binding affinity to a snake venom (cobra venom, russel viper venom, krait venom and saw scale viper venom), comprising the steps of:
a. incubating the pool of antibodies with first venom-coupled beads;
b. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first venom;
c. incubating the supernatant obtained in step (b) with second venom-coupled
beads;
d. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first or second venom; and
e. purifying the supernatant to obtain bispecific antibodies.
In another aspect, the invention provides a lateral-flow immunoassay device for detecting the presence of snake venom in a biological sample, comprising: a lateral flow membrane, a panel of detection antibodies (monospecific or bispecific), a detectable marker conjugated to the detection antibodies and a panel of capture antibodies (monospecific or bispecific). BRIEF DESCRIPTION OF DRAWINGS
The features of the present disclosure will become fully apparent from the following description taken in conjunction with the accompanying figures. With the understanding that the figures depict only several embodiments in accordance with the disclosure and are not to be considered limiting of its scope, the disclosure will be described further through use of the accompanying figures.
Figure 1 depicts SDS-PAGE profiling of crude snake venom. Figure 2 depicts SDS-PAGE profiling of column separated snake venom. Figure 3 depicts the sensitivity of individual anti-venoms antibodies IgG by indirect ELISA; A- Cobra anti-venom IgG, B-Krait anti-venom IgG, C-Russell's viper's viper anti-venom IgG, D- Saw-scale viper anti-venom IgG
Figure 4 depicts the sensitivity of individual anti-venoms antibodies IgY by indirect ELISA; A- Cobra anti-venom IgG, B-Krait anti-venom IgG, C-Russell's viper's viper anti-venom IgG, D- Saw-scale viper anti-venom IgG Figure 5 depicts the venom cross-reactivity by Dot ELISA

Figure 6 depicts the venom cross-reactivity by western blot
Figure 7 depicts the dot ELISA analysis of bispecific antivenom IgY
Figure 8 depicts the dot ELISA analysis of monospecific antivenom IgY
Figure 9 illustrates bi-specificity of anti-venom IgY by lateral flow immunoassay
Figure 10 depicts a schematic representation of lateral flow assay for snake venom detection
kit.
DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the processes and devices belong. Although any process, device or system similar or equivalent to those described herein can also be used in the practice or testing of the process, device or system, representative illustrative methods and compositions are now described.
Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.
It is appreciated that certain features of the processes, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features

which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
The term "monospecific antibodies" in the context of the present invention is to be understood as an antibody which can bind to only one type of snake venom selected from a group comprising cobra venom, russell's viper's viper venom, krait venom or saw scale viper venom. The monospecific antibodies of the present invention have no cross-reactivity to any of the other three snake venoms of the Big Four snakes. The antibodies may be monoclonal or polyclonal.
The term "bispecific antibodies" in the context of the present invention is to be understood as an antibody which can simultaneously bind to two different types of snake venom from amongst snake venoms selected from a group comprising cobra venom, russell's viper's viper venom, krait venom or saw scale viper venom. The term may also refer to an antibody which can bind either to neurotoxic snake venoms (cobra venom and krait venom) or hemotoxic venoms (russell's viper's viper venom and saw scale viper venom). The antibodies may be monoclonal or polyclonal.
The term "venom-coupled beads" as used herein refers to a substrate which can be used for immobilization of snake venom on the surface of substrate. The venom-coupled beads can be used for attachment of antibodies to a specific antigen and subsequent removal of the antibody having reactivity to the antigen.
The terms "sample" or "biological sample" refers to biological fluids, such as whole blood, serum, plasma, tear, saliva, synovial fluid, cerebrospinal fluid, bronchial lavage, ascites fluid, bone marrow aspirate, pleural effusion, urine, as well as tumor tissue or any other bodily constituent or any tissue culture supernatant that could contain the antigen of interest. Samples can be obtained by any appropriate method known in the art.
The term "cobra venom" is intended to encompass any poisonous substance which is transmitted, that is subcutaneously or intramuscularly transmitted, by the bite or sting of a spectacled (Indian) cobra (Naja naja) into a mammal or bird, and which contains various toxins such as, but not limited to, hemotoxins, hemagglutinins, neurotoxins, leukotoxins, and endotheliatoxins.

The term "russell's viper venom" is intended to encompass any poisonous substance which is transmitted, that is subcutaneously or intramuscularly transmitted, by the bite or sting of a Russell's viper (Daboia russelii) into a mammal or bird, and which contains various toxins such as, but not limited to, hemotoxins, hemagglutinins, neurotoxins, leukotoxins, and endotheliatoxins.
The term "krait venom" is intended to encompass any poisonous substance which is transmitted, that is subcutaneously or intramuscularly transmitted, by the bite or sting of a common krait (Bungarus caeruleus) into a mammal or bird, and which contains various toxins such as, but not limited to, hemotoxins, hemagglutinins, neurotoxins, leukotoxins, and endotheliatoxins.
The term "saw scale viper venom" is intended to encompass any poisonous substance which is transmitted, that is subcutaneously or intramuscularly transmitted, by the bite or sting of a saw-scaled viper (Echis carinatus) into a mammal or bird, and which contains various toxins such as, but not limited to, hemotoxins, hemagglutinins, neurotoxins, leukotoxins, and endotheliatoxins.
DETAILED DESCRIPTION OF THE INVENTION
As those in the art will appreciate, the following detailed description describes certain preferred embodiments of the invention in detail and is thus only representative and does not depict the actual scope of the invention. Before describing the present invention in detail, it is understood that the invention is not limited to the particular aspects and embodiments described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention defined by the appended claims.
More specifically, the present invention relates to process for selection of monospecific and bispecific antibodies having binding affinity to one or more of snake venom. Further, the invention relates to lateral flow immunoassay devices developed for onsite detection of snake species involved in envenomation. The devices are highly species specific, sensitive, cost effective, simple, rapid, stable and portable.
For the first time, the inventors have been able to develop a process to develop specific antibodies against snake venoms of spectacled (Indian) cobra (Naja naja), common krait (Bungarus caeruleus), saw-scaled viper (Echis carinatus) and russell's viper (Daboia russelii).

The antibodies developed in this invention are either monospecific (not having cross reactivity to any of the other three species) or bispecific (not having cross reactivity to any of the other two species). Further, the inventors have developed lateral flow immunoassay devices using the antibodies generated.
The present invention represents an advancement over the existing methods for providing monospecific and bi-specific anti-venom antibodies and kits for determination of snake species involved in envenomation. The advances are characterized by the following features:
(a) Affordable: The antibodies and devices developed are highly inexpensive and can be solve the problems of snake envenomation in low and middle-income countries.
(b) Sensitive and Specific: The antibodies and device developed are highly sensitive and very specific to the snake venom responsible for envenomation.
(c) User friendly: The devices developed represent point of care portable solutions for determining the snake species responsible for envenomation. It can be used even by semi-skilled personnel. The determination is not dependent upon the expertise of the person conducting the test. The invention is specifically useful in the rural and semi-urban areas where extensive health-care facilities and expert physicians are not available.
(d) Rapid and Robust: The test results can be immediately ascertained. Further, the device does not require special storage equipment.
(e) Equipment Free: The devices do not require any sophisticated instrument for operation.
(f) Delivery to those who need it: The devices are extremely cheap, easy to transport and use. Hence, these devices can be easily delivered to the rural areas where there is a high requirement of the same.
The inventive approach used in the present invention has led to the development of devices and kits which would help a large proportion of the population across the world.
Before the processes and devices of the present disclosure are described in greater detail, it is to be understood that the invention is not limited to particular embodiments and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the processes and devices will be limited only by the appended claims.

The Big Four snake species spectacled (Indian) cobra (Naja naja), common krait (Bungarus caeruleus), saw-scaled viper (Echis carinatus) and russell's viper are responsible for enormous number of snake envenomation and deaths in India.
The major challenge in treatment of snake envenomation is dependent upon the following three factors:
1. Uncertainty regarding the species of snake involved
2. Due to uncertainty, polyvalent anti-snake venom formulation comprising antibodies of different species are administered which has side effects and causes collateral damage to internal organs
3. Due to complexity and cross-reactivity amongst the Big Four snake venoms, it has not been possible to develop antibodies, specific to a certain species.
Generation of antibodies
In one embodiment, mammalian models are used for production of species specific mammalian anti-venom IgG antibodies. Mammalian models such as mouse, rat, rabbit, equine, goat, guinea pig, sheep are used for production of species specific mammalian anti-venom IgG antibodies.
In another embodiment, avian models are used for production of species specific avian anti-venom IgY antibodies. Avian models such as chicken, duck, and kiwi are used for production of species specific avian anti-venom IgY antibodies. Avian antibodies such as chicken represents a cheaper model for production of antibodies as the antibodies can be abundantly harvested from egg yolk.
In a further embodiment, pre-immunized sera were collected from both mammalian as well as avian species which aided as a negative control.
In yet another embodiment, purified venom from the different species are administered to the models for immunization.
In a further embodiment, the venom along with the adjuvants are administered intramuscularly in avian systems and intravenously in mammalian systems.
In further embodiments, the antibodies are collected from blood in mammalian system and eggs from the avian system. Venom coupled beads for selection of antibodies from antibody pool
In one embodiment, the venom-specific antibodies generated were selected by coupling each venom with CNBr activated sepharose.
In another embodiment, the CNBr sepharose beads were activated in acid and transferred to a coupling buffer for coupling with tubes containing venom.

Selection of bi-specific antibodies from antibody pool
The venom from the Big Four snakes can be broadly classified into two categories:
1. Neurotoxic (venom of cobra and krait)
2. Hemotoxic (venom of russell's viper and saw-scale viper)
In one embodiment, the bi-specific antibodies, having reactivity only to neurotoxic venoms (cobra and krait venom) are selected.
In another embodiment, the bi-specific antibodies, having reactivity only to hemotoxic venoms (venom of russell's viper and saw-scale viper) are selected.
In a further embodiment, bi-specific antibodies, having reactivity only to two species amongst the Big Four snakes are selected.
In another embodiment, for selection of bi-specific antibodies, the pooled antibodies (IgG/IgY) against each venom was incubated with venom coupled beads. The antibodies which have reactivity towards the specific venom are coupled with the beads. The supernatant contains the antibodies which do not have any cross-reactivity. The selection process can be designed to develop bi-specific antibodies.
Selection of monospecific antibodies from antibody pool
In one embodiment, the monospecific antibodies, having reactivity only to a single venom are selected.
In another embodiment, for selection of monospecific antibodies, the pooled antibodies (IgG/IgY) against each venom was incubated with venom coupled beads. The antibodies which have reactivity towards the specific venom are coupled with the beads. The supernatant contains the antibodies which do not have any cross-reactivity. The cycle can be repeated for against venom of the other three species to develop monospecific antibodies. Development of immunoassay devices
Numerous immunoassay formats are known in the art. Immunoassays involve contacting a sample containing or suspected of containing an antigen of interest with at least one antibody that specifically binds to the biomarker.
A signal is then generated indicative of the presence or amount of complexes formed by the binding of antigens in the sample to the antibody (or other class of detection reagent). The signal is then related to the presence or amount of the antigen in the sample.

In one embodiment, the devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive immunoassay formats to generate a signal that is related to the presence or amount of the antigen(s) of interest.
Antibodies or other detection reagents may be immobilized onto a variety of solid supports for use in assays. Examples of suitable solid phases, include, but are not limited to nitrocellulose membranes, membrane filters, cellulose-based papers, beads (including polymeric, latex, and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, PEG gels, SPOCC gels, and multiple-well plates.
Antibodies or other detection reagents may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding.
Biological assays require labels for detection. In one embodiment, the detection label used is gold nanoparticle.
In another embodiment, the detection label is selected from a group comprising radioactive isotopes, enzymes, fluorophore, chemiluminescence markers and the like.
In one embodiment, a plurality of snake venom-associated capture antibodies is immobilized on the substrate in order to detect a plurality of different antigens in a single multiplex assay.
In another embodiment, the lateral flow immunoassay device comprises monospecific and bispecific antibodies as developed in this invention.
In yet another embodiment, the device detects a plurality of antigens selected from a group comprising cobra venom, krait venom, russell's viper venom and saw-scale viper venom.
In another embodiment, the lateral flow immunoassay device comprises sample pad, conjugate release pad, test lines, control line and absorbent pad.
In another embodiment, a housing configured to substantially enclose the device is provided. The housing is used to expose the sample application zone, maintain proper alignment of the materials and indicate positions of the test and control lines. EXAMPLES
Example 1: Preparation of venom antigens for immunization
The snake venoms of spectacled (Indian) cobra (Naja naja), common krait (Bungarus caeruleus), saw-scaled viper (Echis carinatus) and russell's viper (Daboia russelii) were obtained from the Irula foundation, Chennai, Tamilnadu, India.

CM-Sephadex C-50® resins (Sigma Aldrich) were prepared as per the manufacturer's instructions and the 20 mg venom was reconstituted in equilibration buffer and loaded onto the column.
The unadsorbed fractions were washed off with equilibration buffer and the adsorbed fractions were eluted using NaCl gradient. The fractions were analyzed by SDS-PAGE and the size fractioned venoms using cut-off filters were analyzed through HPLC for its purity. Purified venom components were quantified and emulsified with adjuvants for immunization.
Figure 1 depicts SDS-PAGE profiling of crude snake venom and Figure 2 depicts SDS-PAGE profiling of column separated snake venom.
Example 2: Generation of antivenom antibodies
The animal models like mouse, rat, rabbit, equine, goat, guinea pig, sheep were used for the production of species specific mammalian anti-venom IgG antibodies. Further, species specific avian IgY anti-venom antibodies were developed in chicken, duck, and kiwi.
Pre-immunized sera were collected from both mammalian as well as avian species which aided as a negative control. For immunization, 100 ug of purified venom from the different species with equal volumes of Freund's complete adjuvant was administered in avian systems (intramuscularly) and mammalian systems (intravenously).
After 14 days of priming, booster immunizations were administered in the increasing concentration from 125 ug - 200 ug of the venoms with an equal amount of Incomplete Freund's adjuvant at 12-day intervals.
At the end of fifth immunization, blood from the mammalian system and the eggs from the avian system were collected. Further, the blood and eggs were checked for specific antibody raised against the specific venom, and the antibodies were collected.
Example 3: Characterization of antivenom antibodies
The purified IgG and IgY anti-snake venom antibodies were checked for its concentration and purity by Lowry's protein estimation and SDS-PAGE. The developed antibodies were also assessed for its titer value against their respective venoms by indirect ELISA. Briefly, the microtiter plates were coated with 1 ug/well of respective venoms in bicarbonate buffer. Plates were incubated at 37 °C for 1 h and blocked with 3% BSA in PBS at 45 °C for 1 hr. Serial dilutions of respective anti-snake venom antibodies (IgY & IgG) and

negative control (Pre-immune antibody) (1:1000 - 1:1,28,000) were used in triplicates and incubated at 37 °C for 1 hr.
Following PBS-T and PBS wash, 100 ul of secondary antibodies (horseradish peroxidase conjugated) was added to the respective primary antibodies (anti-rabbit IgG/anti-chicken IgY) and incubated at 37°C for 45 min. Plates were washed as mentioned above and developed with coloring substrate TMB - H2O2. The reactions were stopped with 50 ul of 1 N H2SO4 and absorbance was read at 450 nm.
The purified antivenom IgY/IgG antibodies were further characterized by western blotting. Briefly, the venoms were blotted on to 0.45 um nitrocellulose membrane and it was incubated with 5% (w/v) skim milk in PBS for 1 h at 45°C to block unbound sites. Post blocking and PBS-T wash, the membrane was incubated with respective anti-snake venom IgG/IgY antibodies for 1 hour at room temperature. Following the primary antibody incubation, the membrane was washed thrice with PBS-T and incubated with anti-rabbit FIRP/anti-chicken HRP conjugate for 1 hour at room temperature. Subsequent to PBS-T wash, the membrane was developed with diaminobenzidine (Sigma, US) in Tris HC1 (pH 7.0) and hydrogen peroxide (Sampaio et al., 2014).
Figure 3 depicts the sensitivity of individual anti-venoms antibodies IgG by indirect ELISA; A- Cobra anti-venom IgG, B-Krait anti-venom IgG, C-Russell's viper anti-venom IgG, D- Saw-scale viper anti-venom IgG. Figure 4 depicts the sensitivity of individual anti-venoms antibodies IgY by indirect ELISA; A- Cobra anti-venom IgG, B-Krait anti-venom IgG, C-Russel's viper anti-venom IgG, D- Saw-scale viper anti-venom IgG.
Example 4: Preparation of venom coupling beads
The venom-specific antibodies generated in Example 2 were selected using column based systematic evolution of ligands by exponential enrichment procedures by coupling each venom with CNBr activated sepharose.
Briefly, the CNBr sepharose beads were activated in 1 mM HC1 for 15 min at room temperature. The beads were transferred to sintered glass funnel and washed with 200 mL of 1 mM HC1. The beads were washed with 5 mL of coupling buffer and it was transferred to the tubes containing venom, the tubes were rotated gently for 2 h at room temperature. The beads were centrifuged for 1 min at 2000 rpm and the supernatants were taken and the optical density

of the venom was recorded at 280 nm. The venom coupled beads were washed with 3x coupling buffer.
The coupling buffer was removed, and the beads were incubated with blocking buffer for 2 hr at room temperature. The beads were washed four times alternatively with low pH wash buffer and coupling buffer and the optical density of the supernatant was recorded. The gel was poured into the column and washed with five column volumes of sterile PBS and the column was stored in sterile PBS.
Example 5: Preparation of bi-specific antibodies
The venom-specific antibodies generated in Example 2 were selected using the venom coupled beads prepared in Example 4.
The basis of preparation of bi-specific antibodies include incubating the pool of antibodies with first venom-coupled beads and collecting the supernatant, wherein the supernatant comprises antibodies not having cross-reactivity with the first venom. This cycle is repeated once again against the other venom-coupled beads to receive antibodies not having cross-reactivity with the first and second venom.
0.8 mL of pooled antibodies (IgG/IgY) against each venom was incubated with 0.4 mL venom coupled beads for overnight, for a period in the range of 5-15 hrs at a temperature in the range of 0-10°C, preferably 4°C. The volume ratio of antibody to venom-coupled bead ranges from 4:1 to 1:4. Then, the tubes were centrifuged at 12000 rpm for 5 mins and the supernatant was incubated with another venom coupled bead as exhibited in Table 1. The cycle was followed alternatively until bi-specific antibodies were obtained without any cross-reactivity.



The bi-specific antibodies, so collected can be used for treatment of snakebite envenomation.
Figure 5 depicts the venom cross-reactivity by Dot ELISA. Figure 6 depicts the venom cross-reactivity by western blot. Figure 7 depicts the dot ELISA analysis of bispecific antivenom IgY.
Example 6: Preparation of monospecific antibodies
The basis of preparation of monospecific antibodies include incubating the pool of antibodies with first venom-coupled beads and collecting the supernatant, wherein the supernatant comprises antibodies not having cross-reactivity with the first venom. This cycle is repeated twice against the other two venom-coupled beads to receive antibodies not having cross-reactivity with the first, second or third venom.
The 0.8 mL bi-specific antibodies as obtained from the Experimental setups 1, 2, 3 and 4 in Table 1 were incubated with 0.4 mL of venom coupled beads as exhibited in Table 2 for overnight at 4°C. Then, the tubes were centrifuged at 12000 rpm for 5 mins and the supernatant was collected. The process was continued until antibodies were obtained without cross-reactivity.



Figure 8 depicts the dot ELISA analysis of monospecific antivenom IgY.
Example 7: Characterization of venom-specific antibodies
The venom-specific antibodies were assessed by dot ELISA. 1 ug venoms of cobra, krait, russell's viper and saw-scaled viper were coated onto a nitrocellulose membrane. After coating the membrane was blocked with 5% milk solution at 45C for 1 hr.
The primary anti-snake-venom IgG and IgY antibodies were added to the membrane and incubated at room temperature for 1 hr. Following PBST and PBS wash, the secondary antibodies conjugated with HRP corresponding to the respective primary antibodies (anti-rabbit IgG/anti-chicken IgY) were added to the membrane. Then, the membrane was washed with PBST and PBS to remove unbound secondary antibodies. The coloring substrate DAB-H2O2 (Aristogene, India) solution was added for color development.

The mono-specific and bi-specific antivenom antibodies (as exhibited in Table 1 and 2) generated against each snake venom with no cross-reactivity with other species was taken for the development of gold nanoparticle (GNP) conjugated species specific antivenom IgG/IgY antibodies.
Standard conjugation protocol was followed for this assay (Venkataramana et al., 2014). Briefly, diluted antibody (12ul) was added to 42 ul of reaction buffer in a microfuge tube and mixed gently. Antibody mixture (45 ul) was added to the gold nanoparticle and incubated for 15 mins at room temperature. The reaction was stopped by the addition of 5 ul of the quencher. The conjugated antibody GNP was characterized by photoluminescence and dynamic light scattering techniques. The antibody GNP conjugates were employed in the development of bi-specific and mono-specific venom detection kit.
A snake venom detection kit comprising a lateral flow immunoassay device was designed to detect the presence of the venoms of cobra, krait, russell's viper and saw-scale viper.
The device comprises a lateral flow membrane made up of nitrocellulose.
The mono-specific and bi-specific antivenom antibodies (as exhibited in Table 1 and 2) generated against each snake venom with no cross-reactivity with other species was taken for the development of gold nanoparticle (GNP) conjugated species specific antivenom IgG/IgY antibodies.
The membrane consists of different testing zones, such as, sample pad, conjugate release pad, test lines, control line and absorbent pad.
A biological sample is added to the proximal end of the strip at the sample pad. The sample migrates through this region to the conjugate release pad, where a conjugate, which is a detectable marker has been immobilized. The detection particles are conjugated to one of the specific antibodies.
The detection antibodies are anti-cobra venom antibodies, anti-krait venom antibodies, anti-russell's viper venom antibodies and anti-russell's viper venom antibodies. The detectable
1 1

marker is gold nanoparticles. The detection antibodies are present in a concentration of 100 ng/mL to 1 mg/mL.
In other embodiments radioactive isotopes, enzymes, fluorogenic reporters and chemiluminescence markers are used as detectable markers.
The sample re-mobilizes the dried conjugate detection antibodies. The biomarker in the sample interacts with the conjugate as both migrate into the next zone of the strip, which is the reaction matrix.
This reaction matrix is a porous membrane, onto which the other specific anti-bodies of the assay has been immobilized. These anti-bodies are known as capture anti-bodies. The capture anti-bodies immobilized on the membrane are anti-cobra venom antibodies, anti-krait venom antibodies, anti-russell's viper venom antibodies and anti-russell's viper venom antibodies. The capture antibodies are present in a concentration of 100 ng/mL to 1 mg/mL.
Excess reagents move past the capture lines and are entrapped in the wick or absorbent pad.
For detection of venoms, the ELISA principle is used. The presence of a visible signals on the test lines for cobra venom, krait venom, russell' s viper venom or saw-scale viper venom gives a conclusive determination.
Figure 9 illustrates bi-specificity of anti-venom IgY by lateral flow immunoassay.
The membrane is placed into proper housings. The housing is configured to substantially enclose the lateral flow membrane, panel of detection antibodies, detectable markers, capture antibodies, optionally in a disposable one-time use package. The housing is used to expose the sample pad, maintain proper alignment of the materials, and indicate positions of the test and control lines. The housing is made out of a suitable material, preferably plastic.
A schematic view of the device of the present invention is depicted in Figure 10. Example 9: Evaluation of developed immune assay method for venom detection
Developed method of test kit was evaluated for its efficacy and sensitivity using different venoms under varied conditions including concentration variations and mixing with other saliva and blood related components, which are usually mix with the venom samples at bite site as well as biological fluids which can be preferably used for test samples for snakebite envenomation victims. Developed kit successfully identified the target venoms of the study used.

1. A process for selection of monospecific antibodies from a pool of antibodies having
binding affinity to a snake venom selected from a group comprising cobra venom,
russell's viper venom, krait venom and saw scale viper venom, comprising the steps of:
a. incubating the pool of antibodies with first venom-coupled beads;
b. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first venom;
c. incubating the supernatant obtained in step (b) with second venom-coupled
beads;
d. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first or second venom;
e. incubating the supernatant obtained in step (d) with third venom-coupled beads;
f collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first, second or third venom; and g. purifying the supernatant to obtain monospecific antibodies.
2. A process for selection of bispecific antibodies from a pool of antibodies having binding
affinity to a snake venom selected from a group comprising cobra venom, russell's
viper venom, krait venom and saw scale viper venom, comprising the steps of:
a. incubating the pool of antibodies with first venom-coupled beads;
b. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first venom;
c. incubating the supernatant obtained in step (b) with second venom-coupled
beads;
d. collecting the supernatant, wherein the supernatant comprises antibodies not
having cross-reactivity with the first or second venom; and

e. purifying the supernatant to obtain bispecific antibodies.
3. The process as claimed in claim 1 or claim 2, wherein the antibodies are incubated with the venom-coupled beads at a temperature in the range of 0-10°C.
4. The process as claimed in claim 1 or claim 2, wherein the antibodies are incubated with the venom-coupled beads for a period of 5-15 hours.
5. The process as claimed in claim 1 or claim 2, wherein the volume ratio of antibody to venom-coupled beads ranges from 1:4 to 4:1.
6. The process as claimed in claim 1 or claim 2, wherein the beads are CNBr sepharose beads.
7. The process as claimed in claim 1 or claim 2, wherein the antibodies are avian or mammalian antibodies.
8. The process as claimed in claim 1 or claim 2, wherein the antibodies are polyclonal antibodies.
9. A lateral-flow immunoassay device for detecting the presence of snake venom in a biological sample, comprising:
a. a lateral flow membrane;
b. a panel of detection antibodies obtained using a process as claimed in claim 1
or claim 2;
c. a detectable marker conjugated to the detection antibodies; and
d. a panel of capture antibodies obtained using a process as claimed in claim 1 or
claim 2;
wherein the antibodies have binding affinity to cobra venom, russell's viper venom, krait venom and saw scale viper venom.
10. The device as claimed in claim 9, wherein the lateral flow membrane is a nitrocellulose
membrane.

11. The device as claimed in claim 9, wherein the detection antibodies and capture antibodies are present at a concentration in the range of 100 ng/mL to 1 mg/mL.
12. The device as claimed in claim 9, wherein the detectable marker is selected from a group comprising radioactive isotopes, enzymes, fluorogenic reporters, chemiluminescence markers and gold nanoparticles.
13. The device as claimed in claim 9, further comprising a housing configured to enclose the lateral flow membrane, panel of detection antibodies, detectable markers and capture antibodies.
14. A method for detecting snake venom using the device as claimed in claim 9, comprising the steps of:
a. adding a biological sample to the sample pad;
b. detecting the signals at the test lines configured for detecting cobra venom,
russell's viper venom, krait venom and saw scale viper venom;
c. correlating the signals at the test lines for presence of cobra venom, russell's
viper venom, krait venom or saw scale viper venom.