WO2017109660A1

WO2017109660A1 - One step rapid snake envenomation detection and differentiation kit

Info

Publication number: WO2017109660A1
Application number: PCT/IB2016/057751
Authority: WO
Inventors: Medha SONAVANE
Original assignee: RAJAS, Amit
Priority date: 2015-12-23
Filing date: 2016-12-17
Publication date: 2017-06-29

Abstract

The present invention relates to a rapid detection kit for determining the presence of venom toxin in the blood sample in one minute using an improvised lateral flow immunoassay. Further, it helps to differentiate the venom toxin as neurotoxin and hemotoxin. The components of the lateral flow immunoassay in the present invention are arranged in a defined manner to obtain the detection result in less time, like one minute.

Description

-:NON-PROVISIONAL APPLICATION FOR PATENT:-

ONE STEP RAPID SNAKE ENVENOMATION DETECTION AND DIFFERENTIATION KIT

FIELD OF THE INVENTION

The present invention relates to a rapid detection kit for differentiating neurotoxic and hemotoxic venomous snakebite. More particularly, a rapid detection kit determines the presence of venom toxin in the blood sample in one minute using an improvised lateral flow immunoassay and further, helps in discriminating the venom toxin as neurotoxin and hemotoxin. The components of the lateral flow immunoassay in the present invention are arranged in a defined manner to obtain the detection result in less time, like one minute BACKGROUND OF THE INVENTION

Snakebite is one of the leading medical crises in many countries. Venomous snakes account for a large number of deaths and injuries, worldwide. Snakes alone are estimated to inflict approximately 2.5 million venomous bites each year, resulting in more than 100,000 deaths. The actual number may be much larger. Southeast Asia, India, Brazil, and areas of Africa have the most deaths due to snakebite.

In venomous snakes, some have little effect in certain body tissues but some have such a toxic protein composition, which can cause life threatening effects or paralysis of specific tissue. Venom is complex mixture of proteins and enzymes. Venom is broadly classified in to four groups depending upon their action, Neurotoxic, Hemotoxic, Myotoxic and Cytotoxic. Neurotoxic venom directly affects nervous system which result into nervous dysfunction and death occurs due to respiratory paralysis if treatment is not given on time. Hemotoxic venom had its effect on red blood cells (RBC's) which causes damages the cells leads to inflammation, pain, tissue damage, internal bleeding and death if not treated. Myotoxic venom has its effect on muscles. Moreover, cytotoxic venom destroys any cell that comes in its way that may be skin, blood vessels, blood cells etc.

The most common and widely known snake species for snakebite incidents include Cobra (Naja naja), Krait (Bungarus caeruleus), Russell's viper (Vipera russelli), and Saw Scaled Viper (Echis carinatus.) are known as Big Four. Out of which Cobra and Krait has neurotoxic venom and Russell's viper and Saw Scaled viper has hemotoxic venom.

The post snakebite treatment is crucial to save lives as much depends on the efficiency of the primary/secondary health care and efficacy of the anti-venom administered, most of the times, in haste. Therefore, the administration of snakebite treatment based on the snake's venom type identified shall be the advantage to prevent deaths. Moreover, the identification of venom type at early stage also leads to quick treatment of snakebite patients. Therefore, there is a genuine and urgent need for a diagnostic test to identify snake species (venomous) which will enable prompt and accurate treatment to a victim of snakebite.

Thus, the present invention proposes a rapid identification kit for distinguishing neurotoxic and hemotoxic venomous snakebites. In addition, this kit can be used by a layperson and is not limited to medical practitioner. This kit has potential to go up to species-specific differentiation. In the present invention, rapid identification kit gives the differentiation output in less than a minute thus helping in early treatment of patients with snakebite. Accordingly, the present invention proposes one-step rapid snake-envenomation detection kit, which can provide solution to the above listed limitations.

OBJECTS OF THE INVENTION:

A primary object of this invention is to make a rapid one-step detection kit for determines the presence of venom toxin in the victim's blood sample in one minute using an improvised lateral flow immunoassay.

Another object of this invention is provide a rapid detection kit such that it can discriminates the venom toxin as neurotoxin and hemotoxin. Another object of the invention is to provide a detection kit that can be used by even a layperson and is not restricted to medical practitioners.

Another object of the invention is to provide rapid one-step detection kit, which is simple and efficient in operation and, cost effective. SUMMARY OF THE INVENTION:

Before the present invention is described, it is to be understood that present invention is not limited to particular methodologies and materials described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention.

The present invention relates to a rapid one-step detection kit for determining the presence of venom toxin in the victim's blood sample and further distinguishing the venom toxins as neurotoxic and hemotoxic in one-minute duration. The present invention provides a rapid one-step detection kit designed in such a manner that the result is achieved in one-minute duration. Therefore, the rapid detection kit of the present invention works on lateral flow immunoassay, wherein the immunoassay components are arranged in a defined manner to obtain the detection result in less time, like one minute. Everyone can use the rapid detection kit.

According to the present invention, a rapid one-step detection kit for determining the presence of venom toxin in the victim's blood sample and further distinguishing the venom toxins as neurotoxic and hemotoxic in one minute using lateral flow immunoassay comprising of a lateral flow strip wherein said strip has on its surface lateral flow components arranged in a defined manner to obtain the detection result in said one minute; at-least one sample pad and a conjugate pad to receive said whole blood sample and transferring it to nitrocellulose membrane. This nitrocellulose membrane can have multiple indicator lines laterally separated from each other being placed on the said membrane at specified distance with each other. These indicator lines have immobilized primary antibodies, wherein one of the said indicator line has said antibodies reactive with neurotoxic sample or a derivative thereof and another indicator line has specific antibodies reactive to hemotoxic sample or derivative thereof. The said membrane also has a one indicator line which has immobilized secondary antibodies reactive with primary antibodies, serving as a reading means for permitting the indicator lines to be read, thus permitting measuring the venomous toxin/antigen (neurotoxic or hemotoxic) in the blood sample by reading said indicator lines. According to the present invention, the lateral flow strip components are placed in a manner where components extend over each other in succession from sample pad, to conjugate pad, to nitrocellulose membrane, to absorbent pad. According to the present invention, the lateral flow strip components are arranged in defined manner such that, the said sample pad extends over said conjugate pad at- least 0.3cm of length and said nitrocellulose membrane extends over said conjugate pad at-least 0.3 cm of length of the conjugate pad and absorbent pad is at the distal end of the nitrocellulose membrane extending over at-least 0.3cm of the total length of the strip.

According to the present invention, the said indicator lines consisting of two test lines and a control line are placed separated by at least 0.7 cm from each other on the nitrocellulose membrane of the strip.

According to the present invention, the said antibodies immobilized are preferably species-specific primary antibody specific to venom. For identifying neurotoxins in the blood sample, antibodies that are raised against Cobra (Naja-Naja) and Common Krait (Bungarus caeruleus) in rabbit and called as antineurotoxic rabbit antibodies as these snakes are known to have neurotoxins in their venom. Similarly, primary antibodies specific to hemotoxin are developed using snakes like Russell's Viper (Vipera russelli) and Saw-Scaled Viper (Echis carinatus) as they are known for have hemotoxins in their venom. Thus, the primary antibodies are developed in rabbit against the two toxins. Further, for raising these antibodies whole venom or different isolated antigenic proteins can be used.

According to the present invention, the secondary antibodies immobilized are preferably secondary antibodies raised in goat against the primary antibodies and are referred as anti-rabbit goat antibodies.

According to the present invention, the said conjugate pad contains the said primary antibodies labeled with nanoparticle size 20-40 nm selected from a group consisting of colloidal gold particles or latex bead or cellulose bead or silver nanoparticles or polystyrene latex beads or silica particles and beads or combination thereof. According to the present invention, the said primary and secondary antibodies can be raised in any animal model.

Other objects, features and advantages of the present invention will become apparent from the following description when taken in conjunction with the accompanying drawings wherein like characters of reference designate corresponding parts throughout the several views.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

These and other features, aspects and advantages of the present invention are better understood when the following detailed description of the invention is read with reference to the accompanying drawings, in which:

Figure 1 of the accompanying drawings illustrates a schematic of the lateral flow immunoassay.

Figure 2 illustrates the principle of lateral flow immunoassay of Figure 1 .

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described, it is to be understood that this invention is not limited to particular methodologies and materials described, as these may vary as per the person skilled in the art. It is also to be understood that the terminology used in the description is for the purpose of describing the particular embodiments only, and is not intended to limit the scope of the present invention.

Before the present invention is described, it is to be understood that 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 this invention belongs. Further, it is to be understood that the present invention is not limited to the methodologies and materials similar, equivalent to those described herein can be used in the practice, or testing of the present invention, the preferred methods and materials are described, as these may vary within the specification indicated. Unless stated to the contrary, any use of the words such as "including," "containing," "comprising," "having" and the like, means "including without limitation" and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but may be implemented in various combinations. The described embodiments of the invention and the disclosed examples are given for the purpose of illustration rather than limitation of the invention as set forth the appended claims. Further, the terms disclosed embodiments are merely exemplary methods of the invention, which may be embodied in various forms.

According to one of the embodiment of the present invention, the one-step detection kit for determining the presence of venom toxins in the victim's blood sample and further distinguishing the venom toxins as neurotoxic and hemotoxic in one-minute duration. The said rapid detection kit is designed in such a manner that the result is achieved in one minute and it is easy to use even by layperson. The present invention relates to lateral flow immunoassay in which the immunoassay components are arranged in a defined manner to obtain the detection result in less time, like one minute. The present invention further describes the detection kit, which will help to distinguish between neurotoxic and hemotoxic venomous snakebite. Accordingly, the present invention relates to an improved lateral flow immunoassay that detects and distinguishes between neurotoxic and hemotoxic venomous snakebites in the victim's blood sample. According to another embodiment of the present invention, the rapid detection kit practices Lateral Flow Immunoassay (LFIA) based on antigenantibody agglutination reaction. It has less reagent requirement, cost effectiveness and, has suitability for collecting samples outside a laboratory, which makes it significant over other tests. The known working principle of the lateral flow immunoassay i.e. antigen antibody reaction is achieved with the help of capillary flow action due to which bio molecule involved in the reaction travels across the nitrocellulose membrane as indicated by a color band from attached gold beads.

In the present invention, the components of said lateral flow are arranged in a defined manner to obtain the detection result in one minute. Several factors like flow rate, length of membrane, membrane capacity etc., are standardized to achieve the detection results in one minute. Thus, the further embodiment mentions the preferred parameters that are desirable to achieve the venomous neurotoxic and hemotoxic toxins detection result in one minute. PRINCIPLE AND TEST PROCEDURE

According to the present invention, the lateral flow immunoassay is used for rapid detection of venom in blood sample and further to differentiate the venom toxins as neurotoxic and hemotoxic. As illustrated in Fig.1 of the accompanying drawings, the kit includes an immuno-chromatographic strip (hereinafter 'strip') comprising of sample pad (1), a gold conjugate pad(2), nitrocellulose membrane(7), and absorbing pad(6). The materials make contact with each other in succession from sample pad, to conjugate pad, to nitrocellulose membrane, to absorbent pad. The blood sample is added to the sample pad, the materials comprising the strip act as wick and cause the sample to travel laterally across the strip until it reach the distal end, the absorbent pad(6). Conjugate and sample pads should be hydrophobic and importantly have good adsorption capacity. The nitrocellulose material consists of three lines referred as control line (5) and test line 1 (3) for neurotoxin and test line 2 (4) for hemotoxin.

As illustrated in Figure 2 of the present invention, the sample(1) is passed from sample pad (6) to conjugate pad having antigen specific antibody labeled with nanoparticle, size of 20-40nm (2) where the sample venom antigen reacts with the conjugated primary antibodies and forms a conjugate antibody-antigen complex (hereinafter the conjugate complex). This conjugate complex along-with the unbound conjugate antibodies (7) travels towards nitrocellulose membrane via capillary movement (3). As already known, unbound sites of antigens can bind with further primary antibodies, thus the test lines contains primary antibodies specific to neurotoxic toxin/antigen in test line 1 and hemotoxic toxin/antigen in test line 2. As the conjugate complex travels further, the primary antibody captures the conjugate complex. The conjugate complex on binding with primary antibodies contains antigen sandwiched between labelled antibody and primary antibody forming primary antibody-conjugate complex (10). At the same time, unbound conjugate antibody travels further and is captured at control line by secondary antibody (1 1) to form secondary antibody conjugate formation. The color indication at control line indicates the travelling of sample across the membrane. The excessive sample and conjugate antibodies are further collected in absorbent pad (9).

The primary antibodies are anti-snake venom antibodies raised against venomous snakes. Further, for raising these antibodies whole venom or different isolated antigenic proteins can be used. The colored line in the test line (12) denotes presence of neurotoxin venom toxin in the sample and colored line in test line (13) denotes presence of hemotoxin venom toxin in the sample, while control line (14) should always appear regardless of the presence or absence of antigen in the sample. After passing these reaction lines, the sample enters the final porous material (9), the absorbent pad that simply acts as a waste container.

According to one of the embodiments of the present invention, the lateral flow immunoassay works on an immune-chromatographic principle. In the present invention, a whole blood sample containing the venom toxin collected from the victim's affected area is introduced on the sample pad. The sample travels laterally via capillary movement towards conjugate pad. The venom antigen (if present) will form a complex. The conjugate pad has immobilized primary antibodies coated with gold nanoparticles (i.e. specific antivenom antibodies raised against the specific venomous snakes) on the conjugate pad. This so formed antigen+conjugate-antibody complex, travels towards the nitrocellulose membrane. The nitrocellulose membrane has three indicator lines referred as test Iine1 , test line 2 and control line. The test line 1 has immobilized primary antibodies specific to neurotoxic venom components and test line 2 is immobilized primary antibodies specific to hemotoxic venom components, which has affinity to bind with venom antigen. As known in prior art, antigen has multiple binding sites, thus the primary antibodies on test lines are bound to antigen complexed with conjugate antibodies. This binding results in colorations. The binding forms a red coloration on the test line indicating the presence of venom antigen. The intensity of the red coloration on the test line (T1 or T2) is directly proportional to the amount of neurotoxin or hemotoxin venom toxins present in the sample respectively. An unbound conjugate antibody travels further on the membrane and is captured at control line by secondary antibody. These secondary antibodies are produced specific to primary antibodies. In the control line, the conjugated antibodies forms complex with the secondary antibodies resulting in red coloration. The red line on the control line acts as a positive control to assure that functional, conjugated antibody has travelled throughout the system. A colored line in the control line should always appear regardless of the presence or absence of venom antigen in the whole blood sample.

The said primary antibodies used in the present invention are raised in rabbit against known four snakes i.e. (Indian cobra, Naja naja; Common krait, Bungarus caeruleus; Russell's viper, Daboia russelii; Saw-scaled viper, Echis carinatus). The said primary antibodies (1) raised against Cobra (Naja-Naja) and Common Krait (Bungarus caeruleus), are anti-neurotoxic rabbit antibodies, whereas primary antibodies (2) raised against Russell's Viper (Vipera russelli) and Saw-Scaled Viper (Echis carinatus) are anti- hemotoxic rabbit antibodies. Further, for raising these antibodies whole venom or different isolated antigenic proteins can be used. The said primary antibodies are further labeled with nanoparticle, size of 20-40nm, preferably colloidal gold particles which are immobilized on conjugate pad. These antibodies are referred as 'Anti snake venom antibodies' (ASVS), while secondary antibodies used are species specific antibodies specific to these primary antibodies. The said secondary antibodies raised in goat against primary antibodies i.e. anti-rabbit goat antibodies.

ASSEMBLY OF IMMUNOCHROMATOGRAPHIC STRIP:-

In the present invention, sample pad is made up of glass-fiber membrane. The desired thickness of sample pad is kept as 370 μηι and length and breadth of sample pad is effectively kept as 1 .5cm X 3mm. Similarly, conjugate pad is made from glass fiber membrane with the preferable 0.5cm x 3 mm dimensions.

Nitrocellulose membranes with the dimension of 3cm x 3mm while wicking/absorbent pad made from cotton linters is of 1 .9 cm in length. The distance between test line 1 , test line 2 and control line is 0.7 cm respectively. All these membranes are assembled on the backing material. The backing materials are typically polystyrene or other plastic materials coated with a medium to high tack adhesive. All components of the lateral flow assay are laminated to the backing material to provide rigidity and easy handling of the strip. The backing material is coated with a pressure-sensitive adhesive to hold the various components in place.

Determining the arrangement of the lateral flow immunoassay components, specifically the partial extending over of the components on each other is critical to the success and reproducibility of a test strip. It is important for the success of a test that the components extending over each other are correctly defined in a way compatible with the dimensions of the test strip at the same time it takes into consideration the limits of the tolerance. Thus, in the present inventions the sample pad partly extends over the conjugate pad of 0.3 cm of length. Similarly, 0.3 cm of nitrocellulose membrane is placed under conjugate pad. The distance between control line and test Iine1 and test line 2 is 0.7cm. Absorbent pad is placed at the end towards the control line with again extending 0.3cm over nitrocellulose membrane (As illustrated in Fig. 1 of the accompanying drawing). It should be obvious to the person skilled in the art that variation in the positioning of the materials changes the degree of overlap and, consequently, the flow properties of the finished strip. While considerable emphasis has been placed herein on the specific elements of the preferred embodiment, it will be appreciated that many alterations can be made and that many modifications can be made in preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiments of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

Claims

We claim,

1 ] A rapid one-step detection kit for detecting the presence of hemotoxin and neurotoxin venom toxins in the whole blood sample in one minute using toxin specific primary antibodies in lateral flow immunoassay.

2] The rapid one-step detection kit of claim 1 , wherein the said lateral flow immunoassay comprises of a lateral flow strip having a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad arranged in defined manner to obtain the result in said one minute.

3] The rapid one-step detection kit of claim 1 , wherein the said nitrocellulose membrane of the said lateral flow strip has at least three indicator lines laterally separated from each other such that the resultant coloration of the lateral flow immunoassay are distinctly visible.

4] The rapid one-step detection kit of claim 1 , wherein at least one of the said indicator line on the said nitrocellulose membrane of said lateral flow immunoassay acts as a color indicator for presence of neurotoxin.

5] The rapid one-step detection kit of claim 1 , wherein at least one of the said indicator line on the said nitrocellulose membrane of said lateral flow immunoassay acts as a color indicator for presence of hemotoxin.

6] The rapid one-step detection kit of claim 1 , wherein at least one of the said indicator line of the said nitrocellulose membrane of said lateral flow immunoassay acts as a reading means for completion of test.

7] The rapid one-step detection kit of claim 1 , wherein the said indicator line consists of primary antibodies raised using whole venom or fraction of venom or isolated venom protein as antigen, specific to neurotoxin venom in blood sample or derivative.

8] The rapid one-step detection kit of claim 5, wherein the said indicator line consists of primary antibodies raised using whole venom or fraction of venom or isolated venom protein as antigen, specific to hemotoxin venom in blood sample or a derivative thereof. ] A rapid one-step detection kit of claim 1 , wherein the said primary antibody specific to neurotoxin venom can be raised in an animal model using whole venom or fractions of venom or isolated venom proteins as antigen, not limiting to rabbit; against the venomous snakes selected from Cobra (Naja- Naja), Common Krait (Bungarus caeruleus). 0] A rapid one-step detection kit of claim 1 , wherein the said primary antibody specific to hemotoxin venom is raised in an animal model using whole venom or fractions of venom or isolated venom proteins as antigen, not limiting to rabbit; against venomous snakes selected from Russell's Viper (Vipera russelli), Saw-

Scaled Viper (Echis carinatus).