FORM 2
THE PATENT ACT 1970
(39 of 1970)
AND
The Patent Rules 2003
(sec. 10; rule 13)
COMPLETE SPECIFICATION
1. TITLE: HUMAN PLASMA PROTEINS-GNp CONJUGATE -
AN ALTERNATIVE NOVEL POLYVALENT ANTI SNAKE VENOM (ASV)

DESCRIPTION
The following describes the nature of an invention, its characteristics and method of its production and the manner in which it is to be used.
3.1 TITLE:
Human plasma proteins-GNp conjugate - An alternative novel Polyvalent Anti Snake Venom (ASV).
3.2. FIELD OF INVENTION Biophysics
The invention involves in vitro development of a Gold nanoparticle (GNp)-protein conjugate that has potential of being used as an alternative to conventional Anti Snake Venoms (ASV) derived from immunized animals like horse and sheep.
■r /
3.3 PREAMBLE
It is estimated that globally more than 5 million persons per year are bitten by snakes. In India alone the mortality is believed to be around 30,000. In West Bengal an annual incidence of snakebite of 0.16% and a mortality rate of 0.016% per year is reported. Maharashtra reports an incidence of mortality from snake bites to be 2.4 per 100,000 persons per year. Other Indian states with high incidence of snakebites cases are Tamil Nadu, Uttar Pradesh, and Kerala.

In south Asian countries the situation is similar. In Myanmar (Burma) Russell's vipers are responsible for 90% of snake bite cases. In Bangladesh, a survey of 10% of the country in 1988-1989 revealed 764 bites with 168 deaths over the one-year period. In Vietnam there are about 30,000 bites per year. Among the Malayan rubber plantation workers the snake bite fatality was 22%, while in Pakistan an estimated 20,000 snakebite deaths occur each year. In Nepal there is an estimated number of 20,000 snakebites each year, mainly in the Terai region. Fishermen are also occasionally bitten by sea snakes but they rarely reach hospitals alive.
There are three major families of venomous snakes; the Elapidae that includes cobra, king cobra, krait and coral snake; the Viperidae that includes all varieties of Vipers and the Hydrophidae family of sea snakes.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700615/https: //www.ncbi.nlm.nih.gov/pmc/articles/PMC2700615/ (downloaded 19.02.2020)
Snake venoms comprise a mixture of biologically active proteins and poly-peptides along with other non-protein components including carbohydrates, lipids, amines, and inorganic salts. Proteins and polypeptides can be classified into enzymes like phospholipase A2 (PLA2), serine metalloproteases (SVMP), serine proteases (SVSP) and L-amino acid oxidases (LAAO)]. The non-enzymatic Substances are three-finger toxins

(3FTx), kunitz peptides (KUN) and disintegrins (DIS). The actual composition of snake venoms varies depending on the variety of snake family, genus and species, geographical location and age and size of the snake. For instance, 3FTx and PLA2 generally predominate in venoms of elapid snakes, while PLA2, SVMP, and SVSP are the most common in vipers. Variations in snake venom composition are important and affect development, production, and modality of treatments using anti-venoms.
The treatment of snake envenomation principally involves the administration of specific anti-venoms for the snake species. The snake anti-venoms are antibodies or antibody fragments which are derived from the plasma of animals typically horses and sheep that have been immunized with several species of snake venom. The intravenously injected antibodies IgG immunoglobulin or antibody fragments bind with and neutralize free venom in the patient's plasma which reverses or prevents further toxic effects.
However, animal-derived anti-venoms being foreign proteins also carry a risk of hypersensitivity reactions which can result in cutaneous and multi-organ reactions that are potentially life-threatening. The present invention is an attempt to overcome this limitation.
Anti-venoms also remain expensive and their supply is often limited in some regions. An alternative and equally effective

anti-venom when developed may improve their easy availability and cost-effectiveness. The present invention addresses this problem as well.
Snake Anti Venoms (ASV) derived from animals has several uncertainties in respect of their efficacy dependence on the source animal species. The present invention does not require any animals for its manufacture and would thereby not involve this uncertainty.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848174/ (Downloaded 19.02.2020)
SUMMARY
According to WHO report, snake envenomation is considered to be one of the most neglected health hazards mainly among the farmers, forest guards, plantation workers, laborers etc. Snake bite victims die due to lack of first aid measures and delayed medical attention and administration of anti-snake venom (ASV) and its non-specific reaction. Often fatalities occur due to snake bite due to problems that are associated with the treatment itself. The presently available ASV contains polyvalent antibodies against venom of several snake species which are not species specific. It also contains equine proteins that are not suitable for human system. Thus there is a need to develop an alternative ASV complex which be compatible to human body and which does not show any hypersensitive reactions. The present invention is an attempt in this direction.

As gold nano-particles hold great medicinal properties an attempt has been made to develop soft and hard BSA-GNp conjugates and to explore their potential as ASV. The characterization of gold nano-particles and BSA-GNp conjugates was carried out using DLS, UV-visible spectroscopy and fluorescence spectrometry. Their anti-venom activity against cobra and viper venoms was confirmed in vitro through biochemical assays like haemolysis of human erythrocytes, protease activity and acidimetry.
Bovine Serum Albumin (BSA) being an animal protein, human plasma protein-GNp conjugates were prepared. Its biophysical characteristics as studied using above spectroscopic techniques revealed strong interaction between gold nano-particles and human plasma proteins. This conjugate was assessed in vitro for anti-venom activity. It was found that hemolytic activity of cobra and viper venom was reduced significantly by 94.16% and 93.19% respectively. Protease activity of cobra and viper venom was also reduced by 61.15% and 66.06% respectively. Phospholipase A2 activity was also reduced by 16.18% and 46.05 % respectively. Biocompatibility of soft and hard conjugates was assessed using simple agglutination test. It is concluded that the invention product human plasma protein-GNp conjugates in general and particularly the soft conjugates hold great anti-snake venom potential.

COMPLETE SPECIFICATION
4.1 Introduction
Snake bite is one of the major health hazards in tropical countries accounting for about 2.7 million envenomations with about 1.37 lakh deaths per year over the world. Most of the evenomations occur in Africa, Asia and Latin America. In India around 50,000 snake bites induced deaths occur every year. Major venomous snake species in India are Indian cobra (Naja naja), common Krait (Bungarus caeruleus\ Russell's viper (Daboia russelii) and Saw scaled viper {Echis carinatus). The venom is salivary secretion of venomous species secreted to immobilize and digest the prey.
4.2 Scientific Background
Snake venoms contain various enzyme proteins, polypeptides, nerve growth factors, nucleotides etc. The major components responsible of the pharmacological activity of venoms are proteins and peptides (Casewell N. R. et. al. 2014) including acetylcholinesterase(AChE), phospholipase A2, hyaluronidases, L-amino acid oxidases, phosphodiesterases, serine & metalloproteases, C-lectins, disintegrins, prothrombin activators etc. (Xiao H.etal. 2017).
Snake bites cause acute medical emergencies including haemorrhage, disruption of haemostasis, neuromuscular

paralysis, tissue necrosis, myolysis, cardiotoxicity, kidney failure, thrombosis and death if not treated in time. Following snake bites many patients face permanent disabilities including blindness, restricted mobility, extensive scarring, contractures and amputations. These pharmacological effects are mainly due to Phospholipase A2, Proteases, AChE, hyaluronidase, prothrombin activator and oxidases.
In rural areas, lack or delay of medical services increases the mortality rate and morbidity due to snake bites. Several folk medicines and herbs have been investigated for their anti-venom activity by researchers. Such herbal compounds have been hypothesized to act by protein precipitation, enzyme inactivation, chelation, adjuvant action, antioxidant action, protein folding hypothesis and combined action of all these reactions among which protein precipitation-inactivation hypothesis is the most accepted one [Gomes A. 2010].
However the only medically approved effective treatment available is anti-snake venom (ASV), enzyme (pepsin) refined Fab fragments of IgG antibodies isolated from serum of animals like horse or sheep that have been immunized with venom of one or more species. The ASV can be monovalent that can neutralize venom of only one species or polyvalent, having ability to neutralize venoms of several species of venomous snakes.

In India only polyvalent ASV is available. The ethanolic extract of Andrographis paniculata is shown to have anti-cobra venom potency by Premendran S. J. et. al. in 2011. Its anti-venom activity was shown to be increased when used along with ASV. However about 20% of the ASV treated patients suffer early (anaphylactic and pyrogenic reactions) or late reactions like delayed serum sickness, pruritus, urticaria, arthralgia, lymphadenopathy and encephalopathy because of the ASV heterogeneity. Thus there is a need to develop effective and reliable treatment for snake envenomation.
Nano-science has shown promising outcomes in the field of medicine [Sarmento B. 2019]. The present invention describes anti-venom activity of gold nano-particles when conjugated with proteins like BSA (commercially available Bovine Serum Albumin) and whole human plasma proteins against cobra and viper venoms as confirmed in vitro their anti-venom activity on hemolysis, acidimetry, and protease assay. Further the conjugates were analyzed for their biocompatibility using standard agglutination test.
4.3 Prior Art
Anti-venom effects of C-60 fullerene (carbon nanoparticles) have been studied by Karain et. al. [2016]. The study revealed that C-60 fullerene prolongs survival of crickets envenomed

with Crotalusoreganushelleri venom. However in this study the crickets were injected with C-60 fullerene 48 hours before the administration of venom. One more study by Suman B. et. al. [2018] contributes to the use of nano-materials against venom. The study exhibits the anti-venom effect of green synthesized silver nano-particles (by Trichodesmaindicum) against Cobra venom.
Being a precious metal, gold has caught the attention of medical scientists. Colloidal gold has been studied extensively for its therapeutic values. 'Quinta essentiaauri' by Paracelsus dictates medicinal importance of colloidal gold. He used potable gold, gold chloride reduced by vegetable dig outs in alcohol or oils. He used it to treat several mental diseases and syphilis [Daraee H. et. al. 2014]. A study by Saha K. and Gomes A. [2014] illustrates the potential of GNp conjugated with 2-hydroxy-4-methoxy benzoic acid (HMBA) against nephrotoxicity, myotoxicity and hepatotoxicity induced by Russell's viper venom in albino mice.
Present Invention 4.4.1 Materials
Crude lyophilized Naja naja (Indian cobra) & Doboia russelli
(Russell's viper) venom was procured from Haffkine institute, Parel, Mumbai.
BSA (Himedia), Chloroauric acid (Molychem), Casein (SDFCL), Tri-Sodium citrate (SRL), Calcium chloride

(SDFCL), Sodium chloride (Himedia), Sodium deoxycholate (Himedia), Trichloroacetic acid (LOBA Chemie), Sodium hydroxide (SDFCL), Sodium carbonate (LOBA Chemie), Copper sulphate (SRL), Sodium potassium tartarate (SDFCL), Folin ciocalteu reagent (SDFCL) were obtained from market and were of AR grade.
4.4.2 Methods
Venom (10mg/ml stock solution) and GNPs were prepared in double distilled water. Saline pH 7.4 was used as diluents for hemolysis and whole blood clotting test, whereas for acidimetry and protease activity double distilled water was used as diluents. All experiments were performed in triplicates.
4.4.2.1 Synthesis of gold nano-particles:
Gold nanoparticles (GNps) were synthesized using chemical reduction method [Mariam J. J. et. al. 2016] previously introduced by Turkevitch [1951] with slight modification. Herein 0.25mM gold salt (HAuCl4) was heated to 82°C, with continued stirring on hot plate magnetic stirrer. One percent (1 %) tri-sodium citrate (3mL) was added drop-wise once temperature reached to 82°C. Reaction was stopped as the colour of solution was changed to purple and finally to deep wine red. Synthesized GNps were cooled and stored in dark. Characterization was carried out using UV-visible spectroscopy

temperature reached to 82°C. Reaction was stopped as the colour of solution.was changed to purple and finally to deep wine red. Synthesized GNps were cooled and stored in dark. Characterization was carried out using UV-visible spectroscopy (Implen nanophotometer) and Zetasizer nano ZS90 dynamic light scattering (Malvern) system.
The concentration of nano-particles was calculated in molar units following the method of Lui X. et. al. (2006). Total number of gold atoms (Nt) initially added to the solution was divided by average number of gold atoms per nano-particle (N) by assuming complete reduction of Au+3 to Au° (Eq.l).

Where, V is volume and NA is Avogrado's constant. Average number of gold atoms in each nano-particle is 30.89602D , where D is the diameter of nano-particle.
4.4.2.2 Preparation of protein nano-particle conjugates:
Protein (BSA) nano-particle conjugates were prepared by simply incubating gold nano-particles of particular concentration with proteins. Gold nano-particles (8.32nM) were incubated with 3 mg/ml BSA for half an hour and it was denoted as conjugate-SC2 (soft conjugate -2), this was centrifuged at 15,000 rpm for 15 minutes at room temperature and the supernatant was decanted. The residue pellet was designated HC2 (hard conjugate 2) and reconstituted to original volume in distilled water for further analysis.

temperature and clear plasma was separated. The plasma was diluted with normal saline in 1:5 ratios. 8.32 nM GNps were incubated with 200µL diluted blood plasma for 30 minutes, which was designated and used as conjugate-SC1. On centrifugation at 15,000 rpm the residue pellet was recovered and designated as Conjugate-HC1 (hard conjugate 1), which was reconstituted to original volume with distilled water.
Here onwards the prefix 'SC denotes the whole liquid material of the GNp-proteins conjugate with plasma or BSA whereas 'HC' denotes the residue pellet after centrifugation of SC. The post-fix '1' represents the human plasma sample while '2' denotes the BSA
4.4.2.4 UV-Visible spectroscopy:
UV visible measurements of various venoms were recorded in the range of 200-600nm. 40µ.g/ml Cobra venom and 60µg/ml Viper venom were incubated with increasing concentrations of GNps for 30 minutes and changes in the spectra venom were analyzed using UV-visible spectroscopy.
4.4.2.5 Fluorescence spectroscopy:
The fluorescence spectroscopy (Varian, Cary Eclipse) was performed by exciting venom at 280nm and the emission spectra were recorded in the range of 310-500nm by setting the excitation and emission slit width at 10 nm. PMT voltage was set to 530V and path length to 10 nm. Interaction of venom with

increasing concentration GNps (2.3nM to 19.18nM) was studied by recording fluorescence spectra of venom after 30 minutes of incubation.
4.4.2.6 Haemolytic activity:
Hemolysis was assayed according to the method described by
Malagoli D. (2007) with some modifications. Whole blood obtained from a healthy volunteer was centrifuged at 2000 rpm to separate Red blood cells (RBCs). The pellet RBCs were washed thrice with normal saline (0.9% NaCl, pH 7.4). A 5 per cent RBCs suspension was prepared in normal saline. Further venom was diluted to various concentrations varying between 10|ig/mL and 100n,g/mL and was incubated with 500^iL RBCs for lhour. After 1 hour incubation it was centrifuged at 2000 rpm and absorbance of supernatant was recorded at 580nm. The percentage haemolysis was calculated using Eq.2

Saline was used as negative control and distilled water as positive control. The HU5o (haemolytic unit 50) the concentration of venom at which 50% haemolysis occurs was determined and was used for further inhibition studies.
f. Inhibition study was carried out by pre-incubating venom with
gold nanq-particles conjugates 1 & 2 for 1 hour and then with

500(iL RBCs the percentage haemolysis was calculated using same procedure mentioned above.
4.4.2.7 Protease assay:
Proteolysis of casein protein by venom proteases (Greenberg,
1955) was performed at pH 7.4. Casein (200µg/ml) and Venom (200µg/ml) and 2mM Calcium chloride were incubated in 2ml distilled water for an hour at 37°C. The reaction was terminated by adding 100µl Trichloroacetic acid (TCA). The mixture was filtered and 1ml of filtrate was used to determine the concentration of tyrosine released in the mixture. Tyrosine concentration was determined by Folin-Lowry method using L-tyrosine as a standard. Enzyme activity was expressed in unit/hr where 1 unit corresponds to 0.02µM tyrosine release. Assay was performed for all venom complexes to understand their inhibition profile.
4.4.2.8 Acidimetry assay (phospholipase A2 activity)
Acidimetric assay was performed using a method described by Tan N. H. & Tan C. S. (1988) with slight modifications. Phospholipase A2 activity was studied using egg yolk, a rich source of lecithin. Egg yolk suspension was prepared by mixing equal volumes of egg yolk, 18 mM calcium chloride and 8.1 mM sodium deoxycholate. The pH of this suspension was adjusted to 8.00 using 0.1N sodium hydroxide. A 15ml egg yolk suspension was added to 100µl of reaction mixture containing venom (200µg/ml) in order to initiate hydrolysis and pH change

was recorded as a function of time. A unit decrease in pH corresponds to fatty acid release. Enzyme activity was
calculated using Eq.3.

Appropriate control was used in place of reaction
mixture. Inhibition studies were carried out by pre-incubating venom with GNPs, soft coronas & hard coronas.
4.4.3 Results
4.4.3.1 Characterization of Gold Nano-particles
GNps were synthesized as described above. The change in colour of solution from colourless to purple and to deep wine red confirmed the synthesis of nano-particles. Plasmon absorbance is responsible for characteristic colour of gold nano-material. Its intensity and surface plasmon band gives information of about size, shape and dispersity of GNps, UV-visible spectroscopy and is widely used to study their optical properties. Surface plasmon resonance peak for gold nano-particles was observed at 520 nm [Fig. 1A] and DLS (Dynamic Light Scattering) showed the average hydrodynamic size of the nano-particles to be 18 nm [Fig.IB]. Concentration of synthesized GNps was found to be 27.74 nM as calculated using Eq. 1 as describe above.
Figure 1- A. shows the absorption at different wavelength varying from 200 to 800 nm
Figure 1 -B shows the DLS intensity as a function of particle size

4.4.3.2 Characterization of venoms interaction with GNp (i) UV-Visible Spectroscopy
UV-visible spectra of both cobra and viper venom is given in Figure.2. Since venom is a cocktail of proteins, it gives an absorbance maximum at 276 nm, which is predominantly due to tyrosine amino acid and at 520 nm. Cobra venom showed increase in absorbance intensity with increasing concentration of GNps at 276 nm accompanied by a blue shift of 2-6 nm. Similarly viper venom exhibited increase in absorbance intensity with increasing concentration of GNps at 277 nm with a blue shift of 2-4 nm. Increase in absorbance intensity of venom upon interaction with GNps establishes formation of ground state complexes. This increase in the intensity indicates change in the microenvironment of proteins. Amino acids are buried in the hydrophobic core of the protein and when they become exposed to the aqueous environment; blue shift occurs (Schmid F. X. 2001). The observed results indicated perturbation in the protein conformation. Similarly absorbance at 520 nm was found to be increased with increasing concentration of GNps with a red shift of about 25 nm and the peak was also widened. This bathochromic shift further confirms the interaction between GNps and venom proteins (MariamJ. 2016).
(ii) Fluorescence spectroscopy
Snake venoms exhibit endogenous fluorescence due to the
presence of aromatic amino acids (Petrilla V. et. al. 2015).

Intrinsic fluorescence spectra for venom and venom in presence of GNps in the wavelength range of 310 to 500 nm are shown in Fig. 3. Emission peaks for cobra and viper venom were observed at 340 nm and 346 nm respectively upon excitation at 280 nm.
Upon incubation with GNps the fluorescence spectra of both venoms were found to be quenched accompanied by 1-5 nm red shift in case cobra venom and about 1-3 nm blue shift in case of viper venom. Red shift predicts the exposure of fluorophore to hydrophilic environment.
Fluorescence quenching can occur due to several mechanisms that involve ground state complex formation, excited state reactions, molecular rearrangements, energy transfer and collisional encounters etc. One possible mechanism can be hypothesized that tryptophan fluorescence might have been rendered due to its interaction with charged polar molecules like the GNps that are capped with negatively charged citrate ions. Since quencher molecules do not freely penetrate the hydrophobic core of proteins, thus only those tryptophan residues which are present on the surface of the protein are quenched. Quenching is of two types dynamic quenching, where the - fluorescence is ceased upon the contact of fluorophore with quencher in excited state and static quenching, where non-fluorescent complex is formed between fluorophore

and quencher. Both interactions to occur need contact between fluorophore and quencher.
Fig. 3 [A] Fluorescence spectra of cobra venom
Fig. 3 [B] Stern-Volmer plot
Fig. 3[C] Double logarithmic plot of cobra venom-
GNP interaction
Fig. 3[D] Fluorescence spectra of viper venom
Fig. 3 [E] Stern-Volmer plot
Fig. 3[F] Double logarithmic plot of viper venom-
GNP interaction
Fluorescence quenching was analysed using Stem-Volmer equation [Eq. 4] to study the quenching behaviour of the quencher.

Where, F0 and F are fluorescence intensities in presence and absence of quencher respectively, kq is the biomolecular quenching rate constant, is the life time of fluorophore and
for biopolymers it is 10" , [Q] is concentration of the quencher and Ksv is Stem-Volmer quenching constant given by KSv = Thus biomolecular quenching rate constant can be
calculated using formula,
Linearity of Stem-Volmer plot indicates the presence of only one class of fluorophores having equal accessibility quencher. Deviation from linearity towards X-axis reveals the

presence of more thaii one class of fluorophores having unequal accessibility. Quenching constant for dynamic quenching is usually close to 1 xlO10 M'V1 and for static quenching it
becomes higher (Lakowicz J. R. 2006). The data herein presented demonstrate predominance of static quenching in the interaction of venom proteins with GNps. Fluorescence of both venom proteins was effectively quenched by GNps at 9-1 InM.
Number of binding sites (n) and binding constant (K) of venom for GNps were calculated using double logarithmic plot (log [(F0 - F)/F] versus log [Q]) which yields Eq.5. Here slope equals to n and intercept on Y-axis equals to log K. Double logarithmic plot showed that cobra venom has 1.0434 binding sites and viper venom has 0.7452 binding sites for GNps. Binding constants for cobra and viper venom were found to be 1.83 x 108 and 7.47 x: 105 respectively.

Table-1 below gives fluorescence quenching data: Stern-Volmer quenching constant, biding constant and number of binding sites of venom for GNps

Table 1

Sample Venom
+GNp Ksv K (M'V1) Binding sites (n) Binding constant
Cobra venom 8.5 x 107 8.5 x 1015 1.0434 1.83 x108
Viper venom 6.35 x 109 6.35 x 1017 0.7452 7.47 x 105
4.4.4 Biochemical functions of the Invention
The results contained in the above Table clearly establish that
the GNps interact with proteinous components of snake venoms causing therein macromolecular conformation changes. The GNps and/or venom protein-GNp complexes may compete with other components of the venom and thereby act as ASVs by inhibiting the biochemical processes following venomous snake bites. The present invention is based on the later hypothesis that the GNp-Plasma protein conjugates would be effective alternatives to currently used monovalent or polyvalent ASVs.
4.4.4.1 Haemolytic activity:
Phospholipase A2 (PLA2) is the major toxin in venoms augmenting several pharmacological effects of envenomation. It is present in cobra and viper venoms in multiple forms. Cobra venom has group IA and viper venom has group IIB form of PLA2. It hydrolyses sn2 bond in glycerophospholipids of plasma membrane freeing lysophosphatadyl choline and fatty acids. It acts on RBC membrane causing haemolysis. This

haemolytic activity of PLA2 was measured in haemolytic unit HU50 (casing 50% haemolysis) which was found to be 40µg/ml and 60µ,g/ml for Cobra and Viper venoms respectively.
Inhibitory effects of GNps, conjugate -1 & 2 were analysed and
■are presented in Fig.4. Both conjugate 1 & 2 were found to be almost equally effective having near about 92% reduction in haemolytic activity of both the venoms.
Table - 2 Percent reduction in the haemolytic activity of venoms by GNps, SC1, SC2. HC1 and HC2
Samples
Cobra Venom Viper Venom
GNP 31.96 GNp 37.60
GNp Conjugate SCI 94.16 GNp Conjugate SCI 93.19
GNp Conjugate SC2 88.90 GNp Conjugate SC2 91.09
GNp Conjugate HC1 32.99 GNp Conjugate HC1 41.09
GNp Conjugate HC2 23.49 GNp Conjugate HC2 44.97
Samples
Cobra Venom Viper Venom
GNP 131.96 GNp 137.60
GNp Conjugate SC1 94.16 GNp Conjugate SCI 93.19 GNp Conjugate SC2 88.90 GNp Conjugate SC2 91.09 GNp Conjugate HC1 32.99 GNp Conjugate HC1 41.09 GNp Conjugate HC2 [ 23.49 [ GNp Conjugate HC2 [ 44.97
4.4.4.2 Protease activity:
Protease activity of snake venom is devoted to metalloproteases
and PLA2, responsible for degradation of extracellular matrix, connective tissues, local blood vessles leading to bleeding, haemorrhage, oedema etc (Vineetha M. S., Janardhan B., More S. S. 2017). The proteolytic activity of cobra and viper venom was studied according to the method described by Greenberg with some modifications. It involves incubation of venom with substrate casein that results in release of tyrosin and fragmneted peptides. The reaction is stopped by adding terminator TCA, which forms complexes with undigested casein. Complexed

casein can be separated by filtration and free tyrosin present in fitrate can be measured spectrometrically using Folin-Lowry method. Here phenolic group of tyrosin complexes with copper present in Folin's reagent, which further reduces phosphomolybdate giving a blue coloured compound that absorbas at 660 nm. Intesity of this colour is directly proportional to the concentration of tyrosin residues. Proetase activity of venom can thus be calculated as one unit enzyme activity corresponds to 0.02µM tyrosin released. Protease activity of venom alone, with GNp and GNp-Conjugatess (SC1, SC2, HC1 and HC2) was determined using this method and is given in Figure 5.
Fig. 5 shows Protease activity of [A] cobra venom and [B] viper venom in presence of GNps, conjugate-SCl, conjugate-SC2, conjugate-HCl and. Conjugate-HC2
The percentage reduction in protease activity of venom of each of these complexes is given in Table-3. SCI showed as much as 61% reduction for cobra venom and 66% reduction in viper venom protease activity.
Table-3 Percentage reduction in the protease activity of venom by GNps, conjugate-SCl, conjugate-SC2, conjugate-HCl and conjugate-HC2

Table - 3 Percent reduction in the Protease activities of venoms by GNps, and its conjugates
Samples
Cobra Venom Viper Venom
GNP 43.57 GNp 38.53
GNp Conjugate SCI 61.15 GNp Conjugate SCI 66.06
GNp Conjugate SC2 38.32 GNp Conjugate HC2 53.63
GNp Conjugate HC1 40.43 GNp Conjugate HC1 45.46
GNp Conjugate HC2 43.83 GNp Conjugate HC2 43.72
Samples
Cobra Venom Viper Venom
GNP I 43.57 GNp I 38.53
GNp Conjugate SC1 61.15 GNp Conjugate SC1 66.06 GNp Conjugate SC2 38.32 GNp Conjugate HC2 53.63 GNp Conjugate HC1 40.43 GNp Conjugate HC1 45.46 GNp Conjugate HC2 [ 43.83 | GNp Conjugate HC2 | 43.72"
4.4.4.3 Acidimetry assay:
The Acidimetry assay was performed to study the activity of
PLA2 on egg phospholipids present in egg yolk lecithin. Here
the principal mechanism of action of PLA2 is the same as on
RBC membrane, the difference is just in substrate provided.
Substrate egg yolk lecithin is a rich source of phopsholipids.
PLA2 requires Ca2+ as a cofactor and acts on sn2 bond of
phospholipids releasing lysophospholipids and free fatty acids.
Released fatty acids were measured acidimetrycally on pH
meter and enzyme activity was calculated using Eq.3. A marked
reduction in PLA2 activity of both cobra and viper venoms was
observed.
Fig.6 PLA2 activity of [A] cobra venom and [B] viper venom in presence of GNps, and its conjugates
Percentage reduction in the PLA2 activity of both the venom was calculated and is shown in Table-4. It was seen that PLA2 activity of cobra venom was not sufficienty reduced by GNPs and other complexes. Here reduction was seen with only

conjugate -1, which was found to be 16%. However PLA2 activity of viper venom was reduced and conjugate -1 showed about 46% in PLA2 activity.
Table-4 gives percentage reduction in the phospholipase A2 activity of venom by GNps, conjugate -1, conjugate-2
Table - 4 Table-4 Percentage reduction in the phospholipase A2 activity of venom by GNps and its conjugates
Samples
Cobra Venom Viper Venom
GNp 1.64 GNp 14.47
GNp Conjugate SCI 1.43 GNp Conjugate SCI 11.18
GNp Conjugate SC2 1.02 GNp Conjugate SC2 NA
GNp Conjugate HC1 16.18 GNp Conjugate HC1 46.05
GNp Conjugate HC2 2.57 GNp Conjugate HC2 19.07
Samples
Cobra Venom Viper Venom
GNp 1 1.64 GNp 1 14.47
GNp Conjugate SC1 1.43 GNp Conjugate SCI 11.18 GNp Conjugate SC2 1.02 GNp Conjugate SC2 NA GNp Conjugate HC1 16.18 GNp Conjugate HC1 46.05 GNp Conjugate HC2 | 2.57 | GNp Conjugate HC2 | 19.07
Though soft conjugates were not efficient in reducing phospholipase A2 activity on lecithin substrate, they were found to have greater inhibitory effect on hemolytic activity (exhibited by the same enzyme phospholipase A2). This indicates that soft conjugates are more efficient to neutralize the PLA2 activity of venom on RBCs than on lecithin.
4.4.4.4 Biocompatibility:
Any component to be given intravenously needs to
biocompatible i.e. it should not show any immunological reaction. Venom inhibitory complexes were analysed for biocompatibility using simple agglutination test {or all the blood groups. It was found that protein-nano-particle conjugates

with plasma proteins and BSA were biocompatible in vitro i.e. they didn't exhibit any agglutination reaction as shown in Figure 7.
4.4,5 Conclusions - Implications of the above results
The aforesaid description evidences that the extensive Experiments that have been carried out using Gold Nano-particles(GNps) and experimental proteins like Bovine Serum Albumin (BSA) exhibited anti-venom properties. It was further observed that proteins contained in whole human plasma conjugated with GNps also demonstrated effective inactivation Cobra and Viper venoms and such complexes particularly GNp-human plasma conjugate can be safely exploited as a polyvalent ASV for treatment of Snake bites. Such conjugates can be prepared on site simply by mixing the nano-particle suspension with human plasma stored and preserved or freshly prepared on site.
Utility
The utility of the invention is firmly established from the in vitro results herein presented that the invention product Protein-GNp conjugates possess "high biological activity" and can effectively inhibit hemolytic activity and several other enzymatic processes induced on snake bites. In human blood samples these are the main and the most devastating effects of snake bites. Though direct data on practical utility is lacking and clinical trials on human subjects after snake bites would be

ethically very complicated, nay impossible, satisfying strict FDA approved criteria but that is not a prerequisite for assessing the utility of such a product for its patentability within the purview of Indian Patent Laws. A person skilled in art would hold that the in vitro results herein presented clearly establish the usefulness of the invention for treatment of snake bites and therefore satisfy the Utility criterion under the Indian Patent Act 1971 as amended 2005.
IN VIEW OF THE ABOVE THE PRESENT INVENTION IS NOVEL AND INVOLVED SEVERAL INVENTIVE STEPS. THE INVENTION IS THEREFORE PATENTABLE.
4.6 References
Casewell N. R., Wagstaff S. C, Wuster W., Cook D. A. N., Bolton F. M. S., King S. L, Pla D., Sanz L., Calvete J. J. and Harrison R. A. Medically important differences in snake venom composition are dictated by distinct postgenomic mechanisms. Proceedings of the national academy of sciences of the United States of America. 2014; 111(25); 9205-9210
Daraee H., Eatemadi A., Abbasi E., Aval S. F., Kouhi M. and Akbarzadeh A. Application of gold nanoparticles in biomedical and drug delivery. Artificial cell, nanomedicine and biotechnology. 2016; 44; 410-422
Gomes A., Das R., Sarkhel S, Mishra R., Mukherjee S., Bhattacharya S. and Gomes A. Herbs and herbal constituents active against snake bite. Indian journal of experimental biology. 2010; 48; 865-878
Greenberg D. M. Plant proteolytic enzymes. Methods in enzymology. 1955; 2;54-63

Karain B. D., Lee M. K. H., Glaser C. D. R J. J. and Hwang Y. Y. C60 Fullerenes as a novel treatment for poisoning and envenomation: A proof-of-concept study for snakebite. Journal of nanoscience and nanotechnology. 2016; 16; 1-8
Lakowicz J. R. Principles of fluorescence spectroscopy, 3 rd edition. 2006
Lui X., Atwater M., Wang J. and Huo Q. Extinction coefficient of gold nanoparticles with different sizes and different capping ligands. Colloidal and surface B: Biointerfaces. 2007; 58; 3-7
Malagoli D. A full-length protocol to test hemolytic activity of palytoxin on human erythrocytes. Invertebrate survival journal. 2007; 4; 92-94
Mariam J. J., Sivakami S. and Dongre P. M. Elucidation of structure and functional properties of albumin bound to gold nanoparticles. Journal of biomolecular structure and dynamics. 2016;
Petrilla V., Tomeckova V., Komanicky V., Lichardusova L., Sutorova M., Petrillova M. and Sopkova D. Fluorescent profiling of venom-selected cobra species. Spectroscopy letters: An international journal for rapid communication. 2015; 47; 1-5
Premendran S. J., Salwe K. J., Pathak S., Brahmane R. and Manimekalai K. Anti-cobra venom activity of plant Andrographis paniculata and its comparison with polyvalent anti-snake venom. Journal of natural science, biology and medicine. 2011; 2(2); 198-204.
Saha K. and Gomes A. Russell's viper venom induced nephrotoxicity, myotoxicity, hepatotoxicity - Neutralization with gold nanoparticle conjugated to 2-hydroxy-4-methoxy benzoic acid in vivo. Indian journal of experimental biology. 2017; 55; 7-14
Sanhajariya S., Duffull S. B. and Isbister G. K. Pharmacokinetics of snake venom. Toxins. 2018; 10(73)

Sarmento B. Have nanomedicines progressed as much as we'd hoped for in drug discovery and development? Expert opinion on drug discovery. 2019; 14; 723-725
Schmid F. X. Biological macromolecules: UV-visible spectrometry. Encyclopedia of life sciences. 2001;
Suman B., Eswari B., Divya B. J., Pallavi C., Venkataswamy M., Kemparaj K and Thyagaraju K. Effect of silver nano particles synthesized of Trichodesma indicum against Naja naja (Cobra) venom. International journal of pharmaceutical sciences and research. 2018; 9(8); 3291-3296.
Tan N. H. and Tan C. S. Acidimetry assay for phospholipase A2 using egg yolk suspension. Analytical biochemistry. 1988; 170; 282-288 Turkevich J., Stevenson P. C. and Hillier J. A study of the nucleation and growth process in the synthesis of colloidal gold. Discussions of the Faraday society. 1951; 11; 55-75
Vineetha M. S., Janardhan B. and More S. S. Biochemical and pharmacological neutralization of Indian saw-scaled viper snake venom by Canthium parvoflorum extract. Indian journal of biochemistry & biophysics. 2017; 54; 173-185.
World Health Organization, Global snakebite burden-Report by the Director-General. Seventy-first World Health Assembly (2018) Xiao H., Pan H., Liao., Yang M. and Huang C. Snake venom PLA2, a promising target for broad-spectrum antivenom drug development. BioMed Research International. (2017)

5. CLAIMS
We claim:
1. Gold Nanoparticle-Protein conjugates as polyvalent anti snake venoms (ASV)
(a) The average size of the gold nano-particles in claim 1 to be preferably 18 nm or thereabout (13-25nm)
(b) The proteins in claim 1 to be preferably the whole human plasma proteins
(c) The conjugates in claim 1 may be prepared by incubating 8.32 nM GNp with plasma (fivefold diluted) in distilled
Water
(d) The conjugate in claim 1 to be efficient against other
venom released by poisonous animal including
scorpions, amphibians and insects.