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Toxicon X
Toxicon X
Toxicon: X
2590-1710
Elsevier

S2590-1710(24)00020-1
10.1016/j.toxcx.2024.100203
100203
Clinical studies, antivenoms and epidemiology
Development of a gold nanoparticle-based novel diagnostic prototype for in vivo detection of Indian red scorpion (Mesobuthus tamulus) venom
Puzari Upasana a
Khan Mojibur R. b
Mukherjee Ashis K. akm@tezu.ernet.in
ashmukh@yahoo.co.uk
ab⁎
a Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, School of Sciences, Tezpur University, Tezpur, 784028, Assam, India
b Division of Life Sciences, Institute of Advanced Study in Science and Technology, Vigyan Path Garchuk, Paschim Boragaon, Guwahati, 781035, Assam, India
⁎ Corresponding author. Institute of Advanced Study in Science and Technology, Guwahati, 781035, Assam, India. akm@tezu.ernet.inashmukh@yahoo.co.uk
18 8 2024
9 2024
18 8 2024
23 1002032 7 2024
10 8 2024
14 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Indian red scorpion Mesobuthus tamulus is responsible for substantial mortality in India and Sri Lanka; however, no specific diagnostic method is available to detect the venom of this scorpion in envenomed plasma or body fluid. Therefore, we have proposed a novel, simple, and rapid method for detecting M. tamulus venom (MTV) in the plasma of envenomed animals using polyclonal antibodies (PAb) raised against three modified custom peptides representing the antigenic epitopes of K+ (Tamapin) and Na+ (α-neurotoxin) channel toxins, the two major MTV toxins identified by proteomic analysis. The optimum PAb formulation containing PAb 1, 2, and 3 in proportion (1:1:1, w/w/w) acted synergistically, demonstrating significantly higher immunological recognition of MTV than anti-scorpion antivenom (developed against native toxins) and individual antibodies against peptide immunogens. The PAb formulation could detect MTV optimally in envenomed rat plasma (intravenous and subcutaneous routes) at 30–60 min post-injection. The acetonitrile precipitation method developed in this study to augment the MTV detection sensitivity enriched the low molecular mass peptide toxins in envenomed rat plasma, which was ascertained by mass spectrometry analysis. The gold nanoparticles conjugated PAb formulation, characterised by biophysical techniques such as Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM), demonstrated their interaction with low molecular mass MTV peptide toxins in envenomed rat plasma. This interaction results in the accumulation of the gold nanoparticles, thus leading to signal change in absorbance spectra that can be discerned within 10 min. From a standard curve of MTV spiked plasma, the quantity of MTV in envenomed rat plasma could be determined by gold nanoparticle-PAb formulation conjugate.

Graphical abstract

Image 1

Highlights

• Polyclonal antibodies raised against antigenic custom peptides of two major toxins of Mesobuthus tamulus venom.

• Antibody formulation showed higher recognition Mesobuthus tamulus venom than commercial scorpion antivenom.

• Antibody formulation detected Mesobuthus tamulus venom in envenomed rat plasma.

• Antibody formulation detected Mesobuthus tamulus venom in low molecular mass peptide toxins enriched envenomed plasma.

• Antibody formulation conjugated to gold nanoparticles showed visual Mesobuthus tamulus venom detection within 5–10 min.

Keywords

Scorpion envenomation diagnosis
Indian red scorpion venom
Toxin-epitope specific antibodies
Gold nanoparticles
LSPR
Handling editor: Ray Norton
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pmcAbbreviations

ACN Acetonitrile

AFM Atomic force microscopy

ANOVA Analysis of variance

ASA Anti-scorpion antivenom

AuNP Gold nanoparticle

BSA Bovine serum albumin

CPs custom peptides

DMF dimethyl formamide

DTT dithiothreitol

ECL, Enhanced Chemiluminescence

EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

ELISA Enzyme-linked immunosorbent assay

FDS False discovery rate

FTIR Fourier-transform infrared spectroscopy

HRP Horse-radish peroxidase

IAA Iodoacetamide

KLH Keyhole Limpet Hemocyanin

LC-MS/MS Liquid Chromatography with tandem mass spectrometry

LMMPT low molecular mass peptide toxins

LOD Limit of detection

LSPR Localised surface plasmon resonance

MBS m-maleimidobenzoyl-Nhydroxysuccinimide ester

MTV Mesobuthus tamulus venom

MUA Mercaptoundecanoic acid

NCBI National Centre for Biotechnology Information

NHS N-Hydroxysuccinimide

NnV Naja naja venom

OECD Organisation for Economic Co-operation and Development

PAb PAb formulation

PAb Polyclonal purified antibodies

PBS Phosphate buffer saline

PSVPL, Premium Serum and Vaccines Pvt. Ltd.

PVDF Polyvinylidene difluoride

RVV Daboia russelii venom

SDS-PAGE Sodium dodecyl-sulfate polyacrylamide gel electrophoresis

SPR Surface plasmon resonance

TBS tris buffer saline

TEM Transmission electron microscope

TMB 3,3′,5,5′-Tetramethylbenzidine

UV–Vis Ultraviolet–visible

1 Introduction

Across the globe, approximately 1.23 million scorpion stings worldwide result in about 3250 deaths yearly; therefore, scorpion sting envenomation is considered a neglected public health disease in tropical and sub-tropical countries (Bawaskar, 1984; Chippaux and Goyffon, 2008). Among the scorpion species found in India, only the Indian red scorpion (Mesobuthus tamulus) and Indian black scorpion (Heterometrus bengalensis) pose a significant threat to humans; however, clinical reports show that the M. tamulus venom (MTV) is more toxic compared to any venomous scorpion in this subcontinent (Badhe et al., 2007; Bawaskar, 1984; Bhadani et al., 2006; Das et al., 2021; Kularatne et al., 2015; Senthilvelan et al., 2015).

Most of those affected by scorpion envenomation are elderly adults, immuno-compromised individuals, and small children whose immune systems are still maturing (Badhe et al., 2007; Das et al., 2020; Laustsen et al., 2016; Ortiz et al., 2015; Santos et al., 2016; Tiwari and Deshpande, 1993). The stung patients from rural areas often arrive at health centres 1–2 h after the incident due to their initial preference for traditional medicine or inaccessible health centres. Unfortunately, there is no commercial diagnostic tool or technique for identifying scorpion venom in human bodily fluids like blood. As a result, doctors mostly follow the traditional diagnosis approach, which concentrates on the clinical signs of scorpion stings (Ailani et al., 1999; Sivak et al., 1983; Sofer and Gueron, 1988). However, because the symptoms of the affected patients change gradually, these diagnosis methods may result in an inadequate treatment plan (Krifi et al., 1998).

Administration of anti-scorpion antivenom (ASA) and alpha-adrenergic receptor inhibitors such as prazosin have been used to treat scorpion envenomation (Bawaskar and Bawaskar, 2007, 2011; Chippaux, 2012; Soulaymani Bencheikh et al., 2007). However, an accurate diagnosis method for scorpion venom toxins is needed to determine the quantity of venom injected in patients for efficient therapy against scorpion stings (Mars et al., 2018; Mazhdi and Hamidi, 2021).

In some parts of the world, efforts have been made to detect the venom of several scorpion species. For example, the ELISA method has been described for detecting venoms of Tityus serrulatus, Androctonus australis garzonii, and Buthus occitanus tunetanus; however, to our knowledge, none of them has found clinical usage to date (Chávez-Olórtegui et al., 1994; De Rezende et al., 1996; Krifi et al., 1998; Rezende et al., 1995). In the recent decade, a group of researchers has developed an amperometric biosensor to identify the toxic fraction within the Androctonus australis hector venom exhibiting rapid body diffusion using the bispecific nanobody format (Mars et al., 2018). In another study, a sensitive two-site immunometric assay was designed to detect A. a. hector scorpion venom toxin in envenomed experimental animals' biological fluids (Devaux et al., 2002); however, no studies have been documented to create a method for identifying and measuring MTV in India.

Considerations like price, storage, and transport stability at room temperature are crucial when establishing a detection method or kit for developing countries. In addition, the diagnosis procedure needs to be quick and accurate in identifying and measuring the venom injected into the patient's bodily fluid (Puzari and Mukherjee, 2020). Above all, the diagnosis technique must be easy enough for nurses or other general healthcare professionals in rural healthcare centres to utilise without requiring a technical expert (Puzari and Mukherjee, 2020). Therefore, in the present study, we proposed a simple method for rapidly diagnosing and quantifying MTV in the serum or plasma of envenomed patients.

Proteomic analysis of MTV revealed the predominance of low molecular mass K+ and Na+ channel toxins, accounting for 38.2% and 38.4% of the proteome, respectively (Das et al., 2020). The Na+ channel toxins are mainly responsible for the neurotoxic effects of scorpion stings. The K+ channel toxins, short peptides comprised of 30–40 amino acids, show a high affinity toward one or more subtypes of potassium channels (Das et al., 2020). Production and application of antibodies specific to these toxins may be preferred for detecting MTV in patient body fluid. However, producing polyclonal antibodies against venom toxins is not feasible with the costly and time-consuming purification of the toxins. Therefore, a more cost-effective and straightforward method was to use the unique peptide epitope or antigenic determinant of these two toxins and use them to design synthetic peptides by partially modifying their sequence to enhance their antigenicity. After that, polyclonal antibodies against modified toxin epitopes (custom peptides) were raised in rabbits. The immune reactivity of the rabbit custom peptide antibodies (PAb) was determined in vitro and then with MTV-envenomed rat plasma (in vivo). A formulation of PAbs demonstrated better immune recognition of MTV than individual PAbs. This formulation holds great promise for diagnosing Indian red scorpion venom in patient plasma. Furthermore, for sensitive detection of these low molecular mass peptide toxins of MTV present in low concentrations in plasma, a process has also been proposed for depleting high-abundance proteins present in the plasma by acetonitrile (ACN) precipitation.

Colourimetric assays offer a straightforward visual detection technique without complex equipment. Because of their simplicity of synthesis, low cost, and distinctive optical features, metal nanoparticle-based colourimetric assays, especially gold nanoparticle-based assays, are frequently utilised to detect disease (Jazayeri et al., 2018; Oliveira et al., 2019). Our study used absorbance spectra and localised surface plasmon resonance (LSPR) detection by observing the colour change in the gold nanoparticle (AuNP) colloidal suspension upon the interaction between MTV toxins and the PAb formulation. Notably, this is the first report to detect Indian red scorpion venom in envenomed animal plasma, and it holds great promise to detect its venom in the patient's body fluid.

2 Materials and methods

2.1 Materials

Lyophilised MTV was a gift from Premium Serum and Vaccines Pvt. Ltd. (PSVPL), Pune, India. Lyophilised commercial ASA was procured from PSVPL, Pune, India (batch No.: SS170401). All the other analytical grade chemicals and reagents used in the study were purchased from Sigma Aldrich, USA, and HiMedia, India. Synthesis of toxin-epitope-specific custom peptides and production of antibodies against the custom peptides in rabbits were outsourced to S. Biochem Pvt. Ltd., India, and BioBharati Pvt. Ltd., Kolkata, India, respectively.

2.2 Animals

Laboratory inbreeds, pathogen-free Wistar strain albino rats (220 ± 10 g) used in this study were purchased from M/S Chakrabarty Enterprise, Kolkata. Animal experimentations were approved by the institutional animal ethics committee (IASST/IAEC/2022/09). Rats were maintained and used according to the OECD (Organisation for Economic Co-operation and Development) guidelines for chemical safety and animal welfare. BioBharati Pvt. Ltd. followed OECD guidelines for maintaining the white New Zealand rabbits (Oryctolagus cuniculus) used for antibody production.

2.3 A bioinformatics approach for identifying and designing the antigenic epitopes of the major toxins of MTV

The amino acid sequences of the two major toxins of MTV, viz. Na+ and K+ ion-channel toxins were retrieved from the National Centre for Biotechnology Information (NCBI) (https://ncbi.nlm.nih.gov) and UniProt (https://uniprot.org) servers. After that, these sequences were submitted to the Immunomedicine Group: Predicted Antigenic Peptides online server (http://imed.med.ucm.es/Tools/antigenic.pl) for antigenic region determination (Kolaskar and Tongaonkar, 1990). Predictions of antigenic regions are based on the occurrence of amino acid residues in segmental epitopes known experimentally. The epitopes with antigenic propensity greater than 1.0 were determined as antigenic regions for raising antibodies (Kolaskar and Tongaonkar, 1990). Therefore, antigenic peptides with antigenic propensity >1 with 16–20 amino acid residues on the surface of the toxin were selected and modified to raise polyclonal antibodies in rabbits. Notably, peptides of length >20 residues increase the risk of non-specificity, and shorter peptides of length <10 residues may not be able to produce antibodies that would recognise the native protein (toxin) with adequate affinity (Lee et al., 2016).

In CP1, the peptide sequence was initiated from the Trp residue, and a terminal Cys residue was added before the Trp residue. In the case of CP2, an additional terminal Cys residue was included. For CP3, a Ser residue was added, while the terminal Cys residue remained unchanged. These alterations were implemented in the peptide design to account for the peptide synthesis, purification procedure, and subsequent KLH conjugation of peptides.

2.4 Raising polyclonal antibodies against the toxin-epitope-specific custom peptides (CPs)

2.4.1 KLH conjugation of the CPs

The CPs were conjugated with Keyhole Limpet Hemocyanin (KLH) as described by (Van Regenmortel, 1988). Briefly, m-maleimidobenzoyl-Nhydroxysuccinimide ester (MBS) was dissolved in 200 μL dimethyl formamide (DMF), and the solution was gently stirred at room temperature for 30 min after adding 70 μL of this solution to a 10 mg/mL KLH solution prepared in 10 mM phosphate buffer, pH 7.0. Sephadex G25 size exclusion chromatography was used to remove the free crosslinker (MBS-DMF). Firstly, column equilibration was done in 50 mM phosphate buffer (pH 7.0). The same buffer was used to load the KLH reaction mixture into the column and then elute it. The KLH with a molecular weight of ∼400 kDa was eluted in the void volume. The CPs dissolved in 100 μL of DMF were rapidly mixed with the purified KLH/MBS. The mixture was vortexed, and 2N NaOH was used to adjust the pH to 7.0–7.2. The mixture was stirred overnight at 4 °C to allow the peptides to conjugate to KLH. The next day, the solution was lyophilised after adding 0.1 M ammonium bicarbonate. The coupling efficiency of the synthetic peptides was determined using the Cysteine Standard Assay (Riddles et al., 1979). The lyophilised conjugates were outsourced to raise antibodies in rabbits.

2.4.2 Raising and purifying custom peptide-specific antibodies by immunising rabbits with KLH-conjugated CPs

The previously described method was followed to raise the polyclonal antibodies in rabbits against the KLH-conjugated CPs (Puzari et al., 2023). After drawing 2 mL blood as pre-immune serum from the white New Zealand rabbits, primary immunisation was performed 24 h later by injecting ∼200 μg of the KLH-conjugated peptides in Freund's complete adjuvant subcutaneously. A first booster dose of 100 μg KLH-conjugated peptides in Freund's incomplete adjuvant was administered to the rabbits and kept under observation 15 days later.

On the 10th day after administration of the first dose, a second booster dose (in Freund's incomplete adjuvant) was injected into the rabbits. Again, on the following 10th day, a test bleed was done from the ear vein; serum was separated, and an ELISA was performed to check the antibody titer. On the 10th day post the second booster dose, a third booster dose in Freund's incomplete adjuvant was administered to the rabbits. After that, a test bleed was done from the ear vein again, and ELISA was performed with the separated serum to determine the antibody titer.

For the ELISA, 100 μL solution of 2 μg/mL native peptide (non-KLH conjugated custom peptide) in phosphate buffer saline (PBS) (200 ng/well) was used to coat the microtiter wells and left overnight at 4 °C. After that, 250 μL of 1% BSA in PBS was added and incubated for 1 h at room temperature. After washing the wells thrice with wash buffer (PBS with 0.05% Tween 20), diluted serum samples (100 μL) were incubated for 30 min at room temperature. The wells were washed thrice with wash buffer again, and 100 μL of Protein A-HRP (Zymed, USA) was diluted to 1/60,000 in blocking buffer (PBS with 5% BSA). The wells were incubated for 30 min at room temperature, washed with wash buffer, and then incubated in the dark with 100 μL of TMB/H2O2 substrate solution at room temperature for 30 min. Lastly, 50 μL of 2M H2SO4 was used to stop the reaction, and then absorbance was recorded at 450 nm (primary wavelength) and 630 nm (reference wavelength) against the reagent blank.

Affinity chromatography was employed to purify each polyclonal antibody from the rabbit antisera (Ayyar et al., 2012; Hober et al., 2007; Page and Thorpe, 1998). Dry CnBr-activated sepharose 4B resin was swelled and activated using 100 mM carbonate buffer (pH 8.5). The native peptide (2–5 mg) was separately dissolved into the 100 mM carbonate buffer for coupling. Each peptide was added to the resin separately and incubated 16 h at 4 °C. Any unreacted peptide was washed from the resin using 100 mM carbonate buffer (pH 8.5). The peptide-coupled resin was incubated with 100 mM Tris-Cl buffer pH 8 for 2 h at room temperature. The resin was washed thrice using PBS. After that, 5 mL of antisera was mixed with 5 mL PBS (1:1) and incubated with the peptide-coupled resin for 16 h at 4 °C. The resin was collected and washed thrice using PBS. Then, the bound antibody was eluted using 100 mM Glycine, pH 2.5, and after collection, the pH was neutralised using 3M Tris-Cl pH 8.8.

The immune reactivity of the PAbs against their respective CP (against which they were raised) and KLH was assessed by dot blot analysis (Puzari et al., 2023). Briefly, the polyvinylidene fluoride (PVDF) membrane was activated with 100% methanol and then equilibrated with 1X TBS (tris buffer saline) with 0.05% tween-20 (TBS-T). After that, 2 μg (1 μL) of the KLH conjugated CP/KLH was spotted onto the activated membrane and air-dried. Non-specific binding was blocked by 5% fat-free skimmed milk and incubated at room temperature for 1 h, with gentle shaking. The membrane was then washed with TBS-T and incubated with the PAbs (1 μg/μL PAbs) at a dilution of 1:1000. Anti-rabbit HRP-conjugated secondary antibody at 1:2000 dilutions were used to detect the primary antibodies (PAbs).

The blot was developed using Enhanced Chemiluminescence (ECL) substrate (Cat no. 1705060, Bio-Rad) using the ChemiDoc imaging system with Image Lab software (Bio-Rad, USA), and the intensity of the dots was measured using ImageJ software (https://imagej.nih.gov/ij/). The detailed methodology for dot intensity measurement using ImageJ is included in the supplementary document. BSA was used as a negative control in the analysis.

2.5 Determination of in vitro immune cross-reactivity of PAb formulation (PAbF) and commercial ASA towards MTV

2.5.1 Indirect ELISA

For the immune cross-reactivity studies, we combined the PAbs in a formulation where the individual PAbs 1, 2, and 3 (1:1:1, w/w/w) showed synergistic activity in immune recognition of MTV. The indirect ELISA was performed per the protocol (Kakati et al., 2022; Patra et al., 2019), followed by some modifications. Briefly, different doses of MTV (6.25–100 ng protein) were coated onto 96 well microtiter plates (in triplicate) and incubated overnight at 4 °C. The next day, the wells were washed thrice with 1X PBS wash buffer and then incubated at room temperature for 2 h with PAbF/commercial ASA as primary antibodies in 1:40 (MTV: PAbF/commercial ASA, protein: protein) ratio, followed by washing with wash buffer. Next, the wells were incubated at room temperature for 2 h with anti-rabbit IgG HRP conjugated (for PAbF)/anti-horse IgG HRP conjugated (for commercial ASA) secondary antibody of 1:2000 to detect the PAbF/commercial ASA. After three consecutive washes with wash buffer, the wells were incubated with 100 μL 1X TMB/H2O2 for another 30 min in dark conditions, and 50 μL of 2M H2SO4 was added to stop the reaction. Absorbance was measured at 492 nm in MultiskanGO (Thermo Scientific, USA) microplate reader against reagent blank. The analysis was performed in triplicates.

2.5.2 Dot blot assay

Dot blot analysis was performed as described by our previous protocol with modifications (Puzari et al., 2023). The antibodies were spotted on the activated PVDF membrane, with venom samples sandwiched between the spotted capture antibody and primary antibody). The PVDF membranes were marked with spots, pre-treated with 100% methanol for 2 min, and washed with TBS-T for 15–30 min. The activated membranes were then air-dried at 37 °C and spotted with 2 μg PAbF/commercial ASA, followed by blocking of non-specific binding using 5% BSA in TBS-T solution. After that, the membranes were incubated with MTV (0.3, 0.15, and 0.075 ng/μL)-spiked rat plasma for 30 min at room temperature. This concentration of MTV was calculated considering that an adult M. tamulus can inject 1.5 mg of venom into an adult human with approximately 5 L of blood; therefore, the concentration of MTV in blood comes out to be 0.3 ng/μL (Khadse, 2016).

After washing, the membranes were incubated with PAbF/commercial ASA at 1:1000 dilutions for 45 min. The anti-rabbit IgG HRP detected the primary antibody conjugated (for PAbF)/anti-horse IgG HRP conjugated (for commercial ASA) secondary antibody (1:2000 dilutions), and the blots were developed by ECL substrate as described above. ImageJ software was used to analyse the dot intensities. Since we have used the same antibody for capture and detection, we performed another dot blot assay where we let the secondary antibody directly bind to the capture antibody (control without antigen). The dot intensities of the blots obtained from the above sandwich assay were normalised against the dot intensities of the control without antigen. The analysis was performed in triplicates.

In another set of experiments, the potency of the PAbF (1 μg/μL) to recognise the different concentrations of MTV (0.3, 0.15, and 0.075 ng/μL) spiked rat plasma was studied by dot blot assay. The analysis was performed in triplicates.

2.5.3 Western blot analysis

Western blot analysis evaluated immunorecognition of MTV against PAbF, and the protocol was adopted from our previous reports (Das et al., 2020; Kakati et al., 2022; Kalita et al., 2017). Briefly, 80 μg MTV proteins were separated in a 15 % SDS-PAGE under reduced conditions, and the proteins were transferred to a methanol-activated PVDF membrane. Post-transfer of the proteins, transfer efficacy was determined by staining the PVDF membrane with 0.5% Ponceau-S red. The membrane was incubated overnight with 5% BSA in TBS-T at 4 °C (to prevent non-specific binding of PAbF). The next day, after washing the membrane thrice with TBS-T, PAbF was added (1:1000 dilution) as the primary antibody and incubated at room temperature for 2 h. After three consecutive washes, the membrane was incubated with anti-rabbit IgG-HRP conjugated antibody (1:4000 dilution), and the immunoblot was developed with ECL substrate as described above.

2.5.4 A comparison of the interaction between MTV and ASA or PAbF

The methodology used to study the MTV-PAbF interaction by spectrofluorometric analysis was described previously (Das et al., 2022; Patra et al., 2021). Briefly, graded concentrations of PAbF (10–160 μg/mL) were incubated at room temperature with a fixed concentration of MTV (10 μg/mL). The analysis of the reaction mixture was conducted in the Varioskan LUX Multimode Microplate reader (Thermo Fisher Scientific, Denmark) by setting the excitation wavelength at 280 nm, temperature at 25 °C, emission slits set at 5 nm, and emission spectra was monitored from 300 to 500 nm. Fluorescence spectra of MTV/PAbF were determined as a control and compared with the relative fluorescence spectra intensity (λmax) of venom-PAbF interaction. The change in intensity (Δλmax) was plotted against the PAbF concentration (μg/mL) using GraphPad Prism 5.0 software (Dutta et al., 2019; Mukherjee et al., 2016), and the KD value was calculated. The KD value obtained was compared with the reported kd value of commercial ASA-MTV interaction under identical conditions in our laboratory (Das et al., 2022). Furthermore, the percentage of venom-specific antibodies in PAbF was compared to commercial ASA venom-specific antibodies reported previously (Das et al., 2022).

2.6 Determination of MTV in the plasma of experimentally envenomed rats

The detailed protocol of MTV determination in the plasma of envenomed rats is shown in a schematic diagram (Fig. 1), and a description of methods is given below.Fig. 1 Flow diagram of a methodology for MTV envenomation simulation of Wistar rats, by intravenous (i.v.) and subcutaneous (s.c.) routes, and detection of MTV in the plasma of envenomed rats. Schematic representations were generated using Biorender (©BioRender: biorender.com).

Fig. 1

2.6.1 Experimental envenomation of rodents with MTV

Envenomation of albino Wistar strain rats was simulated by adopting the method of Brunda et al., in 2006 with slight modifications (Brunda et al., 2006). The albino Wistar strain rats were divided into four groups (220 ± 10 g, n = 20). As already explained, an adult of an average 60 kg weight can receive a maximum of 1.5 mg MTV in a sting equivalent to 25 μg/kg (5 μg/200 g). Therefore, the group II rats (n = 5) were intravenously injected with 25 μg (approximately five times higher than the amount of MTV entering the bloodstream of an average adult human in one sting) of MTV dissolved in 200 μL of 1X PBS. Meanwhile, group IV rats (n = 5) were subcutaneously injected with 100 μg MTV dissolved in 200 μL of 1X PBS (four times higher than the venom injected intravenously). Group I (n = 5) and group II (n = 5) were intravenously and subcutaneously injected with only 1X PBS (untreated control), respectively.

2.6.2 Determination of MTV in envenomed rat plasma

Blood was collected from the retro-orbital veins of groups I-IV rats at 30 min and 60 min after venom injection in tubes containing heparin as an anticoagulant (5% of total blood volume collected). Blood was collected by cardiac puncture after 120 min of venom injection from all the groups of rats. The tubes were centrifuged on the fixed rotor centrifuge at 4 °C with 4300 rpm for 15 min (Eppendorf refrigerated Centrifuge 5804 R) to separate the plasma and stored in aliquots at −20 °C until use. However, the storage was never longer than seven days.

Dot blot analysis was performed by spotting the PAbF on the activated PVDF membrane, with envenomed and control plasma samples sandwiched between the PAbF as the capture antibody and the PAbF as the primary antibody. Briefly, the activated PVDF membranes spotted with the PAbF (4 μg) were incubated at room temperature with 10 μL of the plasma (group I-IV) for 15 min. After washing, the membranes were incubated with PAbF as the primary antibody at 1:500 dilutions for 45 min, which was further detected by the anti-rabbit HRP-conjugated antibody (1:1000). The blots were developed by ECL substrate, and their intensities were measured using ImageJ software. The dot intensities obtained were normalised against those obtained from control without antigen. The analysis was performed in triplicates.

2.6.3 A process for enriching low molecular mass peptides in plasma

A slightly modified ACN precipitation method, which may be effective in precipitating large abundant proteins and disrupting the binding of low molecular mass MTV toxins to their carrier proteins, may help enrich the plasma with only our desired proteins (Kay et al., 2008; Liu et al., 2014; Polson et al., 2003). We developed a modified method for isolating low molecular mass peptide toxins (LMMPT) to provide a more sensitive diagnosis of MTV in the envenomed plasma samples. The envenomed plasma samples were collected 60 min after MTV injection (i.v. and s.c). ACN was added to the plasma at a ratio of 1:2.5 (plasma: ACN, v/v) and incubated in ice for 30 min. The mixture was centrifuged at room temperature at 10000 rpm for 5 min in a microcentrifuge (Eppendorf MiniSpin). The supernatant containing low molecular mass peptides was collected and concentrated in a water bath at an optimised temperature of 60 °C. The semi-solid portions left behind (having LMMPT) were reconstituted in 30 μL of phosphate buffer, pH 7.4. Determination of MTV toxins in the non-enriched envenomed plasma and reconstituted LMMPT by PAbF was done by dot blot and sandwich ELISA following the abovementioned procedures. All the analyses were performed in triplicates.

2.6.4 Proteomics analysis to determine the presence of MTV major toxins in LMMPT fraction of envenomed plasma

Proteomic analysis was done to validate the presence of Na+ and K+ channel toxins in the LMMPT-enriched MTV-treated rat plasma. The control and MTV-treated plasma were treated with ACN (section 2.7), and isolated peptides were identified for Na+ and K+ channel toxins. Briefly, 40 μg of the LMMPT-enriched peptides of control and MTV-treated plasma underwent reduction with 10 mM dithiothreitol (DTT) and alkylated with 55 mM iodoacetamide (IAA) at room temperature in dark conditions. After reduction and alkylation, the peptides were incubated at 37 °C with proteomics-grade trypsin for about 16 h. The ZipTip C18 (EMD Millipore) tips were used to desalt and concentrate the tryptic peptides. Data acquisition was done by Orbitrap Fusion™ mass spectrometer (Thermo Fisher Scientific, Inc.) coupled to an EASY-nLC™ 1200 nano-flow LC system (Thermo Fisher Scientific, Inc.) equipped with EASY-Spray column (50 cm × 75 μm ID; PepMap C18 column). The MS and MS/MS spectra were acquired at 375–1500 m/z (Das et al., 2020; Kakati et al., 2022; Kalita et al., 2017).

The raw MS/MS data were analysed using Proteome Discoverer software (version 2.2; Thermo Fisher Scientific, Inc) with a false discovery rate (FDR) set to <1%. Two missed cleavages, and at least one unique peptide per toxin entry were allowed. The raw data were searched against the nonredundant NCBI databases' Buthidae family (Taxonomy ID: 6856) and M. tamulus (Taxonomy ID: 34647) protein entries. Precursor and product ion tolerances were 10 ppm and 0.05 Da, respectively. Further, the oxidation of methionine residues was marked as a flexible modification, and carbamidomethylation of cysteine was kept fixed (Das et al., 2020; Kakati et al., 2022; Kalita et al., 2017). The coverage percentages of identified sequences ≤5% have not been considered for protein identification. The amino acid sequences of the proteins and peptides identified were aligned by multiple sequence alignment to find conserved residues and confirm the presence/absence of the targeted MTV toxins.

2.6.5 Synthesis of AuNPs and conjugation of PAbF to the AuNP

2.6.5.1 Synthesis and characterisation of AuNP

With minor modifications, AuNP synthesis was carried out using the sodium citrate reduction method described previously (Nam et al., 2014; Pong et al., 2007). Briefly, 50 mL chloroauric acid solution (HAuCl4) (1 mM) was heated to reflux under stirring conditions at 100 rpm, and then 5 mL of 38 mM sodium citrate solution was added promptly. The solution was heated until the solution's colour transformed from pale yellow to wine red and then cooled down to room temperature with continuous stirring. The solution was stored in dark abler bottles at 4 °C. The AuNPs were characterised by UV–Vis (ultraviolet–visible) spectrophotometer, Transmission electron microscope (TEM), Zetasizer, Fourier-transform infrared spectroscopy (FTIR), and Atomic force microscopy (AFM) (Borse et al., 2020; Busch et al., 2019; da Silva et al., 2022; Oliveira et al., 2019). The detailed methodology has been described in the supplementary file.

2.6.5.2 Conjugation of AuNPs with PAbF

The minimum concentration of PAbF required for maintaining the conjugated AuNPs' stability was determined by adding NaCl (Rayavarapu et al., 2007; Schmitz et al., 2023; Zhang et al., 2020). Briefly, the suitable concentration of NaCl for the purpose was optimised by mixing 100 μL AuNP with graded concentrations (60, 120, and 250 mM) of NaCl and incubated for 15 min. The recorded UV–Vis spectrophotometer (400–800 nm) recorded the spectra. In the next experiment, 100 μL AuNPs were incubated for 20 min (at room temperature), with 2 μL of graded concentrations (0.05, 0.1, 0.25, 0.5, and 1 μg/μL) of PAbF in a microtiter plate. Next, 100 μL of 250 mM NaCl was added, and the plate was shaken for 5 min in the spectrophotometer before recording the absorbance at a UV–Vis spectrophotometer (400–800 nm).

The PAbF was conjugated to surface-modified AuNPs by covalent coupling (Oliveira et al., 2019), with slight modifications in the next step. The surface of AuNP was functionalised with a thiolated mercaptoundecanoic acid linker (MUA), with 11 carbon atoms between the COOH and SH groups. Briefly, 1 mL of 10 mM MUA was added to 5 mL AuNPs and incubated under stirring conditions at 100 rpm at 35 °C for 24 h. After washing the surface-modified AuNPs thrice by centrifugation (10000 rpm, 30 min), they were suspended in ultrapure water. The PAbF was conjugated to the surface-modified AuNPs via EDC/NHS [EDC: 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS: N-Hydroxysuccinimide] coupling chemistry, where the PAbF forms a pre-activated carboxylic acid amide bond by covalently binding to surface-modified AuNPs via their amino groups. Briefly, 2 mL of the surface-modified AuNPs was added to 200 μL EDC (50 μM) and incubated under stirring conditions for 30 min at 150 rpm. After that, 200 μL of NHS (75 μM) was added to the mixture and stirred for 30 min. Subsequently, 20 μg of PAbF (1 μg/μL) was allowed to react with the reaction mixture at room temperature for 30 min. The reaction mixture was left overnight at 4 °C, and the next day, it was centrifuged at 10000 rpm for 30 min. The pellet obtained was suspended in 100 μL of phosphate buffer (10 mM, pH 7.4) after adding 1% (w/v) BSA to functionalise the non-antibody coated areas, forming the AuNP-PAbF conjugate. Protein estimation was done using the Bradford method, using BSA as a standard to determine the concentration of unbound antibodies remaining in the supernatant after the conjugation. The binding efficiency of the PAbF was calculated using the following equation (1),(1) Efficiency (%) = ([PAbF]0 – [PAbF])/ [PAbF]0 * 100

where [PAbF]0 is the initial concentration of PAbF added to the AuNP solution and [PAbF] is the concentration from the supernatant after three consecutive washes.

The AuNP-PAbF conjugates were also characterised by UV–Vis spectrophotometer, FTIR, Zetasizer, TEM, and AFM by the above methods (Borse et al., 2020; Busch et al., 2019; da Silva et al., 2022; Oliveira et al., 2019).

2.7 Determination of selectivity of the AuNP-PAbF conjugate with the MTV spiked rat plasma (in vitro) and LMMPT-enriched plasma from envenomed animals (in vivo)

Detection sensitivity of the AuNP-PAbF conjugate was determined by adding 10 μL of this conjugate to 30 μL of MTV spiked rat plasma (0.3 ng/μL) and incubated for 10 min. Only rat plasma (untreated) was used as a control, and Naja naja venom (NnV) and Daboia russelii venom (RVV) samples (50 ng/μL) spiked rat plasma were treated as negative controls. The LSPR peak intensity was recorded by UV–Vis spectrophotometer (400–700 nm).

Under in vivo conditions, to determine the detection sensitivity of the AuNP-PAbF conjugate, 10 μL of this conjugate was added to the 30 μL of the reconstituted LMMPT enriched plasmas (control and envenomed) and incubated for 10 min. Upon interaction with the MTV toxins in plasma, LSPR peak shifting was determined by recording peak intensity using a UV–Vis spectrophotometer (400–700 nm). To analyse the sensitivity of the AuNP-PAbF conjugate-based LSPR, MTV spiked rat plasma (1–5 ng/μL) was incubated with 20 μL AuNP-PAbF conjugate for 5 min. Based on the LSPR peak absorbance, a calibration curve was obtained and used to quantify the amount of MTV detected in LMMPT-enriched MTV-treated plasma.

2.8 Statistical analysis

All data have been represented as mean ± standard deviation (S.D.) of independent triplicate experiments. The significance of differences between control and test values was analysed by the Student's t-test in Sigma Plot 11.0 for Windows (version 10.0). For more than two groups, the significance of differences was examined with a one-way analysis of variance (ANOVA) followed by post hoc analysis in GraphPad Prism software. The p-value ≤0.05 was considered statistically significant.

3 Results

3.1 Rabbit polyclonal antibodies against the antigenic CPs

Table 1 represents the predominant toxins of MTV, the antigenic regions of these toxins, and the CPs designed from these antigenic epitopes.Table 1 The antigenic epitopes and the antigenic propensity of the native and modified epitopes are predominant toxins in the MTV proteome.

Table 1Name of toxin	Antigenic epitope(s)	The antigenic propensity of toxin epitopes	Epitope-based synthetic peptides. Modified residues are underlined, and the peptide length is shown in parenthesis.	Designation of custom peptide	The antigenic propensity of modified peptides	
Na+ channel toxin (α- neurotoxin)	36CDWWVPYGVVCWCEDLPTPVPIR58	1.1089	CWWVPYGVVSWSEDLPTPVP (20)	CP1	1.0907	
12CTYICTFNNYCHALCTD28	1.1045	YISTFNNYSHALSTDC (16)	CP2	1.0418	
K+ channel toxin (Tamapin)	4NLRRCELSCRSLGLLGKC21	1.0797	SNLRRSELSSRSLGLLGKC (19)	CP3	1.0340	

Polyclonal antibodies against KLH-conjugated CPs were raised in rabbits and affinity purified. A high titer of antibodies was observed in rabbit sera (Supplementary Figs. S1A–C). The purified antibodies did not show recognition towards the KLH carrier protein. Further, the dot blot study also showed that PAbs could recognise the CPs against which they were raised to varying degrees under identical experimental conditions (Supplementary Figs. S2A and B). However, cross-reactivity among CPs and PAbs could not be observed.

3.2 The PAbF demonstrated better in vitro immune cross-reactivity towards MTV as compared to commercial ASA

The dose-dependent ELISA demonstrated that the PAbF could recognise MTV even at a low dose of 6.25 ng (Fig. 2A). Moreover, PAbF showed 2.9–7.8 fold higher immune cross-reactivity (p < 0.05) than that of the commercial ASA towards MTV under identical experimental conditions (Fig. 2A).Fig. 2 (A) Comparison of the immune cross-reactivity between PAbF/commercial ASA towards MTV determined by Indirect ELISA. There is a significant difference in the fold change value between the immune-reactivity of commercial ASA and PAbF at all the MTV doses, *p ≤ 0.05; (B) Dot blot assay of MTV (0.3 ng/μL) spiked rat plasma using the PAbF and commercial ASA; (C) Image analyses of dot intensities of immune-reactivity were performed using ImageJ software. The dot intensities have been normalised against intensities of control without antigen. Significance of difference of commercial ASA compared to PAbF, *p < 0.05; (D) Dot blot assay of MTV (0.3 ng/μL, 0.15 ng/μL and 0.075 ng/μL) spiked rat plasma using the PAbF. Blot 1 was incubated with MTV (0.075 ng/μL) spiked rat plasma; Blot 2 was incubated with MTV (0.15 ng/μL) spiked rat plasma, and Blot 3 was incubated with MTV (0.3 ng/μL) spiked rat plasma; (E) Image analyses of dot intensities were performed using ImageJ software. The dot intensities have been normalised against intensities of control without antigen. Significance of difference of 0.15 ng/μL dose of MTV compared to 0.3 ng/μL dose of MTV *p < 0.05. Error bars indicate mean ± S.D. (n = 3).

Fig. 2

Instead of using the same antibody for both capture and detection, we performed a dot blot assay where it was observed that the secondary antibody (anti-rabbit IgG-HRP/anti-horse IgG-HRP) showed weaker immune-recognition towards the capture antibody (dot intensities designated as control without antigen) (Supplementary Figs. S3C and D); these blots have been called control without antigen henceforth. The secondary antibody may have an affinity for the Fc region of the primary antibody. Due to non-directed immobilisation, fewer Fc regions may become available while depositing the primary antibody onto the PVDF membrane. Thus, after normalising the dot intensities against the control without antigen intensities, we found the superior immune cross-reactivity of PAbF towards MTV-spiked rat plasma compared to commercial ASA under similar experimental conditions (Fig. 3B and C). Thus, a higher proportion of Na+ and K+ channel-specific antibodies in the PAbF showed significantly higher potency in immune recognition of MTV than commercial ASAs under identical experimental conditions.Fig. 3 (A) Dot blot assay comparing the immune-recognition of MTV in the plasma of the group I-IVrats by PAbF and commercial ASA when the blood was collected at 30 min, 60 min, and 120 min after the injection (i.v. and s.c.). Blots 1–3 incubated with control plasma (i.v.) collected after 30 min, 60 min, and 120 min recognised by PAbF; Blots 4–6 incubated with MTV-treated plasma (i.v.) collected after 30 min, 60 min, and 120 min recognised by PAbF; Blots 7–9 incubated with control plasma (s.c.) collected after 30 min, 60 min, and 120 min recognised by PAbF; Blots 10–12 incubated with MTV-treated plasma (s.c.) collected after 30 min, 60 min and 120 min recognised by PAbF; Blots 13–15 incubated with control plasma (s.c.) collected after 30 min, 60 min and 120 min recognised by commercial ASA; Blots 16–18 incubated with MTV-treated plasma (s.c.) collected after 30 min, 60 min and 120 min recognised by commercial ASA; (B) Image analyses of dot intensities of the group I-IV rats' plasma detection by PAbF and commercial ASA. The dot intensities have been normalised against intensities of control without antigen. Significance of difference in recognition of MTV-treated plasma collected at 30 min, 60 min, and 120 min by PAbF compared to recognition by commercial ASA *p < 0.05; recognition of MTV-treated plasma (s.c.) collected at 60 min and 120min PAbF compared to recognition of MTV-treated plasma collected at 30 min ɣp<0.05; recognition of MTV-treated plasma (s.c.) collected at 30 min and 120min PAbF compared to recognition of MTV-treated plasma collected at 60 min ωp<0.05; recognition of MTV-treated plasma (i.v.) collected at 30 min and 60 min by PAbF compared to recognition of MTV-treated plasma collected at 120 minψp<0.05. Error bars indicate mean ± S.D. (n = 3).

Fig. 3

The ELISA and dot blot results demonstrating the immune cross-reactivity of the individual PAbs and PAbF are included in the supplementary (Supplementary Figs. S3A, B, E, F).

The dot blot analysis showed that PAbF could show excellent immune recognition of 0.3 ng/μL MTV spiked serum and also an appreciable immune recognition of 0.15 ng/μL MTV spiked serum; however, below this concentration of MTV, immune recognition was found to be non-existent after normalisation of the dot intensities measured (Fig. 2D and E).

The Ponceau-S red staining ensured the efficient transfer of MTV proteins to the PVDF membrane (Supplementary Fig. S3G). The result of the Western blot analysis also confirmed the immune recognition of the low molecular mass toxins of MTV by the PAbF (Supplementary Fig. S3H).

3.3 The spectrofluorometric analysis also demonstrated better interaction of PAbF with MTV compared to commercial ASA

Through spectrofluorometric titration, the Kd value for PAbF was determined at 63.46 ± 6.64 μg protein/mL (Supplementary Fig. S4), which was much lower than the reported Kd value of the same batch of commercial ASA (321.5 ± 16 μg protein/mL) under identical conditions (Das et al., 2022). This observation may be due to the presence of a significantly higher amount of MTV-specific antibodies (66.67%) in 1 mL of the PAbF (Supplementary Table S1) compared to commercial ASA (6.3%) (Das et al., 2022).

3.4 The PAbF could better immune recognise the MTV enriched plasma sample compared to the non-enriched plasma sample from the envenomed Wistar rats

The dot blot analysis showed that PAbF detected the MTV in the plasma of the intravenously envenomed Wistar rats in a time-dependent manner; the highest recognition was shown 30 min and 60 min post-injection of MTV, and after that, the intensity of the signal (immune cross-reactivity between PAbF and MTV in plasma) decreased (Fig. 3A and B). However, in the case of the subcutaneously envenomed Wistar rats, the immune recognition of MTV was found to increase from 30 min to 60 min and then decrease after 60 min (Fig. 3A and B). Moreover, as expected, the PAbF showed much better recognition for MTV than the immunorecognition by commercial ASA (Fig. 3A and B).

Dot blot analysis showed that the unique protocol developed in this study to enrich the envenomed plasma with low molecular mass MTV toxins is very potent (about 2 folds higher) in immune recognising the MTV in LMMPT-enriched MTV-treated plasma compared to the determination of MTV in non-enriched plasma (Fig. 4A and B). The sandwich ELISA also demonstrated MTV's superior immune recognition (about 2-fold higher) in LMMPT-enriched MTV-treated plasma compared to non-enriched MTV-treated plasma by PAbF (Fig. 4C).Fig. 4 (A) Comparison of immune cross-reactivity of PAbF towards MTV in the LMMPT-enriched MTV-treated and non-enriched plasma of envenomed rats. Blot 1 incubated with control non-enriched plasma; Blot 2 incubated with non-enriched MTV-treated plasma; Blot 3 incubated with control LMMPT-enriched MTV-treated plasma; Blot 4 incubated with LMMPT-enriched MTV-treated plasma; (B) Image analyses of dot intensities of the plasma detection by the PAbF. The dot intensities have been normalised against intensities of control without antigen. Significance of difference in recognition of LMMPT-enriched MTV-treated plasma compared to non-enriched MTV-treated plasma ɣp<0.05; (C) Immune-reactivity of the PAbF towards MTV-treated non-enriched plasma and MTV-treated-LMMPT-enriched plasma determined by Sandwich ELISA. The absorbance values have been normalised against control without antigen. Significance of difference of recognition of MTV-treated-LMMPT-enriched plasma compared to MTV-treated non-enriched plasma *p < 0.05. Error bars indicate mean ± S.D. (n = 3).

Fig. 4

3.5 Mass spectrometry analysis demonstrated the protocol developed in this study enriches the low molecular mass MTV toxins in MTV-treated rat plasma

The LC-MS/MS analysis of LMMPT-enriched MTV-treated rat plasma demonstrated the presence of scorpion venom K+ and Na+ channel toxins against the Buthidae family and M. tamulus databases. In contrast, these toxins could not be identified in control (untreated) plasma, which is obvious. In the MTV-treated rat plasma sample, the other protein identified against the Buthidae family and M. tamulus databases was the structural protein. The amino acid sequence alignment of the identified protein sequences with the K+ and Na + channel toxins of MTV used for the custom peptide synthesis showed conserved residues of these toxins (Supplementary Fig. S5).

3.6 Biophysical characterisation demonstrated conjugation of PAbF with AuNPs

During the synthesis of AuNP by citrate reduction of Au (III) in water, citrate plays the role of a reducing agent by facilitating the reduction of Au (III) to Au (0) and as a protecting agent by providing growth limitation to the nanoparticles and inhibiting aggregation (Frens, 1973; Herizchi et al., 2016; Kimling et al., 2006). The synthesised AuNPs displayed a wine-red colour. The UV–Vis spectra recorded in the 400–700 nm range depicted a plasmonic peak at around 524 nm for the bare AuNPs. Before proceeding to the antibody conjugation process, the optimum binding concentration of PAbF to the AuNPs was determined using NaCl at a concentration of 250 mM. NaCl aggregated the AuNPs at this concentration, changing the colour of the AuNP colloidal solution from red wine to purple/blue (Supplementary Fig. S6A). The PAbF formed the most stable complex with AuNPs at a concentration of 1 μg/μL (Supplementary Fig. S6B). Therefore, at 1 μg/μL concentration of PAbF, the antibody can bind entirely to the AuNP surface and maintain the dispersion.

For antibody conjugation to the AuNPs, MUA functionalisation to the AuNPs showed a bathochromic shift of plasmonic peak from 524 nm to 528 nm. The peak showed a further bathochromic change to 530 nm after the conjugation of PAbF to the functionalised AuNPs (Supplementary Fig. S6C).

The infrared spectra of AuNP obtained from FTIR showed peaks at 3452 cm−1, 2922 cm−1, and 1636 cm−1, characteristic of –O–H stretching, –C–H stretching, and –C=O stretching for the functional group of the citrate group stabilising the AuNPs (Pramanik et al., 2021; Stuart, 2004; Vechia et al., 2020). When functionalised with MUA, peaks were observed at 2924 and 2854 cm−1, corresponding to symmetric and asymmetric -C-H elongation vibrations, respectively. Furthermore, the conjugation of the PAbF to functionalised AuNP resulted in a peak at 3433 cm−1, which represents amide NH2 asymmetric stretching (3500 and 2800 cm−1), another peak at 1644 cm−1, which falls under the amide I region (1600–1700 cm−1), and a broad band at 682 cm−1 for N–H wagging (750–650 cm−1) (Ji et al., 2020; Pramanik et al., 2021; Stuart, 2004)(Supplementary Fig. S6D). On the other hand, the zeta potential (mV) of the bare citrate-capped AuNPs and AuNP-PAb conjugate was measured as −38.25 ± 0.07 mV and −30.31 ± 0.01 mV, respectively (Supplementary Fig. S6E).

TEM analysis depicted monodisperse shapes for the bare citrate-capped AuNPs; however, when PAbF was conjugated to the AuNPs, the particles got aggregated (Supplementary Figs. S6F and G). The particles' average diameter was 15.33 ± 0.12 nm and 16.44 ± 0.63 nm for bare AuNPs and AuNP-PAbF conjugate, respectively (Supplementary Figs. S6H and I). Further, the AFM analysis determined the height of the bare AuNP as 13.01 ± 0.03 nm, and after PAb adsorption, the size of the AuNP increased to 17.29 ± 0.17 nm (Supplementary Figs. S6J, K, L, M). The PAbF adsorption efficiency to the AuNP was 55.8 %, as determined via the calibrated Bradford protein assay curve (Supplementary Fig. S7).

3.7 Detection of MTV in spiked serum (in vitro) and enriched plasma from envenomed Wistar strain rats (in vivo) by AuNP-PAbF conjugate and quantitation of MTV in envenomed plasma

The UV–Vis spectrum result suggests that under in vitro conditions, the AuNP-PAbF conjugate showed precise immune recognition of MTV spiked rat plasma (0.3 ng/μL); however, it did not show immune cross-reactivity towards negative controls NnV and RVV (Supplementary Fig. S8). For control, NnV, and RVV samples, the LSPR peak was observed at 530 nm. In contrast, for MTV, the peak shifted to 579 nm (Supplementary Fig. S8).

In the case of LMMPT-enriched MTV-treated plasmas from groups III and IV rats, bathochromic shifts in absorption maxima (λmax) from 530 nm of AuNP-PAbF conjugate to 538 nm were observed, suggesting the immunoreaction occurrence; however, no such wavelength shift or increase in absorption intensity was observed for control plasma from group I and III rats (Fig. 5A). The protocol developed in this study can rapidly detect MTV in the plasma of envenomed experimental rats within 5–10 min, indicating this method's suitability in the rural health centres for detecting MT envenomation.Fig. 5 (A) Absorbance spectra of the AuNP-PAbF conjugate in the presence of control (untreated, group I and II) and MTV-treated-LMMPT-enriched plasma from group III and IV rats; (B) Absorbance spectrum for MTV spiked rat plasma detection. Absorbance curves correspond to plasma samples containing 1–5 ng/μL MTV; (C) Calibration curve for MTV spiked rat plasma detection at concentrations 1–5 ng/μL. Error bars indicate mean ± S.D. (n = 3).

Fig. 5

The AuNP-PAbF conjugate was treated with MTV concentrations ranging from 1 ng/μL to 5 ng/μL, and UV–Vis measurements were recorded. From Fig. 5B, it was observed that with an increase in MTV concentration, the characteristic peak of the AuNPs at 524 nm decreased, and after aggregation of AuNPs, there was a shift in the band to around 540 nm. The MTV was quantified per previously available protocols (Bala et al., 2016; Sittiwong and Unob, 2015). The absorbance ratio of the aggregated peak to the characteristic AuNP peak, i.e., A540/A524, was plotted, and the calibration curve thus obtained was fitted to the concentration of MTV. It was found that A540/A524 was proportional to the concentration of MTV with a regression coefficient R2 = 0.9975 (Fig. 5C). The sensing method's detection limit (LOD) was calculated to be 0.3 ng/μL using formula 3.3σ/S where σ is the standard deviation of the response, S is the slope of the calibration curve. The limit of quantitation was calculated to be 0.91 ng/μL as per formula 10σ/S. By the above method, the venom quantity detected in the intravenously and subcutaneously injected LMMPT-enriched MTV-treated plasma was 0.64 ng/μL (38.8 ± 1.0%) and 3.05 ng/μL (48.3 ± 0.5%), respectively. Here, we would like to state that in vitro condition of venom detection differs from in vivo venom detection because several factors may interfere during in vivo venom detection.

4 Discussion

Several clinical symptoms are reported in cases of scorpion envenomation, and current diagnosis techniques based on these clinical manifestations can occasionally harm patients' health if they are misdiagnosed. Therefore, using this assay to confirm venomous bites in clinical samples where the diagnosis is unclear may be helpful. Synthetic peptides have been the subject of increased investigation in recent years. Our investigation used their adaptability to raise polyclonal antibodies against the CPs specific to the MTV toxin epitope.

Obtaining scorpion venom is challenging since, after laborious procedures, only a minimal amount of venom can be extracted from each specimen, separating toxins from the entire venom complex. Additionally, purifying toxins is time-consuming, expensive, and yield-dependent. As a result, it is preferred to identify, design, and synthesise bespoke peptides because they can be produced consistently and with long-term stability. Additionally, synthetic peptides facilitate the measurement of antibodies specific to specific antigens, resulting in more precise assays (Meloen et al., 1997; Puzari et al., 2023).

The highest antigenic propensity epitopes of two major toxins of MTV viz. Na+ and K+ channel toxins were used to design the peptides that mimic the antigenic epitope of these two major toxins of MTV to raise polyclonal antibodies against them. Since the rapid oxidation of cysteine residues in the native peptide sequence can affect peptide synthesis and purification by adversely influencing the cleavage of protecting groups (Spears et al., 2021), the cysteine residues were substituted with serine while designing toxin-epitope-specific custom peptides. There is very little difference between these two amino acids. Therefore, there are only minor differences in bond lengths and angles between these two amino acids, but the conversion of cysteine to serine can ease the peptide synthesis process (Catalano et al., 2021). Usually, the small size of peptides does not induce an immune response to produce high-titre antibodies in host animals (Hermanson, 2008). Thus, the peptides were conjugated with carrier protein KLH via a bifunctional linker to raise a high titer of PAbs in rabbits (Houen et al., 2003; Schaaper et al., 1989).

Animal models have significantly contributed to advancing biomedical research by helping us understand various biological and pathological processes (Domínguez-Oliva et al., 2023). Due to their physiological similarity to humans, rodents have recently become the most commonly used species in biomedical research (Carbone, 2021; Makowska and Weary, 2019). Their use can advance our understanding of various processes, including therapeutics, diagnostic methods, and biological development (Carbone, 2021; Domínguez-Oliva et al., 2023; Makowska and Weary, 2019).

Detection at the concentration of 0.15 ng/μL may be beneficial to determine MTV in the plasma/serum of envenomed animals or humans, as reports have suggested that the maximum venom volume injected per sting of M. tamulus is 1.5 mg in approximately 5L of blood for an adult human (concentration of 0.3 ng/μL). Interestingly, PAbF showed synergistic action in significantly higher immune recognition of MTV than the individual PAbs. Such synergistic action of PAbs for MTV detection or any other venom protein detection has yet to be reported.

Moreover, a higher proportion of Na+ and K+ channel-specific antibodies in the PAbF showed significantly higher potency in immune recognition of MTV than commercial ASAs under identical experimental conditions. Furthermore, PAbF did not show immune cross-reactivity towards the snake venoms tested, ruling out that they could give false positive results against neurotoxic snakebites. Based on the results of the immune cross-reactivity of individual PAbs and PAbF with MTV as demonstrated by ELISA and dot blot assay, PAbF was used further to detect MTV in the plasma of envenomed rats.

The pharmacokinetics studies of other scorpion venoms (A. australis garzonii, T. serrulatus, and B. occitanus tunetanus) in rodents have demonstrated that the peak concentration of venom reaches in blood between 30 and 60 min after venom injection, and after that, the venom is distributed in the organs and eliminated rapidly from the blood (Krifi et al., 2001, 2005; Revelo et al., 1996). Our latest study on the blood glucose levels in rats after MTV treatment has reported a significant increase in the levels 30–120 min post venom injection (Das et al., 2023), and the finding of this study is in close agreement with these reports. Additionally, the signal intensity (immune cross-reactivity between PAbF and MTV in plasma) for the subcutaneously envenomed animal was higher than intravenously envenomed, which may be because more venom was injected via the subcutaneous route. Since the venom reaches blood at a slower rate via this route, the dispersion of venom from the bloodstream is slower than intravenous administration of MTV.

One of the mandates of our study was to develop a sensitive method that can detect a low concentration of MTV in the plasma of envenomed patients. However, the low molecular mass MTV toxins (or other peptides) in plasma are often veiled by the presence of high-abundance proteins (HAPs), which comprise more than 99% of the amount of protein in plasma (Polaskova et al., 2010; Righetti et al., 2005); consequently, it was an obstacle in our intention of developing of a sensitive kit for MTV detection. To overcome this problem, we developed a new technique to enrich LMMPT in MTV-envenomed plasma to detect scorpion envenomation better. The PAbF showed better immune recognition of MTV in LMMPT-enriched MTV-treated plasma than non-enriched MTV-treated plasma, thereby indicating that this technique can be helpful for the development of a sensitive, cost-effective, and rapid method for diagnosing and quantifying MTV in scorpion sting patients.

In snake and scorpion envenomation cases, there is often a considerable time gap between bite incidence and the arrival of most patients at health centres (Das et al., 2021; Krifi et al., 2001; Puzari and Mukherjee, 2020; Selvanayagam and Gopalakrishnakone, 1999). Since venom detection after a certain time interval post-sting is a difficult task due to the fast elimination of the venom, one of the objectives of this study was to develop a simple method that a technician can use in rural tropical primary and secondary health centres without the requirement of costly equipment for rapid diagnosis of scorpion envenomation. Immune-recognition techniques such as ELISA and Western blot based on expensive chemicals and instruments may serve a different purpose. Therefore, we considered developing a rapid and straightforward visual detection method for MTV envenomation. During the last couple of decades, AuNPs have shown tremendous promise in detecting biomaterials and various other applications (Chen et al., 2008; Li and Chen, 2015; O'Neal et al., 2004; Ramalingam, 2019).

AuNPs possess tuneable and unique optical properties due to the possession of a physical phenomenon called surface plasmon resonance (SPR). AuNPs are strongly absorbed in the visible light region, and the oscillating electromagnetic field of the light induces a collective coherent oscillation of the conduction band electrons on the NP surface (Huang and El-Sayed, 2010; Ramalingam, 2019). The detection sensitivity by AuNP also depends on the size of the particle. There have been reports that AuNPs with larger sizes have better sensitivity but are not as stable as AuNPs of approximately 13–20 nm size (Guo et al., 2011; Lou et al., 2012).

In our study, the synthesised AuNPs were monodisperse with a size of around 13–15 nm, and the size of the AuNP-PAbFs increased marginally (16–17 nm). The amide bonds observed in the infrared spectroscopic studies confirmed the conjugation and the increase in zeta potential when compared to bare AuNPs. Because NaCl concentration plays a vital role in choosing the minimum concentration of PAbF required for stabilising the AuNP- PAbF complex, its optimum concentration was studied. NaCl aggregates AuNPs, changing the colour of the AuNP colloidal solution from red wine to purple/blue. The optimum binding concentration of PAbF to AuNPs surface to maintain the uniform dispersion was determined at 1 μg/μL, which is good enough to determine the low concentration of MTV in a scorpion-envenomed plasma sample. Furthermore, the AuNPs utilised in this study could be substituted with more sensitive detection systems, such as AuNP-coupled semiconductor quantum dots or fluorophore dyes, to increase the detection limit of MTV. However, the significant advantage of this protocol developed in this study is that it can rapidly detect MTV in the plasma of envenomed experimental rats within 5–10 min, indicating this method's suitability in the rural health centres for detecting MT envenomation.

The detection method proposed in our study necessitates using a visible range spectrophotometer to detect the LSPR peak shifting. The detection method may help quantify the circulating venom and assess the antivenom required to eliminate the venom from the blood. There have been a few reports of venom detection of scorpion species that are not endemic to India (Krifi et al., 1998; Mars et al., 2018). However, the method suggested in this study is the first report of an analytical method using species-specific antibodies for sensitive detection of MTV within 5–10 min. However, this method is a proof-of-concept, and further investigations are warranted to improve the technique's sensitivity based on nanoparticle conjugate. In the future, it may be feasible to design devices/kits based on image analysis, microfluidics, or lateral flow assays for rapid detection of MTV at the point of care. Moreover, a thorough investigation of samples from a large cohort of stung patients following M. tamulus sting will also have to be executed to facilitate the use of the proposed diagnostic method in clinical settings.

5 Conclusion

A prompt diagnosis of scorpion envenomation is essential to preserve patients' lives because the condition might have several complications. This approach should also be affordable and straightforward. In this work, we generated rabbit polyclonal antibodies against four CPs based on the main toxins of MTV. The reported antibodies in the formulation have demonstrated synergistic effects in both in vitro and in vivo recognition of MTV. As a result, a detection kit for MTV detection at rural health clinics across the country might be created using this antibody composition. However, more research utilising the plasma of M. tamulus envenomed patients is required to validate its application in clinical settings for either the antibody formulation or the discovered diagnostic approach.

Ethical statement

The authors hereby stated that all procedures involving animals were conducted in ethical manner. Ethical approvals were obtained from institutional animal ethics committee of Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam, India vide approval number IASST/IAEC/2022/09. Animals at IASST and BioBharati Pvt. Ltd. were maintained and used according to the OECD guidelines for chemical safety and animal welfare.

CRediT authorship contribution statement

Upasana Puzari: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Mojibur R. Khan: Writing – review & editing. Ashis K. Mukherjee: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Ashis Kumar Mukherjee has patent Antibodies for the Detection of Indian Red Scorpion Venom and Composition Thereof pending to Institute of Advanced Study in Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Acknowledgements

UP is the recipient of a research fellowship from Tezpur University. The authors thank the SAIC of IASST and TU for the instrument help. This study received partial financial support from the core grants of 10.13039/501100024589 IASST , Guwahati. The authors thank Dr. A. Bala, IASST, and Dr. B. Das, Tezpur University, for their help and guidance during the in vivo experiments. The authors thank Dr. Srikanth Rapole, NCCS, Pune, for LC-MS/MS sample processing and Dr. A. Patra, IASST, for helping with the mass spectroscopy data analysis. A graphical abstract was created with BioRender.com.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.toxcx.2024.100203.
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