www.nature.com/scientificreports OPEN An in vitro potency assay using nicotinic acetylcholine receptor binding works well with antivenoms Received: 12 January 2018 Accepted: 29 May 2018 against Bungarus candidus and Published: xx xx xxxx Naja naja Kavi Ratanabanangkoon1,2,3, Pavinee Simsiriwong1, Kritsada Pruksaphon4, Kae Yi Tan5, Bunkuea Chantrathonkul1, Sukanya Eursakun1 & Choo Hock Tan6 In order to facilitate/expedite the production of efective and afordable snake antivenoms, a novel in vitro potency assay was previously developed. The assay is based on an antiserum’s ability to bind to postsynaptic neurotoxin (PSNT) and thereby inhibit the PSNT binding to the nicotinic acetylcholine receptor (nAChR). The assay was shown to work well with antiserum against Thai Naja kaouthia which produces predominantly the lethal PSNTs. In this work, the assay is demonstrated to work well with antiserum/antivenom against Bungarus candidus (BC), which also produces lethal presynaptic neurotoxins, as well as antivenom against Sri Lankan Naja naja (NN), which produces an abundance of cytotoxins. The in vitro and in vivo median efective ratios (ER50s) for various batches of antisera against BC showed a correlation (R2) of 0.8922 (p < 0.001) while the corresponding value for the anti-NN antivenom was R2 = 0.7898 (p < 0.01). These results, together with the known toxin profles of various genera of elapids, suggest that this in vitro assay could be used with antisera against other species of Bungarus and Naja and possibly other neurotoxic snake venoms worldwide. The assay should signifcantly save numerous lives of mice and accelerate production of life-saving antivenoms. Snake envenomation is an important yet neglected medical problem in various developing countries with an estimated annual envenomation world wide of about 5.5 million cases1,2. Efective and afordable antivenoms (AV) which are the mainstay for treatment remain unavailable in several parts of the world while research eforts are undertaken to solve this problem3. In the development and production of an AV, an important step involves the in vivo assay to evaluate the potency of the produced antiserum/antivenom. Te standard murine lethality neutralizing assay is considered by WHO as an essential AV potency assay. Tis assay is used to fnd frst, the median lethal dose (LD50) that determines the lethality of the venom, and the median efective dose (ED50) of the AV 4,5. Te assay is expensive, laborious and, due to biological variation, ofen give highly variable results. In addition, some murine lethality results might not be consistent with the relevant efcacy outcomes in humans6,7. In Tailand, as well as in many Buddhist countries, it is very difcult to fnd researchers or students who would agree to do these in vivo experiments. Because of these reasons, various in vitro assays have been developed to reduce or replace the murine lethality assay. Te most widely used assay is based mainly on ELISA8–10 but some of these assays have ofen been shown to give poor correlation with the in vivo assay11,12. Moreover, the antigen-antibody ‘binding’ reaction of ELISA may not result in the ‘neutralization’ of the antigen, and therefore alternative in vitro assay should be developed. 1Laboratory of Immunology, Chulabhorn Research Institute, Bangkok, 10210, Thailand. 2Chulabhorn Graduate Institute, Bangkok, 10210, Thailand. 3Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok, 10400, Thailand. 4Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand. 5Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia. 6Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia. Correspondence and requests for materials should be addressed to K.R. (email: [email protected]) or C.H.T. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:9716 | DOI:10.1038/s41598-018-27794-3 1 www.nature.com/scientificreports/ Figure 1. Inhibition of nAChR binding by B. candidus and N. naja venoms to NK3-coated plate. Data were means ± SD of 3 determinations. Recently, a novel in vitro assay using solubilized, purifed nicotinic acetylcholine receptor (nAChR) binding has been developed for AV potency assay against the Tai cobra Naja kaouthia13. Tis elapid snake produces mainly postsynaptic neurotoxins (PSNT) which binds specifcally and with high afnity to the nAChR at the mus- cle endplate and thereby inhibits the neuro-muscular transmission causing death in animals. Te in vitro assay therefore closely mimics the toxicological reactions in vivo; and the in vivo and in vitro potency assays showed a correlation R2 = 0.9807, p < 0.0001. Te assay should also work well with AVs against elapids in the genera other than Naja which produce mainly or exclusively post-synaptic toxins (PSNTs) e.g., King cobra, Ophiophagus hannah14. However, snakes in the genus Bungarus (kraits) also produce, in addition to PSNTs, the lethal presynaptic neurotoxins (β-neurotoxins) which in elapids, belong to the group 1 phospholipase A2 enzymes. Tese toxins do not bind to nAChR but react with receptors on the membrane of the motor nerve terminals which contain the acetylcholine vesicles. Te toxins hydrolyze the phospholipids of the plasma membrane, resulting in the loss of synaptic vesicles in the nerve terminal. Eventually the nerve terminals degenerate with the failure of the neuro- 15 16 muscular transmission . Te LD50 of the β-neurotoxins is about 10 ng/g which is considerable lower than that of the PSNT (0.18 μg/g mouse)17. It is therefore conceivable that in the case of some Bungarus venoms, death could be caused, at least in part, by the β-neurotoxins; but this efect would not be measured by the nAChR binding of the in vitro potency assay. Consequently the developed in vitro assay might not be useful for assay of AV against Bungarus spp. Another interesting case is the Naja naja (Indian cobra) which is a WHO category 1 medically most important elapid in India, Pakistan and Sri Lanka. Envenomation by this snake resulted in muscle weakness and death by respiratory failure which is likely the efect of the venom PSNTs. Interestingly, the venom of the Sri Lankan snake was shown by proteomics study to contain 71.55% of cytotoxins (cardiotoxins)18. Tese three fnger toxins (3FTs) cause severe local tissue necrosis in most (88%) of the victims18 and could possibly contribute to the venom lethal- ity. It is therefore interesting to investigate whether the developed in vitro nAChR binding assay could be used for potency assay of AV against this cobra. We report here that the in vitro nAChR binding assay, when used in the potency determinations of AVs against Bungarus candidus (Tailand) and Naja naja (Sri Lanka), gave high correlation with the corresponding in vivo murine lethality neutralization assays. Results Studies on the optimal conditions of the in vitro AV potency assay. Te optimal concentrations of NK3, nAChR, rat anti-nAChR antibody and goat-anti-rat HRP conjugate used in the assay. Te optimal con- centrations of these four reagents used in the in vitro potency assay were described in a previous study13. Te optimal concentration of NK3 for coating the plates was 15 µg/ml, and 0.707 µg/ml of nAChR for binding to the NK3 coated plate. Rat anti-nAChR serum and goat anti-rat-IgG conjugated HRP were used at 1:1600 dilution and 1:4500 dilution, respectively. Inhibition of nAChR binding to NK3-coated plate by B. candidus or N. naja venom. Crude B. candidus and N. naja venoms were separately used to determine the 50% inhibition of the nAChR binding (in vitro IC50). In the frst step, various concentrations of crude B. candidus (or N. naja) venom and the purifed nAChR (0.707 µg/ml) were incubated for 1 h at 25 °C. Afer the incubation, the solution was added to the NK3 coated plates. Te con- centration of the venom that blocked the binding of nAChR by 50% was the IC50. Te results (Fig. 1) showed that the IC50s of B. candidus and N. naja venoms were 0.1625 ± 0.0172 µg/ml and 0.4067 ± 0.0292 µg/ml, respectively. Neutralization of B. candidus or N. naja venom by horse monospecifc antisera against B. candidus or by Vins antivenom against N. naja as determined by nAChR binding to the NK3-coated plate. Nine horse monovalent anti-B. candidus sera were 2-fold diluted from 1:500 to 1:512,000. Tese diluted sera of diferent horses were separately incubated with 5xIC50 of B. candidus venom (1.4029 µg/ml) in the ‘Pre-incubation 1′ experiment. Te reaction mixtures were ultrafltered and subjected to ‘Pre-incubation 2′. Te resulting reaction mixtures were then added to the NK3-coated plates. Te nAChR binding to the plate was read at OD450nm and the results which SCIENTIFIC REPORTS | (2018) 8:9716 | DOI:10.1038/s41598-018-27794-3 2 www.nature.com/scientificreports/ Figure 2. Neutralization of B. candidus venom by horse anti-B. candidus sera (A) and neutralization of N. naja venom by Vins anti-N. naja antivenom (B) as determined by nAChR binding to NK3-coated plate. Te results were means ± SD of 3 determinations. Horse In vitro ER50 ± SD In vivo ER50 Number sera# (µg venom/µl antiserum) (mg venom/ml antiserum) 1 Bc-A 0.6698 ± 0.1091 1.19 (1.59–1.86) 2 Bc-B 0.3982 ± 0.0397 0.94 (0.62–1.46) 3 Bc-C 0.6131 ± 0.0203 1.25 (0.83–1.94) 4 Bc-D 0.6597 ± 0.0639 1.25 (0.83–1.94) 5 Bc-E 0.3184 ± 0.0690 0.75 (0.50–1.17) 6 Bc-F 0.2161 ± 0.0230 0.39 (0.26–0.60) 7 Bc-G 0.1206 ± 0.0170 0.33 (0.22–0.51) 8 Bc-H 0.0662 ± 0.0027 0.13 (0.09–0.20) 9 Bc-I 1.1083 ± 0.2009 1.50 (1.00–2.33) # Table 1.