Toxicon 77 (2014) 1–5
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Development of an in vitro potency assay for antivenom against Malayan pit viper (Calloselasma rhodostoma) Duangporn Pornmuttakun a,1, Kavi Ratanabanangkoon b, c, * a
Department of Microbiology, Faculty of Science, Mahidol University, Thailand Laboratory of Immunology, Chulabhorn Research Institute, Thailand c Chulabhorn Graduate Institute, Bangkok, Thailand b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2013 Received in revised form 15 October 2013 Accepted 17 October 2013 Available online 30 October 2013
An in vitro potency assay of antivenom against Malayan pit viper (Calloselasma rhodostoma, CR) has been developed. The assay is based on the neutralizing activity of the antivenom against the coagulant activity of the venom. The minimum coagulant dose (MCD) of CR venom was 22.12 0.25 mg/ml. The coagulation time induced by 2MCD of the venom was used as the control for calculating the neutralizing activity of each batch of antivenom. The in vitro potency of antivenom, expressed as effective dose (ED), was the antivenom/venom ratio at which the coagulation time was increased three fold of that induced by 2MCD of the venom. Eleven batches of the antivenom were assayed for their lethality neutralizing activity (ED50) by the in vivo assay using mice as well as the developed in vitro assay. The correlation coefficient (r) between the in vitro neutralizing activities (ED) and in vivo neutralizing activities (ED50) was 0.957, (p value < 0.001). This simple and rapid in vitro assay of C. rhodostoma antivenom should be a good alternative method for the assessment of antivenom potency during the immunization program and fractionation process. The assay should be adaptable for use with antivenoms against other similar procoagulant venoms. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Calloselasma rhodostoma In vitro potency assay Antivenom Coagulation inhibition
1. Introduction Antivenom production usually involves immunization, fractionation of the antibody and quality control of the final product. In each of these processes, assay of the neutralizing antivenom antibody is monitored to assess the performance of the process. The potency of antivenom is traditionally assessed by the in vivo neutralization of Abbreviations: CR, Calloselasma rhodostoma; MCD, minimum coagulant dose; ED50, median effective dose; ED, in vitro effective dose. * Corresponding author. Laboratory of Immunology, Chulabhorn Research Institute, 54 Kampangpetch 6 Road, Lak-si, Bangkok 10210, Thailand. Tel.: þ66 86 974 8874; fax: þ66 2 553 8563. E-mail addresses: [email protected], [email protected] (K. Ratanabanangkoon). 1 Present address: Queen Saovabha Memorial Institute, Rama IV Road, Bangkok, Thailand. 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.10.021
venom lethality in mice (World Health Organization, 1981). However, the in vivo assay is expensive, gives variable results and is often opposed on ethical or religious grounds. Thus, alternative in vitro immunoassays, e.g., ELISA have been developed (Tan et al., 1992; Theakston and Reid, 1979). ELISA is easy to develop and perform. It works well when the principal lethal toxin of the venom is known and used as the antigen of the ELISA (Rungsiwongse and Ratanabanangkoon, 1991). Such an assay quantitates only the antibody against the lethal toxin of the venom. This is the case with many elapid venoms where the cause of death is due to the postsynaptic neurotoxins. However, some of the ELISA results of antivenom potency assays do not correlate well with those of in vivo assays (Ibrahim and Farid, 2009). The reason for this is that most snake venoms contain up to a hundred different proteins, most of which
2
D. Pornmuttakun, K. Ratanabanangkoon / Toxicon 77 (2014) 1–5
are non-toxic. Thus, when crude venoms are used as antigens, the ELISAs quantitate the total antibodies against all the venom proteins regardless of their toxicity. This is especially true for antivenoms against viper venoms in which various toxic enzymes work in combination (Markland, 1998; White, 2005). In these cases, fractionations of the venoms to find the most appropriate venom antigens of the ELISA have to be carried out, which is laborious (Heneine et al., 1998). As an alternative to ELISA, an interesting in vitro assay involving the inhibition of enzymatic activity of venom was reported by Gutiérrez et al. (1988). Polyvalent antivenom against Bothops asper was shown to neutralize phospholipase A2 indirect hemolytic activity of the venom using a sensitive plate test. The enzyme inhibitory activity and the in vivo neutralization activity of the antivenom were found to have highly significant correlation. Malayan pit viper (Calloselasma rhodostoma, CR) is the most medically important snake in Malaysia, Thailand and other Southeast Asian countries (Ho et al., 1986; Reid et al., 1963a; Tan and Ponnudurai, 1996). It accounts for the majority of snake envenomations. At the Queen Saovabha Memorial Institute, Bangkok, the production of antivenom against CR accounted for 34% of the total 85,774 vials of 7 monospecific antivenoms produced in 2007. The CR venom contains a number of enzymes acting on haemostasis. The principal in vivo effects of the CR venom are defibrination, hemorrhage and local tissue necrosis (Ho et al., 1986; Reid et al., 1963b). The crude venom has dual effects on blood coagulation: the thrombin-like enzymes which bring about clotting in vitro but defibrination (anticoagulantion) in vivo (Reid et al., 1963b). The patient’s blood fails to clot because of the depletion of clotting factors as a result of the venom procoagulant activity. The thrombin-like enzyme converts fibrinogen to fibrin and has powerful coagulant properties. The well-known thrombin-like enzyme from CR, Arvin or Ancrod, has been used clinically as a defibrinating agent. The objective of this study was to develop a simple and reliable in vitro assay to estimate the potency of antivenom against CR. The in vitro assay, based on the neutralization of the coagulation induced by the venom, was found to have a high correlation coefficient with the in vivo neutralization assay. This simple and inexpensive assay should be applicable to antivenoms against many similar procoagulant venoms.
99.16 9.19 mg/ml. Sheep blood in 3.8% sodium citrate used as anticoagulant was purchased from National Animal Laboratory Center, Nakorn Prathom. Sheep blood was centrifuged at 2100 g at 4 C for 20 min; the plasma was separated and stored at 20 C. 2.2. In vitro assay of antivenom potency 2.2.1. Determination of minimum coagulant dose (MCD) The MCD was determined according to the method described by Theakston and Reid (1983) with some modifications. Various concentrations of fresh CR venom were prepared in 0.05 M Tris–HC1 pH 7.2. Each venom solution (100 ml) was mixed thoroughly with 200 ml of citrated solution of sheep plasma previously incubated at 37 C. The clotting time was determined visually and recorded in seconds. Each test was run in triplicate. The logarithm of venom concentration was plotted against the clotting time to estimate the clotting activity. The MCD is defined as the concentration of venom (in mg/ml) which induces coagulation of sheep plasma in 60 s under the described conditions. 2.2.2. Neutralization of the coagulant activity The method described by Gene et al. (1989) was used. A constant amount of venom (20 MCD) was incubated with several dilutions of antivenom for 30 min at 37 C in a final volume of 1.0 ml. Then, 100 ml of the mixture which contained 2 MCD of the venom was added to 200 ml of citrated solution of sheep plasma. Controls included sheep plasma incubated with either venom alone or antivenom alone. Neutralization was expressed as effective dose (ED). ED is defined as the antivenom/venom ratio at which coagulation time is increased three fold when compared to the coagulation time of plasma incubated with venom alone. 2.3. In vivo assay of antivenom potency
2.1. Chemicals and biochemical
2.3.1. Determination of median lethal dose (LD50) The LD50 assay was based on the method described by Theakston and Reid (1983). Groups of five ICR mice weighing 20 2 g were injected intra-peritoneally with CR venom at doses ranging from 2 to 10 mg/g mouse. The venom was prepared in normal saline and the volume of injection was kept constant at 200 ml/20 g mouse. Control animals were injected with normal saline only. The lethality, expressed in percent death of animals, was recorded 48 h after injection. The medium lethal dose (LD50) of the venom was calculated by the method of Reed and Muench (1938).
Chemicals were of reagent grade and were purchased from Sigma Chemical Company, St. Louis, MO, USA, except as indicated. Pooled fresh crude CR venom was purchased from Queen Saovabha Memorial Institute, Bangkok. The venom was kept at 20 C until used. Eleven batches of crude antivenom sera against CR, obtained from horses during varying stages of immunization programs and thus with varying antibody titers, were from Queen Saovabha Memorial Institute. The sera were separately stored at 4 C, and were filtered through 0.45 micron membrane before use. The protein concentration of the sera was
2.3.2. Determinations of median effective dose (ED50) The in vivo neutralization activity of CR antivenom was expressed as median effective dose (ED50), defined as microliters of the antivenom per mg of venom required to prevent death in 50% of injected mice. The procedure was similar to that described above for LD50 determination, but 2.5LD50 dose of CR venom in 20 ml of normal saline was mixed with different doses of horse antivenom against CR. Normal horse serum was added to make the volume up to 200 ml. These mixtures were incubated at room temperature for 30 min before injection. The
2. Materials and methods
D. Pornmuttakun, K. Ratanabanangkoon / Toxicon 77 (2014) 1–5
A Coagulation time (sec.)
volume of injection was kept constant at 200 ml/20 g mouse. Control mice were injected with venom solution (in sterile normal saline) or horse serum. The number of surviving mice was recorded 48 h after injection. ED50s were calculated using the probit analysis method of Finney (1971). It should be mentioned that at the QSMI horse farm, neutralization test of horse antivenom sera is often carried out by i.v. injection at the mouse tail vein. In order to avoid inaccuracies caused by leakage of the solution at the injection site, the present test was done using i.p. injection. Experiments involving animals were reviewed and approved by the Animal Care and Use Committee of Faculty of Science, Mahidol University (Protocol No. MUSC 56-008-271).
3
80 70 60 50 40
22.12
30 0 15
20
25
30
35
40
50
CR venom concentration (µg/ml)
Protein concentration was determined by the procedure described by Lowry et al. (1951) using bovine serum albumin as the standard. 2.5. Statistical analysis ED50 of antivenoms are expressed as means with 95% confidence intervals (C.I.) and were calculated using the probit analysis method of Finney (1971). Correlation between the ED50 and ED values, obtained from the in vivo and the in vitro assays, respectively, was determined by means of linear regression analysis with the correlation coefficient of Pearson and Lee (1903). 3. Results 3.1. Determination of minimum coagulant dose (MCD) and the neutralization of coagulant activity by antivenom The coagulant activity of fresh CR venom as a function of venom concentration is shown in Fig. 1A. MCD, defined as the final concentration of CR venom (mg/ml) which induces coagulation of plasma in 60 s, was determined in 3 separated experiments and shown to be 22.12 0.25 mg/ml. The average coagulation time of 2MCD of the venoms was 34.1 3.39 s. The in vitro neutralizing activities of 11 batches of horse antivenom against CR venom were determined (Materials and methods). The CR venom at 2MCD was included in every test to serve as a control to correct day to day variations. The coagulation time induced by 2MCD venom was used as the reference value for calculating the neutralization capacities of antivenoms. The coagulation time induced by the venom in the presence of each batch of antivenom at various dilutions was plotted against the antivenom/venom ratio (Fig. 1B). The effective dose (ED) of each batch of antivenom was determined at the antivenom/venom ratio (ml/mg) at which coagulation time is increased three fold when compared to the coagulation time induced by 2MCD of venom alone. As an example of such determination, the coagulation time of 2MCD of the venom in the control tube in Fig. 1A was 34.82 s. The ED of antivenom batch #1, the ratio of antivenom/venom giving coagulation time of
B
160 140
Coagulation time (sec.)
2.4. Protein determination
104.46
120 100 80 60
ED=389.91 µl/mg
40 0 0
280
320
360
400
440
480
Antivenom/venom ratio (µl/mg) Fig. 1. A. A plot of clotting time against various concentrations of CR venom. B. The neutralization of coagulant activity of antivenom batch #1. ED was the ratio of antivenom/venom at which coagulation time is increased three folds when compared to the coagulation time induced by 2 MCD of venom alone. The coagulation times of 2 MCD of venom alone was 34.82 s.
3 34.82 ¼ 104.56 s was, from the graph, 389.91 ml/mg. The EDs of 11 batches of antivenom are shown in Table 1. Horse antivenoms and normal horse serum were also tested for their coagulant activity in the absence of CR venom. There was no coagulant activity observed with sheep plasma within 10 min. 3.2. Median effective dose (ED50) of various batches of antivenom The LD50 of the CR venom was determined to be 5.41 mg/ g mouse (95% confidence interval 2.98–6.57 mg/g mouse). This lot of venom was used in all determinations of ED50 of 11 batches of horse antivenoms against CR venom. The antivenom potencies of 11 batches of CR antivenom sera determined by the in vivo neutralization assay using mice, the ED50, together with the 95% confidence intervals are shown in Table 1. 3.3. Correlation between in vitro neutralizing activities and in vivo neutralizing activities of 11 batches of CR antivenom The in vivo neutalizing activities, ED50, and in vitro neutalizing activities, ED, of 11 batches of antivenom were
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Table 1 The in vitro and in vivo neutralization activities of various batches of CR antivenom as expressed as effective dose (ED) and ED50, respectively. Antivenom In vitro effective dose (ED) In vivo effective dose (ED50) (ml serum/mg venom) No. (ml serum/mg venom) 1 2 3 4 5 6 7 8 9 10 11
388.56 566.43 152.97 188.12 399.14 211.85 320.60 180.88 347.39 254.87 340.21
1.91 13.59 4.62 10.89 2.06 15.61 9.78 9.32 19.81 2.03 21.90
356.00 495.50 169.00 218.50 422.50 263.50 333.00 248.50 377.00 332.00 373.50
(297.00–422.50) (426.00–532.50) (97.50–193.00) (164.00–300.50) (394.00–481.00) (205.00–343.50) (266.00–407.50) (173.00–357.00) (257.50–483.00) (256.50–442.50) (291.00–432.50)
compared by means of linear regression analysis. A plot of ED50 vs ED is shown in Fig. 2. The correlation coefficient (r) of these two assays is 0.957 (p < 0.001). 4. Discussion The potency of an antivenom is generally determined by its ability to save the lives of experimental animals injected with the homologous venom. The in vivo assay of antivenom potency can give variable results caused by a wide range of factors, e.g., species, strain, weight, sex and routes of injection. The assay is also time consuming, laborious and expensive. It is often difficult for some snake venoms and antivenoms especially those of vipers, to obtain good dose response curve unless a large number of animals are used. Moreover, the in vivo assays have been opposed on ethical grounds, and in Buddhist countries, most laboratory personnel refuse to perform such assays. Because of these shortcomings, attempts have been made to find alternative in vitro assays to the use of live 600
In vitro neutralization (µl serum/mg venom) ED
500
400
300
200
100
0 0
100
200
300
400
500
600
In vivo neutralization (µl serum/mg venom) ED50 Fig. 2. Correlation between in vivo neutralizing activity (ED50) and in vitro neutralizing activity (ED). The correlation coefficient (r) was 0.957 (p value < 0.001).
animals. ELISA has been widely used to quantitate the antibody content in antivenom preparations (Rungsiwongse and Ratanabanangkoon, 1991; Tan et al., 1992; Theakston and Reid, 1979). This assay showed very high correlation with in vivo assay with snake venom containing a major lethal toxin such as that found in elapid venoms. When the venom lethal component is used as antigen, the ELISA quantitates only the antibody against the lethal component and therefore gives results which correlate highly with those obtained from in vivo assays (Rungsiwongse and Ratanabanangkoon, 1991). However, viper and pit viper venoms are composed of diverse proteins that have different enzymatic activities acting synergistically on substrates and the overall toxicity is not the resultant or the summation of toxicity of all separate substances. Consequently ELISA that quantitates the antibody against all venom proteins does not usually have a good correlation with the results obtained from in vivo assays (Heneine et al., 1998). The present in vitro assay of antivenom potency was based on the neutralization of the coagulation induced by the venom. For CR venom, the thrombin-like serine protease. (Ancrod or Arvin) acts on fibrinogen causing coagulation in vitro and leads to defibrinating syndrome in vivo (Nolan et al., 1976; Pirkle, 1998; Reid et al., 1963b). Although the venom contains hemorrhagin and other enzymes (Chung et al., 1996; Tan et al., 1986), the thrombin-like enzyme may also contribute to the pathological effect of this venom. The in vitro assay based on the inhibition of this enzyme reaction seems to be a reasonable choice. As observed by Gene et al. (1989), antivenoms alone could induce coagulation of fibrinogen and plasma. However, it was found here that CR equine antiserum per se had no coagulant effect on sheep plasma. One important criterion in the development of in vitro assay is how good its results correlate with those obtained from the in vivo assay. In this study, good correlation between the in vitro activity and the in vivo activity was obtained; the correlation coefficient (r) was 0.957 (p < 0.001). However, as mentioned by Gutiérrez et al. (1988), even when an in vitro test showed high correlation, it did not necessarily mean that the enzyme was responsible for the lethal toxic effect of the venom. Rather, it suggested that the antibody production against the enzyme paralleled those against the predominant lethal toxins of the venom. Compared with the in vivo test, the amounts of venom and antivenom used in the experiment are smaller, requiring 440 mg of venom for MCD and 50–500 ml of antivenom for ED determinations while in in vivo assays more than 1 mg of venom and 2 ml of antivenom were needed for each batch of antivenom. This observation can be an important consideration with some very scarce venom. The in vitro assay took less than 1 h to obtain the required result. The results reported here indicated that the in vitro assay using the neutralization of coagulant effect of CR venom could be used as an alternative assay for potency assessment of CR antivenom. The procedure is simple, rapid and inexpensive. However, it does not mean that this method can be used to totally replace the in vivo rodent assay which remains the requirement in many pharmacopoeias for standardization of commercial antivenoms. The
D. Pornmuttakun, K. Ratanabanangkoon / Toxicon 77 (2014) 1–5
in vitro assay, however, could be employed to evaluate the potency of antivenom at various steps of antivenom production, for example, during immunization and in different stages of plasma fractionation. The in vitro assay could reduce the use of live animals. Apart from the antivenom against C. rhodostoma, the neutralization of coagulant activity may be adopted for assessing the potency of antivenoms against other procoagulant venoms. Funding This study was supported by a research grant from Thailand Research Fund. Ethical statement This manuscript presents an experimental study performed following the standard procedures of scientific ethics, including the use and care of experimental animal. I certify that the result of the submitted for publication in Toxicon manuscript are original and are not submitted elsewhere for publication. Acknowledgments The authors thank Assistant Professor Maurice Broughton for valuable suggestions and Ms Lalida Arsa for assistance in preparing the manuscript. Conflict of interest statement None declared. References Chung, M.C., Ponnudurai, G., Kataoka, M., Shimizu, S., Tan, N.H., 1996. Structural studies of a major hemorrhagin (rhodostoxin) from the venom of Calloselasma rhodostoma (Malayan pit viper). Arch. Biochem. Biophys. 325, 199–208. Finney, D.J., 1971. Probit Analysis, third ed., vol. 60. Cambridge University Press, p. 1432. Journal of Pharmaceutical Sciences. Gene, J.A., Roy, A., Rojas, G., Gutierrez, J.M., Cerdas, L., 1989. Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic
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activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841–848. Gutiérrez, J., Avila, C., Rojas, E., Cerdas, L., 1988. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411–413. Heneine, L.G.D., Carvalho Jr., Á.d.D., Barbosa, C.d.f., arávjo dos Santos, M.R., 1998. Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom. Toxicon 36, 1363–1370. Ho, M., Warrell, D.A., Looareesuwan, S., Phillips, R.E., Chanthavanich, P., Karbwang, J., Supanaranond, W., Viravan, C., Hutton, R.A., Vejcho, S., 1986. Clinical significance of venom antigen levels in patients envenomed by the Malayan pit viper (Calloselasma rhodostoma). Am. J. Trop. Med. Hyg. 35, 579–587. Ibrahim, N.M., Farid, N.M., 2009. Comparison between two in vitro ELISAbased assays in the determination of antivenom potency. J. Appl. Sci. Res. 5, 1223–1229. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Markland, F.S., 1998. Snake venoms and the hemostatic system. Toxicon 36, 1749–1800. Nolan, C., Hall, L.S., Barlow, G.H., 1976. Ancrod, the coagulating enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom. Methods Enzymol. 45, 205–213. Pearson, K., Lee, A., 1903. On the laws of inheritance in man: I. Inheritance of physical characters. Biometrika 2, 357–462. Pirkle, H., 1998. Thrombin-like enzymes from snake venoms: an updated inventory. Thromb. Haemost. 79, 675–683. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Epidemiol. 27, 493–497. Reid, H.A., Chan, K.E., Thean, P.C., 1963b. Prolonged coagulation defect (defibrination syndrome) in Malayan viper bite. Lancet 1, 621–626. Reid, H.A., Thean, P.C., Chan, K.E., Baharom, A.R., 1963a. Clinical effects of bites by Malayan viper (Ancistrodon rhodostoma). Lancet 1, 617–621. Rungsiwongse, J., Ratanabanangkoon, K., 1991. Development of an ELISA to assess the potency of horse therapeutic antivenom against Thai cobra venom. J. Immunol. Methods 136, 37–43. Tan, N.-H., Ponnudurai, G., 1996. The toxinology of Calloselasma rhodostoma (Malayan pit viper) venom. J. Toxicol. Toxin Rev. 15, 1–17. Tan, N.-H., Yeo, K.-H., Nik Jaafar, M.I., 1992. The use of enzyme-linked immunosorbent assay for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom and venom antibodies. Toxicon 30, 1609–1620. Tan, N.H., Kanthimathi, M.S., Tan, C.S., 1986. Enzymatic activities of Calloselasma rhodostoma (Malayan pit viper) venom. Toxicon 24, 626– 630. Theakston, R.D., Reid, H.A., 1983. Development of simple standard assay procedures for the characterization of snake venom. Bull. World Health Organ 61, 949–956. Theakston, R.D.G., Reid, H.A., 1979. Enzyme-linked Immunosorbent Assay (ELISA) in assessing antivenom potency. Toxicon 17, 511–515. White, J., 2005. Snake venoms and coagulopathy. Toxicon 45, 951–967. World Health Organization, 1981. Progress in the Characterization of Venoms and Standardization of Antivenoms. World Health Organization, Geneva.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Development of an in vitro potency assay for antivenom against Malayan pit viper (Calloselasma rhodostoma) Duangporn Pornmuttakun a,1, Kavi Ratanabanangkoon b, c, * a
Department of Microbiology, Faculty of Science, Mahidol University, Thailand Laboratory of Immunology, Chulabhorn Research Institute, Thailand c Chulabhorn Graduate Institute, Bangkok, Thailand b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 July 2013 Received in revised form 15 October 2013 Accepted 17 October 2013 Available online 30 October 2013
An in vitro potency assay of antivenom against Malayan pit viper (Calloselasma rhodostoma, CR) has been developed. The assay is based on the neutralizing activity of the antivenom against the coagulant activity of the venom. The minimum coagulant dose (MCD) of CR venom was 22.12 0.25 mg/ml. The coagulation time induced by 2MCD of the venom was used as the control for calculating the neutralizing activity of each batch of antivenom. The in vitro potency of antivenom, expressed as effective dose (ED), was the antivenom/venom ratio at which the coagulation time was increased three fold of that induced by 2MCD of the venom. Eleven batches of the antivenom were assayed for their lethality neutralizing activity (ED50) by the in vivo assay using mice as well as the developed in vitro assay. The correlation coefficient (r) between the in vitro neutralizing activities (ED) and in vivo neutralizing activities (ED50) was 0.957, (p value < 0.001). This simple and rapid in vitro assay of C. rhodostoma antivenom should be a good alternative method for the assessment of antivenom potency during the immunization program and fractionation process. The assay should be adaptable for use with antivenoms against other similar procoagulant venoms. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Calloselasma rhodostoma In vitro potency assay Antivenom Coagulation inhibition
1. Introduction Antivenom production usually involves immunization, fractionation of the antibody and quality control of the final product. In each of these processes, assay of the neutralizing antivenom antibody is monitored to assess the performance of the process. The potency of antivenom is traditionally assessed by the in vivo neutralization of Abbreviations: CR, Calloselasma rhodostoma; MCD, minimum coagulant dose; ED50, median effective dose; ED, in vitro effective dose. * Corresponding author. Laboratory of Immunology, Chulabhorn Research Institute, 54 Kampangpetch 6 Road, Lak-si, Bangkok 10210, Thailand. Tel.: þ66 86 974 8874; fax: þ66 2 553 8563. E-mail addresses: [email protected], [email protected] (K. Ratanabanangkoon). 1 Present address: Queen Saovabha Memorial Institute, Rama IV Road, Bangkok, Thailand. 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.10.021
venom lethality in mice (World Health Organization, 1981). However, the in vivo assay is expensive, gives variable results and is often opposed on ethical or religious grounds. Thus, alternative in vitro immunoassays, e.g., ELISA have been developed (Tan et al., 1992; Theakston and Reid, 1979). ELISA is easy to develop and perform. It works well when the principal lethal toxin of the venom is known and used as the antigen of the ELISA (Rungsiwongse and Ratanabanangkoon, 1991). Such an assay quantitates only the antibody against the lethal toxin of the venom. This is the case with many elapid venoms where the cause of death is due to the postsynaptic neurotoxins. However, some of the ELISA results of antivenom potency assays do not correlate well with those of in vivo assays (Ibrahim and Farid, 2009). The reason for this is that most snake venoms contain up to a hundred different proteins, most of which
2
D. Pornmuttakun, K. Ratanabanangkoon / Toxicon 77 (2014) 1–5
are non-toxic. Thus, when crude venoms are used as antigens, the ELISAs quantitate the total antibodies against all the venom proteins regardless of their toxicity. This is especially true for antivenoms against viper venoms in which various toxic enzymes work in combination (Markland, 1998; White, 2005). In these cases, fractionations of the venoms to find the most appropriate venom antigens of the ELISA have to be carried out, which is laborious (Heneine et al., 1998). As an alternative to ELISA, an interesting in vitro assay involving the inhibition of enzymatic activity of venom was reported by Gutiérrez et al. (1988). Polyvalent antivenom against Bothops asper was shown to neutralize phospholipase A2 indirect hemolytic activity of the venom using a sensitive plate test. The enzyme inhibitory activity and the in vivo neutralization activity of the antivenom were found to have highly significant correlation. Malayan pit viper (Calloselasma rhodostoma, CR) is the most medically important snake in Malaysia, Thailand and other Southeast Asian countries (Ho et al., 1986; Reid et al., 1963a; Tan and Ponnudurai, 1996). It accounts for the majority of snake envenomations. At the Queen Saovabha Memorial Institute, Bangkok, the production of antivenom against CR accounted for 34% of the total 85,774 vials of 7 monospecific antivenoms produced in 2007. The CR venom contains a number of enzymes acting on haemostasis. The principal in vivo effects of the CR venom are defibrination, hemorrhage and local tissue necrosis (Ho et al., 1986; Reid et al., 1963b). The crude venom has dual effects on blood coagulation: the thrombin-like enzymes which bring about clotting in vitro but defibrination (anticoagulantion) in vivo (Reid et al., 1963b). The patient’s blood fails to clot because of the depletion of clotting factors as a result of the venom procoagulant activity. The thrombin-like enzyme converts fibrinogen to fibrin and has powerful coagulant properties. The well-known thrombin-like enzyme from CR, Arvin or Ancrod, has been used clinically as a defibrinating agent. The objective of this study was to develop a simple and reliable in vitro assay to estimate the potency of antivenom against CR. The in vitro assay, based on the neutralization of the coagulation induced by the venom, was found to have a high correlation coefficient with the in vivo neutralization assay. This simple and inexpensive assay should be applicable to antivenoms against many similar procoagulant venoms.
99.16 9.19 mg/ml. Sheep blood in 3.8% sodium citrate used as anticoagulant was purchased from National Animal Laboratory Center, Nakorn Prathom. Sheep blood was centrifuged at 2100 g at 4 C for 20 min; the plasma was separated and stored at 20 C. 2.2. In vitro assay of antivenom potency 2.2.1. Determination of minimum coagulant dose (MCD) The MCD was determined according to the method described by Theakston and Reid (1983) with some modifications. Various concentrations of fresh CR venom were prepared in 0.05 M Tris–HC1 pH 7.2. Each venom solution (100 ml) was mixed thoroughly with 200 ml of citrated solution of sheep plasma previously incubated at 37 C. The clotting time was determined visually and recorded in seconds. Each test was run in triplicate. The logarithm of venom concentration was plotted against the clotting time to estimate the clotting activity. The MCD is defined as the concentration of venom (in mg/ml) which induces coagulation of sheep plasma in 60 s under the described conditions. 2.2.2. Neutralization of the coagulant activity The method described by Gene et al. (1989) was used. A constant amount of venom (20 MCD) was incubated with several dilutions of antivenom for 30 min at 37 C in a final volume of 1.0 ml. Then, 100 ml of the mixture which contained 2 MCD of the venom was added to 200 ml of citrated solution of sheep plasma. Controls included sheep plasma incubated with either venom alone or antivenom alone. Neutralization was expressed as effective dose (ED). ED is defined as the antivenom/venom ratio at which coagulation time is increased three fold when compared to the coagulation time of plasma incubated with venom alone. 2.3. In vivo assay of antivenom potency
2.1. Chemicals and biochemical
2.3.1. Determination of median lethal dose (LD50) The LD50 assay was based on the method described by Theakston and Reid (1983). Groups of five ICR mice weighing 20 2 g were injected intra-peritoneally with CR venom at doses ranging from 2 to 10 mg/g mouse. The venom was prepared in normal saline and the volume of injection was kept constant at 200 ml/20 g mouse. Control animals were injected with normal saline only. The lethality, expressed in percent death of animals, was recorded 48 h after injection. The medium lethal dose (LD50) of the venom was calculated by the method of Reed and Muench (1938).
Chemicals were of reagent grade and were purchased from Sigma Chemical Company, St. Louis, MO, USA, except as indicated. Pooled fresh crude CR venom was purchased from Queen Saovabha Memorial Institute, Bangkok. The venom was kept at 20 C until used. Eleven batches of crude antivenom sera against CR, obtained from horses during varying stages of immunization programs and thus with varying antibody titers, were from Queen Saovabha Memorial Institute. The sera were separately stored at 4 C, and were filtered through 0.45 micron membrane before use. The protein concentration of the sera was
2.3.2. Determinations of median effective dose (ED50) The in vivo neutralization activity of CR antivenom was expressed as median effective dose (ED50), defined as microliters of the antivenom per mg of venom required to prevent death in 50% of injected mice. The procedure was similar to that described above for LD50 determination, but 2.5LD50 dose of CR venom in 20 ml of normal saline was mixed with different doses of horse antivenom against CR. Normal horse serum was added to make the volume up to 200 ml. These mixtures were incubated at room temperature for 30 min before injection. The
2. Materials and methods
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A Coagulation time (sec.)
volume of injection was kept constant at 200 ml/20 g mouse. Control mice were injected with venom solution (in sterile normal saline) or horse serum. The number of surviving mice was recorded 48 h after injection. ED50s were calculated using the probit analysis method of Finney (1971). It should be mentioned that at the QSMI horse farm, neutralization test of horse antivenom sera is often carried out by i.v. injection at the mouse tail vein. In order to avoid inaccuracies caused by leakage of the solution at the injection site, the present test was done using i.p. injection. Experiments involving animals were reviewed and approved by the Animal Care and Use Committee of Faculty of Science, Mahidol University (Protocol No. MUSC 56-008-271).
3
80 70 60 50 40
22.12
30 0 15
20
25
30
35
40
50
CR venom concentration (µg/ml)
Protein concentration was determined by the procedure described by Lowry et al. (1951) using bovine serum albumin as the standard. 2.5. Statistical analysis ED50 of antivenoms are expressed as means with 95% confidence intervals (C.I.) and were calculated using the probit analysis method of Finney (1971). Correlation between the ED50 and ED values, obtained from the in vivo and the in vitro assays, respectively, was determined by means of linear regression analysis with the correlation coefficient of Pearson and Lee (1903). 3. Results 3.1. Determination of minimum coagulant dose (MCD) and the neutralization of coagulant activity by antivenom The coagulant activity of fresh CR venom as a function of venom concentration is shown in Fig. 1A. MCD, defined as the final concentration of CR venom (mg/ml) which induces coagulation of plasma in 60 s, was determined in 3 separated experiments and shown to be 22.12 0.25 mg/ml. The average coagulation time of 2MCD of the venoms was 34.1 3.39 s. The in vitro neutralizing activities of 11 batches of horse antivenom against CR venom were determined (Materials and methods). The CR venom at 2MCD was included in every test to serve as a control to correct day to day variations. The coagulation time induced by 2MCD venom was used as the reference value for calculating the neutralization capacities of antivenoms. The coagulation time induced by the venom in the presence of each batch of antivenom at various dilutions was plotted against the antivenom/venom ratio (Fig. 1B). The effective dose (ED) of each batch of antivenom was determined at the antivenom/venom ratio (ml/mg) at which coagulation time is increased three fold when compared to the coagulation time induced by 2MCD of venom alone. As an example of such determination, the coagulation time of 2MCD of the venom in the control tube in Fig. 1A was 34.82 s. The ED of antivenom batch #1, the ratio of antivenom/venom giving coagulation time of
B
160 140
Coagulation time (sec.)
2.4. Protein determination
104.46
120 100 80 60
ED=389.91 µl/mg
40 0 0
280
320
360
400
440
480
Antivenom/venom ratio (µl/mg) Fig. 1. A. A plot of clotting time against various concentrations of CR venom. B. The neutralization of coagulant activity of antivenom batch #1. ED was the ratio of antivenom/venom at which coagulation time is increased three folds when compared to the coagulation time induced by 2 MCD of venom alone. The coagulation times of 2 MCD of venom alone was 34.82 s.
3 34.82 ¼ 104.56 s was, from the graph, 389.91 ml/mg. The EDs of 11 batches of antivenom are shown in Table 1. Horse antivenoms and normal horse serum were also tested for their coagulant activity in the absence of CR venom. There was no coagulant activity observed with sheep plasma within 10 min. 3.2. Median effective dose (ED50) of various batches of antivenom The LD50 of the CR venom was determined to be 5.41 mg/ g mouse (95% confidence interval 2.98–6.57 mg/g mouse). This lot of venom was used in all determinations of ED50 of 11 batches of horse antivenoms against CR venom. The antivenom potencies of 11 batches of CR antivenom sera determined by the in vivo neutralization assay using mice, the ED50, together with the 95% confidence intervals are shown in Table 1. 3.3. Correlation between in vitro neutralizing activities and in vivo neutralizing activities of 11 batches of CR antivenom The in vivo neutalizing activities, ED50, and in vitro neutalizing activities, ED, of 11 batches of antivenom were
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Table 1 The in vitro and in vivo neutralization activities of various batches of CR antivenom as expressed as effective dose (ED) and ED50, respectively. Antivenom In vitro effective dose (ED) In vivo effective dose (ED50) (ml serum/mg venom) No. (ml serum/mg venom) 1 2 3 4 5 6 7 8 9 10 11
388.56 566.43 152.97 188.12 399.14 211.85 320.60 180.88 347.39 254.87 340.21
1.91 13.59 4.62 10.89 2.06 15.61 9.78 9.32 19.81 2.03 21.90
356.00 495.50 169.00 218.50 422.50 263.50 333.00 248.50 377.00 332.00 373.50
(297.00–422.50) (426.00–532.50) (97.50–193.00) (164.00–300.50) (394.00–481.00) (205.00–343.50) (266.00–407.50) (173.00–357.00) (257.50–483.00) (256.50–442.50) (291.00–432.50)
compared by means of linear regression analysis. A plot of ED50 vs ED is shown in Fig. 2. The correlation coefficient (r) of these two assays is 0.957 (p < 0.001). 4. Discussion The potency of an antivenom is generally determined by its ability to save the lives of experimental animals injected with the homologous venom. The in vivo assay of antivenom potency can give variable results caused by a wide range of factors, e.g., species, strain, weight, sex and routes of injection. The assay is also time consuming, laborious and expensive. It is often difficult for some snake venoms and antivenoms especially those of vipers, to obtain good dose response curve unless a large number of animals are used. Moreover, the in vivo assays have been opposed on ethical grounds, and in Buddhist countries, most laboratory personnel refuse to perform such assays. Because of these shortcomings, attempts have been made to find alternative in vitro assays to the use of live 600
In vitro neutralization (µl serum/mg venom) ED
500
400
300
200
100
0 0
100
200
300
400
500
600
In vivo neutralization (µl serum/mg venom) ED50 Fig. 2. Correlation between in vivo neutralizing activity (ED50) and in vitro neutralizing activity (ED). The correlation coefficient (r) was 0.957 (p value < 0.001).
animals. ELISA has been widely used to quantitate the antibody content in antivenom preparations (Rungsiwongse and Ratanabanangkoon, 1991; Tan et al., 1992; Theakston and Reid, 1979). This assay showed very high correlation with in vivo assay with snake venom containing a major lethal toxin such as that found in elapid venoms. When the venom lethal component is used as antigen, the ELISA quantitates only the antibody against the lethal component and therefore gives results which correlate highly with those obtained from in vivo assays (Rungsiwongse and Ratanabanangkoon, 1991). However, viper and pit viper venoms are composed of diverse proteins that have different enzymatic activities acting synergistically on substrates and the overall toxicity is not the resultant or the summation of toxicity of all separate substances. Consequently ELISA that quantitates the antibody against all venom proteins does not usually have a good correlation with the results obtained from in vivo assays (Heneine et al., 1998). The present in vitro assay of antivenom potency was based on the neutralization of the coagulation induced by the venom. For CR venom, the thrombin-like serine protease. (Ancrod or Arvin) acts on fibrinogen causing coagulation in vitro and leads to defibrinating syndrome in vivo (Nolan et al., 1976; Pirkle, 1998; Reid et al., 1963b). Although the venom contains hemorrhagin and other enzymes (Chung et al., 1996; Tan et al., 1986), the thrombin-like enzyme may also contribute to the pathological effect of this venom. The in vitro assay based on the inhibition of this enzyme reaction seems to be a reasonable choice. As observed by Gene et al. (1989), antivenoms alone could induce coagulation of fibrinogen and plasma. However, it was found here that CR equine antiserum per se had no coagulant effect on sheep plasma. One important criterion in the development of in vitro assay is how good its results correlate with those obtained from the in vivo assay. In this study, good correlation between the in vitro activity and the in vivo activity was obtained; the correlation coefficient (r) was 0.957 (p < 0.001). However, as mentioned by Gutiérrez et al. (1988), even when an in vitro test showed high correlation, it did not necessarily mean that the enzyme was responsible for the lethal toxic effect of the venom. Rather, it suggested that the antibody production against the enzyme paralleled those against the predominant lethal toxins of the venom. Compared with the in vivo test, the amounts of venom and antivenom used in the experiment are smaller, requiring 440 mg of venom for MCD and 50–500 ml of antivenom for ED determinations while in in vivo assays more than 1 mg of venom and 2 ml of antivenom were needed for each batch of antivenom. This observation can be an important consideration with some very scarce venom. The in vitro assay took less than 1 h to obtain the required result. The results reported here indicated that the in vitro assay using the neutralization of coagulant effect of CR venom could be used as an alternative assay for potency assessment of CR antivenom. The procedure is simple, rapid and inexpensive. However, it does not mean that this method can be used to totally replace the in vivo rodent assay which remains the requirement in many pharmacopoeias for standardization of commercial antivenoms. The
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in vitro assay, however, could be employed to evaluate the potency of antivenom at various steps of antivenom production, for example, during immunization and in different stages of plasma fractionation. The in vitro assay could reduce the use of live animals. Apart from the antivenom against C. rhodostoma, the neutralization of coagulant activity may be adopted for assessing the potency of antivenoms against other procoagulant venoms. Funding This study was supported by a research grant from Thailand Research Fund. Ethical statement This manuscript presents an experimental study performed following the standard procedures of scientific ethics, including the use and care of experimental animal. I certify that the result of the submitted for publication in Toxicon manuscript are original and are not submitted elsewhere for publication. Acknowledgments The authors thank Assistant Professor Maurice Broughton for valuable suggestions and Ms Lalida Arsa for assistance in preparing the manuscript. Conflict of interest statement None declared. References Chung, M.C., Ponnudurai, G., Kataoka, M., Shimizu, S., Tan, N.H., 1996. Structural studies of a major hemorrhagin (rhodostoxin) from the venom of Calloselasma rhodostoma (Malayan pit viper). Arch. Biochem. Biophys. 325, 199–208. Finney, D.J., 1971. Probit Analysis, third ed., vol. 60. Cambridge University Press, p. 1432. Journal of Pharmaceutical Sciences. Gene, J.A., Roy, A., Rojas, G., Gutierrez, J.M., Cerdas, L., 1989. Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogenolytic
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activities of Costa Rican crotaline snake venoms and their neutralization by a polyvalent antivenom. Toxicon 27, 841–848. Gutiérrez, J., Avila, C., Rojas, E., Cerdas, L., 1988. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 26, 411–413. Heneine, L.G.D., Carvalho Jr., Á.d.D., Barbosa, C.d.f., arávjo dos Santos, M.R., 1998. Development of an ELISA to assess the potency of horse therapeutic polyvalent antibothropic antivenom. Toxicon 36, 1363–1370. Ho, M., Warrell, D.A., Looareesuwan, S., Phillips, R.E., Chanthavanich, P., Karbwang, J., Supanaranond, W., Viravan, C., Hutton, R.A., Vejcho, S., 1986. Clinical significance of venom antigen levels in patients envenomed by the Malayan pit viper (Calloselasma rhodostoma). Am. J. Trop. Med. Hyg. 35, 579–587. Ibrahim, N.M., Farid, N.M., 2009. Comparison between two in vitro ELISAbased assays in the determination of antivenom potency. J. Appl. Sci. Res. 5, 1223–1229. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Markland, F.S., 1998. Snake venoms and the hemostatic system. Toxicon 36, 1749–1800. Nolan, C., Hall, L.S., Barlow, G.H., 1976. Ancrod, the coagulating enzyme from Malayan pit viper (Agkistrodon rhodostoma) venom. Methods Enzymol. 45, 205–213. Pearson, K., Lee, A., 1903. On the laws of inheritance in man: I. Inheritance of physical characters. Biometrika 2, 357–462. Pirkle, H., 1998. Thrombin-like enzymes from snake venoms: an updated inventory. Thromb. Haemost. 79, 675–683. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Epidemiol. 27, 493–497. Reid, H.A., Chan, K.E., Thean, P.C., 1963b. Prolonged coagulation defect (defibrination syndrome) in Malayan viper bite. Lancet 1, 621–626. Reid, H.A., Thean, P.C., Chan, K.E., Baharom, A.R., 1963a. Clinical effects of bites by Malayan viper (Ancistrodon rhodostoma). Lancet 1, 617–621. Rungsiwongse, J., Ratanabanangkoon, K., 1991. Development of an ELISA to assess the potency of horse therapeutic antivenom against Thai cobra venom. J. Immunol. Methods 136, 37–43. Tan, N.-H., Ponnudurai, G., 1996. The toxinology of Calloselasma rhodostoma (Malayan pit viper) venom. J. Toxicol. Toxin Rev. 15, 1–17. Tan, N.-H., Yeo, K.-H., Nik Jaafar, M.I., 1992. The use of enzyme-linked immunosorbent assay for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom and venom antibodies. Toxicon 30, 1609–1620. Tan, N.H., Kanthimathi, M.S., Tan, C.S., 1986. Enzymatic activities of Calloselasma rhodostoma (Malayan pit viper) venom. Toxicon 24, 626– 630. Theakston, R.D., Reid, H.A., 1983. Development of simple standard assay procedures for the characterization of snake venom. Bull. World Health Organ 61, 949–956. Theakston, R.D.G., Reid, H.A., 1979. Enzyme-linked Immunosorbent Assay (ELISA) in assessing antivenom potency. Toxicon 17, 511–515. White, J., 2005. Snake venoms and coagulopathy. Toxicon 45, 951–967. World Health Organization, 1981. Progress in the Characterization of Venoms and Standardization of Antivenoms. World Health Organization, Geneva.
