Pergamon Toxicon, Vol. 32, No. 3, pp. 351-363, 1994

Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved

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C A P R Y L I C A C I D F R A C T I O N A T I O N O F H Y P E R I M M U N E

H O R S E P L A S M A : D E S C R I P T I O N O F A S I M P L E P R O C E D U R E

F O R A N T I V E N O M P R O D U C T I O N

GUSTAVO ROJAS, Jos~. MANUEL JIM~NEZ and Josl~ MARIA GUTII~RREZ Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San Josf, Costa Rica

(Received 28 May 1993; accepted 17 September 1993)

G. ROJAS, J. M. JIM~NEZ and J. M. GUTII~RREZ. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon 32, 351-363, 1994.--A simple methodology for hyper- immune horse plasma fractionation, based on caprylic acid precipitation, is described. Optimal conditions for fractionation were studied; the method gives best results when concentrated caprylic acid was added to plasma, whose pH had been adjusted to 5.8, until a final caprylic acid concentration of 5% was reached. The mixture was vigorously stirred during caprylic acid addition and then for 60 min; afterwards the mixture was filtered. Non-immunoglobulin proteins precipitated in these conditions, whereas a highly enriched im- munoglobulin preparation was obtained in the filtrate, which was then dialysed to remove caprylic acid before the addition of NaC1 and phenol. Thus, antivenom was produced after a single precipitation step followed by dialysis. In order to compare this methodology with that based on ammonium sulfate fractionation, a sample of hyperimmune plasma was divided into two aliquots which were fractionated in parallel by both methods. It was found that caprylic acid-fractionated antivenom was superior in terms of yield, production time, albumin/globulin ratio, turbidity, protein aggregates, electrophoretic pattern and neutralizing potency against several activities of Bothrops asper venom. Owing to its efficacy and simplicity, this method could be of great value in antivenom and antitoxin production laboratories.

INTRODUCTION

ANTIVENOMS obtained from hyperimmune horse plasma constitute the mainstay in the treatment of snakebite envenomations, being produced in many laboratories around the world (THEAKSTON and WARRELL, 1991). However, despite the well-demonstrated clinical efficacy of antivenoms, their administration induces a number of unwanted allergic reactions (CoRRIGAN et al., 1978; MALASIT et al., 1986; CuPo et al., 1991), in part due to the presence of non-immunoglobulin plasma proteins which are not eliminated during fractionation.

Most protocols for antivenom production are based on ammonium sulfate precipitation with or without pepsin digestion (LATIrI, 1978; RUSSELL, 1988; RAW et al., 1991). These

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352 G. ROJAS et al.

procedures have undergone little innovation during the last decades, making the intro- duction of new methodologies that would give products of higher quality a priority. Several new alternatives have been proposed, such as affinity and ion-exchange chromatography, either alone or in combination with saline fractionation and enzyme digestion (RUSSELL et al., 1985; DIAS et al., 1989; SMITH et al., 1992). Although highly adequate in terms of quality of the product, these methods are difficult to adapt to production laboratories. Thus, there is a need to develop new procedures for antivenom production which would combine technical as well as economical advantages.

The use of caprylic (octanoic) acid in the purification of IgG from various sources has been described (STEINBUCH and AUDRAN, 1969; MCKINNEY and PARKINSON, 1987; PEROSA et al., 1990). This method is used mainly for small-scale purification, with very good results. Dos SANTOS et al. (1989) described the use of caprylic acid, in combination with pepsin digestion, for the purification of F(ab')2 antivenom in Brazil. We have attempted to find optimal conditions for the use of caprylic acid in antivenom production. This work describes a simple method based on a single precipitation step, which gives a highly enriched IgG preparation with high yield and good neutralizing ability. Owing to its efficacy, simplicity, low cost and reduced production time, this method could be of great value in the industrial production of antivenoms and antitoxins.

MATERIALS AND METHODS

Venoms and hyperimmune plasma The venoms used in this work were obtained from snakes collected in Costa Rica and maintained at Instituto

Clodomiro Picado. Once obtained, venoms were lyophilized and stored at -20°C. Plasma was collected from horses that had been immunized with a mixture of equal parts of the venoms of Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys. The immunization schedule described by ESTRADA et al. (1992) was followed for the production of polyvalent antivenom (BOLA~OS and CEROAS, 1980).

Preparation of antivenom by caprylic acid fractionation Standardization of the method. Four variables were studied in order to find optimal conditions: caprylic acid

concentration, pH, stirring time and stirring intensity.

Caprylic acid concentration. Aliquots of 50 ml of hyperimmune horse plasma were brought to pH 4.5 by adding 1.76 N acetic acid; this pH was recommended by MCKINrCEV and PAItrdNSON (1987). Then, caprylic acid (Sigma Chemical Co., St Louis, MO, U.S.A.) was added to each aliquot to reach final concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 10% and 16% (v/v). Acid was added slowly with constant stirring and the mixture was then stirred for 1 hr at 22-24°C. The mixtures were then filtered through Whatman 2 V paper (Whatman, U.K.) and the filtrates were dialyzed for 48 hr against distilled water. Afterwards, NaC1 was added to a final concentration of 0.15 M, the solutions were filtered and their pH adjusted to 7.2 by addition of 1 N NaOH.

pH. Aliquots of 50 ml of hyperimmune plasma were prepared and their pH adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 with 1.76 N acetic acid. Then, caprylic acid was added to give a final concentration of 5% and plasma was fractionated as described above.

Stirring time and intensity. Aliquots of 50 ml of hyperimmune plasma were prepared and their pH was brought to 4.5 with 1.76 N acetic acid. Then, caprylic acid was added slowly to a final concentration of 5%. In order to standardize stirring time, samples were stirred vigorously for either 30 min or 60 min. To standardize stirring intensity, samples were stirred for 60 min in four different conditions: vigorous stirring, moderate stirring, mild stirring and no stirring. The rest of the fractionation was carried out as described.

Fractionation of hyperimmune plasma with caprylic acid and with ammonium sulfate Caprylic acidfractionation. Once the optimal conditions for caprylic acid fractionation were established, l liter

of hyperimmune plasma was collected and fractionated as follows: pH was adjusted to 5.8 + 0.1 with 1.76 N acetic acid. Caprylic acid was then added slowly and with vigorous stirring to give a final concentration of 5%. Constant

Caprylic Acid Fractionation of Antivenom 353

vigorous stirring was maintained for 1 hr at 22-24°C and the mixture was filtered through Whatman 2 V paper. The filtrate was dialyzed for 48 hr at 22-24°C against distilled water. Afterwards, NaC1 and phenol were added to give final concentrations of 0.15 M and 0.15 g/dl, respectively. The mixture was filtered again and its pH adjusted to 7.2 with 1 N NaOH.

Ammonium sulfate fractionation. The procedure routinely used at Instituto Clodomiro Picado was followed (OPS, 1977). Briefly, 1 liter of a 24 g/dl ammonium sulfate solution, containing 0. ! 5 M NaCI and 0.2 g/dl phenol was added to 1 liter o f hyperimmune plasma and the mixture was stirred vigorously for 1 hr at 22-24°C. Twenty hours later the mixture was filtered and, immediately, ammonium sulfate was added to the filtrate to obtain a final concentration of 24 g/dl. After 1 hr of vigorous stirring at 22-24°C, the mixture was left unstirred for 20 hr before filtration. Precipitate was dissolved and dialyzed against distilled water. Dialyzed antivenom was acidified to pH 5.4 with 1 N acetic acid, stirred vigorously for I hr and left at 22-24°C for 20 hr. Afterwards, the preparation was filtered and its pH adjusted to 7.2 with I N NaOH. Then, NaCI and phenol were added to obtain final concentrations of 0.15 M and 0.15 g/dl, respectively.

Comparative analysis of antivenoms prepared by caprylic acid and ammonium sulfate fractionations In order to compare antivenoms prepared by these two procedures, neutralization of the lethality of B. asper

venom was studied as described below. Antivenoms were then diluted with 0.15 M NaCI in order to attain the same neutralizing potency against lethality (expressed as effective dose 50%, EOs0). Once standardized in terms of EDs0, the following parameters were compared: yield, turbidity, total protein concentration, albumin concentration, presence of protein aggregates, electrophoretic profile and neutralizing ability against myotoxic, edema-forming, hemorrhagic, coagulant and indirect hemolytic activities of B. asper venom. In addition, the titer o f anti-B, asper myotoxin and anti-crude venom antibodies were also determined by enzyme immunoassay.

Protein and albumin concentrations. Total protein concentration was determined by the Biuret method, as described by SCHOSINSKV et al. (1983). Albumin was determined by the bromocresol green method, as modified by SCHOSINSKY et al. (1983).

Turbidity and protein aggregates. Turbidity of antivenoms was assessed either by visual inspection or by recording the absorbances at 600nm in a Zhimadzu UV-160 spectrophotometer (ROJAS et aL, 1993). To detect the presence of protein aggregates, samples of 2 ml of each antivenom were applied to a Sepharose 6B column (83 x 2 cm). The column was equilibrated and eluted with phosphate-buffered saline solution, pH 7.2. As mol. wt standards, the following markers were used: bovine serum albumin (mol. wt 68,000), bovine IgG (150,000) and dextran blue (2,000,000). Elution profiles were determined by recording absorbances at 280 nm (JANSON and RYDEN, 1989).

Electrophoresis o f antivenoms. Electrophoreses were carried out on cellulose polyacetate membranes (Sephrapore III, Gelman Instruments, Michigan, U.S.A.) in barbital buffer, pH 8.6, for 20-25 min at 180 V. Strips were stained with Amidoblack and destained with 0.9 N acetic acid. Densitometric analysis was performed with a Hoeffer Scientific Instruments (California, U.S.A.) apparatus using the GS365W Microsoft Windows Program.

Neutralization studies Mixtures of fixed doses of B. asper venom and various dilutions of antivenoms were prepared and incubated

at 37°C for 30 min. Then, several pharmacological activities of the mixtures were tested in several assay systems and neutralizing ability of antivenoms was determined and expressed as effective dose 50% (EDeo), defined as the ratio #1 antivenom/mg venom at which the effect of venom was reduced by 50%. In the case of coagulant effect, neutralizing ability was expressed as effective dose (see below).

Neutralization of lethality. Mixtures of venom and antivenom were incubated and injected i.p. in Swiss-Webster mice (n = 6; 16-18 g). The challenge venom dose injected per mouse was 250/~g, corresponding to 4 x LDs0. Deaths were recorded for 48 hr and Eta50 was estimated by Probits (FIr~NEV, 1964).

Neutralization o f myotoxic activity. The method of GtrrI~RREZ et al. (1981) was followed. After incubation, mixtures of venom and antivenom were injected i.m. in Swiss-Webster mice (n = 5; 18-20 g). The challenge dose of venom per mouse was 50/~g, and controls were injected with either PBS alone or 50 #g venom alone. Three hours after injection, mice were bled by cutting the tip of the tail, and blood was collected into heparinized

354 G. ROJAS et al.

capillary tubes. Creatine kinase (CK; EC 2.7.3.2) levels in plasma were determined by using the Sigma kit 520 and expressed as units/ml, one unit resulting in the phosphorylation of 1 nanomole of creatine per min at 25°C.

Neutralization of hemorrhagic activity. The method of KONDO et al. (1960) was used, with the modifications introduced by Gtrrn~sRez et al. (1985). After incubation, 100 #1 of each antivenom-venom mixture were injected intradermally in Swiss-Webster mice (n = 4; 18-20 g). The challenge dose of venom injected per mouse was 25 #g, corresponding to 10 minimum hemorrhagic doses (MHD) (GuTI~R~Z et al., 1985). Controls received either PBS alone or 10 MHD of venom alone. Two hours after injection, mice were sacrificed by ether inhalation, their skins were removed and the diameter of hemorrhagic areas was determined.

Neutralization of edema-forming activity. The method of YAMAKAWA et al. (1976) was used, as modified by GtrnC:RREZ et al. (1986a). After incubation, 50 #l of each antivenom-venom mixture were injected s.c. in the right foot pad of Swiss-Webster mice (n = 4; 18-20 g), whereas the left foot pad received 50/d of PBS. The challenge dose of venom injected per mouse was 10 #g and controls received 10/tg of venom alone. After 3 hr, mice were sacrificed by cervical dislocation, and their feet were cut and weighed. Edema was expressed as percentage of increment in the weight of the right foot as compared to the left one.

Neutralization of coagulant effect. The method of GEN~ et al. (1989) was followed. After incubation, 100/~1 of each antivenom-venom mixture were added to 200/~ 1 of citrated sheep plasma and clotting times were recorded with a Fibrometer (BBL, Maryland, U.S.A.). Challenge dose of venom was 5/~g, corresponding to 2 minimum coagulant doses (MCD). In control samples, plasma was incubated with either PBS or venom alone. Neutralizing ability was expressed as effective dose, defined as the ratio/zl antivenom/mg venom where clotting time was prolonged three times when compared to Clotting time of plasma samples incubated with venom alone (GENI~ et al., 1989).

Neutralization of indirect hemolytic effect. Indirect hemolysis was determined according to GUTII:RREZ et al. (1988). After incubation, 15 #1 of each antivenom-venom mixture were added to wells made in agarose-egg yolk-erythrocyte gels. The challenge dose of venom (0.4 #g) corresponded to 1 MHD, i.e. amount of venom that induces a hemolytic halo of 15 mm diameter. Controls of either PBS or venom alone were included. Gels were incubated for 20 hr at 37°C and the diameters of hemolytic halos measured.

Determination of anti-B, asper crude venom and anti-myotoxin antibody titers. Bothrops asper myotoxin I was isolated according to GUTI~.REZ et al. (1986b). For antibody titer determination, the enzyme immunoassay described by LOMONTE et al. (1991) was followed, using as antigens either 1 #g of crude B. asper venom or 0.4 #g of B. asper myotoxin I. Several dilutions of antivenom were used in order to obtain titration curves.

Statistical analysis The Student's t-test was used to determine the significance of the differences between the mean values of two

experimental groups.

RESULTS

Determinat ion o f the op t imal conditions f o r p lasma f rac t ionat ion with caprylic acid T a b l e 1 d e p i c t s the r e su l t s o f p l a s m a f r a c t i o n a t i o n u s i n g v a r i o u s c o n c e n t r a t i o n s o f

c a p r y l i c ac id . I t w as o b s e r v e d t h a t p r o t e i n a n d a l b u m i n c o n c e n t r a t i o n s we re s i m i l a r w h e n

w o r k i n g w i t h c a p r y l i c ac id c o n c e n t r a t i o n s r a n g i n g f r o m 4 % to 7 % . H o w e v e r , w h e n o t h e r

p a r a m e t e r s we re t a k e n i n t o c o n s i d e r a t i o n , a c o n c e n t r a t i o n o f 5 % gave be s t resul t s , s ince

ve loc i ty o f f i l t r a t i o n w as r a p i d a n d a yie ld o f 59 + 6 % was o b t a i n e d . I n a d d i t i o n , t he f ina l

p r e p a r a t i o n was d e v o i d o f t u r b i d i t y . C a p r y l i c ac id c o n c e n t r a t i o n s l o w e r t h a n 4 % r e n d e r e d

a f ina l p r o d u c t w i t h excess ive t u r b i d i t y a n d re l a t ive ly h i g h a l b u m i n c o n t a m i n a t i o n .

H o w e v e r , w h e n w o r k i n g w i t h c a p r y l i c ac id c o n c e n t r a t i o n s h i g h e r t h a n 5 % , t he ve loc i ty

o f f i l t r a t i o n was excess ive ly s low a n d t he yie ld d e c r e a s e d ( T a b l e 1).

Caprylic Acid Fractionation of Antivenom

TABLE 1. EFFECT OF CAPRYLIC ACID CONCENTRATION ON THE FRACTIONATION OF HYPERIMMUNE PLASMA FOR ANTIVENOM PRODUCTION

Caprylic acid Protein Albumin Velocity concentration concentration concentration Yield of % (v/v) (g/dl) (g/dl) (%) filtration Turbidity

355

I 3.33 + 0.06* n.t.~ 20 4- 4 very slow:~ + + + § 2 3.23 +0.12 0.864-0.04 49-1-6 very slow + + + 3 3.17+0.06 0.574-0.06 584-5 slow + + 4 3.03 4- 0.06 0.25 4- 0.01 59 __+ 7 fast + 5 2.904-0.10 0.244-0.06 594-6 fast - 6 3.00 4- 0.06 0.23 4- 0.02 57 4- 6 slow - 7 2.93 _ 0.06 0.21 4- 0.01 50 4- 3 slow - 8 2.674-0.06 0.184-0.02 4 7 + 5 very slow -

10 2.43 + 0.12 n.t. 46 4- 6 very slow - 16 0.97 4- 0.06 n.t. 27 + 4 very slow -

* Results for protein concentration, albumin concentration and yield are presented as mean 4- S.D. (n = 3).

t n.t., Not tested. :~ Fast: less than 5 min; slow: between 5 and 30 rain; very slow: more than 30 min. These

filtration times refer to experiments in which 50 ml of hyperimmune plasma were fractionated. § ( - ) No turbidity; ( + ) mild turbidity; ( + + ) moderate turbidity; ( + + + ) high turbidity.

R e g a r d i n g t he effect o f p H , T a b l e 2 s h o w s t h a t a l b u m i n c o n c e n t r a t i o n w a s s im i l a r a t

p H 4.5 o r h i g h e r , w h e r e a s p r o t e i n c o n c e n t r a t i o n v a r i e d d e p e n d i n g o n t h e p H . W h e n t a k i n g

i n t o c o n s i d e r a t i o n t he p a r a m e t e r s o f yield , ve loc i ty o f f i l t r a t i o n a n d t u r b i d i t y , t he be s t

r e su l t s w e r e o b t a i n e d a t p H v a l u e s o f 5.5 a n d 6.0 ( T a b l e 2). T h e c o n d i t i o n o f p l a s m a

s t i r r i n g a f t e r c a p r y l i c a c id a d d i t i o n d r a s t i c a l l y a f fec ted o u r resu l t s . W h e n t h e r e was n o

s t i r r i n g o r o n l y a m i l d s t i r r ing , y ie ld w a s v e r y low a n d f i l t r a t i o n ve loc i ty was s l ow ( T a b l e 3).

I n c o n t r a s t , m i x t u r e s t h a t w e r e v i g o r o u s l y s t i r r ed g a v e exce l l en t r e su l t s in t e r m s o f b o t h

y ie ld a n d f i l t r a t i o n ve loc i ty . T h e r e we re n o s i gn i f i c an t d i f f e r ences ( P > 0.05) w h e n s t i r r i n g

was p e r f o r m e d f o r 30 o r 60 m i n . T h u s , v i g o r o u s s t i r r i n g fo r 60 m i n was c h o s e n as

t h e o p t i m a l c o n d i t i o n f o r e x p e r i m e n t s . A f t e r f i l t r a t i on , c a p r y l i c ac id w a s e l i m i n a t e d b y

d i a ly s i s f o r 48 hr . T h i s p r o c e d u r e r e d u c e d c a p r y l i c ac id c o n c e n t r a t i o n to a v a l u e o f

0 .0076___0 .0008%, as d e t e r m i n e d b y gas l i q u i d c h r o m a t o g r a p h y . I n s u m m a r y , t he

TABLE 2. EFFECT OF pH ON THE FRACTIONATION OF HYPERIMMUNE PLASMA WITH CAPRYLIC

ACID FOR ANTIVENOM PRODUCTION

Protein Albumin Velocity concentration concentration Yield of

pH (g/dl) (g/dl) (%) filtration Turbidity

4.0 0.59 + 0.05* n.t.t 10 + 2 slow* + + + § 4.5 2.23 4-0.13 0.20+0.01 59+ 1 slow + + 5.0 2.43 4- 0.09 0.21 _ 0.06 57 4- 2 fast + + 5.5 2.46 4- 0.05 0.22 _ 0.06 60 4- 5 fast - 6.0 2.42 4- 0.04 0.22 4- 0.06 60 4- 2 fast - 6.5 2.25 4- 0.06 0.21 4- 0.01 56 4- 1 fast - 7.0 2.32+0.01 0.20-1-0.06 61 + 4 fast -

* Results for protein concentration, albumin concentration and yield are presented as mean + S.D. (n = 3).

t n.t., Not tested. :~ Fast: less than 5 rain; slow: between 5 min and 30 min. These filtration times refer

to experiments in which 50 ml of hyperimmune plasma were fractionated. § ( - ) No turbidity; ( + ) mild turbidity; ( + + ) moderate turbidity; ( + + + ) high

turbidity.

356 G. ROJAS et al.

TABLE 3. EFFECT OF STIRRING INTENSITY ON THE

ERACTIONAT1ON OF HYPERIMMUNE PLASMA WITH

CAPRYLIC ACID FOR ANTIVENOM PRODUCTION

Stirring Yield Velocity of intensity (%) filtration

Vigorous 60 + 5* fastt Moderate 44 + 3 fast Mild 28 + 3 slow No stirring 0 no filtration

* Results presented as mean + S.D. (n = 3). t Fast: less than 5 rain; slow: between 5 min

and 30min. These filtration times refer to experiments in which 50 ml of hyperimmune plasma were fractionated.

condi t ions selected for f rac t iona t ion o f hype r immune horse p l a sma with capryl ic acid were: p H 5.8, capryl ic acid concen t ra t ion o f 5%, and v igorous st i rr ing for 60 min.

Comparison between antivenoms fractionated with caprylic acid and ammonium sulfate Table 4 presents a compar i son between an t ivenoms p repa red by capryl ic acid and

a m m o n i u m sulfate f rac t ionat ion . In pe r fo rming this compara t ive analysis, bo th ant iven- oms were di luted to have ident ical neut ra l iz ing capac i ty agains t the lethal effect o f B. asper venom. This was done in o rder to s imulate the way an t ivenoms are s t andard ized in p roduc t ion labora tor ies , since they are di luted or concen t ra ted to achieve the same EDs0. Resul ts indicate tha t an t ivenom prepared by capryl ic acid f rac t iona t ion was bet ter in all respects, having higher yield, lower pro te in and a lbumin concentra t ions , lower a lbu- min /g lobu l in ra t io , less tu rb id i ty and shor ter p roduc t ion t ime (Table 4).

The c h r o m a t o g r a p h i c profiles o f bo th an t ivenoms when subjected to gel f i l t rat ion on Sepharose 6B are shown in Fig. 1. Both an t ivenoms presented large peaks cor respond ing to pro te ins with e lut ion volumes s imilar to that o f bovine IgG, in add i t ion to a low mol. wt peak tha t co r responds to phenol . In addi t ion , an t ivenom f rac t iona ted by a m m o n i u m sulfate p rec ip i ta t ion showed a peak which was absent in capryl ic ac id- f rac t iona ted ant ivenom, cor respond ing to prote ins o f high mol. wt, p r o b a b l y pro te in aggregates.

TABLE 4. COMPARISON OF ANTIVENOMS PREPARED BY CAPRYLIC ACID AND AMMONIUM SULFATE

FRACTIONATIONS*

Caprylic acid Ammonium sulfate Parameter fractionated antivenom* fractionated antivenom

Production time (days) 8 _ It 15 + 3

Yield (%) 60 _ 7t 49 + 4

Protein concentration (g/dl) 3.74 + 0.05t 4.18 + 0.04

Albumin concentration (g/dl) 0.17 + 0.02t 0.53 _ 0.05

Albumin/globulin ratio 0.04 + 0.001 t 0.13 _ 0.02

Turbidity (A600)~: 0.020 _+ 0.001t 0.032 _ 0.001

* Results are presented as mean + S.D. (n = 3). t Results were significantly different (P > 0.01) between the two antivenoms. STurbidity was quantificated by recording the absorbance at 600 nm (see Materials and

Methods).

Caprylic Acid Fractionation of Antivenom 357

1 . 0 .

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=

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0.4-.

• ~ 0 . 2

5 10 15 20 25 30 ,35 4O

TUBE NUMBER

E E

o

o

flD <[

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F I G . 1. G E L FILTRATION OF ANTIVENOMS ON SEPHAROSE

6B. Two milliliters of antivenoms prepared by ammonium sulfate precipitation (A) or caprylic acid precipitation (B) were applied to a 83 × 2 cm column. The following mol. wt markers were used: (a) Dextran blue 2000 (mol. wt 2,000,000); (b) bovine IgG (150,000); and (c)

bovine serum albumin (63,000).

a

b

O

F I G . 2 . ELECTROPI-IORESIS OF ANTIVENOMS ON CELLULOSE

POLYACETATE STRIPS.

Electrophoreses were run for 20-25 min at 180 V, using barbital buffer, pH 8.6. Strips were then stained with Amidoblack and destained with 0.9 N acetic acid. (a) Hyperimmune horse serum; (b) caprylic acid-fraction- ated antivenoms; (c) ammonium sulfate-fractionated antivenom. Arrow signals sample application point.

Electrophoret ic compar ison between these ant ivenoms also evidenced conspicuous differ- ences (Figs 2 and 3). Levels o f beta and gamma globulins were similar, but ant ivenom fract ionated with a m m o n i u m sulfate had higher amounts o f alpha globulin and albumin. These two fractions were greatly reduced in ant ivenom fractionated with caprylic acid.

When neutralizing potencies o f these ant ivenoms were compared, no significant differences were observed in the neutralization o f lethal and myotoxic activities o f B. asper venom. In addition, anti-B..asper venom and anti-B, asper myotoxin I an t ibody titers were similar in both ant ivenoms (Fig. 4). Caprylic acid-fractionated ant ivenom was significantly more effective in the neutralization o f indirect hemolytic, hemorrhagic, edema-forming and coagulant effects (Table 5). Neutral izat ion curves for edema-forming and coagulant activities are shown in Figs 5 and 6. The edema-forming activity o f B. asper venom was totally neutralized by caprylic acid-fractionated ant ivenom at an an t ivenom/venom ratio o f 2000/d/ rag , in contrast to a m m o n i u m sulfate-fractionated ant ivenom where neutraliz- at ion was only partial (Fig. 5). Regarding coagulant effect, Fig. 6 shows that, at an t ivenom/venom ratios higher than 250 #l /mg, neutralization o f this effect was much more effective with ant ivenom fractionated with caprylic acid.

358 G. ROJAS et al.

A 5

1 2 3 4

k..,-.-..--

B

C

FIG. 3. DENSITOMETRIC ANALYSIS OF ANTIVENOMS SEPAR- ATED BY ELECTROPFIORESIS ON CELLULOSE POLYACETATE

MEMBRANES, AS DESCRIBED 1N THE LEGEND OF FIG. 2. (A) Hyperimmune horse serum; (B) ammonium sul- fate-fractionated antivenom; (C) caprylic acid-frac- tionated antivenom. The following serum protein fractions were resolved: (l) ?-globulin, (2) fl-glohulin,

(3) a2-globulin, (4) aFglobulin, (5) albumin.

1.6 I A Tx--J i *\T~ o SULF

I. 1.6 2.0 2.4 2.8 3.2 3.6 4.0 4.4 4.8 5.2

LOG ANTIVENOM DILUTION

3.0 I

.~. 2.5"4 I " ~ . ~ 0 SULF u ~ • CAPR

2.0"1 ~ % ~ NORM

1.oq

o.51 " - - ;'"" Q'~

o.o/ , , " , " ~ i ' ~ " 7 " " - T * ~ . ~ 1.6 2.0 2.4 2.8 3,2 3.6 4.0 4.4 4.8 5.2

LOG FJ~TIVENOM DILUTION

FIG. 4. ANTIBODY TITERS AGAINST CRUDE B. asper VENOM (A) AND B. asper MYOTOXIN I (B) 1N ANTIVENOMS PREPARED BY AMMONIUM SULFATE FRACTIONATION ( O ) AND CAPRYLIC ACID FRACTIONATION ( O ) , AS DETER-

MINED BY ENZYME IMMUNOASSAY, Normal horse serum (/X) was used as control.

D I S C U S S I O N

Caprylic acid has been used for the purification of IgG from serum and ascites fluid (STEINBUCH and AUDRAN, 1969; McKINNEY and PARKINSON, 1987; PEROSA et al., 1990). Although the molecular basis of this fractionation remains unclear, it has been shown that caprylic acid precipitates non-IgG serum proteins under particular physicochemical conditions, leaving in solution a highly enriched IgG preparation. This procedure has been

Caprylic Acid Fractionation of Antivenom 359

TABLE 5. COMPARISON OF THE NEUTRALIZING ABILITY OF ANTIVENOMS PREPARED BY CAPRYLIC ACID AND AMMONIUM SULFATE FRACTIONATIONS

Caprylic acid Ammonium sulfate fractionated fractionated

Activity antivenom (EDso)* antivenom (EDs0)*

Lethal 455 _ 21 459 + 48 Indirect hemolytic 420 _ 1 I t 655 _ 23 Hemorrhagic 117 _ 18t 188 _ 27 Edema-forming 927 _ 134t 1457 4- 185 Myotoxic 269 4- 24 277 4- 17 Coagulant 378 4- 21t > 750

* Neutralizing ability is expressed as effective dose 50% (EDso) except in the case of coagulant activity, where it is expressed as effective dose (ED) (see Materials and Methods for details). Results are presented as mean + S.D. (n = 4).

* Results were significantly different (P < 0.01) between the two anti- venoms.

used mainly for experimental purposes, although Dos SANTOS et al. (1989) described its use, in combination with pepsin digestion, for the purification of antivenom F(ab')2 fragments. There is an urgent need to develop new fractionation methods for antivenom production in order to obtain products of higher purity which would induce fewer allergic reactions in snakebitten patients. Our study succeeded in defining optimal experimental conditions for the purification of horse immunoglobulins from hyperimmune plasma by using caprylic acid precipitation. In addition, it was clearly demonstrated that this procedure is superior when compared to the traditional ammonium sulfate fractionation.

Our data clearly demonstrate that good results are obtained when caprylic acid is added directly to undiluted plasma whose pH has been adjusted to 5.8 by the addition of 1.76 N acetic acid, in disagreement with the observations of MCKINNEY and PARKINSON (1987) who proposed that differential precipitation of serum proteins by this acid requires dilutions of serum with acetate buffer. Our finding has obvious practical implications, since dilution of plasma implies the need of an additional concentration step. Instead, if caprylic acid is added to undiluted plasma, the filtrate obtained after precipitation keeps a high

l ~.~ 100, N / q . O SULF

I

~ 20-

o I oJa I;o 125 zo 2.5

m] ANTIVENOM /mg VENOM

FIG. 5. NEUTRALIZATION OF EDEMA-FORMING ACTIVITY OF B. oJper VENOM BY ANTIVENOMS PREPARED BY AMMONIUM SULFATE (O) AND CAPRYLIC ACID ( 0 ) FRACTIONATIONS.

Edema-forming activity was expressed as percentage, taking as 100% the effect induced by venom alone, i.e. controls in which venoms was incubated with no antivenom. Results are presented as

mean _+ S.D. (n = 4).

360 G. ROJAS et al.

175

150-

~ 125-

~-- 100-

75-

~ 0-

o 25 !

1

0

0 SULF" • CAPR

0:2 o14 o16 08 ml ANTIVENOM / m g VENOM

FIG. 6. NEUTRALIZATION OF COAGULANT ACTIVITY OF B. asper VENOM BY ANTIVENOMS PREPARED BY AMMONIUM SULFATE ((~) AND CAPRYLIC ACID ( 0 ) FRACTIONATIONS.

When control samples containing 5 # g of venoms that had been incubated with no ant ivenom were added to sheep citrated plasma, a clotting time of 30sec was recorded. As the ratio /~1 ant ivenom/mg venom increased, clotting times were prolonged. Results are presented as

mean + S.E. (n = 4).

protein concentration, making unnecessary in most cases the introduction of a concen- tration step. If, however, neutralizing titer is reduced after precipitation and dialysis, immunoglobulins could be concentrated by either ultrafiltration or ammonium sulfate precipitation.

In defining optimal conditions for antivenom production, we studied variables such as yield, filtration time, turbidity, presence of protein aggregates, protein and albumin concentrations and electrophoretic profile in samples of antivenom fractionated with caprylic acid under a variety of experimental conditions. In defining such optimal conditions, it was concluded that the pH of plasma has to be adjusted to 5.5 by addition of acetic acid 1.76 N and that concentrated caprylic acid has to be added slowly to acidified plasma, up to a final concentration of 5%, and the mixture stirred vigorously for 1 hr at room temperature before filtration. When these conditions were followed, a highly enriched preparation of IgG was obtained having good neutralizing ability and giving a yield of approximately 60%. These conditions differ from those described as optimal by other workers who fractionated sera of various sources (STEINBUCH and AUDRAN, 1969; MCKINNEY and PARKINSON, 1987). Thus, optimal laboratory conditions have to be defined for each particular product. After establishing our optimal conditions in small-scale experiments, this method was scaled up in the production facility of Instituto Clodomiro Picado with volumes of 50 liters of plasma. Results obtained were very similar to the ones described for small-scale experiments (results not shown).

In order to compare this new method with the one routinely used at Instituto Clodomiro Picado, hyperimmune horse plasma was fractionated in parallel with either caprylic acid or ammonium sulfate. Results clearly indicate that antivenom fractionated with caprylic acid was superior, having shorter production time, higher yield, lower protein and albumin concentration, and lower content of protein aggregates than antivenom prepared by ammonium sulfate fractionation. The higher purity of caprylic acid-fractionated anti- venom, together with its lower protein content, suggest that it would induce fewer allergic reactions in humans, a hypothesis that needs to be addressed clinically.

Caprylic Acid Fractionation of Antivenom 361

In addition to physicochemical parameters, both antivenoms were compared in terms of their ability to neutralize several activities of B. asper venom. Since both antivenoms were diluted to have the same neutralizing potency against lethality, it was expected that neutralization of other activities would be similar. This was the case only with myotoxic effect, as well as with antibody titers against crude venom and against B. asper myotoxin I. Surprisingly, antivenom fractionated with caprylic acid was more effective than ammonium sulfate-precipitated antivenom in the neutralization of hemorrhagic, edema- forming, coagulant and indirect hemolytic activities. The reasons for this finding are not clear at present, but give further support to the usefulness of caprylic acid fractionation. It is tempting to speculate that, since this method yields a highly enriched IgG fraction, antibodies of higher affinity would be present.

In the case of coagulant activity, GENI~ et al. (1989) showed that ammonium sulfate- fractionated antivenom induced plasma coagulation under some experimental con- ditions, probably owing to the presence of traces of thrombin in this preparation. Antivenom fractionated with caprylic acid did not have this problem, probably owing to its higher purity. Moreover, caprylic acid-fractionated antivenom showed higher neutralizing ability against coagulant effect than ammonium sulfate-fractionated anti- venom. It has been observed, however, that polyvalent antivenom is relatively inefficient in the neutralization of edema-forming activity of Central American crotaline venoms (LoMONTE, 1985; GUTII~RREZ et al., 1986a). It was postulated that, owing to the presence of non-immunoglobulin proteins in antivenom fractionated with ammonium sulfate, incubation of venom and antivenom results in the proteolytic release of pharmacologi- cally active peptides from plasma protein precursors, thereby making difficult the neutralization of edema (GuTII~RREZ et al., 1986a). Interestingly, this phenomenon was not observed with antivenom fractionated with caprylic acid, since a good neutralization curve was obtained, edema being totally neutralized at an antivenom/venom ratio of 2000/~l/mg.

In conclusion, this study describes a simple and highly efficient method for the fractionation of hyperimmune horse plasma in antivenom production. The methodology is evidently superior when compared to the ammonium sulfate precipitation currently used. Caprylic acid fractionation gives a highly enriched immunoglobulin preparation with a high yield, adequate neutralizing potency and optimal physicochemical properties. Owing to its efficacy and simplicity, this methodology might be highly useful in antivenom production laboratories. Moreover, since it involves batch precipitation and dialysis, it can be easily adapted to existing production facilities without requiring the introduction of drastic changes in infrastructure and equipment.

Acknowledgements--The authors thank ABEL ROBLES for his support in the laboratory work, as well as CECILIA DiAZ, ALFREDO VARGAS and EDUAKDO SEGURA for their collaboration in different aspects of this project. This work was supported by International Foundation for Science (grant F/0883-3) and by Vicerrectoria de Investigaci6n, Universidad de Costa Rica (project 741-87-057). G. ROJAS is a research fellow of the Costa Rican National Council for Science and Technology (CONICIT).

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