Taxlrnn, Vd. 30, No . 10, pp. 1117-1188, 1992 Printed in Gnat Briuin.

0041-0101/92 53 .00 + .00 ® 1992 PaQamon Pisa Ltd

PRODUCTION OF A MONOCLONAL ANTIBODY AGAINST THE THROMBIN-LIKE ENZYME, HABUTOBIN, FROM TRIMERESURUS FLAVOVIRIDIS VENOM

M. NexwMUnw, K. KINrox and T. Kosval Departmaat of Physiology, School of Medicine, University of the Ryukyus, 207 Uehara, Nlahihara, Okinawa, 903-01 Japan (Receftrd

10 Aprü 1992;

accepted

19

May

1992)

M. Nom, K. Knvrox and T. Kosuol . Production of a monoclonal

antibody against the thrombin-like enzyme, habutobin, from Trimeresurus flavoviridis venom. Toxicon 30, 1177-1188, 1992.-We succeeded in producing a monoclonal antibody to the thrombin-like enzyme, habutobin, which was purified from crude venom of the snake Trirneresurus ,~tavoviridis. The monoclonal antibody obtained belonged to IgGI, and its light chain consisted of a x~hain . The monoclonal antibody reacted specifically with habutobin and crude venom from T.1lavoviridis but did not react with human thrombin or bovine thrombin on Western blotting. The concentration of habutobin and crude venom of T.Jlavoviridis, in vitro, could be measured by means of ELISA rising the monoclonal antibody. Furthermore, the ELISA-double sandwich method employing this monoclonal antibody may represent a reliable method for determining the habutobin levels in the circulating blood.

INTRODUCTION IT x~+s been reported that a thrombin-like enzyme is present in the venom of Trimeresurus flavoviridis (KOSUGI et al., 198 . This thrombin-like enzyme was named habutobin after Trirneresurus 1lavoviridis, the Habu snake (NARAMLTRA et al., 1992). Some thrombin-like enzymes from snake venoms are most useful in the clinical field as antihaemostatic drugs ($1'OCKBR and Mt~R, 1988). Several researchers have examined the release of fibrinopeptide from fibrinogen using thrombin-like enzymes (DOOLII-I'LS, 1965 ; BLO1~Xclc et al., 1966). A classification of thrombin-like enzymes into three types has been made, depending on the kinds of fibrinopeptide released. That is to say, these enzymes from snake venoms can split fibrinopeptide A or B alone, or both A and B, and thereby induce fibrin clot formation (STOCZCEIt and Mist, 1988). In our previous paper (KOBUGI et al., 1986), it was clarified that habutobin belonged to the Type A group. We therefore attempted to produce a monoclonal antibody against habutobin, in order to measure the habutobin concentration in vitro and in vivo, and to elucidate the utility of habutobin in medical research . 1177

1178

M. NAKAMURA er a{.

MATERIALS AND METHODS Animals BALB/c mice weighing from 20 g to 30 g, Wistar rata weighing from 250 g to 300 g, and rabbits (Japanese white) weighing from 2.5 kg to 3.0 kg were purchased from Kyudo Co., Ltd (Kumamoto, Japan) . Dogs weighing from 10 kg to 1S kg were also used in the experiments. RQQ~CntJ Freund's complete adjuvant wen obtained from DIFCO Laboratories (Detroit, MI, U.S.A.). RPMI 1640 medium N~ssui was obtained from NISSUI Pharmaceutical Co ., Ltd (Tokyo, Japan). Fetal bovine serum (Rehatuin F. S.) was obtained from the Biochemical Division, Armour Pharmaceutical Co. (Kenkakee, IL, U.S .A.) . 8-Agzaguanine was obtained from Sigma Chemical Co. (St. Louis, U.S.A .). Polyethylene glyco14000 (for gas chromatography) wan obtained from E. Merck AG (Darmstadt, F.R.G .). The mouse myeloma cell line P3-X63-Ag8-Ul (P3U1) was obtained from American Type Culture Collection (ATCC) . The thrombin-like enzyme, habutobin, was purified from Tibneresurua lfavovlridir by the same method as described previously (Kosucrt et al., 1986). Crude venom of T. Jlarorlridls, and human thrombin were obtained from Sigma Chemical Co . (St. Louis, U.SA.) . Bovine thrombin was obtained from Mochida Pharmaceutical Co., Ltd (Tokyo, Japan) . Glycine, citric acid, sodium chloride, sodium hydroxide, ammonium sulphate, potassium chloride, disodium hydrogen Phosphate 12 H=O, potassium dihydrogen Phosphate, bovine albumin, anhydrous sodium carbonate, sodium hydrogen carbonate, Tween 20, 3,3-diaminobenzidine, Tria (hydroxymethyl)-aminometbane and merthiolate sodium were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Ethylenediamine tetraaatic acid, disodium salt was obtained from Dojindo Lab. (Ktmtamoto, Japan). Hydrogen Peroxide was obtained from Santoku Chemical Ind . Co ., Ltd (Miyagi, Japan) . Pet+oxidase (POD} conjugated goat anti-mouse IgG, peroxidase substrate kits and Moux Typer® sub-isotyping kits were obtained from Bio-Rad laboratories (Richmond, U.S .A.) . POD-0onjugated goat anti-rat IgG was obtained from Cappel Laboratories (NC, U.S .A.) . Protein A-Sepharose CL-4B, Phest gel (giadient 10-15) and Phast gel sodium dodecyl sulphate (SDS) buffer strips were obtained from Pharmacies LKB, Biotechnology AB (Uppsala, Sweden). Avid AL gel was obtained from Bioprobe International, Inc. (California, U.S .A .) . Nembutal® injections were obtained from AHOTT Co . Ltd (Osaka, Japan). Unless otherwise stated, only reagent-grade chemicals were used as other reagents . llfetlrods Cell line. The mouse myeloma cell line P3-X63-Ag8-Ul (P3U1) was incubated in a COz incubator under S% COr 95% moisture and grown in RPMI 1640 medium (containing 0.30 mg/ml of trglutamine, 0.1 mg/ml of pyruvic acid, 0.2 Pg/ml of insulin, 100pg/ml of penicillin and 100Pg/ml of streptomycin), supplemented with 15% fetal bovine serum (final concentration) and 20 pg/ml of 8- azeguA+++nom for 7 days . After 8 days, the cell line was transferred and grown in RPMI 1640 medium without 8-a7aguanine . Mouse Imrmorizattorr . Three male BALB/c mice were immunized by iatraperitoneal injection of 701+8 habutobin emuhûflod in 0.5 ml of Freund's complete adjuvant, three times at intervals of 2-3 weeks. Blood sampling was carried out at 2 weeks after each sensitiTation. The increase of antibody levels in the blood samples was monitored by means of enzymo-linked immunoaorbent assay (ELISA) using habutobin (1 .Opg/ml) . The mouse with the highest titre of antibody was further boosted and 4 days later, spleen cells of the mouse with the increased antibody were removed for use in all fusion . CeIJjusion . The mouse was lightly anaesthetized with ethyl ether, and the spleen was excised immediately after the mouse had been killed by means of bleeding from the carotid artery . The spleen cells were fused with cells from the mouse myeloma line P3-X63-Ag8-Ul (P3U1) for 2min at 37°C with addition of 50% (wt/wt) polyethyleneglyool (mol. wt 4000). The counts of spleen cells and myeloma cells were each adjusted to 2 x 10' cells and 2.7 x 10' alla. After fusion, the ce))s were resuspended in hypoxanthine aminopterm thymidine (HAT) medium and plated at 5 x 10' cells per well onto 96-well plates . The cells were maintained in HAT medium for a minimum of 10 days. Growing clones of hybridoma were defined as wells showing continuing outgrowth at more than 10 days after fusion. Subsequently, the hybridoma with the highest titre of antibody against habutobin was aclaKed, and it was cloned to singlo-celle by limiting dilution using a 96-well plate. At 4 weeks after the hybridoma had been cultured in hypoxanthine thymidine (IiT) medium, the hybridoma cell with the highest titre of antibody was cultured in HT medium by sale of expaaaion. The cultured medium from the hybridoma was collected. The titre of antibody against habutobin was detected by ELISA in cultured medium from the hybridoma. lktermlnation of the meets lnviaoeoglobwlfn fn class mid sub-claw . The immunoglobulin clans and sub~laas of mouse monoclonal antibody (the antibody against habutobin) wen determined using a Mouse Typerm sub-iaotyping kit. Habutobin was adsorbed on the microtitration plate by adding 100 pl of habntobin solution (1 .0 pg/ml) to all wells, and the plate was incubated for 1 hr at room temperature . After incubation, the wells of the plate were washed flue times with Dulbeoco's phosphate buffer saline (PBS) containing 0.05% Tween 20 (Dulbecoo's PHS-Tween 20) at each step. After washing, the plate was incubated for 2 hr at room temperature

Monoclonal Antibody Against Habutobin

1179

with Dulbeoco's PBS containing 1% bovine serum albumin (BSA) at the blocking step . After the incubation and washing, cultured medium from the hybridoma was added to each of the wells and the plate was incubatod for 2hr at room temperature. Further, after incubation and washing, rabbit anti-mouse IgG IgCita, IgGtb, IgGt, IgM, IgA, x~hain and .i-chain were added, respectively, to each well, and the plate was incubated for 2 hr at room temperature. After washing the plate, each well was filled with diluted POD~ogjugated goat anti-rabbit IgG. The plate was then incubated for 1 hr at room temperature, and subsequently washed four times with PBS-Tween 20 . Finally, the plate was washed with Dulbecco'a PBS. At IS min after substrate solution (2,2ezino~i{3-eWylbenzWiazoline-6-sulphonic acidD had been added to each well, colour development was stopped by the addition of 2% oxalic acid . Spectrophotometric readings were then made using .l, = 415 nm, ~ = 492nm wavelength filters of a dual wavelength microplate photometer (MTP-22, Corona Electric Co ., Ltd). Purification of morroclorral antibody . Purification of the monoclonal antibody was perforated using protein A-Sepharose CL-4H according to the method of EY et al. (1978) . The protein A-Sepharose CL-4B was swollen with IO mM (PBS) (pH 8.0). Five millilitres of the protein A-Sepharose gel was packed into a wlumn and aSmity chromatography was carried out at 4°C. Prior to application of the cultured medium, the column was washed with 0.1 M citric acid--S M sodium hydroxide (pH 3.0) and was then equilibrated with 1.5 M glycdno-5 M sodium hydroxide containing 3 M sodium chloride (pH 8.9). Twenty millilitres of the cultured medium was mixed with 20 ml of 1.5 M glycine-5 M sodium hydroxide containing 3 M sodium chloride (pH8.9), and this mixture was applied to the protein A-Sepharose gel column. The flow rate was adjusted to 40 ml/hr. The adsorbed fractions were eluted with 0.1 M citric acid-5 M sodium hydroxide (pH 6.0). Eluates of 4.0 ml per tube wero collected for the non-adsorbed and adsorbed fractions. The protein concentration in each tube was measured with a spectrophotometer (Hitachi) at 280 nm . Western 61ott6~g. Electrophoresis was carried out according to the separation procedure in a Phast System . The sample was incubated overnight at room temperature in 10 mM Tris-HCl (pH 8.0) containing 4.5% SDS, 2 mM ethylen ' .ins tetraacetic acid, disodium salt and 5% 2-mercaptoethanol. Polyacrylamide electrophoresis was performed with a Phast gel gradient 10-15 and Phast gel SDS buffer strips using the Phast System (Pharmacia LKB Biotxhnology) . Transfer of protein from the gels onto paper was carried out according to the method of Towaav et a1. (1979) using the Phast System. The protein of habutobin on the polyacrylamide slab gel was transferred to a nitrocellulose membrane (0 .451rm pore aia in roll form, Advantec Toyo) according to the principle of electrotransfer experiments. After completion of the transfer, the blotting paper was washed three times with Dulbeoco's PBS-Tween 20 (pH 7.2) at each step . After washing, the blotting paper was soaked in Dulbeoco's PHS containing 3% HSA for 1 hr at 37°C . Once the blotting paper had been blocked, it was incubated with the purified monoclonal antibody against habutobin (100 pg/ml) in Dalbxoo's PBS for 30 min at room temperature. After washing, the blotting paper was soaked in POD~onjugated goat anti-mouse Ig(i which was diluted 1:1000 with Dulbeooo'a PHS containing 3% BSA, and was then incubated for 30 min at room temperature. After washing, the blotting paper was soaked in a solution of 2501tg/ml of 3,3~iaminobenzidine tetrahydrocWorido0.01% HzOz-50mM Tris-HCl (pH 7.2). The reaction was terminated after 15 min by washing with distilled water. The blotting paper was finally dried with filter paper. Rat sensitization and pwlfrcatian of rat IgG . Ten rats were sensitiad with 35 pg of habutobin emulsified in 0.4 ml of complete Freund's adjuvant by s.c . injection into the back three times at intervals of 2 days . The degree of increase in antibody levels was monitored by assaying blood samples with ELISA using habutobin. At 1 month after being sensitized, the rats were anaesthetized with ethyl ether and blood samples were taken from the carotid artery using a Gutdown tube . Purification of rat IgG was performed with Avid AL gel (Bio Probe International Inc.) . Five millilitres of the Avid AL gel was packed into a column and aWnity chromatography was carried out at 4°C . Prior to application of the sample, the column was washed with 20% methanol and was Wen equilibrated with 1.5 M glycino-5 M sodium hydroxide containing 3 M sodium chloride (pH 8.9). Serum (0.25 ml) from the rata sensitized wiW habutobinwas mixed wiW 4.75 ml of 1 .5 M glycino-5 M sodium hydroxide containing 3 M sodium chloride (pH 8.9). The mixture was applied to the column of Avid AL gel. The flow rate was adjusted to 30 ml/hr. The adsorbed fractions were eluted wiW 0.1 M citric acid-5 M sodium hydroxide (pH 3.0). At the final step, the column was washed wiW 20% methanol. Eluates of 4.0 ml per tube were collected for We non-adsorbed and adsorbed fractions. The protein concentration in each tube was measured wiW a spectrophotometer (Hitachi) at 280 nm . ELIS.! (tnzyme-linked immrotosarbertt assay) . ELISA jor determining the specificity ojmorroclonal antibody. ELISA was performed according to the meWod of Errcvwr .r . and PFxtetwtvr+ (1972) . Disposable sterile ELISA plates wiW polystyrene 9Crwells (Corning, NY, U.S.A .) were coated wiW antigen (habutobin) in 0.05 M carbonate buffer (pH 9.6) overnight at 4°C. At this and all other steps, the wells were filled to a volume of 100 pl. The wells of the plate were washed five times wiW DulbecGO's PHS-Tween 20 (pH 7.2) and then incubated for 2hr at room temperature wiW Dulbecco's PBS containing 1% BSA at We blocking step . After washing, We antibody (purified monoclonal antibody) was added to each well and We plate was then incubated for 2 hr at room temperature . Following washing wiW Dulbecco's PBS, We plate was incubated wiW POD~onjugated goat anti-mouse IgG which was diluted 1:3000 wiW Dulbecco's PBS-Tween 20 for 1 hr at room temperature. After the incubation, We plate was washed four times wiW Dulbeoco's PBS-Tween 20 . Finally, it wes washed wiW Dulbeooo's PBS only. At 30 min after substrate solution (2,2-azino-di-[3~thyl-beazWiazolino-6-sulphonic acid]) had been added to each well, colour develop-

M. NAKAMURA et al.

1 l80

Monoclonal antibody Control Blank

0 .8 .r a

0 .6

m °~ c~ c m a w

0 .4

0 fA

a c

0 .2 0

s >

r?

s >

s >

rf

rF

â

~ V

s > rf

s >

s >

~s

y

rf

r1

n s > rf

s > rF

~ N

G~7

7

7

F~a. 1 . IlOIUNOCiLOHULIN CLA.ß AND SUB-CIr198 OF Tt~ MONOCLONAL ANI~ODY. The upper picture shows results for the cultured medium from the hybridoma (C-5). The monoclonal antibody reacted wiW IgG, antibody and x~hain antibody . The lower figure shows the absorbante of the monoclonal antibody on ELISA using the MouseTyper® sub-isotyping kit.

meat was stopped by the addition of 2% oxalic acid. Spectrophotometric readings were then made using x, = 415 nm, ~ = 492 nm wavelength filters of a dual wavelength microplate photometer (MTP-22, Corona Electric Co., Ltd). ELISA for eattneating the concentration of habutobfn in bujjer and In the blood of various anirnaLr in vitro. The ELISA-sandwich technique was developed for determining the oonoentration of habutobin in blood by reference to the meWods of No'rt~ANS et al. (1982) and Lwna and MY~ (1982) . ELISA plates were coated with purified monoclonal antibody against hsbutobin. After the plate had been blocked wiW Dulbecco'a PBS, the sample ranging in concentration from 10 ng/ml to 2800 ng/ml was added to each of thewells and the plate was incubated overnight at room temperature . The plate was washed, and then incubated with rat anti-habutobin IgG for 2 hr at room temperature . Following washing with Dulbecco's PBS, the plate was incubated with POD~onjugated goat anti-rat IgG which was diluted at 1:3000 with Dulbaooo's PBS-Tween 20 for I hr at room temperature . For the substrate reaction, the method employed was the same as that used above in the ELISA for determining the specificity of the monoclonal antibody. The net absorbante of the sample was calculated by subtracting the absorbante of the negative control sample from the found absorbante of the test sample obtained with the dual wavelength miaoplate photometer.

RESULTS Immunoglobulin class atzd sttb-class

Of 377 wells with growing clones, 319 (84.6%)indicated a positive reaction to the antigen (habutobin) by means of ELISA. Seven wells of hybridoma with the highest titre of antibody against habutobin were used for the limiting dilution, applying materials to seven plates with 96 wells, and the hybridoma cells were cultured in HT medium. After 4 weeks, one hybridoma cell was selected from the results of determinations of the antibody

Monoclonal Antibody Against Habutobin

A buffer B buffer 5,0

t

1

1181

C buffer

1

1

4.0 3.0 2 .0 -2 .0

1.Oy

0.5r

-0 .5

O

Tube Ib . FIa . Z. hl7RII7CATION OF TII