Vox Sang. 31 (Suppl. 1): 84-95 (1976)
Purification of Hepatitis B Surface Antigen Using Polyethylene Glycol, Pepsin and Tween 80 NRAPENDRA NATH,Scorn MAZZUR,ROBERTLEDMANand CHYANG T. FANG American National Red Cross, Blood Research Laboratory, Bethesda, Md.
Abstract. A simple, inexpensive procedure for the isolation and purification of HBsAg from plasma is described. The technique included precipitation of HB,Ag with PEG and elimination of normal plasma proteins by digestion with pepsin. Tween 80 was used to remove contaminating lipoprotein(s). This technique resulted in about a 200-fold gain in the specific activity of HBsAg and yielded about 2040% recovery. Rabbits immunized with the purified antigen produced type-specific antibodies to HBsAg without detectable reactivity to normal human plasma antigens.
Introduction
In 1965, BLUMBERGet al. [ l ] demonstrated the relationship of Australia antigen (HB,Ag) to serum hepatitis and thus paved the way for further investigations into the nature of the etiological agent of serum hepatitis. Failure to propagate HB,Ag in cell culture or in the usual laboratory animals persuaded many research workers to attempt to purify HB,Ag from human blood. Fortunately, HB,Ag, unlike many human infectious agents, can be isolated from blood by simple physicochemical techniques. Isolation and purification of hepatitis B surface antigen (HB,Ag) has been achieved by many techniques such as isopycnic banding in cesium chloride [ l l , 12, 221, by column chromatography [2, 19, 201, 1 Contribution No. 345 from the American National Red Cross Blood Research Laboratory.
Received: February 6, 1976; accepted: April 5, 1976.
NATH/MAZZUR/LEDMAN/FANG
85
affinity chromatography [5, 241, isoelectric focusing [15, 281, precipitation by ammonium sulfate [3], or polyethylene glycol (PEG) [6-8, 171 and various combinations of these procedures. The most commonly used method is that of isopycnic banding in cesium chloride; however, not many laboratories have the equipment or the resources to be able to use this method effectively for routine production of moderately large batches of immunologically pure HB,Ag. In this communication we have described a method for isolating HB,Ag from plasma, taking advantage of the relative resistance of HB,Ag to pepsin [16] and Tween 80 [12], as compared to that of most normal plasma proteins. By use of these methods we have prepared HB,Ag free of human plasma antigens, by simple laboratory techniques and by a method compatible with a considerable increase in scale.
Materials and Methods Source of Plasma HB,Ag-positive plasma was obtained from the reference collection of the American National Red Cross Blood Research Laboratory and had been stored at -30 OC for over a year before being used in this study. Aliquots from these plasma units were tested for the subtype HBRAg using the passive hemagglutination inhibition (PHAI) technique [23]. Counterelectrophoresis (CEP) was done employing HepaScreen test kits supplied by Spectra Biologicals, Oxnard, Calif. Immunoelectrophoresis (IE) of the test samples was done on lantern slides (10 x 7.5 cm) layered with 12 ml of 1% agarose (Indubiose A45, L’Industrie Biologique, France) in 0.05 M veronal buffer, pH 8.2. A potential of 6 V/cm was applied for a period of 60 min. Plates were incubated at room temperature overnight in a humidified chamber. Slides were washed, dried, and stained with Amido black. Immunodiffusion was performed in 1.1% agarose in 0.02 M, pH 8.2 veronal buffer [21]. The wells were 2 mm in diameter and the distance between them was 3 mm. Passive hemagglutination was done by the method of VYASand SHULMAN [27] using a microtiter system. Human type 0 red cells coupled to HBsAg/ad or HB,Ag/ay with chromic chloride were bought from Electronucleonics, Bethesda, Md. Purification of HBsAg from PIasnia 4 g of polyethylene glycol 4000 (PEG Carbowax, Fisher Scientific, Fair Lawn, N.J.) were added to 50ml of plasma. The mixture was stirred for 60min at room temperature and the precipitate was removed by centrifugation at 1,200g for 20 min in an RC-3 centrifuge (Sorvall, Norwalk, Conn.). An additional 4 g PEG was added to the supernatant solution and the resulting precipitate was separated by centrifugation as above. The precipitate was dissolved in 25 ml of distilled water and added to 100 ml of 0.02% pepsin (Worthington, Free-
hold, N.J.) in 0 . 0 2 ~HCI, pH 2.3. The mixture was left overnight at 4 ° C or incubated at 37 "C for 3 h. At the end of incubation the pH of the mixture was adjusted to 7.4 with 1 N NaOH and Tween 80 (polyoxyethylene-sorbitan-mono-oleate, Sigma, St. Louis, Mo.) was added to the pepsin treated mixture to a final concentration of 2%. The mixture was stirred at room temperature for 1 h and the volume was reduced to 15 ml using an XMlOO ultrafiltration membrane (Diaflo, Amicon, Lexington, Mass.). Washing of HBsAg was accomplished by adding 150ml of 0.05 M Tris, pH 7.2, to the solution in the ultrafiltration cell and again reducing the volume to 15 ml. This step was repeated twice. Final recovery of the purified antigen retained on the membrane was made by rinsing the membrane with several 5-ml aliquots of Tris buffer. Final volume of the purified HB>Ag was 15-20 ml.
Prepurution of Antiseritni Antisera against the purified antigen were prepared in rabbits. Equal parts of antigen (50-75 pg protein) and Freund's complete adjuvant (Difco, Detroit, Mich.) were emulsified and injected intramuscularly i n each rabbit on day one. Booster injections, containing antigen in incomplete adjuvant, were given in the second and sixth week. In the ninth week 150 !/g of antigen was injected intravenously. Animals were test bled 2 weeks later, tested for antibody response, and, if this was adequate, the animals were exsanguinated. Antiseru Used The following antisera were used in CEP and IE to test the presence of normal human serum antigens: antihuman serum (Miles, lot 16; Behring, 1,586 V), antihuman IgG (Miles, lot 15), antihuman serum albumin (Miles-Yeda, lot R-29-l), antihuman haptoglobin (Miles-Yeda, lot R-l), antihuman u,-glycoprotein (Miles-Yeda, lot 78-P-1), anti-Fab (Miles-Yeda, lot 327A), antilactoferrin (Behring, lot 2537P), antihuman cl,-lipoprotein (Behring, lot 2348T), antihuman prealbumin (Behring, lot 2466D) and antihuman IgM (Behring, lot 2077W). Anti-HBsAg (Spectra), antiHBS/ad and anti-HBs/ay (Research Resources Branch, NIAID, Bethesda, Md.) were used to detect the presence of HBsAg in various preparations. and LATZ Polyacrylamide gel electrophoresis (PAGE) as described by STOKLOSA [26] was used to test the purity of HBsAg preparation. 5 % gel was used. Gels were stained with Coomassie blue. Protein Determinution The concentration of protein was calculated by using the spectrophotometric method of Warburg and Christian as modified by LAYNE[18]. The equation used is milligrams of protein per milliliter =(OD280nmx 1.55)- (OD260nmx 0.76).
Results Units of plasma were selected on the basis of their CEP titer against anti-HB, antibody (Spectra). Plasma containing HB,Ag of both ad and ay, subtype specificities were processed by the present method of
Purification of HBHAg
87
Table I . Isolation and purification of HBsAg from plasma Purification step
A B C D E F
HB,Ag - Plasma Supernatant of 8% PEG Precipitate of 16% PEG Pepsin-treated C Tween80+D Purified HBIiAg
Volume, CEP titer in 1 antianti-
50 50 25 125 125 20
Protein mg/ml
HBS
NHS
20 40 40
46.4 33.3 17.8 2.0 negative 1.52 negative 0.23
10 1
20
HBqAgCEP U/mg Purifiprotein' cation index 0.43 1.20 2.24 5 .O 0.65
86.9
1 2.7 5.2 11.6
1.5 202
CEP Unit = Highest dilution of sample giving a detectable precipitin line in CEP.
purification. No significant difference was noted in ad and ay specificities in relation to the yield or degree of purification. In a preliminary experiment it was established that under the conditions of this test, 8-18% PEG gave best recovery of purified HB,Ag. Most of the HB,Ag in the starting plasma could be recovered in the 16% PEG precipitate, as indicated by its CEP titer (table I). Treatment with pepsin resulted in some loss in the titer of HB,Ag. However, Tween 80 did not lower the titer of HB,Ag significantly. About 2 0 4 0 % of the initial HB,Ag activity could be recovered in the final preparation. The purity of purified HB,Ag was tested by four different techniques to detect the presence of contaminating normal human serum (NHS) antigens. The tests used were IE, CEP, PAGE, and immunization of rabbits with the purified material. The IE test showed that there was a gradual elimination of NHS antigens at various stages of purification (fig. 1). Several NHS antigens were detectable in the 16% PEG precipitate but only one precipitin arc was detected after pepsin treatment. Treatment with Tween 80 resulted in elimination of the precipitin line with rabbit antiserum to NHS (Miles); however, a precipitin line was detectable with human anti-HB,Ag antiserum (Spectra), showing that purified HB,Ag was free of contaminating antigens. In some earlier experiments pepsin treatment was followed by heating at 80 "C for 1 h instead of treatment with Tween 80. The HB,Ag preparation by this method was pure on IE but NHS antigens were detectable by the CEP technique.
Fig. 1. Concentration of HB,Ag and elimination of normal plasma antigens by the use of PEG, pepsin and T u e e n 80. Well A was filled with the untreated HB-Ag in plasma while B, C , E, and F correspond to various steps in the purification procedure as outlined in table I . Antihuman serum was applied to the troughs. IE was carried out as described in 'Methods'. Plates were washed, dried and Ftained with Amido blach.
The nature of the impurity in HB,Ag preparation purified by heating was tested by the CEP technique, using antisera to several normal human plasma proteins including prealbumin, IgG, IgM, Fab, haptoglobulin, u-glycoprotein, lactoferrin, albumin, and cr,-lipoprotein. The purified antigen preparation formed a detectable precipitin line with anti-*,lipoprotein and anti-whole serum. All other antisera tested were negative. The CEP titer of purified HB,Ag preparations against anti-rr,-lipoproteins varied greatly among different batches of HB,Ag and it did not correlate with the titer of HB,Ag in the original plasma or in the final preparation. In order to remove the contaminating lipoprotein from the HB,Ag preparations, the effect of organic solvents such as ethyl alcohol or diethyl ether and the detergents Tween 80 and nonidet P-40 (NP-40, Shell) was tested.
Purification of HBsAg
89
Table 11. Effect of lipid solvents and detergents on HBsAg Reagent added to HBSAg*
Saline Diethyl ether Ethyl alcohol Tween 80 NP-40
CEP titer of HBhAg anti-HBh (Spectra)
antihuman serum (Miles)
10
200
10 10 10 1
200 200 negativc 10
1 HBtiAg purified by PEG precipitation, pepsin digestion and heating at 80°C for 60 min.
Table I1 compares titers of HB,Ag and NHS antigen in the purified HB,Ag preparation treated with these chemicals when tested against the respective antisera by the CEP technique. Ethyl alcohol and diethyl ether did not have any effect on either HB,Ag or NHS antigens, whereas NP-40 removed both activities almost completely. Tween 80, on thlother hand, selectively eliminated activity due to NHS antigen without significantly affecting HB,Ag activity. Subsequently, it was determined that Tween 80 in a final dilution of 2% was sufficient to remove the rp maining NHS antigen contaminant from most HB,Ag preparations. The preparation treated with Tween 80 was then washed with 0.05 M Tris, pH 7.5, using XM-100 membrane (Diaflo) and simultaneously the volume was reduced to 20% of the original. Attempts to remove contaminating NHS antigen by a second pepsin treatment of the final product (step F, table I) or reheating it at 80 "C resulted in significant loss of i-iB,Ag activity. Similarly gel filtration on a column of Sepharose GI3 (Pharmacia, Uppsala) also failed to separate HB,Ag activity from NHS antigens. Antigen purified by the method employing Tween 80 was applied on 5 % polyacrylamide gel and subjected to electrophoresis under nonreducing conditions to check for contaminating NHS proteins and also to see if HB,Ag particles had been broken down to their subunits by the action of pepsin and Tween 80. HB,Ag/ad purified by three cycles of isopycnic banding on CsCl [ll] was used as a positive control. In the control, one sharp band was found to migrate about 1 mm into the gel
90
F i g . 2 . A comparison of PEG of PEG-pepsin-Tween 80 purified HBsAg (C), plasma containing HBSAg (A), and CsCl purified HBSAg (B).
(fig. 2) and another faint and diffuse band could be seen below the main band, indicating minor impurity. HB,Ag purified by the PEG-pepsinTween 80 method gave only one band identical to the principal band given by antigen purified by CsCl banding. There was no evidence of any additional proteins. Antisera prepared in rabbits following injections of HB>Ag purified by Tween 80 treatment gave no evidence of antibodies to NHS antigens (fig. 3). Materials from several antigen batches were tested for antigenic stability in immunodiffusion. Purified antigen was placed adjacent to the starting material and both were simultaneously reacted with antiserum produced against that particular purified antigen. A precipitin line of
Purification of HBsAg
91
Fig.3. Absence of antibodies to NHS proteins in antisera prepared in rabbits following immunization with PEG-pepsin-Tween 80 purified HBsAg. Wells A and C were filled with 10 ,uI plasma containing HBsAg; well B was charged with purified HBHAg.Troughs 1 and 2 were filled with 40 ,ul of antiserum from rabbits 18 and 19, respectively. Slides were washed, dried and stained with Amido black.
complete identity was produced which indicated that there was no addition or loss of antigens due to the purification treatment detectable by this method. Antisera prepared by this method were type-specific as determined by antibody subtyping using PHAI [13]. These antisera were easily made monospecific anti-d or anti-y by absorption with the heterologous type antigen for use in subtyping of antigens by the PHAI technique [23]. Some batches of monospecific antisera were of sufficient potency to be useful in antigen subtyping by CEP.
Discussion Partial purification of HB,Ag from human plasma has been achieved using PEG by several workers [6-8, 171. However, in most cases CsCl density gradient centrifugation was included as a necessary last step to achieve acceptable purification. These methods, therefore, could not be utilized in the absence of an ultracentrifuge. We felt the need for a simpler method for economical purification of HB,Ag and decided to utilize some of its known properties. KIM and BISSEL[16] have reported
92
NAI HiMAZZUR/LEDMAN/FANG
that HB,Ag is stable in the presence of 0.02% pepsin at p H 2.3 and it is known to be significantly more heat resistant than most normal plasma proteins. We hoped to be able to denature and break down most of the normal plasma proteins by a combination of these treatments, leaving HB,Ag unaffected. The initial PEG precipitation step allowed us to remove about 708 0 % of the normal plasma proteins, and subsequent treatments with pepsin and heating resulted in a substantially purified IdB,Ag preparation. Using less-sensitive methods such as agar gel diffusion and IE, no contaminating NHS antigen was detected in this product. However, testing the same preparation by the CEP technique revealed contaminating NHS antigen. The contaminant was identified as a,-lipoprotein by testing it in CEP against various antisera. It could be eliminated by treatment with Tween 80 without affecting HB,Ag activity, even though HB,Ag also has associated lipoprotein [25]. It was consistently more difficult to isolate HB,Ag free of contaminating NHS antigen from some specific units of plasma. It was found that retreating the final product with Tween 80 (1:50) usually eliminated the remaining impurity. We found that treatment with Tween 80 does not affect the antigenic activity of HB,Ag, as also shown by GERINet al. [12]. The extent of breakdown of HB,Ag as a result of the purification procedure could not be determined, but breakdown products, if any, which retained HB,Ag antigenic properties were apparently over 100,000 molec. wt because the filtrate from the XM-100 membrane (molec. wt cut off at 100,000) showed less than 1% of the HB,Ag activity retained on the membrane. Recently BOURBONNAIS et al. [3] have described a purification procedure involving digestion with pepsin. These authors used gel filtration on Sepharose 4B for the completion of purification. HB,Ag preparations which we purified using Sepharose 4B resulted in the production of antibodies to NHS antigens when injected into rabbits. However, HB,Ag purified by our method was free from contaminants by this criterion. CHAIREZ et al. [4], using P-mercaptoethanol and guanidine for reducing CsCl purified HB,Ag, have described polypeptides ranging from 19,000 to 120,000 molec. wt. At least six of these polypeptides are under 100,000 daltons. On the other hand, HOWARDand ZUCKERMAN [14] have reported two major polypeptides (82,000 and 90,000) from 20 nm HB,Ag particles. A single major band on PAGE obtained with HB,Ag purified by isopycnic banding in CsCl as well as HB,Ag purified by our method, indicated the absence of low molecular weight polypeptides in
Purification of HB,Ag
93
the purified HB,Ag. Most polypeptides produced by reducing with BME have been shown to possess HB,Ag specificity [lo] and are therefore detectable by the usual serological tests. It is possible that pepsin and/or Tween 80 selectively inactivated smaller peptides leaving only large polypeptides intact; however, there is no evidence for it. On the other hand, a 200-fold gain in the specific activity and 40% recovery of total HB,Ag activity suggest that it is unlikely that extensive inactivation of HB,Ag took place during purification. The purification procedure did not affect the antigenic determinants on HB,Ag as was shown by the fact that subtypes of the purified antigen remained unchanged through the purification when tested by PHAI and ID. The immunodiffusion test also gave evidence that no antigenic determinants of HB,Ag were lost and no new antigens were generated by this procedure. The untreated plasma which contained HB,Ag was placed adjacent to the antigen purified from that plasma and reacted against rabbit antiserum produced in response to the same purified antigen. The resulting immunoprecipitin line was a smooth line of identity with no evidence of spur formation which would be expected if the two preparations were not antigenically identical. Failure of rabbits to produce detectable antibodies to NHS antigens following repeated injections of the purified antigen, is strong evidence for the high degree of purity achieved by this method. It is interesting to note that antibody response in rabbits injected with HB,Ag purified by PEG-pepsin-Tween 80 was consistently type-specific. It may be speculated that some determinants type-specific in HB,Ag were unmasked during the purification procedure. The increase in the relative ratio of the type-specific determinants to the group determinant ‘a’ may have helped in inducing type-specific antibody response in rabbits. That such unmasking is possible by the action of pepsin on HB,Ag has been suggested recently by DODD[9]. The purification procedure described here is rapid and inexpensive, and does not require expensive equipment such as an ultracentrifuge. In addition, this method can be easily adapted for the production of either small or very large batches of purified HB,Ag. A cknowledgements This investigation was supported by Public Health Service contract No. NO1-A122522 from the National Institute of Allergy and Infectious Diseases.
94
NATH/MAZZUR/~~EDMAN/FANG
References I BWMBERG,B. S.; ALTER,H. J., and VISNICH, S.: A new antigen in leukemia sera. J. Am. rned. Ass. 191: 541-546 (1965). 2 BOOTH, J. R.; WISEMAN,I . C., and LEE, D.: Separation of hepatitis-associated antigen (HAA) from human plasma for the production of rabbit anti-HAA. VOX Sang. 27: 227-231 (1974). R.; GUEVIN,R. M., and DELVIN,E. E.: Australia antigen ‘ad’ and 3 BOURBONNAIS, ‘ay’ subtypes. Purification and partial characterization. Vox Sang. 29: 269-279 (1975). 4 CHAIREZ, R.; HOLLINGER, F. B.; BRUNSCHWIG, J. P., and DRESSMAN, G. R.: Comparative biophysical studies of hepatitis B antigen, subtypes adw and ayw. J . Virol. 15: 182-190 (1975). 5 CHARM,S. E. and WONC, B. L.: An immunoadsorbent process for removing hepatitis antigen from blood and plasma. Biotech. Bioeng. 16: 593-607 (1974). A,; MULLER,R., and DEICHER,H.: A simple procedure for purifica6 CRUCEANU, tion of hepatitis B antigen. Med. Microbiol. Immun. 159: 83-88 (1973). A,; REPANOVICI, R.; BABES,V.T.; MULL,,, R., and DEICHER,H.: 7 CRUCEANU, Considerations on several methods for the purification of viral hepatitis antigen (HBAg). Rev. Rourn. Virol. 25: 127-131 (1974). 8 Rizzo, E. DE; PANDEY, R.; WALLIS,C., and MELNICK,J. L.: Concentration and purification of hepatitis B antigen with polyethylene glycol and polyelectrolyte 60, a cross-linked copolymer of isobutylene maleic anhydride. Infec. Immunity 6: 335-338 (1972). 9 DODD, R. Y.: Gel filtration of hepatitis B surface antigen. Increased size of the native particle (personal cornmun.). 10 DRESSMAN, G. R.; CHAIREZ,R.; SUAREZ,M.; HOLLINGER, F. B.; COURTNEY, R.J., and MELNICK,J. L.: Production of antibody to individual polypeptide derived from purified hepatitis B surface antigen. J . Virol. 16: 508-515 (1975). 11 GERIN,J. L.; FAUST, R . M., and HOLLAND, P. V.: Biophysical characterization of the adr subtype of hepatitis B antigen and preparation of anti-r sera in rabbits. J. Irnmun. 115: 100-105 (1975). 12 GERIN, J . L.; PURCELL, R. H.; HOGGAN,M. D.; HOLLAND, P. V., and CHANOCK, R. M.: Biophysical properties of Australia antigen. J. Vitrol. I I : 763-768 (1969). 13 GOLD,J. W. M.; ALTER,H. J.; HOLLAND, P. V.; GERIN,J. L., and PURCELL, R. H.: Passive hernagglutination assay for antibody to subtypes of hepatitis B antigen. J. Immun. 112: 1100-1106 (1974). A. J.: Characterization of hepatitis B antigen 14 HOWARD,C. R. and ZUCKERMAN, polypeptides. Intervirology 4: 31-44 (1974). S.; VESTERBERC, O., and WADSTROM, T.: Separation of Australia anti15 JEANSSON, gen by isoelectric focusing in acrylamide gel. Life Sci. //: 929-937 (1972). 16 KIM, C. Y. and BISSEL,D. M.: Stability of the lipid and protein of hepatitisassociated (Australia) antigen. J. infect. Dis. 123; 470-476 (1971). 17 KIM, C. Y. and TILLES,J. G.: Purification and biophysical characterization of hepatitis B antigen. J. d i n . Invest. 52: 1176-1186 (1973). E.: Spectrophotometric and turbidornetric methods for measuring proteins; 18 LAYNE,
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20
21 22
23
24 25
26
27 28
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in COLOWICK and KAPLAN Methods in enzymology, vol. 3, pp. 447-454 (Academic Press, New York 1951). LUZZIO,A. J.: Isolation and purification of hepatitis B antigen by electrochromatography. J. infect. Dis. 131: 359-366 (1975). MADALINSKI, K.; SZTACHELSKA-BUDKOWSKA, A., and BRZOSKO,W. F.: DEAEcellulose chromatography. A method for dissociation of soluble immune complexes of hepatitis B antigen. J. infect. Dis. 129: 371-375 (1974). MAZZUR,S.: The detection of Australia antigen by immunodiffusion and counterelectrophoresis. Am. J. med. Tech. 38: 1-7 (1972). NEURATH,A. R.; COSIO, L.; PRINCE,A.M., and LIPPIN,A.: Purification of hepatitis B antigen associated particles. Use of a reorienting gradient rotor. Proc. SOC. exp. Biol. Med. 144: 384-390 (1973). PRINCE,A. M.; BROTMAN, B., and IKRAM,H.: Hemagglutination assay. Subtyping by hemagglutination inhibition, an ultrasensitive identity test for H B antigen; in VYAS et al. Hepatitis and blood transfusion, pp. 147-154 (Grune & Stratton, New York 1972). SHIRAISHI, H.; SUKENO, N.: SHIRACHI, R., and ISHIDA,N.: Affinity chromatography of hepatitis B antigen. Tohoku J. exp. Med. 113: 393-394 (1974). STEINER,S.; HEUBNER,M. T., and DRESSMAN, G. R.: Major polar lipids of hepatitis B antigen preparations. Evidence for the presence of glycosphingolipid. J. Virol. 14: 572-577 (1974). STOKLOSA,J. T. and LATZ, H. W.: Molecular weight determinations of proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate in just the sample solution. Biochem. biophys. Res. Commun. 58: 74-79 (1974). VYAS,G. N. and SHULMAN, N . R.: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332-333 (1970). WADSTROM, T.: Separation of Australia antigen and some bacterial proteins by isoelectric focussing in polyacrylamide gels. Ann. N.Y. Acad. Sci. 209: 405-414 (1973).
Dr. N. NATH, Hepatitis Testing Laboratory, Blood Research Laboratory, American National Red Cross, 9312 Old Georgetown Road, Bethesda, MD 20014 (USA)
Purification of Hepatitis B Surface Antigen Using Polyethylene Glycol, Pepsin and Tween 80 NRAPENDRA NATH,Scorn MAZZUR,ROBERTLEDMANand CHYANG T. FANG American National Red Cross, Blood Research Laboratory, Bethesda, Md.
Abstract. A simple, inexpensive procedure for the isolation and purification of HBsAg from plasma is described. The technique included precipitation of HB,Ag with PEG and elimination of normal plasma proteins by digestion with pepsin. Tween 80 was used to remove contaminating lipoprotein(s). This technique resulted in about a 200-fold gain in the specific activity of HBsAg and yielded about 2040% recovery. Rabbits immunized with the purified antigen produced type-specific antibodies to HBsAg without detectable reactivity to normal human plasma antigens.
Introduction
In 1965, BLUMBERGet al. [ l ] demonstrated the relationship of Australia antigen (HB,Ag) to serum hepatitis and thus paved the way for further investigations into the nature of the etiological agent of serum hepatitis. Failure to propagate HB,Ag in cell culture or in the usual laboratory animals persuaded many research workers to attempt to purify HB,Ag from human blood. Fortunately, HB,Ag, unlike many human infectious agents, can be isolated from blood by simple physicochemical techniques. Isolation and purification of hepatitis B surface antigen (HB,Ag) has been achieved by many techniques such as isopycnic banding in cesium chloride [ l l , 12, 221, by column chromatography [2, 19, 201, 1 Contribution No. 345 from the American National Red Cross Blood Research Laboratory.
Received: February 6, 1976; accepted: April 5, 1976.
NATH/MAZZUR/LEDMAN/FANG
85
affinity chromatography [5, 241, isoelectric focusing [15, 281, precipitation by ammonium sulfate [3], or polyethylene glycol (PEG) [6-8, 171 and various combinations of these procedures. The most commonly used method is that of isopycnic banding in cesium chloride; however, not many laboratories have the equipment or the resources to be able to use this method effectively for routine production of moderately large batches of immunologically pure HB,Ag. In this communication we have described a method for isolating HB,Ag from plasma, taking advantage of the relative resistance of HB,Ag to pepsin [16] and Tween 80 [12], as compared to that of most normal plasma proteins. By use of these methods we have prepared HB,Ag free of human plasma antigens, by simple laboratory techniques and by a method compatible with a considerable increase in scale.
Materials and Methods Source of Plasma HB,Ag-positive plasma was obtained from the reference collection of the American National Red Cross Blood Research Laboratory and had been stored at -30 OC for over a year before being used in this study. Aliquots from these plasma units were tested for the subtype HBRAg using the passive hemagglutination inhibition (PHAI) technique [23]. Counterelectrophoresis (CEP) was done employing HepaScreen test kits supplied by Spectra Biologicals, Oxnard, Calif. Immunoelectrophoresis (IE) of the test samples was done on lantern slides (10 x 7.5 cm) layered with 12 ml of 1% agarose (Indubiose A45, L’Industrie Biologique, France) in 0.05 M veronal buffer, pH 8.2. A potential of 6 V/cm was applied for a period of 60 min. Plates were incubated at room temperature overnight in a humidified chamber. Slides were washed, dried, and stained with Amido black. Immunodiffusion was performed in 1.1% agarose in 0.02 M, pH 8.2 veronal buffer [21]. The wells were 2 mm in diameter and the distance between them was 3 mm. Passive hemagglutination was done by the method of VYASand SHULMAN [27] using a microtiter system. Human type 0 red cells coupled to HBsAg/ad or HB,Ag/ay with chromic chloride were bought from Electronucleonics, Bethesda, Md. Purification of HBsAg from PIasnia 4 g of polyethylene glycol 4000 (PEG Carbowax, Fisher Scientific, Fair Lawn, N.J.) were added to 50ml of plasma. The mixture was stirred for 60min at room temperature and the precipitate was removed by centrifugation at 1,200g for 20 min in an RC-3 centrifuge (Sorvall, Norwalk, Conn.). An additional 4 g PEG was added to the supernatant solution and the resulting precipitate was separated by centrifugation as above. The precipitate was dissolved in 25 ml of distilled water and added to 100 ml of 0.02% pepsin (Worthington, Free-
hold, N.J.) in 0 . 0 2 ~HCI, pH 2.3. The mixture was left overnight at 4 ° C or incubated at 37 "C for 3 h. At the end of incubation the pH of the mixture was adjusted to 7.4 with 1 N NaOH and Tween 80 (polyoxyethylene-sorbitan-mono-oleate, Sigma, St. Louis, Mo.) was added to the pepsin treated mixture to a final concentration of 2%. The mixture was stirred at room temperature for 1 h and the volume was reduced to 15 ml using an XMlOO ultrafiltration membrane (Diaflo, Amicon, Lexington, Mass.). Washing of HBsAg was accomplished by adding 150ml of 0.05 M Tris, pH 7.2, to the solution in the ultrafiltration cell and again reducing the volume to 15 ml. This step was repeated twice. Final recovery of the purified antigen retained on the membrane was made by rinsing the membrane with several 5-ml aliquots of Tris buffer. Final volume of the purified HB>Ag was 15-20 ml.
Prepurution of Antiseritni Antisera against the purified antigen were prepared in rabbits. Equal parts of antigen (50-75 pg protein) and Freund's complete adjuvant (Difco, Detroit, Mich.) were emulsified and injected intramuscularly i n each rabbit on day one. Booster injections, containing antigen in incomplete adjuvant, were given in the second and sixth week. In the ninth week 150 !/g of antigen was injected intravenously. Animals were test bled 2 weeks later, tested for antibody response, and, if this was adequate, the animals were exsanguinated. Antiseru Used The following antisera were used in CEP and IE to test the presence of normal human serum antigens: antihuman serum (Miles, lot 16; Behring, 1,586 V), antihuman IgG (Miles, lot 15), antihuman serum albumin (Miles-Yeda, lot R-29-l), antihuman haptoglobin (Miles-Yeda, lot R-l), antihuman u,-glycoprotein (Miles-Yeda, lot 78-P-1), anti-Fab (Miles-Yeda, lot 327A), antilactoferrin (Behring, lot 2537P), antihuman cl,-lipoprotein (Behring, lot 2348T), antihuman prealbumin (Behring, lot 2466D) and antihuman IgM (Behring, lot 2077W). Anti-HBsAg (Spectra), antiHBS/ad and anti-HBs/ay (Research Resources Branch, NIAID, Bethesda, Md.) were used to detect the presence of HBsAg in various preparations. and LATZ Polyacrylamide gel electrophoresis (PAGE) as described by STOKLOSA [26] was used to test the purity of HBsAg preparation. 5 % gel was used. Gels were stained with Coomassie blue. Protein Determinution The concentration of protein was calculated by using the spectrophotometric method of Warburg and Christian as modified by LAYNE[18]. The equation used is milligrams of protein per milliliter =(OD280nmx 1.55)- (OD260nmx 0.76).
Results Units of plasma were selected on the basis of their CEP titer against anti-HB, antibody (Spectra). Plasma containing HB,Ag of both ad and ay, subtype specificities were processed by the present method of
Purification of HBHAg
87
Table I . Isolation and purification of HBsAg from plasma Purification step
A B C D E F
HB,Ag - Plasma Supernatant of 8% PEG Precipitate of 16% PEG Pepsin-treated C Tween80+D Purified HBIiAg
Volume, CEP titer in 1 antianti-
50 50 25 125 125 20
Protein mg/ml
HBS
NHS
20 40 40
46.4 33.3 17.8 2.0 negative 1.52 negative 0.23
10 1
20
HBqAgCEP U/mg Purifiprotein' cation index 0.43 1.20 2.24 5 .O 0.65
86.9
1 2.7 5.2 11.6
1.5 202
CEP Unit = Highest dilution of sample giving a detectable precipitin line in CEP.
purification. No significant difference was noted in ad and ay specificities in relation to the yield or degree of purification. In a preliminary experiment it was established that under the conditions of this test, 8-18% PEG gave best recovery of purified HB,Ag. Most of the HB,Ag in the starting plasma could be recovered in the 16% PEG precipitate, as indicated by its CEP titer (table I). Treatment with pepsin resulted in some loss in the titer of HB,Ag. However, Tween 80 did not lower the titer of HB,Ag significantly. About 2 0 4 0 % of the initial HB,Ag activity could be recovered in the final preparation. The purity of purified HB,Ag was tested by four different techniques to detect the presence of contaminating normal human serum (NHS) antigens. The tests used were IE, CEP, PAGE, and immunization of rabbits with the purified material. The IE test showed that there was a gradual elimination of NHS antigens at various stages of purification (fig. 1). Several NHS antigens were detectable in the 16% PEG precipitate but only one precipitin arc was detected after pepsin treatment. Treatment with Tween 80 resulted in elimination of the precipitin line with rabbit antiserum to NHS (Miles); however, a precipitin line was detectable with human anti-HB,Ag antiserum (Spectra), showing that purified HB,Ag was free of contaminating antigens. In some earlier experiments pepsin treatment was followed by heating at 80 "C for 1 h instead of treatment with Tween 80. The HB,Ag preparation by this method was pure on IE but NHS antigens were detectable by the CEP technique.
Fig. 1. Concentration of HB,Ag and elimination of normal plasma antigens by the use of PEG, pepsin and T u e e n 80. Well A was filled with the untreated HB-Ag in plasma while B, C , E, and F correspond to various steps in the purification procedure as outlined in table I . Antihuman serum was applied to the troughs. IE was carried out as described in 'Methods'. Plates were washed, dried and Ftained with Amido blach.
The nature of the impurity in HB,Ag preparation purified by heating was tested by the CEP technique, using antisera to several normal human plasma proteins including prealbumin, IgG, IgM, Fab, haptoglobulin, u-glycoprotein, lactoferrin, albumin, and cr,-lipoprotein. The purified antigen preparation formed a detectable precipitin line with anti-*,lipoprotein and anti-whole serum. All other antisera tested were negative. The CEP titer of purified HB,Ag preparations against anti-rr,-lipoproteins varied greatly among different batches of HB,Ag and it did not correlate with the titer of HB,Ag in the original plasma or in the final preparation. In order to remove the contaminating lipoprotein from the HB,Ag preparations, the effect of organic solvents such as ethyl alcohol or diethyl ether and the detergents Tween 80 and nonidet P-40 (NP-40, Shell) was tested.
Purification of HBsAg
89
Table 11. Effect of lipid solvents and detergents on HBsAg Reagent added to HBSAg*
Saline Diethyl ether Ethyl alcohol Tween 80 NP-40
CEP titer of HBhAg anti-HBh (Spectra)
antihuman serum (Miles)
10
200
10 10 10 1
200 200 negativc 10
1 HBtiAg purified by PEG precipitation, pepsin digestion and heating at 80°C for 60 min.
Table I1 compares titers of HB,Ag and NHS antigen in the purified HB,Ag preparation treated with these chemicals when tested against the respective antisera by the CEP technique. Ethyl alcohol and diethyl ether did not have any effect on either HB,Ag or NHS antigens, whereas NP-40 removed both activities almost completely. Tween 80, on thlother hand, selectively eliminated activity due to NHS antigen without significantly affecting HB,Ag activity. Subsequently, it was determined that Tween 80 in a final dilution of 2% was sufficient to remove the rp maining NHS antigen contaminant from most HB,Ag preparations. The preparation treated with Tween 80 was then washed with 0.05 M Tris, pH 7.5, using XM-100 membrane (Diaflo) and simultaneously the volume was reduced to 20% of the original. Attempts to remove contaminating NHS antigen by a second pepsin treatment of the final product (step F, table I) or reheating it at 80 "C resulted in significant loss of i-iB,Ag activity. Similarly gel filtration on a column of Sepharose GI3 (Pharmacia, Uppsala) also failed to separate HB,Ag activity from NHS antigens. Antigen purified by the method employing Tween 80 was applied on 5 % polyacrylamide gel and subjected to electrophoresis under nonreducing conditions to check for contaminating NHS proteins and also to see if HB,Ag particles had been broken down to their subunits by the action of pepsin and Tween 80. HB,Ag/ad purified by three cycles of isopycnic banding on CsCl [ll] was used as a positive control. In the control, one sharp band was found to migrate about 1 mm into the gel
90
F i g . 2 . A comparison of PEG of PEG-pepsin-Tween 80 purified HBsAg (C), plasma containing HBSAg (A), and CsCl purified HBSAg (B).
(fig. 2) and another faint and diffuse band could be seen below the main band, indicating minor impurity. HB,Ag purified by the PEG-pepsinTween 80 method gave only one band identical to the principal band given by antigen purified by CsCl banding. There was no evidence of any additional proteins. Antisera prepared in rabbits following injections of HB>Ag purified by Tween 80 treatment gave no evidence of antibodies to NHS antigens (fig. 3). Materials from several antigen batches were tested for antigenic stability in immunodiffusion. Purified antigen was placed adjacent to the starting material and both were simultaneously reacted with antiserum produced against that particular purified antigen. A precipitin line of
Purification of HBsAg
91
Fig.3. Absence of antibodies to NHS proteins in antisera prepared in rabbits following immunization with PEG-pepsin-Tween 80 purified HBsAg. Wells A and C were filled with 10 ,uI plasma containing HBsAg; well B was charged with purified HBHAg.Troughs 1 and 2 were filled with 40 ,ul of antiserum from rabbits 18 and 19, respectively. Slides were washed, dried and stained with Amido black.
complete identity was produced which indicated that there was no addition or loss of antigens due to the purification treatment detectable by this method. Antisera prepared by this method were type-specific as determined by antibody subtyping using PHAI [13]. These antisera were easily made monospecific anti-d or anti-y by absorption with the heterologous type antigen for use in subtyping of antigens by the PHAI technique [23]. Some batches of monospecific antisera were of sufficient potency to be useful in antigen subtyping by CEP.
Discussion Partial purification of HB,Ag from human plasma has been achieved using PEG by several workers [6-8, 171. However, in most cases CsCl density gradient centrifugation was included as a necessary last step to achieve acceptable purification. These methods, therefore, could not be utilized in the absence of an ultracentrifuge. We felt the need for a simpler method for economical purification of HB,Ag and decided to utilize some of its known properties. KIM and BISSEL[16] have reported
92
NAI HiMAZZUR/LEDMAN/FANG
that HB,Ag is stable in the presence of 0.02% pepsin at p H 2.3 and it is known to be significantly more heat resistant than most normal plasma proteins. We hoped to be able to denature and break down most of the normal plasma proteins by a combination of these treatments, leaving HB,Ag unaffected. The initial PEG precipitation step allowed us to remove about 708 0 % of the normal plasma proteins, and subsequent treatments with pepsin and heating resulted in a substantially purified IdB,Ag preparation. Using less-sensitive methods such as agar gel diffusion and IE, no contaminating NHS antigen was detected in this product. However, testing the same preparation by the CEP technique revealed contaminating NHS antigen. The contaminant was identified as a,-lipoprotein by testing it in CEP against various antisera. It could be eliminated by treatment with Tween 80 without affecting HB,Ag activity, even though HB,Ag also has associated lipoprotein [25]. It was consistently more difficult to isolate HB,Ag free of contaminating NHS antigen from some specific units of plasma. It was found that retreating the final product with Tween 80 (1:50) usually eliminated the remaining impurity. We found that treatment with Tween 80 does not affect the antigenic activity of HB,Ag, as also shown by GERINet al. [12]. The extent of breakdown of HB,Ag as a result of the purification procedure could not be determined, but breakdown products, if any, which retained HB,Ag antigenic properties were apparently over 100,000 molec. wt because the filtrate from the XM-100 membrane (molec. wt cut off at 100,000) showed less than 1% of the HB,Ag activity retained on the membrane. Recently BOURBONNAIS et al. [3] have described a purification procedure involving digestion with pepsin. These authors used gel filtration on Sepharose 4B for the completion of purification. HB,Ag preparations which we purified using Sepharose 4B resulted in the production of antibodies to NHS antigens when injected into rabbits. However, HB,Ag purified by our method was free from contaminants by this criterion. CHAIREZ et al. [4], using P-mercaptoethanol and guanidine for reducing CsCl purified HB,Ag, have described polypeptides ranging from 19,000 to 120,000 molec. wt. At least six of these polypeptides are under 100,000 daltons. On the other hand, HOWARDand ZUCKERMAN [14] have reported two major polypeptides (82,000 and 90,000) from 20 nm HB,Ag particles. A single major band on PAGE obtained with HB,Ag purified by isopycnic banding in CsCl as well as HB,Ag purified by our method, indicated the absence of low molecular weight polypeptides in
Purification of HB,Ag
93
the purified HB,Ag. Most polypeptides produced by reducing with BME have been shown to possess HB,Ag specificity [lo] and are therefore detectable by the usual serological tests. It is possible that pepsin and/or Tween 80 selectively inactivated smaller peptides leaving only large polypeptides intact; however, there is no evidence for it. On the other hand, a 200-fold gain in the specific activity and 40% recovery of total HB,Ag activity suggest that it is unlikely that extensive inactivation of HB,Ag took place during purification. The purification procedure did not affect the antigenic determinants on HB,Ag as was shown by the fact that subtypes of the purified antigen remained unchanged through the purification when tested by PHAI and ID. The immunodiffusion test also gave evidence that no antigenic determinants of HB,Ag were lost and no new antigens were generated by this procedure. The untreated plasma which contained HB,Ag was placed adjacent to the antigen purified from that plasma and reacted against rabbit antiserum produced in response to the same purified antigen. The resulting immunoprecipitin line was a smooth line of identity with no evidence of spur formation which would be expected if the two preparations were not antigenically identical. Failure of rabbits to produce detectable antibodies to NHS antigens following repeated injections of the purified antigen, is strong evidence for the high degree of purity achieved by this method. It is interesting to note that antibody response in rabbits injected with HB,Ag purified by PEG-pepsin-Tween 80 was consistently type-specific. It may be speculated that some determinants type-specific in HB,Ag were unmasked during the purification procedure. The increase in the relative ratio of the type-specific determinants to the group determinant ‘a’ may have helped in inducing type-specific antibody response in rabbits. That such unmasking is possible by the action of pepsin on HB,Ag has been suggested recently by DODD[9]. The purification procedure described here is rapid and inexpensive, and does not require expensive equipment such as an ultracentrifuge. In addition, this method can be easily adapted for the production of either small or very large batches of purified HB,Ag. A cknowledgements This investigation was supported by Public Health Service contract No. NO1-A122522 from the National Institute of Allergy and Infectious Diseases.
94
NATH/MAZZUR/~~EDMAN/FANG
References I BWMBERG,B. S.; ALTER,H. J., and VISNICH, S.: A new antigen in leukemia sera. J. Am. rned. Ass. 191: 541-546 (1965). 2 BOOTH, J. R.; WISEMAN,I . C., and LEE, D.: Separation of hepatitis-associated antigen (HAA) from human plasma for the production of rabbit anti-HAA. VOX Sang. 27: 227-231 (1974). R.; GUEVIN,R. M., and DELVIN,E. E.: Australia antigen ‘ad’ and 3 BOURBONNAIS, ‘ay’ subtypes. Purification and partial characterization. Vox Sang. 29: 269-279 (1975). 4 CHAIREZ, R.; HOLLINGER, F. B.; BRUNSCHWIG, J. P., and DRESSMAN, G. R.: Comparative biophysical studies of hepatitis B antigen, subtypes adw and ayw. J . Virol. 15: 182-190 (1975). 5 CHARM,S. E. and WONC, B. L.: An immunoadsorbent process for removing hepatitis antigen from blood and plasma. Biotech. Bioeng. 16: 593-607 (1974). A,; MULLER,R., and DEICHER,H.: A simple procedure for purifica6 CRUCEANU, tion of hepatitis B antigen. Med. Microbiol. Immun. 159: 83-88 (1973). A,; REPANOVICI, R.; BABES,V.T.; MULL,,, R., and DEICHER,H.: 7 CRUCEANU, Considerations on several methods for the purification of viral hepatitis antigen (HBAg). Rev. Rourn. Virol. 25: 127-131 (1974). 8 Rizzo, E. DE; PANDEY, R.; WALLIS,C., and MELNICK,J. L.: Concentration and purification of hepatitis B antigen with polyethylene glycol and polyelectrolyte 60, a cross-linked copolymer of isobutylene maleic anhydride. Infec. Immunity 6: 335-338 (1972). 9 DODD, R. Y.: Gel filtration of hepatitis B surface antigen. Increased size of the native particle (personal cornmun.). 10 DRESSMAN, G. R.; CHAIREZ,R.; SUAREZ,M.; HOLLINGER, F. B.; COURTNEY, R.J., and MELNICK,J. L.: Production of antibody to individual polypeptide derived from purified hepatitis B surface antigen. J . Virol. 16: 508-515 (1975). 11 GERIN,J. L.; FAUST, R . M., and HOLLAND, P. V.: Biophysical characterization of the adr subtype of hepatitis B antigen and preparation of anti-r sera in rabbits. J. Irnmun. 115: 100-105 (1975). 12 GERIN, J . L.; PURCELL, R. H.; HOGGAN,M. D.; HOLLAND, P. V., and CHANOCK, R. M.: Biophysical properties of Australia antigen. J. Vitrol. I I : 763-768 (1969). 13 GOLD,J. W. M.; ALTER,H. J.; HOLLAND, P. V.; GERIN,J. L., and PURCELL, R. H.: Passive hernagglutination assay for antibody to subtypes of hepatitis B antigen. J. Immun. 112: 1100-1106 (1974). A. J.: Characterization of hepatitis B antigen 14 HOWARD,C. R. and ZUCKERMAN, polypeptides. Intervirology 4: 31-44 (1974). S.; VESTERBERC, O., and WADSTROM, T.: Separation of Australia anti15 JEANSSON, gen by isoelectric focusing in acrylamide gel. Life Sci. //: 929-937 (1972). 16 KIM, C. Y. and BISSEL,D. M.: Stability of the lipid and protein of hepatitisassociated (Australia) antigen. J. infect. Dis. 123; 470-476 (1971). 17 KIM, C. Y. and TILLES,J. G.: Purification and biophysical characterization of hepatitis B antigen. J. d i n . Invest. 52: 1176-1186 (1973). E.: Spectrophotometric and turbidornetric methods for measuring proteins; 18 LAYNE,
Purification of HBsAg
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20
21 22
23
24 25
26
27 28
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in COLOWICK and KAPLAN Methods in enzymology, vol. 3, pp. 447-454 (Academic Press, New York 1951). LUZZIO,A. J.: Isolation and purification of hepatitis B antigen by electrochromatography. J. infect. Dis. 131: 359-366 (1975). MADALINSKI, K.; SZTACHELSKA-BUDKOWSKA, A., and BRZOSKO,W. F.: DEAEcellulose chromatography. A method for dissociation of soluble immune complexes of hepatitis B antigen. J. infect. Dis. 129: 371-375 (1974). MAZZUR,S.: The detection of Australia antigen by immunodiffusion and counterelectrophoresis. Am. J. med. Tech. 38: 1-7 (1972). NEURATH,A. R.; COSIO, L.; PRINCE,A.M., and LIPPIN,A.: Purification of hepatitis B antigen associated particles. Use of a reorienting gradient rotor. Proc. SOC. exp. Biol. Med. 144: 384-390 (1973). PRINCE,A. M.; BROTMAN, B., and IKRAM,H.: Hemagglutination assay. Subtyping by hemagglutination inhibition, an ultrasensitive identity test for H B antigen; in VYAS et al. Hepatitis and blood transfusion, pp. 147-154 (Grune & Stratton, New York 1972). SHIRAISHI, H.; SUKENO, N.: SHIRACHI, R., and ISHIDA,N.: Affinity chromatography of hepatitis B antigen. Tohoku J. exp. Med. 113: 393-394 (1974). STEINER,S.; HEUBNER,M. T., and DRESSMAN, G. R.: Major polar lipids of hepatitis B antigen preparations. Evidence for the presence of glycosphingolipid. J. Virol. 14: 572-577 (1974). STOKLOSA,J. T. and LATZ, H. W.: Molecular weight determinations of proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate in just the sample solution. Biochem. biophys. Res. Commun. 58: 74-79 (1974). VYAS,G. N. and SHULMAN, N . R.: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332-333 (1970). WADSTROM, T.: Separation of Australia antigen and some bacterial proteins by isoelectric focussing in polyacrylamide gels. Ann. N.Y. Acad. Sci. 209: 405-414 (1973).
Dr. N. NATH, Hepatitis Testing Laboratory, Blood Research Laboratory, American National Red Cross, 9312 Old Georgetown Road, Bethesda, MD 20014 (USA)
