Vox Sang. 2Y: 269- 279 (1975)
Australia Antigen ‘ad’ and ‘ay’ Subtypes Purification and Partial Characterization
R. BOURBONNAIS, R. M. GUEVINand E. E. DELVIN Canadian Rcd Cross Blood Transfusion Service, Montreal, Que.
Abstracf. The two commonly-found Australia antigen subtypes have been purified by simple inexpensive, reproducible, physico-chemical methods involving ammonium sulfate precipitation, peptic digestion, calcium phosphate gel fractionation and molecular sieving on Sepharose 4B. The overall recovery of the two subtypes varied between 50 and 60°/0 of the total antigen content of the starting serum. The purified antigens were found homogeneous by discontinuous gel electrophoresis, analytical ultracentrifugation, and isopycnic banding ultracentrifugation. N o contaminating serum proteins could be detected by immunoelectrophoresis. The two purified antigen subtypes have similar Stokes radii and buoyant densities. The sedimentation coefficients for the ‘ad‘ and ‘ay’ subtypes were found t o be 33.0 and 40.1 x sec, respectively.
Introduction The purification and characterization of the two commonly-found ‘ad’ and ‘ay’ Australia antigens (HBsAg) are the prime requirements in the understanding of their nature, origin and infectivity. A variety of purification methods, including isopycnic banding in cesium chloride, sedimentation rate ultracentrifugation [2, 3, 5 , 61, and, more recently, affinity chromatography [7, 131 have been described. Preparative ultracentrifugation techniques are, however, limited to laboratories having liberal access to the expensive equipment required, while affinity chromatography can only be part of an elaborate purification procedure if it can be used on a large scale.
Received: November 21, 1974; accepted: January 17, 1975.
210
The present report proposes a relatively simple, inexpensive, and high yield method for the purification of the ‘ad’ and ‘ay’ Australia antigens, easily adaptable to large scale production. Physicochemical and immunological properties of the two purified subtypes are also compared.
Material and Methods Purification of Australia Antigen All steps of the purification were performcd at room temperature in burners or solutions containing 0.02 O/O sodium azide as bacteriostatic agent. 200 rnl of serum (fraction I) obtained by recalcification of HBsAg-positive plasina
[12] was fractionated with ammonium sulfate at p H 6.5. The fraction precipitating between 30- and 45-percent saturation was kept. The precipitate collected by centrifugation was then washed once with 200ml of ammonium sulfate at 45-percent saturation, dissolved in isotonic saline, and dialyzed overnight (fraction 11). Protein concentration was adjusted to 10 mg/ml by diluting the fraction with distilled water. Pepsin digestion (25 mg of pepsin ‘Pentex crystallized’ per gram of protein to be hydrolyzed) was carried out at pH 3.0 for 3 h at 37 OC. The p H was kept near 3.0 by periodic addition of 1N HCI. The proteolysis was stopped by raising the p H to 7.4 with 1N NaOH. The solution was immediately concentrated on Amicon membrane XM-100A, washed with 100ml of 0 . 0 5 ~sodium phosphate buffer pH7.4, brought to a final volume of approximately 20 ml, and dialyzed overnight against 2 liters of the same buffer (fraction 111). This fraction was further purified by negative absorption on calcium phosphate gel 191 (2 rng of gel per milligram of protein) equilibrated with 0.05 M phosphate buffer pH 7.4 at a protein concentration of 10 rng/ml. The slurry was stirred for 30 min. The gel was then removed by centrifugation at 3,000 g for 10min and washed 3 times with a volume of 0 . 0 5 ~ phosphate buffer pH7.4 equal to that of the first eluate. The eluted fractions were pooled and concentrated on Amicon membrane XM-100A to a final volume of approximately 5 rnl (fraction IV). Finally, the concentrate was chromatographed on a Sepharose 4B (Pharrnacia) column (78 x 2.5 cm) equilibrated in PBS (0.15 M NaCI, 0.025 M sodium phosphate, pH 7.4, 0.001 M Na,EDTA) with an upward flow of 17 ml/h. The HBsAg-positive fractions were pooled and concentrated by ultrafiltration (fraction V). Analytical Methods
The protein content of each fraction was estimated by the method of Warburg and Christian modified by LAYNE[ll] in a 1-cm path length spectrophotometric cell. The concentration of protein was calculated by the following equation: milligram of protein per rl;jlliliter = (1.55 x OD 280 nm) - (0.76 x OD 260 nm). The extinction co= 3.76) was not used since the proportion of Australia antigen was efficient (E:& constantly changing during the purification procedure. The amount of Australia antigen was measured by electrophoresis on agarose gel [lo] method. Guinea pig anti-HBsAg containing antibody according to LAURELL’S (0.1 ml) was mixed with 10 ml of warmed (45-50 “ C ) 0.85 O/O agarose in 0.05 M Trist~
Australia Antigen Subtypes Purification and Characterization
27 1
barbital buffer pH 8.6 and immediately poured on a (8 x 10 cm) glass plate. Each well, 4 mm in diameter, received l o p 1 of sample. Electrophoresis was performed at 30 mA per plate a t room temperature for 3 h in a 0.05 M Tris-barbital buffer p H 8.6 as the electrode buffer. After electrophoresis, the gels were washed with saline, followed by distilled water, blotted with filter paper and stained with amido-black. The height of the precipitation peak is proportional to the amount of the Australia antigen present. Tube discontinuous polyacrylamide gel electrophoresis was performed according to the method of ORNsrErN and DAVIS[14] in a refrigerated Buchler Polyanalyst cell. A 5-percent acrylamide i n a 0 . 3 8 ~Tris-HC1 buffer pH 8.9 and a 4-percent acrylamide in a 0.06 M Tris-HCI buffer pH 6.7 served as running and stacking gels, respectively. The electrophoresis was allowed to proceed at 3 m A per tube for 1h using 0.05 M Tris-glycine p H 8.3 as the electrode buffer. Immunoelectrophoresis was performed according to the microslide technique of SCHEIDECCER [16] adapted to the Gelman-LKB equipment without any modification. Sedimentation velocities were measured with a Spinco model E analytical ultracentrifuge equipped with the absorption system at 280 nm. Samples were diszolved in PBS to a n initial protein concentration ranging between 0.5 and 0.8 mg/mI. After the rotor reached 25,980 rpm at 20 “C, scannings were taken at 8-min intervals. Sedimentation coefficients were calculated by the method of SCHACHMAN [15] and corrected for viscosity and density of water at 20 OC. The relative viscosity of PBS ( ) / / T I , , = 1.04) was measured with a rotating cylinder viscosimeter and its density (pzo = 1.008 g/cm3) was measured by pycnometry. The isopycnic banding ultracentrifugation was carried out in a continuous cesium chloride density gradient ranging between 1.1 and 1.4g/ml. Aliquots of 0.5rnl in PBS were layered onto 4.7 ml of 26Oio (w/w) CsC1,. After centrifuging the samples in a Beckrnan SW 50.1 rotor at 47,000 rpm for 24 h at 25 OC, fractions of 10 drops were collected automatically, starting from the bottom of each tube. Each fraction was assayed for protein content and Australia antigen activity. The specific gravity of each fraction was measured by the refractive index in duplicate CsCI, gradients. Partial specific volumes (7) of the purified antigens were calculated from the buoyant densities. The Stokes radii (‘a’) of the purified ‘ad’ and ‘ay’ antigens were determined according to the method described by ACKERS[l] on a Sepharose 4B column (78 x 2.5 cm) equilibrated in PBS at room temperature. Thyroglobulin (Sigma), apoferritin (SchwarzMann), catalase (Sigma), aldolase and bovine serum albumin (Pharmacia) were used as standard proteins. The void volume (‘Vo’) was determined with blue dextran (Pharmacia) and the inner volume (‘Vi’), with tritiated water (NEN). All samples were applied to the bottom of the column and eluted upward at a flow rate of 17 ml/h. The column effluent was monitored by measuring the optical density a t 280nm. Distribution coefficients (‘Kd’) were calculated using the basic equation formulated by GELOTTE[4], taking as elution volume the inflection point of each peak. Published values of Stokes radii of standard proteins were substituted in the equation developed by ACKERS111, to calculate the mean gel pore radius (‘r’) of Sepharose 4B. Thc diffusion coefficients (DZ0,) of the purified Australia antigens were calculated by using the STOKES-EINSTEIN I181 equation and taking the experimentally-determined values for the Stokes radii.
272
B~LJRB~NNAIS/GUEVIN/DELVIN
The molecular weights of the Australia antigens were calculated by SVEDBERC and PEDERSON’S [17] equation, using the calculated diffusion and sedimentation coefficients and the partial specific volumes obtained from the buoyant densities.
Results Purification of Australia Antigen Data from typical Australia antigen purifications are summarized in table I. Specific and total activities, shown in the second and third columns for ‘ad’ and fifth and sixth for by’ subtypes, respectively, are measured at each step by the LAURELL [lo] method as shown by figure 1. The amount of HBsAg in each fraction is estimated by measuring the height of the peak. The specific activity is expressed in centimeters per milligram of total protein applied. A slight enhancement of the total antigenic activity after the peptic digestion was noticed in all purifications. This is probably due to the hydrolysis of coating proteins which mask antigenic deter-
Fig. I . LAURELL [lo] rocket method for determination of HBsAg at each main step of purification (as described in Material and Merhods). Left to right: ‘ay’ and ‘ad’ antigen fractions of incrcasing purity. Total protein content of each well is expressed as micrograms. ‘ay’ antigen fraction I: 587, fraction 11: 255, fraction 111: 52, fraction 1V: 15 and fraction V: 2.2; ‘ad’ antigen fraction I: 556, fraction IT: 220, fraction 111: 73, fraction 1V: 17.7 and fraction V: 2.0.
Australia Antigen Subtypes Purification and Characterization
213
n
I 1
,
0
50
IM
150
200
250
300
350
400
Elution volume, ml
Fig. 2. Chromatography of the ‘ad’ antigen fraction IV o n Sepharose 4B. The solid line represents the optical density at 280 nm and the dashed line represents the [lo] Australia antigen activity in millimeters of migration as measured by the LAURELL method.
F i g . 3. Polyacrylamide disc electrophoresis of the purified ‘ay’ and ‘ad’ Australia antigens stained with amido-black.
214
BOURBONNAIS/GUEVIN/DELVIN
Fig. 4 . lmmunoclcctrophoresis. CI Whole serum against rabbit anti-human serum (Behringwerke AG). h Purified ‘ad’ and ‘ay’ antigens against rabbit anti-human serum. c Purified ‘ad’ and ‘ay’ antigens against human anti-HBsAg.
minants on the surface of the Australia antigens. The calcium phosphate gel fractionation is a very important step to remove proteins and aggregates that interfere seriously with chromatographic resolution. Figure 2 shows a typical elution profile obtained by chromatographing the fraction IV of an ‘ad’ antigen on Sepharose 4B. A similar profile is obtained for ‘ay’ subtypes. The final recovery of the antigen is in the range of 50-600/0. The purification factor, however, varies inversely with the titer obtained in the starting material.
Criteria of Purity The two purified Australia antigen subtypes, ‘ad’ and ‘ay’, migrated as single components on polyacrylamide gel disc electrophoresis as depicted by figure 3. These unique protein bands also stained positively for lipo-
Australia Antigen Subtypes Purification and Characterization
275
Fig.5. Sedimentation pattern of purified ‘ay’ antigen. Scannings taken at 16-min intervals are shown. The direction of sedimentation is from left to right. Sedimentation pattern obtained for the purified ‘ad’ antigen is almost the same.
\ \
F r a c t i o n number
Fig. 6. Optical density pattern obtained from isopycnic banding ultracentrifugation in cesium chloride of purified ‘ad’ (dotted line) and ‘ay’ (solid line) Australia antigens. Dashed line represents the density gradient. Antigenic activity corresponds to the optical density peaks.
BOLJRBONNAIS/GUEVIN/DELVIN
276
Table I . Purification of Australia antigens
Purification steps
'ad' antigen
'ay' antigen
specific activity cm/mg
total activity cm
recovery "in
specific activity cm/mg
total activity cm
recovery Oio
0.72 2.96 12.3 56.5 550.0
9,150 8,970 10,000 6,500 4,630
100 98 110 71 51
1.53 5.1 28.8 105.5 590.0
15,650 14,100 15,300 10,300 8,900
100 90 98 66 57
serum (NH4),S0, 30-45 "in saturation Ill: peptic digestion IV: calcium phosphate gel V: sepharose 4B chromatography t: 11:
~
~
~~
~
_
_
_
Specific activity refers to the ratio of the distance from origin at which the peak of precipitation of Ag-Ab complexes are visualized to the amount of protein present in the well.
Table 11. Physical properties of Australia antigens
Physical properties
'ad' antigen means f SD
'ay' antigen means f SD
PI
Stokes radius (a), nm Diffusion coefficient (DZOw),cm' sec-1 Sedimentation coefficient (S,,,), 10-13 sec Buoyant density (&, g/ml Molecular weight (Mw), daltons
10.6 5 0.67 2.01 It: 0.13 33.0 k 2.3 1.19 2.5 x los k 0.17
11.0 f 0.69 1.94 f 0.12 40.1 It: 2.8 1.20 3.0 x 108 k 0.21
n.s. n.s. < 0.01 1
< 0.01
n s . = Not significant. Significance level for difference in means calculated with the Student t-test for paired variates (d.f. = N - 1 = 2).
protein and were active towards human anti-HBsAg. Immunoelectrophoresis (fig. 4b) indicated that there were no detectable immunological reactions between purified 'ad' and 'ay' antigens and an anti-human serum. However, immunological reaction was clearly seen when the purified subtypes were run in the presence of human anti-HBsAg (fig.4~).Analytical centrifugation (fig. 5 ) and isopycnic banding ultracentrifugation in CsCl, (fig. 6) revealed only one component, confirming the homogeneity of our preparations.
Australia Antigen Subtypes Purification and Characterization
27 I
Physical Properties of the ‘ad‘ and ‘ay’ Australia Antigens The Stokes radii (‘a’) as calculated by the method of ACKERS[l] and diffusion coefficients calculated by the STOKES-EINSTEIN [181 equation show no significant difference between the two Australia antigen subtypes as shown in table 11. These values were obtained from a mean gel pore radius of a calibrated Sepharose 4B column of 43.6 nm (C of V = 6.3 “/o) as estimated by the method of ACKERS[l]. Buoyant densities (table 11) were also similar and agreed with the value published by GERINet al. [ 5 ] . Finally, as table I1 shows, sedimentation coefficients and molecular weights, calculated therefrom, seemed to be significantly different (p < 0.01) for the two purified subtypes.
Discussion Physico-chemical methods for the purification of Australia antigen et al. [8] have recently have seldom given satisfactory results, as HOUWEN stressed. The present report outlines a simple and reproducible method for the separation of ‘ad’ and ‘ay’ HBsAg subtypes from serum proteins. As is clearly shown in table I, the recovery of the antigenic fraction is of the order of 50-60(’/0, the purification factor depending on the antigen titer of the starting serum. The purified antigens seem homogeneous and free of contaminants by disc electrophoresis (fig. 3) when amido-black is used as the stain. Trace contamination by serum proteins having the same molecular weight and net charge cannot, however, be excluded by this method. The only protein band observed also stains positively for lipoproteins using oil red, and it is found to be antigenically active against human anti-HBsAg. Immunoelectrophoresis (fig. 4b) did not show human serum proteins accompanying the purified antigens. Different mobilities for the two antigen subtypes are observed by immunoelectrophoresis when compared to normal serum proteins. The ‘ad’ antigen has a mobility between ,&lipoprotein and a2-macroglobulin while the ‘ay’ antigen migrates as an intermediate between a2macroglobulin and haptoglobin (fig. 4c). Analytical ultracentrifugation (fig. 5) and isopycnic banding ultracentrifugation (fig. 6) both indicate homogeneity of the purified antigens. The buoyant densities obtained are similar, within the limits of error, to those obtained by other groups [3, 5, 61 and are not significantly different for the two antigen subtypes.
218
BOURBONNAIS/GUEVIN/DELVIN
The two antigens furthermore have similar Stokes radii as measured by gel filtration (table 11) and are close to the value obtained by HOUWEN et al. [8] using electron microscopy. Sedimentation coefficients being different (p
Australia Antigen ‘ad’ and ‘ay’ Subtypes Purification and Partial Characterization
R. BOURBONNAIS, R. M. GUEVINand E. E. DELVIN Canadian Rcd Cross Blood Transfusion Service, Montreal, Que.
Abstracf. The two commonly-found Australia antigen subtypes have been purified by simple inexpensive, reproducible, physico-chemical methods involving ammonium sulfate precipitation, peptic digestion, calcium phosphate gel fractionation and molecular sieving on Sepharose 4B. The overall recovery of the two subtypes varied between 50 and 60°/0 of the total antigen content of the starting serum. The purified antigens were found homogeneous by discontinuous gel electrophoresis, analytical ultracentrifugation, and isopycnic banding ultracentrifugation. N o contaminating serum proteins could be detected by immunoelectrophoresis. The two purified antigen subtypes have similar Stokes radii and buoyant densities. The sedimentation coefficients for the ‘ad‘ and ‘ay’ subtypes were found t o be 33.0 and 40.1 x sec, respectively.
Introduction The purification and characterization of the two commonly-found ‘ad’ and ‘ay’ Australia antigens (HBsAg) are the prime requirements in the understanding of their nature, origin and infectivity. A variety of purification methods, including isopycnic banding in cesium chloride, sedimentation rate ultracentrifugation [2, 3, 5 , 61, and, more recently, affinity chromatography [7, 131 have been described. Preparative ultracentrifugation techniques are, however, limited to laboratories having liberal access to the expensive equipment required, while affinity chromatography can only be part of an elaborate purification procedure if it can be used on a large scale.
Received: November 21, 1974; accepted: January 17, 1975.
210
The present report proposes a relatively simple, inexpensive, and high yield method for the purification of the ‘ad’ and ‘ay’ Australia antigens, easily adaptable to large scale production. Physicochemical and immunological properties of the two purified subtypes are also compared.
Material and Methods Purification of Australia Antigen All steps of the purification were performcd at room temperature in burners or solutions containing 0.02 O/O sodium azide as bacteriostatic agent. 200 rnl of serum (fraction I) obtained by recalcification of HBsAg-positive plasina
[12] was fractionated with ammonium sulfate at p H 6.5. The fraction precipitating between 30- and 45-percent saturation was kept. The precipitate collected by centrifugation was then washed once with 200ml of ammonium sulfate at 45-percent saturation, dissolved in isotonic saline, and dialyzed overnight (fraction 11). Protein concentration was adjusted to 10 mg/ml by diluting the fraction with distilled water. Pepsin digestion (25 mg of pepsin ‘Pentex crystallized’ per gram of protein to be hydrolyzed) was carried out at pH 3.0 for 3 h at 37 OC. The p H was kept near 3.0 by periodic addition of 1N HCI. The proteolysis was stopped by raising the p H to 7.4 with 1N NaOH. The solution was immediately concentrated on Amicon membrane XM-100A, washed with 100ml of 0 . 0 5 ~sodium phosphate buffer pH7.4, brought to a final volume of approximately 20 ml, and dialyzed overnight against 2 liters of the same buffer (fraction 111). This fraction was further purified by negative absorption on calcium phosphate gel 191 (2 rng of gel per milligram of protein) equilibrated with 0.05 M phosphate buffer pH 7.4 at a protein concentration of 10 rng/ml. The slurry was stirred for 30 min. The gel was then removed by centrifugation at 3,000 g for 10min and washed 3 times with a volume of 0 . 0 5 ~ phosphate buffer pH7.4 equal to that of the first eluate. The eluted fractions were pooled and concentrated on Amicon membrane XM-100A to a final volume of approximately 5 rnl (fraction IV). Finally, the concentrate was chromatographed on a Sepharose 4B (Pharrnacia) column (78 x 2.5 cm) equilibrated in PBS (0.15 M NaCI, 0.025 M sodium phosphate, pH 7.4, 0.001 M Na,EDTA) with an upward flow of 17 ml/h. The HBsAg-positive fractions were pooled and concentrated by ultrafiltration (fraction V). Analytical Methods
The protein content of each fraction was estimated by the method of Warburg and Christian modified by LAYNE[ll] in a 1-cm path length spectrophotometric cell. The concentration of protein was calculated by the following equation: milligram of protein per rl;jlliliter = (1.55 x OD 280 nm) - (0.76 x OD 260 nm). The extinction co= 3.76) was not used since the proportion of Australia antigen was efficient (E:& constantly changing during the purification procedure. The amount of Australia antigen was measured by electrophoresis on agarose gel [lo] method. Guinea pig anti-HBsAg containing antibody according to LAURELL’S (0.1 ml) was mixed with 10 ml of warmed (45-50 “ C ) 0.85 O/O agarose in 0.05 M Trist~
Australia Antigen Subtypes Purification and Characterization
27 1
barbital buffer pH 8.6 and immediately poured on a (8 x 10 cm) glass plate. Each well, 4 mm in diameter, received l o p 1 of sample. Electrophoresis was performed at 30 mA per plate a t room temperature for 3 h in a 0.05 M Tris-barbital buffer p H 8.6 as the electrode buffer. After electrophoresis, the gels were washed with saline, followed by distilled water, blotted with filter paper and stained with amido-black. The height of the precipitation peak is proportional to the amount of the Australia antigen present. Tube discontinuous polyacrylamide gel electrophoresis was performed according to the method of ORNsrErN and DAVIS[14] in a refrigerated Buchler Polyanalyst cell. A 5-percent acrylamide i n a 0 . 3 8 ~Tris-HC1 buffer pH 8.9 and a 4-percent acrylamide in a 0.06 M Tris-HCI buffer pH 6.7 served as running and stacking gels, respectively. The electrophoresis was allowed to proceed at 3 m A per tube for 1h using 0.05 M Tris-glycine p H 8.3 as the electrode buffer. Immunoelectrophoresis was performed according to the microslide technique of SCHEIDECCER [16] adapted to the Gelman-LKB equipment without any modification. Sedimentation velocities were measured with a Spinco model E analytical ultracentrifuge equipped with the absorption system at 280 nm. Samples were diszolved in PBS to a n initial protein concentration ranging between 0.5 and 0.8 mg/mI. After the rotor reached 25,980 rpm at 20 “C, scannings were taken at 8-min intervals. Sedimentation coefficients were calculated by the method of SCHACHMAN [15] and corrected for viscosity and density of water at 20 OC. The relative viscosity of PBS ( ) / / T I , , = 1.04) was measured with a rotating cylinder viscosimeter and its density (pzo = 1.008 g/cm3) was measured by pycnometry. The isopycnic banding ultracentrifugation was carried out in a continuous cesium chloride density gradient ranging between 1.1 and 1.4g/ml. Aliquots of 0.5rnl in PBS were layered onto 4.7 ml of 26Oio (w/w) CsC1,. After centrifuging the samples in a Beckrnan SW 50.1 rotor at 47,000 rpm for 24 h at 25 OC, fractions of 10 drops were collected automatically, starting from the bottom of each tube. Each fraction was assayed for protein content and Australia antigen activity. The specific gravity of each fraction was measured by the refractive index in duplicate CsCI, gradients. Partial specific volumes (7) of the purified antigens were calculated from the buoyant densities. The Stokes radii (‘a’) of the purified ‘ad’ and ‘ay’ antigens were determined according to the method described by ACKERS[l] on a Sepharose 4B column (78 x 2.5 cm) equilibrated in PBS at room temperature. Thyroglobulin (Sigma), apoferritin (SchwarzMann), catalase (Sigma), aldolase and bovine serum albumin (Pharmacia) were used as standard proteins. The void volume (‘Vo’) was determined with blue dextran (Pharmacia) and the inner volume (‘Vi’), with tritiated water (NEN). All samples were applied to the bottom of the column and eluted upward at a flow rate of 17 ml/h. The column effluent was monitored by measuring the optical density a t 280nm. Distribution coefficients (‘Kd’) were calculated using the basic equation formulated by GELOTTE[4], taking as elution volume the inflection point of each peak. Published values of Stokes radii of standard proteins were substituted in the equation developed by ACKERS111, to calculate the mean gel pore radius (‘r’) of Sepharose 4B. Thc diffusion coefficients (DZ0,) of the purified Australia antigens were calculated by using the STOKES-EINSTEIN I181 equation and taking the experimentally-determined values for the Stokes radii.
272
B~LJRB~NNAIS/GUEVIN/DELVIN
The molecular weights of the Australia antigens were calculated by SVEDBERC and PEDERSON’S [17] equation, using the calculated diffusion and sedimentation coefficients and the partial specific volumes obtained from the buoyant densities.
Results Purification of Australia Antigen Data from typical Australia antigen purifications are summarized in table I. Specific and total activities, shown in the second and third columns for ‘ad’ and fifth and sixth for by’ subtypes, respectively, are measured at each step by the LAURELL [lo] method as shown by figure 1. The amount of HBsAg in each fraction is estimated by measuring the height of the peak. The specific activity is expressed in centimeters per milligram of total protein applied. A slight enhancement of the total antigenic activity after the peptic digestion was noticed in all purifications. This is probably due to the hydrolysis of coating proteins which mask antigenic deter-
Fig. I . LAURELL [lo] rocket method for determination of HBsAg at each main step of purification (as described in Material and Merhods). Left to right: ‘ay’ and ‘ad’ antigen fractions of incrcasing purity. Total protein content of each well is expressed as micrograms. ‘ay’ antigen fraction I: 587, fraction 11: 255, fraction 111: 52, fraction 1V: 15 and fraction V: 2.2; ‘ad’ antigen fraction I: 556, fraction IT: 220, fraction 111: 73, fraction 1V: 17.7 and fraction V: 2.0.
Australia Antigen Subtypes Purification and Characterization
213
n
I 1
,
0
50
IM
150
200
250
300
350
400
Elution volume, ml
Fig. 2. Chromatography of the ‘ad’ antigen fraction IV o n Sepharose 4B. The solid line represents the optical density at 280 nm and the dashed line represents the [lo] Australia antigen activity in millimeters of migration as measured by the LAURELL method.
F i g . 3. Polyacrylamide disc electrophoresis of the purified ‘ay’ and ‘ad’ Australia antigens stained with amido-black.
214
BOURBONNAIS/GUEVIN/DELVIN
Fig. 4 . lmmunoclcctrophoresis. CI Whole serum against rabbit anti-human serum (Behringwerke AG). h Purified ‘ad’ and ‘ay’ antigens against rabbit anti-human serum. c Purified ‘ad’ and ‘ay’ antigens against human anti-HBsAg.
minants on the surface of the Australia antigens. The calcium phosphate gel fractionation is a very important step to remove proteins and aggregates that interfere seriously with chromatographic resolution. Figure 2 shows a typical elution profile obtained by chromatographing the fraction IV of an ‘ad’ antigen on Sepharose 4B. A similar profile is obtained for ‘ay’ subtypes. The final recovery of the antigen is in the range of 50-600/0. The purification factor, however, varies inversely with the titer obtained in the starting material.
Criteria of Purity The two purified Australia antigen subtypes, ‘ad’ and ‘ay’, migrated as single components on polyacrylamide gel disc electrophoresis as depicted by figure 3. These unique protein bands also stained positively for lipo-
Australia Antigen Subtypes Purification and Characterization
275
Fig.5. Sedimentation pattern of purified ‘ay’ antigen. Scannings taken at 16-min intervals are shown. The direction of sedimentation is from left to right. Sedimentation pattern obtained for the purified ‘ad’ antigen is almost the same.
\ \
F r a c t i o n number
Fig. 6. Optical density pattern obtained from isopycnic banding ultracentrifugation in cesium chloride of purified ‘ad’ (dotted line) and ‘ay’ (solid line) Australia antigens. Dashed line represents the density gradient. Antigenic activity corresponds to the optical density peaks.
BOLJRBONNAIS/GUEVIN/DELVIN
276
Table I . Purification of Australia antigens
Purification steps
'ad' antigen
'ay' antigen
specific activity cm/mg
total activity cm
recovery "in
specific activity cm/mg
total activity cm
recovery Oio
0.72 2.96 12.3 56.5 550.0
9,150 8,970 10,000 6,500 4,630
100 98 110 71 51
1.53 5.1 28.8 105.5 590.0
15,650 14,100 15,300 10,300 8,900
100 90 98 66 57
serum (NH4),S0, 30-45 "in saturation Ill: peptic digestion IV: calcium phosphate gel V: sepharose 4B chromatography t: 11:
~
~
~~
~
_
_
_
Specific activity refers to the ratio of the distance from origin at which the peak of precipitation of Ag-Ab complexes are visualized to the amount of protein present in the well.
Table 11. Physical properties of Australia antigens
Physical properties
'ad' antigen means f SD
'ay' antigen means f SD
PI
Stokes radius (a), nm Diffusion coefficient (DZOw),cm' sec-1 Sedimentation coefficient (S,,,), 10-13 sec Buoyant density (&, g/ml Molecular weight (Mw), daltons
10.6 5 0.67 2.01 It: 0.13 33.0 k 2.3 1.19 2.5 x los k 0.17
11.0 f 0.69 1.94 f 0.12 40.1 It: 2.8 1.20 3.0 x 108 k 0.21
n.s. n.s. < 0.01 1
< 0.01
n s . = Not significant. Significance level for difference in means calculated with the Student t-test for paired variates (d.f. = N - 1 = 2).
protein and were active towards human anti-HBsAg. Immunoelectrophoresis (fig. 4b) indicated that there were no detectable immunological reactions between purified 'ad' and 'ay' antigens and an anti-human serum. However, immunological reaction was clearly seen when the purified subtypes were run in the presence of human anti-HBsAg (fig.4~).Analytical centrifugation (fig. 5 ) and isopycnic banding ultracentrifugation in CsCl, (fig. 6) revealed only one component, confirming the homogeneity of our preparations.
Australia Antigen Subtypes Purification and Characterization
27 I
Physical Properties of the ‘ad‘ and ‘ay’ Australia Antigens The Stokes radii (‘a’) as calculated by the method of ACKERS[l] and diffusion coefficients calculated by the STOKES-EINSTEIN [181 equation show no significant difference between the two Australia antigen subtypes as shown in table 11. These values were obtained from a mean gel pore radius of a calibrated Sepharose 4B column of 43.6 nm (C of V = 6.3 “/o) as estimated by the method of ACKERS[l]. Buoyant densities (table 11) were also similar and agreed with the value published by GERINet al. [ 5 ] . Finally, as table I1 shows, sedimentation coefficients and molecular weights, calculated therefrom, seemed to be significantly different (p < 0.01) for the two purified subtypes.
Discussion Physico-chemical methods for the purification of Australia antigen et al. [8] have recently have seldom given satisfactory results, as HOUWEN stressed. The present report outlines a simple and reproducible method for the separation of ‘ad’ and ‘ay’ HBsAg subtypes from serum proteins. As is clearly shown in table I, the recovery of the antigenic fraction is of the order of 50-60(’/0, the purification factor depending on the antigen titer of the starting serum. The purified antigens seem homogeneous and free of contaminants by disc electrophoresis (fig. 3) when amido-black is used as the stain. Trace contamination by serum proteins having the same molecular weight and net charge cannot, however, be excluded by this method. The only protein band observed also stains positively for lipoproteins using oil red, and it is found to be antigenically active against human anti-HBsAg. Immunoelectrophoresis (fig. 4b) did not show human serum proteins accompanying the purified antigens. Different mobilities for the two antigen subtypes are observed by immunoelectrophoresis when compared to normal serum proteins. The ‘ad’ antigen has a mobility between ,&lipoprotein and a2-macroglobulin while the ‘ay’ antigen migrates as an intermediate between a2macroglobulin and haptoglobin (fig. 4c). Analytical ultracentrifugation (fig. 5) and isopycnic banding ultracentrifugation (fig. 6) both indicate homogeneity of the purified antigens. The buoyant densities obtained are similar, within the limits of error, to those obtained by other groups [3, 5, 61 and are not significantly different for the two antigen subtypes.
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The two antigens furthermore have similar Stokes radii as measured by gel filtration (table 11) and are close to the value obtained by HOUWEN et al. [8] using electron microscopy. Sedimentation coefficients being different (p
