Journal of Immunological Methods, 20 (1978) 117--129 © Elsevier/North-Holland Biomedical Press
117
A NOVEL IMMUNOADSORBENT: USE FOR THE PREPARATION OF MONOSPECIFIC ANTIBODIES TO THE HEPATITIS B ANTIGEN
R.Y. DODD and J.A. KOBITA
American National Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014, U.S.A., and Department of Microbiology, George Washington University Medical Center, 2300 Eye Street, NW, Washington, DC 20037, U.S.A. (Received 9 August 1977, accepted 20 September 1977)
Controlled pore glass (CPG) adsorbs hepatitis B surface antigen (HBsAg) from whole plasma with a high degree of specificity. The resultant complex is stable at acid pH and in the presence of high concentrations of sodium thiocyanate. The adsorbed HBsAg is qualitatively and quantitatively similar to the soluble material in its ability to bind antibodies to HBsAg (anti-HBs). The HBsAg in 1 ml of strongly reactive plasma is adsorbed by 100 mg of CPG, which can then specifically bind 32,000 passive hemagglutination units of anti-HBs. Bound antibody can be eluted in 77% yield by acid or by chaotropic ions and the CPG-HBsAg complex can be reused in further adsorption-elution cycles. Antibody to HBsAg can be purified 144-fold in a single step by using this technique. The preparation of monospecific subtyping reagents for HBsAg and of immunochemically purified anti-HBs is described.
INTRODUCTION
Hepatitis B surface antigen (HBsAg) is a virally specified antigen associated with hepatitis B. It is an effective, b u t not exclusive, marker for infectivity in donor blood (Gocke, 1972). The epidemiology of hepatitis B has been greatly clarified by the discovery of the antigenic diversity of HBsAg. In brief, at least 7 antigenic subdeterminants have been described and 10 discrete combinations of these determinants were recognized at a recent conference (Courouc~ et al., 1976). Each sample of HBsAg is thought to express the c o m m o n , or a determinant, plus one determinant from each of the pairs d, y, and w, r, although there are exceptions (Mazzur et al., 1975; Courouc~ et al., 1976). The individual determinants of HBsAg are generally identified by the analStatement on nomenclature: Abbreviations for hepatitis B surface antigen and the corresponding antibody follow the recommendations of the World Health Organization Expert Committee on Viral Hepatitis (WHO Technical Report Series, 602). HBsAg = hepatitis B surface antigen; anti-HBs = antibody to hepatitis B surface antigen; HBsAg/ad = HBsAg known to carry the a and d determinants, etc.; anti-HBs/ad = anti-HBs reacting with the a and d determinants, etc.
118 ysis of spurring patterns in agar gel diffusion, since most natural or experimental preparations of antibody to HBsAg (anti-HBs) react with a (Holland, 1975). Other methods require the use of monospecific antisera which must be prepared by cross absorption. This is generally done in liquid phase; a method which results in dilution, and the presence of immune complexes in the absorbed reagent. Such preparations may also be unstable (Shorey, personal communication). Therefore, it is desirable to develop a simple method for insolubilizing HBsAg so that monospecific anti-HBs may be prepared by solid phase immunoadsorption techniques. HBsAg binds strongly and stably to silica (Siebke et al., 1972) or to glass (Pert and Verch, 1975), offering potential for the preparation of a simple, HBsAg-specific immunoadsorbent (Traavik, 1975). We have investigated the conditions for the preparation of such an immunoadsorbent using controlled pore glass (CPG) and unpurified HBsAg. The immunoadsorbent has been used for the preparation of antiHBs/d and anti-HBs/y by cross-adsorption, and anti-HBs/a by elution. Careful choice of elution conditions results in anti-HBs preparations with a high degree of immunochemical purity. MATERIALS AND METHODS
Immunologic reagents Human plasma containing HBsAg was obtained from voluntary blood donations, previously found positive by counterelectrophoresis (CEP; Hepascreen®, Spectra Biologicals, Oxnard, CA). Experiments which did not relate to subtype specificity were performed using a recalcified (Moghaddam, 1971) pool of several hundred such plasma samples; previously subtyped plasma samples from single donors were used for the preparation of subtype specific immunoadsorbents. Human anti-HBs was derived from a recalcified pool of anti-HBs positive plasma from volunteer blood donors, titer 1 : 16,000 by the passive hemagglutination technique of Vyas and Shulman (1970). Subtype reactive antisera were guinea pig anti-HBs/ad (V-801-502058) and anti-HBs/ay (V-802-501-558), generously provided by the Research Resources Branch, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD. Reference antigens were kindly donated by Dr. Scott Mazzur. Rabbit or goat antisera to human serum proteins were obtained from Behring Diagnostics, Somerville, NJ.
Preparation of immunoadsorbent Controlled pore glass (Bioglas® 1500) was obtained from BioRad Laboratories, Richmond, CA. A known weight of CPG was suspended and degassed in a known volume of 1% (W/V) aqueous polyethylene glycol, approximate molecular weight 4000 daltons. Aliquots of the suspension containing known weights of CPG were transferred to 3, 10 or 50 ml stoppered
119 glass vials for further steps. The CPG was extensively washed in adsorption buffer (0.1 M glycine--HC1, pH 3.0). HBsAg-positive or-negative (control) plasma was dialysed overnight against adsorption buffer at 0--4°C. The retained material was diluted to twice its starting volume with adsorption buffer and mixed with the washed CPG for 5 h at room temperature. The equivalent of 100 mg (dry weight) of CPG was used to adsorb each 1 ml of undiluted plasma. After adsorption, the CPG-HBsAg complex (hereafter referred to as CPG-HBsAg) was washed by mixing with 10 ml of distilled water per 100 mg of CPG for 10 min, allowed to settle and decanted. Three further wash cycles were carried out, with phosphate buffered saline (PBS; NaC1, 0.073 M, KH:PO,, 0.018 M, Na~HPO4, 0.057 M, pH 7.2). Control immunoadsorbents (CPG-NHP) were prepared in the same fashion from HBsAg-negative, normal human plasma. The following S~rensen buffers were used to study the effect of pH upon the adsorption of HBsAg to CPG: 0.1 M glycine--HC1, pH 2, pH 3; 0.1 M sodium citrate--HC1, pH 4, pH 5; 0.067 M phosphate, pH 6, pH 7, pH 8.
Adsorption of antibody onto CPG-HBsAg Batch procedures were used to adsorb anti-HBs onto CPG-HBsAg at pH 7.2. Each 100 mg of CPG-HBsAg previously equilibrated with PBS, was mixed with 0.5 ml of antiserum and 2.0 ml of PBS for 2½ h at room temperature. After adsorption of anti-HBs, the CPG-HBsAg was allowed to sediment and the supernatant was recovered. The CPG-HBsAg was then exhaustively washed with PBS. Procedures used to elute bound anti-HBs from the washed CPG-HBsAg are described below.
Test procedures A commercial solid phase radioimmunoassay (RIA) technique (AusRIAII ®, Abbott Laboratories, North Chicago, IL) was used to detect HBsAg. A relative measure of the HBsAg content of test fractions could be obtained by comparison of the linear portions of dilution curves in AusRIA II (Crovari and De Flora, 1975). Counterelectrophoresis was performed with commercially available plates and equipment (Hepascreen®). Passive hemagglutination (PHA) was used to assay anti-HBs according to the method of Vyas and Shulman (1970). Human erythrocytes coated with HBsAg/adw or HBsAg/ayw were obtained from Electronucleonics Laboratories, Inc., Bethesda, MD. Agar gel diffusion was performed according to Mazzur (1972). Immunoelectrophoresis was performed on 25 × 75 mm glass slides, cleaned in ethanol and precoated by immersion in boiling 0.1% agar, followed by air-drying; 2.7 ml of 1% agarose in barbital buffer, pH 8.4 (barbital, 0.003M, sodium barbital, 0.017M, calcium lactate, 0.017M) was
120 applied to each slide. Slides were run at a constant 200 V for 105 min, average current 2 mA per slide; the bridge buffer was barbital, 0.0075 M, sodium barbital 0.043 M, calcium lactate, 0.0012 M, pH 8.6. Slides were photographed after 18 to 24 h development. They were then washed, dried and stained with amido black. Protein assays were performed essentially according to Lowry et al. (1951), with a bovine serum albumin standard. RESULTS
Effect of pH upon the adsorption of HBsAg to CPG Preliminary experiments suggested that, for a given HBsAg-CPG ratio, pH was the most important variable in the adsorption of HBsAg to CPG. Since HBsAg dissociates from glass at high pH (Pert and Verch, 1975), we examined adsorption in neutral and acidic conditions only. The antigen adsorption stage was performed as described above, using the pH 2, 3, 4, 5, 6, 7, and 8 buffers. Samples of the dialysed HBsAg preparations were taken before adsorption. In each case, this initial sample and the supernatants and wash solutions from the adsorption stage, were retained, dialysed against PBS and titrated by AusRIA II. The percentage difference between the total antigen in the supernatants and washes and that in the initial sample was considered to be the proportion of antigen adsorbed onto the CPG. The extent of adsorption of antigen at each pH value is shown in fig. 1. The results indicate that the maximum adsorption of HBsAg takes place at pH 3. The resultant CPG-HBsAg preparations were used to adsorb anti-HBs at pH 7.2. 0.5 ml of anti-HBs, diluted to 2.5 ml in PBS, was added to each 100 mg of CPG-HBsAg and mixed for 2½ h at room temperature. The supernatant was recovered and the CPG-HBsAg was exhaustively washed with PBS. The anti-HBs content of each supernatant and wash was determined; the difference between the a m o u n t (volume × titer) applied and the amount recovered was taken as the a m o u n t adsorbed. The percentage of applied antiHBs adsorbed b y each CPG-HBsAg preparation is shown in fig. 1. In general, the extent of adsorption of antibody parallels the amount of HBsAg b o u n d in each preparation. CPG-HBsAg prepared at pH 3.0 adsorbs the maximum a m o u n t of antibody.
Selectivity of CPG for HBsAg Immunoelectrophoretic experiments Were done in order to see whether CPG adsorbed major serum components in addition to HBsAg. HBsAgpositive plasma was dialysed against pH 3.0 glycine--HC1 buffer and adsorbed onto CPG as described above. Samples of the HBsAg-positive plasma before and after the dialysis and after adsorption with CPG were dialysed against PBS overnight, then applied to t h e wells of immunoelectrophoresis
121 100
0
95
o 9o
85 2
3
4 5 6 pH of A d s o r p t i o n B u f f e r
7
8
Fig. 1. Effect of pH on adsorption of HBsAg to CPG. HBsAg was adsorbed onto CPG at each of the stated pH values and the proportion of retained antigen was determined. Each preparation was washed with PBS and exposed to a standard amount of anti-HBs at pH 7.2; the retention of anti-HBs was also determined. • = percentage of applied HBsAg adsorbed at the stated pH; [] = percentage of applied anti-HBs adsorbed by CPG-HBsAg prepared at the stated pH.
slides. After electrophoresis, the troughs were filled with antibody to h u m a n serum. The resulting patterns are shown in fig. 2. It may be seen that, after dialysis at pH 3, a number of serum components were no longer detectable. There was very little further change as a result of adsorption onto CPG.
Stability of the immunoadsorbent CPG-HBsAg was exposed for 1 h to low pH and to high molarities of sodium thiocyanate; conditions which are used to elute specifically adsorbed antibody. Supernatants were collected, dialysed against PBS and tested by RIA. No HBsAg was f o u n d in the supernatants from CPG-HBsAg exposed to pH values between 2 and 8, to 4 M sodium thiocyanate at neutrality or to 2 M sodium thiocyanate at pH 3.5 or 2.0.
Adsorption of anti-HBs by CPG-HBsAg When human anti-HBs was adsorbed onto CPG-HBsAg as described earlier, 100 mg of the immunoadsorbent removed 99% of the detectable anti-HBs from 2.5 ml of an anti-HBs preparation with a PHA titer of 1 : 1600; that is, about 4000 PHA units (titer × volume). By repeating the adsorption several times we established that 100 mg of CPG-HBsAg would adsorb a m a x i m u m
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Fig. 2. Effect of HBsAg adsorption procedure upon normal plasma constituents. HBsAg positive plasma was dialysed overnight against glycine-HC1 buffer, pH 3.0. One aliquot was adsorbed with CPG for 5 h at room temperature; a control aliquot was exposed to the same conditions in the absence of CPG. All plasma samples were dialyzed against PBS, pH 7.2 before immunoelectrophoresis. Troughs: anti-whole serum; wells: 1, HBsAgpositive plasma; 2, HBsAg-positive plasma exposed to pH 3 for 5 h in the absence of CPG; 3, HBsAg-positive plasma after adsorption onto CPG.
o f a b o u t 3 0 , 0 0 0 P H A units of anti-HBs. This a d s o r p t i o n a p p e a r e d to be specific, as c o n t r o l i m m u n o a d s o r b e n t s p r e p a r e d f r o m H B s A g - n e g a t i v e p l a s m a did n o t a d s o r b anti-HBs significantly (see tables 1 a n d 2).
Re-use o f CPG-HBsAg We f o u n d t h a t 50 t o 1 0 0 % o f t h e a d s o r b e d h u m a n anti-HBs activity c o u l d b e r e c o v e r e d f r o m C P G - H B s A g b y washing w i t h 0.1 M glycine--HC1 b u f f e r , p H 3. A f t e r this t r e a t m e n t a n d r e t u r n to PBS, t h e C P G - H B s A g w o u l d a d s o r b a n t i b o d y to the s a m e e x t e n t as t h e freshly p r e p a r e d i m m u n o a d s o r b e n t . We o b s e r v e d n o change in a d s o r p t i o n or r e c o v e r y o f anti-HBs in t h r e e such cycles o f a single p r e p a r a t i o n o f CPG-HBsAg.
Elu tion of specific anti-HBs O n e g r a m o f C P G - H B s A g was used to a d s o r b m a x i m a l a m o u n t s (15 ml) o f h u m a n anti-HBs, as d e s c r i b e d above. C o n t r o l s w e r e p r e p a r e d in parallel; C P G - H B s A g was used to a d s o r b n o r m a l h u m a n p l a s m a ( N H P ) a n d a CPGN H P c o n j u g a t e was used t o a d s o r b anti-HBs. A f t e r t h e a d s o r p t i o n stage, each i m m u n o a d s o r b e n t was e x h a u s t i v e l y w a s h e d w i t h PBS. T h e t o t a l a m o u n t o f u n a d s o r b e d anti-HBs was d e t e r m i n e d in o r d e r to e s t i m a t e the q u a n t i t y a d s o r b e d b y t h e C P G - H B s A g or C P G - N H P . In o r d e r t o e l u t e a d s o r b e d a n t i b o d y , t h e w a s h e d i m m u n o a d s o r b e n t s were
* P e r c e n t a g e o f t o t a l applied. ** N.D. = n o t d o n e . *** -- Negative w h e n t e s t e d u n d i l u t e d .
Fraction Anti-HBs, s t a r t i n g m a t e r i a l (15 m l ) A d s o r p t i o n stage Anti-HBs, s u p e r n a t a n t PBS washes E l u t i o n stages pH 5, p h o s p h a t e , 4 0 m i n , e l u a t e pH 4, citrate--HCl, 10 rain, e l u a t e p H 3.5, citrate--HC1, 70 rain, e l u a t e pH 3.0, citrate--HC1, 30 m i n , e l u a t e Water, 10 m i n , final w a s h Total eluted
E l u t i o n o f anti-HBs f r o m CPG-HBsAg.
TABLE 1
% *
0.94 0.2 1.79 0.91 1.34 5.18
0.1 0.02 0.18 0.09 0.14 0.53
5840 8000 31680 3720 2406 51646
49984 3203
605 59
62 6
120000
975
4.6 6.7 26.4 3.1 2.0 43.0
41.7 2.7
0.89 0.18 1.84 0.97 1.31 5.19
674 64
975
units
mg
% *
Protein (mg)
Titer
Protein
--
79600 9253
120000
Titer (units)
C P G - N H P + anti-HBs
CPG-HBsAg + anti-HBs
Complex
***
0.76 0.21 1.85 0.92 1.31 5.05
702 66
N.D. **
CPG-HBsAg + NHP Protein (mg)
50
124 TABLE 2 Preparation of monospecific anti-HBs: passive hemagglutination of adsorbed antisera as expressed upon erythrocytes coated with HBsAg/adw or HBsAg/ayw. Antibody *
anti anti anti anti anti anti anti anti
Adsorbent
HBs/ad HBs/ay
---
HBs/ad HBs/ay
CPG-NHP CPG-NHP
HBs/ad HBs/ay
CPG-HBsAg/ay CPG-HBsAg/ad
HBs/ad
HBsAg/ay *** HbsAg/ad ***
HBs/ay
PHA units ** adw
ayw
64000 5000 16322 5138 7232 74 8000 5
4000 16000 5148 16000 64 3756 10 4000
* See text. ** Titer X volume. *** Liquid phase.
e x p o s e d t o a stepwise series o f S~brensen buffers o f decreasing pH, as s h o w n in table 1. B a t c h p r o c e d u r e s were used and each 1 g o f i m m u n o a d s o r b e n t was e l u t e d with 10 ml o f b u f f e r f o r 10 min at r o o m t e m p e r a t u r e . This was f o l l o w e d b y a second, 30 min elution step with t h e same b u f f e r ; this was o m i t t e d at pH 4. A s e c o n d 30 min e l u t i o n step was a d d e d at pH 3.5. Each eluate was dialysed o v e r n i g h t against PBS and its a n t i b o d y c o n t e n t and protein c o n t e n t were m e a s u r e d . Table 1 gives the results o f a typical experim e n t ; o t h e r e x p e r i m e n t s gave essentially similar results. It can be seen f r o m table 1 t h a t the stepwise acid e l u t i o n p r o c e d u r e resulted in t h e r e c o v e r y o f 43% o f the t o t a l starting a n t i b o d y in 0.53% o f the p r o t e i n . H o w e v e r , since o n l y 56% o f the t o t a l a n t i b o d y was a d s o r b e d , acid e l u t i o n r e c o v e r e d 77% o f t h e b o u n d a n t i b o d y . T h e m a j o r part (61%) was e l u t e d at p H 3.5. This f r a c t i o n c o n t a i n e d 26% o f t h e t o t a l starting a n t i b o d y in 0.18% o f the p r o t e i n , a 1 4 4 - f o l d single step p u r i f i c a t i o n . Each e l u a n t released a discrete q u a n t i t y o f a n t i b o d y which was n o t increased b y f u r t h e r e x p o s u r e t o t h a t eluant. Assuming t h a t this is due t o the variation in affinity o f b o u n d a n t i b o d y , these results indicate t h a t a one-step e l u t i o n at pH 3.5 w o u l d result in t h e r e c o v e r y o f a b o u t 75% o f a d s o r b e d a n t i b o d y , with a lesser degree o f p u r i f i c a t i o n . We find t h a t this is i n d e e d t h e case w h e n such an e l u t i o n p r o c e d u r e is used. T a b l e 1 also shows t h a t similar a m o u n t s o f protein, b u t n o a n t i b o d y , were e l u t e d f r o m c o n t r o l i m m u n o a d s o r b e n t s . P o r t i o n s o f t h e eluates were c o n c e n t r a t e d f i f t y - f o l d b y u l t r a f i l t r a t i o n (Minicon ®, A m i c o n C o r p o r a t i o n , L e x i n g t o n , MA) and subjected to i m m u n o electrophoresis. Fig. 3 shows i m m u n o e l e c t r o p h o r e t o g r a m s o f the pH 3.5 eluate and c o r r e s p o n d i n g c o n t r o l f r o m o n e e x p e r i m e n t . It s h o u l d be n o t e d
125
Fig. 3. Elution of human anti-HBs from CPG-HBsAg at pH 3.5. CPG-HBsAg was exposed to excess anti-HBs, and washed thoroughly with PBS. Anti-HBs was eluted with S~brensen buffers of pH 5, 4 and 3.5. The pH 3.5 eluate was concentrated and compared with unadsorbed anti-HBs in immunoelectrophoresis. A control eluate was prepared in the same conditions, but using CPG-NHP instead of CPG-HBsAg. Troughs: 1, anti-whole serum; 2, anti-IgG; 3, HBsAg-positive plasma. Wells: 4, human anti-HBs; 5, concentrated pH 3.5 eluate from CPG-HBsAg; 6, concentrated pH 3.5 eluate from CPG-NHP. t h a t t h e c o n c e n t r a t i o n o f a n t i b o d y in t h e s t a r t i n g s e r u m w a s i n s u f f i c i e n t t o precipitate with HBsAg, whereas the concentrated eluate gave a sharp, clear a r c in t h e 7 r e g i o n . C h a o t r o p i c i o n s w e r e a l s o e f f e c t i v e in e l u t i n g b o u n d a n t i - H B s . A p r e l i m i -
126
Fig. 4. Preparation of monospecific anti-HBs. Guinea pig anti-HBs/ad was adsorbed with HBsAg/ay in liquid phase and with CPG-HBsAg]ay; similarly, anti-HBs/ay was adsorbed with HBsAg]ad in liquid phase and with CPG-HBsAg/ad. Adsorbed antibody was eluted from the CPG preparations with 4 M NaSCN. Adsorbed antibodies or eluates were concentrated and tested against standard antigens in immunodiffusion. The unadsorbed anti-HBs preparations were also tested against the same antigens. 1, adsorbing y antigen; 2, adsorbing d antigen ; 3, ad w ; 4, adr; 5, ay w; A, anti-d, solid phase; B, anti-y, solid phase; C, anti-d, liquid phase; D, anti-y, liquid phase; E, unadsorbed anti-ad; F, unadsorbed anti-ay; G, anti-a derived from anti-ad by elution; H, anti-a derived from anti-ay by elution.
127 nary experiment showed that a combination of lowered pH and moderate concentrations of sodium thiocyanate (pH 3.5 citrate--HC1, 1 M NaSCN) when used in conjunction with a selected sequence of wash steps, released anti-HBs of high purity. The product gave a single IgG precipitin arc in immunoelectrophoresis and a single band in polyacrylamide gel electrophoresis. Lower pH values or high molarities of NaSCN were necessary to elute animal anti-HBs, presumably because of the high affinities of antibodies produced by intensive immunization schedules.
Preparation of monospecific subtyping reagents S u b t y p e specific CPG-HBsAg was prepared using plasma samples which had been previously t y p e d for d or y determinants. S u b t y p e reactive guinea pig antisera were cross-adsorbed with the heterologous CPG-HBsAg preparation, using exactly the conditions described above for human anti-HBs. Comparable absorptions were also carried o u t in liquid phase, using the same antigen-containing plasma samples, which were mixed with the heterologous antibody preparations. Proportions were selected to correspond with the antigen--antibody ratio in the solid phase adsorption. After adsorption, each preparation was concentrated by ultrafiltration (Minicon®). The solid phase reagents were concentrated to the original volume of antibody. Because of their protein content, it was impossible to concentrate the liquid phase reagents to the same extent; they were reduced to two to three times the starting volume of antibody. The adsorbed reagents were tested in PHA against erythrocytes coated with HBsAg/adw and HBsAg/ayw (table 2). The content of subtypespecific antibody was remarkably similar in the reagents produced b y solidor liquid-phase adsorption, although the anti-HBs/a content of the solid phase reagents was somewhat higher than that of the liquid phase preparations. It should be noted that CPG-NHP does not adsorb anti-HBs significantly. Adsorbed antibody was eluted from the CPG-HBsAg with 4 M NaSCN. All adsorbed and eluted reagents were evaluated against standard antigens in agar gel diffusion (fig. 4). It may be seen that the solid phase adsorption procedure resulted in a highly specific preparation of anti-HBs/d or anti-HBs/y. Liquid phase absorption resulted in a weaker, but equally specific antiHBs/d, b u t the putative anti-HBs/y did n o t give any precipitin lines. The eluted antibody reacted in a manner consistent with anti-HBs/a. Finally, the difficulty of interpreting spurring patterns obtained with unadsorbed antisera is clearly demonstrated in fig. 4. DISCUSSION Direct adsorption to a surface is an established method of preparing insolubilized antigens or enzymes. A particular advantage of this approach is
128 that the adsorption of solute is itself a purification step. However, it is accepted that affinity media prepared in this fashion may not be sufficiently stable for effective use (Zaborsky, 1973). We have shown that HBsAg is adsorbed to controlled pore glass and the antigen remains b o u n d in conditions suitable for adsorption and elution of antibody. Controlled pore glass does n o t bind HBsAg exclusively; we have noted that CPG-HBsAg also adsorbs an interfering antilipoprotein specificity from an experimental anti-HBs prepared in goats (Nath, personal communication). Nevertheless, the adsorption is sufficiently selective to allow us to prepare a satisfactory HBsAg-specific immunoadsorbent from whole plasma. Estimates of the a m o u n t of HBsAg present in carrier plasma samples (Kim and Tilles, 1973) suggest that 100 mg of CPG adsorb a b o u t 50 pg of HBsAg. This method of insolubilizing HBsAg does not significantly alter its quantitative, or qualitative, binding capacity for anti-HBs. We have shown that the CPGHBsAg immunoadsorbent may be used for the preparation of monospecific anti-d and anti-y, and that b o u n d antibody can be eluted in good yield and with a reasonable degree of immunochemical purity. The products of the solid phase immunoadsorption technique were superior to reagents prepared in liquid phase, when tested in AGD and CEP. In particular, we were unable to concentrate liquid phase anti-y sufficiently to react in gel precipitation tests, because relatively large amounts of whole, HBsAg/ad positive plasma were used for adsorption. When tested in PHA, the reagents produced by solid phase adsorption were a little less well absorbed than the liquid phase reagents, as shown by their titers against the heterologous antigen (table 2). Adjustment of the antibody to CPG-HBsAg ratio would presumably reduce this effect. Recovery of anti-a adsorbed to CPG-HBsAg suggests that appropriate combinations of adsorption and elution could be used to isolate monospecific antibodies to any of the subdeterminants of HBsAg. Human anti-HBs adsorbed to CPG-HBsAg at pH 7.2 was eluted by lowering the pH. Stepwise reduction of the pH resulted in the elution of discrete fractions of the total b o u n d antibody, indicating that our anti-HBs was heterogeneous with respect to affinity. Appropriate elution conditions could be used to select for antibodies of a given affinity, for maximum yields or for the highest degree of purification. Such monospecific anti-HBs reagents should be of considerable use in studies of the nature of the antigenic determinants of HBsAg and in the routine subtyping of HBsAg specimens. Similarly, immunochemically purified anti-HBs is required for a number of test procedures such as radioimmunoassay and reversed passive hemagglutination, and is a valuable reagent for studies of the antigenic nature of HBsAg. ACKNOWLEDGEMENTS We thank Dr. N. Nath and his staff for AusRIA assays. This work was performed during studies leading to the degree of Ph.D. in the Department
129
of Microbiology of the George Washington University. Publication number 371 from the American National Red Cross. REFERENCES Couroucd, A.M., P.V. Holland, J.Y. Muller and J.P. Soulier (eds.), 1976, HBs Antigen Subtypes, Bibl. Haematol. 42 (Karger, Basel). Crovari, P. and S. De Flora, 1975, Boll. Ist. Sieroter. Milan. 54, 149. Gocke, D.J., 1972, J. Am. Med. Ass. 219, 1165. Holland, P.V., 1975, Am. J. Med. Sci. 270, 161. Kim, C.Y. and J.G. Tilles, 1973, J. Clin. Invest. 52, 1176. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Mazzur, S., 1972, Am. J. Med. Technol. 38,343. Mazzur, S., S. Burgert and G. Le Bouvier, 1975, J. Immunol. 114, 1510. Moghaddam, M., K.L.G. Goldsmith and R.A. Kekwick, 1971, Vox Sang. 20,277. Pert, J.H. and R.L. Vetch, 1975, Vox Sang. 29, 157. Siebke, J.C., E. Kjeldsberg and T. Traavik, 1972, Acta Pathol. Microbiol. Scand. B 80, 935. Traavik, T., 1975, Acta Pathol. Microbiol. Scand. B 8 3 , 1 5 3 . Vyas, G.N. and N.R. Shulman, 1970, Science 170, 332. Zaborsky, O.R., 1973, in: Immobilized Enzymes (C.R.C. Press, Cleveland) p. 75.
117
A NOVEL IMMUNOADSORBENT: USE FOR THE PREPARATION OF MONOSPECIFIC ANTIBODIES TO THE HEPATITIS B ANTIGEN
R.Y. DODD and J.A. KOBITA
American National Red Cross, Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014, U.S.A., and Department of Microbiology, George Washington University Medical Center, 2300 Eye Street, NW, Washington, DC 20037, U.S.A. (Received 9 August 1977, accepted 20 September 1977)
Controlled pore glass (CPG) adsorbs hepatitis B surface antigen (HBsAg) from whole plasma with a high degree of specificity. The resultant complex is stable at acid pH and in the presence of high concentrations of sodium thiocyanate. The adsorbed HBsAg is qualitatively and quantitatively similar to the soluble material in its ability to bind antibodies to HBsAg (anti-HBs). The HBsAg in 1 ml of strongly reactive plasma is adsorbed by 100 mg of CPG, which can then specifically bind 32,000 passive hemagglutination units of anti-HBs. Bound antibody can be eluted in 77% yield by acid or by chaotropic ions and the CPG-HBsAg complex can be reused in further adsorption-elution cycles. Antibody to HBsAg can be purified 144-fold in a single step by using this technique. The preparation of monospecific subtyping reagents for HBsAg and of immunochemically purified anti-HBs is described.
INTRODUCTION
Hepatitis B surface antigen (HBsAg) is a virally specified antigen associated with hepatitis B. It is an effective, b u t not exclusive, marker for infectivity in donor blood (Gocke, 1972). The epidemiology of hepatitis B has been greatly clarified by the discovery of the antigenic diversity of HBsAg. In brief, at least 7 antigenic subdeterminants have been described and 10 discrete combinations of these determinants were recognized at a recent conference (Courouc~ et al., 1976). Each sample of HBsAg is thought to express the c o m m o n , or a determinant, plus one determinant from each of the pairs d, y, and w, r, although there are exceptions (Mazzur et al., 1975; Courouc~ et al., 1976). The individual determinants of HBsAg are generally identified by the analStatement on nomenclature: Abbreviations for hepatitis B surface antigen and the corresponding antibody follow the recommendations of the World Health Organization Expert Committee on Viral Hepatitis (WHO Technical Report Series, 602). HBsAg = hepatitis B surface antigen; anti-HBs = antibody to hepatitis B surface antigen; HBsAg/ad = HBsAg known to carry the a and d determinants, etc.; anti-HBs/ad = anti-HBs reacting with the a and d determinants, etc.
118 ysis of spurring patterns in agar gel diffusion, since most natural or experimental preparations of antibody to HBsAg (anti-HBs) react with a (Holland, 1975). Other methods require the use of monospecific antisera which must be prepared by cross absorption. This is generally done in liquid phase; a method which results in dilution, and the presence of immune complexes in the absorbed reagent. Such preparations may also be unstable (Shorey, personal communication). Therefore, it is desirable to develop a simple method for insolubilizing HBsAg so that monospecific anti-HBs may be prepared by solid phase immunoadsorption techniques. HBsAg binds strongly and stably to silica (Siebke et al., 1972) or to glass (Pert and Verch, 1975), offering potential for the preparation of a simple, HBsAg-specific immunoadsorbent (Traavik, 1975). We have investigated the conditions for the preparation of such an immunoadsorbent using controlled pore glass (CPG) and unpurified HBsAg. The immunoadsorbent has been used for the preparation of antiHBs/d and anti-HBs/y by cross-adsorption, and anti-HBs/a by elution. Careful choice of elution conditions results in anti-HBs preparations with a high degree of immunochemical purity. MATERIALS AND METHODS
Immunologic reagents Human plasma containing HBsAg was obtained from voluntary blood donations, previously found positive by counterelectrophoresis (CEP; Hepascreen®, Spectra Biologicals, Oxnard, CA). Experiments which did not relate to subtype specificity were performed using a recalcified (Moghaddam, 1971) pool of several hundred such plasma samples; previously subtyped plasma samples from single donors were used for the preparation of subtype specific immunoadsorbents. Human anti-HBs was derived from a recalcified pool of anti-HBs positive plasma from volunteer blood donors, titer 1 : 16,000 by the passive hemagglutination technique of Vyas and Shulman (1970). Subtype reactive antisera were guinea pig anti-HBs/ad (V-801-502058) and anti-HBs/ay (V-802-501-558), generously provided by the Research Resources Branch, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD. Reference antigens were kindly donated by Dr. Scott Mazzur. Rabbit or goat antisera to human serum proteins were obtained from Behring Diagnostics, Somerville, NJ.
Preparation of immunoadsorbent Controlled pore glass (Bioglas® 1500) was obtained from BioRad Laboratories, Richmond, CA. A known weight of CPG was suspended and degassed in a known volume of 1% (W/V) aqueous polyethylene glycol, approximate molecular weight 4000 daltons. Aliquots of the suspension containing known weights of CPG were transferred to 3, 10 or 50 ml stoppered
119 glass vials for further steps. The CPG was extensively washed in adsorption buffer (0.1 M glycine--HC1, pH 3.0). HBsAg-positive or-negative (control) plasma was dialysed overnight against adsorption buffer at 0--4°C. The retained material was diluted to twice its starting volume with adsorption buffer and mixed with the washed CPG for 5 h at room temperature. The equivalent of 100 mg (dry weight) of CPG was used to adsorb each 1 ml of undiluted plasma. After adsorption, the CPG-HBsAg complex (hereafter referred to as CPG-HBsAg) was washed by mixing with 10 ml of distilled water per 100 mg of CPG for 10 min, allowed to settle and decanted. Three further wash cycles were carried out, with phosphate buffered saline (PBS; NaC1, 0.073 M, KH:PO,, 0.018 M, Na~HPO4, 0.057 M, pH 7.2). Control immunoadsorbents (CPG-NHP) were prepared in the same fashion from HBsAg-negative, normal human plasma. The following S~rensen buffers were used to study the effect of pH upon the adsorption of HBsAg to CPG: 0.1 M glycine--HC1, pH 2, pH 3; 0.1 M sodium citrate--HC1, pH 4, pH 5; 0.067 M phosphate, pH 6, pH 7, pH 8.
Adsorption of antibody onto CPG-HBsAg Batch procedures were used to adsorb anti-HBs onto CPG-HBsAg at pH 7.2. Each 100 mg of CPG-HBsAg previously equilibrated with PBS, was mixed with 0.5 ml of antiserum and 2.0 ml of PBS for 2½ h at room temperature. After adsorption of anti-HBs, the CPG-HBsAg was allowed to sediment and the supernatant was recovered. The CPG-HBsAg was then exhaustively washed with PBS. Procedures used to elute bound anti-HBs from the washed CPG-HBsAg are described below.
Test procedures A commercial solid phase radioimmunoassay (RIA) technique (AusRIAII ®, Abbott Laboratories, North Chicago, IL) was used to detect HBsAg. A relative measure of the HBsAg content of test fractions could be obtained by comparison of the linear portions of dilution curves in AusRIA II (Crovari and De Flora, 1975). Counterelectrophoresis was performed with commercially available plates and equipment (Hepascreen®). Passive hemagglutination (PHA) was used to assay anti-HBs according to the method of Vyas and Shulman (1970). Human erythrocytes coated with HBsAg/adw or HBsAg/ayw were obtained from Electronucleonics Laboratories, Inc., Bethesda, MD. Agar gel diffusion was performed according to Mazzur (1972). Immunoelectrophoresis was performed on 25 × 75 mm glass slides, cleaned in ethanol and precoated by immersion in boiling 0.1% agar, followed by air-drying; 2.7 ml of 1% agarose in barbital buffer, pH 8.4 (barbital, 0.003M, sodium barbital, 0.017M, calcium lactate, 0.017M) was
120 applied to each slide. Slides were run at a constant 200 V for 105 min, average current 2 mA per slide; the bridge buffer was barbital, 0.0075 M, sodium barbital 0.043 M, calcium lactate, 0.0012 M, pH 8.6. Slides were photographed after 18 to 24 h development. They were then washed, dried and stained with amido black. Protein assays were performed essentially according to Lowry et al. (1951), with a bovine serum albumin standard. RESULTS
Effect of pH upon the adsorption of HBsAg to CPG Preliminary experiments suggested that, for a given HBsAg-CPG ratio, pH was the most important variable in the adsorption of HBsAg to CPG. Since HBsAg dissociates from glass at high pH (Pert and Verch, 1975), we examined adsorption in neutral and acidic conditions only. The antigen adsorption stage was performed as described above, using the pH 2, 3, 4, 5, 6, 7, and 8 buffers. Samples of the dialysed HBsAg preparations were taken before adsorption. In each case, this initial sample and the supernatants and wash solutions from the adsorption stage, were retained, dialysed against PBS and titrated by AusRIA II. The percentage difference between the total antigen in the supernatants and washes and that in the initial sample was considered to be the proportion of antigen adsorbed onto the CPG. The extent of adsorption of antigen at each pH value is shown in fig. 1. The results indicate that the maximum adsorption of HBsAg takes place at pH 3. The resultant CPG-HBsAg preparations were used to adsorb anti-HBs at pH 7.2. 0.5 ml of anti-HBs, diluted to 2.5 ml in PBS, was added to each 100 mg of CPG-HBsAg and mixed for 2½ h at room temperature. The supernatant was recovered and the CPG-HBsAg was exhaustively washed with PBS. The anti-HBs content of each supernatant and wash was determined; the difference between the a m o u n t (volume × titer) applied and the amount recovered was taken as the a m o u n t adsorbed. The percentage of applied antiHBs adsorbed b y each CPG-HBsAg preparation is shown in fig. 1. In general, the extent of adsorption of antibody parallels the amount of HBsAg b o u n d in each preparation. CPG-HBsAg prepared at pH 3.0 adsorbs the maximum a m o u n t of antibody.
Selectivity of CPG for HBsAg Immunoelectrophoretic experiments Were done in order to see whether CPG adsorbed major serum components in addition to HBsAg. HBsAgpositive plasma was dialysed against pH 3.0 glycine--HC1 buffer and adsorbed onto CPG as described above. Samples of the HBsAg-positive plasma before and after the dialysis and after adsorption with CPG were dialysed against PBS overnight, then applied to t h e wells of immunoelectrophoresis
121 100
0
95
o 9o
85 2
3
4 5 6 pH of A d s o r p t i o n B u f f e r
7
8
Fig. 1. Effect of pH on adsorption of HBsAg to CPG. HBsAg was adsorbed onto CPG at each of the stated pH values and the proportion of retained antigen was determined. Each preparation was washed with PBS and exposed to a standard amount of anti-HBs at pH 7.2; the retention of anti-HBs was also determined. • = percentage of applied HBsAg adsorbed at the stated pH; [] = percentage of applied anti-HBs adsorbed by CPG-HBsAg prepared at the stated pH.
slides. After electrophoresis, the troughs were filled with antibody to h u m a n serum. The resulting patterns are shown in fig. 2. It may be seen that, after dialysis at pH 3, a number of serum components were no longer detectable. There was very little further change as a result of adsorption onto CPG.
Stability of the immunoadsorbent CPG-HBsAg was exposed for 1 h to low pH and to high molarities of sodium thiocyanate; conditions which are used to elute specifically adsorbed antibody. Supernatants were collected, dialysed against PBS and tested by RIA. No HBsAg was f o u n d in the supernatants from CPG-HBsAg exposed to pH values between 2 and 8, to 4 M sodium thiocyanate at neutrality or to 2 M sodium thiocyanate at pH 3.5 or 2.0.
Adsorption of anti-HBs by CPG-HBsAg When human anti-HBs was adsorbed onto CPG-HBsAg as described earlier, 100 mg of the immunoadsorbent removed 99% of the detectable anti-HBs from 2.5 ml of an anti-HBs preparation with a PHA titer of 1 : 1600; that is, about 4000 PHA units (titer × volume). By repeating the adsorption several times we established that 100 mg of CPG-HBsAg would adsorb a m a x i m u m
122
Fig. 2. Effect of HBsAg adsorption procedure upon normal plasma constituents. HBsAg positive plasma was dialysed overnight against glycine-HC1 buffer, pH 3.0. One aliquot was adsorbed with CPG for 5 h at room temperature; a control aliquot was exposed to the same conditions in the absence of CPG. All plasma samples were dialyzed against PBS, pH 7.2 before immunoelectrophoresis. Troughs: anti-whole serum; wells: 1, HBsAgpositive plasma; 2, HBsAg-positive plasma exposed to pH 3 for 5 h in the absence of CPG; 3, HBsAg-positive plasma after adsorption onto CPG.
o f a b o u t 3 0 , 0 0 0 P H A units of anti-HBs. This a d s o r p t i o n a p p e a r e d to be specific, as c o n t r o l i m m u n o a d s o r b e n t s p r e p a r e d f r o m H B s A g - n e g a t i v e p l a s m a did n o t a d s o r b anti-HBs significantly (see tables 1 a n d 2).
Re-use o f CPG-HBsAg We f o u n d t h a t 50 t o 1 0 0 % o f t h e a d s o r b e d h u m a n anti-HBs activity c o u l d b e r e c o v e r e d f r o m C P G - H B s A g b y washing w i t h 0.1 M glycine--HC1 b u f f e r , p H 3. A f t e r this t r e a t m e n t a n d r e t u r n to PBS, t h e C P G - H B s A g w o u l d a d s o r b a n t i b o d y to the s a m e e x t e n t as t h e freshly p r e p a r e d i m m u n o a d s o r b e n t . We o b s e r v e d n o change in a d s o r p t i o n or r e c o v e r y o f anti-HBs in t h r e e such cycles o f a single p r e p a r a t i o n o f CPG-HBsAg.
Elu tion of specific anti-HBs O n e g r a m o f C P G - H B s A g was used to a d s o r b m a x i m a l a m o u n t s (15 ml) o f h u m a n anti-HBs, as d e s c r i b e d above. C o n t r o l s w e r e p r e p a r e d in parallel; C P G - H B s A g was used to a d s o r b n o r m a l h u m a n p l a s m a ( N H P ) a n d a CPGN H P c o n j u g a t e was used t o a d s o r b anti-HBs. A f t e r t h e a d s o r p t i o n stage, each i m m u n o a d s o r b e n t was e x h a u s t i v e l y w a s h e d w i t h PBS. T h e t o t a l a m o u n t o f u n a d s o r b e d anti-HBs was d e t e r m i n e d in o r d e r to e s t i m a t e the q u a n t i t y a d s o r b e d b y t h e C P G - H B s A g or C P G - N H P . In o r d e r t o e l u t e a d s o r b e d a n t i b o d y , t h e w a s h e d i m m u n o a d s o r b e n t s were
* P e r c e n t a g e o f t o t a l applied. ** N.D. = n o t d o n e . *** -- Negative w h e n t e s t e d u n d i l u t e d .
Fraction Anti-HBs, s t a r t i n g m a t e r i a l (15 m l ) A d s o r p t i o n stage Anti-HBs, s u p e r n a t a n t PBS washes E l u t i o n stages pH 5, p h o s p h a t e , 4 0 m i n , e l u a t e pH 4, citrate--HCl, 10 rain, e l u a t e p H 3.5, citrate--HC1, 70 rain, e l u a t e pH 3.0, citrate--HC1, 30 m i n , e l u a t e Water, 10 m i n , final w a s h Total eluted
E l u t i o n o f anti-HBs f r o m CPG-HBsAg.
TABLE 1
% *
0.94 0.2 1.79 0.91 1.34 5.18
0.1 0.02 0.18 0.09 0.14 0.53
5840 8000 31680 3720 2406 51646
49984 3203
605 59
62 6
120000
975
4.6 6.7 26.4 3.1 2.0 43.0
41.7 2.7
0.89 0.18 1.84 0.97 1.31 5.19
674 64
975
units
mg
% *
Protein (mg)
Titer
Protein
--
79600 9253
120000
Titer (units)
C P G - N H P + anti-HBs
CPG-HBsAg + anti-HBs
Complex
***
0.76 0.21 1.85 0.92 1.31 5.05
702 66
N.D. **
CPG-HBsAg + NHP Protein (mg)
50
124 TABLE 2 Preparation of monospecific anti-HBs: passive hemagglutination of adsorbed antisera as expressed upon erythrocytes coated with HBsAg/adw or HBsAg/ayw. Antibody *
anti anti anti anti anti anti anti anti
Adsorbent
HBs/ad HBs/ay
---
HBs/ad HBs/ay
CPG-NHP CPG-NHP
HBs/ad HBs/ay
CPG-HBsAg/ay CPG-HBsAg/ad
HBs/ad
HBsAg/ay *** HbsAg/ad ***
HBs/ay
PHA units ** adw
ayw
64000 5000 16322 5138 7232 74 8000 5
4000 16000 5148 16000 64 3756 10 4000
* See text. ** Titer X volume. *** Liquid phase.
e x p o s e d t o a stepwise series o f S~brensen buffers o f decreasing pH, as s h o w n in table 1. B a t c h p r o c e d u r e s were used and each 1 g o f i m m u n o a d s o r b e n t was e l u t e d with 10 ml o f b u f f e r f o r 10 min at r o o m t e m p e r a t u r e . This was f o l l o w e d b y a second, 30 min elution step with t h e same b u f f e r ; this was o m i t t e d at pH 4. A s e c o n d 30 min e l u t i o n step was a d d e d at pH 3.5. Each eluate was dialysed o v e r n i g h t against PBS and its a n t i b o d y c o n t e n t and protein c o n t e n t were m e a s u r e d . Table 1 gives the results o f a typical experim e n t ; o t h e r e x p e r i m e n t s gave essentially similar results. It can be seen f r o m table 1 t h a t the stepwise acid e l u t i o n p r o c e d u r e resulted in t h e r e c o v e r y o f 43% o f the t o t a l starting a n t i b o d y in 0.53% o f the p r o t e i n . H o w e v e r , since o n l y 56% o f the t o t a l a n t i b o d y was a d s o r b e d , acid e l u t i o n r e c o v e r e d 77% o f t h e b o u n d a n t i b o d y . T h e m a j o r part (61%) was e l u t e d at p H 3.5. This f r a c t i o n c o n t a i n e d 26% o f t h e t o t a l starting a n t i b o d y in 0.18% o f the p r o t e i n , a 1 4 4 - f o l d single step p u r i f i c a t i o n . Each e l u a n t released a discrete q u a n t i t y o f a n t i b o d y which was n o t increased b y f u r t h e r e x p o s u r e t o t h a t eluant. Assuming t h a t this is due t o the variation in affinity o f b o u n d a n t i b o d y , these results indicate t h a t a one-step e l u t i o n at pH 3.5 w o u l d result in t h e r e c o v e r y o f a b o u t 75% o f a d s o r b e d a n t i b o d y , with a lesser degree o f p u r i f i c a t i o n . We find t h a t this is i n d e e d t h e case w h e n such an e l u t i o n p r o c e d u r e is used. T a b l e 1 also shows t h a t similar a m o u n t s o f protein, b u t n o a n t i b o d y , were e l u t e d f r o m c o n t r o l i m m u n o a d s o r b e n t s . P o r t i o n s o f t h e eluates were c o n c e n t r a t e d f i f t y - f o l d b y u l t r a f i l t r a t i o n (Minicon ®, A m i c o n C o r p o r a t i o n , L e x i n g t o n , MA) and subjected to i m m u n o electrophoresis. Fig. 3 shows i m m u n o e l e c t r o p h o r e t o g r a m s o f the pH 3.5 eluate and c o r r e s p o n d i n g c o n t r o l f r o m o n e e x p e r i m e n t . It s h o u l d be n o t e d
125
Fig. 3. Elution of human anti-HBs from CPG-HBsAg at pH 3.5. CPG-HBsAg was exposed to excess anti-HBs, and washed thoroughly with PBS. Anti-HBs was eluted with S~brensen buffers of pH 5, 4 and 3.5. The pH 3.5 eluate was concentrated and compared with unadsorbed anti-HBs in immunoelectrophoresis. A control eluate was prepared in the same conditions, but using CPG-NHP instead of CPG-HBsAg. Troughs: 1, anti-whole serum; 2, anti-IgG; 3, HBsAg-positive plasma. Wells: 4, human anti-HBs; 5, concentrated pH 3.5 eluate from CPG-HBsAg; 6, concentrated pH 3.5 eluate from CPG-NHP. t h a t t h e c o n c e n t r a t i o n o f a n t i b o d y in t h e s t a r t i n g s e r u m w a s i n s u f f i c i e n t t o precipitate with HBsAg, whereas the concentrated eluate gave a sharp, clear a r c in t h e 7 r e g i o n . C h a o t r o p i c i o n s w e r e a l s o e f f e c t i v e in e l u t i n g b o u n d a n t i - H B s . A p r e l i m i -
126
Fig. 4. Preparation of monospecific anti-HBs. Guinea pig anti-HBs/ad was adsorbed with HBsAg/ay in liquid phase and with CPG-HBsAg]ay; similarly, anti-HBs/ay was adsorbed with HBsAg]ad in liquid phase and with CPG-HBsAg/ad. Adsorbed antibody was eluted from the CPG preparations with 4 M NaSCN. Adsorbed antibodies or eluates were concentrated and tested against standard antigens in immunodiffusion. The unadsorbed anti-HBs preparations were also tested against the same antigens. 1, adsorbing y antigen; 2, adsorbing d antigen ; 3, ad w ; 4, adr; 5, ay w; A, anti-d, solid phase; B, anti-y, solid phase; C, anti-d, liquid phase; D, anti-y, liquid phase; E, unadsorbed anti-ad; F, unadsorbed anti-ay; G, anti-a derived from anti-ad by elution; H, anti-a derived from anti-ay by elution.
127 nary experiment showed that a combination of lowered pH and moderate concentrations of sodium thiocyanate (pH 3.5 citrate--HC1, 1 M NaSCN) when used in conjunction with a selected sequence of wash steps, released anti-HBs of high purity. The product gave a single IgG precipitin arc in immunoelectrophoresis and a single band in polyacrylamide gel electrophoresis. Lower pH values or high molarities of NaSCN were necessary to elute animal anti-HBs, presumably because of the high affinities of antibodies produced by intensive immunization schedules.
Preparation of monospecific subtyping reagents S u b t y p e specific CPG-HBsAg was prepared using plasma samples which had been previously t y p e d for d or y determinants. S u b t y p e reactive guinea pig antisera were cross-adsorbed with the heterologous CPG-HBsAg preparation, using exactly the conditions described above for human anti-HBs. Comparable absorptions were also carried o u t in liquid phase, using the same antigen-containing plasma samples, which were mixed with the heterologous antibody preparations. Proportions were selected to correspond with the antigen--antibody ratio in the solid phase adsorption. After adsorption, each preparation was concentrated by ultrafiltration (Minicon®). The solid phase reagents were concentrated to the original volume of antibody. Because of their protein content, it was impossible to concentrate the liquid phase reagents to the same extent; they were reduced to two to three times the starting volume of antibody. The adsorbed reagents were tested in PHA against erythrocytes coated with HBsAg/adw and HBsAg/ayw (table 2). The content of subtypespecific antibody was remarkably similar in the reagents produced b y solidor liquid-phase adsorption, although the anti-HBs/a content of the solid phase reagents was somewhat higher than that of the liquid phase preparations. It should be noted that CPG-NHP does not adsorb anti-HBs significantly. Adsorbed antibody was eluted from the CPG-HBsAg with 4 M NaSCN. All adsorbed and eluted reagents were evaluated against standard antigens in agar gel diffusion (fig. 4). It may be seen that the solid phase adsorption procedure resulted in a highly specific preparation of anti-HBs/d or anti-HBs/y. Liquid phase absorption resulted in a weaker, but equally specific antiHBs/d, b u t the putative anti-HBs/y did n o t give any precipitin lines. The eluted antibody reacted in a manner consistent with anti-HBs/a. Finally, the difficulty of interpreting spurring patterns obtained with unadsorbed antisera is clearly demonstrated in fig. 4. DISCUSSION Direct adsorption to a surface is an established method of preparing insolubilized antigens or enzymes. A particular advantage of this approach is
128 that the adsorption of solute is itself a purification step. However, it is accepted that affinity media prepared in this fashion may not be sufficiently stable for effective use (Zaborsky, 1973). We have shown that HBsAg is adsorbed to controlled pore glass and the antigen remains b o u n d in conditions suitable for adsorption and elution of antibody. Controlled pore glass does n o t bind HBsAg exclusively; we have noted that CPG-HBsAg also adsorbs an interfering antilipoprotein specificity from an experimental anti-HBs prepared in goats (Nath, personal communication). Nevertheless, the adsorption is sufficiently selective to allow us to prepare a satisfactory HBsAg-specific immunoadsorbent from whole plasma. Estimates of the a m o u n t of HBsAg present in carrier plasma samples (Kim and Tilles, 1973) suggest that 100 mg of CPG adsorb a b o u t 50 pg of HBsAg. This method of insolubilizing HBsAg does not significantly alter its quantitative, or qualitative, binding capacity for anti-HBs. We have shown that the CPGHBsAg immunoadsorbent may be used for the preparation of monospecific anti-d and anti-y, and that b o u n d antibody can be eluted in good yield and with a reasonable degree of immunochemical purity. The products of the solid phase immunoadsorption technique were superior to reagents prepared in liquid phase, when tested in AGD and CEP. In particular, we were unable to concentrate liquid phase anti-y sufficiently to react in gel precipitation tests, because relatively large amounts of whole, HBsAg/ad positive plasma were used for adsorption. When tested in PHA, the reagents produced by solid phase adsorption were a little less well absorbed than the liquid phase reagents, as shown by their titers against the heterologous antigen (table 2). Adjustment of the antibody to CPG-HBsAg ratio would presumably reduce this effect. Recovery of anti-a adsorbed to CPG-HBsAg suggests that appropriate combinations of adsorption and elution could be used to isolate monospecific antibodies to any of the subdeterminants of HBsAg. Human anti-HBs adsorbed to CPG-HBsAg at pH 7.2 was eluted by lowering the pH. Stepwise reduction of the pH resulted in the elution of discrete fractions of the total b o u n d antibody, indicating that our anti-HBs was heterogeneous with respect to affinity. Appropriate elution conditions could be used to select for antibodies of a given affinity, for maximum yields or for the highest degree of purification. Such monospecific anti-HBs reagents should be of considerable use in studies of the nature of the antigenic determinants of HBsAg and in the routine subtyping of HBsAg specimens. Similarly, immunochemically purified anti-HBs is required for a number of test procedures such as radioimmunoassay and reversed passive hemagglutination, and is a valuable reagent for studies of the antigenic nature of HBsAg. ACKNOWLEDGEMENTS We thank Dr. N. Nath and his staff for AusRIA assays. This work was performed during studies leading to the degree of Ph.D. in the Department
129
of Microbiology of the George Washington University. Publication number 371 from the American National Red Cross. REFERENCES Couroucd, A.M., P.V. Holland, J.Y. Muller and J.P. Soulier (eds.), 1976, HBs Antigen Subtypes, Bibl. Haematol. 42 (Karger, Basel). Crovari, P. and S. De Flora, 1975, Boll. Ist. Sieroter. Milan. 54, 149. Gocke, D.J., 1972, J. Am. Med. Ass. 219, 1165. Holland, P.V., 1975, Am. J. Med. Sci. 270, 161. Kim, C.Y. and J.G. Tilles, 1973, J. Clin. Invest. 52, 1176. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Mazzur, S., 1972, Am. J. Med. Technol. 38,343. Mazzur, S., S. Burgert and G. Le Bouvier, 1975, J. Immunol. 114, 1510. Moghaddam, M., K.L.G. Goldsmith and R.A. Kekwick, 1971, Vox Sang. 20,277. Pert, J.H. and R.L. Vetch, 1975, Vox Sang. 29, 157. Siebke, J.C., E. Kjeldsberg and T. Traavik, 1972, Acta Pathol. Microbiol. Scand. B 80, 935. Traavik, T., 1975, Acta Pathol. Microbiol. Scand. B 8 3 , 1 5 3 . Vyas, G.N. and N.R. Shulman, 1970, Science 170, 332. Zaborsky, O.R., 1973, in: Immobilized Enzymes (C.R.C. Press, Cleveland) p. 75.
