Journal oflmmunological Methods, 25 (1979) 177--183 © Elsevier/North-Holland Biomedical Press
177
S U B T Y P I N G O F H E P A T I T I S B S U R F A C E A N T I G E N BY RADIOIMMUNOASSAY
PENTTI UKKONEN and VESA KOISTINEN Laboratory o f Viral Immunopathology, Department o f Virology, University o f Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, and Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, SF-00310 Helsinki 31, Finland (Received 11 July 1978, accepted 25 July 1978)
A solid-phase radioimmunoassay used for HBsAg screening of blood donors at the Finnish Red Cross Blood Transfusion Service (FRC-RIA) was modified for subtyping of HBsAg. The anti-HBs coated test tubes of FRC-RIA served as the solid-phase anti-HBs. Monospecific 12SI-labelled antibodies were prepared by absorption of the labelled antiHBs used in FRC-RIA with partially purified HBsAg coupled to Sepharose 4B. Labelled anti-y detected 4 ng/ml of ay antigen and 160 ng/ml of ad antigen; the reagent was 40 times more sensitive for the homologous subtype. Labelled anti-d detected 3 ng/ml of ad antigen, but it did not bind to ay antigen. Thus, anti-d was completely monospecific. The standard FRC-RIA was only about 5 times more sensitive than the subtype RIA; 96% of HBsAg positive blood donor samples screened by FRC-RIA could by subtyped by RIA. The important feature of this method is the absorption of the antibodies in the presence of carrier protein after radioactive labelling. This has certain advantages: (1) No extra radioactive labelling is needed for subtyping, because the labelled anti-HBs of standard RIAs for HBsAg can be utilized. (2) Small amounts of labelled monospecific antibodies can be prepared at a time, which promotes economical use of reagents. (3) Only crude antigen preparation (e.g. polyethyleneglycol precipitate from plasma) is needed for the absorption of antibodies.
INTRODUCTION T h e a n t i g e n i c s u b t y p e s o f h e p a t i t i s B s u r f a c e a n t i g e n ( H B s A g ) are i m p o r t a n t in t h e e p i d e m i o l o g i c s t u d y o f h e p a t i t i s B virus ( H B V ) i n f e c t i o n . F o u r m a i n s u b t y p e s , a d w , a y w , a d r a n d a y r ( L e B o u v i e r , 1 9 7 1 ; B a n c r o f t e t al., 1 9 7 2 ) s h o w d i f f e r e n c e s in d i s t r i b u t i o n a c c o r d i n g t o t h e g e o g r a p h i c a l o r i g i n and the clinical form of HBV infection (Nordenfelt, 1975). S u b t y p i n g has been p e r f o r m e d by i m m u n o d i f f u s i o n (ID), c o u n t e r i m m u n o e l e c t r o p h o r e s i s (CIEP}, i n h i b i t i o n o f h a e m a g g l u t i n a t i o n (HI) a n d r a d i o i m m u n o a s s a y ( R I A ) . T h e d i s a d v a n t a g e o f ID a n d CIEP is t h e i r p o o r sensitivity; only strongly positive samples can be subtyped by these methods. V a r i o u s R I A t e s t s h a v e b e e n u s e d f o r s u b t y p i n g . T h e s e are c o n s i d e r a b l y m o r e s e n s i t i v e t h a n t h e ' f i r s t g e n e r a t i o n ' ID a n d CIEP tests, b u t o f t e n r e q u i r e
178 extra procedures and reagents as compared with ordinary HBsAg screening by RIA. We have developed a simple modification of our routine HBsAg RIA (Koistinen, 1978), for screening blood donors at the Finnish Red Cross Blood Transfusion Service, which permits distinction between the subtypes ad and ay. The m e t h o d does not require additional radioactive labelling, reagents or equipment. In this paper, the test is described and its sensitivity and specificity are assessed. METHODS AND MATERIAL
The subtype radioimmunoassay The subtype RIA is a modification of the HBsAg radioimmunoassay of the Finnish Red Cross Blood Transfusion Service (FRC-RIA), described by Koistinen (1978). The same reagents and test procedures are used for subtyping as in the standard FRC-RIA. Solid-phase anti-HBs. Sheep anti-HBs gamma-globulin, containing both anti-ad and anti-ay antibodies, was adsorbed to polystyrene test tubes (Koistinen, 1978). The tubes were used within a few days. Monospecific 12SI-labelled anti-HBs was prepared by absorbing the stock solution of the radiolabelled anti-ad (or anti-ay) used in FRC-RIA with partially purified HBsAg/ay (or ad) coupled to Sepharose 4B. The procedure yielded 12SI-labelled anti-d (or anti-y). The immunoadsorbent was prepared in the following way: HBsAg (ad or ay) was precipitated from plasma by 11% (w/v) polyethyleneglycol (PEG 6000), the precipitate was dissolved in 0.1 M sodium bicarbonate and coupled to Sepharose 4B by the CNBr method (Cuatrecasas, 1970). Immunoadsorbents made from purified HBsAg (Koistinen, 1978) were also tried. To make radioactive anti-d (anti-d*), 400 ~l of packed immunoadsorbent (HBsAg/ay) was suspended in an equal volume of phosphate-buffered saline, and 10 pCi (300--400 ~l) of anti-ad* was added to the suspension which also contained about 1% bovine serum albumin (to prevent non-specific absorption). The mixture was incubated overnight at +4°C in a centrifuge tube which was continuously mixed in a cell mixer. After incubation, the tube was centrifuged at low speed, and the supernatant containing anti-d* was removed. For anti-y*, 10 ~Ci (500--900 ~1) of anti-ay* was absorbed with 800 ~1 of immunoadsorbent (HBsAg/ad). The absorbed labelled antibody was used at a dilution which gave 80-90,000 cpm/0.2 ml. Test procedure. The test was performed in the same way as FRC-RIA except that two labelled antibody reagents were used as follows. 0.2 ml of a sample was added to two test tubes. After incubation overnight at room temperature (alternatively 2 h at +40°C), the tubes were emptied by aspira-
179
tion and washed twice with 0.9% saline. 0.2 ml of anti-d* was added to one tube and 0.2 ml of anti-y* to the other one. After incubation of 1 h at +40°C, the tubes were emptied and washed twice with 0.9% saline, and counted in a gamma counter (Rackgamma, Wallac, Finland). Two positive controls at two dilutions (subtypes ad and ay) and 7 negative controls were measured with both anti-d* and anti-y* at each run. The cut-off level (discriminating between positivity and negativity) for each reagent was defined as 2.3 times the mean cpm of the negative controls, as currently used in FRC-RIA.
Other methods A micromodification of immunodiffusion (ID) was applied for subtyping of HBsAg, as described earlier (Ukkonen et al., 1977). Counterimmunoelectrophoresis (CIEP) was used for HBsAg screening of blood donors (Helske, 1974). Patient material was tested for HBsAg by complement fixation (CF) (Ukkonen et al., 1977) and AusRIA I or H (Abbott Laboratories, U.S.A.). HBsAg used in the titration experiment was purified as previously described (Koistinen, 1978). The protein concentration of the HBsAg preparation was determined by the m e t h o d of Lowry. RESULTS
Specificity and sensitivity of the subtype RIA The radiolabelled antibody reagents differed in some respects. When anti-d* was used, a positive result was obtained only for samples containing HBsAg/ad, while samples of subtype ay were negative. Thus, the anti-d* reagent was completely monospecific. Anti-y* was not totally monospecific for ay antigen: most ad samples (except very weakly positive ones) were also weakly positive with anti-y*. However, using anti-y* the count rates were much lower for ad than for ay samples, the proportion being about 1/5-1/10. We failed to produce a completely monospecific anti-y*, although we tried several immunoadsorbents made from different antigen preparations (crude or purified HBsAg of different origin). Even the same HBsAg/ad serum from which the immunoadsorbent was prepared was positive when tested with the resulting anti-y*. Another difference between the two labelled reagents was that anti-y* usually gave notably higher maximal values (about 10,000 cpm) than did anti-d* (about 2000 cpm). This difference did not, however, affect the sensitivity or the interpretation of the test. The criteria for the subtyping were as follows: A sample positive with anti-d* (and weakly positive or negative with anti-y*) was regarded as subtype ad. A sample negative with anti-d* but positive with anti-y* was considered to be subtype ay. The sensitivity of the test was determined by a titration where 5-fold
180
1200
1200
lOOC
I000
80C
800
60C
u
600
40C
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20C
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anti- d" T
I0000
400 2000 80
16
06 32
003
I0000
01
HBsAg/ad (ng/ml)
, , , li~ ' ~ - - ~ - - ~ 16 06 003 4O0 32 0.I 2000 80
HBsAg/ay (ng/rnl)
Fig. 1. Titration o f purified HBsAg (A: subtype ad; and B: subtype ay) with radiolabelled anti-HBs reagents anti-d* and anti-y*. The intercept o f the curve and the cut-off line indicates the sensitivity of each test system.
dilutions of purified HBsAg (diluted in normal human plasma) were assayed with different labelled anti-HBs reagents (Figs. 1 and 2). The intercept of the titration curve and the corresponding cut-off line indicates the minimum amount of antigen detectable by the test system. The results are summarized in Table 1. The sensitivity of the subtype RIA was 3--4 ng/ml while unabsorbed antibody reagents detected less than 1 ng/ml of antigen. Thus, the sensitivity of the subtype RIA was about 1/4--1/6 of that of the ordinary FRC-RIA. Anti-d* was about 50 times more sensitive for ad than anti-y*. I0000
I00001
B anti ay" anti-ad" anli-ay"
anli ad '
1000
IOOC
I
J
[oo
I00
20,L_~ I0000
400 2000 80
HBsAg/ad
16
32
06
(ng/ml)
OI
0 03
20 I0000
~L 400 2000 80
HBsAg/ay
J !6
, ~-06 32
,
oI
0 03
(ng/ml)
Fig. 2. Titration of purified HBsAg (A: subtype ad ; and B: subtype ay ) with radiolabelled unabsorbed anti-HBs reagents anti-ad* and anti-ay*. The intercept of the curve and the cut-off line indicates the sensitivity o f each test system.
181 TABLE 1 THE SENSITIVITY OF THE DIFFERENT RADIOLABELLED anti-HBs PREPARATIONS IN DETECTING HBsAg The results are based on the titration curves illustrated in Figs. 1 and 2. Subtype of HBsAg tested
ad ay
Minimum amount of HBsAg (ng/ml) detectable by anti-ad*
anti-ay*
0.6 0.7
0.8 0.6
anti-d* 3 > 104
anti-y* 160 4
A n t i - y * , w h i c h was n o t e n t i r e l y m o n o s p e c i f i c , was a b o u t 40 t i m e s m o r e s e n s i t i v e f o r ay t h a n f o r ad a n t i g e n . 1 0 / ~ g / m l was t h e h i g h e s t c o n c e n t r a t i o n o f p u r i f i e d H B s A g / a y t e s t e d a n d it was n e g a t i v e w i t h a n t i - d * , i n d i c a t i n g complete monospecificity.
Comparison with subtyping by ID T w e n t y - f o u r s a m p l e s f r o m p a t i e n t m a t e r i a l p r e v i o u s l y s u b t y p e d b y ID w e r e a s s a y e d b y t h e s u b t y p e R I A . T w e n t y - t w o gave i d e n t i c a l r e s u l t s b y b o t h m e t h o d s ( 1 0 ad a n d 12 ay). O n e s a m p l e w e a k l y p o s i t i v e in ID a n d i n t e r p r e t e d as d o u b t f u l l y ad was d e f i n i t e l y s u b t y p e ay b y R I A . A n o t h e r s a m p l e , s u b t y p e ay b y ID, gave high c o u n t s w i t h a n t i - y * b u t it was also w e a k l y p o s i t i v e w i t h a n t i - d * . T h e s a m p l e was r e g a r d e d as s u b t y p e ay, a l t h o u g h it m i g h t h a v e h a d an u n c o m m o n s u b t y p e c o n f i g u r a t i o n ady.
TABLE 2 TYPABILITY OF DIFFERENT MATERIALS POSITIVE FOR HBsAg ND = not done; N = number of samples. Result in HBsAg testing by different methods
Total tested
Typable by RIA
Blood donors (N = 163) FRC-RIA pos., CIEP neg. FRC-RIA pos., CIEP ND CIEP pos., FRC-RIA ND Patients (N = 33) AusRIA pos., CF neg. CF and AusRIA pos. a Anticomplementary serum.
ID
N
%
12 27 124
7 26 124
58 96 100
8 25
8 25
100 100
N
% ND ND ND
1a 23
13 92
182
Typability of different HBsAg positive samples One hundred and sixty-three samples from HBsAg positive blood donors were subtyped by RIA (Table 2). All of the samples positive by CIEP were typable by RIA at the 1 : 10 dilution routinely used for strongly positive samples. Of 12 samples negative by CIEP but weakly positive in FRC-RIA screening, 5 (42%) were negative in the subtype RIA. The patient material consisted of 33 samples positive for HBsAg by CF or AusRIA (Table 2). All were typable by RIA, including 8 CF-negative samples. 73% could be subtyped by ID; all CF-negative samples (except one strongly anticomplementary sample) were negative by ID. DISCUSSION The radioimmunological methods used to determine HBsAg subtypes may be grouped in 3 categories: (1) A competitive RIA, in which the antigen in the sample competes for the anti-HBs antibody with labelled antigen of known subtype (Aach et al., 1973). (2) A modification of the sandwich-type solid-phase antibody RIA, in which the antigen in the sample binds the subtype specific soluble antibody, thus preventing its binding to the solid-phase antigen and leaving the subsequently added labelled antigen in solution (Ng et al., 1976; Hoofnagle et al., 1977). (3) A sandwich-type antigen RIA, in which labelled antibodies more or less subtype specific are used (Ling et al., 1973; Miyakawa et al., 1975). Our m e t h o d belongs to the last group. It differs, however, in one important respect: the liquid-phase antibodies are made monospecific after radiolabelling. Technically, this procedure has obvious advantages over those where the antibodies are absorbed to monospecificity before labelling: First, the test can be easily set up as a modification of the ordinary sandwich-type RIA, because no extra labelling of antibodies is needed for subtyping. Second, small amounts of monospecific antibody can be prepared at a time. This is possible because enough carrier protein (bovine serum albumin in this work} can be used to prevent non-specific absorption, and, consequently, very low amounts of antibody can be absorbed without significant loss. When subtyping is done in a routine diagnostic laboratory, the number of subtyping tests carried o u t is only a minute fraction of the number of HBsAg screening tests. Consumption of labelled monospecific antibodies is thus very small compared with the amount of labelled unabsorbed antibodies used in screening. In our method, waste of antibodies and radioactive iodine is avoided. Third, only crude antigen preparation is required for immunoadsorption. It is not necessary to maximize the capacity per unit volume of immunoadsorbent, as low amounts of antibody (about 1 pg in this work) can be used in the presence of carrier protein. We used a PEG precipitate from HBsAg positive plasma which was as effective as purified HBsAg.
183 F o u r t h , it is apparent t hat commercial RIAs or e n z y m e immunoassays may be modified in the way described here for FRC-RIA. T he only additional reagent needed is the solid-phase (crude) antigen easily prepared in any laboratory. Why we could n o t produce completely monospecific anti-y* remained unclear. This did n o t invalidate the m e t h o d , because anti-y* gave considerably higher c o u n t rates and bet t er sensitivity for ay than for ad samples. It is apparent also from earlier reports that totally monospecific reagent~ are n o t necessary for subtyping (Ginsberg et al., 1972; Aach et al., 1973; Hoofnagle et al., 1977). A b o u t one-fifth o f the sensitivity of the ordinary FRC-RIA was obtained when the anti-a specific antibodies were absorbed out. The sensitivity of FRC-RIA is comparable with that of the most sensitive commercial methods, e.g. AusRIA II ( U kkone n et al., 1977; Koistinen, 1978). Other reports on subtype RIA do not state the sensitivity in absolute units (ng/ml) o f HBsAg, and, therefore, the comparison is difficult. Hoofnagle et al. (1977) used a modification of AusAb ( A b b o t t Laboratories, U.S.A.), the sensitivity of which was only 1 / 2 0 - - 1 / 1 0 0 o f that of AusRIA II. We s u b ty p ed 27 blood d o n o r samples HBsAg positive by routine FRCRIA screening: as m any as 96% (26/27) were typable by RIA. This percentage illustrates the practical efficiency of the test, as the d o n o r group tested represented unselected new HBsAg carriers. However, some samples are t o o weak to be s u b typed by any m e t h o d , because sensitivity is usually somewhat reduced when an HBsAg test is modified for subtyping. This is evident from the work we have quoted. ACKNOWLEDGEMENTS The study was supported by grants from the Finnish Medical F o u n d a t i o n and the Orion F o u n d a t i o n , and by a grant from the Sigrid Jus~lius Foundation to Prof. K. Penttinen. T he e xpe r t technical assistance of Sinikka Hakkarainen is gratefully acknowledged. REFERENCES Aach, R.D., E.J. Hacker and C.W. Parker, 1973, J. Immunol. 111,381. Bancroft, W.H., F.K. Mundon and P.K. Russell, 1972, J. Immunol. 109,842. Cuatrecasas, P., 1970, J. Biol. Chem. 245, 3059. Ginsberg, A.L., W.H. Bancroft and M.E. Conrad, 1972, J. Lab. Clin. Med. 80,291. Helske, T., 1974, Scand. J. Haematol. Suppl. 22. Hoofnagle, J.H., R.J. Gerety, L.A. Smallwood and L.F. Barker, 1977, Gastroenterology 72,290. Koistinen, V.U., 1978, Vox Sang. 34,321. LeBouvier, G., 1971, J. Infect. Dis. 123,671. Ling, C.M., H. Irace, R. Decker and L.R. Overby, 1973, Science 180,203. Miyakawa, Y., M. Imai and M. Mayumi, 1975, J. Immunol. 114, 1135. Ng, P.L., L.W. Powell, J.W. Halliday, K.L. Gera, L. McKeering and C.B. Campbell, 1976, Am. J. Gastroenterol. 65,127. Nordenfelt, E., 1975, Scand. J. Infect. Dis. 7, 147. Ukkonen, P., V. Keistinen and K. Penttinen, 1977, J. Immunol. Methods 15, 343.
177
S U B T Y P I N G O F H E P A T I T I S B S U R F A C E A N T I G E N BY RADIOIMMUNOASSAY
PENTTI UKKONEN and VESA KOISTINEN Laboratory o f Viral Immunopathology, Department o f Virology, University o f Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, and Finnish Red Cross Blood Transfusion Service, Kivihaantie 7, SF-00310 Helsinki 31, Finland (Received 11 July 1978, accepted 25 July 1978)
A solid-phase radioimmunoassay used for HBsAg screening of blood donors at the Finnish Red Cross Blood Transfusion Service (FRC-RIA) was modified for subtyping of HBsAg. The anti-HBs coated test tubes of FRC-RIA served as the solid-phase anti-HBs. Monospecific 12SI-labelled antibodies were prepared by absorption of the labelled antiHBs used in FRC-RIA with partially purified HBsAg coupled to Sepharose 4B. Labelled anti-y detected 4 ng/ml of ay antigen and 160 ng/ml of ad antigen; the reagent was 40 times more sensitive for the homologous subtype. Labelled anti-d detected 3 ng/ml of ad antigen, but it did not bind to ay antigen. Thus, anti-d was completely monospecific. The standard FRC-RIA was only about 5 times more sensitive than the subtype RIA; 96% of HBsAg positive blood donor samples screened by FRC-RIA could by subtyped by RIA. The important feature of this method is the absorption of the antibodies in the presence of carrier protein after radioactive labelling. This has certain advantages: (1) No extra radioactive labelling is needed for subtyping, because the labelled anti-HBs of standard RIAs for HBsAg can be utilized. (2) Small amounts of labelled monospecific antibodies can be prepared at a time, which promotes economical use of reagents. (3) Only crude antigen preparation (e.g. polyethyleneglycol precipitate from plasma) is needed for the absorption of antibodies.
INTRODUCTION T h e a n t i g e n i c s u b t y p e s o f h e p a t i t i s B s u r f a c e a n t i g e n ( H B s A g ) are i m p o r t a n t in t h e e p i d e m i o l o g i c s t u d y o f h e p a t i t i s B virus ( H B V ) i n f e c t i o n . F o u r m a i n s u b t y p e s , a d w , a y w , a d r a n d a y r ( L e B o u v i e r , 1 9 7 1 ; B a n c r o f t e t al., 1 9 7 2 ) s h o w d i f f e r e n c e s in d i s t r i b u t i o n a c c o r d i n g t o t h e g e o g r a p h i c a l o r i g i n and the clinical form of HBV infection (Nordenfelt, 1975). S u b t y p i n g has been p e r f o r m e d by i m m u n o d i f f u s i o n (ID), c o u n t e r i m m u n o e l e c t r o p h o r e s i s (CIEP}, i n h i b i t i o n o f h a e m a g g l u t i n a t i o n (HI) a n d r a d i o i m m u n o a s s a y ( R I A ) . T h e d i s a d v a n t a g e o f ID a n d CIEP is t h e i r p o o r sensitivity; only strongly positive samples can be subtyped by these methods. V a r i o u s R I A t e s t s h a v e b e e n u s e d f o r s u b t y p i n g . T h e s e are c o n s i d e r a b l y m o r e s e n s i t i v e t h a n t h e ' f i r s t g e n e r a t i o n ' ID a n d CIEP tests, b u t o f t e n r e q u i r e
178 extra procedures and reagents as compared with ordinary HBsAg screening by RIA. We have developed a simple modification of our routine HBsAg RIA (Koistinen, 1978), for screening blood donors at the Finnish Red Cross Blood Transfusion Service, which permits distinction between the subtypes ad and ay. The m e t h o d does not require additional radioactive labelling, reagents or equipment. In this paper, the test is described and its sensitivity and specificity are assessed. METHODS AND MATERIAL
The subtype radioimmunoassay The subtype RIA is a modification of the HBsAg radioimmunoassay of the Finnish Red Cross Blood Transfusion Service (FRC-RIA), described by Koistinen (1978). The same reagents and test procedures are used for subtyping as in the standard FRC-RIA. Solid-phase anti-HBs. Sheep anti-HBs gamma-globulin, containing both anti-ad and anti-ay antibodies, was adsorbed to polystyrene test tubes (Koistinen, 1978). The tubes were used within a few days. Monospecific 12SI-labelled anti-HBs was prepared by absorbing the stock solution of the radiolabelled anti-ad (or anti-ay) used in FRC-RIA with partially purified HBsAg/ay (or ad) coupled to Sepharose 4B. The procedure yielded 12SI-labelled anti-d (or anti-y). The immunoadsorbent was prepared in the following way: HBsAg (ad or ay) was precipitated from plasma by 11% (w/v) polyethyleneglycol (PEG 6000), the precipitate was dissolved in 0.1 M sodium bicarbonate and coupled to Sepharose 4B by the CNBr method (Cuatrecasas, 1970). Immunoadsorbents made from purified HBsAg (Koistinen, 1978) were also tried. To make radioactive anti-d (anti-d*), 400 ~l of packed immunoadsorbent (HBsAg/ay) was suspended in an equal volume of phosphate-buffered saline, and 10 pCi (300--400 ~l) of anti-ad* was added to the suspension which also contained about 1% bovine serum albumin (to prevent non-specific absorption). The mixture was incubated overnight at +4°C in a centrifuge tube which was continuously mixed in a cell mixer. After incubation, the tube was centrifuged at low speed, and the supernatant containing anti-d* was removed. For anti-y*, 10 ~Ci (500--900 ~1) of anti-ay* was absorbed with 800 ~1 of immunoadsorbent (HBsAg/ad). The absorbed labelled antibody was used at a dilution which gave 80-90,000 cpm/0.2 ml. Test procedure. The test was performed in the same way as FRC-RIA except that two labelled antibody reagents were used as follows. 0.2 ml of a sample was added to two test tubes. After incubation overnight at room temperature (alternatively 2 h at +40°C), the tubes were emptied by aspira-
179
tion and washed twice with 0.9% saline. 0.2 ml of anti-d* was added to one tube and 0.2 ml of anti-y* to the other one. After incubation of 1 h at +40°C, the tubes were emptied and washed twice with 0.9% saline, and counted in a gamma counter (Rackgamma, Wallac, Finland). Two positive controls at two dilutions (subtypes ad and ay) and 7 negative controls were measured with both anti-d* and anti-y* at each run. The cut-off level (discriminating between positivity and negativity) for each reagent was defined as 2.3 times the mean cpm of the negative controls, as currently used in FRC-RIA.
Other methods A micromodification of immunodiffusion (ID) was applied for subtyping of HBsAg, as described earlier (Ukkonen et al., 1977). Counterimmunoelectrophoresis (CIEP) was used for HBsAg screening of blood donors (Helske, 1974). Patient material was tested for HBsAg by complement fixation (CF) (Ukkonen et al., 1977) and AusRIA I or H (Abbott Laboratories, U.S.A.). HBsAg used in the titration experiment was purified as previously described (Koistinen, 1978). The protein concentration of the HBsAg preparation was determined by the m e t h o d of Lowry. RESULTS
Specificity and sensitivity of the subtype RIA The radiolabelled antibody reagents differed in some respects. When anti-d* was used, a positive result was obtained only for samples containing HBsAg/ad, while samples of subtype ay were negative. Thus, the anti-d* reagent was completely monospecific. Anti-y* was not totally monospecific for ay antigen: most ad samples (except very weakly positive ones) were also weakly positive with anti-y*. However, using anti-y* the count rates were much lower for ad than for ay samples, the proportion being about 1/5-1/10. We failed to produce a completely monospecific anti-y*, although we tried several immunoadsorbents made from different antigen preparations (crude or purified HBsAg of different origin). Even the same HBsAg/ad serum from which the immunoadsorbent was prepared was positive when tested with the resulting anti-y*. Another difference between the two labelled reagents was that anti-y* usually gave notably higher maximal values (about 10,000 cpm) than did anti-d* (about 2000 cpm). This difference did not, however, affect the sensitivity or the interpretation of the test. The criteria for the subtyping were as follows: A sample positive with anti-d* (and weakly positive or negative with anti-y*) was regarded as subtype ad. A sample negative with anti-d* but positive with anti-y* was considered to be subtype ay. The sensitivity of the test was determined by a titration where 5-fold
180
1200
1200
lOOC
I000
80C
800
60C
u
600
40C
4O0
20C
2OO
t
,_
ly
•
anti- d" T
I0000
400 2000 80
16
06 32
003
I0000
01
HBsAg/ad (ng/ml)
, , , li~ ' ~ - - ~ - - ~ 16 06 003 4O0 32 0.I 2000 80
HBsAg/ay (ng/rnl)
Fig. 1. Titration o f purified HBsAg (A: subtype ad; and B: subtype ay) with radiolabelled anti-HBs reagents anti-d* and anti-y*. The intercept o f the curve and the cut-off line indicates the sensitivity of each test system.
dilutions of purified HBsAg (diluted in normal human plasma) were assayed with different labelled anti-HBs reagents (Figs. 1 and 2). The intercept of the titration curve and the corresponding cut-off line indicates the minimum amount of antigen detectable by the test system. The results are summarized in Table 1. The sensitivity of the subtype RIA was 3--4 ng/ml while unabsorbed antibody reagents detected less than 1 ng/ml of antigen. Thus, the sensitivity of the subtype RIA was about 1/4--1/6 of that of the ordinary FRC-RIA. Anti-d* was about 50 times more sensitive for ad than anti-y*. I0000
I00001
B anti ay" anti-ad" anli-ay"
anli ad '
1000
IOOC
I
J
[oo
I00
20,L_~ I0000
400 2000 80
HBsAg/ad
16
32
06
(ng/ml)
OI
0 03
20 I0000
~L 400 2000 80
HBsAg/ay
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, ~-06 32
,
oI
0 03
(ng/ml)
Fig. 2. Titration of purified HBsAg (A: subtype ad ; and B: subtype ay ) with radiolabelled unabsorbed anti-HBs reagents anti-ad* and anti-ay*. The intercept of the curve and the cut-off line indicates the sensitivity o f each test system.
181 TABLE 1 THE SENSITIVITY OF THE DIFFERENT RADIOLABELLED anti-HBs PREPARATIONS IN DETECTING HBsAg The results are based on the titration curves illustrated in Figs. 1 and 2. Subtype of HBsAg tested
ad ay
Minimum amount of HBsAg (ng/ml) detectable by anti-ad*
anti-ay*
0.6 0.7
0.8 0.6
anti-d* 3 > 104
anti-y* 160 4
A n t i - y * , w h i c h was n o t e n t i r e l y m o n o s p e c i f i c , was a b o u t 40 t i m e s m o r e s e n s i t i v e f o r ay t h a n f o r ad a n t i g e n . 1 0 / ~ g / m l was t h e h i g h e s t c o n c e n t r a t i o n o f p u r i f i e d H B s A g / a y t e s t e d a n d it was n e g a t i v e w i t h a n t i - d * , i n d i c a t i n g complete monospecificity.
Comparison with subtyping by ID T w e n t y - f o u r s a m p l e s f r o m p a t i e n t m a t e r i a l p r e v i o u s l y s u b t y p e d b y ID w e r e a s s a y e d b y t h e s u b t y p e R I A . T w e n t y - t w o gave i d e n t i c a l r e s u l t s b y b o t h m e t h o d s ( 1 0 ad a n d 12 ay). O n e s a m p l e w e a k l y p o s i t i v e in ID a n d i n t e r p r e t e d as d o u b t f u l l y ad was d e f i n i t e l y s u b t y p e ay b y R I A . A n o t h e r s a m p l e , s u b t y p e ay b y ID, gave high c o u n t s w i t h a n t i - y * b u t it was also w e a k l y p o s i t i v e w i t h a n t i - d * . T h e s a m p l e was r e g a r d e d as s u b t y p e ay, a l t h o u g h it m i g h t h a v e h a d an u n c o m m o n s u b t y p e c o n f i g u r a t i o n ady.
TABLE 2 TYPABILITY OF DIFFERENT MATERIALS POSITIVE FOR HBsAg ND = not done; N = number of samples. Result in HBsAg testing by different methods
Total tested
Typable by RIA
Blood donors (N = 163) FRC-RIA pos., CIEP neg. FRC-RIA pos., CIEP ND CIEP pos., FRC-RIA ND Patients (N = 33) AusRIA pos., CF neg. CF and AusRIA pos. a Anticomplementary serum.
ID
N
%
12 27 124
7 26 124
58 96 100
8 25
8 25
100 100
N
% ND ND ND
1a 23
13 92
182
Typability of different HBsAg positive samples One hundred and sixty-three samples from HBsAg positive blood donors were subtyped by RIA (Table 2). All of the samples positive by CIEP were typable by RIA at the 1 : 10 dilution routinely used for strongly positive samples. Of 12 samples negative by CIEP but weakly positive in FRC-RIA screening, 5 (42%) were negative in the subtype RIA. The patient material consisted of 33 samples positive for HBsAg by CF or AusRIA (Table 2). All were typable by RIA, including 8 CF-negative samples. 73% could be subtyped by ID; all CF-negative samples (except one strongly anticomplementary sample) were negative by ID. DISCUSSION The radioimmunological methods used to determine HBsAg subtypes may be grouped in 3 categories: (1) A competitive RIA, in which the antigen in the sample competes for the anti-HBs antibody with labelled antigen of known subtype (Aach et al., 1973). (2) A modification of the sandwich-type solid-phase antibody RIA, in which the antigen in the sample binds the subtype specific soluble antibody, thus preventing its binding to the solid-phase antigen and leaving the subsequently added labelled antigen in solution (Ng et al., 1976; Hoofnagle et al., 1977). (3) A sandwich-type antigen RIA, in which labelled antibodies more or less subtype specific are used (Ling et al., 1973; Miyakawa et al., 1975). Our m e t h o d belongs to the last group. It differs, however, in one important respect: the liquid-phase antibodies are made monospecific after radiolabelling. Technically, this procedure has obvious advantages over those where the antibodies are absorbed to monospecificity before labelling: First, the test can be easily set up as a modification of the ordinary sandwich-type RIA, because no extra labelling of antibodies is needed for subtyping. Second, small amounts of monospecific antibody can be prepared at a time. This is possible because enough carrier protein (bovine serum albumin in this work} can be used to prevent non-specific absorption, and, consequently, very low amounts of antibody can be absorbed without significant loss. When subtyping is done in a routine diagnostic laboratory, the number of subtyping tests carried o u t is only a minute fraction of the number of HBsAg screening tests. Consumption of labelled monospecific antibodies is thus very small compared with the amount of labelled unabsorbed antibodies used in screening. In our method, waste of antibodies and radioactive iodine is avoided. Third, only crude antigen preparation is required for immunoadsorption. It is not necessary to maximize the capacity per unit volume of immunoadsorbent, as low amounts of antibody (about 1 pg in this work) can be used in the presence of carrier protein. We used a PEG precipitate from HBsAg positive plasma which was as effective as purified HBsAg.
183 F o u r t h , it is apparent t hat commercial RIAs or e n z y m e immunoassays may be modified in the way described here for FRC-RIA. T he only additional reagent needed is the solid-phase (crude) antigen easily prepared in any laboratory. Why we could n o t produce completely monospecific anti-y* remained unclear. This did n o t invalidate the m e t h o d , because anti-y* gave considerably higher c o u n t rates and bet t er sensitivity for ay than for ad samples. It is apparent also from earlier reports that totally monospecific reagent~ are n o t necessary for subtyping (Ginsberg et al., 1972; Aach et al., 1973; Hoofnagle et al., 1977). A b o u t one-fifth o f the sensitivity of the ordinary FRC-RIA was obtained when the anti-a specific antibodies were absorbed out. The sensitivity of FRC-RIA is comparable with that of the most sensitive commercial methods, e.g. AusRIA II ( U kkone n et al., 1977; Koistinen, 1978). Other reports on subtype RIA do not state the sensitivity in absolute units (ng/ml) o f HBsAg, and, therefore, the comparison is difficult. Hoofnagle et al. (1977) used a modification of AusAb ( A b b o t t Laboratories, U.S.A.), the sensitivity of which was only 1 / 2 0 - - 1 / 1 0 0 o f that of AusRIA II. We s u b ty p ed 27 blood d o n o r samples HBsAg positive by routine FRCRIA screening: as m any as 96% (26/27) were typable by RIA. This percentage illustrates the practical efficiency of the test, as the d o n o r group tested represented unselected new HBsAg carriers. However, some samples are t o o weak to be s u b typed by any m e t h o d , because sensitivity is usually somewhat reduced when an HBsAg test is modified for subtyping. This is evident from the work we have quoted. ACKNOWLEDGEMENTS The study was supported by grants from the Finnish Medical F o u n d a t i o n and the Orion F o u n d a t i o n , and by a grant from the Sigrid Jus~lius Foundation to Prof. K. Penttinen. T he e xpe r t technical assistance of Sinikka Hakkarainen is gratefully acknowledged. REFERENCES Aach, R.D., E.J. Hacker and C.W. Parker, 1973, J. Immunol. 111,381. Bancroft, W.H., F.K. Mundon and P.K. Russell, 1972, J. Immunol. 109,842. Cuatrecasas, P., 1970, J. Biol. Chem. 245, 3059. Ginsberg, A.L., W.H. Bancroft and M.E. Conrad, 1972, J. Lab. Clin. Med. 80,291. Helske, T., 1974, Scand. J. Haematol. Suppl. 22. Hoofnagle, J.H., R.J. Gerety, L.A. Smallwood and L.F. Barker, 1977, Gastroenterology 72,290. Koistinen, V.U., 1978, Vox Sang. 34,321. LeBouvier, G., 1971, J. Infect. Dis. 123,671. Ling, C.M., H. Irace, R. Decker and L.R. Overby, 1973, Science 180,203. Miyakawa, Y., M. Imai and M. Mayumi, 1975, J. Immunol. 114, 1135. Ng, P.L., L.W. Powell, J.W. Halliday, K.L. Gera, L. McKeering and C.B. Campbell, 1976, Am. J. Gastroenterol. 65,127. Nordenfelt, E., 1975, Scand. J. Infect. Dis. 7, 147. Ukkonen, P., V. Keistinen and K. Penttinen, 1977, J. Immunol. Methods 15, 343.
