- Email: [email protected]
Enzyme potentiated radioimmunoassay (EPRIA): A sensitive third-generation test for the detection of hepatitis B surface antigen
Journal of Immunological Methods, 47 (1981) 145--159 Elsevier/North-Holland Biomedical Press
145
ENZYME POTENTIATED RADIOIMMUNOASSAY (EPRIA): A SENSITIVE THIRD-GENERATION TEST FOR THE DETECTION OF HEPATITIS B SURFACE ANTIGEN
HOWARD A. FIELDS, CANDACE L. DAVIS, GORDON R. DREESMAN, DANIEL W. BRADLEY and JAMES E. MAYNARD Hepatitis Laboratories Division *, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Phoenix, AZ, and Department of Virology and Epidemiology, Baylor College of Medicine, Houston, TX, U.S.A. (Received 3 April 1981, accepted 15 July 1981) A sensitive, specific immunoassay for detection of hepatitis B surface antigen (HBsAg) is described. The assay combines enzyme-linked immunosorbent assay and solid-phase radioimmunoassay and is termed enzyme potentiated radioimmunoassay (EPRIA). HBsAg was quantitated by enzymatic conversion of L-[14C]glutamic acid to 14CO2 and gamma-aminobutyric acid by glutamate decarboxylase (GDC) conjugated with goat antiHBs IgG. Conjugation of IgG and GDC was by a thiol-disulfide bond exchange reaction after reacting N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) with each reagent. A positive/negative ratio of 2.2 was established as significant by examination of 40 normal sera negative for HBsAg. This value was the mean cpm plus 3 standard deviations. By an identical statistical analysis of sensitivity, EPRIA was found to be approximately 100-fold more sensitive than Ausria II (Abbott Laboratories, North Chicago, IL). INTRODUCTION R a d i o i m m u n o a s s a y ( R I A ) has b e e n u s e d e x t e n s i v e l y f o r d e t e c t i o n o f h e p a t i t i s a s s o c i a t e d antigens a n d a n t i b o d i e s in sera, biological tissues, a n d f e c e s ( H o l l i n g e r et al., 1 9 7 1 , 1 9 7 5 b; Ling a n d O v e r b y , 1 9 7 2 ; Fields e t al., 1 9 7 8 ) . R a d i o i m m u n o a s s a y s w h i c h use ~:SI-labeled r e a g e n t s are l i m i t e d b y a relatively s h o r t shelf life a n d r a d i a t i o n h a z a r d . T h e d e v e l o p m e n t o f e n z y m e l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) (Engvall a n d P e r l m a n n , 1 9 7 1 ) h a s obv i a t e d t h e s e difficulties. E L I S A s y s t e m s are q u a n t i t a t e d b y s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f e n z y m e s u b s t r a t e r e a c t i o n s . D e s p i t e t h e i r amplif y i n g n a t u r e , h o w e v e r , t h e y d o n o t u s u a l l y e x c e e d t h e sensitivity o f R I A (Watson, 1 9 7 6 ; Jarvis, 1 9 7 9 ) . This r e p o r t d e s c r i b e s an assay t e r m e d e n z y m e p o t e n t i a t e d r a d i o i m m u n o assay ( E P R I A ) w h i c h c o m b i n e s t h e p r i n c i p l e s o f E L I S A a n d R I A . 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 ) is q u a n t i t a t e d b y e n z y m a t i c c o n v e r s i o n o f [14C]g l u t a m i c acid to ~4CO2 a n d g a m m a - a m i n o b u t y r i c acid b y g l u t a m a t e decar* World Health Organization Collaborating Centre for Reference and Research on Viral Hepatitis. 0022-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
146
boxylase (GDC) conjugated to IgG. The product, ~4CO2, is measured by liquid scintillation beta spectrometry. This gives a significant increase in sensitivity compared with RIA. MATERIALS AND METHODS
Reagents L-[ 1J4C]Glutamic acid (40--50 mCi/mmol), Protosol and Econofluor were from New England Nuclear (Boston, MA). The substrate was diluted to contain l 0 s cpm per 0.2 ml of 0.01 M sodium acetate buffer, pH 3.8. GDC type V and 2,2'-dipyridyl disulfide (2-PDS) were from Sigma Chemical Company (St. Louis, MO). GDC was further purified on a 2.6 cm X 90 cm column of Sephacryl S-300 (Pharmacia Fine Chemicals, Piscataway, NJ) equilibrated with phosphate buffered saline, pH 7.4, containing 1 : 4000 NaN3 (PBSN). As an alternative, GDC was purified from Escherichia coli (Yang and Metzler, 1979). N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was from Pharmacia Fine Chemicals. HBsAg positive serum was obtained by plasmapheresis of a chronic asymptomatic chimpanzee carrier. Normal human sera (NHS) and acute phase sera were from hepatitis A and non A/non B (NANB) infected individuals. All sera were stored at --20°C.
Purification of HBsAg HBsAg was purified from plasma by ultracentrifugation (Dreesman et al., 1972). Antigen was twice pelleted, sonicated, treated with a pH 2.4 buffer, and subjected to two isopycnic ultracentrifugation bandings in CsC1 followed by rate-zonal ultracentrifugation in CsCi. HBsAg was localized by rocket immunoelectrophoresis (Laurell, 1967) with an LKB Multiphor unit and power supply.
Immunization of guinea pigs Twenty pg purified HBsAg was mixed with Freund's complete adjuvant and four guinea pigs (g.p.) were injected intradermally in each rear foot pad. The animals were boosted by the intramuscular route every 2 weeks with antigen in Freund's incomplete adjuvant and exsanguinated when the antiHBs titer exceeded 1 : 1 X 106 by Ausab (Abbott Laboratories, North Chicago, IL). The IgG was fractionated from these sera by precipitation with saturated ammonium sulfate at 4°C followed by DEAE-cellulose chromatography (DE-52, Whatman, Clifton, NJ). Goat anti-HBs serum was obtained from the World Health Organization (WHO) and the IgG fraction prepared as described above for g.p. antisera. Immunospecific goat anti-HBs IgG was purified by affinity chromatography. Purified HBsAg was coupled to CNBr-activated Sepharose according to the manufacturer's recommendations (Pharmacia Fine Chemicals). Six ml of antiserum was chromatographed at 2--3 ml/h (0.9 cm X 10 cm column) with
147 0.1 M phosphate buffer, pH 7.0, containing 0.5 M NaC1. When the O.D.280 returned to baseline, specific a n t i b o d y was eluted with 0.2 M glycine, pH 2.5. The eluted fractions were neutralized and stored at 4°C. Purity was ascertained by immunoelectrophoresis with rabbit anti-goat serum.
SPDP thiolation Purified GDC and IgG were thiolated at different SPDP : protein molar ratios. The degree of substitution (number of thiol groups) was determined by the m e t h o d of Grossetti and Murray (1967). Briefly, after reacting GDC or IgG with SPDP for 30 min, the modified protein was desalted on a 1.6 cm × 90 cm column of Sephadex G-25 (Pharmacia Fine Chemicals} equilibrated with 0.1 M sodium acetate buffer, pH 4.6, containing 0.1 M NaC1 (buffer A). One molar dithiothreitol (DTT) was added to the modified protein preparation to a final DTT concentration of 50 mM. After incubation for 30 min at room temperature, the reaction mixture was desalted on a second Sephadex G-25 column equilibrated with 0.1 M Tris-HC1 buffer, pH 7.4, containing 0.5 M KC1 and 1 mM EDTA (buffer B). The eluted proteins were concentrated to a volume of 3.5 ml by ultrafiltration, by means of a 25 mm diameter YM10 ultrafilter and an 8 MC stirred cell (Amicon, Lexington, MA). Equal volumes of thiolated protein and 1.5 mM 2-PDS were added to a cuvette and the concentration of liberated 2-thiopyridone (2-TP) was determined spectrophotometrically at a wavelength of 343 nm after subtracting absorbance due to thiolated protein alone and 2-PDS alone. One mole of liberated 2-TP is equivalent to 1 thiol group.
Covalent chromatography To verify that GDC and IgG had been thiolated by SPDP, the thiolated proteins were chromatographed on thiopropyl Sepharose 6B (Pharmacia Fine Chemicals) according to the manufacturer's recommendations. All proteins to which more than 1 thiol group had been introduced by reaction with SPDP were covalently bound to the column and were subsequently eluted with a buffer containing 50 mM DTT.
Conjugation o f IgG and GDC Conjugates designed to be composed of one molecule of IgG and one molecule of GDC were prepared by mixing the respective SPDP modified proteins. Each protein was substituted with 1--2 residues/mole of protein. IgG was modified to contain 1--2 residues of 2-pyridyl disulfide per mole by the addition of 2.4 moles of SPDP/mole of IgG. The modified protein was desalted on G-25 equilibrated with buffer B and concentrated to 4 ml by ultrafiltration. GDC was modified to contain 1--2 thiol residues/mole by addition of 75 moles of SPDP/mole of GDC. The substituted protein preparation was desalted on G-25 equilibrated with buffer A, reduced with 50 mM DTT, and desalted again on G-25 equilibrated with buffer B. The thiolated protein was concentrated to 4 ml by ultrafiltration and used the
148
same day. The substituted proteins were then mixed together at a molar ratio of 6--10 moles IgG/mole GDC and rotated for 22 h at 27°C. Finally, the reaction mixture was fractionated by gel filtration chromatography on a 2.6 cm × 90 cm column of Bio-Rad A-1.5 m (Richmond, CA) equilibrated with PBSN at 4°C. Protein assay Protein concentration was determined by the Bio-Rad protein assay against the protein standard I for IgG and protein standard II for GDC supplied. Iodination Iodination of IgG with l~sI was by the chloramine-T method of G r e e n w o o d et al. (1963), as modified b y Hollinger et al. (1975b). Briefly, purified IgG was reacted with 0.5 mCi Na12SI in the presence of chloramineT (25 ~g) for 15 sec at room temperature and the reaction was stopped by addition of sodium metabisulfite (50 ]~g). Separation of b o u n d 12sI from free 12sI was accomplished on a PD-10 column of Sephadex G-25 (Pharmacia Fine Chemicals). The iodinated protein had a specific activity of 15--20 ~Ci/ /~g. E n z y m e assay One hundred microliters of protosol were added to a liquid scintillation vial containing a 2.4 cm GF/C (Whatman, Clifton, NJ) glass microfiber disc. Acetate buffer (0.2 ml) containing 25 mM L-glutamic acid and l 0 s cpm of L-[1-14C]glutamic acid was added to Immulon Removawells (Dynatech, Alexandria, VA) placed within 1/2-dram shell vials (Kimble). After addition of 10 ~1 of GDC, the shells were transferred to liquid scintillation vials. Each vial was capped with a Polyseal (Polyseal Corp., Iselin, NJ) cone-shaped screw cap and incubated at 37°C (Fig. 1). After incubation, the reaction was stopped by removing the shell. Fifteen ml of Econofluor containing 15% methanol were added to each vial which was then counted in a Delta 300 (Tracor Analytic) with the samples channel ratio set at 30%. Constant quenching eliminated the need for a quench correction. Units of enzyme (~moles/min) were determined by calculating the ]~moles of 14CO2 evolved. This value was multiplied b y the ratio of unlabeled : labeled substrate and added to the moles of radiolabeled p r o d u c t formed per minute at 37°C. EPRIA Optimal dilutions for coating Immulon micro-wells with antibody and for determining the optimal concentration o f conjugate were ascertained by checkerboard titration. Serial 2-fold dilutions of g.p. anti-HBs IgG prepared in 0.05 M Tris buffer, pH 8.4, containing 0.3 M KC1 and 2 mM EDTA (TKE) were used to coat the wells. After the wells had been washed, a dilution of
149
Liquid ScintillationVbal
'
" ......
½Dram Sh~l
Enzyme Reaction
~ 2 . 4 ~--Containing
cm Diameter GI=/C Filter P~er O.Iml Protosol
Fig. 1. Schematic representation of the reaction vessel for EPRIA.
HBsAg-positive plasma was added and tested against 2-fold dilutions of conjugate. The diluent for the conjugate was composed of equal volumes of NHS, normal goat serum, normal g.p. serum, and 0.05 M Tris buffer adjusted to a pH value of 7.5. Positive to negative ratios (P/N) were determined by calculating a quotient from the cpm for the test specimen divided by a mean cpm determined for at least 6 negative serum controls. The assay was performed by coating the wells with 250 ~1 of diluted g.p. anti-HBs IgG and washing each well 4 times with 0.5% bovine serum albumin (BSA) in PBSN (PBSN-BSA). Two hundred microliters of test sera or HBsAg-negative control sera diluted 1 : 8 0 0 in PBSN containing 1% BSA were added and incubated for 1 h at 45°C unless stated otherwise. After another wash cycle, 200 pl of conjugate were added and incubated for different times and at different temperatures. After a final wash cycle the wells were transferred to 1/2 dram shells (Kimble). Two h u n d r e d microliters of 0.01 M acetate buffer, pH 3.8 (unless stated otherwise), containing 10 s cpm of L-[1-14C]glutamic acid were added to the I m m u l o n wells which were then placed in liquid scintillation vials. Each vial was capped with a Polyseal cone-shaped screw cap (Fig. 1) and incubated at 37°C for 16 h. The reaction was stopped by removing the shell. Econofluor-methanol cocktail was added and the vials c o u n t e d in a Delta 300 liquid scintillation counter (see Materials and Methods on enzyme assay).
Specificity F o r t y normal h u m a n sera f o u n d to be negative for HBsAg by Ausria II were assayed by EPRIA. The values obtained were used to calculate a mean iX) cpm value and a standard deviation (S.D.). All 40 sera tested had a cpm value less than the X + 3.0 S.D. of X.
Sensitivity Serial 2-fold dilutions of HBsAg positive sera were made in PBSN-BSA.
150
Each dilution was tested for HBsAg by EPRIA, Ausria II and a modified micro-solid phase immunoradiometric assay (m-SPIRA). EPRIA end-point titers were determined by calculating a X + S.D. cpm value. The highest dilution cpm value which exceeded the X + 3.0 S.D. was considered as the endpoint titer. Ausria II and modified m-SPIRA endpoints were based upon a P/N value of 2.1. Conditions for the m-SPIRA were as previously described for detection of hepatitis core antigen (Fields et al., 1977) except t h a t the conjugate diluent was identical to the EPRIA conjugate diluent. RESULTS
Conjugation of IgG to GDC Each protein was reacted with SPDP at various molar ratios. The degree of substitution was ascertained by measuring the concentration of released 2-TP spectrophotometrically after reacting the thiolated proteins with 2-PDS. A calibration curve was constructed for each protein (Fig. 2). A ratio of approximately 2.4 moles of SPDP per mole of IgG was required to introduce between 1 and 2 thiol groups per mole of IgG. GDC required the addition of 75 moles of SPDP per mole to achieve approximately the same degree of thiolation. In both cases there was a linear relationship between the molar ratio of SPDP to protein and the degree of substitution. In an a t t e m p t to prepare a conjugate composed of one molecule of IgG and one molecule of GDC, each protein was reacted with SPDP to achieve a degree of substitution between 1 and 2. Preliminary experiments with GDC -?
140 s •
120E3 I 0 0 (_9
-5
o
"6 80-
n"~o
~5 ~
4020O-
J
-39.
f
I
I
I
I
2
3
I
1
0
Degree of Substitution (Number of Thiol Groups per mole of protein)
Fig. 2. Degree of substitution of GDC (e with various molar ratios of SPDP.
e) and IgG (o
o) after treatment
151
showed no reduction in enzyme activity after incubation with 50 mM DTT (data not shown). The specific activity of thiolated/GDC, however, was reduced by 35% as determined by calculating units of enzyme per mg of protein before and after thiolation. Thiolated GDC was mixed with 2-pyridyl disulfide substituted IgG at a molar ratio of 6--10 moles of IgG/mole of GDC to drive the thiol-disulfide bond exchange reaction to completion. The two protein solutions were rotated end-over-end at 27°C for 22 h. The reaction mixture was then chromatographed on a Bio-Rad A-1.5 m gel filtration column. Iodinated goat anti-HBs IgG (approximately 3 X 107 cpm) was mixed with the purified IgG fraction before SPDP modification to localize the IgG during the fractionation procedure. Each fraction was assayed for cpm by gamma spectrometry and for enzyme activity, except t h a t unlabeled glutamate was omitted. The fractionation profile is shown in Fig. 3. Two optical density (O.D.:80) peaks were observed preceding elution of unconjugated IgG. While each of these peaks showed approximately the same a m o u n t of radiolabeled IgG activity, the second peak (Pool III) had substantially more enzyme activity. No further attempts were made to determine the molecular composition of the conjugate. A small a m o u n t of enzyme activity was seen at fraction 150 accompanying an optical density shoulder. This shoulder may have been due to monomeric GDC subunits of molecular weight 50,000. The peak at fraction 157 was n o t characterized. All the enzyme was consumed in the forma55 5O o
.05
-
.04
I00
G~
-80 ~
2O -0
-600
t5
-40
I0
X 0 ,03-
...- i
II ci ~.02-
•: '
!,
.."
I
I':
I
0
- Z O v~
.01
V 0
I
40
,"" I
I
60
80
I I I I
5
,I :,,
if~ I
I00
I
120
I
140
i
160
i
-0
180
Froct~n Number (4ml/froc.) Fig. 3. F r a c t i o n a t i o n o f G D C - I g G c o n j u g a t e s o n a 2 . 6 c m × 9 0 c m c o l u m n o f B i o - R a d A-1.5m equilibrated with PBSN ( , O.D.280; ...... , GDC activity; ...... , [ 12 s I ] I g G a c t i v i t y ) . T h e f l o w r a t e w a s a d j u s t e d t o 1 2 m l / h a n d 4 - m l f r a c t i o n s w e r e collected.
152
tion of conjugate, as shown by the absence of an optical density or enzyme activity shoulder between the second conjugate peak and the free IgG peak. Previous experiments with equimolar a m o u n t of IgG and GDC repeatedly demonstrated free GDC activity around fraction 111.
Checkerboard titration Pools were made as in Fig. 3 and each pool was assayed for performance in the EPRIA system by checkerboard titration. Serial 2-fold dilutions of g.p. anti-HBs IgG were made in TKE buffer and I m m u l o n wells were coated for 1 h at 4°C. Each coating dilution was tested against 2-fold dilutions of each pool as shown in Table 1. Pool II was assayed on two separate occasions and both times it was found that a 1 : 25 (3.5 pg/well) dilution of g.p. antiHBs IgG and a 1 : 32 dilution of conjugate were optimal. The difference in P/N values between trial 1 and trial 2 was due to differences in the d e n o m i n a t o r (N value) in which small changes have very large effects on quotients. Pool II consistently yielded the highest P/N value. Values in tests with pool I were n o t tabulated because of insufficient enzyme activity. Since pool IV was composed of both pool II and III, its performance was intermediate.
Optimal conditions for Immulon coating Guinea pig anti-HBs IgG was diluted in TKE buffer and 250 pl were added to I m m u l o n wells. The wells were incubated for various lengths of time at TABLE 1 Determination of optimal reagent concentration. Pool
Conjugate dilution
P/N D i l u t i o n o f g.p. anti-HBs IgG 1:12.5
1:25
1:50
1:100
1:200
II
1 : 8 1 : 16 1 : 32
176 201 333
205 265 381 b
235 269 308
NT NT NT
NT a NT NT
II
1 : 16 1 : 32 1 : 64
NT NT NT
225 249 140
199 248 97
191 180 177
50 64 48
III
1 :
16 1 : 32 1 : 64
NT NT NT
39 61 34
29 32 29
35 33 18
10 8 6
IV
1 : 16 1 : 32 1 : 64
NT NT NT
157 200 167
169 182 129
192 163 195
55 65 42
a Not tested. b U n d e r s c o r e i n d i c a t e s m a x i m u m P/N o b t a i n e d for each p o o l .
153
4°C and at 25°C. The g.p. anti-HBs IgG preparation was obtained from a different animal from that used for evaluation of the various pools. As Fig. 4 shows, optimal conditions for coating were incubation of the wells at 4°C for 60 min with a 1 : 200 dilution of IgG. This dilution corresponds to 0.56 pg of protein per well. This g.p. antibody and these conditions for coating were used for the remainder of the study. Effect o f conjugate time and temperature incubation conditions on E P R I A Optimal reagent dilutions were established by checkerboard titration of pool TI incubated for 2 h at 37°C. Conjugate incubation times and temperatures were tested as indicated in Fig. 5. The original conditions for conjugate incubation of pool II were shown to be optimal, i.e., 2 h at 37°C. The results were expressed as functions of the P/N value. Examination of the individual P and N values (data n o t shown) showed that the P value increased for the first 2 h and then remained relatively constant; the N value continued to rise t h r o u g h o u t the incubation period. These findings indicated that equilibrium for the conjugate was reached in 2 h. Effect o f p H on E P R I A To determine the optimal pH for the enzyme assay, coated Immulon wells were incubated with a constant a m o u n t of HBsAg diluted (1 : 800) in PBSNBSA, washed, and then reacted with conjugate. After a final wash, 0.20 ml of 0.01 M acetate buffer containing 10 s cpm of substrate adjusted to the pH values shown in Fig. 6 were added to each well. P/N values were determined with NHS diluted (1 : 800) in PBSN-BSA as the negative control. As shown in Fig. 6, there was a broad plateau of activity from pH 3.4 to 4.4, with a pH o p t i m u m at 3.8. The activity d r o p p e d precipitously at higher pH values. These results are similar to those of Shukuya and Schwert (1960) for free
I00
..% 25°C
80
. . . . . .3o,~. --"
..."",,,-
. . ;,•.
,.
•
',,,
•
•
Z v
•
N
",,.,., \
2O 0
i 1:50
I:100
u 1:200
I
1:50 Dilution of Coating IgG 1:400
I
HO0
I
1:200
I
1:400
Fig. 4. E f f e c t o f t i m e a n d t e m p e r a t u r e o n passive a d s o r p t i o n o f g.p. a n t i - H B s IgG. V a r i o u s c o n c e n t r a t i o n s o f IgG were a d s o r b e d t o I m m u l o n wells a t 25°C a n d 4°C for 30, 60 a n d 1 2 0 rain. O p t i m a l c o n d i t i o n s were a s c e r t a i n e d b y determining P / N values•
154 8070-
60I00 -
50•o.
Z
80-
h: 4o-
37°C ." ~-
"I.~,.,
60-
30-
40-
20-
20-
tO-
Z
O-
I I
I 2
I 3
Time (hours)
I 4
O'
3.4
\ I
I
I
I
3B
4.2
4.6
5D
I ,5.4
pH
Fig. 5. E f f e c t of time and t e m p e r a t u r e o f conjugate i n c u b a t i o n on P/N values as determ i n e d by EPRIA. Optimal c o n c e n t r a t i o n s of coating g.p. anti-HBs IgG and conjugate were previously established by c h e c k e r b o a r d titration• Three temperatures (m _, 25Oc; • . . . . . . • , 37°C; • . . . . . .A, 45OC) were e x a m i n e d at 1-h intervals for 4 h. Optimal c o n d i t i o n s were ascertained by determining P/N values• Fig. 6. E f f e c t of pH on P/N values as d e t e r m i n e d by E P R I A . Optimal conditions for coating g.p. anti-HBs IgG and conjugate dilution were previously established by checkerboard titration• Substrate diluted in 0.01 M acetate buffer at various pH values was added to I m m u l o n wells and incubated. O p t i m u m pH was ascertained by determining P/N values•
GDC using either pyridine-pyridine h y d r o c h l o r i d e or glycine-glycine hydrochloride buffers.
Specificity of EPRIA A f r e q u e n c y distribution for 40 normal h u m a n sera shown t o be negative for HBsAg when tested by an Ausria II assay, was det erm i ned from the values o b tain ed by testing each serum by EPRIA. The values are pl ot t ed in Fig. 7. The X value obtained after subtraction o f the background was 191 cpm with a S.D. value of +-76 cpm. All the sera tested fell below the X + 3 S.D. value (424), which corresponds to a 99.9% confidence level with a one-tailed test. Thirty-eight o u t of 40 sera fell below the X + 2 S.D. units. The X + 3 S.D. units (424 cpm) is equivalent to a P/N value of 2.2. E xamin atio n of 142 sera including Ausria II negative NHS and sera from patients with acute hepatitis A or acute hepatitis NANB gave only 4 false
155 -i S.D
(115)
1
~
. I S.D.
• 2 SD
*3 .~D.
(~)
(267)
(,~47)
(424)
1
1
itS
1
%
0~
I
1
'
'
'
'
IO0
I
'
'
'
'
200
I
.
.
.
300
.
!
'
4O0
CPM
Fig. 7. F r e q u e n c y d i s t r i b u t i o n o f 4 0 n o r m a l h u m a n sera t e s t e d b y E P R I A . F o r t y Ausria II n e g a t i v e h u m a n sera s p e c i m e n s were t e s t e d b y E P R I A a n d m e a n c p m value a n d stand a r d d e v i a t i o n o f t h e m e a n were d e t e r m i n e d .
TABLE 2 Sensitivity c o m p a r i s o n Of m - S P I R A , A u s r i a II, a n d E P R I A . Dilution (1 : 1 0 0 x 2 n) n
P/N m-SPIRA a
A u s r i a II
0
NT f
3
3.24 g
NT 66.7 42.8 21.6 14.7 7.5 3.7
4 5 6 7 8 9 10. 11 12 13 14 15 16
NT 1.9 NT 1.3 NT 1.2 1.1 NT NT NT NT NT NT
b
2.8
1.7 1.3 1.3 NT NT NT NT
a HBsAg incubation 2 h at 45°C; conjugate HBs [ 1 2 s I ] I g G as p r o b e . b P r o c e d u r e A. c HBsAg incubation 1 h at 45°C; conjugate HBs IgG c o n j u g a t e d t o G D C as p r o b e . d HBsAg incubation 1 h at 45°C; conjugate r a p h y p u r i f i e d g o a t a n t i - H B s IgG c o n j u g a t e d e HBsAg incubation 2 h at 45°C; conjugate r a p h y p u r i f i e d g o a t a n t i - H B s IgG c o n j u g a t e d f Not tested. g Underscore indicates end-point.
EPRIA c
EPRIA d
EPRIA e
283.5 186.8 141.3 72.5 50.6 21.5 12.4 8.5 4.04
2 153.0 550.2 413.6 206.8 118.1 66.6 31.7 18.9 9.2 5.9 4.2 3.0 2.0
NT NT NT NT NT NT NT NT 19.1 10.1 5.0 4.1 2.8
2.35
1.66 1.7 1.1 NT NT
1.80
2.3
0.9
1.7
i n c u b a t i o n 2 h a t 4 5 ° C ; p o l y c l o n a l g o a t anti-
i n c u b a t i o n 2 h a t 3 7 ° C ; p o l y c l o n a l g o a t antiincubation 2 h at 37°C; affinity chromatogt o G D C as probe. incubation 2 h at 37°C; affinity chromatogt o G D C as p r o b e .
156
positives (2.8%), none of which exceeded a P/N value of 3.9. These sera could not be neutralized with WHO reference goat anti-HBs and were subsequently classified as negatives.
Sensitivity of EPRIA Serial 2-fold dilutions of an HBsAg-positive serum were made in PBSNBSA. Each dilution was tested in duplicate. P/N values were determined using NHS diluted in PBSN-BSA as a negative control. The X plus 3 S.D. units of this negative control correspond to a P/N value of 1.8. The endpoint was considered to be the dilution at which the P/N value obtained fell below 1.8. Identical dilutions were tested for endpoint titer by a modified m-SPIRA, Ausria II, as well as under three different conditions for EPRIA (Table 2). The end-point titer of an HBsAg-positive specimen as determined by Ausria II was 1 0 0 × 29 (1 : 51,200). Ausria II utilizes affinity chromatography purified h u m a n anti-HBs IgG radiolabeled with 12sI as the probe. When a polyclonal goat anti-HBs IgG radiolabeled with 12sI was used as in the modified m-SPIRA (Table 2) then the sensitivity was reduced 64-fold. This is indicated by comparison of the end-point titers observed with Ausria II and m-SPIRA. It was of considerable interest t h a t utilization of polyclonal goat anti-HBs IgG conjugated to GDC in EPRIA exceeded the sensitivity afforded
706050-
z
403020EPRIA t
I00 7
EPRIA 2"
t,~iol I ~ _ I
I
I
I
I
I
I
8
9
I0
II
12
13
14
1 I
I
15
16
n (dilution= IOOx 2") Fig. 8. S e n s i t i v i t y o f E P R I A . O p t i m a l c o n d i t i o n s for c o a t i n g g.p. a n t i - H B s IgG a n d conjugate d i l u t i o n s were previously d e t e r m i n e d b y c h e c k e r b o a r d t i t r a t i o n . Serial 2-fold dilut i o n s o f HBsAg-positive sera were m a d e in PBSN-BSA. E a c h d i l u t i o n was t e s t e d in duplicate b y Ausria II a n d E P R I A . P / N values were d e t e r m i n e d w i t h t h e m e a n c p m o b t a i n e d f r o m a t least 6 negative c o n t r o l s in t h e d e n o m i n a t o r . F o o t n o t e s 1 a n d 2 c o r r e s p o n d to f o o t n o t e s c a n d d in T a b l e 2, respectively.
157
by Ausria II 4-fold. Moreover, utilization of affinity chromatography purified goat anti-HBs IgG conjugated to GDC in EPRIA exceeded the sensitivity of Ausria II 64-fold when the antigen was incubated for 1 h at 45°C and almost 128-fold when the antigen was incubated for 2 h at 45°C. A portion of these data are illustrated graphically in Fig. 8. DISCUSSION
EPRIA combines in one test the amplification of ELISA and the sensitivity afforded by RIA instrumentation. This combination resulted in a novel assay for detection of HBsAg approximately 100-fold more sensitive than Ausria II. Because of the microtiter f o r m a t the assay is easy to perform and large numbers of samples may be processed at one time. The gaseous product of the enzyme reaction (~4CO2) separates from the aqueous phase substrate w i t h o u t the introduction of a tedious biochemical separation. Despite its long half life, the use of 14C represents a significant reduction in the biological hazard as compared with other isotopes such as '2sI and 32p (National Institutes of Health Radiation Safety Guide, 1972). Numerous attempts to conjugate GDC to IgG by the glutaraldehyde method (Avrameas, 1969~ Avrameas and Ternynck, 1971) were unsuccessful. GDC was consistently refractory to glutaraldehyde activation and as a consequence h o m o p o l y m e r s of IgG were preferentially produced. GDC does not possess any known carbohydrate m o i e t y and this precluded the metaperiodate oxidation m e t h o d developed by Wilson and Nakane (1978). A new m e t h o d of conjugating two proteins was developed by Carlsson et al. (1978) based upon a thiol-disulfide bond exchange reaction. This m e t h o d has the distinct advantage of allowing control over the molecular size of the conjugate and, therefore, the specific activity. In addition, conjugates prepared by this m e t h o d are stable but m a y be cleaved with reducing agents such as DTT. Covalent chromatography and direct determination of thiol groups (Grossetti and Murray, 1967) revealed t h a t native GDC does n o t contain sulfhydryl groups accessible for reaction in a thiol-disulfide bond exchange reaction. Therefore, to conjugate GDC to IgG by this reaction it was necessary to introduce a thiol group by prior modification with SPDP. To achieve a degree of thiolation of between 1 and 2 residues per mole of GDC a molar ratio of SPDP to GDC of 75 was necessary. Since SPDP attacks the e-amino group of lysine, these amino groups were apparently n o t as readily accessible for modification as t h e y were for IgG. This observation was consistent with our inability to conjugate GDC to IgG by the glutaraldehyde m e t h o d which also utilizes the e-amino groups of lysine. Conjugates prepared by the thiol-disulphide bond exchange reaction were fractionated by gel filtration chromatography (Bio-Rad A-1.5 m). Pools were made and evaluated by EPRIA. Pools II, III, and IV all demonstrated EPRIA activity. Pool IV was rechromatographed on a Bio-Rad A-0.5 m column
158 which excluded proteins of molecular weight exceeding 5.0 × 10 s. This fractionation procedure resulted in the removal of free IgG which contaminated pools III and IV and c o m p e t e d for available antigen sites in the test. We found that different lots of g.p. anti-HBs IgG required different concentrations of IgG per well for o p t i m u m performance. Factors such as differences in antibody avidity, antibody affinity for the solid phase matrix, and passive adsorption properties of the wells may all be important. Before each assay was performed o p t i m u m conditions were established by checkerboard titration to minimize these differences. The specificity of the test was demonstrated to be acceptable although n o t as good as commercial kits (personal observations). Our observed frequency of 2.8% false positives may be a necessary compromise for the increased sensitivity generated by EPRIA. We are studying the effects on specificity and sensitivity of immunoabsorbing g.p. anti-HBs IgG before passive adsorption on polystyrene. Since HBsAg hyperimmunized laboratory animals demonstrate anti-NHS activity, removal of this activity before coating of Immulon wells may improve the specificity. Nevertheless, false positives are easily identified by a simple neutralization step before retesting. The most important application of EPRIA is in providing a sensitive serologic test in the absence of affinity chromatography purified antibody. Table 2 clearly demonstrates that a modified m-SPIRA for the detection of HBsAg using iodinated polyclonal IgG resulted in a test severely limited by its lowered sensitivity. Comparing the sensitivity obtained b y m-SPIRA with that of EPRIA using polyclonal antibody for each assay showed that EPRIA was 256 times more sensitive. Thus, EPRIA affords excellent sensitivity without the requirement for immunospecific antibody. This m a y be important for the development of tests for viral antigens, such as those in NANB hepatitis, the concentrations of which are apparently much lower than for plasmas from patients infected with hepatitis type B virus, or for which it is n o t at present possible to prepare immunospecific antibody. The sensitivity of EPRIA is limited by the specificity of the reagents. Conjugates must be diluted so that the background is within acceptable limits. An examination of the effect of conjugate dilutions on P/N ratio revealed that the denominator (-N value) decreased more rapidly than the numerator (P value) (data not shown). Thus, as the conjugate was diluted the quotient became higher until it reached a maximum value and then d r o p p e d rapidly. Reactions contributing to the denominator value, i.e., non-specific results, are diluted out much more readily than the specific reactions. If the nonspecific reactions could be reduced further, higher concentrations of conjugate could be used, giving specific positive responses of greater magnitude. We are investigating the possibility of immuno-specifically purifying coating antibody and of using monoc!onal antibodies to improve specificity. Until more specific reagents are available for detection of HBsAg, the development of other ultrasensitive tests such as those described by Harris et al. (1979) and Shalev et al. (1980) may n o t be possible.
159 REFERENCES Avrameas, S., 1969, Immunochemistry 8, 336. Avrameas, S. and T. Ternynck, 1971, Immunochemistry 8, 1175. Carlsson, J., H. Drevin and R. Axen, 1978, Biochem. J. 173,723. Dreesman, G.R., F.B. Hollinger, R.B. McCombs and J.L. Melnick, 1972, Infect. Immun. 5, 213. Engvall, E. and P. Perlmann, 1971, Immunochemistry 8, 871. Fields, H.A., F.B. Hollinger, J. Desmyter, J.L. Melnick and G.R. Dreesman, 1977, Intervirology 8, 336. Fields, H.A., D.W. Bradley, C.L. Davis, B.L. Murphy, C.A. Schable and J.E. Maynard, 1978, J. Immunol. 121, 930. Greenwood, F.C., W.M. Hunter and J.S. Glever, 1963, Biochem. J. 89, 114. Grossetti, D.R. and J.F. Murray, 1967, Arch. Biochem. Biophys. 119, 41. Harris, C.C., R.H. Yolken, H. Krokan and I.C. Hsu, 1979, Proc. Natl. Acad. Sci. USA 76, 5336. Hollinger, F.B., V. Vorndam and G.R. Dreesman, 1971, J. Immunol. 107, 1099. Hollinger, F.B., D.W. Bradley, J.E. Maynard, G.R. Dreesman and J.L. Melnick, 1975a, J. Immunol. 115, 5. Hollinger, F.B., M. Morrison, R. Chairez and G.R. Dreesman, 1975b, J. Immunol. Methods 8, 67. Jarvis, R.F., 1979, Antiobiot. Chemother. 26, 105. Laurell, C.-B., 1967, in: Protein in Biological Fluids, Vol. 14, ed. H. Peters (Elsevier, Amsterdam) p. 499. Ling, C.M. and L.R. Overby, 1972, J. Immunol. 109, 834. Radiation Safety Staff, Department of Nuclear Medicine, Clinical Center, 1972, in: The National Institutes of Health Radiation Safety Guide (U.S. Department of Health, Education and Welfare). Shalev, A., A.H. Greenberg and P.J. McAlpine, 1980, J. Immunol. Methods 38, 125. Shukuya, R. and G.W. Schwert, 1960, J. Biol. Chem. 235, 1649. Watson, D., 1976, Lancet ii, 570. Wilson, M.B. and P.K. Nakane, 1978, in: Immunofluorescence and Related Staining Techniques, eds. W. Knapp, K. Holvbar and G. Wick (Elsevier/North-Holland Biomedical Press, Amsterdam) p. 215. Yang, B.I. and D.E. Metzler, 1979, in: Methods in Enzymology, Vol. 62, eds. D.B. McCormick and L.D. Wright (Academic Press, New York) p. 528.
145
ENZYME POTENTIATED RADIOIMMUNOASSAY (EPRIA): A SENSITIVE THIRD-GENERATION TEST FOR THE DETECTION OF HEPATITIS B SURFACE ANTIGEN
HOWARD A. FIELDS, CANDACE L. DAVIS, GORDON R. DREESMAN, DANIEL W. BRADLEY and JAMES E. MAYNARD Hepatitis Laboratories Division *, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Phoenix, AZ, and Department of Virology and Epidemiology, Baylor College of Medicine, Houston, TX, U.S.A. (Received 3 April 1981, accepted 15 July 1981) A sensitive, specific immunoassay for detection of hepatitis B surface antigen (HBsAg) is described. The assay combines enzyme-linked immunosorbent assay and solid-phase radioimmunoassay and is termed enzyme potentiated radioimmunoassay (EPRIA). HBsAg was quantitated by enzymatic conversion of L-[14C]glutamic acid to 14CO2 and gamma-aminobutyric acid by glutamate decarboxylase (GDC) conjugated with goat antiHBs IgG. Conjugation of IgG and GDC was by a thiol-disulfide bond exchange reaction after reacting N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) with each reagent. A positive/negative ratio of 2.2 was established as significant by examination of 40 normal sera negative for HBsAg. This value was the mean cpm plus 3 standard deviations. By an identical statistical analysis of sensitivity, EPRIA was found to be approximately 100-fold more sensitive than Ausria II (Abbott Laboratories, North Chicago, IL). INTRODUCTION R a d i o i m m u n o a s s a y ( R I A ) has b e e n u s e d e x t e n s i v e l y f o r d e t e c t i o n o f h e p a t i t i s a s s o c i a t e d antigens a n d a n t i b o d i e s in sera, biological tissues, a n d f e c e s ( H o l l i n g e r et al., 1 9 7 1 , 1 9 7 5 b; Ling a n d O v e r b y , 1 9 7 2 ; Fields e t al., 1 9 7 8 ) . R a d i o i m m u n o a s s a y s w h i c h use ~:SI-labeled r e a g e n t s are l i m i t e d b y a relatively s h o r t shelf life a n d r a d i a t i o n h a z a r d . T h e d e v e l o p m e n t o f e n z y m e l i n k e d i m m u n o s o r b e n t assay ( E L I S A ) (Engvall a n d P e r l m a n n , 1 9 7 1 ) h a s obv i a t e d t h e s e difficulties. E L I S A s y s t e m s are q u a n t i t a t e d b y s p e c t r o p h o t o m e t r i c d e t e r m i n a t i o n o f e n z y m e s u b s t r a t e r e a c t i o n s . D e s p i t e t h e i r amplif y i n g n a t u r e , h o w e v e r , t h e y d o n o t u s u a l l y e x c e e d t h e sensitivity o f R I A (Watson, 1 9 7 6 ; Jarvis, 1 9 7 9 ) . This r e p o r t d e s c r i b e s an assay t e r m e d e n z y m e p o t e n t i a t e d r a d i o i m m u n o assay ( E P R I A ) w h i c h c o m b i n e s t h e p r i n c i p l e s o f E L I S A a n d R I A . 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 ) is q u a n t i t a t e d b y e n z y m a t i c c o n v e r s i o n o f [14C]g l u t a m i c acid to ~4CO2 a n d g a m m a - a m i n o b u t y r i c acid b y g l u t a m a t e decar* World Health Organization Collaborating Centre for Reference and Research on Viral Hepatitis. 0022-1759/81/0000--0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
146
boxylase (GDC) conjugated to IgG. The product, ~4CO2, is measured by liquid scintillation beta spectrometry. This gives a significant increase in sensitivity compared with RIA. MATERIALS AND METHODS
Reagents L-[ 1J4C]Glutamic acid (40--50 mCi/mmol), Protosol and Econofluor were from New England Nuclear (Boston, MA). The substrate was diluted to contain l 0 s cpm per 0.2 ml of 0.01 M sodium acetate buffer, pH 3.8. GDC type V and 2,2'-dipyridyl disulfide (2-PDS) were from Sigma Chemical Company (St. Louis, MO). GDC was further purified on a 2.6 cm X 90 cm column of Sephacryl S-300 (Pharmacia Fine Chemicals, Piscataway, NJ) equilibrated with phosphate buffered saline, pH 7.4, containing 1 : 4000 NaN3 (PBSN). As an alternative, GDC was purified from Escherichia coli (Yang and Metzler, 1979). N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was from Pharmacia Fine Chemicals. HBsAg positive serum was obtained by plasmapheresis of a chronic asymptomatic chimpanzee carrier. Normal human sera (NHS) and acute phase sera were from hepatitis A and non A/non B (NANB) infected individuals. All sera were stored at --20°C.
Purification of HBsAg HBsAg was purified from plasma by ultracentrifugation (Dreesman et al., 1972). Antigen was twice pelleted, sonicated, treated with a pH 2.4 buffer, and subjected to two isopycnic ultracentrifugation bandings in CsC1 followed by rate-zonal ultracentrifugation in CsCi. HBsAg was localized by rocket immunoelectrophoresis (Laurell, 1967) with an LKB Multiphor unit and power supply.
Immunization of guinea pigs Twenty pg purified HBsAg was mixed with Freund's complete adjuvant and four guinea pigs (g.p.) were injected intradermally in each rear foot pad. The animals were boosted by the intramuscular route every 2 weeks with antigen in Freund's incomplete adjuvant and exsanguinated when the antiHBs titer exceeded 1 : 1 X 106 by Ausab (Abbott Laboratories, North Chicago, IL). The IgG was fractionated from these sera by precipitation with saturated ammonium sulfate at 4°C followed by DEAE-cellulose chromatography (DE-52, Whatman, Clifton, NJ). Goat anti-HBs serum was obtained from the World Health Organization (WHO) and the IgG fraction prepared as described above for g.p. antisera. Immunospecific goat anti-HBs IgG was purified by affinity chromatography. Purified HBsAg was coupled to CNBr-activated Sepharose according to the manufacturer's recommendations (Pharmacia Fine Chemicals). Six ml of antiserum was chromatographed at 2--3 ml/h (0.9 cm X 10 cm column) with
147 0.1 M phosphate buffer, pH 7.0, containing 0.5 M NaC1. When the O.D.280 returned to baseline, specific a n t i b o d y was eluted with 0.2 M glycine, pH 2.5. The eluted fractions were neutralized and stored at 4°C. Purity was ascertained by immunoelectrophoresis with rabbit anti-goat serum.
SPDP thiolation Purified GDC and IgG were thiolated at different SPDP : protein molar ratios. The degree of substitution (number of thiol groups) was determined by the m e t h o d of Grossetti and Murray (1967). Briefly, after reacting GDC or IgG with SPDP for 30 min, the modified protein was desalted on a 1.6 cm × 90 cm column of Sephadex G-25 (Pharmacia Fine Chemicals} equilibrated with 0.1 M sodium acetate buffer, pH 4.6, containing 0.1 M NaC1 (buffer A). One molar dithiothreitol (DTT) was added to the modified protein preparation to a final DTT concentration of 50 mM. After incubation for 30 min at room temperature, the reaction mixture was desalted on a second Sephadex G-25 column equilibrated with 0.1 M Tris-HC1 buffer, pH 7.4, containing 0.5 M KC1 and 1 mM EDTA (buffer B). The eluted proteins were concentrated to a volume of 3.5 ml by ultrafiltration, by means of a 25 mm diameter YM10 ultrafilter and an 8 MC stirred cell (Amicon, Lexington, MA). Equal volumes of thiolated protein and 1.5 mM 2-PDS were added to a cuvette and the concentration of liberated 2-thiopyridone (2-TP) was determined spectrophotometrically at a wavelength of 343 nm after subtracting absorbance due to thiolated protein alone and 2-PDS alone. One mole of liberated 2-TP is equivalent to 1 thiol group.
Covalent chromatography To verify that GDC and IgG had been thiolated by SPDP, the thiolated proteins were chromatographed on thiopropyl Sepharose 6B (Pharmacia Fine Chemicals) according to the manufacturer's recommendations. All proteins to which more than 1 thiol group had been introduced by reaction with SPDP were covalently bound to the column and were subsequently eluted with a buffer containing 50 mM DTT.
Conjugation o f IgG and GDC Conjugates designed to be composed of one molecule of IgG and one molecule of GDC were prepared by mixing the respective SPDP modified proteins. Each protein was substituted with 1--2 residues/mole of protein. IgG was modified to contain 1--2 residues of 2-pyridyl disulfide per mole by the addition of 2.4 moles of SPDP/mole of IgG. The modified protein was desalted on G-25 equilibrated with buffer B and concentrated to 4 ml by ultrafiltration. GDC was modified to contain 1--2 thiol residues/mole by addition of 75 moles of SPDP/mole of GDC. The substituted protein preparation was desalted on G-25 equilibrated with buffer A, reduced with 50 mM DTT, and desalted again on G-25 equilibrated with buffer B. The thiolated protein was concentrated to 4 ml by ultrafiltration and used the
148
same day. The substituted proteins were then mixed together at a molar ratio of 6--10 moles IgG/mole GDC and rotated for 22 h at 27°C. Finally, the reaction mixture was fractionated by gel filtration chromatography on a 2.6 cm × 90 cm column of Bio-Rad A-1.5 m (Richmond, CA) equilibrated with PBSN at 4°C. Protein assay Protein concentration was determined by the Bio-Rad protein assay against the protein standard I for IgG and protein standard II for GDC supplied. Iodination Iodination of IgG with l~sI was by the chloramine-T method of G r e e n w o o d et al. (1963), as modified b y Hollinger et al. (1975b). Briefly, purified IgG was reacted with 0.5 mCi Na12SI in the presence of chloramineT (25 ~g) for 15 sec at room temperature and the reaction was stopped by addition of sodium metabisulfite (50 ]~g). Separation of b o u n d 12sI from free 12sI was accomplished on a PD-10 column of Sephadex G-25 (Pharmacia Fine Chemicals). The iodinated protein had a specific activity of 15--20 ~Ci/ /~g. E n z y m e assay One hundred microliters of protosol were added to a liquid scintillation vial containing a 2.4 cm GF/C (Whatman, Clifton, NJ) glass microfiber disc. Acetate buffer (0.2 ml) containing 25 mM L-glutamic acid and l 0 s cpm of L-[1-14C]glutamic acid was added to Immulon Removawells (Dynatech, Alexandria, VA) placed within 1/2-dram shell vials (Kimble). After addition of 10 ~1 of GDC, the shells were transferred to liquid scintillation vials. Each vial was capped with a Polyseal (Polyseal Corp., Iselin, NJ) cone-shaped screw cap and incubated at 37°C (Fig. 1). After incubation, the reaction was stopped by removing the shell. Fifteen ml of Econofluor containing 15% methanol were added to each vial which was then counted in a Delta 300 (Tracor Analytic) with the samples channel ratio set at 30%. Constant quenching eliminated the need for a quench correction. Units of enzyme (~moles/min) were determined by calculating the ]~moles of 14CO2 evolved. This value was multiplied b y the ratio of unlabeled : labeled substrate and added to the moles of radiolabeled p r o d u c t formed per minute at 37°C. EPRIA Optimal dilutions for coating Immulon micro-wells with antibody and for determining the optimal concentration o f conjugate were ascertained by checkerboard titration. Serial 2-fold dilutions of g.p. anti-HBs IgG prepared in 0.05 M Tris buffer, pH 8.4, containing 0.3 M KC1 and 2 mM EDTA (TKE) were used to coat the wells. After the wells had been washed, a dilution of
149
Liquid ScintillationVbal
'
" ......
½Dram Sh~l
Enzyme Reaction
~ 2 . 4 ~--Containing
cm Diameter GI=/C Filter P~er O.Iml Protosol
Fig. 1. Schematic representation of the reaction vessel for EPRIA.
HBsAg-positive plasma was added and tested against 2-fold dilutions of conjugate. The diluent for the conjugate was composed of equal volumes of NHS, normal goat serum, normal g.p. serum, and 0.05 M Tris buffer adjusted to a pH value of 7.5. Positive to negative ratios (P/N) were determined by calculating a quotient from the cpm for the test specimen divided by a mean cpm determined for at least 6 negative serum controls. The assay was performed by coating the wells with 250 ~1 of diluted g.p. anti-HBs IgG and washing each well 4 times with 0.5% bovine serum albumin (BSA) in PBSN (PBSN-BSA). Two hundred microliters of test sera or HBsAg-negative control sera diluted 1 : 8 0 0 in PBSN containing 1% BSA were added and incubated for 1 h at 45°C unless stated otherwise. After another wash cycle, 200 pl of conjugate were added and incubated for different times and at different temperatures. After a final wash cycle the wells were transferred to 1/2 dram shells (Kimble). Two h u n d r e d microliters of 0.01 M acetate buffer, pH 3.8 (unless stated otherwise), containing 10 s cpm of L-[1-14C]glutamic acid were added to the I m m u l o n wells which were then placed in liquid scintillation vials. Each vial was capped with a Polyseal cone-shaped screw cap (Fig. 1) and incubated at 37°C for 16 h. The reaction was stopped by removing the shell. Econofluor-methanol cocktail was added and the vials c o u n t e d in a Delta 300 liquid scintillation counter (see Materials and Methods on enzyme assay).
Specificity F o r t y normal h u m a n sera f o u n d to be negative for HBsAg by Ausria II were assayed by EPRIA. The values obtained were used to calculate a mean iX) cpm value and a standard deviation (S.D.). All 40 sera tested had a cpm value less than the X + 3.0 S.D. of X.
Sensitivity Serial 2-fold dilutions of HBsAg positive sera were made in PBSN-BSA.
150
Each dilution was tested for HBsAg by EPRIA, Ausria II and a modified micro-solid phase immunoradiometric assay (m-SPIRA). EPRIA end-point titers were determined by calculating a X + S.D. cpm value. The highest dilution cpm value which exceeded the X + 3.0 S.D. was considered as the endpoint titer. Ausria II and modified m-SPIRA endpoints were based upon a P/N value of 2.1. Conditions for the m-SPIRA were as previously described for detection of hepatitis core antigen (Fields et al., 1977) except t h a t the conjugate diluent was identical to the EPRIA conjugate diluent. RESULTS
Conjugation of IgG to GDC Each protein was reacted with SPDP at various molar ratios. The degree of substitution was ascertained by measuring the concentration of released 2-TP spectrophotometrically after reacting the thiolated proteins with 2-PDS. A calibration curve was constructed for each protein (Fig. 2). A ratio of approximately 2.4 moles of SPDP per mole of IgG was required to introduce between 1 and 2 thiol groups per mole of IgG. GDC required the addition of 75 moles of SPDP per mole to achieve approximately the same degree of thiolation. In both cases there was a linear relationship between the molar ratio of SPDP to protein and the degree of substitution. In an a t t e m p t to prepare a conjugate composed of one molecule of IgG and one molecule of GDC, each protein was reacted with SPDP to achieve a degree of substitution between 1 and 2. Preliminary experiments with GDC -?
140 s •
120E3 I 0 0 (_9
-5
o
"6 80-
n"~o
~5 ~
4020O-
J
-39.
f
I
I
I
I
2
3
I
1
0
Degree of Substitution (Number of Thiol Groups per mole of protein)
Fig. 2. Degree of substitution of GDC (e with various molar ratios of SPDP.
e) and IgG (o
o) after treatment
151
showed no reduction in enzyme activity after incubation with 50 mM DTT (data not shown). The specific activity of thiolated/GDC, however, was reduced by 35% as determined by calculating units of enzyme per mg of protein before and after thiolation. Thiolated GDC was mixed with 2-pyridyl disulfide substituted IgG at a molar ratio of 6--10 moles of IgG/mole of GDC to drive the thiol-disulfide bond exchange reaction to completion. The two protein solutions were rotated end-over-end at 27°C for 22 h. The reaction mixture was then chromatographed on a Bio-Rad A-1.5 m gel filtration column. Iodinated goat anti-HBs IgG (approximately 3 X 107 cpm) was mixed with the purified IgG fraction before SPDP modification to localize the IgG during the fractionation procedure. Each fraction was assayed for cpm by gamma spectrometry and for enzyme activity, except t h a t unlabeled glutamate was omitted. The fractionation profile is shown in Fig. 3. Two optical density (O.D.:80) peaks were observed preceding elution of unconjugated IgG. While each of these peaks showed approximately the same a m o u n t of radiolabeled IgG activity, the second peak (Pool III) had substantially more enzyme activity. No further attempts were made to determine the molecular composition of the conjugate. A small a m o u n t of enzyme activity was seen at fraction 150 accompanying an optical density shoulder. This shoulder may have been due to monomeric GDC subunits of molecular weight 50,000. The peak at fraction 157 was n o t characterized. All the enzyme was consumed in the forma55 5O o
.05
-
.04
I00
G~
-80 ~
2O -0
-600
t5
-40
I0
X 0 ,03-
...- i
II ci ~.02-
•: '
!,
.."
I
I':
I
0
- Z O v~
.01
V 0
I
40
,"" I
I
60
80
I I I I
5
,I :,,
if~ I
I00
I
120
I
140
i
160
i
-0
180
Froct~n Number (4ml/froc.) Fig. 3. F r a c t i o n a t i o n o f G D C - I g G c o n j u g a t e s o n a 2 . 6 c m × 9 0 c m c o l u m n o f B i o - R a d A-1.5m equilibrated with PBSN ( , O.D.280; ...... , GDC activity; ...... , [ 12 s I ] I g G a c t i v i t y ) . T h e f l o w r a t e w a s a d j u s t e d t o 1 2 m l / h a n d 4 - m l f r a c t i o n s w e r e collected.
152
tion of conjugate, as shown by the absence of an optical density or enzyme activity shoulder between the second conjugate peak and the free IgG peak. Previous experiments with equimolar a m o u n t of IgG and GDC repeatedly demonstrated free GDC activity around fraction 111.
Checkerboard titration Pools were made as in Fig. 3 and each pool was assayed for performance in the EPRIA system by checkerboard titration. Serial 2-fold dilutions of g.p. anti-HBs IgG were made in TKE buffer and I m m u l o n wells were coated for 1 h at 4°C. Each coating dilution was tested against 2-fold dilutions of each pool as shown in Table 1. Pool II was assayed on two separate occasions and both times it was found that a 1 : 25 (3.5 pg/well) dilution of g.p. antiHBs IgG and a 1 : 32 dilution of conjugate were optimal. The difference in P/N values between trial 1 and trial 2 was due to differences in the d e n o m i n a t o r (N value) in which small changes have very large effects on quotients. Pool II consistently yielded the highest P/N value. Values in tests with pool I were n o t tabulated because of insufficient enzyme activity. Since pool IV was composed of both pool II and III, its performance was intermediate.
Optimal conditions for Immulon coating Guinea pig anti-HBs IgG was diluted in TKE buffer and 250 pl were added to I m m u l o n wells. The wells were incubated for various lengths of time at TABLE 1 Determination of optimal reagent concentration. Pool
Conjugate dilution
P/N D i l u t i o n o f g.p. anti-HBs IgG 1:12.5
1:25
1:50
1:100
1:200
II
1 : 8 1 : 16 1 : 32
176 201 333
205 265 381 b
235 269 308
NT NT NT
NT a NT NT
II
1 : 16 1 : 32 1 : 64
NT NT NT
225 249 140
199 248 97
191 180 177
50 64 48
III
1 :
16 1 : 32 1 : 64
NT NT NT
39 61 34
29 32 29
35 33 18
10 8 6
IV
1 : 16 1 : 32 1 : 64
NT NT NT
157 200 167
169 182 129
192 163 195
55 65 42
a Not tested. b U n d e r s c o r e i n d i c a t e s m a x i m u m P/N o b t a i n e d for each p o o l .
153
4°C and at 25°C. The g.p. anti-HBs IgG preparation was obtained from a different animal from that used for evaluation of the various pools. As Fig. 4 shows, optimal conditions for coating were incubation of the wells at 4°C for 60 min with a 1 : 200 dilution of IgG. This dilution corresponds to 0.56 pg of protein per well. This g.p. antibody and these conditions for coating were used for the remainder of the study. Effect o f conjugate time and temperature incubation conditions on E P R I A Optimal reagent dilutions were established by checkerboard titration of pool TI incubated for 2 h at 37°C. Conjugate incubation times and temperatures were tested as indicated in Fig. 5. The original conditions for conjugate incubation of pool II were shown to be optimal, i.e., 2 h at 37°C. The results were expressed as functions of the P/N value. Examination of the individual P and N values (data n o t shown) showed that the P value increased for the first 2 h and then remained relatively constant; the N value continued to rise t h r o u g h o u t the incubation period. These findings indicated that equilibrium for the conjugate was reached in 2 h. Effect o f p H on E P R I A To determine the optimal pH for the enzyme assay, coated Immulon wells were incubated with a constant a m o u n t of HBsAg diluted (1 : 800) in PBSNBSA, washed, and then reacted with conjugate. After a final wash, 0.20 ml of 0.01 M acetate buffer containing 10 s cpm of substrate adjusted to the pH values shown in Fig. 6 were added to each well. P/N values were determined with NHS diluted (1 : 800) in PBSN-BSA as the negative control. As shown in Fig. 6, there was a broad plateau of activity from pH 3.4 to 4.4, with a pH o p t i m u m at 3.8. The activity d r o p p e d precipitously at higher pH values. These results are similar to those of Shukuya and Schwert (1960) for free
I00
..% 25°C
80
. . . . . .3o,~. --"
..."",,,-
. . ;,•.
,.
•
',,,
•
•
Z v
•
N
",,.,., \
2O 0
i 1:50
I:100
u 1:200
I
1:50 Dilution of Coating IgG 1:400
I
HO0
I
1:200
I
1:400
Fig. 4. E f f e c t o f t i m e a n d t e m p e r a t u r e o n passive a d s o r p t i o n o f g.p. a n t i - H B s IgG. V a r i o u s c o n c e n t r a t i o n s o f IgG were a d s o r b e d t o I m m u l o n wells a t 25°C a n d 4°C for 30, 60 a n d 1 2 0 rain. O p t i m a l c o n d i t i o n s were a s c e r t a i n e d b y determining P / N values•
154 8070-
60I00 -
50•o.
Z
80-
h: 4o-
37°C ." ~-
"I.~,.,
60-
30-
40-
20-
20-
tO-
Z
O-
I I
I 2
I 3
Time (hours)
I 4
O'
3.4
\ I
I
I
I
3B
4.2
4.6
5D
I ,5.4
pH
Fig. 5. E f f e c t of time and t e m p e r a t u r e o f conjugate i n c u b a t i o n on P/N values as determ i n e d by EPRIA. Optimal c o n c e n t r a t i o n s of coating g.p. anti-HBs IgG and conjugate were previously established by c h e c k e r b o a r d titration• Three temperatures (m _, 25Oc; • . . . . . . • , 37°C; • . . . . . .A, 45OC) were e x a m i n e d at 1-h intervals for 4 h. Optimal c o n d i t i o n s were ascertained by determining P/N values• Fig. 6. E f f e c t of pH on P/N values as d e t e r m i n e d by E P R I A . Optimal conditions for coating g.p. anti-HBs IgG and conjugate dilution were previously established by checkerboard titration• Substrate diluted in 0.01 M acetate buffer at various pH values was added to I m m u l o n wells and incubated. O p t i m u m pH was ascertained by determining P/N values•
GDC using either pyridine-pyridine h y d r o c h l o r i d e or glycine-glycine hydrochloride buffers.
Specificity of EPRIA A f r e q u e n c y distribution for 40 normal h u m a n sera shown t o be negative for HBsAg when tested by an Ausria II assay, was det erm i ned from the values o b tain ed by testing each serum by EPRIA. The values are pl ot t ed in Fig. 7. The X value obtained after subtraction o f the background was 191 cpm with a S.D. value of +-76 cpm. All the sera tested fell below the X + 3 S.D. value (424), which corresponds to a 99.9% confidence level with a one-tailed test. Thirty-eight o u t of 40 sera fell below the X + 2 S.D. units. The X + 3 S.D. units (424 cpm) is equivalent to a P/N value of 2.2. E xamin atio n of 142 sera including Ausria II negative NHS and sera from patients with acute hepatitis A or acute hepatitis NANB gave only 4 false
155 -i S.D
(115)
1
~
. I S.D.
• 2 SD
*3 .~D.
(~)
(267)
(,~47)
(424)
1
1
itS
1
%
0~
I
1
'
'
'
'
IO0
I
'
'
'
'
200
I
.
.
.
300
.
!
'
4O0
CPM
Fig. 7. F r e q u e n c y d i s t r i b u t i o n o f 4 0 n o r m a l h u m a n sera t e s t e d b y E P R I A . F o r t y Ausria II n e g a t i v e h u m a n sera s p e c i m e n s were t e s t e d b y E P R I A a n d m e a n c p m value a n d stand a r d d e v i a t i o n o f t h e m e a n were d e t e r m i n e d .
TABLE 2 Sensitivity c o m p a r i s o n Of m - S P I R A , A u s r i a II, a n d E P R I A . Dilution (1 : 1 0 0 x 2 n) n
P/N m-SPIRA a
A u s r i a II
0
NT f
3
3.24 g
NT 66.7 42.8 21.6 14.7 7.5 3.7
4 5 6 7 8 9 10. 11 12 13 14 15 16
NT 1.9 NT 1.3 NT 1.2 1.1 NT NT NT NT NT NT
b
2.8
1.7 1.3 1.3 NT NT NT NT
a HBsAg incubation 2 h at 45°C; conjugate HBs [ 1 2 s I ] I g G as p r o b e . b P r o c e d u r e A. c HBsAg incubation 1 h at 45°C; conjugate HBs IgG c o n j u g a t e d t o G D C as p r o b e . d HBsAg incubation 1 h at 45°C; conjugate r a p h y p u r i f i e d g o a t a n t i - H B s IgG c o n j u g a t e d e HBsAg incubation 2 h at 45°C; conjugate r a p h y p u r i f i e d g o a t a n t i - H B s IgG c o n j u g a t e d f Not tested. g Underscore indicates end-point.
EPRIA c
EPRIA d
EPRIA e
283.5 186.8 141.3 72.5 50.6 21.5 12.4 8.5 4.04
2 153.0 550.2 413.6 206.8 118.1 66.6 31.7 18.9 9.2 5.9 4.2 3.0 2.0
NT NT NT NT NT NT NT NT 19.1 10.1 5.0 4.1 2.8
2.35
1.66 1.7 1.1 NT NT
1.80
2.3
0.9
1.7
i n c u b a t i o n 2 h a t 4 5 ° C ; p o l y c l o n a l g o a t anti-
i n c u b a t i o n 2 h a t 3 7 ° C ; p o l y c l o n a l g o a t antiincubation 2 h at 37°C; affinity chromatogt o G D C as probe. incubation 2 h at 37°C; affinity chromatogt o G D C as p r o b e .
156
positives (2.8%), none of which exceeded a P/N value of 3.9. These sera could not be neutralized with WHO reference goat anti-HBs and were subsequently classified as negatives.
Sensitivity of EPRIA Serial 2-fold dilutions of an HBsAg-positive serum were made in PBSNBSA. Each dilution was tested in duplicate. P/N values were determined using NHS diluted in PBSN-BSA as a negative control. The X plus 3 S.D. units of this negative control correspond to a P/N value of 1.8. The endpoint was considered to be the dilution at which the P/N value obtained fell below 1.8. Identical dilutions were tested for endpoint titer by a modified m-SPIRA, Ausria II, as well as under three different conditions for EPRIA (Table 2). The end-point titer of an HBsAg-positive specimen as determined by Ausria II was 1 0 0 × 29 (1 : 51,200). Ausria II utilizes affinity chromatography purified h u m a n anti-HBs IgG radiolabeled with 12sI as the probe. When a polyclonal goat anti-HBs IgG radiolabeled with 12sI was used as in the modified m-SPIRA (Table 2) then the sensitivity was reduced 64-fold. This is indicated by comparison of the end-point titers observed with Ausria II and m-SPIRA. It was of considerable interest t h a t utilization of polyclonal goat anti-HBs IgG conjugated to GDC in EPRIA exceeded the sensitivity afforded
706050-
z
403020EPRIA t
I00 7
EPRIA 2"
t,~iol I ~ _ I
I
I
I
I
I
I
8
9
I0
II
12
13
14
1 I
I
15
16
n (dilution= IOOx 2") Fig. 8. S e n s i t i v i t y o f E P R I A . O p t i m a l c o n d i t i o n s for c o a t i n g g.p. a n t i - H B s IgG a n d conjugate d i l u t i o n s were previously d e t e r m i n e d b y c h e c k e r b o a r d t i t r a t i o n . Serial 2-fold dilut i o n s o f HBsAg-positive sera were m a d e in PBSN-BSA. E a c h d i l u t i o n was t e s t e d in duplicate b y Ausria II a n d E P R I A . P / N values were d e t e r m i n e d w i t h t h e m e a n c p m o b t a i n e d f r o m a t least 6 negative c o n t r o l s in t h e d e n o m i n a t o r . F o o t n o t e s 1 a n d 2 c o r r e s p o n d to f o o t n o t e s c a n d d in T a b l e 2, respectively.
157
by Ausria II 4-fold. Moreover, utilization of affinity chromatography purified goat anti-HBs IgG conjugated to GDC in EPRIA exceeded the sensitivity of Ausria II 64-fold when the antigen was incubated for 1 h at 45°C and almost 128-fold when the antigen was incubated for 2 h at 45°C. A portion of these data are illustrated graphically in Fig. 8. DISCUSSION
EPRIA combines in one test the amplification of ELISA and the sensitivity afforded by RIA instrumentation. This combination resulted in a novel assay for detection of HBsAg approximately 100-fold more sensitive than Ausria II. Because of the microtiter f o r m a t the assay is easy to perform and large numbers of samples may be processed at one time. The gaseous product of the enzyme reaction (~4CO2) separates from the aqueous phase substrate w i t h o u t the introduction of a tedious biochemical separation. Despite its long half life, the use of 14C represents a significant reduction in the biological hazard as compared with other isotopes such as '2sI and 32p (National Institutes of Health Radiation Safety Guide, 1972). Numerous attempts to conjugate GDC to IgG by the glutaraldehyde method (Avrameas, 1969~ Avrameas and Ternynck, 1971) were unsuccessful. GDC was consistently refractory to glutaraldehyde activation and as a consequence h o m o p o l y m e r s of IgG were preferentially produced. GDC does not possess any known carbohydrate m o i e t y and this precluded the metaperiodate oxidation m e t h o d developed by Wilson and Nakane (1978). A new m e t h o d of conjugating two proteins was developed by Carlsson et al. (1978) based upon a thiol-disulfide bond exchange reaction. This m e t h o d has the distinct advantage of allowing control over the molecular size of the conjugate and, therefore, the specific activity. In addition, conjugates prepared by this m e t h o d are stable but m a y be cleaved with reducing agents such as DTT. Covalent chromatography and direct determination of thiol groups (Grossetti and Murray, 1967) revealed t h a t native GDC does n o t contain sulfhydryl groups accessible for reaction in a thiol-disulfide bond exchange reaction. Therefore, to conjugate GDC to IgG by this reaction it was necessary to introduce a thiol group by prior modification with SPDP. To achieve a degree of thiolation of between 1 and 2 residues per mole of GDC a molar ratio of SPDP to GDC of 75 was necessary. Since SPDP attacks the e-amino group of lysine, these amino groups were apparently n o t as readily accessible for modification as t h e y were for IgG. This observation was consistent with our inability to conjugate GDC to IgG by the glutaraldehyde m e t h o d which also utilizes the e-amino groups of lysine. Conjugates prepared by the thiol-disulphide bond exchange reaction were fractionated by gel filtration chromatography (Bio-Rad A-1.5 m). Pools were made and evaluated by EPRIA. Pools II, III, and IV all demonstrated EPRIA activity. Pool IV was rechromatographed on a Bio-Rad A-0.5 m column
158 which excluded proteins of molecular weight exceeding 5.0 × 10 s. This fractionation procedure resulted in the removal of free IgG which contaminated pools III and IV and c o m p e t e d for available antigen sites in the test. We found that different lots of g.p. anti-HBs IgG required different concentrations of IgG per well for o p t i m u m performance. Factors such as differences in antibody avidity, antibody affinity for the solid phase matrix, and passive adsorption properties of the wells may all be important. Before each assay was performed o p t i m u m conditions were established by checkerboard titration to minimize these differences. The specificity of the test was demonstrated to be acceptable although n o t as good as commercial kits (personal observations). Our observed frequency of 2.8% false positives may be a necessary compromise for the increased sensitivity generated by EPRIA. We are studying the effects on specificity and sensitivity of immunoabsorbing g.p. anti-HBs IgG before passive adsorption on polystyrene. Since HBsAg hyperimmunized laboratory animals demonstrate anti-NHS activity, removal of this activity before coating of Immulon wells may improve the specificity. Nevertheless, false positives are easily identified by a simple neutralization step before retesting. The most important application of EPRIA is in providing a sensitive serologic test in the absence of affinity chromatography purified antibody. Table 2 clearly demonstrates that a modified m-SPIRA for the detection of HBsAg using iodinated polyclonal IgG resulted in a test severely limited by its lowered sensitivity. Comparing the sensitivity obtained b y m-SPIRA with that of EPRIA using polyclonal antibody for each assay showed that EPRIA was 256 times more sensitive. Thus, EPRIA affords excellent sensitivity without the requirement for immunospecific antibody. This m a y be important for the development of tests for viral antigens, such as those in NANB hepatitis, the concentrations of which are apparently much lower than for plasmas from patients infected with hepatitis type B virus, or for which it is n o t at present possible to prepare immunospecific antibody. The sensitivity of EPRIA is limited by the specificity of the reagents. Conjugates must be diluted so that the background is within acceptable limits. An examination of the effect of conjugate dilutions on P/N ratio revealed that the denominator (-N value) decreased more rapidly than the numerator (P value) (data not shown). Thus, as the conjugate was diluted the quotient became higher until it reached a maximum value and then d r o p p e d rapidly. Reactions contributing to the denominator value, i.e., non-specific results, are diluted out much more readily than the specific reactions. If the nonspecific reactions could be reduced further, higher concentrations of conjugate could be used, giving specific positive responses of greater magnitude. We are investigating the possibility of immuno-specifically purifying coating antibody and of using monoc!onal antibodies to improve specificity. Until more specific reagents are available for detection of HBsAg, the development of other ultrasensitive tests such as those described by Harris et al. (1979) and Shalev et al. (1980) may n o t be possible.
159 REFERENCES Avrameas, S., 1969, Immunochemistry 8, 336. Avrameas, S. and T. Ternynck, 1971, Immunochemistry 8, 1175. Carlsson, J., H. Drevin and R. Axen, 1978, Biochem. J. 173,723. Dreesman, G.R., F.B. Hollinger, R.B. McCombs and J.L. Melnick, 1972, Infect. Immun. 5, 213. Engvall, E. and P. Perlmann, 1971, Immunochemistry 8, 871. Fields, H.A., F.B. Hollinger, J. Desmyter, J.L. Melnick and G.R. Dreesman, 1977, Intervirology 8, 336. Fields, H.A., D.W. Bradley, C.L. Davis, B.L. Murphy, C.A. Schable and J.E. Maynard, 1978, J. Immunol. 121, 930. Greenwood, F.C., W.M. Hunter and J.S. Glever, 1963, Biochem. J. 89, 114. Grossetti, D.R. and J.F. Murray, 1967, Arch. Biochem. Biophys. 119, 41. Harris, C.C., R.H. Yolken, H. Krokan and I.C. Hsu, 1979, Proc. Natl. Acad. Sci. USA 76, 5336. Hollinger, F.B., V. Vorndam and G.R. Dreesman, 1971, J. Immunol. 107, 1099. Hollinger, F.B., D.W. Bradley, J.E. Maynard, G.R. Dreesman and J.L. Melnick, 1975a, J. Immunol. 115, 5. Hollinger, F.B., M. Morrison, R. Chairez and G.R. Dreesman, 1975b, J. Immunol. Methods 8, 67. Jarvis, R.F., 1979, Antiobiot. Chemother. 26, 105. Laurell, C.-B., 1967, in: Protein in Biological Fluids, Vol. 14, ed. H. Peters (Elsevier, Amsterdam) p. 499. Ling, C.M. and L.R. Overby, 1972, J. Immunol. 109, 834. Radiation Safety Staff, Department of Nuclear Medicine, Clinical Center, 1972, in: The National Institutes of Health Radiation Safety Guide (U.S. Department of Health, Education and Welfare). Shalev, A., A.H. Greenberg and P.J. McAlpine, 1980, J. Immunol. Methods 38, 125. Shukuya, R. and G.W. Schwert, 1960, J. Biol. Chem. 235, 1649. Watson, D., 1976, Lancet ii, 570. Wilson, M.B. and P.K. Nakane, 1978, in: Immunofluorescence and Related Staining Techniques, eds. W. Knapp, K. Holvbar and G. Wick (Elsevier/North-Holland Biomedical Press, Amsterdam) p. 215. Yang, B.I. and D.E. Metzler, 1979, in: Methods in Enzymology, Vol. 62, eds. D.B. McCormick and L.D. Wright (Academic Press, New York) p. 528.