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Effect of nonspecific interactions of the hepatitis B surface antigen with the solid phase on its detection by two-site immunoradiometric assay
Journal of Virological Methods, 15 (1987) 303-312 Elsevier
303
JVM 00571
of the hepatitis Effect of nonspecific interactions B surface antigen with the solid phase on its detection by two-site immunoradiometric assay Ridha
Khelifa
and Theresa
MO
Viral Hepatitis Section. Viral Diagnostic Services Division, Bureau of Microbiology, Laborator> Cenrre for Disease Control. Health md W’rlfure Canada. Ottcrw~~,Onlario. Cmudu (Accepted
13 November
19X6)
Two-site immunoradiometric assay (Ausria 11-125. procedure B. Abbott) was used to detect hepatitis B surface antigen (HBsAg) in chronic carrier sera from clinically healthy subjects or from kidney transplant or hemodialysis patients. The titration curves of some high-titered sera showed a prozonelike effect: this corresponded in two such sera LB and MA. to 40 c/r inhibition of HBsAg detection in undiluted serum relative to a 1:800 dilution. The nature of the inhibition in the above two sera was investigated. We found that the inhibition depended on the,time of first incubation but not on the affinity of capture antibodies. Nonspecific adsorption of HBsAg to the solid phase during the first incubation with inhibitory sera and elution of the thus adsorbed antigen during the Fecond incubation. with the resulting neutralization of “‘I-labeled anti-HBs. appeared to cause the inhibition of detection observed. The inhibition was prevented by addition of the detergent Triton X-100 to serum or by elution of adsorbed antigen prior to the second incubation, thus indicating that further improvements of the HBsAg immunoradiometric assay may be possible. HBsAg;
Two-site
immunoradiometric
assay:
Quantitative
determination;
Inhibition
of detection
Introduction The quantitative determination of the hepatitis B surface antigen (HBsAg) in paired sera allows to predict the outcome of acute hepatitis B virus infection. A significant reduction in HBsAg concentration within a few weeks after onset of illness indicates a high probability for HBsAg clearance. Whereas the absence of such a decrease indicates progression towards chronicity (Gerlich et al., 1977; Frosner et al., 1982; Frosner and France, 1984; Joller-Jemelka et al., 1985). The most sensitive and widely used technique for the detection of HBsAg is twoCorrespondence 10: R. Khelifa. Viral Hepatitis Section, Viral Diagnostic Services Division. Bureau of Microbiology. Laboratory Centre for Disease Control. Health and Welfare Canada. Ottawa, Ontario, Canada KlA 0L2. 01609341871$03,50
0
1987 Elsevier
Science
Publishers
B.V. (Biomedical
Division)
site immunoradiometric assay (Miller and Overby, 1974; Decker and Overby, 1977). Although the quantitative use of such assays to determine the approximate level of HBsAg in undiluted sera has been reported (Nath et al., 1980), such quantitation is in principle possible only if unknown samples are analyzed at two or more dilutions, because of the possibility of prozone or high-dose hook effects (Miles, 1977; Rodbard et al., 1978). Such effects result in a paradoxical fall in assay responses at saturating antigen concentrations. Only the understanding of the nature, and the prevention of such inhibitory effects would allow a clear quantitative interpretation of assay responses obtained with undiluted test samples. Here, we report investigations on the nature of a prozone-like inhibition affecting the detection of HBsAg by two-site immunoradiometric assay in some chronic carrier sera and show that this inhibition may be prevented by a better control of nonspecific adsorption of HBsAg to the solid phase.
Materials
and Methods
Sera Normal human serum (NHS) was a pool of sera collected from normal healthy donors negative for all hepatitis B markers. HBsAg-containing sera were obtained either from healthy, HBeAg-negative chronic carriers (MF, PG), or from HBeAgpositive hemodialysis (LB, LE, HM, AL) or kidney transplant (MA) patients. All the serum samples used were negative for anti-HBs (Ausab, Abbott Laboratories). H BsAg assay Two hundred-k1 aliquots of the different sera, either undiluted or diluted in NHS, were assayed for HBsAg using procedure B of the commercial two-site immunoradiometric assay Ausria II-125 (Abbott Laboratories) as recommended by the manufacturer. Briefly, polystyrene beads coated with guinea pig anti-HBs were incubated with control or test samples for 18 h at 22°C. The beads were then washed 5 times with distilled water and reacted with 200 t~_lof human “‘I-labeled anti-HBs for 1 h at 45°C. Finally, the beads were washed 5 times and counted in a gamma counter. To assay for nonspecific adsorption of HBsAg to polystyrene, noncoated polystyrene beads (6 mm diameter) were used instead of the above anti-HBs-coated beads, under similar conditions as in the HBsAg assay. In addition, incubation with llSI-labeled anti-HBs was performed either at 45°C for 1 h or at 22°C for 4 h. In the kinetic experiment, the HBsAg assay was performed on undiluted serum LB or MA with first incubation varying from 5 min to 18 h. The various first incubations were performed in the same reaction tray and were stopped simultaneously; the assay was then continued as above. The results of the above assays were expressed as S/N values which represent the ratio of cpm of samples (S) and the mean cpm of negative controls (N).
305
HBsAg-coated polystyrene beads (Ausab, Abbott Laboratories) were used instead of anti-HBs-coated beads in a procedure identical to the HBsAg assay. The 5% inhibition for each test serum was calculated as a % reduction of counts relative to mean cpm obtained with NHS. In this inhibition assay, as well as in the above assays. the specificity of ‘““I-labeled anti-HBs binding to HBsAg on the solid phase was confirmed by specific neutralization of the antigen with an appropriate dilution of rabbit antiserum to HBsAg (Behring Diagnostics).
Anti-HBs-coated beads (Ausria II-123 were incubated with 200 ~1 of serum LB or MA or with NHS as in the HBsAg assay. The beads were then washed as above and incubated at 22°C for 4 h with 200 JJJ of ““I-labeled HBsAg (Ausab). In parallel, similar beads were preincubated at 22°C for 4 h with ‘2sI-Iabeled HBsAg, then washed and incubated at 22°C for 18 h with serum LB, or NHS or PBS, to test for displacement of bound labeled antigen. At the end of incubation, the beads were washed and counted. Preelution experiments For the preelution experiments, either in the HBsAg assay or in the inhibition assay, an eiution step was included between the first and the second incubations. After the first incubation, the washed beads were incubated in 200 ~1 of PBS either for 1 h at 45°C or for 4 h at 22°C. The beads were then washed 5 times and the corresponding assay was continued as above.
Results Sample-related vuriations in HBsAg detectabilit? The data represented in Fig. 1 show three different types of titration curves which we have encountered while testing for HBsAg in serial dilutions of different sera from chronic HBsAg carriers. The first type, exemplified by the curve of serum AL had a sigmoid shape, in line with the ‘ideal’ dose-response curve characteristic of two-site immunoradiometric assays (Rodbard et al., 1978). The second type represented by the titration curve of a low-titered serum, MF, apparently corresponded to the subplateau portion of the ‘ideal’ dose-response curve. The third type was obtained with hightitered sera such as serum LB and showed a high-dose hook or prozone-like effect indicative of a low HBsAg detectability in undiluted serum and of a gradual increase in detectability upon dilution. This effect of dilution indicated that HBsAg
306
0
2
1
4
3
Log Dilution
5
7
6
Factor
Fig. 1. Titration of different sera for HBsAg. Serial dilutions of sera in NHS were tested for HBsAg II-125 assay (Abbott). SIN = ratio of cpm of sample (S) and the mean of cpm values of negative control human serum (N). 0-0. serum MF; A-A. serum AL; 0-o. serum LB. by the Ausria
detection was inhibited in undiluted LB-like (inhibitory) sera. This inhibition might be due to either the presence of high antigen concentration or to other inhibitory factors, or to both. Mechanism of inhibition of HBsAg detection In order to investigate the possible mechanisms of the prozone-like inhibition of HBsAg detection. we first looked for the site of this inhibition. Blocking experiments using two inhibitory sera (LB and MA) and “‘I-labeled HBsAg were performed to see whether the inhibition is exerted at the level of HBsAg capture. The data obtained (Table 1) show that anti-HBs-coated beads preincubated with either
TABLE
1
Blocking
of “51-HBsAg
binding
and displacement
of bound
First incubation” of anti-HBscoated polystyrene beads
Second incubation” coated polystyrene
“I-HBsAg “‘I-HBsAg “I-HBsAg Serum LB Serum MA NHS’
PBS Serum LB NHS “‘1-HBaAg “‘I-HBsAg “‘I-HBsAg
“51-HBsAg of anti-HBsbeads
by unlabeled
HBsAg
CPMh “I-HBsAg
in serum
bound
38167 26755 37032 6X0 708 37945
‘I Incubation of beads with “‘I-HBsAg was for 4 h at 2?“C. and incubation with PBS and the different sera was for 8 h at 22°C. h The figures shown are rounded mean values of two separate determinations, ’ Negative for all hepatitis B markers.
307
II II-n -
5 min
15 Inin
min
2h
lh
16h
Tlme ot First Incubation
Fig. 2. Kinetics of the inhibitory reactions occurring in the HBsAg assay with serum LB. The HBsAg assay was performed on undiluted serum LB as described in Materials and Methods except for the time of first incubation which was varied as indicated.
serum LB or serum MA could not bind any significant amounts of labeled antigen. On the other hand, serum LB was not able to remove or displace more than 30% of precaptured labeled HBsAg. Therefore, the blocking of “‘I-labeled HBsAg binding by the inhibitory sera appears to be mainly due to saturation of the capture antibody by unlabeled antigen and not to any other effect of these sera on the capture antibody. It is noteworthy that in control experiments (data not shown) noninhibitory serum AL as well as optimal dilutions of inhibitory sera (LB, MA) did not inhibit significantly the binding of ‘2’I-labeled HBsAg to anti-HBs-coated beads. This indi-
E 2 m,
6000
$? I4 .?." 'E-
a & 2
4000
0
PBS NHS MF PG LE
HM AL
LB MA
Fig. 3. Inhibition assay using HBsAg-coated beads. The inhibition assay was performed as indicated in Materials and Methods. PBS = phosphate-buffered saline; NHS = normal human serum negative for all hepatitis B markers; MF, PG, LE. HM, AL. LB, and MA = serum samples from various chronic HBsAg carriers with different HBsAg titers and completely negative for anti-HBs (Ausab, Abbott). The titration curve of serum PG was similar to MF, that of LE and HM was similar to AL, and that of MA was similar to LB (see Fig. 1).
I‘ABLE
2
Nonspecific
adsorption
Samples’
Serum Serum NHS’
of HBaAg
to polystyrene
bends
CPM” (S/N) “‘I-anti-HBs
AL LB
bound
at
22°C. 4 h
WC,
I l-15(5.4) I1 l-12(52.3)
607 (4.7) 2376 (1X.6) 128 (1.0)
213 (1.0)
1h
,’ Serum samples were incubated with noncoated polystyrene beads for 18 h at 22°C. ” The CPM shown are rounded mean value5 from a sin@ experiment in duplicates. These figures were used to calculate the S!N values shown in brackets. ( Negative for all hepatitis B markers.
cates a high capacity for HBsAg capture of these beads and implies that the saturation plateau shown by the dilution curve of serum AL was most likely due to anti-HBs in the HBsAg assay. the use of a limiting amount of “SI-labeled The above data indicate that the inhibitory reactions were occurring at a different site than specific HBsAg capture. This conclusion was further substantiated by a kinetic experiment using serum LB (Fig. 2) or serum MA (not shown), which indicated that HBsAg detection in undiluted serum increased when the time of first incubation was significantly reduced. These data sugggested that some inhibitory reactions that were slower than specific HBsAg capture were affecting the detection of captured HBsAg, and led us to examine the effect of the inhibitory sera on the detection of HBsAg coated onto polystyrene beads. in the absence of capture antibodies (inhibition assay). The data obtained (Fig. 3) show that sera LB or MA drastically inhibited the detection of HBsAg by approximately 80%. Whereas five HBsAg-containing sera not showing the prozone-like effect in the HBsAg assay. did not cause any significant inhibition. This showed that the capture antibodies were not involved in the inhibition and prompted us to examine the possible role of nonspecific adsorption of HBsAg to the solid phase. We therefore assessed the ability of HBsAg in serum LB or AL to adsorb to noncoated polystyrene beads. The data obtained (Table 2) show that, under either detection condition, significantly more HBsAg was found to be adsorbed to beads incubated with serum LB (S/N = 52.3 with second incubation at 22”C, and 18.6 at 45°C) than to beads incubated with serum AL (SIN = 5.4 with second incubation at 22”C, and 4.7 at 45’C). The results of beads incubated with serum LB also show that approximately 3 times less HBsAg on beads could be detected at 45°C (S/N = 18.6) than at 22°C (S/N = 52.3). thus indicating significant elution at 45°C of the adsorbed HBsAg. Such an adsorptionielution event may account at least in part, for the observed inhibition of HBsAg detection, since it would result in partial neutralization of the “51-labeled anti-HBs. Rernowl
of the
inhihitor~~ recrctiom
In order to assess the importance of the above mechanism, we investigated the effect on HBsAg detection of three different treatments intended to decrease non-
309
0 Dilution Factor: Pre-Elution:
--
-800 1
1
800
l--.._lI 1
lh : 45°C
4h-
NONE
0 800
(preeluFig. 4. Effect of elution of adsorbed HBsAg prior to incubation with ‘2sI-labeled anti-HBs tion). The SIN values and the c/c inhibition of HBsAg detection were determined separately by the HBsAg assay and by the inhibition assay. respectively. Serum LB was used either undiluted or diluted 12300 in NHS as indicated. Preelution was performed as described in Materials and Methods.
specific adsorption. The first treatment was aimed at eliminating the effect of any HBsAg adsorption to the wells of the reaction tray during the HBsAg assay, and consisted of a transfer of the assay beads to fresh wells after the first incubation. This procedure resulted only in a slight (~107’ o ) increase in the S/N values of inhibitory sera, thus indicating that the wells of the reaction tray were not significantly involved in the observed inhibition. The second and third treatments were aimed at removing nonspecifically adsorbed HBsAg by elution prior to incubation with ‘*“I-labeled anti-HBs (preelution), and at prevention of nonspecific adsorption of HBsAg to the solid phase using the nonionic detergent Triton X-100, respectively. In these experiments, the percentages of inhibition of HBsAg detection (7% inhibition) were determined separately by the inhibition assay which was performed in parallel to the HBsAg assay. The data of the preelution experiments (Fig. 4) show that with undiluted serum LB, preelution at 22°C for 4 h produced
Dilution Factor:
1
Diluent:
_
NHS
OVA/PBS
TX/PBS
NHS
Fig. 5. Effect of Triton X-100 on HBsAg detection in the HBsAg assay and in the inhibition assay. The S/N values and the C/o inhibition of HBsAg detection were determined separately by the HBsAg assay and by the inhibition assay. respectively. Serum LB was used either undiluted or diluted in NHS or 3% ovalbumin in PBS (OVA/PBS). or 0.15% Triton X-100 in PBS (TX/PBS). as indicated.
310
a small (~40%) increase in SIN value (HBsAg assay) with a concomitant (218%) decrease in inhibition of HBsAg detection (inhibition assay), whereas preelution at 45°C for 1 h restored the responses obtained with undiluted serum LB in both assays to practically the same level as that obtained with the 1:X00 dilution. The effect of Triton X-100 treatment is summarized in Fig. 5. These data show that. when serum LB was diluted 1:lO in PBS containing 3% ovalbumin or in NHS, only a slight increase in SIN value was obtained relative to that of undiluted serum and no effect was observed in the inhibition assay. However, when 0.15% Triton X-100 in PBS was used as a diluent, the S/N value of the 1:lO dilution reached the same level as that of the 1:800 dilultion in NHS; the inclusion of Triton X-100 in the diluent also completely removed the inhibition of HBsAg detection as indicated by the results of the inhibition assay. Validity of the HBsAg treatmerlts
assay after its modification
by preelution
or Triton X-100
The above preelution and Triton X-100 treatments were applied to serial 1:lO dilutions in NHS of the noninhibitory serum AL. It was found (data not shown) that preelution had no significant effect on HBsAg determination at any serum dilution, while Triton X-100 treatment decreased the sensitivity of the assay as shown by a 2 to 3 x decrease in S/N values a high serum dilutions (lo-’ and 10m4). Therefore, it appears that preelution, but not Triton X-100 treatment, may be applied indiscriminately to test sera regardless of their HBsAg concentration.
Discussion The present study indicates that the mechanism underlying the prozone-like inhibition of HBsAg detection by immunoradiometric assay in some high-titered sera, involves nonspecific interactions of the antigen with the solid phase. These interactions appeared to consist of (a) the nonspecific adsorption of HBsAg to the solid phase during the first incubation (with serum), followed by (b) elution of the adsorbed antigen especially during the second incubation (with ‘2sI-labeled anti-HBs); this then resulted in partial neutralization of the labeled antibody and in a decrease in the amount which remains bound to the solid phase. This mechanism is similar to that postulated by Chau et al. (1983) to explain the prozoning exhibited by an immunoradiometric assay for anti-HBc IgM; but it is different than the mechanism of high-dose hook effect postulated by Rodbard et al. (1978). The latter showed in a theoretical kinetic study that the presence of different affinity classes in the capture antibody would lead to an inhibition of detection at high antigen concentration due to dissociation of the antigen captured by low-affinity antibodies. However. as shown here by the inhibition assay. the inhibition of HBsAg detection by the inhibitory sera could take place even when the detected HBsAg was directly coated onto polystyrene beads, in the complete absence of capture antibodies; thus, the role of antibody heterogeneity in this inhibition can be safely ruled
311
out. The data of the kinetic experiment also implied that the inhibitory reactions were much slower than specific antigen capture thus suggesting secondary interaction of the captured HBsAg with some ‘inhibitor’ in serum, or nonspecific interaction of HBsAg with the solid phase. However, the inhibitor explanation is unlikely in view of the data of the adsorption (Table 2) and preelution (Fig. 4) experiments. If the effect of preelution were to remove a putative inhibitor and not to elute adsorbed HBsAg, the detection of HBsAg adsorbed to polystyrene (Table 2) should have been higher under conditions (1 h, 45°C) of more elution of the putative inhibitor as indicated by the preelution data (Fig. 4). On the contrary, significantly less adsorbed HBsAg was detected under these conditions. Therefore, the eluted material appears most likely to be HBsAg and not an inhibitor that might be masking the captured antigen. In light of this conclusion, the removal of the inhibition of HBsAg detection by the nonionic detergent Triton X100 is more likely to be due to the abrogation of nonspecific adsorption of HBsAg rather than to the removal of a putative inhibitor by the detergent. Taken together, our data confirm the possibility that HBsAg was interacting nonspecifically with the solid phase and are in line with the adsorption/elution mechanism. The ability of HBsAg from inhibitory sera to adsorb to the solid phase in spite of its presence in undiluted human serum poses the question of whether this adsorption was due to high antigen concentration alone or in association with other factors such as differences in antigenic structure (Wands et al., 1984) or abnormal serum components (Neurath et al., 1973). The data shown here do not answer this question, but preliminary experiments (not shown) indicated that the HBsAg in serum LB adsorbed even to beads coated with NHS thus suggesting that some normal serum components may mediate HBsAg adsorption to the solid phase. This is in line with the known ability of HBsAg to interact with various serum components (Imai et al., 1979; Shih et al., 1980; Craxi et al., 1985). Our data do not explain the difference in S/N values obtained at saturation with undiluted serum AL and with diluted serum LB (Fig. 1). This type of difference in HBsAg detectability could not be related to antigen concentration or to nonspecific adsorption. It might correspond to different antigenic structures or to different degrees of association of HBsAg with serum components (Burrell, 1975; Shih et al., 1980) or to both. Further investigations on this important aspect are warranted. In conclusion, our data emphasize the influence of intrinsic sample properties on the detectability of HBsAg by immunoradiometric assay and indicate that the prozone-like inhibition of HBsAg detection may be prevented through a better control of HBsAg adsorption to the solid phase. Further understanding of the factors affecting HBsAg detectability might lead to a better quantitation of this antigen.
312
Acknowledgements We are grateful to Dr. R.K. Chaudhary for helpful advice. We also thank Dr. J.M. Weber and Dr. D.A. Kennedy for kindly reviewing, and Mrs. L. Edstrom for typing the manuscript. References Burrell, C.J. (1975) .I. Gen. Virol. 27, 117. Chau, K.H., Hargie. M.P.. Decker. R.H., Mushahwar. I.K. and Overhy, L.R. (1983) Hepatology 3, 142. Craxi. A.. Magrin, S., Greco. J.. Vinci, M., Tine. F., Raimondo. G., Lonzo, G. and Pagliaro L. (1985) J. Med. Virol. 15. 383. Decker, R.N. and Overhy. L.R. (1977) In: Quality Control in Nuclear Medicine (Rhodes, B.A., ed.), p. 427. C.V. Moshy, St Louis, MO. Friisner. G.G.. Schomerus. H.. Wiedmann, K.H.. Zachoval, R., Bayerl, B., Backer. U.. Gathof, G.A. and Sugg, U. (1982) Eur. J. Clin. Microhiol. 1. 52. Friisner. G.G. and France. E. (1984) In: Viral Hepatitis and Liver Disease. (Vyas, G.N., Dienstag, J.L. and Hoofnagle, J.H. eds.). p. 487. Grune and Stratton. New York and London. Gerlich. W., Stamm, B. and Thomssen, R. (1977) Verh. Dtsch. Ges. Inn. Med. 83. 544. Imai, M., Yanase. Y., Nojiri, T.. Myakawa. Y. and Mayumi M. (1979) Gastroenterology 76, 242. Joller-Jemelka, H.I., Pfister. H.F. and Groh. P.J. (1985) Schweiz. Med. Wochenschr. 115. 1249. Miles. L.E.M. (1977) In: Handbook of Radioimmunoassay. (Abraham. G.E., ed.). p, 131. Marcel Dekker, New York and Basel. Miller. J.P. and Overhy, L.R. (1974) In: Nuclear Medicine in Vitro. (Rothfeld, B., ed.), p. 363. J.B. Lippincott. Philadelphia. PA. Nath, N.. Fang, C.T., Berherian. H., Bastiaans. M.J.. Dodd, R.Y., Sandier, S.G. and Barker. L.F. (1980) VOX Sang, 39-73. Neurath, A.R., Prince. A.M., Lippin, A. and Chen. M. (1973) Lancet ii, 1394. Rodhard, D.. Feldman, Y.. Jaffe, M.L. and Miles, L.E.M. (1978) Immunochemistry 15. 77. Shih. J. W.K., Tan, P.L. and Gerin. J.L. (1980) Infect. Immun. 28, 459. Wands, J.R., Wang, M.A., Shorey. J.. Brown. R.D.. Marciniak. R.A. and Isselhacher, K.J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81. 2237.
303
JVM 00571
of the hepatitis Effect of nonspecific interactions B surface antigen with the solid phase on its detection by two-site immunoradiometric assay Ridha
Khelifa
and Theresa
MO
Viral Hepatitis Section. Viral Diagnostic Services Division, Bureau of Microbiology, Laborator> Cenrre for Disease Control. Health md W’rlfure Canada. Ottcrw~~,Onlario. Cmudu (Accepted
13 November
19X6)
Two-site immunoradiometric assay (Ausria 11-125. procedure B. Abbott) was used to detect hepatitis B surface antigen (HBsAg) in chronic carrier sera from clinically healthy subjects or from kidney transplant or hemodialysis patients. The titration curves of some high-titered sera showed a prozonelike effect: this corresponded in two such sera LB and MA. to 40 c/r inhibition of HBsAg detection in undiluted serum relative to a 1:800 dilution. The nature of the inhibition in the above two sera was investigated. We found that the inhibition depended on the,time of first incubation but not on the affinity of capture antibodies. Nonspecific adsorption of HBsAg to the solid phase during the first incubation with inhibitory sera and elution of the thus adsorbed antigen during the Fecond incubation. with the resulting neutralization of “‘I-labeled anti-HBs. appeared to cause the inhibition of detection observed. The inhibition was prevented by addition of the detergent Triton X-100 to serum or by elution of adsorbed antigen prior to the second incubation, thus indicating that further improvements of the HBsAg immunoradiometric assay may be possible. HBsAg;
Two-site
immunoradiometric
assay:
Quantitative
determination;
Inhibition
of detection
Introduction The quantitative determination of the hepatitis B surface antigen (HBsAg) in paired sera allows to predict the outcome of acute hepatitis B virus infection. A significant reduction in HBsAg concentration within a few weeks after onset of illness indicates a high probability for HBsAg clearance. Whereas the absence of such a decrease indicates progression towards chronicity (Gerlich et al., 1977; Frosner et al., 1982; Frosner and France, 1984; Joller-Jemelka et al., 1985). The most sensitive and widely used technique for the detection of HBsAg is twoCorrespondence 10: R. Khelifa. Viral Hepatitis Section, Viral Diagnostic Services Division. Bureau of Microbiology. Laboratory Centre for Disease Control. Health and Welfare Canada. Ottawa, Ontario, Canada KlA 0L2. 01609341871$03,50
0
1987 Elsevier
Science
Publishers
B.V. (Biomedical
Division)
site immunoradiometric assay (Miller and Overby, 1974; Decker and Overby, 1977). Although the quantitative use of such assays to determine the approximate level of HBsAg in undiluted sera has been reported (Nath et al., 1980), such quantitation is in principle possible only if unknown samples are analyzed at two or more dilutions, because of the possibility of prozone or high-dose hook effects (Miles, 1977; Rodbard et al., 1978). Such effects result in a paradoxical fall in assay responses at saturating antigen concentrations. Only the understanding of the nature, and the prevention of such inhibitory effects would allow a clear quantitative interpretation of assay responses obtained with undiluted test samples. Here, we report investigations on the nature of a prozone-like inhibition affecting the detection of HBsAg by two-site immunoradiometric assay in some chronic carrier sera and show that this inhibition may be prevented by a better control of nonspecific adsorption of HBsAg to the solid phase.
Materials
and Methods
Sera Normal human serum (NHS) was a pool of sera collected from normal healthy donors negative for all hepatitis B markers. HBsAg-containing sera were obtained either from healthy, HBeAg-negative chronic carriers (MF, PG), or from HBeAgpositive hemodialysis (LB, LE, HM, AL) or kidney transplant (MA) patients. All the serum samples used were negative for anti-HBs (Ausab, Abbott Laboratories). H BsAg assay Two hundred-k1 aliquots of the different sera, either undiluted or diluted in NHS, were assayed for HBsAg using procedure B of the commercial two-site immunoradiometric assay Ausria II-125 (Abbott Laboratories) as recommended by the manufacturer. Briefly, polystyrene beads coated with guinea pig anti-HBs were incubated with control or test samples for 18 h at 22°C. The beads were then washed 5 times with distilled water and reacted with 200 t~_lof human “‘I-labeled anti-HBs for 1 h at 45°C. Finally, the beads were washed 5 times and counted in a gamma counter. To assay for nonspecific adsorption of HBsAg to polystyrene, noncoated polystyrene beads (6 mm diameter) were used instead of the above anti-HBs-coated beads, under similar conditions as in the HBsAg assay. In addition, incubation with llSI-labeled anti-HBs was performed either at 45°C for 1 h or at 22°C for 4 h. In the kinetic experiment, the HBsAg assay was performed on undiluted serum LB or MA with first incubation varying from 5 min to 18 h. The various first incubations were performed in the same reaction tray and were stopped simultaneously; the assay was then continued as above. The results of the above assays were expressed as S/N values which represent the ratio of cpm of samples (S) and the mean cpm of negative controls (N).
305
HBsAg-coated polystyrene beads (Ausab, Abbott Laboratories) were used instead of anti-HBs-coated beads in a procedure identical to the HBsAg assay. The 5% inhibition for each test serum was calculated as a % reduction of counts relative to mean cpm obtained with NHS. In this inhibition assay, as well as in the above assays. the specificity of ‘““I-labeled anti-HBs binding to HBsAg on the solid phase was confirmed by specific neutralization of the antigen with an appropriate dilution of rabbit antiserum to HBsAg (Behring Diagnostics).
Anti-HBs-coated beads (Ausria II-123 were incubated with 200 ~1 of serum LB or MA or with NHS as in the HBsAg assay. The beads were then washed as above and incubated at 22°C for 4 h with 200 JJJ of ““I-labeled HBsAg (Ausab). In parallel, similar beads were preincubated at 22°C for 4 h with ‘2sI-Iabeled HBsAg, then washed and incubated at 22°C for 18 h with serum LB, or NHS or PBS, to test for displacement of bound labeled antigen. At the end of incubation, the beads were washed and counted. Preelution experiments For the preelution experiments, either in the HBsAg assay or in the inhibition assay, an eiution step was included between the first and the second incubations. After the first incubation, the washed beads were incubated in 200 ~1 of PBS either for 1 h at 45°C or for 4 h at 22°C. The beads were then washed 5 times and the corresponding assay was continued as above.
Results Sample-related vuriations in HBsAg detectabilit? The data represented in Fig. 1 show three different types of titration curves which we have encountered while testing for HBsAg in serial dilutions of different sera from chronic HBsAg carriers. The first type, exemplified by the curve of serum AL had a sigmoid shape, in line with the ‘ideal’ dose-response curve characteristic of two-site immunoradiometric assays (Rodbard et al., 1978). The second type represented by the titration curve of a low-titered serum, MF, apparently corresponded to the subplateau portion of the ‘ideal’ dose-response curve. The third type was obtained with hightitered sera such as serum LB and showed a high-dose hook or prozone-like effect indicative of a low HBsAg detectability in undiluted serum and of a gradual increase in detectability upon dilution. This effect of dilution indicated that HBsAg
306
0
2
1
4
3
Log Dilution
5
7
6
Factor
Fig. 1. Titration of different sera for HBsAg. Serial dilutions of sera in NHS were tested for HBsAg II-125 assay (Abbott). SIN = ratio of cpm of sample (S) and the mean of cpm values of negative control human serum (N). 0-0. serum MF; A-A. serum AL; 0-o. serum LB. by the Ausria
detection was inhibited in undiluted LB-like (inhibitory) sera. This inhibition might be due to either the presence of high antigen concentration or to other inhibitory factors, or to both. Mechanism of inhibition of HBsAg detection In order to investigate the possible mechanisms of the prozone-like inhibition of HBsAg detection. we first looked for the site of this inhibition. Blocking experiments using two inhibitory sera (LB and MA) and “‘I-labeled HBsAg were performed to see whether the inhibition is exerted at the level of HBsAg capture. The data obtained (Table 1) show that anti-HBs-coated beads preincubated with either
TABLE
1
Blocking
of “51-HBsAg
binding
and displacement
of bound
First incubation” of anti-HBscoated polystyrene beads
Second incubation” coated polystyrene
“I-HBsAg “‘I-HBsAg “I-HBsAg Serum LB Serum MA NHS’
PBS Serum LB NHS “‘1-HBaAg “‘I-HBsAg “‘I-HBsAg
“51-HBsAg of anti-HBsbeads
by unlabeled
HBsAg
CPMh “I-HBsAg
in serum
bound
38167 26755 37032 6X0 708 37945
‘I Incubation of beads with “‘I-HBsAg was for 4 h at 2?“C. and incubation with PBS and the different sera was for 8 h at 22°C. h The figures shown are rounded mean values of two separate determinations, ’ Negative for all hepatitis B markers.
307
II II-n -
5 min
15 Inin
min
2h
lh
16h
Tlme ot First Incubation
Fig. 2. Kinetics of the inhibitory reactions occurring in the HBsAg assay with serum LB. The HBsAg assay was performed on undiluted serum LB as described in Materials and Methods except for the time of first incubation which was varied as indicated.
serum LB or serum MA could not bind any significant amounts of labeled antigen. On the other hand, serum LB was not able to remove or displace more than 30% of precaptured labeled HBsAg. Therefore, the blocking of “‘I-labeled HBsAg binding by the inhibitory sera appears to be mainly due to saturation of the capture antibody by unlabeled antigen and not to any other effect of these sera on the capture antibody. It is noteworthy that in control experiments (data not shown) noninhibitory serum AL as well as optimal dilutions of inhibitory sera (LB, MA) did not inhibit significantly the binding of ‘2’I-labeled HBsAg to anti-HBs-coated beads. This indi-
E 2 m,
6000
$? I4 .?." 'E-
a & 2
4000
0
PBS NHS MF PG LE
HM AL
LB MA
Fig. 3. Inhibition assay using HBsAg-coated beads. The inhibition assay was performed as indicated in Materials and Methods. PBS = phosphate-buffered saline; NHS = normal human serum negative for all hepatitis B markers; MF, PG, LE. HM, AL. LB, and MA = serum samples from various chronic HBsAg carriers with different HBsAg titers and completely negative for anti-HBs (Ausab, Abbott). The titration curve of serum PG was similar to MF, that of LE and HM was similar to AL, and that of MA was similar to LB (see Fig. 1).
I‘ABLE
2
Nonspecific
adsorption
Samples’
Serum Serum NHS’
of HBaAg
to polystyrene
bends
CPM” (S/N) “‘I-anti-HBs
AL LB
bound
at
22°C. 4 h
WC,
I l-15(5.4) I1 l-12(52.3)
607 (4.7) 2376 (1X.6) 128 (1.0)
213 (1.0)
1h
,’ Serum samples were incubated with noncoated polystyrene beads for 18 h at 22°C. ” The CPM shown are rounded mean value5 from a sin@ experiment in duplicates. These figures were used to calculate the S!N values shown in brackets. ( Negative for all hepatitis B markers.
cates a high capacity for HBsAg capture of these beads and implies that the saturation plateau shown by the dilution curve of serum AL was most likely due to anti-HBs in the HBsAg assay. the use of a limiting amount of “SI-labeled The above data indicate that the inhibitory reactions were occurring at a different site than specific HBsAg capture. This conclusion was further substantiated by a kinetic experiment using serum LB (Fig. 2) or serum MA (not shown), which indicated that HBsAg detection in undiluted serum increased when the time of first incubation was significantly reduced. These data sugggested that some inhibitory reactions that were slower than specific HBsAg capture were affecting the detection of captured HBsAg, and led us to examine the effect of the inhibitory sera on the detection of HBsAg coated onto polystyrene beads. in the absence of capture antibodies (inhibition assay). The data obtained (Fig. 3) show that sera LB or MA drastically inhibited the detection of HBsAg by approximately 80%. Whereas five HBsAg-containing sera not showing the prozone-like effect in the HBsAg assay. did not cause any significant inhibition. This showed that the capture antibodies were not involved in the inhibition and prompted us to examine the possible role of nonspecific adsorption of HBsAg to the solid phase. We therefore assessed the ability of HBsAg in serum LB or AL to adsorb to noncoated polystyrene beads. The data obtained (Table 2) show that, under either detection condition, significantly more HBsAg was found to be adsorbed to beads incubated with serum LB (S/N = 52.3 with second incubation at 22”C, and 18.6 at 45°C) than to beads incubated with serum AL (SIN = 5.4 with second incubation at 22”C, and 4.7 at 45’C). The results of beads incubated with serum LB also show that approximately 3 times less HBsAg on beads could be detected at 45°C (S/N = 18.6) than at 22°C (S/N = 52.3). thus indicating significant elution at 45°C of the adsorbed HBsAg. Such an adsorptionielution event may account at least in part, for the observed inhibition of HBsAg detection, since it would result in partial neutralization of the “51-labeled anti-HBs. Rernowl
of the
inhihitor~~ recrctiom
In order to assess the importance of the above mechanism, we investigated the effect on HBsAg detection of three different treatments intended to decrease non-
309
0 Dilution Factor: Pre-Elution:
--
-800 1
1
800
l--.._lI 1
lh : 45°C
4h-
NONE
0 800
(preeluFig. 4. Effect of elution of adsorbed HBsAg prior to incubation with ‘2sI-labeled anti-HBs tion). The SIN values and the c/c inhibition of HBsAg detection were determined separately by the HBsAg assay and by the inhibition assay. respectively. Serum LB was used either undiluted or diluted 12300 in NHS as indicated. Preelution was performed as described in Materials and Methods.
specific adsorption. The first treatment was aimed at eliminating the effect of any HBsAg adsorption to the wells of the reaction tray during the HBsAg assay, and consisted of a transfer of the assay beads to fresh wells after the first incubation. This procedure resulted only in a slight (~107’ o ) increase in the S/N values of inhibitory sera, thus indicating that the wells of the reaction tray were not significantly involved in the observed inhibition. The second and third treatments were aimed at removing nonspecifically adsorbed HBsAg by elution prior to incubation with ‘*“I-labeled anti-HBs (preelution), and at prevention of nonspecific adsorption of HBsAg to the solid phase using the nonionic detergent Triton X-100, respectively. In these experiments, the percentages of inhibition of HBsAg detection (7% inhibition) were determined separately by the inhibition assay which was performed in parallel to the HBsAg assay. The data of the preelution experiments (Fig. 4) show that with undiluted serum LB, preelution at 22°C for 4 h produced
Dilution Factor:
1
Diluent:
_
NHS
OVA/PBS
TX/PBS
NHS
Fig. 5. Effect of Triton X-100 on HBsAg detection in the HBsAg assay and in the inhibition assay. The S/N values and the C/o inhibition of HBsAg detection were determined separately by the HBsAg assay and by the inhibition assay. respectively. Serum LB was used either undiluted or diluted in NHS or 3% ovalbumin in PBS (OVA/PBS). or 0.15% Triton X-100 in PBS (TX/PBS). as indicated.
310
a small (~40%) increase in SIN value (HBsAg assay) with a concomitant (218%) decrease in inhibition of HBsAg detection (inhibition assay), whereas preelution at 45°C for 1 h restored the responses obtained with undiluted serum LB in both assays to practically the same level as that obtained with the 1:X00 dilution. The effect of Triton X-100 treatment is summarized in Fig. 5. These data show that. when serum LB was diluted 1:lO in PBS containing 3% ovalbumin or in NHS, only a slight increase in SIN value was obtained relative to that of undiluted serum and no effect was observed in the inhibition assay. However, when 0.15% Triton X-100 in PBS was used as a diluent, the S/N value of the 1:lO dilution reached the same level as that of the 1:800 dilultion in NHS; the inclusion of Triton X-100 in the diluent also completely removed the inhibition of HBsAg detection as indicated by the results of the inhibition assay. Validity of the HBsAg treatmerlts
assay after its modification
by preelution
or Triton X-100
The above preelution and Triton X-100 treatments were applied to serial 1:lO dilutions in NHS of the noninhibitory serum AL. It was found (data not shown) that preelution had no significant effect on HBsAg determination at any serum dilution, while Triton X-100 treatment decreased the sensitivity of the assay as shown by a 2 to 3 x decrease in S/N values a high serum dilutions (lo-’ and 10m4). Therefore, it appears that preelution, but not Triton X-100 treatment, may be applied indiscriminately to test sera regardless of their HBsAg concentration.
Discussion The present study indicates that the mechanism underlying the prozone-like inhibition of HBsAg detection by immunoradiometric assay in some high-titered sera, involves nonspecific interactions of the antigen with the solid phase. These interactions appeared to consist of (a) the nonspecific adsorption of HBsAg to the solid phase during the first incubation (with serum), followed by (b) elution of the adsorbed antigen especially during the second incubation (with ‘2sI-labeled anti-HBs); this then resulted in partial neutralization of the labeled antibody and in a decrease in the amount which remains bound to the solid phase. This mechanism is similar to that postulated by Chau et al. (1983) to explain the prozoning exhibited by an immunoradiometric assay for anti-HBc IgM; but it is different than the mechanism of high-dose hook effect postulated by Rodbard et al. (1978). The latter showed in a theoretical kinetic study that the presence of different affinity classes in the capture antibody would lead to an inhibition of detection at high antigen concentration due to dissociation of the antigen captured by low-affinity antibodies. However. as shown here by the inhibition assay. the inhibition of HBsAg detection by the inhibitory sera could take place even when the detected HBsAg was directly coated onto polystyrene beads, in the complete absence of capture antibodies; thus, the role of antibody heterogeneity in this inhibition can be safely ruled
311
out. The data of the kinetic experiment also implied that the inhibitory reactions were much slower than specific antigen capture thus suggesting secondary interaction of the captured HBsAg with some ‘inhibitor’ in serum, or nonspecific interaction of HBsAg with the solid phase. However, the inhibitor explanation is unlikely in view of the data of the adsorption (Table 2) and preelution (Fig. 4) experiments. If the effect of preelution were to remove a putative inhibitor and not to elute adsorbed HBsAg, the detection of HBsAg adsorbed to polystyrene (Table 2) should have been higher under conditions (1 h, 45°C) of more elution of the putative inhibitor as indicated by the preelution data (Fig. 4). On the contrary, significantly less adsorbed HBsAg was detected under these conditions. Therefore, the eluted material appears most likely to be HBsAg and not an inhibitor that might be masking the captured antigen. In light of this conclusion, the removal of the inhibition of HBsAg detection by the nonionic detergent Triton X100 is more likely to be due to the abrogation of nonspecific adsorption of HBsAg rather than to the removal of a putative inhibitor by the detergent. Taken together, our data confirm the possibility that HBsAg was interacting nonspecifically with the solid phase and are in line with the adsorption/elution mechanism. The ability of HBsAg from inhibitory sera to adsorb to the solid phase in spite of its presence in undiluted human serum poses the question of whether this adsorption was due to high antigen concentration alone or in association with other factors such as differences in antigenic structure (Wands et al., 1984) or abnormal serum components (Neurath et al., 1973). The data shown here do not answer this question, but preliminary experiments (not shown) indicated that the HBsAg in serum LB adsorbed even to beads coated with NHS thus suggesting that some normal serum components may mediate HBsAg adsorption to the solid phase. This is in line with the known ability of HBsAg to interact with various serum components (Imai et al., 1979; Shih et al., 1980; Craxi et al., 1985). Our data do not explain the difference in S/N values obtained at saturation with undiluted serum AL and with diluted serum LB (Fig. 1). This type of difference in HBsAg detectability could not be related to antigen concentration or to nonspecific adsorption. It might correspond to different antigenic structures or to different degrees of association of HBsAg with serum components (Burrell, 1975; Shih et al., 1980) or to both. Further investigations on this important aspect are warranted. In conclusion, our data emphasize the influence of intrinsic sample properties on the detectability of HBsAg by immunoradiometric assay and indicate that the prozone-like inhibition of HBsAg detection may be prevented through a better control of HBsAg adsorption to the solid phase. Further understanding of the factors affecting HBsAg detectability might lead to a better quantitation of this antigen.
312
Acknowledgements We are grateful to Dr. R.K. Chaudhary for helpful advice. We also thank Dr. J.M. Weber and Dr. D.A. Kennedy for kindly reviewing, and Mrs. L. Edstrom for typing the manuscript. References Burrell, C.J. (1975) .I. Gen. Virol. 27, 117. Chau, K.H., Hargie. M.P.. Decker. R.H., Mushahwar. I.K. and Overhy, L.R. (1983) Hepatology 3, 142. Craxi. A.. Magrin, S., Greco. J.. Vinci, M., Tine. F., Raimondo. G., Lonzo, G. and Pagliaro L. (1985) J. Med. Virol. 15. 383. Decker, R.N. and Overhy. L.R. (1977) In: Quality Control in Nuclear Medicine (Rhodes, B.A., ed.), p. 427. C.V. Moshy, St Louis, MO. Friisner. G.G.. Schomerus. H.. Wiedmann, K.H.. Zachoval, R., Bayerl, B., Backer. U.. Gathof, G.A. and Sugg, U. (1982) Eur. J. Clin. Microhiol. 1. 52. Friisner. G.G. and France. E. (1984) In: Viral Hepatitis and Liver Disease. (Vyas, G.N., Dienstag, J.L. and Hoofnagle, J.H. eds.). p. 487. Grune and Stratton. New York and London. Gerlich. W., Stamm, B. and Thomssen, R. (1977) Verh. Dtsch. Ges. Inn. Med. 83. 544. Imai, M., Yanase. Y., Nojiri, T.. Myakawa. Y. and Mayumi M. (1979) Gastroenterology 76, 242. Joller-Jemelka, H.I., Pfister. H.F. and Groh. P.J. (1985) Schweiz. Med. Wochenschr. 115. 1249. Miles. L.E.M. (1977) In: Handbook of Radioimmunoassay. (Abraham. G.E., ed.). p, 131. Marcel Dekker, New York and Basel. Miller. J.P. and Overhy, L.R. (1974) In: Nuclear Medicine in Vitro. (Rothfeld, B., ed.), p. 363. J.B. Lippincott. Philadelphia. PA. Nath, N.. Fang, C.T., Berherian. H., Bastiaans. M.J.. Dodd, R.Y., Sandier, S.G. and Barker. L.F. (1980) VOX Sang, 39-73. Neurath, A.R., Prince. A.M., Lippin, A. and Chen. M. (1973) Lancet ii, 1394. Rodhard, D.. Feldman, Y.. Jaffe, M.L. and Miles, L.E.M. (1978) Immunochemistry 15. 77. Shih. J. W.K., Tan, P.L. and Gerin. J.L. (1980) Infect. Immun. 28, 459. Wands, J.R., Wang, M.A., Shorey. J.. Brown. R.D.. Marciniak. R.A. and Isselhacher, K.J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81. 2237.