Clin. exp. Immunol. (1992) 87, 298-303
HBsAg in urine: a new approach for the detection of urinary antigens S. BADUR*, L. GRANGEOT-KEROS* & J. PILLOT*t *Service de Microbiologie et d'Immunologie, and INSERM U 131 H6pital Antoine B&clre, Clamart, and tUnitW d'Immunologie Microbienne, Institut Pasteur, Paris, France
(Acceptedfor publication 21 August 1991)
SUMMARY In order to define the optimal conditions for detection of microbial antigens in urine, urinary HBsAg excreted during hepatitis B was chosen as a model. Using commercial kits, which mainly involve antidiscontinuous epitopes, we found urinary HBsAg in only 50% of patients with HBsAg in their sera. In contrast, with an inhibition method involving a monoclonal antibody recognizing a continuous epitope, urinary HBsAg was found in 100% of these patients. Structural analysis of HBsAg showed that urinary HBsAg is denatured; it can escape detection by commercial kits well fitted for detection of native serum HBsAg. General implications for the revelation of urinary microbial antigens are discussed.
Keywords urinary HBsAg antigenuria microbial antigen degradation INTRODUCTION
Diagnosis of infectious diseases by characterization of microbial antigens in urine can be hampered by the limited concentration of investigated material. Such a failure could also be related to inappropriate conditions of the molecular state of the antigens, as shown for pneumococcal polysaccharidic antigens during human infection [1,2]. Little is known about the molecular state of urinary microbial antigens, as far as proteins are concerned. A lipoprotein representing the viral envelope of the hepatitis B virus (HBV) is produced in large excess (HBsAg) in sera of infected subjects. This situation is a good approach to investigate the optimal conditions for urinary microbial protein antigen detection and to analyse the molecular structure of the urinary antigen, comparatively to its serum counterpart. The optimal detection of antigenaemia is obtained by radioimmunoassay (RIA) or by enzyme immunoassay (EIA). Nevertheless with such techniques, data concerning the presence of HBsAg in urines vary according to the authors. Villajeros et al. [3] found antigenuria in three of 130 HBsAg carriers, while Vittal [4] showed antigenuria in one out of 13 asymptomatic seropositive carriers, but urinary HBsAg was detected neither in 12 patients with acute hepatitis, nor in five subjects with chronic active hepatitis. In another study, HBsAg was detected in urines of seven out of nine renal transplant recipients [5] and did not correlate with the presence of hidden or apparent haematuria. Human and monkey anti-HBs IgG are currently used for HBsAg detection and it is well established that these antibodies recognize discontinuous (conformational) epitopes and do not bind denatured HBsAg [6,7]. Under these conditions, the lack of
detection by the most sensitive assays (RIA, EIA) could be related to denaturation of the HBsAg excreted into the urines. In this study the presence of HBsAg in urines is re-evaluated by antibody-binding inhibition allowing the characterization of degradation products. In this work we establish that urinary HBsAg can be detected in all patients bearing HBsAg in their serum. Furthermore, in some cases where native HBsAg could be detected, this antigen appeared as a complex mixture of native and degraded viral envelope products. PATIENTS AND METHODS
Patients Forty patients with acute hepatitis B (AHB) and ten patients with chronic hepatitis B (CHB) were investigated. Urine and serum samples were stored at - 250C until investigation. The samples were not concentrated in order to prevent aggregation and to maintain urinary proteins as native as possible. HBV markers Serum HBsAg, hepatitis Be antigen (HBeAg), anti-HBe antibodies, anti-HBc IgM, and urinary HBsAg, were tested by commercial EIA kits (Organon Teknika, Boxtel, The Netherlands). Positive/negative (P/N) ratios for HBsAg were established by using AUSRIA II (Abbott Laboratories, Chicago, IL).
Preparation of monoclonal antibodies to discontinuous and continuous epitopes Monoclonal antibodies (MoAb) recognizing a discontinuous epitope of the HBsAg group a specificity have previously been described [8]. The anti-discontinuous MoAb IgGI chosen for this study recognizes highly purified HBsAg [9] in precipitation test, EIA and RIA. It did not recognize the reduced and
Correspondence: Professor Jacques Pillot, Hopital Antoine Beclere, 157 rue de Ia Porte de Trivaux, 92141 Clamart, France.
298
299
Urinary antigen detection alkylated purified HBsAG either in liquid phase or after adsorption on solid phase. Obtaining a MoAb recognizing a HBsAg continuous epitope is a rare event under usual conditions of MoAb preparation. Indeed, we have observed that a large majority of anti-HBs antibody molecules produced against HBsAg in man and animals are selectively directed against tertiary structures of HBsAg [7]. Therefore, mice usually failed to produce MoAb specific for a linear structure of amino acids. Such a MoAb specificity could nevertheless be obtained by immunizing with the major HBsAg polypeptide subunit. HBsAg was purified from plasma [9]. Polypeptides were resolved by SDS-PAGE according to a discontinuous buffer system in 12 5% acrylamide gel. For identification of polypeptides, the gel was treated with 1 mM dithiothreitol (DTT) in cold distilled water, stained for 5 min with 0-25 M KCl, 1 mm DTT, then fixed for 1 h with cold DTT solution. Polypeptide bands remained clear and the background became opaque. The 24-kD polypeptide (p24) was removed with a razor blade and stored at -250C. Before immunization of mice, the frozen gel was ground and homogenized. Then, the thawed mixture was centrifuged and the supernatant used for intrasplenic immunization [10]; 0-1 ml containing 0-2 mg of p24 was used for each animal. Splenocytes were collected after a unique intrasplenic injection, then fused with myeloma X63 cells. Each culture medium, in which cell colonies developed, was tested on the same plate by EIA against both native and denatured HBsAg. Denaturation of HBsAg for EIA was performed by reduction at 60°C for 10 min, with 5% of 2-mercaptoethanol. This concentration of 2-mercaptoethanol did not hamper the immunological reaction. Plates were coated with 0.1 ml of a solution containing 2 yg/ml of native or reduced HBsAg, washed with phosphate-buffered saline (PBS) containing 0 1% (w/v) of Tween 20 (PBS-T) and overcoated with PBS-T containing 2% of bovine serum albumin (BSA), at 37°C for 3 h. Then, 0 1 ml of each cell culture supernatant was incubated for I h at 37°C. After washing, the presence of MoAb reacting with reduced HBsAg was detected with goat anti-mouse IgG peroxidase conjugate (I pg/ml) produced in our laboratory. EIA inhibition Two types of assays were performed. In the first, native HBsAg was directly adsorbed on the solid phase; in the second, an antiHBs rabbit IgG layer was inserted between the antigen and the solid phase. Coating was performed with 5 pg/ml of our antiHBs IgG preparation and 2 ,g/ml of HBsAg. After washings, 0.1 ml of the HBsAg solution or of urine was mixed with an equal volume of the diluted anti-discontinuous MoAb or an equal volume of MoAb recognizing preferentially the reduced HBsAg and named anti-continuous MoAb. The mixtures were incubated for I h at 37°C, and then 0 I ml was transferred onto HBsAg-coated plates. Fixation of anti-HBs MoAb was revealed with goat anti-mouse IgG peroxidase conjugate. An inhibition beyond 50% was considered significant. Urine analysis
Eleven urine samples were analysed by Western blot. Before electrophoresis, urines were heated at 60°C for 10 min with sample buffer (0 125 M Tris HCl, pH 6 8) containing 2% SDS and 5%/ of 2-mercaptoethanol. Following SDS-PAGE, proteins were transferred to a nitrocellulose sheet [11]. Bands were
detected using rabbit anti-HBs serum from our laboratory (reacting with continuous and discontinuous epitopes) and 125f1 labelled monkey anti-rabbit immunoglobulin (Amersham International, Amersham, UK); the bands were visualized by autoradiography using an X-ray film (CORONEX4, Du Pont de Nemours, Frankfurt/Main, Germany). RESULTS
Specificity of monoclonal antibody selected for its capacity to recognize denatured HBsAg Specificity of MoAbs obtained by immunization of mice with the p24 polypeptide band was controlled by EIA as described in Materials and Methods by using native HBsAg and the same HBsAg reduced with 5% of mercaptoethanol. The anti-discontinuous MoAb was used as control. Under these conditions, one MoAb which reacted with reduced HBsAg twice as much as with native antigen was selected. The same reduced antigen did not react with the anti-discontinuous MoAb (Table 1). The anti-continuous specificity of this MoAb was confirmed by Western blot. After HBsAg reduction, SDS-PAGE and immunoblotting, this MoAb reacted only with the p24 polypeptide whereas the anti-discontinuous MoAb appeared unreactive with any band of the viral envelope (p24, p28, gp 31, gp 33) (results not shown). When reduced HBsAg was mixed with anti-continuous or anti-discontinuous MoAb, inhibition was only observed with the anti-continuous MoAb, indicating that anti-discontinuous MoAb did not recognize the reduced HBsAg (Table 2). Optimization of the anti-continuous monoclonal antibody inhibition test for urine samples Assuming that HBsAg could be present in urine as denatured protein and unreactive in the classical sandwich RIA or EIA, we developed an inhibition test involving the monoclonal antibody recognizing a continuous epitope. For this assay, native HBsAg was presented directly adsorbed on the solid phase or retained by anti-HBs IgG coating the solid phase. As shown in Table 3, the HBsAg presentation did not influence the capacity of native or reduced HBsAg artificially introduced in urine, but influenced the capacity of true urinary HBsAg to block the reaction of anti-continuous MoAb with solid phase HBsAg. If a strong inhibition was observed when urinary HBsAg was used to inhibit the reaction with antigen presented by means of an additional antibody layer, a weak reaction was observed when the antigen was directly adsorbed. Furthermore, native HBsAg Table 1. Enzymeimmunoassay reactivity of anti-continuous epitope MoAb with native or reduced HBsAg
Native HBsAg*
Reduced HBsAgt
epitope (dil. 1/1000) Anti-continuous HBsAg
>2
0297
epitope (dii. 1/1000)
0-535
1-060
MoAb Anti-discontinuous HBsAg
The coating of HBsAg on solid phase was performed in absence* or in presencet of 2-mercaptoethanol. Results are expressed as optical absorbance at 492 nm.
S. Badur, L. Grangeot-Keros & J. Pillot
300
Table 2. Enzymeimmunoassay inhibition of anti-discontinuous and anti-continuous MoAb by reduced HBsAg Dilution of MoAb
Anti-discontinuous epitope
Anti-continuous epitope
10-2
1-3 10o-4 1o-5 10-2 10-3 1O-4 l0-
>2
1-957 1-989 1-700
No antigen
>2
1-980 1 387 1-049
Reduced HBsAg
1 895 1-832 1-723 1-581 0-125 0-140 0-155 0-137
Native HBsAg was used for direct coating. For inhibition, reduced HBsAg was used at 2 jug/ml. Results are expressed as optical absorbance at 492 nm.
Table 3. Otpimization of the inhibition test involving MoAb to a continuous epitope
HBsAg Urine samples
samples coming from the 50 serum HBsAg positive patients (including the 23 urine samples found to be negative by RIA) appeared positive. No correlation was found between the amount of HBsAg in RIA and the inhibitory rate for the anticontinuous MoAb. Furthermore, no correlation was observed between the EIA inhibition test and the other parameters studied in sera. Urine samples found negative for HBsAg by classical RIA and positive by the inhibition test involving anticontinuous MoAb appeared negative when anti-discontinuous MoAb was used (results not shown).
On solid phase
Captured by anti-HBs IgG
Normal A Normal B Normal A+native HBsAg Normal A+ reduced H BsAg
1-090
0-823
1-178 0-627 0 049
1-009 0 573 0.164
Patient 2 Patient 3 Patient 5 Patient 14 Patient 44
0 974 0-825 0-890 0.558 0 628
0.161 0-206 0-258 0-147 0 158
HBsAg was added to the urine at a concentration of 2 ,g/ml. For
inhibition, a dilution of anti-continuous MoAb was chosen to give an optical density of about 1 (10-5). Results are expressed as optical absorbance at 492 nm.
only partially inhibited anti-continuous epitope reactivity, confirming the weak activity of anti-continuous MoAb with native HBsAg. Comparison between serum and urine HBsAg detection As shown in Table 4, among 40 HBsAg-positive patients suffering from acute hepatitis, antigen was found by classical EIA and RIA in urine from 22 subjects. No correlation appeared between the antigenaemia and antigenuria levels of HBsAg. For example, in patient 13 a high P/N ratio (141) was observed with almost the same P/N ratio (105) in urine. Another patient (39) with a P/N ratio of 147 in his serum, had a P/N of 12 in his urine. For the 10 chronic cases (Table 5) a high antigenaemia was observed in RIA whereas HBsAg antigenuria appeared low by the same technique. For the inhibition test using the anti-continuous MoAb, a dilution giving a signal of about 1 optical density was chosen. By this new test, all urine
Polypeptide composition of urinary HBsAg
Plppiecmoiino rnr ~~ The observation that HBsAg could only be detected by an inhibition technique and only with the anti-continuous MoAb, was indicative of a loss of tertiary structure of the HBsAg molecule. Therefore, for I I urines showing various activities in EIA inhibition, the structure of urinary HBsAg was studied by Western blot; reduced and non-reduced samples were examined. Antigenic material was specifically detected as a 62-kD band in only 3/11 patients when reduction was omitted. In the other urines, HBsAg did not enter into the gels (Table 6, Fig. 1). After reduction, antigenic polypeptides appeared generally as several bands in all samples. The analysis of these bands showed that urinary HBsAg was heterogeneous. As many as 10 bands ranging from 14 kD to 67 kD were observed in the different samples (Table 6). All the polypeptides characterized in the urine panel were found in only one out of 11 samples studied (urine no. 2, Fig. 1). The 62-kD band was the most constant since it was found in seven of the 11 samples (64%). DISCUSSION
In infectious diseases renal elimination of microbial antigens is currently observed, but all tests appeared unequally effective for urinary antigen detection [2]. Therefore, we put forward the hypothesis that discrepant results observed for urinary HBsAg could be relevant not only to the sensitivity of the test but also to the molecular state of the antigen which is filtered through the kidney. In our study, the most sensitive serum tests (EIA or RIA) were used for urinary HBsAg detection. These tests involved human polyclonal and mice monoclonal antibodies coated on the solid phase. Indeed, in hepatitis B convalescing patients, or in vaccinated healthy donors, it previously has been shown that anti-polypeptide antibodies, recognizing denatured HBsAg proteins, are rarely found [7]. The human or mouse immune response is generally directed against discontinuous epitopes and denatured HBsAg is not recognized by such tests. Under these conditions, it seems possible that the microbial antigen is largely modified, before or during renal filtration, and could not always be trapped on the solid phase by antibodies only specific for the quaternary or tertiary structures of the protein antigen. This hypothesis was fully confirmed for urinary HBsAg by using a MoAb strongly reactive to a continuous epitope of HBsAg and by using an anti-discontinuous HBsAg epitope MoAb as a control. For immunochemical analysis, a drastically purified HBsAg preparation was used. The purification process of serum HBsAg involved a step of trypsin digestion in order to eliminate the tightly bound serum proteins [9]; such a purified antigen contained only traces of albumin and reacted with six different
301
Urinary antigen detection Table 4. Hepatitis B virus markers in urine samples as compared with serum in acute hepatitis B
Urine
Serum Patient no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 *23 24 25 26 27 28 29 30 31 32 33 34
35 36 37 38 39 40
HBsAg (EIA)
HBeAg (EIA)
Anti-HBe (EIA)
Anti-HBcIgM (EIA)
+ +
+ +
+ -
+ +
+ + + + + -
-
+
-
-
+ + +
-
+
+ + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + +
-
+ + + + + +
-
-
-
+ -
-
-
+ + -
-
-
+ -
HBsAg (RIA)
P/N*
HBsAg (RIA) P/N*
Inhibition
testt
19 171 2-3
0-143 0-161 0-206
62 3-7
0240 0-258
109
3-1
0 291
123 130 137 114 104 109 141 111 86 71 88 69 83 125 79 108 114 93 89 81 11 106 13 39 86 89 76 92 103 75 39 76 147 107
29 1 2-4 2-8 3 2-8 105 2-2 1-8 2-2 19 18 1-2 1-6 1-8 2 5-8 4 2 4-8 2-5 35 19 2 19 7-9
0-112 0-348 0-083 0-071 0-231 0-173 0-146 0-147 0 213 0-183 0-267 0-197 0-327 0-140 0-197 0-267
89 102 7-2 110 119
22 2-1
1-9 22 14 1-8 1-2 3
0-162 0-195 0-178 0-217 0416 0118 0 189 0-062 0 358 0 290 0 190 0057 0-040 0-457
0091 0-095 0-183 0 216
+, Positive results;-, negative results. * P/N is considered positive when above 2 1. t OD with normal urine: 0-823.
MoAbs specific to discontinuous a epitope of H BsAg [8]. Proofs of anti-continuous specificity of the prepared MoAb allowing constant HBsAg detection were: (i) enhanced detection by EIA of reduced HBsAg compared with native antigen; (ii) total inhibition by reduced HBsAg and only partial inhibition by native HBsAg. Under the same conditions, reduced HBsAg was never detected by anti-discontinuous MoAb and reduced HBsAg did not inhibit anti-discontinuous MoAb [8]; (iii) reactivity with p24 polypeptide of the reduced HBsAg after SDS-PAGE, electrophoresis and immunoblotting.
Lack of correlation of parallel measurement in serum and urine cannot be explained and deserves further investigation. Dilution factor does not seem involved. In chronic cases results appear more uniform: high HBsAg levels in sera, low denatured HBsAg levels in urines (P< 5 10 6). The low concentration of HBsAg in urines could be due to the production of anti-HBs antibodies insufficient to clear HBsAg but sufficient to produce immune complexes eliminated by another route. For ETA inhibition assays with urines, native HBsAg directly bound to a solid phase appeared to be less reactive than
302
S. Badur, L. Grangeot-Keros & J. Pillot Table 5. Hepatitis B virus markers in urine samples as compared with serum in chronic hepatitis B
Serum Patient no. 41 42 43 44 45 46 47 48 49 50
Urine
HBsAg (EIA)
HBeAg (EIA)
Anti-HBe (EIA)
Anti-HBcIgM (EIA)
+ + + + + + + + + +
-
+ + + + + + + +
+
-
-
-
-
-
HBsAg (RIA) (P/N* 154 105 131 146 103 114 122 144 102 107
HBsAg (RIA) P/N* 4-2 2
1-8 19 2 3-5 1-4 2-9 25 3-4
Inhibition testt 0-338 0 524 0-468 0-158 0 402 0 520 0-378 0-191 0-603 0 534
+, Positive results;-, negative results. P/N is considered positive when above 2- 1. t OD with normal urine: 0-823. *
Table 6. Molecular analysis of urinary HBs by SDS-PAGE kD
Patient Urine (kD)* no. without reduction 2 7 12
13 17 18 19 23 35 43 44
67, 62 62 62 -
Urine after reduction (kD)*
67,62,58,54,48,31,20,18,17,14 -20,14 62,48,20 62, 20, 14 67,62,58,48,31,20
67 62 58 5
48
-67,62,48,20 -67, 62, 58, 48, 31, 20, 18, 14 62, 48, 31, 20, 18, 14
-31,14
1
-20 -18
* All results correspond to bands immunologically revealed. 20
HBsAg presented bound to an additional antibody layer. The reason for this phenomenon is not clear. Indeed, denaturation of epitopes directly adsorbed on the solid phase cannot be incriminated since the antigen is still recognized by the MoAb. Another hypothesis is that directly adsorbed HBsAg can bind MoAb with a stronger affinity than urinary antigen does. HBsAg appeared present in urine from all patients with antigenaemia, irrespective of other HBV markers. Urinary HBsAg was only fully reactive in EIA and in RIA for 3/50 patients. Under the same conditions, traces of HBsAg were detectable in 24/50 urines. In conclusion, urinary HBsAg remained undetectable with classical means in about 50% of HBsAg carriers as well as in patients suffering from acute hepatitis as in patients with chronic infection, The structure of urinary antigen appeared to be very polymorphic. Classically, Western blot of reduced serum HBsAg displays seven bands (7]; six of them (24, 28, 31l, 33, 39, 41 kD) correspond to S, pre-S2 and pre-Sl polypeptides with or
T17 14
Fig. 1. Western blot profile of urine no. 2. without glycosylation; another band of 68 kD was constantly observed with albumin. In the present study of urinary HBsAg, only one band (31 kD) corresponded to bands appearing from native serum HBsAg. Material below 24 kD (20, 18, 14 kD) was largely represented indicating a partial cleavage of the minimal constitutive polypeptides.
Urinary antigen detection A large majority of the urine polypeptides were detected by Western blot only after treatment by a reducing agent; nevertheless the native urinary antigen was not recognized by an antidiscontinuous MoAb. Therefore constant HBsAg denaturation appears to accompany renal filtration, largely affecting the conformation of the HBsAg molecule, without complete subunit resolution. In conclusion, denatured urinary HBsAg can constantly be detected in urine of HBsAg carriers when an appropriate technique is used. Our observations made with an antigenic viral protein could possibly be extended to other microbial antigens. Latex agglutination and sandwich EIA are usually performed in urine in order to characterize antigenic material from infectious agents eliminated by kidneys. Because it recognizes a unique epitope, MoAb must be preferred to polyclonal antibodies in inhibition tests when the size of the antigen molecule has been reduced. Furthermore, when antigen is largely denatured, a MoAb recognizing a continuous epitope is to be preferred to another MoAb recognizing discontinuous structures.
2 3 4 5
6 7
8
9
ACKNOWLEDGMENTS This work was supported by Caisse Nationale d'Assurance Maladie des Travailleurs Salaries, grant no. 87-3-14 OOC. We wish to thank Dr S. Iscaki (Institut Pasteur) for helpful discussion, Richard Keros (Kabi Pharmacia France) for careful reviewing of the manuscript and Myriam Magneney and Sylvie Pascal for their typing.
10
11
REFERENCES 1 Coonrod JD. Physical and immunologic properties of pneumococ-
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cal capsular polysaccharide produced during human infection. J Immunol 1974; 112:2193-201. Lebrun L, Guibert M, Pillot J. Failure of tests to detect pneumococcal antigens in urine. Eur J Clin Microbiol Infect Dis 1988; 7:83. Villarejos VM, Visna KA, Grutissez J, Rodriguez A. Role of saliva, urine and feces in the transmission of type B hepatitis. New Engl J Med 1974; 291:1375-7. Vittal SBW, Dourdourekas D, Steingmann F. Hepatitis B antigen in saliva, urine and tears. Am J Gastroenterol 1974; 61:133-5. Kaiser L, Thomas JK, Patterson MJ, Sanchez TV, Shapiro RS, Mayor GH. Hepatitis B surface antigen in urine of renal transplant recipients. Ann Int Med 1981; 94:783-4. Neurath AR, Kent SBH, Strick N, Taylor P, Stevens CE. Hepatitis B virus containing pre S gene encoded domains. Nature 1985; 315:1546. Petit MA, Maillard P. Capel F, Pillot J. Immunochemical structure of the hepatitis B surface antigen vaccine. II. Analysis of antibody responses in human sera against the envelope proteins. Mol Immunol 1986; 23:511-23. Pillot J, Riottot MM, Geneste C, Phalente L, Mangalo R. Analysis of the vaccinating group specificity a of HBsAg with monoclonal antibodies. Identification of discontinuous epitopes. Develop Biol Standard 1984; 57:299-304. Pillot J, Petit MA. Immunochemical structure of the hepatitis B surface antigen vaccine. I. Treatment of immobilized HBsAg by dissociation agents with or without enzymatic digestion and identification of polypeptides by protein blotting. Mol Immunol 1984; 21:53-60. Nilsson OB, Svalender PC, Larsson A. Immunization of mice and rabbits by intrasplenic deposition of nanogram quantities of protein attached to sepharose beads or nitrocellulose paper strips. J Immunol Meth 1987; 99:67-75. Towbin A, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979; 76:4350-4.
HBsAg in urine: a new approach for the detection of urinary antigens S. BADUR*, L. GRANGEOT-KEROS* & J. PILLOT*t *Service de Microbiologie et d'Immunologie, and INSERM U 131 H6pital Antoine B&clre, Clamart, and tUnitW d'Immunologie Microbienne, Institut Pasteur, Paris, France
(Acceptedfor publication 21 August 1991)
SUMMARY In order to define the optimal conditions for detection of microbial antigens in urine, urinary HBsAg excreted during hepatitis B was chosen as a model. Using commercial kits, which mainly involve antidiscontinuous epitopes, we found urinary HBsAg in only 50% of patients with HBsAg in their sera. In contrast, with an inhibition method involving a monoclonal antibody recognizing a continuous epitope, urinary HBsAg was found in 100% of these patients. Structural analysis of HBsAg showed that urinary HBsAg is denatured; it can escape detection by commercial kits well fitted for detection of native serum HBsAg. General implications for the revelation of urinary microbial antigens are discussed.
Keywords urinary HBsAg antigenuria microbial antigen degradation INTRODUCTION
Diagnosis of infectious diseases by characterization of microbial antigens in urine can be hampered by the limited concentration of investigated material. Such a failure could also be related to inappropriate conditions of the molecular state of the antigens, as shown for pneumococcal polysaccharidic antigens during human infection [1,2]. Little is known about the molecular state of urinary microbial antigens, as far as proteins are concerned. A lipoprotein representing the viral envelope of the hepatitis B virus (HBV) is produced in large excess (HBsAg) in sera of infected subjects. This situation is a good approach to investigate the optimal conditions for urinary microbial protein antigen detection and to analyse the molecular structure of the urinary antigen, comparatively to its serum counterpart. The optimal detection of antigenaemia is obtained by radioimmunoassay (RIA) or by enzyme immunoassay (EIA). Nevertheless with such techniques, data concerning the presence of HBsAg in urines vary according to the authors. Villajeros et al. [3] found antigenuria in three of 130 HBsAg carriers, while Vittal [4] showed antigenuria in one out of 13 asymptomatic seropositive carriers, but urinary HBsAg was detected neither in 12 patients with acute hepatitis, nor in five subjects with chronic active hepatitis. In another study, HBsAg was detected in urines of seven out of nine renal transplant recipients [5] and did not correlate with the presence of hidden or apparent haematuria. Human and monkey anti-HBs IgG are currently used for HBsAg detection and it is well established that these antibodies recognize discontinuous (conformational) epitopes and do not bind denatured HBsAg [6,7]. Under these conditions, the lack of
detection by the most sensitive assays (RIA, EIA) could be related to denaturation of the HBsAg excreted into the urines. In this study the presence of HBsAg in urines is re-evaluated by antibody-binding inhibition allowing the characterization of degradation products. In this work we establish that urinary HBsAg can be detected in all patients bearing HBsAg in their serum. Furthermore, in some cases where native HBsAg could be detected, this antigen appeared as a complex mixture of native and degraded viral envelope products. PATIENTS AND METHODS
Patients Forty patients with acute hepatitis B (AHB) and ten patients with chronic hepatitis B (CHB) were investigated. Urine and serum samples were stored at - 250C until investigation. The samples were not concentrated in order to prevent aggregation and to maintain urinary proteins as native as possible. HBV markers Serum HBsAg, hepatitis Be antigen (HBeAg), anti-HBe antibodies, anti-HBc IgM, and urinary HBsAg, were tested by commercial EIA kits (Organon Teknika, Boxtel, The Netherlands). Positive/negative (P/N) ratios for HBsAg were established by using AUSRIA II (Abbott Laboratories, Chicago, IL).
Preparation of monoclonal antibodies to discontinuous and continuous epitopes Monoclonal antibodies (MoAb) recognizing a discontinuous epitope of the HBsAg group a specificity have previously been described [8]. The anti-discontinuous MoAb IgGI chosen for this study recognizes highly purified HBsAg [9] in precipitation test, EIA and RIA. It did not recognize the reduced and
Correspondence: Professor Jacques Pillot, Hopital Antoine Beclere, 157 rue de Ia Porte de Trivaux, 92141 Clamart, France.
298
299
Urinary antigen detection alkylated purified HBsAG either in liquid phase or after adsorption on solid phase. Obtaining a MoAb recognizing a HBsAg continuous epitope is a rare event under usual conditions of MoAb preparation. Indeed, we have observed that a large majority of anti-HBs antibody molecules produced against HBsAg in man and animals are selectively directed against tertiary structures of HBsAg [7]. Therefore, mice usually failed to produce MoAb specific for a linear structure of amino acids. Such a MoAb specificity could nevertheless be obtained by immunizing with the major HBsAg polypeptide subunit. HBsAg was purified from plasma [9]. Polypeptides were resolved by SDS-PAGE according to a discontinuous buffer system in 12 5% acrylamide gel. For identification of polypeptides, the gel was treated with 1 mM dithiothreitol (DTT) in cold distilled water, stained for 5 min with 0-25 M KCl, 1 mm DTT, then fixed for 1 h with cold DTT solution. Polypeptide bands remained clear and the background became opaque. The 24-kD polypeptide (p24) was removed with a razor blade and stored at -250C. Before immunization of mice, the frozen gel was ground and homogenized. Then, the thawed mixture was centrifuged and the supernatant used for intrasplenic immunization [10]; 0-1 ml containing 0-2 mg of p24 was used for each animal. Splenocytes were collected after a unique intrasplenic injection, then fused with myeloma X63 cells. Each culture medium, in which cell colonies developed, was tested on the same plate by EIA against both native and denatured HBsAg. Denaturation of HBsAg for EIA was performed by reduction at 60°C for 10 min, with 5% of 2-mercaptoethanol. This concentration of 2-mercaptoethanol did not hamper the immunological reaction. Plates were coated with 0.1 ml of a solution containing 2 yg/ml of native or reduced HBsAg, washed with phosphate-buffered saline (PBS) containing 0 1% (w/v) of Tween 20 (PBS-T) and overcoated with PBS-T containing 2% of bovine serum albumin (BSA), at 37°C for 3 h. Then, 0 1 ml of each cell culture supernatant was incubated for I h at 37°C. After washing, the presence of MoAb reacting with reduced HBsAg was detected with goat anti-mouse IgG peroxidase conjugate (I pg/ml) produced in our laboratory. EIA inhibition Two types of assays were performed. In the first, native HBsAg was directly adsorbed on the solid phase; in the second, an antiHBs rabbit IgG layer was inserted between the antigen and the solid phase. Coating was performed with 5 pg/ml of our antiHBs IgG preparation and 2 ,g/ml of HBsAg. After washings, 0.1 ml of the HBsAg solution or of urine was mixed with an equal volume of the diluted anti-discontinuous MoAb or an equal volume of MoAb recognizing preferentially the reduced HBsAg and named anti-continuous MoAb. The mixtures were incubated for I h at 37°C, and then 0 I ml was transferred onto HBsAg-coated plates. Fixation of anti-HBs MoAb was revealed with goat anti-mouse IgG peroxidase conjugate. An inhibition beyond 50% was considered significant. Urine analysis
Eleven urine samples were analysed by Western blot. Before electrophoresis, urines were heated at 60°C for 10 min with sample buffer (0 125 M Tris HCl, pH 6 8) containing 2% SDS and 5%/ of 2-mercaptoethanol. Following SDS-PAGE, proteins were transferred to a nitrocellulose sheet [11]. Bands were
detected using rabbit anti-HBs serum from our laboratory (reacting with continuous and discontinuous epitopes) and 125f1 labelled monkey anti-rabbit immunoglobulin (Amersham International, Amersham, UK); the bands were visualized by autoradiography using an X-ray film (CORONEX4, Du Pont de Nemours, Frankfurt/Main, Germany). RESULTS
Specificity of monoclonal antibody selected for its capacity to recognize denatured HBsAg Specificity of MoAbs obtained by immunization of mice with the p24 polypeptide band was controlled by EIA as described in Materials and Methods by using native HBsAg and the same HBsAg reduced with 5% of mercaptoethanol. The anti-discontinuous MoAb was used as control. Under these conditions, one MoAb which reacted with reduced HBsAg twice as much as with native antigen was selected. The same reduced antigen did not react with the anti-discontinuous MoAb (Table 1). The anti-continuous specificity of this MoAb was confirmed by Western blot. After HBsAg reduction, SDS-PAGE and immunoblotting, this MoAb reacted only with the p24 polypeptide whereas the anti-discontinuous MoAb appeared unreactive with any band of the viral envelope (p24, p28, gp 31, gp 33) (results not shown). When reduced HBsAg was mixed with anti-continuous or anti-discontinuous MoAb, inhibition was only observed with the anti-continuous MoAb, indicating that anti-discontinuous MoAb did not recognize the reduced HBsAg (Table 2). Optimization of the anti-continuous monoclonal antibody inhibition test for urine samples Assuming that HBsAg could be present in urine as denatured protein and unreactive in the classical sandwich RIA or EIA, we developed an inhibition test involving the monoclonal antibody recognizing a continuous epitope. For this assay, native HBsAg was presented directly adsorbed on the solid phase or retained by anti-HBs IgG coating the solid phase. As shown in Table 3, the HBsAg presentation did not influence the capacity of native or reduced HBsAg artificially introduced in urine, but influenced the capacity of true urinary HBsAg to block the reaction of anti-continuous MoAb with solid phase HBsAg. If a strong inhibition was observed when urinary HBsAg was used to inhibit the reaction with antigen presented by means of an additional antibody layer, a weak reaction was observed when the antigen was directly adsorbed. Furthermore, native HBsAg Table 1. Enzymeimmunoassay reactivity of anti-continuous epitope MoAb with native or reduced HBsAg
Native HBsAg*
Reduced HBsAgt
epitope (dil. 1/1000) Anti-continuous HBsAg
>2
0297
epitope (dii. 1/1000)
0-535
1-060
MoAb Anti-discontinuous HBsAg
The coating of HBsAg on solid phase was performed in absence* or in presencet of 2-mercaptoethanol. Results are expressed as optical absorbance at 492 nm.
S. Badur, L. Grangeot-Keros & J. Pillot
300
Table 2. Enzymeimmunoassay inhibition of anti-discontinuous and anti-continuous MoAb by reduced HBsAg Dilution of MoAb
Anti-discontinuous epitope
Anti-continuous epitope
10-2
1-3 10o-4 1o-5 10-2 10-3 1O-4 l0-
>2
1-957 1-989 1-700
No antigen
>2
1-980 1 387 1-049
Reduced HBsAg
1 895 1-832 1-723 1-581 0-125 0-140 0-155 0-137
Native HBsAg was used for direct coating. For inhibition, reduced HBsAg was used at 2 jug/ml. Results are expressed as optical absorbance at 492 nm.
Table 3. Otpimization of the inhibition test involving MoAb to a continuous epitope
HBsAg Urine samples
samples coming from the 50 serum HBsAg positive patients (including the 23 urine samples found to be negative by RIA) appeared positive. No correlation was found between the amount of HBsAg in RIA and the inhibitory rate for the anticontinuous MoAb. Furthermore, no correlation was observed between the EIA inhibition test and the other parameters studied in sera. Urine samples found negative for HBsAg by classical RIA and positive by the inhibition test involving anticontinuous MoAb appeared negative when anti-discontinuous MoAb was used (results not shown).
On solid phase
Captured by anti-HBs IgG
Normal A Normal B Normal A+native HBsAg Normal A+ reduced H BsAg
1-090
0-823
1-178 0-627 0 049
1-009 0 573 0.164
Patient 2 Patient 3 Patient 5 Patient 14 Patient 44
0 974 0-825 0-890 0.558 0 628
0.161 0-206 0-258 0-147 0 158
HBsAg was added to the urine at a concentration of 2 ,g/ml. For
inhibition, a dilution of anti-continuous MoAb was chosen to give an optical density of about 1 (10-5). Results are expressed as optical absorbance at 492 nm.
only partially inhibited anti-continuous epitope reactivity, confirming the weak activity of anti-continuous MoAb with native HBsAg. Comparison between serum and urine HBsAg detection As shown in Table 4, among 40 HBsAg-positive patients suffering from acute hepatitis, antigen was found by classical EIA and RIA in urine from 22 subjects. No correlation appeared between the antigenaemia and antigenuria levels of HBsAg. For example, in patient 13 a high P/N ratio (141) was observed with almost the same P/N ratio (105) in urine. Another patient (39) with a P/N ratio of 147 in his serum, had a P/N of 12 in his urine. For the 10 chronic cases (Table 5) a high antigenaemia was observed in RIA whereas HBsAg antigenuria appeared low by the same technique. For the inhibition test using the anti-continuous MoAb, a dilution giving a signal of about 1 optical density was chosen. By this new test, all urine
Polypeptide composition of urinary HBsAg
Plppiecmoiino rnr ~~ The observation that HBsAg could only be detected by an inhibition technique and only with the anti-continuous MoAb, was indicative of a loss of tertiary structure of the HBsAg molecule. Therefore, for I I urines showing various activities in EIA inhibition, the structure of urinary HBsAg was studied by Western blot; reduced and non-reduced samples were examined. Antigenic material was specifically detected as a 62-kD band in only 3/11 patients when reduction was omitted. In the other urines, HBsAg did not enter into the gels (Table 6, Fig. 1). After reduction, antigenic polypeptides appeared generally as several bands in all samples. The analysis of these bands showed that urinary HBsAg was heterogeneous. As many as 10 bands ranging from 14 kD to 67 kD were observed in the different samples (Table 6). All the polypeptides characterized in the urine panel were found in only one out of 11 samples studied (urine no. 2, Fig. 1). The 62-kD band was the most constant since it was found in seven of the 11 samples (64%). DISCUSSION
In infectious diseases renal elimination of microbial antigens is currently observed, but all tests appeared unequally effective for urinary antigen detection [2]. Therefore, we put forward the hypothesis that discrepant results observed for urinary HBsAg could be relevant not only to the sensitivity of the test but also to the molecular state of the antigen which is filtered through the kidney. In our study, the most sensitive serum tests (EIA or RIA) were used for urinary HBsAg detection. These tests involved human polyclonal and mice monoclonal antibodies coated on the solid phase. Indeed, in hepatitis B convalescing patients, or in vaccinated healthy donors, it previously has been shown that anti-polypeptide antibodies, recognizing denatured HBsAg proteins, are rarely found [7]. The human or mouse immune response is generally directed against discontinuous epitopes and denatured HBsAg is not recognized by such tests. Under these conditions, it seems possible that the microbial antigen is largely modified, before or during renal filtration, and could not always be trapped on the solid phase by antibodies only specific for the quaternary or tertiary structures of the protein antigen. This hypothesis was fully confirmed for urinary HBsAg by using a MoAb strongly reactive to a continuous epitope of HBsAg and by using an anti-discontinuous HBsAg epitope MoAb as a control. For immunochemical analysis, a drastically purified HBsAg preparation was used. The purification process of serum HBsAg involved a step of trypsin digestion in order to eliminate the tightly bound serum proteins [9]; such a purified antigen contained only traces of albumin and reacted with six different
301
Urinary antigen detection Table 4. Hepatitis B virus markers in urine samples as compared with serum in acute hepatitis B
Urine
Serum Patient no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 *23 24 25 26 27 28 29 30 31 32 33 34
35 36 37 38 39 40
HBsAg (EIA)
HBeAg (EIA)
Anti-HBe (EIA)
Anti-HBcIgM (EIA)
+ +
+ +
+ -
+ +
+ + + + + -
-
+
-
-
+ + +
-
+
+ + + + + + +
+ + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + + + +
-
+ + + + + +
-
-
-
+ -
-
-
+ + -
-
-
+ -
HBsAg (RIA)
P/N*
HBsAg (RIA) P/N*
Inhibition
testt
19 171 2-3
0-143 0-161 0-206
62 3-7
0240 0-258
109
3-1
0 291
123 130 137 114 104 109 141 111 86 71 88 69 83 125 79 108 114 93 89 81 11 106 13 39 86 89 76 92 103 75 39 76 147 107
29 1 2-4 2-8 3 2-8 105 2-2 1-8 2-2 19 18 1-2 1-6 1-8 2 5-8 4 2 4-8 2-5 35 19 2 19 7-9
0-112 0-348 0-083 0-071 0-231 0-173 0-146 0-147 0 213 0-183 0-267 0-197 0-327 0-140 0-197 0-267
89 102 7-2 110 119
22 2-1
1-9 22 14 1-8 1-2 3
0-162 0-195 0-178 0-217 0416 0118 0 189 0-062 0 358 0 290 0 190 0057 0-040 0-457
0091 0-095 0-183 0 216
+, Positive results;-, negative results. * P/N is considered positive when above 2 1. t OD with normal urine: 0-823.
MoAbs specific to discontinuous a epitope of H BsAg [8]. Proofs of anti-continuous specificity of the prepared MoAb allowing constant HBsAg detection were: (i) enhanced detection by EIA of reduced HBsAg compared with native antigen; (ii) total inhibition by reduced HBsAg and only partial inhibition by native HBsAg. Under the same conditions, reduced HBsAg was never detected by anti-discontinuous MoAb and reduced HBsAg did not inhibit anti-discontinuous MoAb [8]; (iii) reactivity with p24 polypeptide of the reduced HBsAg after SDS-PAGE, electrophoresis and immunoblotting.
Lack of correlation of parallel measurement in serum and urine cannot be explained and deserves further investigation. Dilution factor does not seem involved. In chronic cases results appear more uniform: high HBsAg levels in sera, low denatured HBsAg levels in urines (P< 5 10 6). The low concentration of HBsAg in urines could be due to the production of anti-HBs antibodies insufficient to clear HBsAg but sufficient to produce immune complexes eliminated by another route. For ETA inhibition assays with urines, native HBsAg directly bound to a solid phase appeared to be less reactive than
302
S. Badur, L. Grangeot-Keros & J. Pillot Table 5. Hepatitis B virus markers in urine samples as compared with serum in chronic hepatitis B
Serum Patient no. 41 42 43 44 45 46 47 48 49 50
Urine
HBsAg (EIA)
HBeAg (EIA)
Anti-HBe (EIA)
Anti-HBcIgM (EIA)
+ + + + + + + + + +
-
+ + + + + + + +
+
-
-
-
-
-
HBsAg (RIA) (P/N* 154 105 131 146 103 114 122 144 102 107
HBsAg (RIA) P/N* 4-2 2
1-8 19 2 3-5 1-4 2-9 25 3-4
Inhibition testt 0-338 0 524 0-468 0-158 0 402 0 520 0-378 0-191 0-603 0 534
+, Positive results;-, negative results. P/N is considered positive when above 2- 1. t OD with normal urine: 0-823. *
Table 6. Molecular analysis of urinary HBs by SDS-PAGE kD
Patient Urine (kD)* no. without reduction 2 7 12
13 17 18 19 23 35 43 44
67, 62 62 62 -
Urine after reduction (kD)*
67,62,58,54,48,31,20,18,17,14 -20,14 62,48,20 62, 20, 14 67,62,58,48,31,20
67 62 58 5
48
-67,62,48,20 -67, 62, 58, 48, 31, 20, 18, 14 62, 48, 31, 20, 18, 14
-31,14
1
-20 -18
* All results correspond to bands immunologically revealed. 20
HBsAg presented bound to an additional antibody layer. The reason for this phenomenon is not clear. Indeed, denaturation of epitopes directly adsorbed on the solid phase cannot be incriminated since the antigen is still recognized by the MoAb. Another hypothesis is that directly adsorbed HBsAg can bind MoAb with a stronger affinity than urinary antigen does. HBsAg appeared present in urine from all patients with antigenaemia, irrespective of other HBV markers. Urinary HBsAg was only fully reactive in EIA and in RIA for 3/50 patients. Under the same conditions, traces of HBsAg were detectable in 24/50 urines. In conclusion, urinary HBsAg remained undetectable with classical means in about 50% of HBsAg carriers as well as in patients suffering from acute hepatitis as in patients with chronic infection, The structure of urinary antigen appeared to be very polymorphic. Classically, Western blot of reduced serum HBsAg displays seven bands (7]; six of them (24, 28, 31l, 33, 39, 41 kD) correspond to S, pre-S2 and pre-Sl polypeptides with or
T17 14
Fig. 1. Western blot profile of urine no. 2. without glycosylation; another band of 68 kD was constantly observed with albumin. In the present study of urinary HBsAg, only one band (31 kD) corresponded to bands appearing from native serum HBsAg. Material below 24 kD (20, 18, 14 kD) was largely represented indicating a partial cleavage of the minimal constitutive polypeptides.
Urinary antigen detection A large majority of the urine polypeptides were detected by Western blot only after treatment by a reducing agent; nevertheless the native urinary antigen was not recognized by an antidiscontinuous MoAb. Therefore constant HBsAg denaturation appears to accompany renal filtration, largely affecting the conformation of the HBsAg molecule, without complete subunit resolution. In conclusion, denatured urinary HBsAg can constantly be detected in urine of HBsAg carriers when an appropriate technique is used. Our observations made with an antigenic viral protein could possibly be extended to other microbial antigens. Latex agglutination and sandwich EIA are usually performed in urine in order to characterize antigenic material from infectious agents eliminated by kidneys. Because it recognizes a unique epitope, MoAb must be preferred to polyclonal antibodies in inhibition tests when the size of the antigen molecule has been reduced. Furthermore, when antigen is largely denatured, a MoAb recognizing a continuous epitope is to be preferred to another MoAb recognizing discontinuous structures.
2 3 4 5
6 7
8
9
ACKNOWLEDGMENTS This work was supported by Caisse Nationale d'Assurance Maladie des Travailleurs Salaries, grant no. 87-3-14 OOC. We wish to thank Dr S. Iscaki (Institut Pasteur) for helpful discussion, Richard Keros (Kabi Pharmacia France) for careful reviewing of the manuscript and Myriam Magneney and Sylvie Pascal for their typing.
10
11
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