Electron Microscopy. study was undertaken to confirm the validity of. unrelated to the hepatitis A or B viruses (7, 15), and

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1 INFECTION AND IMMUNITY, Apr. 1976, p Copyright C 1976 American Society for Microbiology Vol. 13, No. 4 Printed in U.S.A. Quantitation of Antibody to Hepatitis A Antigen by Immune Electron Microscopy JULES L. DIENSTAG,* DAVID W. ALLING, AND ROBERT H. PURCELL National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 214 Received for publication 25 November 1975 A set of precipitin reactions was performed by immune electron microscopy (IEM) with hepatitis A antigen (HA Ag) and varying quantities of antibody to HA Ag (anti-ha). Serial dilution of anti-ha resulted in progressive diminution in IEM antibody rating. These data, together with a highly significant correlation between IEM ratings and immune adherence hemagglutination (IAHA) titers on 92 coded serum samples, confirm that quantitative serology can be performed by IEM. To demonstrate the different kinetics of antibody development by IEM and IAHA, we used both techniques to test for anti-ha in longitudinally collected sera from a chimpanzee experimentally infected with hepatitis A virus. Detection of anti-ha was possible by IEM during acute hepatitis, but IAHA anti-ha was not observed until approximately 4 weeks later. Six weeks after acute illness, IEM ratings reached a plateau beyond which IAHA titers continued to rise gradually. Peak titers were achieved 11 months after inoculation. Until recently, neutralization of virus activity in marmosets (9, 26) was the only method of detecting antibody during hepatitis A virus infection. In 1973, Feinstone et al. (6) reported the detection by immune electron microscopy (IEM) of a 27-nm virus-like particle in the stools of volunteers with acute viral hepatitis type A. Hepatitis A antigen (HA Ag), as the particle was designated, was detected in acutephase stool filtrates incubated with convalescent serum containing antibody (anti-ha); for the detection of antibody, HA Ag-positive stool filtrates were incubated with the serum to be tested, and the amount of antibody adhering to HA Ag particles was noted. Quantitation of antibody ratings by IEM for other virus antigens had been standardized previously by Kapikian et al. (12-14). The same rating system, when applied to the IEM assay for anti-ha, distinguished reliably and reproducibly, under code, between preinfection or acute sera and convalescent sera from patients with type A hepatitis. On a scale of to 4+, antibody seroconversions of at least 1+ have been demonstrated in every one of 42 cases of epidemiologically compatible type A hepatitis, but in none of 15 cases of type B hepatitis and in none of more than 55 cases of non-b post-transfusion hepatitis tested in one laboratory alone (28). Similar experiences have been reported from other laboratories as well (8, 2). Despite these data, the specificity and quantitative application of IEM for anti-ha detection have been challenged (1, 31), and the results of serological studies containing IEM antibody ratings have been questioned (31). Moreover, the development of quantitative complement fixation (CF) (27) and immune adherence hemagglutination (IAHA) assays for anti-ha (23) led to a renewed challenge of the validity of 129 quantitative IEM serology (31). The following study was undertaken to confirm the validity of quantitative anti-ha ratings by IEM and to compare IEM ratings with IAHA titers. MATERIALS AND METHODS Sera. Serum samples were obtained at various times during the course of illness from patients with experimental type A hepatitis (3, 17), naturally occurring type A hepatitis (5, 8, 25), naturally occurring type B hepatitis (18), post-transfusion hepatitis unrelated to the hepatitis A or B viruses (7, 15), and viral gastroenteritis (14). In addition, serial sera were collected from a chimpanzee experimentally infected with hepatitis A virus (4). IEM. Anti-HA was detected under code in serum samples by IEM as described previously (5, 6). Two percent human stool filtrates containing approximately 25 to 5 hepatitis A antigen particles per electron microscope grid square served as the source of antigen, and sera were diluted 1:1 in phosphatebuffered saline (.1 M phosphate,.85% sodium chloride, ph 7.4). The amount of antibody adhering to antigen particles was rated on a scale of to 4+, based on the density of the antibody halo (5, 6). IAHA. The IAHA assay was performed on heat-

2 121 DIENSTAG, ALLING, AND PURCELL inactivated (3 min at 56 C) serum samples as described by Moritsugu et al. (24). The assay is a modification of the IAHA test described by Mayumi et al. (21) and Miller et al. (23). Hepatitis A antigen purified from human stool (24) was used as the source of antigen, and sera were diluted in veronalbuffered saline supplemented with.1% bovine serum albumin (11). Statistical calculations. The degree of dependence between IEM ratings and IAHA titers on the same serum samples was estimated by calculating a Spearman rank correlation coefficient (3). A similar computation was made for a comparable set of sera tested by CF and IAHA and reported previously by Provost et al. (27), Miller et al. (23), and Krugman et al. (16). To allow calculation of these correlation coefficients, anti-ha titers measured by IAHA or CF and anti-ha ratings measured by IEM were converted to rank order values. RESULTS To demonstrate the validity of anti-ha quantitation by IEM, we attempted to reproduce a classic set of antigen-antibody precipitin reactions in which antigen concentration was held constant and antibody concentration was varied to yield preparations ranging from antigen excess to antibody excess (2). Convalescent serum from a single patient with naturally occurring type A hepatitis was diluted 1:64 in phosphate-buffered saline and in serial 1-fold dilutions thereafter. The anti-ha titer of the undiluted serum was 1:32, by IAHA, and its anti-ha rating by IEM (performed on a 1-fold dilution of the original serum) was 4+. In the region of extreme antibody excess, individual particles were heavily coated by antibody (rating 3 to 4+) and rarely aggregated. When small immune aggregates were formed, the distance between particles was relatively great. Almost all antigenic sites on the particles were occupied, limiting interparticle aggregation (Fig. 1A). As the antibody was diluted and the mixture approached antigen-antibody equivalence, large aggregates of HA Ag were common, the distance between particles was reduced, and the quantity of antibody was rated 2 to 3+ (Fig. 1B). Further antibody dilution resulted in relative antigen excess, characterized by small aggregates with small interparticle distances and occasional single particles with minimal antibody (rated 1+) (Fig. 1C). Beyond a serum dilution of 1:64,, only a trace of antibody could be detected, and only INFECT. IMMUN. unaggregated single particles were seen (Fig. 1D). On the basis of this and several similar dilution studies, we determined that a 1+ rise in antibody rating was the approximate equivalent of a 1-fold increase in titer. To evaluate the comparability of quantiative anti-ha determinations made by IEM and IAHA, we compared IEM ratings of single (1- fold) serum dilutions with IAHA titers determined on serial (2-fold) serum dilutions under code on 92 serum samples collected from various sources as described in Materials and Methods. The good agreement in antibody quantitation by the two methods is demonstrated in Fig. 2. For these samples the Spearman rank correlation coefficient for agreement between IEM ratings and IAHA titers was +.97 (P <.1). For a comparable set of 97 sera tested by Provost et al. (27), Miller et al. (23), and Krugman et al. (16), the Spearman rank correlation coefficient for a comparison between CF and IAHA titers was only (P <.1). The temporal relationship between the development of IEM and IAHA antibody was evaluated in longitudinalfy collected serum samples from a chimpanzee experimentally infected with hepatitis A virus. Antibody was first detectable by IEM during acute illness, approximately 4 weeks earlier than by IAHA (Fig. 3). Antibody detectable by IEM reached maximum concentration approximately 6 weeks after onset of hepatitis, whereas maximum titers measured by IAHA were not reached until almost a year after onset. Anti-HA measured by both methods was still present at high levels almost 2 years after hepatitis in this chimpanzee. DISCUSSION The demonstration of a set of precipitin reactions for the HA Ag-anti-HA system analogous to a classic quantitative precipitin curve for other antigen-antibody interactions (1) indicates that quantitative estimates of serum anti- HA can be made reliably by IEM. Although evaluation of serial serum dilutions for antibody would be the most quantitative method of performing IEM, the laboriousness of such an approach precludes its use for routine testing. However, the graphic and statistical correlation presented here between IEM ratings and IAHA titers confirms the validity of quantita- FIG. 1. Hepatitis A antigen particles incubated with serially diluted antibody concentrations. (A) Extreme antibody excess; serum diluted 1:64; IEM antibody rating 3 to 4 +. (B) Approximate antigen-antibody equivalence; serum dilution 1:64; antibody rating 2 to 3 +. (C) Relative antigen excess; serum dilution 1:6,4; antibody rating 1+. (D) Extreme antigen excess with trace antibody; serum dilution 1:64,. Note the decrease in interparticle distance and in antibody halo density with successive antibody dilution. All particles measure 27 nm, but those with more antibody appear larger. The bar represents 1 nm. Magnification x114,7 (2% phosphotungstic acid negative stain).

3 VOL. 13, 1976 A B C.s- A.- At1.. <A14. -D ~~~~~~~~~~~~~~~!-.', *4. ^'' b','.-'''' '' '-'- t..,.'.; '.',-f;:',t '.-.' 'es,., < _ e 1 nm.;.->.^ 2....:; ;.-... s.,. > ANTIBODY TO HEPATITIS A ANTIGEN ṡ.~~~~~~~~~~~~. *,P,~ t. :.. *wa,.'.:,ff ;J.- *.;..- ;-.i:..

4 1212 DIENSTAG, ALLING, AND PURCELL tive serological studlies performed on a single for the kinetics of hemagglutination-inhibitdilution of serum by IEM (4-8, 12-15, 2, 28). In ing, neutralizing, and CF antibodies to poliowith results obtained by myelitis viruses (19, 22). Drawn 3 to 4 weeks terms of correlation IAHA, IEM actually fared better than the CF after acute illness, a serum sample from a patient with type A hepatitis may contain almost test. Multiple factors nnay account for the differ- maximal IEM anti-ha yet barely detectable ences among the three serological methods. As IAHA anti-ha. Secondly, IEM antibody deter- 3 and in the previous minations plateau early during convalescence demonstrated in Fig. study by Krugman et al. (16), the kinetics of at a rating of 3 to 4+, beyond which further IEM, IAHA, and ( CF anti-ha development quantitation cannot be made without dilution vary. Similar differeences have been described of the serum. Thus, as shown in Fig. 3, serial 1,31,72 163,84 H 2,48 oc: i, 2,56 I <1 - sera collected 6 weeks after acute illness all had anti-ha ratings by IEM of 3 to 4+, whereas the anti-ha titer determined by IAHA varied from 1:64 to 1:1,24. In addition, the three assays may detect different antibody classes; if non-complement-fix- * ing antibodies are involved, they will not be detected by CF or IAHA but will appear in the IEM assay. Moreover, serum anti-complementary activity occurring early during the course of illness may invalidate the results of CF and IAHA tests on the acute sample of a serum pair. In fact, the unique specificity of IEM derives from the direct visualization of the antigen-antibody complex, independent of indirect indicators of immunoreaction such as complement or hemagglutination of erythrocytes. On 2 3 the other hand, although IEM is the most spe- IEM RATING cific of the three tests, like immunofluores- FIG. 2. Reciprocal cantibody titer determined by cence, IEM is a subjective test whose results LAHA plotted as a fundction ofantibody rating deter- can be influenced by observer bias and, theremined by IEM on a 1:11 dilution of the same serum fore, must be performed under code. sample. Although IAHA is a more practical test for _ 32.CL - 16 *u E 8 I '1 CHIMPANZEE 753 /, f / ' 48 '96 WEEKS AFTER INOCULATION FIG. 3. Anti-HA rated in a single 1:1 serum dilution by IEM and titered in serial twofold dilutions by IAHA in longitudinally collected serum samples from a chimpanzee with experimental hepatitis A virus infection. 4 3 I1 INFECT. IMMUN.

5 VOL. 13, 1976 large-scale screening of sera for anti-ha, IEM may be necessary for early diagnosis, that is, for the detection of antibody early in the course of illness, before the development of IAHA antibody. Furthermore, IEM may be necessary to assess occasional sera with anti-complementary activity or nonspecific reactivity in the IAHA test, and because of problems in obtaining and purifying HA Ag suitable for IAHA and CF, IEM will remain the only available assay for anti-ha in many laboratories. LITERATURE CITED 1. Almeida, J. D., F. W. Gay, and T. G. Wreghitt Pitfalls in the study of hepatitis A. Lancet 2: Almeida, J. D., and A. P. Waterson The morphology of virus-antibody interaction. Adv. Virus Res. 15: Boggs, J. D., J. L. Melnick, M. E. Conrad, and B. F. Felsher Viral hepatitis: clinical and tissue culture studies. J. Am. Med. Assoc. 214: Dienstag, J. L., S. M. Feinstone, R. H. Purcell, J. H. Hoofnagle, L. F. Barker, W. T. London, H. Popper, J. M. Peterson, and A. Z. 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Hyg. 65: Locarnini, S. A., A. A. Ferris, A. C. Stott, and I. D. Gust The relationship between a 27-nm viruslike particle and hepatitis A as demonstrated by immune electron microscopy. Intervirology 4: Mayumi, M., K. Okochi, and K. Nishioka Detection of Australia antigen by means of immune adherence hemagglutination test. Vox Sang. 2: Melnick, J. L., M. Ramos-Alvarez, F. L. Black, A. J. Girardi, and D. Nagaki Poliomyelitis viruses in tissue culture. VII. Experiences with viral and serological diagnostic procedures. Yale J. Biol. Med. 26: Miller, W. J., P. J. Provost, W. J. McAleer,. L. Ittensohn, V. M. Villarejos, and M. R. Hilleman Specific immune adherence assay for human hepatitis A antibody. Application to diagnostic and epidemiologic investigations. Proc. Soc. Exp. Biol. Med. 149: Moritsugu, Y., J. L. Dienstag, J. Valdesuso, D. C. Wong, J. Wagner, J. A. Routenberg, and R. H. 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