Radioimmunoassay for Detection of Hepatitis B e Antigen and its Antibody. Results of Clinical Evaluation

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1 Radioimmunoassay for Detection of Hepatitis B e Antigen and its Antibody. Results of Clinical Evaluation ISA K. MUSHAHWAR, PH.D., LARRY C. MCGRATH, PH.D., JAMES DRNEC, PH.D., AND LACY R. OVERBY, PH.D. Mushahwar, Isa K., McGrath, Larry C, Drnec, James, and Overby, Lacy R.: Radioimmunoassay for detection of Hepatitis B e antigen and its antibody. Results of clinical evaluation. Am J Clin Pathol 76: , The performance of a solid phase radioimmunoassay (ABBOTT-HBe ) for the detection of hepatitis B e antigen (HBeAg;) and its antibody anti-hbe was evaluated in clinical studies. The reagents and procedure were found to be reproducible by seven investigators, and labto-lab variations were minimal, lln HBsAg positive sera, AB BOTT-HBe detected HBeAg or anti-hbe in 9% of the specimens compared to 5% tested by immunodiffusion. During the early acute stage of viral infection, serum HBeAg coincided with the rise and decline of HBsAg and seroconversion to anti- HBe occurred most often prior to loss of HBsAg. Patients with clinical evidence of chronic liver disease showed persistence of HBsAg and HBeAg. Vertical transmission studies of hepatitis B virus infection from mother to newborns, showed that mothers whose sera were positive for both HBsAg and HBeAg resulted in a greater incidence of transmission of hepatitis B virus to their offspring than mothers whose sera were HBsAg positive but HBeAg negative. (Key words: ABBOTT-HBe ; HBeAG; Anti-HBe; Hepatitis B Virus; Hepatitis B serological markers; Acute Hepatitis B infection; Qironic Hepatitis B infection; Radioimmunoassay.) THREE DISTINCT antigen-antibody systems are associated with hepatitis B virus (HBV): hepatitis B surface antigen (HBsAg) and it:> antibody anti-hbs; hepatitis B core antigen (HBeAg) and its antibody anti- HBe; and hepatitis B e antigen (HBeAg) and its antibody anti-hbe. HBsAg is associated with the presence of HBV DNA polymerase and circulating Dane particles in serum ; hence, its presence has been proposed as a marker of ongoing viral replication, liver disease, 3 ' 2 and a high risk of transmission of HBV.'- 214 The presence of anti-hbe is indicative of lower infectivity and a better clinical prognosis. 3 4 ', In the past, agar gel immunodiffusion and counterelectrophoresis 813 methods have been used for detecting HBeAg and anti-hbe in serum. These methods have limited sensitivity and detectability and may lack objectivity due to indistinct precipitation patterns. Since Received January 27, 1981; received revised manuscript and accepted for publication May I, Address reprint requests to Dr. Mushahwar: Hepatitis Research Laboratory, Department 9D, Building L-3, Abbott Laboratories, North Chicago, Illinois 664. Abbott Laboratories, North Chicago, Illinois only about one half of the sera with HBsAg are found to have HBeAg or anti-hbe by these methods, a simpler, more sensitive and specific test was desirable to assess the significance and prevalence of these markers throughout the course of disease. An RIA (ABBOTT- HBe ) was therefore developed' and was tested in clinical studies to verify its performance for clinical laboratory use. The basis for the ABBOTT-HBe test 1 is illustrated in Fig. 1. The test is a direct solid phase RIA using anti-hbe coated 6 mm beads. HBeAg if present in serum will bind to the solid phase antibody. During a second incubation, l25 I-labeled anti-hbe will then bind to the antigen fixed to the solid phase. The resulting count rate of the multiple layer product (sandwich) is in proportion to HBeAg in the original serum. The anti-hbe test uses the same reagents and is based on a neutralization principle. A sample to be tested is mixed with a neutralization solution which contains a known amount of HBeAg. If antibody is present in the sample, neutralization of the added antigen will occur. The amount of antigen not neutralized by the antibody of the sample is then determined as described in the HBeAg test. The amount of antigen bound to the bead is inversely proportional to the antibody content of the sample. Materials and Methods Preparation and Preliminary Testing of Reagents Sera containing HBeAg and anti-hbe were obtained according to the methods described previously. 1 Anti- HBe coated polystyrene beads, l25 I-anti-HBe IgG, negative and positive controls, and neutralizing reagent were prepared and assembled together as lots and were subjected to quality control testing for detectability, sensitivity, and reproducibility. Detectability of each lot was determined by its ability to correctly identify specimens in a panel of HBeAg and anti-hbe positive and negative sera. Sensitivity was determined by testing dilutions of a reference standard serum containing Downloaded from on 9 May 218 ( /81/11/692 $.8 American Society of Clinical Pathologists 692

2 Vol. 76 No. 5 CLINICAL EVALUATION OF ABBOTT-HBe 693 RADIOIMMUNE ASSAY HB e Ag REAGENT SAMPLE COMPLEX,25 l-probe PRODUCT ANTI-HB e HBeAg ANTI-HB e HBeA 9 FIG. 1. Schematic illustration of the direct solid-phase radioimmunoassay for HBeAg. Polystyrene beads of 6 mm diameter were coated with a dilution of human anti-hbe serum, illustrated as the solid phase reagent. The l25 I-probe was purified human anti-hbe IgG, labeled with '"[-iodine. The sample,.2 ml was incubated with the solid phase at room temperature for 18 hours. After washing, the solid phase was incubated with the '"l-antibody at 45 C for three hours. The beads were again washed and counted for l25 l-antibody uptake. HBeAg or anti-hbe; reproducibility was evaluated by testing 3 replicates of both negative and positive control for HBeAg and anti-hbe. The mean, standard deviation, and coefficient of variation for each control was then calculated. Stability of each lot was evaluated by storing portions of each lot for various time intervals and at different storage conditions. For studies performed by the clinical investigators, the reagents were used as described below. Investigators Kits were shipped to seven investigators in the United States, Europe, and Taiwan for evaluation. The following aspects of the reagents were evaluated: Reproducibility Five investigators were sent a panel of 4 serum specimens, containing 24 different samples of varying titers of HBeAg and anti-hbe. There were 16 duplicates within the panel. Each of the five investigators assayed the panel sample a total of five times over five different assay runs. Thus, each of the 16 duplicate panel samples was tested a total of 5 times while the eight single panel samples were assayed 25 times. This protocol permitted assessment of interlaboratory day-to-day variation and laboratory-to-laboratory variations with the same reagents. Clinical Diagnosis All seven investigators were included in this study because they possessed as a part of their ongoing hepatitis programs collections of sera from well-documented patient populations. These included HBsAgpositive and-negative renal dialysis patients, HBsAgcarrier pregnant women and serial serum samples from their infants, HBsAg-positive homosexuals, HBsAgpositive acute and chronic hepatitis patients, HBsAgpositive hemophiliacs, and for controls HBsAg-negative blood donors and patients. Detectability Detectability of HBeAg and anti-hbe with AB BOTT-HBe was compared with agar gel immunodiffusion and counterelectrophoresis 813 by six investigators. For this purpose, 51 HBsAg-positive specimens that had been previously screened by the latter methods were retested by ABBOTT-HBe. Serological Profiles of HBV Patients In order to extend information on the possible prognostic implications of HBeAg and anti-hbe during acute and chronic HBV infection, five investigators retested serum specimens previously obtained serially from 149 patients with clinically apparent acute or chronic hepatitis. The first available specimens for all acute patients were obtained at the onset of the acute phase of illness. RIA Test Procedures Either serum, plasma, or recalcified plasma was used interchangeably in the assays. Each separate analytical run included three negative controls and three positive controls. All investigators performed the tests according to an already described procedure. 1 Samples were considered positive for HBeAg if the positive to negative (P/N) ration was S;2.1. Similarly, a sample was considered positive for anti-hbe if its count rate was lower than the cutoff (equivalent to 5% neutralization). The cutoff is defined as: (cpm) + Negative (cpm)/ 2. An alternative method to determine if a serum sample is positive for anti-hbe is to calculate the percent neutralization using the following formula: CPM (negative control) CPM (specimen) CPM (negative control) CPM (positive control) Results X 1 = % neutralization Quality-control Tests for Performance and Stability For the HBeAg test, the initial net count rates for negative controls of the kits prior to the clinical investigators' evaluation were approximately 234 cpm while Downloaded from on 9 May 218

3 694 MUSHAHWAR, McGRATH ET AL. A.J.C.P. November ( 32 1 ISO >- 16 u 5 uo X 1 I *+ * T 6-7 B I:i-I >21 FIG. 2. Histogram of the distribution of S/N (sample mean cpm/ negative control mean cpm) values obtained when 944 HBsAg negative sera were tested for HBeAg by ABBOTT-HBe. The S/N value for each specimen was calculated, and results were grouped by number of sera in each percentile. rates for the positive controls were about 5221 cpm, giving typical P/N values of about 22. Reproducibility of negative controls ranged from five to 12% coefficient of variation (CV) while reproducibility of positive controls ranged from six to 11%. For the anti-hbe test, the initial net count rates for negative controls of the kits which were evaluated were approximately 5673 cpm while rates for the positive controls were about 292 cpm, giving typical N/P values of about 19. Reproducibility of negative controls ranged from four to 9% CV, while reproducibility of positive controls ranged from five to 1% CV. Precision Five investigators tested a 4 member panel with varying titers of HBeAg and/or anti-hbe. Each investigator tested the panel five times over five different assay runs, for a total of 1, assays. The five investigators correctly identified each specimen as positive or negative for HBeAg in all 1, tests. For anti-hbe the investigators correctly identified 994 of the 1, specimens as positive or negative. Fig. 3 shows a similar distribution frequency for anti- HBe. The mean percent neutralization was 11.1%. There were 15 specimens that were positive for anti- HBe in this population. Each of these specimens however were also positive for anti-hbe and/or anti-hbs indicating a previous exposure or infection with HBV. Analysis of Figs. 2 and 3 show that HBsAg-negative specimens should have S/N ratios for HBeAg of less than 2.1 and a percent neutralization value of less than 5% for anti-hbe. In summary, in a total of 944 HBsAg negative sera, one specimen (.1%) was found to be repeatedly positive for HBeAg. No specimens were found to be repeatedly positive for anti-hbe without the presence of another HBV serological marker. Detectability As summarized in Table 1, six investigators tested a total of 51 HBsAg-positive specimens. ABBOTT- HBe detected the presence of HBeAg or anti-hbe in 458 (9%) of the specimens compared to 257 (5%) of the specimens using rheophoresis or counterimmunophoresis procedures. Reproducibility Thirty replicates of each member of a Dilution Sensitivity Panel for both HBeAg and anti-hbe were tested by ABBOTT-HBe. For each specimen the probability of a positive or negative result was calculated using statistical methods. Additionally, data from the testing of 2 specimens negative for anti-hbe and 269 specimens negative for HBeAg from normal blood donors Specificity Five investigators tested for HBeAg 994 HBsAg negative specimens, and for anti-hbe 953 HBsAg negative specimens. Each investigator used at least two different master lots of reagents, and total of five different master lots of reagents were used in the study. Fig. 2 shows the HBeAg distribution frequency for the negative population. The S/N mean (sample mean cpm/negative control mean cpm) was 1.6. Two specimens were higher than the identified 2.1 cutoff value. -3/-2-1/ 1/2 3/4 5/6 7/8 9/1-4/-3-2O/-1 /1 2/3 4/5 6/7 BO/9 >I PERCENT NEUTRALIZATION FIG. 3. Histogram of the distribution of neutralization values obtained when 953 HBsAg negative sera were tested for anti-hbe by ABBOTT-HBe. The percent neutralization value for each specimen was calculated, and results were grouped by number of sera in each percentile. Downloaded from on 9 May 218

4 Vol. 76 No. 5 Table I. Comparison of Rheophoresis, Counterelectrophoresis (CIEP), and ABBOTT-HBe Radioimmunoassay for the Detection of HBeAg and Anti-HBe by Six Clinical Investigators HBeAg + Anti-HBc + HBeAg +, Anti-HBe + HBeAg -, Anti-HBc - Total ABBOTT-HBe No Percent Rheophoresis or CIEP No. Percent were examined to determine the frequency and distribution of negative results. The results from this study are shown in Tables 2 and 3. The data are expressed in terms of 1, assays. Thus, for anti-hbe Dilution Sensitivity Panel members A, B, and C the frequency of a positive result is 1, of 1, assays. For anti-hbe panel member D, which is a borderline positive sample, the data show that a positive value is predicted in 77 of 1, assays. Therefore, an anti-hbe borderline positive sample would be detected as positive the majority of the time. The last anti-hbe panel member E shows the predicted intermediate results. For HBeAg Dilution Sensitivitity Panel members A through E (Table 3), a positive value is predicted in 1, of 1, assays. The last HBeAg Panel member F is the initial dilution for which negative results are predicted. Thus an HBeAg borderline positive sample has a very high probability of being detected as positive. For the normal blood donor population in both the anti-hbe and HBeAg assays, a negative result was obtained in 1, of 1, assays. This shows that the likelihood of obtaining a false positive result on a true negative specimen with either the anti-hbe or HBeAg assay is extremely low. ABBOTT-HBe Stability and Sensitivity Anti-HBe Master Lot Stability. The changes in N/ P values over an eight week period for all clinical master lots stored at 2-8 C was studied. The rate of decrease in the N/P ratios was calculated as a measure of stability. For these master lots, the N/P ratio decreased from 18.9 to ten after four weeks (about 47%). There was no further significant decrease after eight weeks. A master lot with an N/P value of ^4. is considered acceptable for use. There was no change in extinction titer (1:6,4) of a reference serum with the anti-hbe test over a period of eight weeks storage at 2-8 C. HBeAg Master Lot Stability. As in the case of the anti-hbe test, the changes in P/N values were deter- CLINICAL EVALUATION OF ABBOTT-HBe 695 mined for all clinical master lots stored at 2-8 C for eight weeks. The rate of decrease in the P/N ratio is a measure of stability. For these master lots, the P/N ratio decreased from 22 to 11.4 after four weeks (about 48%) and there was no further significant decrease at eight weeks. Reagents with a P/N value of L4. is considered acceptable for use. A reference HBeAg-positive serum was titered at 1:3,2 to 1:6,4 with all master lots throughout the entire eight weeks of testing. Vertical Transmission Studies One investigator found that children of mothers whose sera were positive for both HBsAg and HBeAg had a greater incidence of infection with HBV than children of mothers whose sera were HBsAg positive but HBeAg negative. Table 4 summarizes the results of an investigator in Taipai, Taiwan. were assayed from 531 babies born to HBsAg positive mothers. Serum samples were obtained from each mother and baby within 24 hours of delivery. The neonates were further tested at regular intervals for periods up to one year. A total of 28 babies were born to mothers who were HBeAg positive; 175 (84.1%) of the babies subsequently developed HBs antigenemia within the first three months following birth. In contrast, only 86 (26.7%) of 323 babies of HBeAg-negative mothers developed HBs antigenemia within the same time period. A large percentage of these 86 mothers (87%) had serum which was anti-hbe positive. Seroconversion of HBeAg to Anti-HBe The investigators tested serial serum samples obtained from a total of 149 patients beginning with the acute stage of infection or prior to the appearance of Table 2. Estimated Number of and Negative Test Results if Were Tested 1, Times for Anti-HBe by ABBOTT-HBe Tested Dilution sensitivity panel A B C D E Normal blood donor population Dilution 1:4 1:8 1:16 1:32 1:64 None Numbei rof Negative , 1, 1, 1, Expected Result Borderline positive Borderline positive Negative Downloaded from on 9 May 218

5 696 MUSHAHWAR, McGRATH ET AL. A.J.C.P. November 1981 HBsAg and continuing through either the recovery phase of acute hepatitis (appearance of anti-hbs) or the establishment of a chronic HBsAg carrier state. During onset of the acute stage of disease, HBeAg was usually detected within three to five days following the appearance of HBsAg and typically persisted for two to six weeks. Seroconversion from HBeAg to anti-hbe was monitored in 117 patients. In 85 patients (73%) seroconversion occurred two to four weeks before the loss of HBsAg, and immediately after HBeAg became undetectable. In 21 patients (18%) HBeAg to anti-hbe seroconversion occurred at approximately the same time as loss of HBsAg (within ± one week after HBsAg disappeared). In 19 (59%) of the patients that developed chronic HBs antigenemia, HBeAg persisted for an average of at least one year (range one to seven) without seroconversion. Another 13 patients developed chronic HBs antigenemia, but this was accompanied by seroconversion of HBeAg to anti-hbe within an average of at least one year (range one to five). Both HBsAg and anti-hbe then persisted. Discussion Seven investigators evaluated ABBOTT-HBe, a commercial RIA for detecting HBeAg and anti-hbe in serum. The studies demonstrated that the reagents and procedures were reproducible, sensitive and specific. and negative results were clearly delineated using the recommended cutoff values based on negative control sera. The frequency of detecting HBeAg and anti-hbe in HBsAg-positive sera was 9% using ABBOTT-HBe compared to only 5% with agar gel diffusion and counterelectrophoresis (Table 1). Table 3. Estimated Number of and Negative Test Results if Were Tested 1, Times for HBeAg by ABBOTT-HBe Number of Expected Tested Dilution Negative Results Dilution sensitivity panel A B C D E F 1:16 1:32 1:64 1:128 1:256 1: , 1, 1, 1, 1, 68 Borderline positive Normal blood donor population None 1, Negative Table 4. Babies HBsAg Outcome in Relation to Mothers HBeAg Baby HBsAg Negative No Mothers HBeAg Percent No Negative Percent Total The first and most widely used marker for HBV infection has been serum HBsAg. More recently, HBeAg has been shown to be associated with the virion nucleocapsid and correlated with the presence of HBV DNA polymerase and the number of Dane particles in serum The free HBeAg in serum has been proposed as a marker for a high risk of infectiousness and transmission of HBV. 1,214 This idea is substantiated in the current studies by the vertical transmission results. The frequency of HBs antigenemia was 84.1% in children of HBeAg-positive mothers but only 26.7% in HBeAg-negative mothers. These results confirm and extend those of other investigators, 214 " who also showed a strong correlation between HBeAg positive mothers and the subsequent development of HBs antigenemia in their babies. These results also indicate that the correlation of HBeAg with infectivity are not absolute because HBs antigenemia developed in the children of mothers who were anti-hbe positive. HBeAg is a complex of soluble proteins found in serum and other biological fluids of people seropositive for HBsAg. 8 " It is believed to be cryptically associated with different morphological forms of HBsAg 19 and also with HBV nucleocapsid. 17 Anti-HBe usually develops in serum during the late acute phase of disease 6 and most often appears immediately after the disappearance of HBeAg 7 or within one week after HBeAg is no longer detectable. Testing of several sera in the current studies by six clinical investigators showed that HBsAg and HBeAg became detectable at nearly the same time when ABBOTT-HBe was used. Also, in patients showing resolution of viremia and recovery, anti-hbe appeared immediately after HBeAg disappearance. Its appearance was coincidental with the decline in titer and eventual loss of detectable HBsAg. ABBOTT-HBe, an RIA for either HBeAg or anti- HBe, was shown to be specific, sensitive, and practically useful in a clinical setting as studied by 7 investigators. Use of the reagents demonstrated that e-antigen expression is a usual expression after HBV infection in humans. More than 9% of all individuals that developed HBs antigenemia were found to also express HBe antigenemia, either in a persisting profile or seroconver- Downloaded from on 9 May 218

6 Vol. 76 No. 5 CLINICAL EVALUATION OF ABBOTT-HBe 697 sion to anti-hbe. This seroconversion was most often associated with resolution of viral replication and convalescence. The persistence of HBeAg for eight to ten weeks after its appearance was associated with infectious viremia and chronicity. References 1. Alter HJ, Scef LB, Kaplan PM, et al: Type B hepatitis: the infectivity of blood positive for e antigen and DNA polymerase after accidental needlestick exposure. N Engl J Med 295:99-913, Beasley RP,TrepoC, StevensCE, et al: Thee antigen and vertical transmission of the hepatitis B surface antigen. Am J Epidemiol 15:94-98, Eleftheriou N, Heathcoat J, Thomas HC, et al: Incidence and clinical significance of e antigen and antibody in acute and chronic liver disease. Lancet ii:l , El Sheikh N, Woolf IL, Galbraith RM, et al: "e" Antigen-antibody systems as indicator of liver damage in patients with hepatitis B antigen. Brit Med J 4: , Hindman SH, Gravelle CR, Murphy BL, et al: "e" Antigen, Dane particles, and serum DNA polymerase activity in HBsAg carriers. Ann Int Med 85:458-46, Krugman S, Overby LR, Mushahwar IK, et al: Viral hepatitis, type B. Studies on natural history and prevention reexamined. N Engl J Med 3:11-16, Ling CM, Mushahwar IK, Overby LR, et al: Hepatitis B e-antigen and its correlation with other serological markers in chimpanzees. Infect Immun 24: , Magnius LO, Espmark JA: New specificities in Australia antigen positive sera distinct from LeBouvier determinants. J Immunol 19: , Magnius LO, Lindholm A, Lundin P, et al: A new antigen-antibody system. Clinical significance in long-term carriers of hepatitis B surface antigen. J Amer Med Assoc 231: , Mushahwar IK, Overby LR, FrOsner G, et al: Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassays. J Med Virol 2:77-87, Mushahwar IK, Overby LR, Ling CM, et al: Characterization of urinary hepatitis B e-antigen. Fed Proc 39:227, Nielsen JO, Dietrichson O, Juhl E: Incidence and meaning of "c" determinant among hepatitis B antigen positive patients with acute and chronic liver disease. Lancet 2: , Nordenfelt E, Kjcllcn L: Dane particles, DNA polymerase, and e antigen in two different categories of hepatitis B antigen carriers. Intervirology 5: , Okada K, Kamiyama I, Inomata M, et al: e-antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N Engl J Med 294: , Stevens CE, Neurath RA, Beasley RP, et al: HBeAg and anti- HBe detection by radioimmunoassay: correlation with vertical transmission of hepatitis B virus in Taiwan. J Med Virol 3: , Takahashi K, Imai M, Tsuda F, et al: Association of Dane particles with e-antigen in the serum of asymptomatic carriers of hepatitis B surface antigen. J Immunol 117:12-15, Takahashi K, Akahane Y, Gotanda T, et al: Demonstration of hepatitis B e antigen in the core of Dane particles. J Immunol 122: , Tong MJ, Stevenson D, Gordon I. Correlation of e antigen, DNA polymerase activity, and Dane particles in chronic benign and chronic active type B hepatitis infections. J Infect Dis 135:98-984, Vnck J, Prince AM, Trepo C, et al: Cryptic association of e- antigen with different morphologic forms of Hepatitis B surface antigen. J Med Virol 4: , 1979 Downloaded from on 9 May 218