Vox Sang. 33: 369-372 (1977)

Radioimmunoassay of Hepatitis B Core Antigen and Antibody with Autologous Reagents1 Girish N . Vyas and Isabel M . Roberts Department of Laboratory Medicine and Liver Research Center, University of California, San Francisco, Calif.

Abstract. Anti-HBc is the most sensitive indicator of current or recent hepatitis B virus infection, hence its detection is valuable in laboratory diagnosis of the infection. Dane particles isolated from the plasma of a single chronic carrier and his autologous IgG labeled with 12sI were utilized in a solid-phase sandwich radioimmunoassay for anti-HBc. The principle of serologic neutralization of core antigen and consequent inhibition of binding of the cores to the solid-phase rendered the method to be a simple, sensitive, specific and rapid means of detecting anti-HBc.

Hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) are two distinct immunologic markers of hepatitis B virus (HBV). While HBsAg occurs commonly in HBV infection as the 16- to 25-nm spherical particles and filamentous forms, HBcAg occurs exclusively on the 27-nm cores exteriorly covered by HBsAg in the relatively uncommon 42 nm Dane particles [l]. Since the DNA polymerase is contained in the cores, its activity has been used as a marker for the cores of the Dane particles [4, lo]. The Dane particles containing DNA polymerase are considered complete HBV but only a minor subpopulation of the Dane particles actually contain a full viral genome [ 2 ] ; the remainder of the Dane particles are either empty or defective viral particles This work was supported by contract N013-3000 with the National Heart and Lung Institute and the Liver Center grant 1 P50 AM18520 from the National Institute of Arthritis and Metabolic Diseases. 1

[7]. HBcAg is resistant to various enzymes and detergents [8], hence it is possible to store stable reagent of purified cores [12]. Hepatitis B core antibodies (anti-HBc) are found not only during the acute and chronic HBV infection, but also during the period of convalescence from the disease, hence its detection is the most sensitive indicator of a current or recent occurrence of HBV infection [6]. Assay of anti-HBc is also valuable in epidemiologic studies, in the evaluation of efficacy of hepatitis B immune globulin, in ascertaining safety of HBV vaccines, in the differential diagnosis of chronic active hepatitis and for screening potentially infectious sources of blood [5, 6, 131. The highest titers of anti-HBc, without detectable anti-HBs, are encountered in the asymptomatic healthy carriers of HBsAg [4, 5, 141; however, the combination of HBsAg and anti-HBc does not indicate whether the infection is acute and recent. The enveloping HBsAg apparently

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TableI. A, B, C and D were sequential 0, 3, 6 and 9 month specimens from subjects who suffered HBV infection as a result of a failure of protection by globulin [3]; results of anti-HBc shown as cpm and interpretations as + or

-

cpm

AntiHBC

Specimen

201/A B C D 202/A B C D 203/A B C

16,824 17,602 356 327 16,057 211 270 267 15,204 299 239

-

D

225

204/A 14,917 B 577 C 451 D 964 205/A 15,003 B 282 C 313 D 305 controls DDserum(t) 357 00913 serum(-) 14,195 saline (blank) 14,944

Spec-

imen

+

+ + + + + + +

cpm

AntiHBc

-

+ + +

-

+ + + + -

prevents the reaction of the HBcAg with autologous anti-HBc. This unique natural phenomenon was taken advantage of in deriving autologous antigen and antibody as reagents for a solid-phase sandwich radioimmunoassay described in this report. Healthy voluntary blood donors positive for HBsAg and permanently rejected from donating blood for transfusion were screened for the DNA polymerase [lo]. Four out of 48 such donors had high DNA polymerase activity. A selected single volunteer was repeatedly plasmapheresed. From 600ml plasma the Dane particles were isolated by removal of the high speed supernatant plasma (HSS) containing anti-HBc and purification of the pelleted particles by three repeated procedures of sedimentation in sucrose (S,) density gradients [4, 101. A yield of 6.0ml of S, was used as the source of cores. Cores were released from the S , by treatment with 5 volumes of a solution containing 0.12 M NH,CI, 0.08 M MgCl,, 0.25% bmercaptoethanol and 0.5% nonionic detergent neodo1 25-9 (Shell Chemicals, New York). This mix-

ture was incubated at 37 OC for 1h to obtain the core reagent (CR). The HSS plasma was subjected to DEAE-Sephadex chromatography using 0.025 M phosphate buffer, pH 7.0 for isolation of IgG. The purified IgG was labeled with 1261 using a modification of chloramine-T oxidation procedure described previously [13]. This was used as the 1251labeled anti-HBc reagent (125I-IgG-HBc). A modification of solid-phase sandwich radioimmunoassay (SPS-RIA) described by Salmon and Smith [ll] and in principle similar to the microSPRIA described by Purcell et al. 191 was employed for the test. Coating of a batch of 10 x 75 mm Falcon polystyrene tubes with antiHBc was accomplished with aliquots of 0.25 ml HSS plasma incubated at 37OC for 1&20h, followed by dilution with 3.0ml phosphate buffered saline, pH 7.2, mixed and left at room temperature for 24 h, washed ten times with distilled water and air-dried. Normal human plasma lacking HBsAg, anti-HBc and anti-HBs was used as a diluent for CR and 115I-IgG-HBc when tested in box titration for the selection of appropriate dilutions of each of the two reagents so that the ratio of the counts per minute (cpm) with and without cores was approximately 40. The test procedure was as follows: (1) aliquots of 1OOyl of test serum (TS) mixed with 50yl of CR and incubated at 37 OC for 30 min; (2) the TSCR mixture was transferred to anti-HBc-coated tubes and incubated for 1 h at 37 OC; (3) the tubes were washed five times with phosphate-buffered saline (PBS); (4) 100,d of iz6I-IgG-HBc added and incubated for 1h at 37 OC; ( 5 ) the tubes were washed five times with PBS and counted in gamma counter.

When the SPS-RIA test was carried out in duplicates on each positive specimen diluted 1 : l O and undiluted, the reproducible results persuaded us to forego replicate testing and also forego the extra step of dilution. The difference in cpm between negative and positive specimens averaged 50 times. Representative positive specimens gave between 200 and 350cpm - whereas the negative specimens uniformly gave 14,000-16,000

Solid Phase RIA for Anti-HBc

371

Table II. Methods used for detection of anti-HBc ~

~~

__

Methods and references

~~

Source of HBc-reagents antigen

immunosupComplement fixation (CF) [6J pressed chimpanzee liver

Micro-SPRIA[9]

HBsAg-positive plasma

antibody not needed

convalescent patient plasma

Approximate test time, h

Sensitivity Remarks about the relative method to CF

4

1

simple and practical; HBcAg from chimpanzee liver limits its use; occasional nonspecific reactions

60

10

simple and specific; long testing time makes it less practical

chronic carrier Immune adherance heagglut- plasma ination [I21

not needed

4

10

simple and practical; nonspecisc reactions can occur

DNA polRIADA [4]

chronic carrier plasma

not needed

10

45

sensitive, specific but complex; impractical for general use

SPS-RIA (reported here)

chronic carrier plasma

autologous HSS plasma

3

80

simple, specific, sensitive, and practical

cpm. SPS-RIA results of anti-HBc testing performed on 54 specimens containing and 196 specimens lacking anti-HBc previously tested by the double antibody RIA using DNA polymerase reaction [4] (DNA polRIADA) showed complete concurrence of the results. Further validation and representative test results on sequential specimens from patients with HBV infection 131 are illustrated in table I. A representative specimen positive for anti-HBc with complement fixation (CF) titer of 256 gave titers of 20,000 and 12,000 in the SPS-RIA and the DNA pol-RIADA respectively. A relative evaluation of various assays for anti-HBc is depicted in table 11. Since the test described here is simple, specific, rapid and sensitive, it is practical to use this assay for anti-HBc in clinical and epidemiologic studies currently underway.

Inherent advantages of the SPS-RIA include use of the empty or defective cores lacking DNA polymerase activity, autologous anti-HBc and cores as reagents virtually eliminate nonspecific reactions, the inhibition of binding of the cores obviates the conventional need for neutralization test for specificity of RIA reactions, and the dichotomy of cpm between positive and negative sera aids an unambiguous interpretation of the results.

Acknowledgements Thanks are due to Dr. George Grady of Tufts University and Dr. Juy Hoofnagk of the US Food and Drug Administration for providing us some of the previously characterized serum specimens. We thank Jennifer Harford and Rivka Zisling for technical assistance.

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References 1 Almeida, J. D. and Waterson, A. P.: Hepatitis B antigen - an incomplete history. Am. J. med. Sci. 270: 105-114 (1975). 2 Gerin, J. L.; Ford, E. C.,and Purcell, R. H.: Biochemical characterization of Australia antigen. Evidence for defective particles of hepatitis B virus. Am. J. Path. 81: 651468 (1975). 3 Grady, G.F. and Lee, V.: Prevention of hepatitis from accidental exposure among medical workers. New Engl. J. Med. 293: 1067-1070 (1975). 4 Greenman, R. L.; Robinson, W. S., and Vyas, G. N.: A sensitive test for antibody against the hepatitis B core antigen. Vox Sang. 29: 77-80 (1975). 5 Hoofnagle, J. H.; Gerety, R. J., and Barker, L. F.: Antibody to hepatitis B core antigen. Am. J. med. Sci. 270: 179-187 (1975). 6 Hoofnagle, J. H.; Gerety, R. J.; Ni, L. Y., et al.: Antibody to hepatitis B core antigen: a sensitive indicator of persistent viral replication. New Engl. J. Med. 290: 1336-1340 (1974). 7 Huang, A. S. and Baltimore, D.: Defective viral particles and viral disease processes. Nature, Lond. 226: 325-326 (1970). 8 Huang, A. S.: Structural and immunoreactive characteristics of hepatitis B core antigen. Am. J. med. Sci. 270: 131-139 (1975). 9 Purcell, R. H.; Gerin, J. L.; Almeida, J. D.,

10

11

12

13

14

et al.: Radioimmunoassay for the detection of the core of the Dane particle and antibody to it. Intervirology 2: 231-243 (1974). Robinson, W.S.: DNA and DNA polymerase in the core of the Dane particle of hepatitis B. Am. J. med. Sci. 270: 151-159 (1975). Salmon, S. E. and Smith, B. A.: Sandwich solid phase radioimmunoassays for the characterization of human immunoglobulins synthesized in vitro. J. Immun. 104: 665-672 (1970). Tsuda, F.; Takahashi, T.; Takahashi, K., et al.: Determination of antibody to hepatitis B core antigen by means of immune adherence hemagglutination. J. Immun. 3: 835-838 (1975). Vyas, G.N.; Roberts, I. M.; Mackay, I. R., et al.: Immunologic mechanisms in hepatitis 13 assayed by antigen binding lymphocytes. Am. J. med. Sci. 270: 241-246 (1975). Vyas, G. N.; Roberts, I. M.; Peterson, D. L., et al.: Nonspecific test reactions for antibodies to hepatitis B surface antigen in chronic HBsAg carriers. J. Lab. clin. Med. (in press).

Received: April 21, 1976 Accepted: September 15, 1976 Dr. Girish N. Vyas, Room 506-M, Department of Laboratory Medicine, University of California School of Medicine, San Francisco, CA 94134 (USA)

Addendum Roelcke, D.; Riesen, W.; Geisen, H . P., and Ebert, W. Serological Identification of the New Cold Agglutinin Specificity anti-Gd Note When this paper was in press, the paper entitled Identification of sialosylparagloboside as the erythrocyte receptor for an ‘anti-p’ antibody of Schwarting, Marcus and Metaxas appeared in this journal [Vox Sang. 32: 257-261, 19771. Erythro-

cytes treated with neuraminidase were no longer agglutinated by this ‘anti-p’ Fol., but the agglutination was not affected by papain treatment, thus resembling the antibodies Kn and Hei. Dr. M . Metaxas, Zurich, was kind enough to test the antibodies Kn and Hei in comparison to the ‘anti-p’ Fol. with PI, P, and p erythrocytes. In contrast to the ‘anti-p’ Fol., the antibody Hei did not show any preference and the antibody Kn only a very slight preference for p erythrocytes. Therefore, ‘anti-p’ and anti-Gd are not identical.

Radioimmunoassay of hepatitis B core antigen and antibody with autologous reagents.

Vox Sang. 33: 369-372 (1977) Radioimmunoassay of Hepatitis B Core Antigen and Antibody with Autologous Reagents1 Girish N . Vyas and Isabel M . Rober...
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