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Detection of Australia Antigen by Radioimmunoassay

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Detection of Australia Antigen by Radioimmunoassay Proc. Nat. Acad. Sci. USA Vol. 68, No. 5, pp. 1056-1060, May 1971 Detection of Australia Antigen by Radioimmunoassay RICHARD D. AACH, JOE W. GRISHAM, AND CHARLES W. PARKER Departments of Internal Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110 Communicated by Carl V. Moore, March 9, 1971 ABSTRACT A double-antibody radioimmunoassay has SW25.1 rotor at 25,000 rpm for 28 hr) in a preformed linear been developed for the detection of Australia antigen and gradient of CsCl (density 1.09-1.30). After rebanding in a antibodies to Australia antigen in human sera. The assay is simple to perform, and can be completed in 18 hr. second CsCl gradient, of density 1.15-1.25, Au antigen was Based on parallel studies of human sera, it is approxi- recovered in a single symmetrical peak (fraction density of mately 2000 times more sensitive than agar gel diffusion about 1.20) of absorbance at 280 nm. 5 ml of serum, containing and 200 times more sensitive than quantitative comple- about 300 mg of protein, yielded about 50 ,ug (1.2 absorbance ment fixation. Australia antigen or antibody to Australia antigen was demonstrated in 22 of 23 single serum sam- units at 280 nm) of protein (range 30-100 Mg, depending on ples obtained from patients acutely ill with the long- the serum) by the Lowry technique (10); bovine serum al- incubation form of hepatitis; eight of the sera were nega- bumin was used as the standard. The purified Au antigen tive when evaluated by less sensitive techniques. preparation formed a single line of precipitation in agar gel diffusion with anti-Au antiserum. Human serum proteins Since the Australia antigen (Au antigen) was first described immunoelectrophoresis when tested in 1964 (1), considerable evidence has accumulated to link were not detected by it with viral hepatitis (2, 3). This antigenic determinant (also against rabbit antiserum of high titer. the Anti-Au antibodies were prepared in guinea pigs by in- called the hepatitis-associated antigen and SH antigen) jections into the foot pad of about 25-100 of Au antigen, appears to reside on a virus-like particle about 200 A in di- ;&g in mixed in an equal volume of complete Freund's adjuvant. ameter (4). It has been observed most frequently patients Animals were injected at about monthly intervals with 10-25 with the long-incubation form of viral hepatitis (2, 5). of Au in most in the Ag antigen protein Freund's adjuvant. Anti-guinea Current methods frequently used testing for pig IgG and anti-human IgG antibodies, for use in the double Au antigen are agar gel diffusion (2, 3), complement fixation radioimmunoassay procedure, were prepared in rabbits by (5, 6), and various electrophoretic techniques (7, 8). We have immunization with the appropriate purified IgG (11). described a radioimmunoassay for the demonstration of Au the Au The Au antigen preparation described above (or, more re- antigen that uses human serum with antibodies to was radio- A more sensitive cently, material isolated by zonal centrifugation*) antigen (anti-Au antibody) (9). radioim- labeled by the Chloramine-T method of Hunter and Green- munoassay, using guinea pig serum that contains anti-Au is described in detail in wood (12). The reaction mixture contained Au antigen (about antibody, has now been developed and 0.004 at 280 1 mCi of and 50 is can be com- absorbance unit nm), K125q, this paper. The method extremely sensitive, in 0.2 M 7.4 volume can be modified to test ,ug of Chloramine-T phosphate, pH (final pleted in less than 24 hr, and readily 250 sodium for the of anti-Au antibodies. 50 Al). After 15 min at 4VC, ,ug of metabisulphite simultaneously presence and 2 mg of KI were added, and free I- was removed on a MATERIAL AND METHODS Sephadex G-25 column equilibrated in phosphate-saline Au antigen and anti-Au antibodies were initially demonstrated (0.15 M NaCl-0.01 M phosphate, pH 7.4) or 0.15 M veronal in human serum both by Ouchterlony agar gel diffusion and buffer, pH 7.4. In selected experiments, reaction conditions quantitative complement fixation (5). Immunologic identity were systematically altered to determine maximum radio- with the Au (1) antigen and anti-Au (1) antibody (2) was labeling efficiency. This included varying the amount of established by immunodiffusion with sera kindly supplied Chloramine-T from 25-200 ,ug with proportionate changes by Dr. Blumberg and his associates (Institute for Cancer Re- of sodium metabisulphite, the use of different buffers and search, Fox Chase, Pa.). pH (ethylenediamine, 0.2 M, pH 7.4; veronal, 0.2 M, pH Au antigen was purified by column chromatography and 7.5; borate, 0.2 M, pH 8.5), and variations in the reaction density gradient ultracentrifugation. Serum was first placed time and temperature. Radiolabeled Au antigen was stored on a column (2.5 X 100 cm) containing Sepharose 2-B, equil- in standard diluents (phosphate-saline or veronal buffer, ibrated with the eluent, 0.15 M NaCl in 0.05% Na deoxy- 0.5% bovine serum albumin) at 40C and -20'C. Under these cholate. Au antigen was eluted well ahead of the major pro- conditions, precipitability with anti-Au antibody remained tein peak. Fractions containing Au antigen were pooled, constant for at least a month. dialyzed to remove detergent, and centrifuged (Spinco * Electronucleonics, Bethesda, Md. 1 ml of this preparation contains 2 X 1014 Au antigen particles; A2wg = 0.740. Human Abbreviation: Au (antigen), Australia antigen (hepatitis-asso- serum proteins were not detected by immunoelectrophoresis or ciated antigen). immunodiffusion with rabbit anti-human serum. 1056 Downloaded by guest on September 30, 2021 Proc. Nat. Acad. Sci. USA 68 (1971) Detection of Australia Antigen 1057 The radioimmunoassay was performed by the double- 80 antibody technique using guinea pig anti-Au (0.05 ml of a a Guinea pig Anti-Au 1:10,000 dilution) as the first antibody and rabbit serum con- K Antigen Antiserum taining antibodies to guinea pig IgG (in excess) as the second -Q antibody. The human serum under evaluation (0.05 ml at ,~60- various dilutions) was incubated with the first antibody and [125I]Au antigen (8000 cpm/tube) for 1 hr at 370C, and then 0 the second antibody was added (final reaction volume 0.35 ml). After another 15 hr at 4VC, the precipitate was isolated 40 by centrifugation, washed twice with ice-cold standard dil- 0 uent, and counted in an automatic gamma scintillation counter. In the presence of Au-antigen-containing serum, up to 98% inhibition of precipitation of radiolabeled Au ';K 20 antigen was obtained (by comparison with the cpm precipi- 0 tated when normal guinea pig serum was substituted for first antibody). The average variation between triplicate samples was or less. In serial with the same W 5% experiments serum, 1:320 1:1280 1:5120 1:20480- 1:16)00 the variation in the mean value for counts precipitated was Dilution of Anti -Australia Antigen Antisera 10% or less. For the detection of anti-Au antibodies in human sera, FIG. 1. Precipitation of [1251]Au antigen (8000 cpm) at vari- the samples were incubated with [125I]Au antigen for 1 hr at ous dilutions of human and guinea pig anti-Au antisera. The 370C, then an excess of rabbit anti-human IgG was added and anti-Au antisera were added in a volume of 0.05 ml. "Normal" the incubation was continued for another 15 hr at 40C. human IgG and guinea pig IgG were added as carriers so that serum the amount of precipitate formed with second antibody was 86 samples, each from a different individual, were constant. evaluated initially by agar gel diffusion and complement fixation. Study groups included: (a) 15 sera from patients with post-transfusion hepatitis, "positive" for Au antigen; in the presence of goat anti-rabbit IgG, precipitated up to (b) 8 sera from patients with post-transfusion hepatitis 20% of the total radioactivity. The presence of radiolabel (drawn during the acute phase of their disease), "negative" on protein other than Au antigen did not alter the specificity for Au antigen; (c) 5 sera, which contained anti-Au anti- of the immunoassay (either with guinea pig or human anti- bodies, from patients with hemophilia; (d) 8 samples from Au antibody), as indicated by the fact that precipitation of patients with other forms of liver disease (alcoholic liver [125I]Au was completely inhibited by Au-antigen-containing disease, 5; drug-induced hepatitis, 3); and (e) 50 control sera sera, whereas various dilutions of normal human serum did obtained from healthy adults and hospitalized patients with not inhibit. However, subsequent studies were conducted no evidence of liver disease as judged by history, physical ex- with radiolabeled Au antigen purified by zonal ultracentrif- amination, and laboratory screening procedures (serum ugation. bilirubin, transaminase, and alkaline phosphatase). Immunized guinea pigs produced a high titer of anti-Au IgG antibodies. A standard titration curve of guinea pig RESULTS anti-Au antiserum is compared to our highest titer human The technique for iodination of Au antigen described above (hemophilia) anti-Au antiserum in Fig. 1. The two antibody results in incorporation of only about 1% of the 1251 used. dilution curves are parallel, indicating that the same antigenic Systematic variation of the reaction conditions failed to determinants are being measured. The linear portion of the increase the labeling efficiency. The limited incorporation of titration curve for guinea pig antiserum fell between dilutions 125I on the Au antigen particles could be the result of a low of 1:10,000 and 1:120,000; precipitation of radiolabeled Au tyrosine content or of low tyrosine reactivity (steric hindrance antigen was still evident at an antibody dilution of 1: 200,000.
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