Vox Sang. 34: 321-328 (1978)

A Coated-Tube Radioimmunoassay (FRC-RIA) for Hepatitis B Surface Antigen Vesu Koistinen Finnish Red Cross Blood Transfusion Centre, Helsinki

Abstract. A sandwich-type solid phase radioimmunoassay, FRC-RIA (Finnish Red CrossRIA) for hepatitis B surface antigen (HB,Ag) is described. Polystyrene test tubes coated with sheep anti-HB, y-globulin serve as the solid phase. The technique is specific and has a sensitivity of about 1 ng/ml for the subtypes ad and uy of HB,Ag when corresponding subtype-specific antibodies are used as reagents. The total incubation time can be shortened from over 20 h to 3 h, with almost equal sensitivity, when the incubation temperature is raised from room temperature to 45 "C. The test is economical in reagents and is applicable to routine use.

Introduction Various radioimmunoassay (RIA) techniques have been applied in the detection of hepatitis B surface antigen (HB,Ag). The so-called sandwich technique has proved particularly sensitive and is widely used in commercial test kits. A disadvantage of the commercial kits is their high cost. The larger the number of samples tested, the more economical it is to use self-made RIA reagents. This paper describes a 'homemade' sandwich-type RIA for HB,Ag, comparable in sensitivity to the commercial RIAs. It is easily set up if antiserum to HB,Ag is available. I have previously introduced a sandwichtype RIA for HB,Ag in which the solid

phase consisted of aminocellulose particles, to which sheep antibodies to HI3,Ag were coupled [7, 81. Later, I found that the results are as good when the coated-tube method [l] is used. Much of the washing and all of the centrifugation needed in the cellulose-based method were eliminated in this modification, which made the test more suitable for larger series. The technical details of FRC-RIA are described in this paper. Several variables were studied to optimize the test conditions. AusRIA 11, a sensitive commercial RIA for HB,Ag, was used as a reference method with which FRC-RIA was compared using groups of sera with different incidences of I-wlAg. FRC-RIA was included in a previous pa-

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per in which enzyme immunoassay (Organon) was compared with some other HB,Ag detection methods [14]. After that stirring during the incubations was omitted as it was found unnecessary.

Materials and Methods Hepatitis B Antigen Hepatitis B antigen (subtypes ad and ay) was isolated by a four-step procedure: precipitation with polyethylene glycol, immunoadsorption, pepsin digestion, and gel filtration. The plasmas used for the isolation were obtained from asymptomatic chronic carriers of HBBAg. About 500800ml of pooled plasma was used at a time. The procedure was briefly as follows: Dane particles were first removed by precipitation with 5.5% polyethylene glycol (PEG) 6,000 at neutral pH [ll]. The HBsAg in the supernatant was then precipitated at a PEG concentration of 11%. The precipitate was dissolved in PBS and incubated with sheep anti-HB, y-globulin coupled to Sepharose 4-B by the cyanogen bromide method of Cuatrecasas [2], the immunoadsorbent was washed with PBS and the antigen was eluted with 5 M NaI. The eluate was digested with pepsin (1 mg/100mg protein) at pH 2.3 for 3 h at 37OC. The pepsin digestion was found necessary for effective purification at the gel filtration step. The digest was clarified by centrifugation and fractionated by gel filtration on Sephadex G-200. The HBsAg-containing fractions coming with the void volume were pooled and concentrated to a protein concentration of about 1 mg/ml. The recovery in HBsAg as measured by RIA was about 40% (the recovery in HB8Ag protein may have been less, since the RIA-titer increased 2- to 3-fold at the pepsin digestion step). The preparations consisted of spherical particles about 20nm in diameter with an occasional short rod when studied with an electron microscope, and contained no normal plasma proteins according to immunodiffusion and immunoelectrophoresis against horse anti-whole normal human serum. However, when sheep were immunized with the

preparations and the sera were studied by immunoelectrophoresis, there were faint precipitation lines of a couple of plasma components. Preparation of Antisera and Labelled Antibodies Two sheep were injected intramuscularly with HBsAg in complete Freunds adjuvant at monthly intervals, each dose containing 125-250 yg of protein. Each animal was immunized with only one subtype. Blood collection was started after the third booster. The sera were absorbed with normal human plasma coupled to Sepharose 4-B, after which no antibodies to normal plasma proteins were detectable. y-Globulin was isolated by ammonium sulphate and caprylic acid precipitation as described by Fine and Steinbuch [3]. Antibodies were isolated from the sheep yglobulin by immunoadsorption with HB8Ag coupled to Sepharose 4-B. The antibodies were eluted with 3.5 M KSCN in PBS at +4 O C . The eluate was dialysed in PBS and concentrated to a protein concentration of 2-3 mg/ml, divided into small aliquots for iodination and stored at -23 *C. The antibody preparation was labelled with 1251 by the chloramine T method of Hunter und Greenwood [6], using 0.02 mCi 12Wyg of protein. The reaction mixture was dialysed for 1 h against PBS and the remaining free iodide was removed by gel filtration on Sephadex G-25. The labelling efficiency was about 15pCi/pg of protein. The labelled antibody was diluted in PBS containing 2% BSA to a specific activity of about 20yCi/ml, divided into aliquots and stored at -23 OC. Coating of the Tubes Polystyrene tubes (12 x 55 mm, C. A. Greiner und Sohne, Korkverarbeitungs- und Kunststoffwerke, Niirtingen, FRG) were coated with sheep anti-HBR y-globulin by a slight modification of the method of Cart et al. [l]. The y-globulin was diluted in 0.2 M sodium carbonate-bicarbonate buffer, pH 9.2.0.2 ml of the solution was pipetted into each tube and the tubes were incubated for 15 min at room temperature. Thereafter, the yglobulin solution was sucked off and the tubes were filled with 0.5% bovine serum albumin in the bicarbonate buffer, allowed to stand for 15 min at room temperature, and rinsed twice with physio-

Coated-Tube Radioimmunoassay for HB,Ag

logical saline. The tubes were stored at -23 OC without liquid. The FRC-RIA Tesr FRC-RIA was performed routinely as follows: 0.2ml of each sample was pipetted into an antibody-coated tube, which was sealed with an adhesive paper sheet. The tubes were incubated overnight at room temperature and washed twice with physiological saline. 0.2 ml of solution of the labelled antibody was added (about 106 dpdO.2 ml), and the tubes were incubated for 4 h at room temperature. Finally, they were washed twice with physiological saline, and the solid phase radioactivity was counted (LKB-Wallac Automatic Gamma Counter). Calcularion of the Cuioff (PosirivelNegarive Limit) The cutoff was obtained by multiplying the mean of the negative controls by the factor 2.1. 7-10 negative and 6 positive (3 strong, 3 weak) controls were included in each series. Confirmarion of Positive Results A specific competitive inhibition experiment was made on all those preliminary positive samples for which results with other methods were not available or which were negative by other methods: FRC-RIA was performed in the usual way except that 0.02ml of sheep antiserum to HBRAg was added together with the labelled antibody. The same amount of normal sheep serum was added to the control tube. If the antiserum rendered the result negative, the sample was considered to contain HBHAg (neutralizable positive). If the count rate was above the cutoff in spite of the antiserum, the sample was assigned the term unspecific positive. Orher Techniques for the Detection of HBsAg AusRIA I1 and AusAB (Abbott Laboratories, Chicago, Ill.) were used according to the instructions of the manufacturer. The polystyrene beads were washed with the Pentawasha multidispenseraspirator connected with a Filamatica liquid filling machine (National Instruments Co., Baltimore, Md.) and a vacuum pump. Counterimmunoelectrophoresis (CIEP) was done in 0.8% agarose in veronal buffer as described by Gocke and Howe [4], adapter for Finnish Red Cross by Helske [5].

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Results Concentration of y-Globulin in the Coating Solution When the concentration of the y-globulin used for the coating of the tubes was varied from 5 to lOOpg/ml, the sensitivity increased up to a concentration of 50pg/ml. This concentration was then used in the routine. The same y-globulin solution could be used up to 16 times without any decrease in sensitivity (this was the highest number tested), which means that less than 0.6 p g of y-globulin is consumed in a single test. Stability of the Coating The stability of the coated tubes was found to vary at refrigerator temperature: as a rule, the sensitivity tended to decrease considerably when the tubes were stored for a few days. The sensitivity remained unaltered for at least 2 weeks when the tubes were kept at -23 "C.However, even when stored frozen, the sensitivity in RIA decreased after 4 weeks. Stability of the Labelled Antibody The same labelled antibody could be used for up to about 2 months without any significant decrease in sensitivity, although there was a pronounced decrease in the count rate at higher antigen concentrations (fig. 1). Composition of the Labelled Antibody Solution The labelled antibody was diluted to the desired specific activity in a solution composed of 60% normal human serum (NHS), 20% normal sheep serum and 20% PBS, pH 7.8, containing 0.2% NaN,. This composition was found to give the lowest back-

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cpm bound

llipoo

l/d,ooo

serum dilution

Fig. 1. The age of the labelled antibody. Each dot represents the mean of the results obtained with three tubes. Abscissa: Dilutions in NHS of an HBBAg positive serum, subtype ad. Ordinate: solid phase radioactivity. 0= Antibody freshly labelled; A = antibody 2 months old.

Fig. 2. Incubation time and temperature. Abscissa: Dilutions in NHS of an HB,Ag positive serum, subtype ad. B = Mean bound radioactivity in the positive sample tubes; Bo = mean bound radioactivity in the negative control tubes. The incubation time and temperature in the first and the second incubation phase were as follows (RT= room temperature): 0= Overnight RT + 4 h RT; A = 2 h 45 O C + 1 h 45 OC; 0 = 1 h 45 O C + 1 h 45 OC; = 2 h R T + 1 h RT.

ground counts. The NHS used did not contain HB,Ag or anti HB, as tested by AusRIA and AusAB, later by FRC-RIA and 'FRCAB' (an antibody-RIA analogous to FRCRIA, in which HB,Ag and '=I-HB,Ag are

used as the solid and the liquid phase reactants, respectively). Because both the solid phase and the labelled antibodies were of sheep origin, it is possible that anti-sheep y-globulin antibodies in the specimen could have given rise to unspecific positive reactions. For this reason, normal sheep serum was included in the labelled antibody solution.

Incubation Time and Temperature In the method as it was used routinely, the incubations were performed at room temperature. Higher incubation temperatures were tried to shorten the incubation time. Figure 2 shows that temperature has a very pronounced effect on the speed of the reaction: when the incubation temperature was 45 OC, almost as good a sensitivity as with the 'routine' method was attained in a total incubation time of 3 h. It was found later that 40 "C is about equally as good, and at this temperature both serum and plasma can be used as samples, whereas at 45 "C, plasma gives a higher background and, consequently, lower sensitivity. Subtype Specificity and Sensitivity All the studies reported above were made with an antiserum specific for the subtype ad. The healthy carriers of HB,Ag in Finland studied so far have been of this subtype [lo]. Recently, we isolated HB,Ag, subtype ay, from the plasma of a drug addict who had had icteric hepatitis twice, and we prepared sheep antiserum also to this subtype. The aim of this study was to find out the effect of the subtype specificity of the reactants on sensitivity. The study was made using (a) whole plasmas of subtypes ad and ay; (b) isolated HB,Ag, and

Coated-Tube Radioimrnunoassay for HB,Ag

b

1/1qooo

l/loo,~

serum dilution

Fig. 3. a Subtype specificity. Abscissa: Dilutions in NHS of an HBsAg positive serum, subtype ad. The subtype specificities of the solid and liquid phase reagents were as follows: 0=Solid phase ay, liquid phase ad; A =solid and liquid phase ad; 0 = solid phase ad + ay 2:1, liquid phase ad + ay 1:l. b Subtype specificity. Abscissa: Dilutions in NHS of an HBsAg positive serum, subtype ay. 0=Solid phase ad, liquid phase ad; A = solid phase ad, liquid phase ay; 0 = solid and liquid phase ay; =solid phase ad + ay 2:1, liquid phase ad + ay 1:l.

(c) an Abbott Diagnostics Division reference panel, prepared by Abbott Laboratories in Chicago. The sensitivity to each subtype was found to be dependent on the subtype specificity of the solid and liquid phase reactants. When a mixture of antibodies for each subtype was used both for the coating and as the labelled antibody, the sensitivity was high for both subtypes at the same time (fig. 3a, b). The combination anti-ad + anti-ay 2: 1 in the solid phase plus anti-ad + anti-ay 1:l

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in the liquid phase was used also to determine the sensitivity of FRC-RIA in weight units of HB,Ag. When the Abbott panel was used, the detection limit of FRC-RIA was found to be about lng/ml for both subtypes. In our hands, AusRIA I1 detected 1.25 ng/ml of HB,Ag subtype ad, and 2.5 ng/ml of subtype ay when the same panel was used. For HBAg preparations of both subtypes isolated by us, the detection limit in FRC-RIA was about 0.5 ng/ml. The difference between the results with the panel and our HBAg preparations may have been due to the fact that the pepsin digestion included in our isolation procedure increased the antigen titer in FRC-RIA 2- to 3-fold, whereas, presumably, the antigen preparations of the Abbott panel were prepared without pepsin digestion. Comparative ScreeningStudy with FRC-RIA and AusRIA II FRC-RIA and AusRIA I1 were compared using five groups of sera with different incidences of HB,Ag: (1) patients of Rinnekoti, an institution for the mentally retarded; (2) staff of Rinnekoti; (3) family members of Hl3,Ag carriers; (4) prisoners, and (5) staff of the Finnish Red Cross Blood Transfusion Service (table I). Groups 1-4 formed part of previously published studies (group 1, 2: Tevaluoto-Aarnio [13]; group 3, 4: Helske [5]). 3,742 normal donor sera and plasmas were studied with FRC-RIA only (table 11). Reagents specific for the HB,Ag subtype ad were used in FRC-RIA. The confirmatory kit for AusRIA was not available. A few samples were positive by one or the other method (table I). By this criterion, neither method can be said to be more sensitive. The overall percentage of repeatable

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Table I. Comparison of FRC-RIA and AusRIA I1 Patients of Rinnekotil

Staff of Family members of Rinnekoti HBaAg carriers

Prisoners

Staff of FRC Blood Transfusion Service

528

293

842

1,413

261

5

0

76

6

2

0

0

6a

0

0

0

0

23

0

0

Unrepeatable positive in AusRIA I1

10

4

29

32

a

Unrepeatable positive in FRC-RIA

3

5

23

28

2

Unspecific positive in FRC-RIA

4

0

1

1

0

Number of samples Positive with both tests FRC-RIA AusRIA -

+,

FRC-RIA-, AusRIA

+

l

a

An institution for mentally retarded. One sample was wepeatably positive with AusRIA 11. Neutralization test not performed.

but non-neutralizable, i.e., non-specific positives was 0.28% in FRC-RIA. It is curious that there were more of these in blood donor sera (13 of 2,000) than in blood donor plasmas (1 of 1,742) (table 11). The percentage of unrepeatable positives was 1.2% in FRCRIA and 2.4% in AusRIA 11. All of the unrepeatable and unspecific positives were weak, i.e., the count rate did not exceed 500 cpm, while the cutoff was usually below 200 cpm in F'RC-RIA.

Discussion

The coated-tube method was used by

Ling and Overby [9] in a sandwich type of radioimmunoassay for HB,Ag that was

later produced commercially (AusRIA I). Later, the coated tubes were replaced by polystyrenebeads as the solid phase (AusRIA 11). At the same time, the sensitivity increased many-fold. The detection limit of FRC-RIA, about 1 ng/ml, is equal to or lower than that of other current methods [15]. So it seems that the structure of the solid phase is not, after all, the crucial point for sensitivity. The cutoff was calculated in FRC-RIA in the same way as it is done in AusRIA, i.e., by multiplying the negative control mean by the factor 2.1. This was found to be, on average, 7.9 and 8.2 SDs above the negative control mean in FRC-RIA and AusRIA 11, respectively. It was concluded that it is justified to use this coefficient when

Coated-Tube Radioimmunoassay for HB=Ag

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Table II. Screening of blood donor plasmas and sera with FRC-RIA

Plasmas

Sera

1,742

2,000

Neutralizable positive (the one that was positive in FRC-RIA was positive with CIEP, too)

1

0

Non-neutralizable (unspecific) positive

1

13

Unrepeatable positive

9

17

Total number of samples tested

comparing FRC-RIA with AusRIA. It is obvious, however, that more positive cases could possibly be found by decreasing the arbitrary limit of positiveness. On the other hand, the coefficient must be sufficiently high to make sure that no series has too many unrepeatable positives, as the number of negative controls is small. In the case of blood transfusion services, where the number of HB,Ag positive samples is generally low, the actual level and dispersion of each individual series would be more accurately approximated, and the cutoff could be correspondingly decreased when a sizable proportion of the (apparently negative) samples would be taken into account in calculating the limit. This poses no problem if the results are analysed by a computer. This study confirms the previous findings [12] that the incubation time can be shortened by raising the incubation temperature. The importance of the subtype specificity of the solid phase and labelled antibody for sensitivity was clearly demonstrated. It is also noteworthy that good sensitivity to both subtypes could be maintained at the same time when a mixture of antibodies

specific for each subtype was used as reagents. When sera and plasmas were compared, there was no difference in favour of serum as to either non-specific repeatable or unrepeatable positives. This is important when the HB,Ag testing is connected with an automatic blood grouping system in which unclotted samples are required.

Acknowledgements I thank Prof. H . R . Nevanlinna, Dr. J . Leikola and Dr. G . Myllyla for advice, Drs. T. Helske and M . Tevaluoto-Aarnio for the patient and donor sera used in the comparative studies, and Abbott Laboratories for the generous supply of the AusRIA and AusAB kits. The skilful technical assistance of Miss Maija Satukangas and Mrs. Sinikka Hakkarainen is gratefully acknowledged.

References Catt, K.J.; Tregear, G. W.; Burger, H. G., and Skermer, C.: Antibody-coated tube method for radioimmunoassay of human growth hormone. Clinica chim. Acta. 27: 267-279 (1970). Cuatrecasas, P.: Protein purification by affinity chromatography. J. biol. Chem. 245: 30593065 (1970). Fine, J. M. and Steinbuch, M.: A simple technique for the isolation of monoclonal IgG and IgA. Revue eur. Etud. din. biol. IS: 1115-1121 (1970). Gocke, D. J. and Howe, C.: Rapid detection of Australia antigen by counterimmunoelectrophoresis. J. Immun. 104: 1031-1032 (1970). Helske, T.: Carriers of hepatitis B antigen and transfusion hepatitis in Finland. Scand. J. Haematol., suppl. 22 (1974). Hunter, W. M. and Greenwood, F.C.: Preparation of iodine-131 labelled human growth hormone of high specific antivity. Nature, Lond. 194: 495-496 (1962).

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7 Koistinen, V. U.: A direct solid phase radioimmunoassay for hepatitis BB antigen. 14th Congr. Int. SOC. Blood Transfusion, Helsinki 1975. 8 Koistinen, V. U.: A direct solid phase radioimmunoassay for hepatitis BB antigen. 15th Meet. Scand. SOC.Clin. Chem. Clin. Physiol., Arhus 1975. Scand. J. clin. Lab. Invest. 35: suppl. 143, p. 87; SUPPI.,pp. 143-187 (1975). 9 Ling. C. M. and Overby, L. R.: Prevalence of hepatitis B virus antigen as revealed by direct radioimmunoassay with 125-I-antibody. J. Immun. 109: 834-841 (1972). 10 Mazzur, S.; Burgert, S., and Blumberg, B. S.: Geographical distribution of Australia antigen determinants d, y and w. Nature, Lond. 247: 38-40 (1974). 11 Neurath, A. R.; Cosio, L.; Prince, A. M.; Lippin, A., and Ikram, H.: Hepatitis B antigens. Separation of two populations differing in particle size distribution. Proc. SOC.exp. Biol. Med. 143: 440-445 (1973). 12 Prince, A.M. and Jass, D.: Kinetic studies on the direct solid phase radioimmunoassay for

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hepatitis B antigen. Vox Sang. 26: 209-221 (1974). 13 Tevaluoto-Aarnio, M.: Epidemiology of hepatitis B antigenemia in an institution for mentally retarded. Scand. J. infect. Dis. 6: 309-313 (1974). 14 Ukkonen, P.; Koistinen, V., and Penttinen, K.: Evaluation of enzyme immunoassay in the detection of hepatitis B surface antigen. J. immunol. Meth. 15: 343-353 (1977). 15 Wolters, G.; Kuijpers, L.; Kacaki, J., and Schuurs, A.: Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen. J. clin. Path. 29: 873-879 (1976).

Received: May 25, 1977 Accepted: August 8, 1977 Vesa Koistinen, MSc, Finnish Red Cross Blood Transfusion Centre, Kivihaantie 7, 00310 Helsinki 31 (Finland)

A coated-tube radioimmunoassay (FRC-RIA) for hepatitis B surface antigen.

Vox Sang. 34: 321-328 (1978) A Coated-Tube Radioimmunoassay (FRC-RIA) for Hepatitis B Surface Antigen Vesu Koistinen Finnish Red Cross Blood Transfus...
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