Vox Sang. 23: 249-255 (1972)

The Purification of Hepatitis-Associated Antigen (HAA/SH/AU) from Human Serum

and the Preparation of a Sheep Anti-HAA Serum

W. J. M. DUIMEL, H. G. J. BRUMMELHUIS and H. W. KRIJNEN

Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam

Absrract. HAA was obtained from a pool of HAAcontaining sera. The purification consisted of the following steps:

Extraction with Freon, adsorption on and elution from Aerosil and gel filtration on a Sepharose 4B column.

After purification, the presence of the HAA was demonstrated in the macro-Ouchter- lony. The purity was tested by immunoelectrophoresis and double immunodiffusion.

Sheep were immunized with the purified HAA. In the macro-Ouchterlony, the presence of anti-HAA antibodies in the adsorbed sheep serum was demonstrated as a single precipitation line, identical with our human HAA anti-HAA precipitation line.

The anti-HAA sheep serum was tested with good results against the HAA panel of the National Institutes of Health, Bethesda, USA.

Introduction

In 1966, ALTER and BLUMBERG [l] described the first HAA purification. In 1969, GERIN et al. [3] described a purification method consisting of 2 isopycnic bandings in a CsCl density gradient, followed by rate zonal centrifugation on a sucrose density gradient. Many modifications of this method have been applied, they all have the disadvantage that a centrifuge tube will only hold 4-15 ml of the serum to be used for the purification of HAA.

This paper describes a method whereby large quantities of HAA are first partially purified by means of adsorption on Aerosil, elution and con- centration of the eluate.

Received: January 11, 1972; accepted: February 23, 1972.

250 DUIMEL/BRUMMELHUIS/KRIJNEN

Purified HAA can be used in newly developed sensitive test methods to detect HAA, such as the haemagglutination inhibition [9] and the radio- immunoassay tests [5, 101.

For the production of heterologeous antisera against HAA, purified HAA is also of importance. The production of a sheep anti-HAA serum is describ- ed in this paper.

Materials and Methods

Purification Sera obtained from donors and patients who were positive for HAA in the macro-

Ouchterlony technique were pooled. The HAA was purified in the following way: 400 mi of serum were extracted with Freon in a ratio of 4 t o I for % h at room temper-

ature (Freon 1 1 3, Dow Chemical). The serum layer was then incubated for 4 h at 37 "C with 2% (w/v) Aerosil (Aerosil380, Degussa, Frankfurt/Main). After centrifugation for 15 min at 3,000 rpm, the Aerosil was washed 4 times with physiological saline (0.154 M NaCI).

The E$Gof the last washing was 0.26.The Aerosil was eluted batchwise twice with 200 ml 0.01 M Borax, pH 9.3, for 30 min at 37°C. After concentrating the pooled eluates against 25% (w/v) polyvinylpyrrolidone to 1/6 of the original volume, 3 ml aliquots were chromatographed on a Sepharose 4B column (5.0 x 25 cm, Pharmacia) and eluted with phosphate-buffered saline (PBS, 0.140 M NaCI, 0.01 13 M Na,HPO,, 0.0017 M NaH,PO,, pH 7.2). The eluate was collected in fractions of 6 ml each and concentrated by means of ultrafiltration.

Imniunizarion Two sheep have been immunized with purified HAA in the following way:

SheepSf IO. Day I : 4 mi of purified HAA ( E % a = 0.6) with an equal volume of Freund's complete adjuvant were injected intramuscularly in 4 places (2 hindlegs and in the back, at the right and the left side of the spinal column). Bleedings were performed on day 15, 22.43, 64,85, 106, and 127; 10, 100, 100, 500,500,500, and 500 ml, respectively, of whole blood were collected without anticoagulant.

All serum samples of these bleedings had an anti-HAA titer in the macro-Ouchterlony of 64.

Sheep 5941. Day 1 and 22: 2 rnl of purified HAA ( E l i 6 = 0.4) were injected as in sheep 51 10.

Bleedings were performed on day 22,43,64,85, and 106; 10,500,500,500 and 500 ml, respectively, of whole blood were collected without anticoagulant. The anti-HAA titer of the first bleeding was 16; after the second injection, the anti-HAA titer in the serum of all other bleedings was 64.

To obtain a specific anti-HAA antiserum, the sheep serum (300 nil) was extracted with 80 rnl of Freon and adsorbed for 2 h at 37°C and 48 h at 4°C with 300 ml supernatant of

The Purification of Hepatitis-Associated Antigen 25 1

human plasma cryoprecipitate [7]. The sheep serum was further adsorbed with 600 ml Aerosil eluate [8] of the cryoprecipitate (2 h at 37 "C, 48 h at 4 T ) , which had been dialysed against physiological saline.

After centrifugation (2 min at 3,000 rpm), the clarified serum was incubated for 1 h at 37°C with washed human erythrocytes to remove heteroagglutinins. Finally, the serum was extracted with Freon and sterilized by filtration (Schleicher and Schull No. 1121).

Reference sera, containing HAA or anti-HAA antibodies, were obtained from donors and patients. The sheep anti-HAA serum was tested against these sera and against the H A A panel from the National Institutes of Health (NIH), Bethesda, USA (kindly supplied by Dr. J. C. WAGNER and Dr. E. B. SELIGMANN, jr.).

Double agargel diffusion (ID, macro-Ouchterlony) was performed according to BRUMMELHUIS [2] in 0.9% agarose. The holes had a diameter of 0.7 cm and the distance from core to core was 1 . 1 cm [2].

Immunoelectrophoresis (IE) was carried out in 1.3% Agar Noble (Difco), Verona1 buffer 0.05 M, pH 8.6, according to PEETOOM 161.

Counterimmunoelectrophoresis (CEP) was performed according to G ~ C K E and HOWE "I].

Results

A greater reproducibility of the HAA yield was obtained by elution of the Aerosil after the extraction of the human positive serum with Freon. About 5-10% of the serum proteins adsorb at 2% Aerosil. After elution, the yield of HAA was about 60%, the yield of serum proteins about 4%, based on extinction at 280 nm. After concentration of the eluate, most of the serum proteins could be demonstrated in IE. Gel filtration on a Sepharose 4B column gave the elution pattern (shown in figure l), based on absorption at 280 nm of the collected fractions. The location ofthe HAA peak correspond- ed with the V, of the column. There was a sharp increase in HAA titer between the fractions 23 + 24 and 25 + 26. The decrease in HAA titer between the fractions 3 1 + 32, 33 + 34 and 35 + 36 was not so sharp.

Contaminating serum proteins were found in increasing amounts starting with fraction 29 + 30. IE of pooled and concentrated fractions gave the results shown in figure 2.

After pooling and concentrating the fractions which gave no precipitation in the IE to E \:: = 1.2, the titer of this purified HAA was 32 in the macro- Ouchterlony. No serum proteins could be detected in the IE against horse anti-total-human serum 1 :4. In the double immunodiffusion against horse anti-total-human serum 1 :4, three weak precipitation lines could be detected; they were identified as caused by IgG, haptoglobin and albumin.

252 DUIMEL./BRUMMELHUIS/KRIJNEN

1.4 - -

1.2 - -

1.0 - -

0.8 - -

0.6 - -

0 4 - -

02 - -

I

i i

X r ,fl+/

X- X’

xx’

Fig. 1. Elution pattern from a Sepharose 4 B column of 3 ml of HAA-positive concen- trated Aerosil eluate. Elution was performed with PBS, the fractions were 6 ml each. + Indicates fraction positive for HAA.

* -

21 22

m-%- 24 26

! 27 ‘ 29 31 33 28 30 32 34

Fig. 2. IE of pooled and concentrated Sepharose 4 B column fractions against horse anti-human total serum (HaHt) 1 :4. The fraction numbers correspond with the fraction numbers in figure 1.

The Purification of Hepatitis-Associated Antigen

D

f .

253

Fig. 3. Demonstration of immunological identity between sheep anti-Au serum and human anti-Au serum. The central well contained human anti-Au serum; c and f control HAA-positive serum; a, b, d, and e sheep anti-Au serum.

e d Fig. 4. Demonstration of immunological specificity of the sheep anti-Au serum. The

central well contained sheep anti-Au serum; c and f control HAA-positive serum; a, d, and e were HAA-positive samples; b was a HAA-negative sample.

254 DUIMEL/BRUMMELHUIS/KRIJNEN

Figure 3 shows the adsorbed sheep anti-HAA serum. The HAA anti- HAA precipitation line was identical with the standard precipitation line. Testing the sheep anti-HAA serum against the HAA panel of the NIH, gave the following results:

The panel consisted of 60 sera of which 47 were HAA-positive by complement fixation (CF) and 13 HAA-negative by CF.

In the macro-Ouchterlony, with the sheep anti-HAA serum 4 times diluted, 46 of these 47 sera were found positive for HAA. The 13 sera found negative for HAA by CF, were also found negative in the macro-Ouchterlony.

In the CEP, with the sheep anti-HAA undiluted, 42 of these 47 sera were found positive, the 13 sera negative by CF were also negative by CEP.

Passive haemagglutination tests to detect the presence of antibodies against IgG, IgM and albumin in the adsorbed sheep anti-HAA serum were negative.

Discussion

Only after treatment of serum with Freon, an organic solvent that extracts part of the lipoids from serum, is the HAA yield and the HAA: serum protein ratio in the Aerosil eluate reproducible. In the Aerosil eluate, the ratio of HAA to serum proteins is about 15 times more than in the start- ing serum, due to a better adsorption and elution of HAA.

No serum proteins could be detected in the IE after the described puri- fication. In the more sensitive ID, however, the presence of at least 3 serum proteins (IgG, haptoglobin and albumin) could be shown. Even after isopycnic banding in a CsCl density gradient of this purified HAA, 2 serum proteins (haptoglobulin and albumin) could be detected in the HAA-positive fractions in the ID.

The sheep anti-HAA serum of sheep 5 I 10 contained before adsorption next to specific antibodies against HAA, antibodies against human proteins. Immunization with a lower dose of HAA, as given to sheep 5941, did not result in an antiserum containing antibodies only against HAA before adsorption.

The preparation of anti-HAA serum with the described method provides us with a permanent stock of a good precipitating, specific anti-HAA serum for routine use.

A comparison of the results obtained with the sheep anti-HAA serum against the NIH panel shows that the macro-Ouchterlony technique is at least as sensitive as the CEP and comparable in sensitivity to the CF test.

The Purification of Hepatitis-Associated Antigen 255

Acknowledgements

The authors wish to thank C.C. DUIVIS-VORST and L. VAN GIJLSWIJK for technical assistance and MIA VAN DER HART for carrying out the passive haemagglutination tests.

References

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2 BRUMMELHUIS, H. G. J.: Technical aspects and results of the determination of hepatitis associated antigen (HAA) and antibody. Vox Sang. 19: 228 (1970).

3 GERIN, J. L.; PURCELL, R. H.; HOGGAN, M. D.; HOLLAND, P. V., and CHANOCK, R. M.: Biophysical properties of Australia antigen. J. Virol. 4: 763 (1969).

4 GOCKE, D. J. and HOWE, C.: Rapid detection of Australia antigen by counterimmuno- electrophoresis. J. Immunol. 104: 1031 (1970).

5 LANDER, J. L.; ALTER, H. J., and PURCELL, R. H.: Frequency of antibody to hepatitis associated antigen as measured by a new radioimmunoassay technique. J. Imrnunol. 106: 1166 (1971).

6 PEETOOM, F.: The agar precipitation technique and its application as a diagnostic and analytical method (Stanfert & Kroese, Leiden 1963).

7 POOL, J. G. and SHANON, A. E.: Production of high potency concentrates of AHF in a closed bag system. New Engl. J. Med. 273: 1443 (1965).

8 STEPHAN, W. und ROKA, L.: Adsorption von Lipoproteiden. Z. klin. Chem. klin. Biochem. 6: 186 (1968).

9 VYAS, G. N. and SHULMAN, N. R.: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332 (1970).

10 WALSH, J. H.; YALOW, R., and BERSON, S. A.: Detection of Australia antigen and anti- body by means of radioimmunoassay techniques. J. infect. Dis. 121: 550 (1970).

Authors’address: Dr. W.J. M. DUIMEL, Dr. H.G.J. BRUMMELHUIS and Dr. H. W. KRIJNEN, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (The Netherlands)