RIOCHEMICAl
MEDICINE
18,
452-454
SHORT Hepatitis
11977)
COMMUNICATION
I3 Surface Millipore
Antigen Detection Filters System
by the
Detection of the Australia antigen in human blood, currently designated as the hepatitis B surface antigen (HBSA) by means of radioimmunoassay is a most sensitive and generally useful technique (I .2). The high specificity of radioimmunoassay for HBSA has been checked by special neutralization experiments (3.4). These presently reported techniques are based on a solid phase radioimmunoassay system which involves plastic tubes or polystyrene beads or pore glass in tablet form, coated with an antibody to hepatitis B surface antigen (anti-HBSA). If HBSA is present in the serum tested it will combine during incubation with the antibodies fixed on the solid base of, for example, the polystyrene beads. Afterward, any excess of HBSA and serum is removed by washing. When anti-HBSA labeled with iodine-125 is added, it binds with any antigen on the bead forming the “sandwich” -an antibody-antigen-antibody. An alternative counting gamma emitting iodine-125 for HBSA has been evaluated (5). In the present paper a simple and rapid method using the labeled anti-HBSA and Millipore filters is described. MATERIALS
AND METHOD
The antibody to hepatitis B surface antigen in solution was obtained through the courtesy of Dr. E. Mullet-, Abbott, Gmbh, Diagnostics Division, Eschborn, Germany. The specific activity of the 1251-labeled antibody was usually about 0.7 &i/ml of protein. Negative human control (nonreactive for HBSA) and positive human control were purchased from the Institute of Haematology, Warsaw and received as a gift from Abbott. The filtration was performed with Millipore filters HAWP 02500, which have a mean pore size of 0.45 pm and are 25 mm in diameter. The flow rates during the experiment were in the range of 5 to 8 ml/min. Using precision pipets, 0.1 ml of anti-HBSA was added to 0. I ml of negative and positive control. After incubation in 45°C water bath for I .5 hr, 0.1 ml of the above mixture was withdrawn on the prewetted Millipore filter. Immediately after filtration, the filter disk was washed with 5 ml of 0.5% SDS in 0.14 M NaCl. This detergent dissolves weak complexes and aggregates 452 Copyright @ 1977 by Academc Press. Inc. All rights of reproduction in any form reserved.
ISSN 0006.2944
453
SHORT COMMUNICATION
of antigen and antibody, except for those formed via strong linkages between the two macromolecules. Better results can be obtained when the washing solution has the temperature of incubation. Then the filter is dried, and the radioactivity retained on the disks is measured in a welltype gamma scintillation counter. RESULTS
AND DISCUSSION
The principle of antigen detection by means of the Millipore filter technique is that the antigen-antibody complex is bound to the filter while the free antigen and antibody pass through. Such behavior is due to the large size of the antigen particles (mean size, 20 nm), which in the electron microscope appeared to be approximately 60s; the antibody was about 7s in the sedimentation measurement (1). Table 1 shows the results of the ratio of net counts positive to net counts negative for 34 serums tested. About 59% of samples have demonstrated ratios in the range from 4.0 to 5.0.
TABLE RATIO
Number of serum samples Net counts positive/negative
I
OF NET COUNTS POSITIVE FOR HBSA TO NEGATIVE SERUM VERSUS THE NUMBER OF SAMPLES
2
3
4
2-3
3-3.5
3.5-4
9 4-4.5
II
3
2
4.5-5
5-5.5
5.5-6
The presented Millipore filter method was compared with one of the most sensitive radioimmunoassays for HBSA in use at present, that is, the AUSRIA II kit produced by Abbott Laboratories. The results obtained by testing the original AUSRIA II kit were compared to the results obtained by counting the radioactivity on the filters for the same samples. Then the positive serum was diluted in a negative one, as shown in Table 2, and the analogous ratios were determined. The main advantages of the Millipore filters method are (1) the assay needs one incubation for 1.5 hr instead of the two incubations of 3 hr total time using a “sandwich principle”; (2) the troublesome two-step washing procedure of polystyrene beads or pore glass in the filter method is avoided, and washing devices are not needed; (3) the determination of hepatitis B antigen in a given serum by the filter method is much cheaper than using the solid phase radioimmunoassay technique.
454
SHORT COMMUNICATION TABLE SENSITIVII Y COMPARISON SOLID PHASE (AUSRIA
AUSRIA Dilution I:2 I:4 I:8 I:16
2
OF THE Two RADIOIMMUNOASSAYS: II) AND MILLIPORE FILTERS SYSTEM
II
Millipore
Net count negative (cpm)
Net count positive tcpm)
Ratio
255
3187 I504 790 408 274
12.5 5.9 3.1 1.6 I.1
Net count negative (cpm) 875
_I_--
filters method Net count positive (cm) 4462 2275 1137 1050
Ratio 4.8 2.5 1.3 1.2
REFERENCES I. 2. 3. 4.
Ling, C. M., and Overby, L. R., J. Immunol. 109, 834 (1972). Hollinger, F. B., Vorndam, V., and Dreesman, G. R., J. Immunol. 107, 1099 (1971). Prince, A. M., Brotman, B.. Jass, 0.. and Ikram, H. Lancer 16, 1346 (1973). Schlicht, I.. Weise, W., Rutsch, W.. and Martin, W., Drsch. Med. Wnchenschr. 99, 671 (1974).
5. Jordan, C. W., Spiehler. V., Haendiges. R.. and Helman, E. Z., C/in.
Chem.
22, 733
(1974).
A. Institute of Medical Chemistry and Physics Silesian School of Medicine Jagielloizska 4 41-200 Sosnowiec, Poland
SUROWIEC
MEDICINE
18,
452-454
SHORT Hepatitis
11977)
COMMUNICATION
I3 Surface Millipore
Antigen Detection Filters System
by the
Detection of the Australia antigen in human blood, currently designated as the hepatitis B surface antigen (HBSA) by means of radioimmunoassay is a most sensitive and generally useful technique (I .2). The high specificity of radioimmunoassay for HBSA has been checked by special neutralization experiments (3.4). These presently reported techniques are based on a solid phase radioimmunoassay system which involves plastic tubes or polystyrene beads or pore glass in tablet form, coated with an antibody to hepatitis B surface antigen (anti-HBSA). If HBSA is present in the serum tested it will combine during incubation with the antibodies fixed on the solid base of, for example, the polystyrene beads. Afterward, any excess of HBSA and serum is removed by washing. When anti-HBSA labeled with iodine-125 is added, it binds with any antigen on the bead forming the “sandwich” -an antibody-antigen-antibody. An alternative counting gamma emitting iodine-125 for HBSA has been evaluated (5). In the present paper a simple and rapid method using the labeled anti-HBSA and Millipore filters is described. MATERIALS
AND METHOD
The antibody to hepatitis B surface antigen in solution was obtained through the courtesy of Dr. E. Mullet-, Abbott, Gmbh, Diagnostics Division, Eschborn, Germany. The specific activity of the 1251-labeled antibody was usually about 0.7 &i/ml of protein. Negative human control (nonreactive for HBSA) and positive human control were purchased from the Institute of Haematology, Warsaw and received as a gift from Abbott. The filtration was performed with Millipore filters HAWP 02500, which have a mean pore size of 0.45 pm and are 25 mm in diameter. The flow rates during the experiment were in the range of 5 to 8 ml/min. Using precision pipets, 0.1 ml of anti-HBSA was added to 0. I ml of negative and positive control. After incubation in 45°C water bath for I .5 hr, 0.1 ml of the above mixture was withdrawn on the prewetted Millipore filter. Immediately after filtration, the filter disk was washed with 5 ml of 0.5% SDS in 0.14 M NaCl. This detergent dissolves weak complexes and aggregates 452 Copyright @ 1977 by Academc Press. Inc. All rights of reproduction in any form reserved.
ISSN 0006.2944
453
SHORT COMMUNICATION
of antigen and antibody, except for those formed via strong linkages between the two macromolecules. Better results can be obtained when the washing solution has the temperature of incubation. Then the filter is dried, and the radioactivity retained on the disks is measured in a welltype gamma scintillation counter. RESULTS
AND DISCUSSION
The principle of antigen detection by means of the Millipore filter technique is that the antigen-antibody complex is bound to the filter while the free antigen and antibody pass through. Such behavior is due to the large size of the antigen particles (mean size, 20 nm), which in the electron microscope appeared to be approximately 60s; the antibody was about 7s in the sedimentation measurement (1). Table 1 shows the results of the ratio of net counts positive to net counts negative for 34 serums tested. About 59% of samples have demonstrated ratios in the range from 4.0 to 5.0.
TABLE RATIO
Number of serum samples Net counts positive/negative
I
OF NET COUNTS POSITIVE FOR HBSA TO NEGATIVE SERUM VERSUS THE NUMBER OF SAMPLES
2
3
4
2-3
3-3.5
3.5-4
9 4-4.5
II
3
2
4.5-5
5-5.5
5.5-6
The presented Millipore filter method was compared with one of the most sensitive radioimmunoassays for HBSA in use at present, that is, the AUSRIA II kit produced by Abbott Laboratories. The results obtained by testing the original AUSRIA II kit were compared to the results obtained by counting the radioactivity on the filters for the same samples. Then the positive serum was diluted in a negative one, as shown in Table 2, and the analogous ratios were determined. The main advantages of the Millipore filters method are (1) the assay needs one incubation for 1.5 hr instead of the two incubations of 3 hr total time using a “sandwich principle”; (2) the troublesome two-step washing procedure of polystyrene beads or pore glass in the filter method is avoided, and washing devices are not needed; (3) the determination of hepatitis B antigen in a given serum by the filter method is much cheaper than using the solid phase radioimmunoassay technique.
454
SHORT COMMUNICATION TABLE SENSITIVII Y COMPARISON SOLID PHASE (AUSRIA
AUSRIA Dilution I:2 I:4 I:8 I:16
2
OF THE Two RADIOIMMUNOASSAYS: II) AND MILLIPORE FILTERS SYSTEM
II
Millipore
Net count negative (cpm)
Net count positive tcpm)
Ratio
255
3187 I504 790 408 274
12.5 5.9 3.1 1.6 I.1
Net count negative (cpm) 875
_I_--
filters method Net count positive (cm) 4462 2275 1137 1050
Ratio 4.8 2.5 1.3 1.2
REFERENCES I. 2. 3. 4.
Ling, C. M., and Overby, L. R., J. Immunol. 109, 834 (1972). Hollinger, F. B., Vorndam, V., and Dreesman, G. R., J. Immunol. 107, 1099 (1971). Prince, A. M., Brotman, B.. Jass, 0.. and Ikram, H. Lancer 16, 1346 (1973). Schlicht, I.. Weise, W., Rutsch, W.. and Martin, W., Drsch. Med. Wnchenschr. 99, 671 (1974).
5. Jordan, C. W., Spiehler. V., Haendiges. R.. and Helman, E. Z., C/in.
Chem.
22, 733
(1974).
A. Institute of Medical Chemistry and Physics Silesian School of Medicine Jagielloizska 4 41-200 Sosnowiec, Poland
SUROWIEC
