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A space-resolved fluorometer and its application to immunoassay
Journal of lmmunological Methods, 112 (1988) 173-176
173
Elsevier JIM 04862
A space-resolved fluorometer and its application to immunoassay W . K . W a n g *, L.T. H o , Y. C h i a n g a n d T.C. C h e n Biophysics Laboratory, Institute of Physics, Academia Sinica, Taipei, Taiwan, China
(Received 23 October, 1985, last revision received 25 March 1988, accepted 28 March 1988)
A metal surface has been added into a fluorometer to reflect the excited light and to reduce the light scattering. Due to the total separation of the fluorescent signal and the noise by directing them into different spaces, the resolution could be improved by several orders of magnitude. The same metal surface could also be used for immunoassays. The detection of a-fetoprotein in normal rat serum and h u m a n standard serum are given as examples. The results indicate that the apparatus and the method have significant advantages. Key words: Space-resolvedfluorometer; Fluoroimmunoassay; a-Fetoprotein
Introduction
Non-radioactive labels such as bacteriophages, enzymes, stable free radicals, fluorescent and chemiluminescent groups have been used to replace radioactive isotopes for immunoassays (Simpson et al., 1979; Schroeder et al., 1981; Petterson et al., 1983). Recently time-resolved fluoroimmunoassay has advanced sufficiently to become commercially available (Hemmil~i, 1985). In addition to the commonly used test tubes in immunoassay tests, metal surfaces have also been reported useful in immunoassays. In the latter case the antibody-antigen reaction takes place at a liquid-solid interface and the results can be detected by the use of an ellipsometer (Rothen, 1973). Giaever (1974) further developed a visual detection system to replace the eUipsometer but only for a qualitative test. In this paper, we will
Correspondence to: W.K. Wang, Biophysics Laboratory, Institute of Physics, Academia Sinica, Taipei, Taiwan, China. * Joint appointment with Institute of Biomedical Engineering, National Yang-Ming Medical College, Taipei.
report the basic principle of a space-resolved fluorometer which can be used to detect trace amounts of a fluorescent dye as well as a few examples of applications to immunoassays to detect antibody and antigen quantitatively.
Materials and methods Instrument
The outline of the apparatus is shown in Fig. 1. The incident light, after proper filtering, hits the fluorescent dye on a metal surface in a black box. The reflected light is trapped in the black box, while the fluorescent emission, after passing through another filter, goes toward the photon counting system. The main purpose of this design is the spatial separation of the fluorescent light from either the incident or the reflected light. Another advantage is the smooth metal surface causing extremely small amount of light scattering. For example, using a xenon lamp as the light source and single-band interference filters with peaks at 488 n m and 520 nm for the incident light and the fluorescent light respectively, the photon
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
174
3
2
Fig. 1. Schematic diagram of the high resolution fluorometer. 1, light source; 2 and 6, filters; 3, sample and sample holder; 4, reflected photon directed toward a photon trap; 5, fluorescent photon; 7, photon counting system.
counts are only about 102/s for a smooth metal surface prepared by high-vacuum sputtering technique. But if the metal surface is replaced by a piece of regular paper, a plastic or a ceramic plate, light scattering increases and the counts reach as high as 2 x 1 0 5 , 2 × 1 0 4 and 6 x 1 0 3 / s , respectively. In order to make sure that the increase originates from scattering and no fluorescence is involved, all the materials were checked by an argon laser excited fluorometer equipped with double monochromator and no significant fluorescence could be detected at wavelength shorter than 540 nm for ceramic and 600 nm for paper and plastic. Adding this space-resolved design to a fluorometer, the signal-to-noise ratio can thus be increased by several orders of magnitude and very high resolution can be obtained. Another way to prove its high resolution is for example, to directly measure the fluorescence arising from the fluorescein isothiocyanate (FITC). FITC was first diluted to 0.1 n g / m l and a drop of 10 ~1 of this solution was then put on the metal surface and let dry. The photon count was about 5 x 1 0 2 / s . Since the count of the metal surface, dried with water only, was about 3 x 102/s, such a small amount of FITC could thus be clearly detected. In other words, the resolution in this case is better than 10 t~l x 0.1 n g / m l = 1.0 pg of FITC.
Irnmunotest procedure To show its high-resolution performance in an immunotest, the detection of a-fetoprotein (from Calbiochem, U.S.A.), and of anti-rabbit IgG anti-
body (from Miles-Yeda, Israel) in normal rat serum which was a mixture of serum from five healthy rats are given as examples. The procedure of the sample preparation was similar to Giaever's visual detection method (Giaever and Laffin, 1974): anti-~-fetoprotein antibody was first diluted to 0.1 m g / m l with saline. A drop of 10 /~1 and about 3 mm in diameter was put on the surface of a glass slide coated with a thin layer of metal. After incubation for about 40 rain, it was rinsed with distilled water and gently blown dry. In this way, a monolayer of c~-fetoprotein antibody was adsorbed on to the metal surface. 2 /xl of normal rat serum, which contained afetoprotein of different concentrations, was then added to the same spot. After incubating for about 4 h in a moist chamber, the same spot was stained with FITC-conjugated goat IgG anti-c~-fetoprotein and read in the fluorometer. To show that the control serum does not significantly affect the results, some experiments measuring human serum standards (from Dai-Chi, Japan) were also performed. In the case of antibody detection, goat antibody anti-rabbit IgG was used as an example. Similar procedures were followed except that the first layer was rabbit IgG and the stain became FITC-conjugated rabbit antibody to goat IgG.
Results
and discussion
The results of some typical tests measuring a-fetoprotein are shown in Table I. Six sets of experimental data were given. In each set, the dependence of the photon count on the amount of c~-fetoprotein can be seen clearly. The numbers shown for photon counts represent the average c o u n t s / s for three tests. The percentual changes in counts as compared to controls are also given. Table II shows results of measurements of antirabbit IgG antibody. The amounts shown represent total IgG (including anti-rabbit IgG antibody and non-specific IgG). Table III shows the results on c~-fetoprotein-containing human serum. Student's t-tests were performed on these data. The data with * means P < 0.05, data with • * means P < 0.01, with *** P < 0.001 as c o r n -
175 TABLE I M E A S U R E M E N T S OF a - F E T O P R O T E I N Set 1
5 ng/ml
Set 3
Set 4
Set 5
Set 6
9351_+ 581 100%
10191_+ 1668 100%
5880_+ 100%
961
4168_+1088 100%
4688_+ 237 100%
4694_+1066 100%
10679_+1824 114%
12013_+ 2411 118%
7662_+ 130%
389 *
5757-+ 874 138%
5606_+ 959 120%
5571_+1040 119%
16954_+2527"* 181%
16830_+ 4 1 7 4 " 165%
8413_+ 515 * * * 179%
7144_+1020" 152%
Control AFP 1 ng/ml
Set 2
10 n g / m l 20ng/ml
45630_+3566"** 488%
44676_+ 3 7 4 0 " * * 438%
50ng/ml
56787_+4585"** 607%
58930_+11213"* 578%
12375+ 2 1 3 8 " * 210%
11819_+2450 ** 284%
26463_+ 2 7 9 9 " * 450%
16677_+1182"** 400%
16891_+5297"* 360%
10281_+3031" 219%
40355_+11168"* 686%
18877+_7693" 453%
29966_+4934"** 639%
18019+5716"* 384%
pared to controls. The large standard deviations observed could arise mainly from two sources of error. (1) Small incubation volumes; several pipet-
ting volumes were < 5/~1, and we did not use any special pipetting instruments. (2) N o quality control enforced; we did not have any criterion to
T A B L E II M E A S U R E M E N T S OF A N T I B O D I E S TO RABBIT IgG Set 1
Set 2
Set 3
Set 4
Set 5
1 6 9 3 2 ± 3457 100%
9362_+ 2793 100%
6272+1395 100%
6737_+ 1453 100%
5 400 _+ 100%
17124-+ 4814 101%
10664 _+2252 114%
7118_+ 920 114%
7123_+ 1855 106%
5 5 4 7 ± 1409 103%
30 n g / m l
19619_+ 4688 116%
11410_+ 1700 122%
6847+1688 110%
8474_+ 2413 126%
6101+ 113%
90 n g / m l
28036_+ 2 8 6 1 " * 166%
14716_+ 5250 157%
8631_+2811 139%
7838_+ 2352 116%
6 3 5 4 + 2357 118%
270 n g / m l
42021_+15679 * 248%
29934_+10113 * 320%
12306_+3412 * 198%
8285_+ 2834 123%
8604_+ 2 5 5 3 " 159%
Control
G-anti-R IgG 10 n g / r n l
764
1597
500 n g / m l
17888_+11627 266%
17403_+ 4954 * * 322%
1/~g/ml
20840-+ 4070 * * 309%
18246_+ 1995 * * * 338%
2.5 # g / m l
23728-+ 9 9 4 1 " 352% 40989_+10963 * * 608% 48984_+ 3 9 2 4 " * * 727%
38072+ 3177"** 705% 41696_+ 2120 * * * 772% 58651_+20229"* 1 086%
5/zg/ml 10 g g / m l
176 T A B L E III M E A S U R E M E N T S ON H U M A N S E R U M C O N T A I N I N G I N C R E A S I N G C O N C E N T R A T I O N S OF a - F E T O P R O T E I N
Control Set 1 Set2
Set3 Set4
Set5 Set6
Set 7 Set8
6742_+1145 100% 7355_+ 648 100% 5763_+1365 100% 3077_+ 105 100% 2249_+ 69 100% 2175_+1154 100% 2205_+ 382 100% 2369_+ 349 100%
3 ng/ml
8993_+4181 122%
3322_+ 3 5 " 108% 2906+ 206"* 129% 3502_+1709 161% 3225_+ 31 * * 146% 3647_+ 4 2 3 * 129%
10 ng/ml
30 ng/ml
100 n g / m l
300 ng/ml
15571_+1761 * 230% 11697_+2165" 159% 8156_+ 9 9 5 " 142% 4073-+1512 132% 6556_+1746"* 292% 4688_+ 1 9 4 " 216% 3751_+ 861 170% 3888_+ 1 3 " * 164%
21332_+2011 * * * 365% 21264+2852"* 289% 12942_+2158"* 225% 13360_+2816 ** 434% 10712_+3254"* 476% 10403_+2212"* 478% 10684_+2263 ** 416% 14323_+3294** 605%
49544_+12631 ** 850% 36484_+ 1925 * 496% 25539_+ 7 1 8 1 " * 443% 19135-+ 8987 * 622% 17175_+ 3905 ** 764% 17323_+ 2 3 2 1 " * * 769% 15313_+ 3585 ** 596% 26044_+ 1 1 0 0 " * 1100%
52010-+6894 * * * 890%
select the substrate, the metal surface, the antibody-covered metal surface. These should be gradually improved. The data shown are for triplicates. In other words, at this stage, we need more than three tests to detect less than 5 n g / m l of a-fetoprotein in normal serum accurately. For the antibody test (Table II), we do not k n o w the exact amount of anti-rabbit IgG antibody in the antiserum. If it contains 5% specific antibody, the lowest detectable amount should be around 10 n g / m l . In sets 4 and 5 assays were performed to detect high concentrations of antibody. In conclusion, the space-resolved fluorometer discussed above has a low noise-to-signal ratio due to its ability to reduce the noise originating from the incident light, the reflected light and the scattering light. Besides its high resolution, which is at least comparable to that of radioimmunoassays, such a fluorometer also has other advantages. The serum and other reagents are used
in small amounts. Furthermore, we used an indirect method to detect IgG. Other immunoglobuline classes could be detected in a similar way.
References Giaever, I. and Laffin, R.J. (1974) Visual detection of hepatitis B antigen. Proc. Natl. Acad. Sci. U.S.A. 71, 4533. Hemmil~i, I. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem. 31, 359. Petterson, K., Siitari, H., Hemmil~i, I., Soini, E., L/3vgren, T., Hanninen, V., Tanner, P. and Stennan, U. (1983) Time-resolved fluoroimmunoassay of human choriogonadotropin. Clin. Chem. 29, 60. Rothen, A. (1973) Immunologic and enzymatic reactions carried out at a solid-liquid interface. Physiol. Chem. Phys. 5, 243.
Schroeder, H.R., Hines, C.M., Osborn, D.D., Moore, R.P., Hurtle, R.L., Wogoman, F.F., Roger, R.W. and Vogelhut, P.P. (1981) Immunochemiluminometric assay for hepatitis B surface antigen. Clin. Chem. 27, 1378. Simpson, J.S.A., Campbell, A.K., Ryall, M.E.T. and Woodhead, J.S. (1979) A stable chemiluminescent-labelled antibody for immunological assays. Nature 279, 646.
173
Elsevier JIM 04862
A space-resolved fluorometer and its application to immunoassay W . K . W a n g *, L.T. H o , Y. C h i a n g a n d T.C. C h e n Biophysics Laboratory, Institute of Physics, Academia Sinica, Taipei, Taiwan, China
(Received 23 October, 1985, last revision received 25 March 1988, accepted 28 March 1988)
A metal surface has been added into a fluorometer to reflect the excited light and to reduce the light scattering. Due to the total separation of the fluorescent signal and the noise by directing them into different spaces, the resolution could be improved by several orders of magnitude. The same metal surface could also be used for immunoassays. The detection of a-fetoprotein in normal rat serum and h u m a n standard serum are given as examples. The results indicate that the apparatus and the method have significant advantages. Key words: Space-resolvedfluorometer; Fluoroimmunoassay; a-Fetoprotein
Introduction
Non-radioactive labels such as bacteriophages, enzymes, stable free radicals, fluorescent and chemiluminescent groups have been used to replace radioactive isotopes for immunoassays (Simpson et al., 1979; Schroeder et al., 1981; Petterson et al., 1983). Recently time-resolved fluoroimmunoassay has advanced sufficiently to become commercially available (Hemmil~i, 1985). In addition to the commonly used test tubes in immunoassay tests, metal surfaces have also been reported useful in immunoassays. In the latter case the antibody-antigen reaction takes place at a liquid-solid interface and the results can be detected by the use of an ellipsometer (Rothen, 1973). Giaever (1974) further developed a visual detection system to replace the eUipsometer but only for a qualitative test. In this paper, we will
Correspondence to: W.K. Wang, Biophysics Laboratory, Institute of Physics, Academia Sinica, Taipei, Taiwan, China. * Joint appointment with Institute of Biomedical Engineering, National Yang-Ming Medical College, Taipei.
report the basic principle of a space-resolved fluorometer which can be used to detect trace amounts of a fluorescent dye as well as a few examples of applications to immunoassays to detect antibody and antigen quantitatively.
Materials and methods Instrument
The outline of the apparatus is shown in Fig. 1. The incident light, after proper filtering, hits the fluorescent dye on a metal surface in a black box. The reflected light is trapped in the black box, while the fluorescent emission, after passing through another filter, goes toward the photon counting system. The main purpose of this design is the spatial separation of the fluorescent light from either the incident or the reflected light. Another advantage is the smooth metal surface causing extremely small amount of light scattering. For example, using a xenon lamp as the light source and single-band interference filters with peaks at 488 n m and 520 nm for the incident light and the fluorescent light respectively, the photon
0022-1759/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
174
3
2
Fig. 1. Schematic diagram of the high resolution fluorometer. 1, light source; 2 and 6, filters; 3, sample and sample holder; 4, reflected photon directed toward a photon trap; 5, fluorescent photon; 7, photon counting system.
counts are only about 102/s for a smooth metal surface prepared by high-vacuum sputtering technique. But if the metal surface is replaced by a piece of regular paper, a plastic or a ceramic plate, light scattering increases and the counts reach as high as 2 x 1 0 5 , 2 × 1 0 4 and 6 x 1 0 3 / s , respectively. In order to make sure that the increase originates from scattering and no fluorescence is involved, all the materials were checked by an argon laser excited fluorometer equipped with double monochromator and no significant fluorescence could be detected at wavelength shorter than 540 nm for ceramic and 600 nm for paper and plastic. Adding this space-resolved design to a fluorometer, the signal-to-noise ratio can thus be increased by several orders of magnitude and very high resolution can be obtained. Another way to prove its high resolution is for example, to directly measure the fluorescence arising from the fluorescein isothiocyanate (FITC). FITC was first diluted to 0.1 n g / m l and a drop of 10 ~1 of this solution was then put on the metal surface and let dry. The photon count was about 5 x 1 0 2 / s . Since the count of the metal surface, dried with water only, was about 3 x 102/s, such a small amount of FITC could thus be clearly detected. In other words, the resolution in this case is better than 10 t~l x 0.1 n g / m l = 1.0 pg of FITC.
Irnmunotest procedure To show its high-resolution performance in an immunotest, the detection of a-fetoprotein (from Calbiochem, U.S.A.), and of anti-rabbit IgG anti-
body (from Miles-Yeda, Israel) in normal rat serum which was a mixture of serum from five healthy rats are given as examples. The procedure of the sample preparation was similar to Giaever's visual detection method (Giaever and Laffin, 1974): anti-~-fetoprotein antibody was first diluted to 0.1 m g / m l with saline. A drop of 10 /~1 and about 3 mm in diameter was put on the surface of a glass slide coated with a thin layer of metal. After incubation for about 40 rain, it was rinsed with distilled water and gently blown dry. In this way, a monolayer of c~-fetoprotein antibody was adsorbed on to the metal surface. 2 /xl of normal rat serum, which contained afetoprotein of different concentrations, was then added to the same spot. After incubating for about 4 h in a moist chamber, the same spot was stained with FITC-conjugated goat IgG anti-c~-fetoprotein and read in the fluorometer. To show that the control serum does not significantly affect the results, some experiments measuring human serum standards (from Dai-Chi, Japan) were also performed. In the case of antibody detection, goat antibody anti-rabbit IgG was used as an example. Similar procedures were followed except that the first layer was rabbit IgG and the stain became FITC-conjugated rabbit antibody to goat IgG.
Results
and discussion
The results of some typical tests measuring a-fetoprotein are shown in Table I. Six sets of experimental data were given. In each set, the dependence of the photon count on the amount of c~-fetoprotein can be seen clearly. The numbers shown for photon counts represent the average c o u n t s / s for three tests. The percentual changes in counts as compared to controls are also given. Table II shows results of measurements of antirabbit IgG antibody. The amounts shown represent total IgG (including anti-rabbit IgG antibody and non-specific IgG). Table III shows the results on c~-fetoprotein-containing human serum. Student's t-tests were performed on these data. The data with * means P < 0.05, data with • * means P < 0.01, with *** P < 0.001 as c o r n -
175 TABLE I M E A S U R E M E N T S OF a - F E T O P R O T E I N Set 1
5 ng/ml
Set 3
Set 4
Set 5
Set 6
9351_+ 581 100%
10191_+ 1668 100%
5880_+ 100%
961
4168_+1088 100%
4688_+ 237 100%
4694_+1066 100%
10679_+1824 114%
12013_+ 2411 118%
7662_+ 130%
389 *
5757-+ 874 138%
5606_+ 959 120%
5571_+1040 119%
16954_+2527"* 181%
16830_+ 4 1 7 4 " 165%
8413_+ 515 * * * 179%
7144_+1020" 152%
Control AFP 1 ng/ml
Set 2
10 n g / m l 20ng/ml
45630_+3566"** 488%
44676_+ 3 7 4 0 " * * 438%
50ng/ml
56787_+4585"** 607%
58930_+11213"* 578%
12375+ 2 1 3 8 " * 210%
11819_+2450 ** 284%
26463_+ 2 7 9 9 " * 450%
16677_+1182"** 400%
16891_+5297"* 360%
10281_+3031" 219%
40355_+11168"* 686%
18877+_7693" 453%
29966_+4934"** 639%
18019+5716"* 384%
pared to controls. The large standard deviations observed could arise mainly from two sources of error. (1) Small incubation volumes; several pipet-
ting volumes were < 5/~1, and we did not use any special pipetting instruments. (2) N o quality control enforced; we did not have any criterion to
T A B L E II M E A S U R E M E N T S OF A N T I B O D I E S TO RABBIT IgG Set 1
Set 2
Set 3
Set 4
Set 5
1 6 9 3 2 ± 3457 100%
9362_+ 2793 100%
6272+1395 100%
6737_+ 1453 100%
5 400 _+ 100%
17124-+ 4814 101%
10664 _+2252 114%
7118_+ 920 114%
7123_+ 1855 106%
5 5 4 7 ± 1409 103%
30 n g / m l
19619_+ 4688 116%
11410_+ 1700 122%
6847+1688 110%
8474_+ 2413 126%
6101+ 113%
90 n g / m l
28036_+ 2 8 6 1 " * 166%
14716_+ 5250 157%
8631_+2811 139%
7838_+ 2352 116%
6 3 5 4 + 2357 118%
270 n g / m l
42021_+15679 * 248%
29934_+10113 * 320%
12306_+3412 * 198%
8285_+ 2834 123%
8604_+ 2 5 5 3 " 159%
Control
G-anti-R IgG 10 n g / r n l
764
1597
500 n g / m l
17888_+11627 266%
17403_+ 4954 * * 322%
1/~g/ml
20840-+ 4070 * * 309%
18246_+ 1995 * * * 338%
2.5 # g / m l
23728-+ 9 9 4 1 " 352% 40989_+10963 * * 608% 48984_+ 3 9 2 4 " * * 727%
38072+ 3177"** 705% 41696_+ 2120 * * * 772% 58651_+20229"* 1 086%
5/zg/ml 10 g g / m l
176 T A B L E III M E A S U R E M E N T S ON H U M A N S E R U M C O N T A I N I N G I N C R E A S I N G C O N C E N T R A T I O N S OF a - F E T O P R O T E I N
Control Set 1 Set2
Set3 Set4
Set5 Set6
Set 7 Set8
6742_+1145 100% 7355_+ 648 100% 5763_+1365 100% 3077_+ 105 100% 2249_+ 69 100% 2175_+1154 100% 2205_+ 382 100% 2369_+ 349 100%
3 ng/ml
8993_+4181 122%
3322_+ 3 5 " 108% 2906+ 206"* 129% 3502_+1709 161% 3225_+ 31 * * 146% 3647_+ 4 2 3 * 129%
10 ng/ml
30 ng/ml
100 n g / m l
300 ng/ml
15571_+1761 * 230% 11697_+2165" 159% 8156_+ 9 9 5 " 142% 4073-+1512 132% 6556_+1746"* 292% 4688_+ 1 9 4 " 216% 3751_+ 861 170% 3888_+ 1 3 " * 164%
21332_+2011 * * * 365% 21264+2852"* 289% 12942_+2158"* 225% 13360_+2816 ** 434% 10712_+3254"* 476% 10403_+2212"* 478% 10684_+2263 ** 416% 14323_+3294** 605%
49544_+12631 ** 850% 36484_+ 1925 * 496% 25539_+ 7 1 8 1 " * 443% 19135-+ 8987 * 622% 17175_+ 3905 ** 764% 17323_+ 2 3 2 1 " * * 769% 15313_+ 3585 ** 596% 26044_+ 1 1 0 0 " * 1100%
52010-+6894 * * * 890%
select the substrate, the metal surface, the antibody-covered metal surface. These should be gradually improved. The data shown are for triplicates. In other words, at this stage, we need more than three tests to detect less than 5 n g / m l of a-fetoprotein in normal serum accurately. For the antibody test (Table II), we do not k n o w the exact amount of anti-rabbit IgG antibody in the antiserum. If it contains 5% specific antibody, the lowest detectable amount should be around 10 n g / m l . In sets 4 and 5 assays were performed to detect high concentrations of antibody. In conclusion, the space-resolved fluorometer discussed above has a low noise-to-signal ratio due to its ability to reduce the noise originating from the incident light, the reflected light and the scattering light. Besides its high resolution, which is at least comparable to that of radioimmunoassays, such a fluorometer also has other advantages. The serum and other reagents are used
in small amounts. Furthermore, we used an indirect method to detect IgG. Other immunoglobuline classes could be detected in a similar way.
References Giaever, I. and Laffin, R.J. (1974) Visual detection of hepatitis B antigen. Proc. Natl. Acad. Sci. U.S.A. 71, 4533. Hemmil~i, I. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem. 31, 359. Petterson, K., Siitari, H., Hemmil~i, I., Soini, E., L/3vgren, T., Hanninen, V., Tanner, P. and Stennan, U. (1983) Time-resolved fluoroimmunoassay of human choriogonadotropin. Clin. Chem. 29, 60. Rothen, A. (1973) Immunologic and enzymatic reactions carried out at a solid-liquid interface. Physiol. Chem. Phys. 5, 243.
Schroeder, H.R., Hines, C.M., Osborn, D.D., Moore, R.P., Hurtle, R.L., Wogoman, F.F., Roger, R.W. and Vogelhut, P.P. (1981) Immunochemiluminometric assay for hepatitis B surface antigen. Clin. Chem. 27, 1378. Simpson, J.S.A., Campbell, A.K., Ryall, M.E.T. and Woodhead, J.S. (1979) A stable chemiluminescent-labelled antibody for immunological assays. Nature 279, 646.