JOURNAL
OF
BIOLUMINESCENCE A N D CHEMILUMINESCENCE VOL 5 11-12
(1990)
A Sensitive and Rapid Luminescence Sandwich Assay for Antibodies to Hepatitis-B Surface Antigen (Anti-HBsAg) Ulrich Missler and William Graham Wood Departments of Neurosurgery an d Internal Medicine, Medical University of Lubeck, D-2400 Lubeck 1, FRG
A cherniluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5 2 6 8 . 1 1 % and has a lower detection limit for hepatitis-B surface antigen of 1.3 UA.
Keywords: Hepatitis B surface antigen antibodies; irnrnunoassay
INTRODUCTION
The aim of the study was to develop a rapid and reliable immunoluminometric assay for AntiHBsAg, which was capable of covering the range found in subjects immunized with HBsAg in two dilution steps. The type of assay chosen was the antigen-antibody-labelled antigen sandwich used in all commercial assays for Anti-HBsAg. An assay was developed which covered the range 1-1OO,OOOU/l with native serum and a 1:lOO dilution-i.e. suitable for monitoring over 99% of all subjects undergoing HBsAg immunization.
MATERIALS AND METHODS Solid-phase-coating
HBs-antigen (80pg; subtype ad and ay 5:l) in 125 ml 0.05 molil phosphate buffer were incubated for 12-18 hours at 37°C with 1000 polystyrene beads (6.4 mm diameter, Spherotech Kugeln, Fulda, FRG). After incubation, the beads were washed three times with phosphate 08863996/90/01001 1-02$05.00
0 1990 by John Wiley & Sons, L t d
buffer. After the third wash step 3 g/l ovalbumin (Gr. 111, Sigma, Munich, FRG) in 0.05mol/l phosphate buffer with 0.015 molil NaN3 (Fa. Merck, Darmstadt, FRG) was used for storage and to stabilize the coated beads. The solid phase can be stored in this buffer for at least 9 months at 4 "C without demonstrable loss in immunological reactivity. Di-acyl-aryl-hydrazide labelling of HBs-antigen
(a) ABEN (7-(4-aminobutyl N-ethyl)-2,3-dihydro-1,4-naphthalazinedione) was succinylated and activated (=ABEN-H). (b) 20pg of a 50pmol/l solution of ABEN-H active ester were incubated with 400 pg HBsantigen (320 pg subtype ad and 80 pg subtype ay) in a solution buffered to pH 9.1 with K2HP04for 90 minutes at ambient temperature in the dark. After incubation, the solution was centrifuged for 90 s at 15,000 rpm to precipitate dicyclohexylurea. In the third step, free ABEN-H was separated by gel-chromatography on a Sephadex G 10 Received 15 October 1988 Revised 28 November 1988
U. MISSLER AND W. G. WOOD
12
column (30 x 1cm) with 0.025 mol/l Tris-HC1 pH 7.5 as elution buffer. The labelled HBs-Ag was diluted 1:30 in 60g/l human serum albumin (Sigma), 125 pg portions being transferred to siliconized vials and lyophilized. Just before use, the lyophilized tracer was reconstituted with 12.5ml of 60 g/l human serum albumin in PBS. The lyophilized tracer is stable for at least 6 months.
Table 1. Stability of standard curve Standard (Un) 0 1.3 4.1 12.3 37 111 333 1000
Assay
Standard or sample (200~1)and an HBs antigen coated bead (6.4 mm diameter) was incubated for 90min at room temperature on a rotator at 170min-'. The bead was washed with 2 x 6ml H 2 0 and then 2 0 0 ~ 1ABEN labelled HBsAg in 60g/l human serum albumin added and the mixture incubated for 60min. After washing, the bead was transferred to a measuring cuvette and covered with 3 0 0 ~ 1catalase. The cuvette was placed in a luminometer (Berthold, LB 952) and the light reaction initiated by injecting an alkaline-peroxide solution (0.5% v/v hydrogen peroxide in 0.3 mol/l sodium hydroxide). The light signal was integrated over 12s. RESULTS AND DISCUSSION
Table 1 shows the stability of the light signal in RLU of the seven standards. The data were collected by measuring the standards under identical conditions in 20 consecutive assays. This table shows that non-commercial luminescence immunoassays can be stabilized to allow a master-curve-function (for example as established in the Berthold Auto-Clinilumat LB 952) to be used. Results obtained on patient samples ( n = 33) compared favourable with those obtained using a commercial AntiHBs EIA (Sorin ABAUQR) (Spearman rank correlation, r = 0.972; regression equations, y = a + bx and x = a + by, uyx = 1.96, by, = 1.13, axy = 0.77, b, = 0.80). The values found with the two methods were not significantly different from each other (Wilcoxon signed rank test, p = 0.07). The lower limit of detection was 1.3U/1 Anti-HBsAg and the measuring range (depending upon standards used) was 1.3-10OOU/l AntiHBsAg. No false positive reactions were found with samples containing HBs-Ag, Anti-HBc, Anti-HBc-IgM, HBe-Ag, Anti-HBe, or HAV-
Mean impulse rate (RLU)
Standard deviation (RLU)
CV (%)
23 35 58 127 31 1 869 2176 4190
0.89 2.25 2.64 7.84 13.7 19.6 93.0 174
3.86 6.40 4.57 6.20 4.41 2.26 4.27 4.14
Table 2. Inter- and intra-assay precision Intra-assay precision U/I Number of data-pairs
Mean CV ( % ) Median CV
1.3-1 0 11-100 101-500 50 1-1 000
74 112 82 46
2.73 2.82 2.95 2.95
1.96 2.34 2.45 2.38
1.3-1000
314
2.85
2.22
Inter-assay precision (measurements in 20 consecutive assays) CV Sample Target Measured Standard value deviation value (U/I) (U/I) (%) (U/I) I II 111
4 40 750
4 38.9 762
0.32 2.89 40.0
8.11 6.92 5.26
marker. The dynamic range of the assay was S1000:S0150:l(signal). A compound precision profile is shown in Table 2. CONCLUSIONS
The assay proved to be stable and capable of measuring samples rapidly and accurately. The dynamic range was large, and the assay was suitable for routine monitoring of hospital personnel undergoing protective immunization with HBsAg.
OF
BIOLUMINESCENCE A N D CHEMILUMINESCENCE VOL 5 11-12
(1990)
A Sensitive and Rapid Luminescence Sandwich Assay for Antibodies to Hepatitis-B Surface Antigen (Anti-HBsAg) Ulrich Missler and William Graham Wood Departments of Neurosurgery an d Internal Medicine, Medical University of Lubeck, D-2400 Lubeck 1, FRG
A cherniluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5 2 6 8 . 1 1 % and has a lower detection limit for hepatitis-B surface antigen of 1.3 UA.
Keywords: Hepatitis B surface antigen antibodies; irnrnunoassay
INTRODUCTION
The aim of the study was to develop a rapid and reliable immunoluminometric assay for AntiHBsAg, which was capable of covering the range found in subjects immunized with HBsAg in two dilution steps. The type of assay chosen was the antigen-antibody-labelled antigen sandwich used in all commercial assays for Anti-HBsAg. An assay was developed which covered the range 1-1OO,OOOU/l with native serum and a 1:lOO dilution-i.e. suitable for monitoring over 99% of all subjects undergoing HBsAg immunization.
MATERIALS AND METHODS Solid-phase-coating
HBs-antigen (80pg; subtype ad and ay 5:l) in 125 ml 0.05 molil phosphate buffer were incubated for 12-18 hours at 37°C with 1000 polystyrene beads (6.4 mm diameter, Spherotech Kugeln, Fulda, FRG). After incubation, the beads were washed three times with phosphate 08863996/90/01001 1-02$05.00
0 1990 by John Wiley & Sons, L t d
buffer. After the third wash step 3 g/l ovalbumin (Gr. 111, Sigma, Munich, FRG) in 0.05mol/l phosphate buffer with 0.015 molil NaN3 (Fa. Merck, Darmstadt, FRG) was used for storage and to stabilize the coated beads. The solid phase can be stored in this buffer for at least 9 months at 4 "C without demonstrable loss in immunological reactivity. Di-acyl-aryl-hydrazide labelling of HBs-antigen
(a) ABEN (7-(4-aminobutyl N-ethyl)-2,3-dihydro-1,4-naphthalazinedione) was succinylated and activated (=ABEN-H). (b) 20pg of a 50pmol/l solution of ABEN-H active ester were incubated with 400 pg HBsantigen (320 pg subtype ad and 80 pg subtype ay) in a solution buffered to pH 9.1 with K2HP04for 90 minutes at ambient temperature in the dark. After incubation, the solution was centrifuged for 90 s at 15,000 rpm to precipitate dicyclohexylurea. In the third step, free ABEN-H was separated by gel-chromatography on a Sephadex G 10 Received 15 October 1988 Revised 28 November 1988
U. MISSLER AND W. G. WOOD
12
column (30 x 1cm) with 0.025 mol/l Tris-HC1 pH 7.5 as elution buffer. The labelled HBs-Ag was diluted 1:30 in 60g/l human serum albumin (Sigma), 125 pg portions being transferred to siliconized vials and lyophilized. Just before use, the lyophilized tracer was reconstituted with 12.5ml of 60 g/l human serum albumin in PBS. The lyophilized tracer is stable for at least 6 months.
Table 1. Stability of standard curve Standard (Un) 0 1.3 4.1 12.3 37 111 333 1000
Assay
Standard or sample (200~1)and an HBs antigen coated bead (6.4 mm diameter) was incubated for 90min at room temperature on a rotator at 170min-'. The bead was washed with 2 x 6ml H 2 0 and then 2 0 0 ~ 1ABEN labelled HBsAg in 60g/l human serum albumin added and the mixture incubated for 60min. After washing, the bead was transferred to a measuring cuvette and covered with 3 0 0 ~ 1catalase. The cuvette was placed in a luminometer (Berthold, LB 952) and the light reaction initiated by injecting an alkaline-peroxide solution (0.5% v/v hydrogen peroxide in 0.3 mol/l sodium hydroxide). The light signal was integrated over 12s. RESULTS AND DISCUSSION
Table 1 shows the stability of the light signal in RLU of the seven standards. The data were collected by measuring the standards under identical conditions in 20 consecutive assays. This table shows that non-commercial luminescence immunoassays can be stabilized to allow a master-curve-function (for example as established in the Berthold Auto-Clinilumat LB 952) to be used. Results obtained on patient samples ( n = 33) compared favourable with those obtained using a commercial AntiHBs EIA (Sorin ABAUQR) (Spearman rank correlation, r = 0.972; regression equations, y = a + bx and x = a + by, uyx = 1.96, by, = 1.13, axy = 0.77, b, = 0.80). The values found with the two methods were not significantly different from each other (Wilcoxon signed rank test, p = 0.07). The lower limit of detection was 1.3U/1 Anti-HBsAg and the measuring range (depending upon standards used) was 1.3-10OOU/l AntiHBsAg. No false positive reactions were found with samples containing HBs-Ag, Anti-HBc, Anti-HBc-IgM, HBe-Ag, Anti-HBe, or HAV-
Mean impulse rate (RLU)
Standard deviation (RLU)
CV (%)
23 35 58 127 31 1 869 2176 4190
0.89 2.25 2.64 7.84 13.7 19.6 93.0 174
3.86 6.40 4.57 6.20 4.41 2.26 4.27 4.14
Table 2. Inter- and intra-assay precision Intra-assay precision U/I Number of data-pairs
Mean CV ( % ) Median CV
1.3-1 0 11-100 101-500 50 1-1 000
74 112 82 46
2.73 2.82 2.95 2.95
1.96 2.34 2.45 2.38
1.3-1000
314
2.85
2.22
Inter-assay precision (measurements in 20 consecutive assays) CV Sample Target Measured Standard value deviation value (U/I) (U/I) (%) (U/I) I II 111
4 40 750
4 38.9 762
0.32 2.89 40.0
8.11 6.92 5.26
marker. The dynamic range of the assay was S1000:S0150:l(signal). A compound precision profile is shown in Table 2. CONCLUSIONS
The assay proved to be stable and capable of measuring samples rapidly and accurately. The dynamic range was large, and the assay was suitable for routine monitoring of hospital personnel undergoing protective immunization with HBsAg.
