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A novel immunoelectrode assay for hepatitis B surface antigen
Sensors and Actuators B, 12 (1993) 49-52
49
A novel immunoelectrode assay for hepatitis B surface antigen Yuqi Li, Kaiquan Chen, Suxuan Wei and Jun He Department of Chemistry, School of Pharmacy, W&t China Universiry of Medical Sciences, Chengdu NW41 (China)
Jiawan Mu Department ofMicrobiology and Immunology,
School of Basic Medical Sciences, West China University of Medical Sciences,
Chengdu 610041 (China)
Abstract A new anti-hepatitis B surface antigen (anti-HBs) membrane disc of silk has been prepared by chemical modification with cyanogen bromide. The disc,is immersed in hepatitis B surface antigen (HBsAg)( + ) serum solution or assayed serum sample at 37 “C for 1 h to make HBsAg couple with anti-HBs, then is washed with working buffer (PBS). This disc is used to make up an immunoelectrode with Ag-AgCl as the internal electrode. The immunoelectrode and a saturated calomel electrode are dipped together into the working b&r. The determination results of 105 samples (61 positive and 44 negative) obtained by this method are more than 85% in agreement with the data obtained by ELISA. The results are linear over the range 20.0-320 ng/ml.
IIltIOdUCtiOll
Though better
results were obtained, such as a difference of response (> 10 mV) for HBsAg( + ) and HBsAg( - ) serum, there were still sign&ant
Hepatitis B is increasingly threatening public health. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (EL&A) have been extensively described for accurate determination of traces of hepatitis B surface antigen (HBsAg) in biological fluids. Boitieux and Thomas suggested oxygen electrode-based enzyme immunoassay for the amperometric determination of HBsAg [ 1,2], but the method is still unsatisfactory. Our aim was to establish a new method which offers the advantages of greater simplicity, safety, speed; specificity and sensitivity. A few years ago (1986), the authors made a preliminary study on an antibody electrode and set up a direct potentiometric method to detect HBsAg by dipping the antibody electrode and an Ag-AgCl reference electrode directly into the patient’s serum samples (Fig. 1).
some problems, for instance, the short lifetime of the electrode, the need for more serum samples and lack of safety for the analyst. In this paper, the authors propose an improved method, in which a silk disc is coupled with anti-HBs and complexed with HBsAg found in the patient’s serum, then used to make up an immunoelectrode. It is immersed together with a saturated calomel electrode (SCE) into the working buffer to detect the change of the membrane potential.
Experimental Apparatus
and reagents
All
Fig. 1. Apparatus for 1986electrode.
potentiometric measurements were made with an Orion model 940 Expandable Ionanalyzer (USA) in conjunction with a YEW-3964 potentiometric pen recorder. Membrane preparation and measurements were performed in a thermostatted cell controlled at 37 and 25 “C, respectively; an Ag-AgCl electrode (internal electrode) and SCE were used. Reagents: (i) cyanogen bromide (Sigma Co.) was diluted to 3.2% (w/w) with 0.1 mol/l Na2C03 solution; (ii) anti-HBs (4.0 mg/ml), HBsAg( + ) sera (containing , HBsAg 1.28 ug/ml), HBsAg( - ) sera and diphtherotoxoid (Dip 800 ugplml), (Institute of Biologicals,
@ 1993- ElsevierSquoia. All rights reserved
M
Chengdu); (iii) Human chorionic gonadotrophin (HCG, 200 pg/ml) and human serum samples (105 cases) (Department of Microbiology and Immunology of our university); (iv) progesterone (Prog, 2 ng/ml, Institute of Biologic&, Shanghai); (v) 1% (w/w) bovine serum albumin solution (BSA, Sigma Co.); (vi) working buffer (0.1 mol/l phosphate buffer solution pH = 7.40, PBS), all antigens were diluted with PBS; (vii) 0.05 mol/l NaHCO, and 0.05 mol/l carbonate buffer solution (pH = 9.60, CBS). Chemicals of analytical reagent grade, silk (native of Chengdu) and doubly distilled water were used throughout.
b > E I
d
1
2
3
4
5
Fig. 3. Potential vs. time curve: a, HBsAg( ) serum; b, HBsAg(-) serum. t
Immobilization of anti-HBs on silk disc
Cyanogen bromide solution, 100 ~1, was added onto a disc for 10min. It was washed with 0.05 mol/l NaHCO, solution, then 50 ~1 anti-HBs of diluent (1:6) with CBS was added onto the activated disc and left overnight at 4 “C to form an antibody disc. After 16 h, the disc was rinsed three times with PBS. Finally the disc was immersed in 1% (w/w) BSA solution at 37 “C for 30min, then washed with PBS and stored at 4°C for use. Preparation of antigen -antibody complex membranes (sample membrane) 50 fl of diluted ( 1:lO) HBsAg( + ) serum or patient’s
serum was added onto the modified antibody disc, wh”lchwas then allowed to stand at 37 “C for 1 h, to make HBsAg couple with anti-HBs. Afterwards the disc was washed three times with PBS. The disc is shown in Fig. 2. Immunoelectrode assembly The immunoelectrode was constructed by fixing the
complex membrane on the bottom of the indicator electrode tube, which has an Ag-AgCl internal electrode and 1 x 10d3 mol/l NaCl as internal solution of the indicator electrode. Determination of membrane potentials
The immunoelectrode and SCE were dipped simultaneously into PBS. The signals of the membrane poten-
5
10
1000
1~npl~WW
Fig. 4. AmVOvs. log CHw.
tials were recorded from O-5 min by a direct potential method at 25 “C. The disc was discarded after each potential determination. Preparation of the calibration graph Diluted HBsAg( + ) serum with PBS was used to
make a series of different concentrations (5,10,20, 40,80,160,320,640 ng/ml) and was used as described above to make the complex membranes. Using PBS diluted (1: 10) HBsAg( - ) serum instead of HBsAg( + ) serum with the same method, blank membranes could be prepared and their potentials measured, respectively. The difference value AmY0 of the initial membrane potential of both the sample and blank membranes (Fig. 3) is used to plot AmVb versus log ‘&A~ (Fig. 4).
Stability and sensitivity The stability and sensitivity of this antibody mem-
Fig. 2. Apparatus for the HBsAg determination.
brane prepared by chemical modification appear to be higher than those prepared by a physical adsorption method (ELISA) (Fig. 5).
51 3
4 30
I
160
I :
158 156 154
..
152
60
90
120
.
150
150
.
;
* f Dip &P:og
time (day)
Fig. 5. Comparison of storage time and activity of membrane: o, chemical modification; p, physical adsorption.
.
l
1 HISAg
tVP9
Fig. 6. Speoifioityof the immtmoeleotrode. Line shows average values of HBsAg( - ) serum.
Because the silk membrane has hydroxyl groups, chemical modification can be accomplished by the following reactions:
yyaBr_
$-O)=NH
)_OH
td
NH:-B
(antibody)
p-:-NH-B >
+
The modged discs can be stored at 4 “C for 135 days, retaining 90% of their initial activity (Fig. 5). Relationship between membrane potential and response time In Fig. 3 a is the response curVe of the complex
membrane using HBsAg( + ) serum and b is the response curve of the blank membrane using HBsAg( ) serum. The difference between the initial membrane potentials of curves a and b was greater than that after 5 min, i.e., AmV, > AmVr . Therefore AmVOcould always be used to give higher sensitivity and to allow a faster determination.
-
Relationship between AmV, and the logarithm of the concentration of HBsAg (Fig. 4)
This relationship is linear over the concentration range 20.0-320 ng/ml. The limit of detection is 18.0 ng/ ml. The logarithmic regression formula is AmVO= 6.38 + 5.13 log C. Reproducibility of the method
Using the method a positive serum sample (1:lO dilution) was determined five times at two-day intervals. The relative standard deviation (RSD) was 10.78% (Table 1).
OH
Specificity of the immunoelectroak
Using Prog. Dip. HCG and HBsAg( - ) serum instead of HBsAg( + ) serum, we have made the complex membrane and ,determined the membrane potentials by the same method. A significant difference in electrode response was observed between HBsAg and each of the antigens or haptene (p < 0.05) (Fig. 6). It may be thought that there is little or no affinity between those antigens and anti-HBs, i.e., they do not form a complex membrane and cannot cause charge transfer and orientational change of dipoles to exist on the antibody membrane. This method has been used in the clinical assay. The results of 105 serum samples by this method are in good agreement with those obtained by ELISA. The positive criterion is AmV, > 3 (Table 2).
cooclll6lons
This method avoids many problems: the antibody need not be labelled with enzyme or radioisotope; the serum sample quantity is much reduced, there is no need for other reagents; the electrode need not be
TABLE 1. Reproducibilityof the method (with the same sample) Date (January 1990) AmV,,(%)
7th day
9th day
10th day
11th day
13thday
RSD
9.4
11.5
9.0
11.3
10.5
10.78
52 TABLE 2. Comparison of immunoelectrode assay (part of serum samples) Sample No.
1
2
3
4
5
6
51
52
53
54
55
56
57
Immunoelectrode a=ay (AmV,) ELISA
6.8 _
0.8 -
0.6 -
0.9 -
1.8 -
1.8
7.1
6.5
9.1
10.7
6.8
13.7
1.5
t
t
t
t
t
+
-
-
dipped directly into the serum of the patient and there is no need for washing, recovery, etc. The entire assay procedure is much simpler than the routine method (ELISA). The antibody discs can be stored for a longer time at 4 “C, so they could be made commercially. The antibody disc is only used once then thrown away, so the lifetime of the electrode is much longer than that of other immunoelectrodes. The methods are not suitable in the determination of antigens and haptenes because they are not conjugated proteins.
Biographies Yuqi Li is an associate professor of analytical chemistry at the West China University of Medical Sciences (WCUMS). Her current research interests include the development and implementation of chemical and biological sensors for use in clinical chemistry, and have resulted in 31 publications.
Kaiquan Chen, is an associate professor of analytical chemistry at WCUMS and has been interested in the clinical applications and development of biochemical sensors since 1985. She has published five papers.
Acknowledgement This project was supported by the National Science
Foundation of China.
References 1 J.-L. Boitieux, G. Desmet and D. Thomas, An antibody electrode, preliinary report on a new approach in enzyme immunoassay, Clin. Chem., 25 (1979) 318. 2 J.-L. Boitieux, G. Desmet and D. Thomas, Oxygen electrodebased enzyme immunoassay for the amperometric determination of hepatitis B surface antigen, Anal. Chim. Acta., 163 (1984) 309. 3 F. Lu, R.-C. Chen and Z. Wan, Study and application of immun&ctrode for HBsAg, Chem. Sensors 9( 1) ( 1989)55 (in Chinese).
Suxuun Wei, a professor in the Organic Chemistry Department, WCUMS, has focused her research interests on enzymatic chemistry and its use in diagnostics. Jan He, a lecturer of organic chemistry at WCUMS, has focused her research interests on biosensor and enzyme assays in recent years. Jiawm Mu, an associate professor of microbiology and immunology at WCUMS, has been interested in the immunocyte of HBV since 1973, especially in the immune regulation of idiotype and anti-idiotypic antibodies against anti-H& in HBV infection. She has published 25 research papers.
49
A novel immunoelectrode assay for hepatitis B surface antigen Yuqi Li, Kaiquan Chen, Suxuan Wei and Jun He Department of Chemistry, School of Pharmacy, W&t China Universiry of Medical Sciences, Chengdu NW41 (China)
Jiawan Mu Department ofMicrobiology and Immunology,
School of Basic Medical Sciences, West China University of Medical Sciences,
Chengdu 610041 (China)
Abstract A new anti-hepatitis B surface antigen (anti-HBs) membrane disc of silk has been prepared by chemical modification with cyanogen bromide. The disc,is immersed in hepatitis B surface antigen (HBsAg)( + ) serum solution or assayed serum sample at 37 “C for 1 h to make HBsAg couple with anti-HBs, then is washed with working buffer (PBS). This disc is used to make up an immunoelectrode with Ag-AgCl as the internal electrode. The immunoelectrode and a saturated calomel electrode are dipped together into the working b&r. The determination results of 105 samples (61 positive and 44 negative) obtained by this method are more than 85% in agreement with the data obtained by ELISA. The results are linear over the range 20.0-320 ng/ml.
IIltIOdUCtiOll
Though better
results were obtained, such as a difference of response (> 10 mV) for HBsAg( + ) and HBsAg( - ) serum, there were still sign&ant
Hepatitis B is increasingly threatening public health. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (EL&A) have been extensively described for accurate determination of traces of hepatitis B surface antigen (HBsAg) in biological fluids. Boitieux and Thomas suggested oxygen electrode-based enzyme immunoassay for the amperometric determination of HBsAg [ 1,2], but the method is still unsatisfactory. Our aim was to establish a new method which offers the advantages of greater simplicity, safety, speed; specificity and sensitivity. A few years ago (1986), the authors made a preliminary study on an antibody electrode and set up a direct potentiometric method to detect HBsAg by dipping the antibody electrode and an Ag-AgCl reference electrode directly into the patient’s serum samples (Fig. 1).
some problems, for instance, the short lifetime of the electrode, the need for more serum samples and lack of safety for the analyst. In this paper, the authors propose an improved method, in which a silk disc is coupled with anti-HBs and complexed with HBsAg found in the patient’s serum, then used to make up an immunoelectrode. It is immersed together with a saturated calomel electrode (SCE) into the working buffer to detect the change of the membrane potential.
Experimental Apparatus
and reagents
All
Fig. 1. Apparatus for 1986electrode.
potentiometric measurements were made with an Orion model 940 Expandable Ionanalyzer (USA) in conjunction with a YEW-3964 potentiometric pen recorder. Membrane preparation and measurements were performed in a thermostatted cell controlled at 37 and 25 “C, respectively; an Ag-AgCl electrode (internal electrode) and SCE were used. Reagents: (i) cyanogen bromide (Sigma Co.) was diluted to 3.2% (w/w) with 0.1 mol/l Na2C03 solution; (ii) anti-HBs (4.0 mg/ml), HBsAg( + ) sera (containing , HBsAg 1.28 ug/ml), HBsAg( - ) sera and diphtherotoxoid (Dip 800 ugplml), (Institute of Biologicals,
@ 1993- ElsevierSquoia. All rights reserved
M
Chengdu); (iii) Human chorionic gonadotrophin (HCG, 200 pg/ml) and human serum samples (105 cases) (Department of Microbiology and Immunology of our university); (iv) progesterone (Prog, 2 ng/ml, Institute of Biologic&, Shanghai); (v) 1% (w/w) bovine serum albumin solution (BSA, Sigma Co.); (vi) working buffer (0.1 mol/l phosphate buffer solution pH = 7.40, PBS), all antigens were diluted with PBS; (vii) 0.05 mol/l NaHCO, and 0.05 mol/l carbonate buffer solution (pH = 9.60, CBS). Chemicals of analytical reagent grade, silk (native of Chengdu) and doubly distilled water were used throughout.
b > E I
d
1
2
3
4
5
Fig. 3. Potential vs. time curve: a, HBsAg( ) serum; b, HBsAg(-) serum. t
Immobilization of anti-HBs on silk disc
Cyanogen bromide solution, 100 ~1, was added onto a disc for 10min. It was washed with 0.05 mol/l NaHCO, solution, then 50 ~1 anti-HBs of diluent (1:6) with CBS was added onto the activated disc and left overnight at 4 “C to form an antibody disc. After 16 h, the disc was rinsed three times with PBS. Finally the disc was immersed in 1% (w/w) BSA solution at 37 “C for 30min, then washed with PBS and stored at 4°C for use. Preparation of antigen -antibody complex membranes (sample membrane) 50 fl of diluted ( 1:lO) HBsAg( + ) serum or patient’s
serum was added onto the modified antibody disc, wh”lchwas then allowed to stand at 37 “C for 1 h, to make HBsAg couple with anti-HBs. Afterwards the disc was washed three times with PBS. The disc is shown in Fig. 2. Immunoelectrode assembly The immunoelectrode was constructed by fixing the
complex membrane on the bottom of the indicator electrode tube, which has an Ag-AgCl internal electrode and 1 x 10d3 mol/l NaCl as internal solution of the indicator electrode. Determination of membrane potentials
The immunoelectrode and SCE were dipped simultaneously into PBS. The signals of the membrane poten-
5
10
1000
1~npl~WW
Fig. 4. AmVOvs. log CHw.
tials were recorded from O-5 min by a direct potential method at 25 “C. The disc was discarded after each potential determination. Preparation of the calibration graph Diluted HBsAg( + ) serum with PBS was used to
make a series of different concentrations (5,10,20, 40,80,160,320,640 ng/ml) and was used as described above to make the complex membranes. Using PBS diluted (1: 10) HBsAg( - ) serum instead of HBsAg( + ) serum with the same method, blank membranes could be prepared and their potentials measured, respectively. The difference value AmY0 of the initial membrane potential of both the sample and blank membranes (Fig. 3) is used to plot AmVb versus log ‘&A~ (Fig. 4).
Stability and sensitivity The stability and sensitivity of this antibody mem-
Fig. 2. Apparatus for the HBsAg determination.
brane prepared by chemical modification appear to be higher than those prepared by a physical adsorption method (ELISA) (Fig. 5).
51 3
4 30
I
160
I :
158 156 154
..
152
60
90
120
.
150
150
.
;
* f Dip &P:og
time (day)
Fig. 5. Comparison of storage time and activity of membrane: o, chemical modification; p, physical adsorption.
.
l
1 HISAg
tVP9
Fig. 6. Speoifioityof the immtmoeleotrode. Line shows average values of HBsAg( - ) serum.
Because the silk membrane has hydroxyl groups, chemical modification can be accomplished by the following reactions:
yyaBr_
$-O)=NH
)_OH
td
NH:-B
(antibody)
p-:-NH-B >
+
The modged discs can be stored at 4 “C for 135 days, retaining 90% of their initial activity (Fig. 5). Relationship between membrane potential and response time In Fig. 3 a is the response curVe of the complex
membrane using HBsAg( + ) serum and b is the response curve of the blank membrane using HBsAg( ) serum. The difference between the initial membrane potentials of curves a and b was greater than that after 5 min, i.e., AmV, > AmVr . Therefore AmVOcould always be used to give higher sensitivity and to allow a faster determination.
-
Relationship between AmV, and the logarithm of the concentration of HBsAg (Fig. 4)
This relationship is linear over the concentration range 20.0-320 ng/ml. The limit of detection is 18.0 ng/ ml. The logarithmic regression formula is AmVO= 6.38 + 5.13 log C. Reproducibility of the method
Using the method a positive serum sample (1:lO dilution) was determined five times at two-day intervals. The relative standard deviation (RSD) was 10.78% (Table 1).
OH
Specificity of the immunoelectroak
Using Prog. Dip. HCG and HBsAg( - ) serum instead of HBsAg( + ) serum, we have made the complex membrane and ,determined the membrane potentials by the same method. A significant difference in electrode response was observed between HBsAg and each of the antigens or haptene (p < 0.05) (Fig. 6). It may be thought that there is little or no affinity between those antigens and anti-HBs, i.e., they do not form a complex membrane and cannot cause charge transfer and orientational change of dipoles to exist on the antibody membrane. This method has been used in the clinical assay. The results of 105 serum samples by this method are in good agreement with those obtained by ELISA. The positive criterion is AmV, > 3 (Table 2).
cooclll6lons
This method avoids many problems: the antibody need not be labelled with enzyme or radioisotope; the serum sample quantity is much reduced, there is no need for other reagents; the electrode need not be
TABLE 1. Reproducibilityof the method (with the same sample) Date (January 1990) AmV,,(%)
7th day
9th day
10th day
11th day
13thday
RSD
9.4
11.5
9.0
11.3
10.5
10.78
52 TABLE 2. Comparison of immunoelectrode assay (part of serum samples) Sample No.
1
2
3
4
5
6
51
52
53
54
55
56
57
Immunoelectrode a=ay (AmV,) ELISA
6.8 _
0.8 -
0.6 -
0.9 -
1.8 -
1.8
7.1
6.5
9.1
10.7
6.8
13.7
1.5
t
t
t
t
t
+
-
-
dipped directly into the serum of the patient and there is no need for washing, recovery, etc. The entire assay procedure is much simpler than the routine method (ELISA). The antibody discs can be stored for a longer time at 4 “C, so they could be made commercially. The antibody disc is only used once then thrown away, so the lifetime of the electrode is much longer than that of other immunoelectrodes. The methods are not suitable in the determination of antigens and haptenes because they are not conjugated proteins.
Biographies Yuqi Li is an associate professor of analytical chemistry at the West China University of Medical Sciences (WCUMS). Her current research interests include the development and implementation of chemical and biological sensors for use in clinical chemistry, and have resulted in 31 publications.
Kaiquan Chen, is an associate professor of analytical chemistry at WCUMS and has been interested in the clinical applications and development of biochemical sensors since 1985. She has published five papers.
Acknowledgement This project was supported by the National Science
Foundation of China.
References 1 J.-L. Boitieux, G. Desmet and D. Thomas, An antibody electrode, preliinary report on a new approach in enzyme immunoassay, Clin. Chem., 25 (1979) 318. 2 J.-L. Boitieux, G. Desmet and D. Thomas, Oxygen electrodebased enzyme immunoassay for the amperometric determination of hepatitis B surface antigen, Anal. Chim. Acta., 163 (1984) 309. 3 F. Lu, R.-C. Chen and Z. Wan, Study and application of immun&ctrode for HBsAg, Chem. Sensors 9( 1) ( 1989)55 (in Chinese).
Suxuun Wei, a professor in the Organic Chemistry Department, WCUMS, has focused her research interests on enzymatic chemistry and its use in diagnostics. Jan He, a lecturer of organic chemistry at WCUMS, has focused her research interests on biosensor and enzyme assays in recent years. Jiawm Mu, an associate professor of microbiology and immunology at WCUMS, has been interested in the immunocyte of HBV since 1973, especially in the immune regulation of idiotype and anti-idiotypic antibodies against anti-H& in HBV infection. She has published 25 research papers.