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Oxygen electrode-based enzyme immunoassay for the amperometric determination of hepatitis B surface antigen
Analytica Chimica Acta, 163 (1984) 309-313 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
Short Communication
OXYGEN ELECTRODE-BASED ENZYME IMMUNOASSAY FOR THE AMPEROMETRIC DETERMINATION OF HEPATITIS B SURFACE ANTIGEN
JEAN-LOUIS
BOITIEUX
and DANIEL THOMAS*
La bomtoire de Technologie Enzymatique, Universitt! de Technologie B.P. 233, 60206 Compiegne Cedex (France)
de Compiegne
GERARD DESMET Labomtoire (Fmnce)
d’Hormonologie,
Centre Hospitalier
Sud, B.P. 3009, 80030 Amiens
Cedex
(Received 29th March 1984)
Summary. Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1-100 pg 1-l. A microcomputer is used for data acquisition and processing.
Numerous methods are available for the accurate determination of traces of antigens and haptens in biological fluids. Radioimmunoassay and enzyme immunoassay (e.i.a.) are the most sensitive. Since the introduction of immunosorbents, many solid-phase techniques aimed at obtaining optimum results with enzyme-linked immunosorbent assays (e.1.i.s.a.) have been extensively described [ 1, 21. Recently, however, protein membranes have been used for the immobilization of enzymes [3]. The binding of immunoglobulins onto gelatin membranes has been described [4] and an enzyme immunoassay technique has been introduced for the determination of hepatitis B surface antigen (HB,A,) in biological fluids [ 51. In this communication, the use of glucose oxidase as the enzyme label is reported; the labelled antibody is bound to a protein membrane. The advantages of the glucose oxidase label have recently been demonstrated [6]. The purpose of this work is to study the possibilities of automation of this type of antigen estimation by using a semi-continuous flow mode, and processing the signals with the aid of a microcomputer. The active membrane is fixed over a modified oxygen electrode, designed in this laboratory. The oxygen consumption of the enzyme catalyzed reaction is measured when the electrode is in contact with a standard glucose solution. The signal from the electrode is directly proportional to the oxygen consumption and to the antigen concentration. 0003-2670/84/$03.00
0 1984 Elsevier Science Publishers B.V.
310
Experimental Chemicals. Glucose oxidase (E.C.1.1.3.4.; Type II, 18400 U g-l; Sigma) and pig skin gelatin (Rousselot Laboratory, 60400 Rib&ourt, France) were used. Glutaraldehyde and other chemicals used were of analytical grade (Merck). For the determination of HB,AB, a panel of the reference center for virus hepatitis at the Institute of Hygiene, Giittingen University, was used by courtesy of Dr. Gerlich. Hepatitis B surface antigen (HB,AJ was isolated and purified from human positive sera by a simple method described elsewhere [ 71. Anti-HB,A, anti. bodies were prepared and purified by preparative electrophoresis followed by chromatography on DEAE cellulose [ 51. Equipment. Amperometric measurements were done with an oxygen analyzer. The microcomputer was an Apple II+ computer with 48K of main memory. The interface card cage was constructed in the Electronics Department, University of Technology of Compiegne. The measurement cell (Fig. 1) was designed in this laboratory. The internal peripheral bus of the computer enabled a variety of peripheral units to be added. A disc controller card and parallel interface cards (Apple) were added for the printer and interface card cage. The interface card cage was mounted on the computer through a ribbon cable and contained circuit boards for signal conditioning and buffering, and analog-to-digital card based upon the Date1 ADC (EK-12B) an oxygen analyzer and a 4-bit logic system. The flow system used for the measurements is shown in Fig. 2. Preparation of the glucose oxidase--antibody conjugate. Glucose oxidase (ex. Aspergillus niger) was conjugated to specific antibodies by use of glutaraldehyde [ 81. Further fractionation on Sephadex G200 equilibrated with 0.01 mol 1-l phosphate buffer, pH 6.8, was used to eliminate the free enzyme. The fractions of the first peak were pooled and distributed in sterile 0.2-ml aliquots. This solution could be stored at 4°C for several months without significant loss in activity. Immobilization of antibodies on pig skin gelatin. The general procedure used to prepare an active membrane and to fix the specific antibodies has
mless steel Ibody
body
membrane
i xygen
electrode
electrolyte RinSlng
Fig. 1. Cross-section of the immunosensor electrode and measuring cell. Fig. 2. Flow system used for the antigen determination.
liquid
Std glucose
311
been described previously [ 41. Antibodies were immobilized on discs (10 mm diameter, ~0.2 mm thick) cut from the activated membranes. The discs were immersed in a 30 g 1-l bovine serum albumin solution for 1 h and thoroughly washed in distilled water. These antibody-coated discs could be stored at -4” C in 0.02 mol 1-l phosphate buffer, pH 6.8, or in the presence of 1 g 1-l of gentamicin solution at 4” C. Procedure for the determination of HB,A,. The gelatin membrane was immersed for 30 min in 0.5 ml of diluted (1:lOOO) HB,A, positive serum, immobilized antibodies G being in excess with respect to the antigen. The membrane was then thoroughly washed. The active membrane was incubated for 1 h in 1 ml of a solution of conjugate (1:200 in 0.01 mol 1-l phosphate buffer, pH 6.8) as in the “sandwich” method [ 91. The membrane was washed with 0.01 mol 1-l sodium phosphate buffer pH 6.8 and fixed on the hydrophobic selective gas membrane of an oxygen sensor by a magnetic device, as shown in Fig. 1. The measuring cell was filled with standard glucose solution. Consumption of oxygen was measured by the oxygen sensor. It was proportional to the enzyme activity retained on the membrane and therefore to the HB,A, concentration. Signal processing. In the direct measurement mode, the program starts by asking for a time interval for the collection or sampling of data. Next, it runs an accuracy test before plotting the data points. This test is done on the 20 data points (N = 20, Fig. 3). If S(ti) (with i = 0, . . ., N) are the measured values, the accuracy test is operated through S(ti) - S(to) = Pi for each sampled point. It checks that P, = Pi - 1. The program calculates the value D which is the concentration to be determined, i.e., D = Pi for i = N (when P, = Pi - 1); ti (the interval between two data points) is obviously an important parameter. A machine language subroutine was written for data acquisition from the ADC. The rest of the software was implemented in Basic in the usual way. 1
end of collect i
Fig. 3. Outline of data acquisition.
312
Results and discussion The effects of experimental conditions on the detection of glucose oxidase activity were first established. A preliminary report [lo] has shown that the coupling of glucose oxidase with antibodies does not affect significantly the Michaelis constant. The stability of glucose oxidase after coupling to the antibodies allowed a good reproducibility of the HB,A, measurement to be achieved. The stoichiometry of the enzyme reaction has been studied, using the computerized enzyme electrode system for glucose determination in blood and serum [ll, 121. The binding capacity and selectivity of the membrane were also considered. A pig-skin gelatin membrane was chosen for linking the antibodies. Previous work based on electron microscopy studies of artificial protein membranes [4] showed the homogeneity of the pig-skin gelatin membrane, which has a uniform thickness, a glossy surface and a regular network. The calibration graph of signal vs. log HB,A, concentration was linear over the range 0.1-100 c(g 1-l. The precision at various concentrations are shown in Table 1. The between-assay relative standard deviation was 7--12X The average recovery of known amounts of HB,A, added to a serum pool was 95%. An unknown sample of HB,A, positive serum was analyzed by the present procedure and by radio-immunoassay with good agreement. These results are encouraging because the limit of detection of a classical enzyme immunoassay is about 2.4 pg 1-l [ 131, and radio-immunoassay has a limit of 3-4/.Lg 1-l [14]. The enzyme immunosensor depends on the immunochemical reaction for its selectivity, because an antibody binds to its corresponding antigen. Accordingly, the antibodies used are highly selective for HB,Ap but are not completely specific. This computerized enzyme immunosensor allowed 30 sample measurements per hour including the washing step between samples, sampling and oxygen measurement. The volume of the sample for each measurement was ca. 50 ~1. The stability of the active membrane when stored at 4°C is very good (not more than 20% loss of initial activity after six months) so that the calibration graph can be reproduced easily with the original membrane. TABLE 1 Precision of the method (10 measurements with the same membrane) HBsA, taken (pg 1-l) Mean found (pg 1-l) S.d.a R.s.d.
1.00
10.0
100
1.01 0.04 4
9.6 0.2 2
94 3 3
aStandard deviation (pg 1-l) and relative standard deviation (%).
313
In conclusion, the computerized continuous flow system described for HB,A, measurements can be used for quantitative immunological reactions. This technology has also been tested for the estimation of the hormone 17-poestradiol; a competitive enzyme-linked immunoassay was used. The first tests with standard oestradiol solutions gave satisfactory results at levels of a few pmol l-l and above. REFERENCES 1 E. EngvaIl and P. Perlmann, Immunochem., 8 (1971) 871. 2 A. H. W. M. Schuurs and B. K. van Weemen, Clin. Chim. Acta, 81 (1977) 1. 3 D. Thomas, in D. Thomas and J. P. Kernevez (Eds.), Analysis and Control of Immobilized Enzyme Systems, Elsevier North-Holland, 1976, p. 115. 4 J. L. Boitieux, G. Desmet and D. Thomas, FEBS Lett., 93 (1978) 133. 5 J. L. Boitieux, G. Desmet and D. Thomas, Clin. Chim. Acta, 88 (1978) 329. 6 R. B. Johnson, R. M. Libby and R. M. Nakamura, J. Immunoassay, 1 (1980) 27 7 G. Desmet and J. L. Boitieux, Clin. Chim. Acta, 74 (1977) 59. 8 S. Avrameas and T. Ternynck, Immunochemistry, 8 (1971) 1175. 9 L. Wide, Acta Endocrinol., 142 (1970) 207. 10 J. L. Boitieux, J. L. Rometle, N. Aubry and D. Thomas, Clin. Chim. Acta, 136 (1984) 19 11 J. L. Rommette, B. Froment and D. Thomas, Clin. Chim. Acta, 95 (1979) 249. 12 J. P. Kernevez, L. Konate and J. L. Romette, Biotechnol. Bioeng., (1982) in press. 13 G. Walters, L. Kuijpens, J. Kaccki and A. Schuurs, J. Clin. Pathol., 28 (1976) 873. 14 W. Gerlich, B. Stamm and R. Thomssen, J. Biol. Stand., 4 (1976) 189.
Short Communication
OXYGEN ELECTRODE-BASED ENZYME IMMUNOASSAY FOR THE AMPEROMETRIC DETERMINATION OF HEPATITIS B SURFACE ANTIGEN
JEAN-LOUIS
BOITIEUX
and DANIEL THOMAS*
La bomtoire de Technologie Enzymatique, Universitt! de Technologie B.P. 233, 60206 Compiegne Cedex (France)
de Compiegne
GERARD DESMET Labomtoire (Fmnce)
d’Hormonologie,
Centre Hospitalier
Sud, B.P. 3009, 80030 Amiens
Cedex
(Received 29th March 1984)
Summary. Specific antibodies labelled with glucose oxidase are immobilized onto a gelatin membrane, which is fixed over an oxygen electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen by the enzyme catalyzed reaction. The response increases linearly with increasing antigen concentration over the range 0.1-100 pg 1-l. A microcomputer is used for data acquisition and processing.
Numerous methods are available for the accurate determination of traces of antigens and haptens in biological fluids. Radioimmunoassay and enzyme immunoassay (e.i.a.) are the most sensitive. Since the introduction of immunosorbents, many solid-phase techniques aimed at obtaining optimum results with enzyme-linked immunosorbent assays (e.1.i.s.a.) have been extensively described [ 1, 21. Recently, however, protein membranes have been used for the immobilization of enzymes [3]. The binding of immunoglobulins onto gelatin membranes has been described [4] and an enzyme immunoassay technique has been introduced for the determination of hepatitis B surface antigen (HB,A,) in biological fluids [ 51. In this communication, the use of glucose oxidase as the enzyme label is reported; the labelled antibody is bound to a protein membrane. The advantages of the glucose oxidase label have recently been demonstrated [6]. The purpose of this work is to study the possibilities of automation of this type of antigen estimation by using a semi-continuous flow mode, and processing the signals with the aid of a microcomputer. The active membrane is fixed over a modified oxygen electrode, designed in this laboratory. The oxygen consumption of the enzyme catalyzed reaction is measured when the electrode is in contact with a standard glucose solution. The signal from the electrode is directly proportional to the oxygen consumption and to the antigen concentration. 0003-2670/84/$03.00
0 1984 Elsevier Science Publishers B.V.
310
Experimental Chemicals. Glucose oxidase (E.C.1.1.3.4.; Type II, 18400 U g-l; Sigma) and pig skin gelatin (Rousselot Laboratory, 60400 Rib&ourt, France) were used. Glutaraldehyde and other chemicals used were of analytical grade (Merck). For the determination of HB,AB, a panel of the reference center for virus hepatitis at the Institute of Hygiene, Giittingen University, was used by courtesy of Dr. Gerlich. Hepatitis B surface antigen (HB,AJ was isolated and purified from human positive sera by a simple method described elsewhere [ 71. Anti-HB,A, anti. bodies were prepared and purified by preparative electrophoresis followed by chromatography on DEAE cellulose [ 51. Equipment. Amperometric measurements were done with an oxygen analyzer. The microcomputer was an Apple II+ computer with 48K of main memory. The interface card cage was constructed in the Electronics Department, University of Technology of Compiegne. The measurement cell (Fig. 1) was designed in this laboratory. The internal peripheral bus of the computer enabled a variety of peripheral units to be added. A disc controller card and parallel interface cards (Apple) were added for the printer and interface card cage. The interface card cage was mounted on the computer through a ribbon cable and contained circuit boards for signal conditioning and buffering, and analog-to-digital card based upon the Date1 ADC (EK-12B) an oxygen analyzer and a 4-bit logic system. The flow system used for the measurements is shown in Fig. 2. Preparation of the glucose oxidase--antibody conjugate. Glucose oxidase (ex. Aspergillus niger) was conjugated to specific antibodies by use of glutaraldehyde [ 81. Further fractionation on Sephadex G200 equilibrated with 0.01 mol 1-l phosphate buffer, pH 6.8, was used to eliminate the free enzyme. The fractions of the first peak were pooled and distributed in sterile 0.2-ml aliquots. This solution could be stored at 4°C for several months without significant loss in activity. Immobilization of antibodies on pig skin gelatin. The general procedure used to prepare an active membrane and to fix the specific antibodies has
mless steel Ibody
body
membrane
i xygen
electrode
electrolyte RinSlng
Fig. 1. Cross-section of the immunosensor electrode and measuring cell. Fig. 2. Flow system used for the antigen determination.
liquid
Std glucose
311
been described previously [ 41. Antibodies were immobilized on discs (10 mm diameter, ~0.2 mm thick) cut from the activated membranes. The discs were immersed in a 30 g 1-l bovine serum albumin solution for 1 h and thoroughly washed in distilled water. These antibody-coated discs could be stored at -4” C in 0.02 mol 1-l phosphate buffer, pH 6.8, or in the presence of 1 g 1-l of gentamicin solution at 4” C. Procedure for the determination of HB,A,. The gelatin membrane was immersed for 30 min in 0.5 ml of diluted (1:lOOO) HB,A, positive serum, immobilized antibodies G being in excess with respect to the antigen. The membrane was then thoroughly washed. The active membrane was incubated for 1 h in 1 ml of a solution of conjugate (1:200 in 0.01 mol 1-l phosphate buffer, pH 6.8) as in the “sandwich” method [ 91. The membrane was washed with 0.01 mol 1-l sodium phosphate buffer pH 6.8 and fixed on the hydrophobic selective gas membrane of an oxygen sensor by a magnetic device, as shown in Fig. 1. The measuring cell was filled with standard glucose solution. Consumption of oxygen was measured by the oxygen sensor. It was proportional to the enzyme activity retained on the membrane and therefore to the HB,A, concentration. Signal processing. In the direct measurement mode, the program starts by asking for a time interval for the collection or sampling of data. Next, it runs an accuracy test before plotting the data points. This test is done on the 20 data points (N = 20, Fig. 3). If S(ti) (with i = 0, . . ., N) are the measured values, the accuracy test is operated through S(ti) - S(to) = Pi for each sampled point. It checks that P, = Pi - 1. The program calculates the value D which is the concentration to be determined, i.e., D = Pi for i = N (when P, = Pi - 1); ti (the interval between two data points) is obviously an important parameter. A machine language subroutine was written for data acquisition from the ADC. The rest of the software was implemented in Basic in the usual way. 1
end of collect i
Fig. 3. Outline of data acquisition.
312
Results and discussion The effects of experimental conditions on the detection of glucose oxidase activity were first established. A preliminary report [lo] has shown that the coupling of glucose oxidase with antibodies does not affect significantly the Michaelis constant. The stability of glucose oxidase after coupling to the antibodies allowed a good reproducibility of the HB,A, measurement to be achieved. The stoichiometry of the enzyme reaction has been studied, using the computerized enzyme electrode system for glucose determination in blood and serum [ll, 121. The binding capacity and selectivity of the membrane were also considered. A pig-skin gelatin membrane was chosen for linking the antibodies. Previous work based on electron microscopy studies of artificial protein membranes [4] showed the homogeneity of the pig-skin gelatin membrane, which has a uniform thickness, a glossy surface and a regular network. The calibration graph of signal vs. log HB,A, concentration was linear over the range 0.1-100 c(g 1-l. The precision at various concentrations are shown in Table 1. The between-assay relative standard deviation was 7--12X The average recovery of known amounts of HB,A, added to a serum pool was 95%. An unknown sample of HB,A, positive serum was analyzed by the present procedure and by radio-immunoassay with good agreement. These results are encouraging because the limit of detection of a classical enzyme immunoassay is about 2.4 pg 1-l [ 131, and radio-immunoassay has a limit of 3-4/.Lg 1-l [14]. The enzyme immunosensor depends on the immunochemical reaction for its selectivity, because an antibody binds to its corresponding antigen. Accordingly, the antibodies used are highly selective for HB,Ap but are not completely specific. This computerized enzyme immunosensor allowed 30 sample measurements per hour including the washing step between samples, sampling and oxygen measurement. The volume of the sample for each measurement was ca. 50 ~1. The stability of the active membrane when stored at 4°C is very good (not more than 20% loss of initial activity after six months) so that the calibration graph can be reproduced easily with the original membrane. TABLE 1 Precision of the method (10 measurements with the same membrane) HBsA, taken (pg 1-l) Mean found (pg 1-l) S.d.a R.s.d.
1.00
10.0
100
1.01 0.04 4
9.6 0.2 2
94 3 3
aStandard deviation (pg 1-l) and relative standard deviation (%).
313
In conclusion, the computerized continuous flow system described for HB,A, measurements can be used for quantitative immunological reactions. This technology has also been tested for the estimation of the hormone 17-poestradiol; a competitive enzyme-linked immunoassay was used. The first tests with standard oestradiol solutions gave satisfactory results at levels of a few pmol l-l and above. REFERENCES 1 E. EngvaIl and P. Perlmann, Immunochem., 8 (1971) 871. 2 A. H. W. M. Schuurs and B. K. van Weemen, Clin. Chim. Acta, 81 (1977) 1. 3 D. Thomas, in D. Thomas and J. P. Kernevez (Eds.), Analysis and Control of Immobilized Enzyme Systems, Elsevier North-Holland, 1976, p. 115. 4 J. L. Boitieux, G. Desmet and D. Thomas, FEBS Lett., 93 (1978) 133. 5 J. L. Boitieux, G. Desmet and D. Thomas, Clin. Chim. Acta, 88 (1978) 329. 6 R. B. Johnson, R. M. Libby and R. M. Nakamura, J. Immunoassay, 1 (1980) 27 7 G. Desmet and J. L. Boitieux, Clin. Chim. Acta, 74 (1977) 59. 8 S. Avrameas and T. Ternynck, Immunochemistry, 8 (1971) 1175. 9 L. Wide, Acta Endocrinol., 142 (1970) 207. 10 J. L. Boitieux, J. L. Rometle, N. Aubry and D. Thomas, Clin. Chim. Acta, 136 (1984) 19 11 J. L. Rommette, B. Froment and D. Thomas, Clin. Chim. Acta, 95 (1979) 249. 12 J. P. Kernevez, L. Konate and J. L. Romette, Biotechnol. Bioeng., (1982) in press. 13 G. Walters, L. Kuijpens, J. Kaccki and A. Schuurs, J. Clin. Pathol., 28 (1976) 873. 14 W. Gerlich, B. Stamm and R. Thomssen, J. Biol. Stand., 4 (1976) 189.