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Application of a mimetic enzyme for the enzyme immunoassay for α-1-fetoprotein
ANALmcA CHIlUfCA
ACE4 Analytica ChimicaActa 300 ( 1995) 273-276
ELSEVIER
Application of a mimetic enzyme for the enzyme immunoassay for a-1-fetoprotein Yun-Xiang Ci *, Yang Qin, Wen-Bao Chang, Yuan-Zong Li Department of Chemistry, Peking University, Beijing 100871, China Received 28 June 1993
Abstract A new mimetic enzyme immunoassay (MEIA) method for cY-1-fetoprotein (AFP) was developed using a mimetic enzyme suIfophenyl)porphine (Mn-TPPS,) as a labelling reagent to catalyze the fluorescence reaction of 4-hydroxyphenylacetic acid (HPA) and hydrogen peroxide. In the sandwich assay standard AFP solution or AFP containing umbilical blood serum Crst reacts with antibody (anti-AFP) coated on a 40-well plate (polystyrene), and then further reacts with MnTPPS,-labelled anti-AFP; the Mn-TPP& on the bound fraction, after removing the free fraction was determined by measuring the fluorescence intensity as a result of the reaction between HPA and H202, catalyzed by bound Mn-TPPS, and anti-AFP conjugate, which was proportional to the concentration of AFP. AFT in the range 0.01-10 pg per well can be determined with a detection limit of 1 ng per well. The method has sufficient sensitivity to be applied to the determination of AFP in umbilical blood serum. Mn( III)-tetra(
Keywords: Enzymatic
methods; Immunoassay;
a-Fetoprotein;
Manganese;
1. Introduction Enzyme-antibody labels are very much used in immunochemistry; they play au important role iu the diagnosis of infectious diseases. Among the different enzymes employed as markers in the heterogeneous enzyme immunoassay, alkaline phosphatase (AP) , horseradish peroxidase (HRP) and galactosidase
(Gal) are most commonly used. It is well known that natural substances having a porphine skeleton in their active centers, that is, peroxidase, heme and hematin [l-3], act as sensitive catalysts for the fluorescence and chemiluminescence reactions, and have been used in immunoassays [ 4,5]. Some synthesized metal complexes [ 61 instead of the above-mentioned natural sub* Corresponding
author.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10003-2670(94)00385-8
Mimetic enzyme immunoassay
stances have been used as catalysts in chemiluminescence immunoassay ( CLlA) with the advantage of cheapness, reproducibility and easy labelling. Recently, we reported the use of a synthesized metal complex catalyst labelling reagent for fluoroimmunoassay of methotrexate. In this paper, Mn-TPP&, which had been previously reported [ 7-91 to show the highest catalytic activity, was used for the analysis of AFP. It was determined by mimetic enzyme immunoassay using Mn-TPP$ as catalyst as follows: AFT from serum a sample first reacts with antibody (antiAFP) coated on a polystyrene 40-well plate, and then further reacts with Mn-TPPS4 labelled anti-AFP. The bound Mn-TPPS, labelled anti-AFP, after removing the free fraction, was detected by its ability to catalyze the formation of the fluorescent product from the reaction between hydrogen peroxide and hydroxypheny-
274
Y.-X. Ci et 01. / Anolytico Chimico Acto 300 (195)
lacetic acid. The fluorescence intensity is thus a measure of the concentration of AFP in the analytical sample.
2. Experimental 2.1. Materials Mn-TPP& was prepared in our laboratory. HPA was purchased from Sigma. AFP and anti-AFT’ were obtained from the Cancer Institute in Beijing (China), PCI, and H20, (30%) were from Beijing Chemical Factory. H,O* (30%) was standardized by titration with standard KMnO,. The 40-well microtitre plate was a product of Qiujin Biochemical Instruments Factory (Shanghai). 2.2. Instruments The fluorescence spectra and relative fluorescence intensity were measured on a Hitachi 850 spectrofluorimeter with a l-cm silica cell. The absorption spectra and absorbances were measured on a Shimadzu UV300 apparatus.
2.3. Synthesis and characteristics of Mn-TPPSC anti-AFP conjugate
Mn-TPPS, was prepared by the procedure described earlier [lo] with a small modification. The synthesis was carried out by the reaction between TPPS, and 5% excess of manganese( II) acetate under reflux in aqueous solution. The product was purified on a cationexchange resin column with acetone-water (l:l, v/v) as eluent. Labelling of anti-AFP with Mn-TPP$ was carried out using PC&. Mn-TPPS, and PC& were ground in a mortar until the reaction was complete. After cooling, a small amount of dimethyl formamide (DMF) was added, which in turn, was added slowly to a neutral Na,CO,-NaHCO, (0.5 N) buffer and stirred for 1 h. During the reaction the pH should be maintained neutral to prevent deactivation of anti-AFP. After the reaction the solution was subjected to gel chromatography on a Sepharose G25 column by use of phosphate-buffered saline (PBS) as eluent; the portion of anti-AFP labelled with Mn-TPPS, was thus obtained.
273-276
Each concentration of the anti-AFP and the MnTPPS, in the labelled anti-AFP was determined by the use of absorbance calibration curves of Mn-TPPS., at 456 nm and anti-AFP at 595 nm after its reaction with Bio-Rad stainer (a protein assay kit containing Coomassie Brilliant Blue G-250)) on the assumption that the absorbance of each constituent remained unchanged before and after labelling. It was concluded from the absorbance measurements that the mole ratio of MnTPPS, to anti-AFP was 20. The concentration of MnTPPS, and anti-AFP in Mn-TPPS,-anti-AFP conjugate were 4 X low4 M and 0.32 mg/ml, respectively.
2.4. Immunoassay (standard procedure)
A polystyrene 40-well microtitre plate was pretreated with 50 ~1 of 0.02% ovalbumin and was allowed to stand for one day. Then, the plate was washed with PBS-Tween 20 3 times and treated with 50 ~1 of 0.2% glutaraldehyde in PBS buffer (pH 7.2) for 30 min at room temperature. The plate was washed with PBSTween 20 buffer 5 times and air-dried for 24 h. 100 ~1 of anti-AFP in 0.05 N Na2C03-NaHC03 buffer (pH 9.5) was used to coat the plate at 4°C overnight. The plate was washed 3 times and blocked with 2% ovalbumin (100 ~1) for 1 h. The plate well was washed 3 times with water and 100 ~1 of unknown or standard AFP sample was transferred into the well and the reaction was allowed to proceed for 1 h. The plate well was washed 3 times with water and 100 ~1 of Mn-TPPS, labelled anti-AFP was added. The immunoassay reaction was allowed to proceed for 1 h. This was followed by washing 3 times with water and addition of a suitable amount of HPA, H,Oz and NaOH solutions (see below). After 13 min reaction the solution was diluted and the fluorescence intensity was measured with excitation and emission wavelengths of 322 and 425 nm, respectively. The optimal fluorescence reaction conditions are: reaction time, 13 min; HPAconcentration, 0.024 M; H,O, concentration, 1.86 X 10e4 M; NaOH concentration, 0.05 M.
Y.-X. Ci et al. I Analytica Chimica Acta 300 (1995) 273-276
3. Results and discussions 3.1. Optimization of coating concentration of antiAFP The polystyrene 40-well plate was incubated at 4°C overnight with anti-AFP, which was prediluted with Na,C03-NaHC03 buffer to different concentrations, followed by incubation with standard AFP and MnTPPS, labelled anti-AFP. The experimental results showed that the optimal concentration for the coating solution ranged from 100 to 500 times dilution of the original anti-AFP solution. 3.2. Optimization of the concentration of Mn-TPPS, labelled anti-AFP The plate well was coated with a 1:200 dilution of antiserum and, in turn, was incubated with standard AFP and Mn-TPPS, labelled anti-AFP (0.32 mglml) prediluted to different concentrations. The 1:lO dilution of the original Mn-TPPS, labelled anti-AFP solution was optimal. 3.3. Immunoassay of AFP in umbilical blood serum Fig. 1 shows a calibration graph obtained by the sandwich method. According to this graph, AFP in the range 0.01-10.0 pg per well can be determined with a detection limit of 1 ng per well (SIN= 3). This method If
215
was used for the determination of AFP in the serum of umbilical blood provided by the Xiehe hospital (Beijing) to give a result of 50 pg/ml AFP. The result was in good agreement with the value determined by the radioimmunoassay method in the Hospital. The coefficient of variation (n = 5) within batch was 10% for the determination of 500 ng/ml AFP. Although the sensitivity of the method is insufficient for routine clinical purposes for cases where the concentration of AFP is very low, it may still be used in a few occasions where the AFP concentrations are high. For example, it may be used for monitoring AFP-producing liver tumours where the concentration of AFP is about 400 ng/ml and for the antenatal screening of birth abnormalities, in which the AFP level in maternal serum during normal pregnancy can reach 450-500 pg/ml and for an abnormal pregnancy the value may be 10 times higher than the normal value [ 111.
4. Conclusions The synthesized manganese complex which functions as a catalyst for the fluorescence reaction between HPA and H202 is useful and promising for immunoassay. The above method has the advantage of being sensitive enough for being used in some clinical assays, and can enable one to select the desired chemical structure of the labelling reagent since it is based on the use of the synthesized metal complex. In this sense, further exploiting of applications in the field of biochemical analysis is promising.
Acknowledgements
*o.-/. I
0
1
2
3
This work was supported by the National Natural Science Foundation of China and the PbD Foundation for the National Education Committee of China. The authors are grateful to Prof. Zhenfu Fan and to The Cancer Institute of Beijing, for kindly providing the AFP samples used in this work. .
I
4
I
Log[AFP] (ng/Wl)
Fig. 1. Calibration graph for the determination of AFP. Conditions: anti-Am, 1:lOO; Mn-TPPS, labelled anti-AFP, 1:lO. If=Fluorescence intensity.
References [ 11 G.G. Guilbault and E. Hieserman, Anal. B&hem., 1.
26 (1968)
276
Y.-X.Ci et al. / Analytica Chimica Acta 300 (1995) 273-276
[ 21 G.G. Guilbault, Handbook of Enzymatic Methods of Analysis,
[3] [4] [5] [6]
Marcel Dekker, New York, 1976. G.F. Zhang and P.K. Dasgupta, Anal. Chem., 64 (1992) 517. Y. Ikariyama and S. Suzuki, Anal. Chem., 54 (1982) 1126. H. Arakawa, M. Maeda and A. Tsuji, Anal. Biochem., 97 (1979) 248. T. Hara, K. Tsukagoshi, A. Arai and Y. Imashiro, Bull. Chem. Sot. Jpn., 62 (1989) 2844.
[ 71 Y.X. Ci and F. Wang, Anal. Chim. Acta, 233 (1990) 299. [S] F. Wang and Y.X. Ci, Analyst, 116 (1991) 297. [9] Y.X. Ci and F. Wang, TaIanta, 37 (1990) 1133. [lo] Y.X. Ci and F. Wang, Fresenius’ J. Anal. Chem., 339 (1991) 46. [ll] G.H. Liu, G.L. Shi and Y.N. Shi, in The Handbook for Commonly used Data in Medicine and Diagnosis Testing, Shannxi Science Press, 1990, pp. 1044-1045.
ACE4 Analytica ChimicaActa 300 ( 1995) 273-276
ELSEVIER
Application of a mimetic enzyme for the enzyme immunoassay for a-1-fetoprotein Yun-Xiang Ci *, Yang Qin, Wen-Bao Chang, Yuan-Zong Li Department of Chemistry, Peking University, Beijing 100871, China Received 28 June 1993
Abstract A new mimetic enzyme immunoassay (MEIA) method for cY-1-fetoprotein (AFP) was developed using a mimetic enzyme suIfophenyl)porphine (Mn-TPPS,) as a labelling reagent to catalyze the fluorescence reaction of 4-hydroxyphenylacetic acid (HPA) and hydrogen peroxide. In the sandwich assay standard AFP solution or AFP containing umbilical blood serum Crst reacts with antibody (anti-AFP) coated on a 40-well plate (polystyrene), and then further reacts with MnTPPS,-labelled anti-AFP; the Mn-TPP& on the bound fraction, after removing the free fraction was determined by measuring the fluorescence intensity as a result of the reaction between HPA and H202, catalyzed by bound Mn-TPPS, and anti-AFP conjugate, which was proportional to the concentration of AFP. AFT in the range 0.01-10 pg per well can be determined with a detection limit of 1 ng per well. The method has sufficient sensitivity to be applied to the determination of AFP in umbilical blood serum. Mn( III)-tetra(
Keywords: Enzymatic
methods; Immunoassay;
a-Fetoprotein;
Manganese;
1. Introduction Enzyme-antibody labels are very much used in immunochemistry; they play au important role iu the diagnosis of infectious diseases. Among the different enzymes employed as markers in the heterogeneous enzyme immunoassay, alkaline phosphatase (AP) , horseradish peroxidase (HRP) and galactosidase
(Gal) are most commonly used. It is well known that natural substances having a porphine skeleton in their active centers, that is, peroxidase, heme and hematin [l-3], act as sensitive catalysts for the fluorescence and chemiluminescence reactions, and have been used in immunoassays [ 4,5]. Some synthesized metal complexes [ 61 instead of the above-mentioned natural sub* Corresponding
author.
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD10003-2670(94)00385-8
Mimetic enzyme immunoassay
stances have been used as catalysts in chemiluminescence immunoassay ( CLlA) with the advantage of cheapness, reproducibility and easy labelling. Recently, we reported the use of a synthesized metal complex catalyst labelling reagent for fluoroimmunoassay of methotrexate. In this paper, Mn-TPP&, which had been previously reported [ 7-91 to show the highest catalytic activity, was used for the analysis of AFP. It was determined by mimetic enzyme immunoassay using Mn-TPP$ as catalyst as follows: AFT from serum a sample first reacts with antibody (antiAFP) coated on a polystyrene 40-well plate, and then further reacts with Mn-TPPS4 labelled anti-AFP. The bound Mn-TPPS, labelled anti-AFP, after removing the free fraction, was detected by its ability to catalyze the formation of the fluorescent product from the reaction between hydrogen peroxide and hydroxypheny-
274
Y.-X. Ci et 01. / Anolytico Chimico Acto 300 (195)
lacetic acid. The fluorescence intensity is thus a measure of the concentration of AFP in the analytical sample.
2. Experimental 2.1. Materials Mn-TPP& was prepared in our laboratory. HPA was purchased from Sigma. AFP and anti-AFT’ were obtained from the Cancer Institute in Beijing (China), PCI, and H20, (30%) were from Beijing Chemical Factory. H,O* (30%) was standardized by titration with standard KMnO,. The 40-well microtitre plate was a product of Qiujin Biochemical Instruments Factory (Shanghai). 2.2. Instruments The fluorescence spectra and relative fluorescence intensity were measured on a Hitachi 850 spectrofluorimeter with a l-cm silica cell. The absorption spectra and absorbances were measured on a Shimadzu UV300 apparatus.
2.3. Synthesis and characteristics of Mn-TPPSC anti-AFP conjugate
Mn-TPPS, was prepared by the procedure described earlier [lo] with a small modification. The synthesis was carried out by the reaction between TPPS, and 5% excess of manganese( II) acetate under reflux in aqueous solution. The product was purified on a cationexchange resin column with acetone-water (l:l, v/v) as eluent. Labelling of anti-AFP with Mn-TPP$ was carried out using PC&. Mn-TPPS, and PC& were ground in a mortar until the reaction was complete. After cooling, a small amount of dimethyl formamide (DMF) was added, which in turn, was added slowly to a neutral Na,CO,-NaHCO, (0.5 N) buffer and stirred for 1 h. During the reaction the pH should be maintained neutral to prevent deactivation of anti-AFP. After the reaction the solution was subjected to gel chromatography on a Sepharose G25 column by use of phosphate-buffered saline (PBS) as eluent; the portion of anti-AFP labelled with Mn-TPPS, was thus obtained.
273-276
Each concentration of the anti-AFP and the MnTPPS, in the labelled anti-AFP was determined by the use of absorbance calibration curves of Mn-TPPS., at 456 nm and anti-AFP at 595 nm after its reaction with Bio-Rad stainer (a protein assay kit containing Coomassie Brilliant Blue G-250)) on the assumption that the absorbance of each constituent remained unchanged before and after labelling. It was concluded from the absorbance measurements that the mole ratio of MnTPPS, to anti-AFP was 20. The concentration of MnTPPS, and anti-AFP in Mn-TPPS,-anti-AFP conjugate were 4 X low4 M and 0.32 mg/ml, respectively.
2.4. Immunoassay (standard procedure)
A polystyrene 40-well microtitre plate was pretreated with 50 ~1 of 0.02% ovalbumin and was allowed to stand for one day. Then, the plate was washed with PBS-Tween 20 3 times and treated with 50 ~1 of 0.2% glutaraldehyde in PBS buffer (pH 7.2) for 30 min at room temperature. The plate was washed with PBSTween 20 buffer 5 times and air-dried for 24 h. 100 ~1 of anti-AFP in 0.05 N Na2C03-NaHC03 buffer (pH 9.5) was used to coat the plate at 4°C overnight. The plate was washed 3 times and blocked with 2% ovalbumin (100 ~1) for 1 h. The plate well was washed 3 times with water and 100 ~1 of unknown or standard AFP sample was transferred into the well and the reaction was allowed to proceed for 1 h. The plate well was washed 3 times with water and 100 ~1 of Mn-TPPS, labelled anti-AFP was added. The immunoassay reaction was allowed to proceed for 1 h. This was followed by washing 3 times with water and addition of a suitable amount of HPA, H,Oz and NaOH solutions (see below). After 13 min reaction the solution was diluted and the fluorescence intensity was measured with excitation and emission wavelengths of 322 and 425 nm, respectively. The optimal fluorescence reaction conditions are: reaction time, 13 min; HPAconcentration, 0.024 M; H,O, concentration, 1.86 X 10e4 M; NaOH concentration, 0.05 M.
Y.-X. Ci et al. I Analytica Chimica Acta 300 (1995) 273-276
3. Results and discussions 3.1. Optimization of coating concentration of antiAFP The polystyrene 40-well plate was incubated at 4°C overnight with anti-AFP, which was prediluted with Na,C03-NaHC03 buffer to different concentrations, followed by incubation with standard AFP and MnTPPS, labelled anti-AFP. The experimental results showed that the optimal concentration for the coating solution ranged from 100 to 500 times dilution of the original anti-AFP solution. 3.2. Optimization of the concentration of Mn-TPPS, labelled anti-AFP The plate well was coated with a 1:200 dilution of antiserum and, in turn, was incubated with standard AFP and Mn-TPPS, labelled anti-AFP (0.32 mglml) prediluted to different concentrations. The 1:lO dilution of the original Mn-TPPS, labelled anti-AFP solution was optimal. 3.3. Immunoassay of AFP in umbilical blood serum Fig. 1 shows a calibration graph obtained by the sandwich method. According to this graph, AFP in the range 0.01-10.0 pg per well can be determined with a detection limit of 1 ng per well (SIN= 3). This method If
215
was used for the determination of AFP in the serum of umbilical blood provided by the Xiehe hospital (Beijing) to give a result of 50 pg/ml AFP. The result was in good agreement with the value determined by the radioimmunoassay method in the Hospital. The coefficient of variation (n = 5) within batch was 10% for the determination of 500 ng/ml AFP. Although the sensitivity of the method is insufficient for routine clinical purposes for cases where the concentration of AFP is very low, it may still be used in a few occasions where the AFP concentrations are high. For example, it may be used for monitoring AFP-producing liver tumours where the concentration of AFP is about 400 ng/ml and for the antenatal screening of birth abnormalities, in which the AFP level in maternal serum during normal pregnancy can reach 450-500 pg/ml and for an abnormal pregnancy the value may be 10 times higher than the normal value [ 111.
4. Conclusions The synthesized manganese complex which functions as a catalyst for the fluorescence reaction between HPA and H202 is useful and promising for immunoassay. The above method has the advantage of being sensitive enough for being used in some clinical assays, and can enable one to select the desired chemical structure of the labelling reagent since it is based on the use of the synthesized metal complex. In this sense, further exploiting of applications in the field of biochemical analysis is promising.
Acknowledgements
*o.-/. I
0
1
2
3
This work was supported by the National Natural Science Foundation of China and the PbD Foundation for the National Education Committee of China. The authors are grateful to Prof. Zhenfu Fan and to The Cancer Institute of Beijing, for kindly providing the AFP samples used in this work. .
I
4
I
Log[AFP] (ng/Wl)
Fig. 1. Calibration graph for the determination of AFP. Conditions: anti-Am, 1:lOO; Mn-TPPS, labelled anti-AFP, 1:lO. If=Fluorescence intensity.
References [ 11 G.G. Guilbault and E. Hieserman, Anal. B&hem., 1.
26 (1968)
276
Y.-X.Ci et al. / Analytica Chimica Acta 300 (1995) 273-276
[ 21 G.G. Guilbault, Handbook of Enzymatic Methods of Analysis,
[3] [4] [5] [6]
Marcel Dekker, New York, 1976. G.F. Zhang and P.K. Dasgupta, Anal. Chem., 64 (1992) 517. Y. Ikariyama and S. Suzuki, Anal. Chem., 54 (1982) 1126. H. Arakawa, M. Maeda and A. Tsuji, Anal. Biochem., 97 (1979) 248. T. Hara, K. Tsukagoshi, A. Arai and Y. Imashiro, Bull. Chem. Sot. Jpn., 62 (1989) 2844.
[ 71 Y.X. Ci and F. Wang, Anal. Chim. Acta, 233 (1990) 299. [S] F. Wang and Y.X. Ci, Analyst, 116 (1991) 297. [9] Y.X. Ci and F. Wang, TaIanta, 37 (1990) 1133. [lo] Y.X. Ci and F. Wang, Fresenius’ J. Anal. Chem., 339 (1991) 46. [ll] G.H. Liu, G.L. Shi and Y.N. Shi, in The Handbook for Commonly used Data in Medicine and Diagnosis Testing, Shannxi Science Press, 1990, pp. 1044-1045.