View Article Online / Journal Homepage / Table of Contents for this issue
1547
ANALYST, OCTOBER 1992, VOL. 117
Spectrophotometric Enzyme-amplified lmmunoassay for Thyroid Stimulating Hormone
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
Robert Wilson Research Centre for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
Thyroid stimulating hormone (TSH) regulates the function of the thyroid gland. Its determination at low concentrations in serum is useful in the diagnosis of hyperthyroidism. In this paper, it is detected using a spectrophotometric enzyme-amplified immunoassay. The reporter enzyme is alkaline phosphatase and its substrate is flavin adenine dinucleotide phosphate (FADP). Reaction with alkaline phosphataseconverts FADP into flavin adenine dinucleotide (FAD), which, unlike FADP, re-activates apo-D-amino acid oxidase (apo-AOD). Re-activation of apo-AOD allows the product of the reporter enzyme to be amplified. The lower limit of detection for TSH by this method is 0.06 pU cm-3. This compares with 0.54 pU cm-3 for an identical assay in which p-nitrophenyl phosphate was the substrate for alkaline phosphatase. Contaminating alkaline phosphatase was removed from the reagents by affinity chromatography. Keywords: Enzyme-amplified immunoassay; thyroid stimulating hormone; flavin adenine dinucleotide phosphate; apo -D-am in0 acid oxidase; affinity chromatography
Enzyme amplification has been used to enhance the speed and sensitivity of various analytical techniques.1-6 In these systems the substrate is either the analyte or a compound present at a concentration proportional to that of the analyte. It is cycled enzymically to increase the amount of a detectable product. In the enzyme-amplified immuno-assisted assay described by Self,' for example, the analyte is placental alkaline phosphatase (PLAP). This is used to dephosphorylate NADP and produce a stoichiometricamount of NAD. The NAD is cycled between alcohol dehydrogenase and diaphorase to produce a coloured dye. The amount of dye produced can be related to the concentration of PLAP by reference to a calibration graph. In this paper, an enzyme-amplification system based on phosphorylated flavin adenine dinucleotide (FADP) is described. Navin adenine dinucleotide (FAD) is the coenzyme of D-amino acid oxidase (AOD). The coenzyme can be removed to yield apo-AOD.7 Removal of FAD is reversible and re-activation of the apo-enzyme has been used in sensitive assays to detect the coenzyme.7.8 If FAD is phosphorylated it is unable to re-activate apo-AOD. Treatment with alkaline phosphatase, however, converts it into FAD. Therefore, FADP and apo-AOD can be used to detect alkaline phosphatase according to the scheme shown in Fig. 1. In the work described here, FADP was prepared by phosphorylating FAD with orthophosphoric acid. It was then used in an enzymeamplified immunoassay for thyroid stimulating hormone (TSH) in which alkaline phosphatase was the enzyme label.
FAD
i
Experimental Materials D-Amino acid oxidase (AOD; E.C. 1.4.3.3) Type I from porcine kidney, alkaline phosphatase (E.C. 3.1.3.1) (4000 DEA units per milligram of enzyme protein [1DEA (diethanolamine) unit is the amount of alkaline phosphatase that will hydrolyse 1 pmol of p-nitrophenyl phosphate per minute at 37 "C, in DEA buffer (1 mol dm-3) that also contains p-nitrophenyl phosphate (15 mmol dm-3) and magnesium chloride (0.5 mmol dm-3)]} from bovine intestinal mucosa, peroxidase (POD; E.C. 1.11.1.7) Type I1 from horseradish, FAD, L-histidyldiazobenzylphosphonic acid attached to agarose, eight-channel multiple pipettes and the enzyme-immunoassay kit for TSH (Catalogue No. SIA 120-A) were all obtained from Sigma (St. Louis, MO, USA). The immunoassay kit contained the following components: microtitre plates with immobilized antibodies to TSH, conjugate solution (antibodies to TSH labelled with alkaline phosphatase), TSH solution (12 pU cm-3 cross-standardized to a World Health Organization primary standard), diluent for the TSH solution (the diluent was pH 7.5 buffered protein solution that contained a surfactant and 0.1% sodium azide as a preservative), and a buffered surfactant wash solution. These components were used in all the immunoassays. All other reagents were of the highest grade commercially available. The plate reader was from Tosoh (Tokyo, Japan). The plate shaker was from Scientific Industries (Bohemia, NY, USA).
AOD
APO-AOD
Dehydroproline
Fig. 1 Schematic representation of enzyme amplified immunoassay for TSH
View Article Online
1548
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
General Methods
Alkaline phosphatase-free peroxidase was prepared by the method of Landt et aZ.9 for phosphatase removal rather than purification; it was used in all assays involving FADP. Alkaline phosphatase solutions were prepared by diluting the commercial solution with 0.1 rnol dm-3 tris(hydroxymethy1)methylamine (Tris) buffer (pH 9.5) that contained bovine serum albumin (BSA) (10 g dm-3), Triton X-100 (0.5 g dm-3), D-proline (50 mmol dm-3), dihydroxybenzenesulfonic acid (DHBS) (5 mmol dm-3) magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). Bovine serum albumin and Triton X-100 helped to prevent adsorption of alkaline phosphatase on the walls of the container. The molarity of alkaline phosphatase in these solutions was based on a relative molecular mass of 140 m a 1 0 and the assumption that all protein (biuret) in the commercial material was alkaline phosphatase. All buffer solutions were adjusted to the correct pH at 25°C and all work was carried out at this temperature. The limit of detection, where calculated, was taken as the analyte concentration equivalent to twice the standard deviation of eight zero calibrators. Preparation of Apo-AOD
Two methods were used to prepare apo-amino acid oxidase (apo-AOD). The first method has been described by Decker and Hinkkanen.7 In the second method, AOD (10 mg) dissolved in 5 cm3 of 10 mmol dm-3 buffer (pH 8.5) was dialysed against 3 x 250 cm3 of 0.1 rnol dm-3 Tris buffer (pH 8.5) that contained potassium bromide (1 rnol dm-3) and ethylenediaminetetraaceticacid (EDTA) (5 mmol dm-3) for a total of 36 h at 4°C in darkness, and then against 3 x 250 cm3 of 0.1 rnol dm-3 Tris buffer (pH 8.5) for 36 h at 4°C in darkness. Standardizationof Apo-AOD
Apo-amino acid oxidase was diluted 1 + 9 with 0.1 rnol dm-3 Tris buffer (pH 8.5) that contained 4-aminoantipyrine (4AP) (0.5 mmol dm-3). Solutions of FAD (0-10 pmol dm-3) were prepared in 0.1 rnol dm-3 Tris buffer (pH 9.5). This buffer contained D-proline (50 mmol dm-3), DHBS (5 mmol dm-3), POD (0.1 mg cm-3), magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). The apo-AOD solution (100 mm3) and FAD solution (100 mm3) were added to the wells of a microtitre plate. After 10 min the absorbance was measured at 492 nm. Stability of Apo-AOD versus pH
The apo-AOD solution was dialysed against 1 dm3 of de-ionized water for 24 h. It was then diluted 1 + 9 with buffer solutions (10 mmol dm-3) that contained 4AP (0.5 mmol dm-3). In the pH range 3-5 the buffer solution was sodium citrate, in the range 6-8 sodium phosphate and in the range 9-10 sodium carbonate. The apo-AOD solutions were maintained at a temperature of 25°C for 24 h in darkness. During this time they were assayed for activity after0,4,8and24 h, by mixing them with 0.1 rnol dm-3 Tris buffer (pH 8.5), that contained POD (0.1 mg cm-3), D-proline (50 mmol dm-3), DHBS (5 mmol dm-3) and FAD (10 pmol dm-3).
ANALYST, OCTOBER 1992, VOL. 117
D-proline (50 mmol dm-3) and DHBS (5 mmol dm-3). The amount of colour development was measured after 60 min. Preparation of FADP
This was prepared according to the method described by Wilson.11 A 50 mg amount of FAD was added to 2 cm3 of anhydrous dimethyl sulfoxide, the mixture was sonicated to dissolve the FAD, and the solvent was evaporated under vacuum at 3040°C. Orthophosphoric acid (1 rnol dm-3) in dimethyl sulfoxide (100 mm3) and N,N-diisopropylethylamine (200 mm3) were then added, with sonication. Next, 100 mm3 of trichloroacetonitrile were added to the solution, followed by further sonication. After 20 min the reaction was terminated by addition of glacial acetic acid to a final concentration of 50% v/v. The product was loaded onto a column packed with diethylaminoethyl (DEAE)-cellulose equilibrated with 50% v/v aqueous acetic acid. Monophosphate derivatives of FAD were eluted with a gradient of 100-400 mmol dm-3 ammonia in 50% v/v aqueous acetic acid. The eluate was dried by rotary evaporation at 30°C and stored at -20°C. Determination of the FAD Liberated From FADP
Alkaline phosphatase converts FADP into FAD by acting as a phosphatase, but this enzyme also has a low level of phosphodiesterase activity. 12 This results in cleavage of FAD and renders the coenzyme unable to re-activate apo-AOD. When determining the amount of FAD that can be liberated from a given amount of FADP, therefore, it is important to keep the phosphodiesterase activity to a minimum. This entails using a small amount of alkaline phosphatase. It is also important to use a low concentration of substrate to ensure that orthophosphate released during the reaction does not inhibit alkaline phosphatase. Assuming that FADP had the same molar absorption coefficient asFAD(E = 11.3dm3mmol-1cm-1)7al pmoldm-3 solution was prepared in 0.1 rnol dm-3 Tris buffer (pH 9.5). This buffer also contained POD (0.1 mg cm-3), D-proline (50 mmol dm-3), DHBS (5 mmol dm-3), magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). The FADP solution was mixed 1 + 1 with an alkaline phosphatase solution (0.1 D E A units (3111-3) made up in the same buffer. At zero time, and at 10 min intervals thereafter, aliquots were assayed for FAD by mixing them 1 + 1 with apo-AOD prepared in 0.1 rnol dm-3 pyrophosphate buffer (pH 8.5) that contained 4AP (0.5 mmol dm-3). The amount of FAD present was determined, with use of the plate reader, at 492 nm, with reference to a calibration graph. The experiment was continued for 90 min, i.e., until the amount of FAD remained constant for five successive measurements. Stability of FADP
The stability of FADP in solution was investigated at 4 and 25°C. The FADP was dissolved in 0.1 rnol dm-3 Tris buffer (pH 8.5) to a concentration of 50 pmol dm-3 ( ~ 4 5 0= 18.0 dm3 mmol-1 cm-1). This buffer also contained 4AP (0.5 mmol dm-3). At appropriate intervals, this solution was assayed for FAD as described previously. Removal of Phosphatase from Apo-AOD
Contaminating phosphatase was removed from apo-AOD by the method of Landt et aZ.9 for phosphatase removal rather than purification. The apo-AOD (1 cm3) was loaded dropwise The apo-AOD was diluted 1 + 9 with de-ionized water that contained 4AP (0.5 mmol dm-3). The rate of re-activation was onto a column packed with L-histidyldiazobenzylphosphonic determined by mixing apo-AOD solution (100 mm3) and acid agarose (1 cm3 of gel in an 8 mm i.d. column). buffer solution (100 mm3) in the wells of a microtitre plate. Phosphatase-free A O D was eluted with 0.1 rnol dm-3 Tris Buffer solutions in the pH range 7.0-9.5 contained Tris (0.2 buffer (pH 8.5) and collected in a volume of 5 cm3. To this was mol dm-3), FAD (100 nmol dm-3), POD (0.1 mg ~ m - ~ ) , added 4AP to a final concentration of 1 mmol dm-3. Re-activation of Apo-AOD versus pH
View Article Online
1549
ANALYST, OCTOBER 1992, VOL. 117
Effect of FADP on the Re-activation of apo-AOD by FAD
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
The apo-AOD solution was used for the assay for FAD in the range 0-250 nmol dm-3 in the presence of FADP at a final concentration of 25 pmol dm-3, according to the method €or standardization of apo-AOD that has already been described. The results were compared with the amount of colour development observed in the absence of FADP.
Optimization of FADP Concentration In an assay for alkaline phosphatase the final FADP concentration was varied between 0 and 50 pmol dm-3. The apo-AOD-AP solutions were diluted 1 + 1 with FADP solutions made up in 0.1 mol dm-3 Tris buffer (pH 8.5) immediately prior to the assay. The product (apo-AODFADP solution) was then mixed 1 1 with a 2 pmol dm-3 alkaline phosphatase solution (100 mm3) and the amount of colour development after 1 h was measured.
+
0
1
2
3
4
5
6
[FADl/pmol dm-3
Fig. 2 Standardization of apo-AOD. The graph was extrapolated as shown and the binding molarity taken as the FAD concentration at the point where the lines crossed (230 nm). This was multiplied by the dilution factor (1 + 19) to find the binding molarity of the original solution (4.6 pmol dm-3)
Re-activation of Apo-AOD by Alkaline Phosphatase versus pH The apo-AOD-FADP solution was prepared as described previously except that FADP was dissolved in, and the apo-enzyme was eluted from the affinity column with, de-ionized water. The rate of re-activation was determined by mixing apo-AOD-FADP solution (100 mm3) and 2 pmol dm-3 phosphatase (100 111111-3) in the pH range 7.0-9.5. The extent of colour development after 1 h was measured.
- 100
-8 80 .-
.-C 60
2 >:..-
Assay for Alkaline Phosphatase Alkaline phosphatase was assayed in the range 0-1 pmol dm-3. To each well of a microtitre plate was added 100 mm3 of alkaline phosphatase solution and 100 mm3 of apo-AOD-FADP solution prepared as described previously. The plate was then covered with aluminium foil. After 1h the absorbance of the solutions in the wells was measured at 492 nm with reference to a reagent blank that contained no D-proline. For comparison, identical amounts of alkaline phosphatase were assayed in 0.1 mol dm-3 diethanolamine buffer (pH 9.8) withp-nitrophenyl phosphate (10 mmol dm-3) as the substrate. This buffer also contained magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). The absorbance was measured at 405 nm.
Immunoassay for TSH Solutions of TSH (100 mm3) in the range 0-5 pU cm-3 were added to the wells of a microtitre plate coated with antibodies to TSH. The plate was then covered with plastic cling film and gently shaken at room temperature for 1h. At the end of this time the wells were washed out five times with a buffered surfactant solution. After each wash, the plate was inverted and vigorously blotted against a clean paper towel to remove as much of the wash solution as possible. After washing, an alkaline phosphatase-labelled antibody solution (100 mm-3) was added to the wells of the plate. It was then incubated for 1 h and washed as described previously. After washing, the extent of colour development associated with known amounts of TSH was measured as described for the alkaline phosphatase assay except that the concentration of apo-AOD was increased by a factor of ten, the FADP was made up in the POD-proline-DHBS solution, and the extent of colour development was measured after 30 min. During this time the plate was gently shaken. For comparison, an immunoassay in which nitrophenyl phosphate was the substrate was carried out as in the alkaline phosphatase assay except that the plate was gently shaken and colour development was measured after 30 min.
40
c.
2
20
I
03
5
7
9
PH
Fig. 3 Variation of the stability of apo-AOD with pH at 25°C. A, 4; B, 8; and C. 24 h
Results and Discussion APO-AOD The method used to prepare apo-AOD is based on that of Decker and Hinkkanen.7 In the original method, sodium pyrophosphate buffer was used instead of Tris. The latter buffer was used in this work because the apo-AOD is used to detect alkaline phosphatase in an immunoassay. In this assay, sodium pyrophosphate, itself a substrate for alkaline phosphatase, would act as a competitive inhibitor, but Tris promotes the reaction by acting as a transphosphorylating agent .I3 The apo-AOD was standardized by plotting absorbance at 492 nm against FAD concentration, as shown in Fig. 2. The lower limit of detection for FAD was 4.3 nmol dm-3. The binding molarity was found by extrapolation, as shown in the diagram. Typically, apo-AOD prepared in Tris buffer had a binding molarity of 4.6 pmol dm-3 prior to dilution. The amount of residual FAD was 46 nmol dm-3 (about 1%of the binding molarity). The apo-AOD prepared in sodium pyrophosphate buffer solution had a lower binding molarity of about 3.7 pmol dm-3. The amount of residual FAD in this material was also about 1% of the binding molarity. A plot of percentage activity remaining versus pH is shown in Fig. 3 from which it can be seen that the apo-enzyme has maximum stability at about pH 8.0. In this work a slightly higher pH (8.5) was used to facilitate comparison with previous work in this area. Investigation of the stability in 0.1 mol dm-3 Tris buffer at this pH showed that there was no detectable loss of activity after 8 h at 25 "C or 1 week at 4 "C.
View Article Online
1550
ANALYST, OCTOBER 1992. VOL. 117
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
FADP
The average yield of FADP was about 18% m/m. A solution with an absorbance of 1.0 at 450 nm in 0.1 mol dm-3 Tris buffer (pH 8.5) contained enough FADP to yield a 56 pmol dm-3 solution of FAD after treatment with alkaline phosphatase. Therefore, a molar absorption coefficient of 18 dm3 mmol-1 cm-1 was assigned to it. The extent of colour development observed when FAD was assayed in the presence of FADP (50 pmol dm-3) was only 50% of that observed in the absence of FADP. This indicates that FAD must compete with FADP for the binding site of apo-AOD. Taking into account the molar absorption coefficient and the 50% decline in colour development, the amount of FAD present in the material, as a percentage of FADP, was calculated to be 0.02%. When an aqueous solution of FADP (50 pmol dm-3) was stored at 25°C for 8 h no hydrolysis to FAD could be detected. After 1week at 4 "C, however, some hydrolysis (about 1%)did occur. Assays for Alkaline Phosphatase and TSH
The optimum concentration of FADP in assays for alkaline phosphatase was about 20 pmol dm-3, as shown in Fig. 4.At concentrations in excess of 25 pmol dm-3 the extent of colour development decreases, presumably because FADP interferes with the re-activation of apo-AOD by FAD. A plot of colour development versus pH for the re-activation of apo-AOD by alkaline phosphatase acting on FADP is shown in Fig. 5. This indicates that the optimum pH is about 9.0, slightly higher than the optimum pH for the re-activation of apo-AOD by FAD, which is about pH 8.5. In the alkaline phosphatase assay this pH was attained by mixing equal volumes of pH 8.5
and pH 9.5 Tris buffer solutions. A plot of colour development versus alkaline phosphatase concentration is shown in Fig. 6. The lower limit of detection for alkaline phosphatase was 12 fmol dm-3 (2.5 amol of phosphatase per well). The lower limit of detection with nitrophenyl phosphate as the substrate was 160 fmol dm-3. When alkaline phosphatase was not removed from the apo-enzyme the background absorbance was about 1.O after 10 min. If the background absorbance increases at a rate per hour of more than 0.2 relative to a reagent blank the apo-AOD should be passed through the affinity column again. When the plate was not covered in aluminium foil there was a slow increase in the background absorbance owing to photoreduction of FADP followed by the formation of hydrogen peroxide (this also occurs with other flavins such as FAD and is not peculiar to FADP). As a result, the background absorbance after 1 h in a well-lit laboratory is more than double that observed when light is excluded. This leads to an increase in the noise-to-signal ratio and a slight decline in the lower limit of detection. The concentration of apo-AOD used in the TSH immunoassay was ten times greater than that used in the alkaline phosphatase assay in order to extend the range over which TSH could be detected. A plot of colour development versus TSH concentration is shown in Fig. 7. The lower limit of detection for TSH in the amplified immunoassay was 0.06 pU cm-3. The precision of the assay was good [relative standard deviation (RSD)
1547
ANALYST, OCTOBER 1992, VOL. 117
Spectrophotometric Enzyme-amplified lmmunoassay for Thyroid Stimulating Hormone
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
Robert Wilson Research Centre for Advanced Science and Technology, University of Tokyo, Tokyo, Japan
Thyroid stimulating hormone (TSH) regulates the function of the thyroid gland. Its determination at low concentrations in serum is useful in the diagnosis of hyperthyroidism. In this paper, it is detected using a spectrophotometric enzyme-amplified immunoassay. The reporter enzyme is alkaline phosphatase and its substrate is flavin adenine dinucleotide phosphate (FADP). Reaction with alkaline phosphataseconverts FADP into flavin adenine dinucleotide (FAD), which, unlike FADP, re-activates apo-D-amino acid oxidase (apo-AOD). Re-activation of apo-AOD allows the product of the reporter enzyme to be amplified. The lower limit of detection for TSH by this method is 0.06 pU cm-3. This compares with 0.54 pU cm-3 for an identical assay in which p-nitrophenyl phosphate was the substrate for alkaline phosphatase. Contaminating alkaline phosphatase was removed from the reagents by affinity chromatography. Keywords: Enzyme-amplified immunoassay; thyroid stimulating hormone; flavin adenine dinucleotide phosphate; apo -D-am in0 acid oxidase; affinity chromatography
Enzyme amplification has been used to enhance the speed and sensitivity of various analytical techniques.1-6 In these systems the substrate is either the analyte or a compound present at a concentration proportional to that of the analyte. It is cycled enzymically to increase the amount of a detectable product. In the enzyme-amplified immuno-assisted assay described by Self,' for example, the analyte is placental alkaline phosphatase (PLAP). This is used to dephosphorylate NADP and produce a stoichiometricamount of NAD. The NAD is cycled between alcohol dehydrogenase and diaphorase to produce a coloured dye. The amount of dye produced can be related to the concentration of PLAP by reference to a calibration graph. In this paper, an enzyme-amplification system based on phosphorylated flavin adenine dinucleotide (FADP) is described. Navin adenine dinucleotide (FAD) is the coenzyme of D-amino acid oxidase (AOD). The coenzyme can be removed to yield apo-AOD.7 Removal of FAD is reversible and re-activation of the apo-enzyme has been used in sensitive assays to detect the coenzyme.7.8 If FAD is phosphorylated it is unable to re-activate apo-AOD. Treatment with alkaline phosphatase, however, converts it into FAD. Therefore, FADP and apo-AOD can be used to detect alkaline phosphatase according to the scheme shown in Fig. 1. In the work described here, FADP was prepared by phosphorylating FAD with orthophosphoric acid. It was then used in an enzymeamplified immunoassay for thyroid stimulating hormone (TSH) in which alkaline phosphatase was the enzyme label.
FAD
i
Experimental Materials D-Amino acid oxidase (AOD; E.C. 1.4.3.3) Type I from porcine kidney, alkaline phosphatase (E.C. 3.1.3.1) (4000 DEA units per milligram of enzyme protein [1DEA (diethanolamine) unit is the amount of alkaline phosphatase that will hydrolyse 1 pmol of p-nitrophenyl phosphate per minute at 37 "C, in DEA buffer (1 mol dm-3) that also contains p-nitrophenyl phosphate (15 mmol dm-3) and magnesium chloride (0.5 mmol dm-3)]} from bovine intestinal mucosa, peroxidase (POD; E.C. 1.11.1.7) Type I1 from horseradish, FAD, L-histidyldiazobenzylphosphonic acid attached to agarose, eight-channel multiple pipettes and the enzyme-immunoassay kit for TSH (Catalogue No. SIA 120-A) were all obtained from Sigma (St. Louis, MO, USA). The immunoassay kit contained the following components: microtitre plates with immobilized antibodies to TSH, conjugate solution (antibodies to TSH labelled with alkaline phosphatase), TSH solution (12 pU cm-3 cross-standardized to a World Health Organization primary standard), diluent for the TSH solution (the diluent was pH 7.5 buffered protein solution that contained a surfactant and 0.1% sodium azide as a preservative), and a buffered surfactant wash solution. These components were used in all the immunoassays. All other reagents were of the highest grade commercially available. The plate reader was from Tosoh (Tokyo, Japan). The plate shaker was from Scientific Industries (Bohemia, NY, USA).
AOD
APO-AOD
Dehydroproline
Fig. 1 Schematic representation of enzyme amplified immunoassay for TSH
View Article Online
1548
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
General Methods
Alkaline phosphatase-free peroxidase was prepared by the method of Landt et aZ.9 for phosphatase removal rather than purification; it was used in all assays involving FADP. Alkaline phosphatase solutions were prepared by diluting the commercial solution with 0.1 rnol dm-3 tris(hydroxymethy1)methylamine (Tris) buffer (pH 9.5) that contained bovine serum albumin (BSA) (10 g dm-3), Triton X-100 (0.5 g dm-3), D-proline (50 mmol dm-3), dihydroxybenzenesulfonic acid (DHBS) (5 mmol dm-3) magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). Bovine serum albumin and Triton X-100 helped to prevent adsorption of alkaline phosphatase on the walls of the container. The molarity of alkaline phosphatase in these solutions was based on a relative molecular mass of 140 m a 1 0 and the assumption that all protein (biuret) in the commercial material was alkaline phosphatase. All buffer solutions were adjusted to the correct pH at 25°C and all work was carried out at this temperature. The limit of detection, where calculated, was taken as the analyte concentration equivalent to twice the standard deviation of eight zero calibrators. Preparation of Apo-AOD
Two methods were used to prepare apo-amino acid oxidase (apo-AOD). The first method has been described by Decker and Hinkkanen.7 In the second method, AOD (10 mg) dissolved in 5 cm3 of 10 mmol dm-3 buffer (pH 8.5) was dialysed against 3 x 250 cm3 of 0.1 rnol dm-3 Tris buffer (pH 8.5) that contained potassium bromide (1 rnol dm-3) and ethylenediaminetetraaceticacid (EDTA) (5 mmol dm-3) for a total of 36 h at 4°C in darkness, and then against 3 x 250 cm3 of 0.1 rnol dm-3 Tris buffer (pH 8.5) for 36 h at 4°C in darkness. Standardizationof Apo-AOD
Apo-amino acid oxidase was diluted 1 + 9 with 0.1 rnol dm-3 Tris buffer (pH 8.5) that contained 4-aminoantipyrine (4AP) (0.5 mmol dm-3). Solutions of FAD (0-10 pmol dm-3) were prepared in 0.1 rnol dm-3 Tris buffer (pH 9.5). This buffer contained D-proline (50 mmol dm-3), DHBS (5 mmol dm-3), POD (0.1 mg cm-3), magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). The apo-AOD solution (100 mm3) and FAD solution (100 mm3) were added to the wells of a microtitre plate. After 10 min the absorbance was measured at 492 nm. Stability of Apo-AOD versus pH
The apo-AOD solution was dialysed against 1 dm3 of de-ionized water for 24 h. It was then diluted 1 + 9 with buffer solutions (10 mmol dm-3) that contained 4AP (0.5 mmol dm-3). In the pH range 3-5 the buffer solution was sodium citrate, in the range 6-8 sodium phosphate and in the range 9-10 sodium carbonate. The apo-AOD solutions were maintained at a temperature of 25°C for 24 h in darkness. During this time they were assayed for activity after0,4,8and24 h, by mixing them with 0.1 rnol dm-3 Tris buffer (pH 8.5), that contained POD (0.1 mg cm-3), D-proline (50 mmol dm-3), DHBS (5 mmol dm-3) and FAD (10 pmol dm-3).
ANALYST, OCTOBER 1992, VOL. 117
D-proline (50 mmol dm-3) and DHBS (5 mmol dm-3). The amount of colour development was measured after 60 min. Preparation of FADP
This was prepared according to the method described by Wilson.11 A 50 mg amount of FAD was added to 2 cm3 of anhydrous dimethyl sulfoxide, the mixture was sonicated to dissolve the FAD, and the solvent was evaporated under vacuum at 3040°C. Orthophosphoric acid (1 rnol dm-3) in dimethyl sulfoxide (100 mm3) and N,N-diisopropylethylamine (200 mm3) were then added, with sonication. Next, 100 mm3 of trichloroacetonitrile were added to the solution, followed by further sonication. After 20 min the reaction was terminated by addition of glacial acetic acid to a final concentration of 50% v/v. The product was loaded onto a column packed with diethylaminoethyl (DEAE)-cellulose equilibrated with 50% v/v aqueous acetic acid. Monophosphate derivatives of FAD were eluted with a gradient of 100-400 mmol dm-3 ammonia in 50% v/v aqueous acetic acid. The eluate was dried by rotary evaporation at 30°C and stored at -20°C. Determination of the FAD Liberated From FADP
Alkaline phosphatase converts FADP into FAD by acting as a phosphatase, but this enzyme also has a low level of phosphodiesterase activity. 12 This results in cleavage of FAD and renders the coenzyme unable to re-activate apo-AOD. When determining the amount of FAD that can be liberated from a given amount of FADP, therefore, it is important to keep the phosphodiesterase activity to a minimum. This entails using a small amount of alkaline phosphatase. It is also important to use a low concentration of substrate to ensure that orthophosphate released during the reaction does not inhibit alkaline phosphatase. Assuming that FADP had the same molar absorption coefficient asFAD(E = 11.3dm3mmol-1cm-1)7al pmoldm-3 solution was prepared in 0.1 rnol dm-3 Tris buffer (pH 9.5). This buffer also contained POD (0.1 mg cm-3), D-proline (50 mmol dm-3), DHBS (5 mmol dm-3), magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). The FADP solution was mixed 1 + 1 with an alkaline phosphatase solution (0.1 D E A units (3111-3) made up in the same buffer. At zero time, and at 10 min intervals thereafter, aliquots were assayed for FAD by mixing them 1 + 1 with apo-AOD prepared in 0.1 rnol dm-3 pyrophosphate buffer (pH 8.5) that contained 4AP (0.5 mmol dm-3). The amount of FAD present was determined, with use of the plate reader, at 492 nm, with reference to a calibration graph. The experiment was continued for 90 min, i.e., until the amount of FAD remained constant for five successive measurements. Stability of FADP
The stability of FADP in solution was investigated at 4 and 25°C. The FADP was dissolved in 0.1 rnol dm-3 Tris buffer (pH 8.5) to a concentration of 50 pmol dm-3 ( ~ 4 5 0= 18.0 dm3 mmol-1 cm-1). This buffer also contained 4AP (0.5 mmol dm-3). At appropriate intervals, this solution was assayed for FAD as described previously. Removal of Phosphatase from Apo-AOD
Contaminating phosphatase was removed from apo-AOD by the method of Landt et aZ.9 for phosphatase removal rather than purification. The apo-AOD (1 cm3) was loaded dropwise The apo-AOD was diluted 1 + 9 with de-ionized water that contained 4AP (0.5 mmol dm-3). The rate of re-activation was onto a column packed with L-histidyldiazobenzylphosphonic determined by mixing apo-AOD solution (100 mm3) and acid agarose (1 cm3 of gel in an 8 mm i.d. column). buffer solution (100 mm3) in the wells of a microtitre plate. Phosphatase-free A O D was eluted with 0.1 rnol dm-3 Tris Buffer solutions in the pH range 7.0-9.5 contained Tris (0.2 buffer (pH 8.5) and collected in a volume of 5 cm3. To this was mol dm-3), FAD (100 nmol dm-3), POD (0.1 mg ~ m - ~ ) , added 4AP to a final concentration of 1 mmol dm-3. Re-activation of Apo-AOD versus pH
View Article Online
1549
ANALYST, OCTOBER 1992, VOL. 117
Effect of FADP on the Re-activation of apo-AOD by FAD
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
The apo-AOD solution was used for the assay for FAD in the range 0-250 nmol dm-3 in the presence of FADP at a final concentration of 25 pmol dm-3, according to the method €or standardization of apo-AOD that has already been described. The results were compared with the amount of colour development observed in the absence of FADP.
Optimization of FADP Concentration In an assay for alkaline phosphatase the final FADP concentration was varied between 0 and 50 pmol dm-3. The apo-AOD-AP solutions were diluted 1 + 1 with FADP solutions made up in 0.1 mol dm-3 Tris buffer (pH 8.5) immediately prior to the assay. The product (apo-AODFADP solution) was then mixed 1 1 with a 2 pmol dm-3 alkaline phosphatase solution (100 mm3) and the amount of colour development after 1 h was measured.
+
0
1
2
3
4
5
6
[FADl/pmol dm-3
Fig. 2 Standardization of apo-AOD. The graph was extrapolated as shown and the binding molarity taken as the FAD concentration at the point where the lines crossed (230 nm). This was multiplied by the dilution factor (1 + 19) to find the binding molarity of the original solution (4.6 pmol dm-3)
Re-activation of Apo-AOD by Alkaline Phosphatase versus pH The apo-AOD-FADP solution was prepared as described previously except that FADP was dissolved in, and the apo-enzyme was eluted from the affinity column with, de-ionized water. The rate of re-activation was determined by mixing apo-AOD-FADP solution (100 mm3) and 2 pmol dm-3 phosphatase (100 111111-3) in the pH range 7.0-9.5. The extent of colour development after 1 h was measured.
- 100
-8 80 .-
.-C 60
2 >:..-
Assay for Alkaline Phosphatase Alkaline phosphatase was assayed in the range 0-1 pmol dm-3. To each well of a microtitre plate was added 100 mm3 of alkaline phosphatase solution and 100 mm3 of apo-AOD-FADP solution prepared as described previously. The plate was then covered with aluminium foil. After 1h the absorbance of the solutions in the wells was measured at 492 nm with reference to a reagent blank that contained no D-proline. For comparison, identical amounts of alkaline phosphatase were assayed in 0.1 mol dm-3 diethanolamine buffer (pH 9.8) withp-nitrophenyl phosphate (10 mmol dm-3) as the substrate. This buffer also contained magnesium nitrate (1 mmol dm-3) and zinc nitrate (0.1 mmol dm-3). The absorbance was measured at 405 nm.
Immunoassay for TSH Solutions of TSH (100 mm3) in the range 0-5 pU cm-3 were added to the wells of a microtitre plate coated with antibodies to TSH. The plate was then covered with plastic cling film and gently shaken at room temperature for 1h. At the end of this time the wells were washed out five times with a buffered surfactant solution. After each wash, the plate was inverted and vigorously blotted against a clean paper towel to remove as much of the wash solution as possible. After washing, an alkaline phosphatase-labelled antibody solution (100 mm-3) was added to the wells of the plate. It was then incubated for 1 h and washed as described previously. After washing, the extent of colour development associated with known amounts of TSH was measured as described for the alkaline phosphatase assay except that the concentration of apo-AOD was increased by a factor of ten, the FADP was made up in the POD-proline-DHBS solution, and the extent of colour development was measured after 30 min. During this time the plate was gently shaken. For comparison, an immunoassay in which nitrophenyl phosphate was the substrate was carried out as in the alkaline phosphatase assay except that the plate was gently shaken and colour development was measured after 30 min.
40
c.
2
20
I
03
5
7
9
PH
Fig. 3 Variation of the stability of apo-AOD with pH at 25°C. A, 4; B, 8; and C. 24 h
Results and Discussion APO-AOD The method used to prepare apo-AOD is based on that of Decker and Hinkkanen.7 In the original method, sodium pyrophosphate buffer was used instead of Tris. The latter buffer was used in this work because the apo-AOD is used to detect alkaline phosphatase in an immunoassay. In this assay, sodium pyrophosphate, itself a substrate for alkaline phosphatase, would act as a competitive inhibitor, but Tris promotes the reaction by acting as a transphosphorylating agent .I3 The apo-AOD was standardized by plotting absorbance at 492 nm against FAD concentration, as shown in Fig. 2. The lower limit of detection for FAD was 4.3 nmol dm-3. The binding molarity was found by extrapolation, as shown in the diagram. Typically, apo-AOD prepared in Tris buffer had a binding molarity of 4.6 pmol dm-3 prior to dilution. The amount of residual FAD was 46 nmol dm-3 (about 1%of the binding molarity). The apo-AOD prepared in sodium pyrophosphate buffer solution had a lower binding molarity of about 3.7 pmol dm-3. The amount of residual FAD in this material was also about 1% of the binding molarity. A plot of percentage activity remaining versus pH is shown in Fig. 3 from which it can be seen that the apo-enzyme has maximum stability at about pH 8.0. In this work a slightly higher pH (8.5) was used to facilitate comparison with previous work in this area. Investigation of the stability in 0.1 mol dm-3 Tris buffer at this pH showed that there was no detectable loss of activity after 8 h at 25 "C or 1 week at 4 "C.
View Article Online
1550
ANALYST, OCTOBER 1992. VOL. 117
Published on 01 January 1992. Downloaded by University of California - Irvine on 31/10/2014 11:14:22.
FADP
The average yield of FADP was about 18% m/m. A solution with an absorbance of 1.0 at 450 nm in 0.1 mol dm-3 Tris buffer (pH 8.5) contained enough FADP to yield a 56 pmol dm-3 solution of FAD after treatment with alkaline phosphatase. Therefore, a molar absorption coefficient of 18 dm3 mmol-1 cm-1 was assigned to it. The extent of colour development observed when FAD was assayed in the presence of FADP (50 pmol dm-3) was only 50% of that observed in the absence of FADP. This indicates that FAD must compete with FADP for the binding site of apo-AOD. Taking into account the molar absorption coefficient and the 50% decline in colour development, the amount of FAD present in the material, as a percentage of FADP, was calculated to be 0.02%. When an aqueous solution of FADP (50 pmol dm-3) was stored at 25°C for 8 h no hydrolysis to FAD could be detected. After 1week at 4 "C, however, some hydrolysis (about 1%)did occur. Assays for Alkaline Phosphatase and TSH
The optimum concentration of FADP in assays for alkaline phosphatase was about 20 pmol dm-3, as shown in Fig. 4.At concentrations in excess of 25 pmol dm-3 the extent of colour development decreases, presumably because FADP interferes with the re-activation of apo-AOD by FAD. A plot of colour development versus pH for the re-activation of apo-AOD by alkaline phosphatase acting on FADP is shown in Fig. 5. This indicates that the optimum pH is about 9.0, slightly higher than the optimum pH for the re-activation of apo-AOD by FAD, which is about pH 8.5. In the alkaline phosphatase assay this pH was attained by mixing equal volumes of pH 8.5
and pH 9.5 Tris buffer solutions. A plot of colour development versus alkaline phosphatase concentration is shown in Fig. 6. The lower limit of detection for alkaline phosphatase was 12 fmol dm-3 (2.5 amol of phosphatase per well). The lower limit of detection with nitrophenyl phosphate as the substrate was 160 fmol dm-3. When alkaline phosphatase was not removed from the apo-enzyme the background absorbance was about 1.O after 10 min. If the background absorbance increases at a rate per hour of more than 0.2 relative to a reagent blank the apo-AOD should be passed through the affinity column again. When the plate was not covered in aluminium foil there was a slow increase in the background absorbance owing to photoreduction of FADP followed by the formation of hydrogen peroxide (this also occurs with other flavins such as FAD and is not peculiar to FADP). As a result, the background absorbance after 1 h in a well-lit laboratory is more than double that observed when light is excluded. This leads to an increase in the noise-to-signal ratio and a slight decline in the lower limit of detection. The concentration of apo-AOD used in the TSH immunoassay was ten times greater than that used in the alkaline phosphatase assay in order to extend the range over which TSH could be detected. A plot of colour development versus TSH concentration is shown in Fig. 7. The lower limit of detection for TSH in the amplified immunoassay was 0.06 pU cm-3. The precision of the assay was good [relative standard deviation (RSD)
