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Development of an affinity-column-mediated enzyme-linked immunosorbent assay for ferritin
Clinica Chimica Acta 273 (1998) 81–87
Short communication
Development of an affinity-column-mediated enzymelinked immunosorbent assay for ferritin Declan G. Spillane, John O’Mullane* Medical Sciences Section, Cork Institute of Technology, Bishopstown, Cork, Republic of Ireland Received 11 July 1997; received in revised form 5 January 1998; accepted 7 January 1998
Keywords: ELISA; Non-competitive; Column regeneration; Enzyme amplification
1. Introduction Enzyme-linked immunosorbent assays (ELISA), first conceived by Engvall and Perlmann in 1971 [1], are renowned for their specificity, sensitivity, and reliability [2]. However, probably their greatest limiting factor is the requirement for prolonged incubations, with assays generally requiring several hours. As an alternative to the conventional microtitre plate ELISA, some authors have turned to affinity chromatography to perform immunological reactions, thereby reducing incubation times [3,4]. The development of a simple, reusable flow-through ELISA for ferritin is described. The assay uses polystyrene affinity columns loaded with Sepharose gel, to which anti-ferritin antibody has been coupled (Fig. 1). Standards and serum samples are introduced to separate columns and pass through the affinity gel under gravity. Ferritin antigen is bound by solid-phase antibody and is subsequently detected using an anti-ferritin-alkaline phosphatase conjugate. Following a washing step to remove unbound material, p-nitrophenyl phosphate ( pNPP) substrate is added and p-nitrophenol ( pNP) is developed in the column at room temperature. Following elution of product from the column, the *Corresponding author. Tel.: 1 353 21 326306; fax: 1 353 21 345191; e-mail: [email protected] 0009-8981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 98 )00018-7
82
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
Fig. 1. Principle of column ELISA for ferritin. Polystyrene affinity columns (0.5–2 ml) loaded with Sepharose gel to which anti-ferritin antibody has been coupled. Binding of ferritin antigen, subsequent detection with anti-ferritin-alkaline phosphatase conjugate, and product formation all occur in the column.
absorbance is measured and the columns are regenerated using a low pH elution. The sensitivity of the present assay can be enhanced using a modification of the enzyme amplification system as described by Johansson et al. [5].
2. Materials and methods
2.1. Materials Mouse monoclonal antibody to human ferritin (clone B8) and mouse monoclonal anti-human ferritin-alkaline phosphatase conjugate (clone B8) were supplied by Biomerieux, Lyon, France. Ferritin, from human liver, was purchased from Calbiochem–Novabiochem, La Jolla, California. pNPP was purchased from Fluka Chemie AG, Buchs, Switzerland. Human serum samples were supplied by the Haematology Department, Cork University Hospital, Cork, Republic of Ireland.
2.2. Methods 2.2.1. Preparation of immunoaffinity columns Cyanogen bromide-activated Sepharose 4B (Pharmacia-Biotech, Uppsala, Sweden) was prepared and coupled to anti-ferritin antibody according to the manufacturer’s instructions. For immobilisation on 1 g (3.5 ml) of Sepharose
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
83
gel, 500 mg of antibody were used. Affinity gel (500 ml)1 was packed into disposable polystyrene columns (Pierce, Rockford, Illinois) according to manufacturer’s instructions. When packed as directed, the columns have a unique stop-flow action, i.e., aqueous solution applied to a column will automatically stop at the disc positioned above the gel when the liquid level reaches it, so the column gel will not dry out if left unattended. In addition, the nature of the system ensures that the quantity of solution applied to the column will always be equal to the amount of eluate.
2.2.2. Assay protocol Columns were equilibrated using at least 3 ml of carrier buffer (50 mmol / l Tris–HCl, pH 7.4, 0.5 mol / l NaCl, 0.1% (w / v) BSA, 0.035% (v / v) Tween 20). Volumes (200 ml) of undiluted samples and standards (ferritin dissolved in carrier buffer) were introduced to different columns and allowed run into the affinity gel. This was followed by 500 ml of carrier buffer to wash protein further into the column. In the next step, 500 ml of conjugate antibody were added to each column at a concentration of 1.96 mg / ml. Following washes with 1 1 1 ml of carrier buffer, 500 ml of freshly prepared substrate solution (2 mg pNPP per ml of 0.1 mol / l Tris–HCl, pH 9.8, 1.5 mol / l NaCl) were added to each column for 30 min at room temperature. p-Nitrophenol product was eluted using 1 ml of carrier buffer and its absorbance was measured at 405 nm in an ELISA plate reader (Dynatech MR7000, Dynatech Laboratories, West Sussex, UK). The timing of substrate incubation was started upon addition of substrate to the first column. To ensure that the time of incubation of substrate within each column was precise, elution buffer was added to the batch of columns in the same sequence as the addition of substrate. Therefore, if one assumes that the speed of pipetting substrate approximates the speed of pipetting elution buffer, the time of incubation of substrate should be the same within each column. 2.2.3. Column regeneration Columns were regenerated using three wash cycles, each cycle consisting of 1 ml of 0.1 mol / l glycine buffer, pH 2.1, followed by 2 ml of carrier buffer.
3. Results
3.1. Assay validation 3.1.1. Working range and limit of detection Precision profiles, performed for ten assays using the same columns, established the working range at 4.1–525 mg / l. The coefficients of variation 1
Gel slurry and distilled water were not degassed as specified in manufacturer’s protocol.
84
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
Table 1 Precision of ferritin determination by column ELISA Mean (mg / l)
S.D. (mg / l)
Within-run (n 5 10) Pool 1 8.98 Pool 2 90.2 Pool 3 350.6 Between-run (n 5 10) Pool 1 10.5 Pool 2 88.22 Pool 3 317.06
C.V. (%)
0.54 4.1 16.98
6.01 4.55 4.84
1.13 6.77 21
10.76 7.67 6.62
(C.V.) of the standards rarely rose above 5% over the ten assays, indicating that the regeneration process affected all columns to the same degree. The limit of detection (L.O.D.), estimated as three times the standard deviation (S.D.) in the measurement of zero-dose response [6], varied between 0.6 and 1.9 mg / l.
3.1.2. Within-run and between-run precision Within-run precision was assessed on fresh columns by analysing three serum pools in replicates of ten (Table 1). To assess between-run precision, the same serum pools were analysed singly for a further nine assays using the same columns (Table 1). 3.1.3. Analytical recovery Human serum devoid of ferritin was spiked with known amounts of ferritin standard and analysed by the present assay on columns regenerated once. Recovery values for eight samples, ranging from 5.0 to 480 mg / l, were 91–110%, with a mean of 101.9% (Table 2). Table 2 Analytical recovery of ferritin Expected Value (mg / l)
Measured Value (mg / l)
% Recovery a
5 20 47 67 93 185 267 480
5.4 22 45 73 87 188 278 437
108 110 97.3 108.9 94.1 101.6 104.1 91
a
% Recovery 5 Measured Value / Expected Value 3 100.
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
85
Fig. 2. Dilution linearity of column ELISA for three human serum samples.
3.1.4. Linearity Three human serum samples with high ferritin concentrations were diluted (1 / 2, 1 / 3, 1 / 5, 1 / 8, 1 / 15, 1 / 30, 1 / 80) in the standard matrix. Neat and diluted samples were measured using the present assay on freshly prepared columns (Fig. 2). 3.1.5. Comparison with chemiluminescence assay Thirty serum samples (8–641 mg / l) were measured using the present assay and the results were compared to those obtained using the chemiluminescence immunoassay on the ACS:180 (Ciba Corning Diagnostics, Medfield, Massachusetts). This yielded a regression equation of y 5 1.084x 2 1.388, r 5 0.995, std. error of y estimate 5 21.35. The columns had been regenerated seven times.
4. Discussion A simple, reliable and reusable affinity-column-mediated enzyme-linked immunosorbent assay for ferritin is described. The assay is based on the
86
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
principles of immunoaffinity chromatography, which ensures rapid interaction between mobile-phase antigen and solid-phase antibody in excess. A notable feature of this assay is the ability to recycle the affinity columns through low pH elution of antigen and conjugate antibody without affecting the integrity of the columns and the assay performance for up to at least ten runs. Presumably, the columns could be used for a greater number of assays. In addition, the regeneration process can efficiently prevent antigen carryover from standards or samples with ferritin concentrations up to 1050 mg / l (210 ng / column); however, the columns retain detectable amounts of antigen for ferritin concentrations greater than 1050 mg / l ( . 210 ng / column). An extra regeneration is required in this case. Standards for the present assay were prepared in Tris–HCl buffer, pH 7.4, containing 0.1% BSA (w / v) and 0.035% (v / v) Tween 20. Due to the differences between the standard matrix and that of serum samples, there was no guarantee that serum samples would be measured accurately. Results obtained indicated the absence of serum matrix effects. Enzyme immunoassays detecting subattomole (subpicogram) amounts of ferritin have been reported elsewhere [7,8], however, such sensitivities were achieved using overnight incubations of antigen with antibody. Using freshly prepared columns, the lower limit of detection for the present assay was estimated at 0.7 mg / l. Determinations were repeated for nine subsequent assays using the same columns, giving detection limits ranging from 0.6 to 1.9 mg / l. While the present assay does not achieve subattomole sensitivity, it does permit the measurement of samples below the lower limit of normal (15 mg / l) in approximately 1.5 h. In addition, the sensitivity of the assay was increased using a modification of the enzyme amplified detection system as described by Johansson et al. [5], without compromising the speed of the assay. From a calibration curve prepared using this system, it appeared that a 1 mg / l sample could be measured with confidence, even after thirteen regenerations of the columns. In addition, the assay volume used was only 50 ml, giving a sensitivity of approximately 50 picograms (1.11 3 10 216 mol). Presumably, greater sensitivity could be achieved using freshly prepared columns.
References [1] Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971;8:871–4. [2] Ekins RP. Current concepts and future developments. In: Collins WP, editor. Alternative Immunoassays. New York: John Wiley and Sons, Inc., 1985:219–37. [3] Freytag JW, Lau HP, Wadsley JJ. Affinity-column-mediated immunoenzymometric assays: Influence of affinity-column ligand and valency of antibody-enzyme conjugates. Clin Chem 1984;30(9):1494–8.
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
87
[4] Lejeune R, Thunus L, Gomez F, Frankenne F, Cloux JL, Hennen G. Subfemtomole enzyme immunoassay for human growth hormone using affinity chromatography and enzyme amplified detection. Anal Biochem 1990;189:217–22. [5] Johansson A, Stanley CJ, Self CH. A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry. Clin Chim Acta 1985;148:119–24. [6] Micallef J, Ahsan R. Immunoassay development. In: Gosling JP, Basso LV, editors. Immunoassay: Laboratory analysis and clinical application. Boston: Butterworth– Heinemann, 1994:51–68. [7] Ishikawa E, Imagawa M, Yoshitake S et al. Major factors limiting sensitivity of sandwich enzyme immunoassay for ferritin, immunoglobulin E, and thyroid-stimulating hormone. Ann Clin Biochem 1982;19:379–84. [8] Hashida S, Ishikawa E. Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay. J Biochem 1990;108:960–4.
Short communication
Development of an affinity-column-mediated enzymelinked immunosorbent assay for ferritin Declan G. Spillane, John O’Mullane* Medical Sciences Section, Cork Institute of Technology, Bishopstown, Cork, Republic of Ireland Received 11 July 1997; received in revised form 5 January 1998; accepted 7 January 1998
Keywords: ELISA; Non-competitive; Column regeneration; Enzyme amplification
1. Introduction Enzyme-linked immunosorbent assays (ELISA), first conceived by Engvall and Perlmann in 1971 [1], are renowned for their specificity, sensitivity, and reliability [2]. However, probably their greatest limiting factor is the requirement for prolonged incubations, with assays generally requiring several hours. As an alternative to the conventional microtitre plate ELISA, some authors have turned to affinity chromatography to perform immunological reactions, thereby reducing incubation times [3,4]. The development of a simple, reusable flow-through ELISA for ferritin is described. The assay uses polystyrene affinity columns loaded with Sepharose gel, to which anti-ferritin antibody has been coupled (Fig. 1). Standards and serum samples are introduced to separate columns and pass through the affinity gel under gravity. Ferritin antigen is bound by solid-phase antibody and is subsequently detected using an anti-ferritin-alkaline phosphatase conjugate. Following a washing step to remove unbound material, p-nitrophenyl phosphate ( pNPP) substrate is added and p-nitrophenol ( pNP) is developed in the column at room temperature. Following elution of product from the column, the *Corresponding author. Tel.: 1 353 21 326306; fax: 1 353 21 345191; e-mail: [email protected] 0009-8981 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 98 )00018-7
82
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
Fig. 1. Principle of column ELISA for ferritin. Polystyrene affinity columns (0.5–2 ml) loaded with Sepharose gel to which anti-ferritin antibody has been coupled. Binding of ferritin antigen, subsequent detection with anti-ferritin-alkaline phosphatase conjugate, and product formation all occur in the column.
absorbance is measured and the columns are regenerated using a low pH elution. The sensitivity of the present assay can be enhanced using a modification of the enzyme amplification system as described by Johansson et al. [5].
2. Materials and methods
2.1. Materials Mouse monoclonal antibody to human ferritin (clone B8) and mouse monoclonal anti-human ferritin-alkaline phosphatase conjugate (clone B8) were supplied by Biomerieux, Lyon, France. Ferritin, from human liver, was purchased from Calbiochem–Novabiochem, La Jolla, California. pNPP was purchased from Fluka Chemie AG, Buchs, Switzerland. Human serum samples were supplied by the Haematology Department, Cork University Hospital, Cork, Republic of Ireland.
2.2. Methods 2.2.1. Preparation of immunoaffinity columns Cyanogen bromide-activated Sepharose 4B (Pharmacia-Biotech, Uppsala, Sweden) was prepared and coupled to anti-ferritin antibody according to the manufacturer’s instructions. For immobilisation on 1 g (3.5 ml) of Sepharose
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
83
gel, 500 mg of antibody were used. Affinity gel (500 ml)1 was packed into disposable polystyrene columns (Pierce, Rockford, Illinois) according to manufacturer’s instructions. When packed as directed, the columns have a unique stop-flow action, i.e., aqueous solution applied to a column will automatically stop at the disc positioned above the gel when the liquid level reaches it, so the column gel will not dry out if left unattended. In addition, the nature of the system ensures that the quantity of solution applied to the column will always be equal to the amount of eluate.
2.2.2. Assay protocol Columns were equilibrated using at least 3 ml of carrier buffer (50 mmol / l Tris–HCl, pH 7.4, 0.5 mol / l NaCl, 0.1% (w / v) BSA, 0.035% (v / v) Tween 20). Volumes (200 ml) of undiluted samples and standards (ferritin dissolved in carrier buffer) were introduced to different columns and allowed run into the affinity gel. This was followed by 500 ml of carrier buffer to wash protein further into the column. In the next step, 500 ml of conjugate antibody were added to each column at a concentration of 1.96 mg / ml. Following washes with 1 1 1 ml of carrier buffer, 500 ml of freshly prepared substrate solution (2 mg pNPP per ml of 0.1 mol / l Tris–HCl, pH 9.8, 1.5 mol / l NaCl) were added to each column for 30 min at room temperature. p-Nitrophenol product was eluted using 1 ml of carrier buffer and its absorbance was measured at 405 nm in an ELISA plate reader (Dynatech MR7000, Dynatech Laboratories, West Sussex, UK). The timing of substrate incubation was started upon addition of substrate to the first column. To ensure that the time of incubation of substrate within each column was precise, elution buffer was added to the batch of columns in the same sequence as the addition of substrate. Therefore, if one assumes that the speed of pipetting substrate approximates the speed of pipetting elution buffer, the time of incubation of substrate should be the same within each column. 2.2.3. Column regeneration Columns were regenerated using three wash cycles, each cycle consisting of 1 ml of 0.1 mol / l glycine buffer, pH 2.1, followed by 2 ml of carrier buffer.
3. Results
3.1. Assay validation 3.1.1. Working range and limit of detection Precision profiles, performed for ten assays using the same columns, established the working range at 4.1–525 mg / l. The coefficients of variation 1
Gel slurry and distilled water were not degassed as specified in manufacturer’s protocol.
84
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
Table 1 Precision of ferritin determination by column ELISA Mean (mg / l)
S.D. (mg / l)
Within-run (n 5 10) Pool 1 8.98 Pool 2 90.2 Pool 3 350.6 Between-run (n 5 10) Pool 1 10.5 Pool 2 88.22 Pool 3 317.06
C.V. (%)
0.54 4.1 16.98
6.01 4.55 4.84
1.13 6.77 21
10.76 7.67 6.62
(C.V.) of the standards rarely rose above 5% over the ten assays, indicating that the regeneration process affected all columns to the same degree. The limit of detection (L.O.D.), estimated as three times the standard deviation (S.D.) in the measurement of zero-dose response [6], varied between 0.6 and 1.9 mg / l.
3.1.2. Within-run and between-run precision Within-run precision was assessed on fresh columns by analysing three serum pools in replicates of ten (Table 1). To assess between-run precision, the same serum pools were analysed singly for a further nine assays using the same columns (Table 1). 3.1.3. Analytical recovery Human serum devoid of ferritin was spiked with known amounts of ferritin standard and analysed by the present assay on columns regenerated once. Recovery values for eight samples, ranging from 5.0 to 480 mg / l, were 91–110%, with a mean of 101.9% (Table 2). Table 2 Analytical recovery of ferritin Expected Value (mg / l)
Measured Value (mg / l)
% Recovery a
5 20 47 67 93 185 267 480
5.4 22 45 73 87 188 278 437
108 110 97.3 108.9 94.1 101.6 104.1 91
a
% Recovery 5 Measured Value / Expected Value 3 100.
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
85
Fig. 2. Dilution linearity of column ELISA for three human serum samples.
3.1.4. Linearity Three human serum samples with high ferritin concentrations were diluted (1 / 2, 1 / 3, 1 / 5, 1 / 8, 1 / 15, 1 / 30, 1 / 80) in the standard matrix. Neat and diluted samples were measured using the present assay on freshly prepared columns (Fig. 2). 3.1.5. Comparison with chemiluminescence assay Thirty serum samples (8–641 mg / l) were measured using the present assay and the results were compared to those obtained using the chemiluminescence immunoassay on the ACS:180 (Ciba Corning Diagnostics, Medfield, Massachusetts). This yielded a regression equation of y 5 1.084x 2 1.388, r 5 0.995, std. error of y estimate 5 21.35. The columns had been regenerated seven times.
4. Discussion A simple, reliable and reusable affinity-column-mediated enzyme-linked immunosorbent assay for ferritin is described. The assay is based on the
86
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
principles of immunoaffinity chromatography, which ensures rapid interaction between mobile-phase antigen and solid-phase antibody in excess. A notable feature of this assay is the ability to recycle the affinity columns through low pH elution of antigen and conjugate antibody without affecting the integrity of the columns and the assay performance for up to at least ten runs. Presumably, the columns could be used for a greater number of assays. In addition, the regeneration process can efficiently prevent antigen carryover from standards or samples with ferritin concentrations up to 1050 mg / l (210 ng / column); however, the columns retain detectable amounts of antigen for ferritin concentrations greater than 1050 mg / l ( . 210 ng / column). An extra regeneration is required in this case. Standards for the present assay were prepared in Tris–HCl buffer, pH 7.4, containing 0.1% BSA (w / v) and 0.035% (v / v) Tween 20. Due to the differences between the standard matrix and that of serum samples, there was no guarantee that serum samples would be measured accurately. Results obtained indicated the absence of serum matrix effects. Enzyme immunoassays detecting subattomole (subpicogram) amounts of ferritin have been reported elsewhere [7,8], however, such sensitivities were achieved using overnight incubations of antigen with antibody. Using freshly prepared columns, the lower limit of detection for the present assay was estimated at 0.7 mg / l. Determinations were repeated for nine subsequent assays using the same columns, giving detection limits ranging from 0.6 to 1.9 mg / l. While the present assay does not achieve subattomole sensitivity, it does permit the measurement of samples below the lower limit of normal (15 mg / l) in approximately 1.5 h. In addition, the sensitivity of the assay was increased using a modification of the enzyme amplified detection system as described by Johansson et al. [5], without compromising the speed of the assay. From a calibration curve prepared using this system, it appeared that a 1 mg / l sample could be measured with confidence, even after thirteen regenerations of the columns. In addition, the assay volume used was only 50 ml, giving a sensitivity of approximately 50 picograms (1.11 3 10 216 mol). Presumably, greater sensitivity could be achieved using freshly prepared columns.
References [1] Engvall E, Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 1971;8:871–4. [2] Ekins RP. Current concepts and future developments. In: Collins WP, editor. Alternative Immunoassays. New York: John Wiley and Sons, Inc., 1985:219–37. [3] Freytag JW, Lau HP, Wadsley JJ. Affinity-column-mediated immunoenzymometric assays: Influence of affinity-column ligand and valency of antibody-enzyme conjugates. Clin Chem 1984;30(9):1494–8.
D.G. Spillane, J. O’ Mullane / Clinica Chimica Acta 273 (1998) 81 – 87
87
[4] Lejeune R, Thunus L, Gomez F, Frankenne F, Cloux JL, Hennen G. Subfemtomole enzyme immunoassay for human growth hormone using affinity chromatography and enzyme amplified detection. Anal Biochem 1990;189:217–22. [5] Johansson A, Stanley CJ, Self CH. A fast highly sensitive colorimetric enzyme immunoassay system demonstrating benefits of enzyme amplification in clinical chemistry. Clin Chim Acta 1985;148:119–24. [6] Micallef J, Ahsan R. Immunoassay development. In: Gosling JP, Basso LV, editors. Immunoassay: Laboratory analysis and clinical application. Boston: Butterworth– Heinemann, 1994:51–68. [7] Ishikawa E, Imagawa M, Yoshitake S et al. Major factors limiting sensitivity of sandwich enzyme immunoassay for ferritin, immunoglobulin E, and thyroid-stimulating hormone. Ann Clin Biochem 1982;19:379–84. [8] Hashida S, Ishikawa E. Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay. J Biochem 1990;108:960–4.