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Immunoradiometric and electroimmuno assay of increased ferritin and apoferritin levels in serum
Pathology (1979), 11, pp. 59-65
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IMMUNORADIOMETRIC AND ELECTROIMMUNO ASSAY OF INCREASED FERRlTlN AND APOFERRITIN LEVELS IN SERUM R. D. BROWN,G. C. MEEHAN,H. ROBINAND H. KRONENBERG Haematology Department, Royal Prince AEfred Hospital, Sydney
Summary Ferritin in serum from patients with increased serum ferritin levels has been studied both quantitatively and qualitatively. All techniques utilized in these studies are suitable to be used as routine screening tests for large numbers of patients. Electroimmuno assay (EIA) has been compared with the solid phase immunoradiometric (IRMA) assay as a technique to determine serum ferritin concentration (r=0.99) and is suggested as a useful. alternative when determining ferritin concentrations above 500 pg/l. Iron stained EIA gels have been used to indicate the iron content of the ferritin molecule in sera. This simple screening test has demonstrated that apoferritin is found more often than iron-rich ferritin in the serum of patients with elevated serum ferritin levels. lmmunoelectrophoresis precipitin bands suggest the heterogeneity of ferritin in serum from different patients.
Ferritin is the major iron storage protein of the body. Each ferritin molecule consists of a spherical protein shell, termed apoferritin and a central core containing a variable amount of ferric hydroxyphosphate. Apoferritin has a molecular weight of 450,000 daltons while ferritin molecules filled with iron may be up to 900,000 daltons. Ferritin is present in most tissues in the form of a characteristic tissue isoferritin. While high concentrations of ferritin are found in the liver and spleen, the concentration of ferritin in serum is normally very low (Crichton, 1971). Serum ferritin levels appear to indicate the size of the reticuloendothelial iron stores in most situations. Recently, a solid phase immunoradiometric assay (IRMA) has been introduced (Miles et al., 1974; Halliday et al., 1975) to determine serum ferritin concentrations. Previous methods could only detect ferritin in serum when the concentration was greatly increased (Reissmann & Dietrich, 1956; Beamish et al., 1971). The IRMA has been particularly useful for the evaluation of iron stores of patients with iron deficiency anaemia (Jacobs et al., 1972; Bentley & Jacobs, 1975), chronic renal failure (Hussein et al., 1975), thalassaemia (Letsky et al., 1974), idiopathic haemochromatosis (Jacobs et al., 1972), rheumatoid arthritis (Bentley & Williams, 1974) and in children (Siimes et al., 1974). However, because increased serum ferritin levels have been found in patients with a wide
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
60 BROWN C t 01. Pathology (19791, 11, January range of disorders, it is now realized that an increased serum ferritin level does not necessarily indicate elevated total iron stores. Bieber & Bieber (1973) have suggested that estimation of serum ferritin may be useful as a non-specific indicator of malignant disease. Recently, it has been demonstrated that the ferritin in serum from 2 patients with haemochromatosis and another with transfusion siderosis had a very low iron content (Arosio e t a / . . 1976: Worwood et a/., 1975). We report here some simple quantitative and qualitative studies of ferritin in sera from patients with increased serum ferritin levels. The immunoradiometric assay is compared with an electroimmuno assay (EIA) technique (Laurell, 1966, 1972) which has been adapted to determine not only the concentration of ferritin in serum, but also to indicate the iron content of the protein molecule.
MATERIALS AND METHODS Puricwr .selrcriorr Serum ferritin levels were determined by solid phase immunoradiometric assay as part of the haematological investipations of more than 800 patients. 30 of whom had serum ferritin levels above 1000 pgil. These 30 patients were the subjects of the studies reported below. All patients with serum ferritin levels above 1000 pg/l were selected for further investigation. This group of patients had a wide range of disorders ( 3 haemachromatosis, 12 acute leukaemia, 2 chronic myeloid leukaemia. 2 myelofibrosis. 4 alcoholic liver disease, 1 myeloma, 1 eosinophilia, 1 Ca lung, 1 lymphoma, 1 chronic lymphatic leukaemia. I Ca stomach and 1 refractory anaemia).
Hutnun liwr f i w i r i i i
Human liver ferritin was prepared from normal livers obtained at autopsy by a modification of the method of Drysdale & Munro (196s). Partially purified liver extract was chromatographed on a Sepharose 6 8 column using 0.02M phosphate buffer ( p H 7.5). Ferritin purity was confirmed by electrophoresis and protein was quantitated by the Lowry technique (Lowry er d.. 1951). .1nti-fcrririn uttti.rer.uni Antiserum was prepared b) injecting rabbits intramuscularly with 1 ml of alumina containing 1 mg of purified ferritin. This was repeated every 10 days for 40 days. Rabbits were then bled after a further 10 days. Purified ferritin was coupled to diazocellulose to provide a ferritin immunoadsorbant. Purified anti-ferritin antibody was prepared by incubation with ferritin immunoadsorbant. After iodination of the immunoadsorbant antibod) complex with I mCiiZ5lin the presence of chioralnine T. the purified labelled antibody was eluted off the immunoadsorbant at pH 2. Polystlrene tubes were coated with rabbit antiserum (500 pl) diluted 1 in 10,000in 0.2M N a H C 0 3 at pH 9.2 for 24 h at 4 C. The tubes were washed 3 times with 0.5M veronal buffered saline p H 8.0 containing 0.4",, bovine seruni albumin (BSA) and then 3 times writh distilled water. Inmn~icncira~i~~riIrrr.ic. us.sa:a?1 I R M A : The assay was performed b\ pipetting 200 pl of ferritin standard or I in 20 diluted serum into the bottom of the antibody coated tubes. Sera with high ferritin values were diluted I in 200. 1 in 2000 or 1 in 20,000 if necessary in BSA buffer. After 6 h incubation at 37 C the tubes were washed 3 times with ESA buffer and then 200 pl of '*'I labelled anti-ferritin antibody containing 10.000c.p.m. was added to each tube. The tubes were incubated at 4°C for 15 h and washed 3 times with 0.04M phosphate buffer prior to counting in a gamma counter (Packard). A logit-log transformation o f the dose-response curve was plotted for each assay. Elrc~rr.oirnniunoussuj' i EIA 1
Electroimmunodirfusion was performed according to the technique of Laurell (1966, 1972). Wells (3 mm) were punched in 0.9",, agarose gels (70 mm x 70 mm) in 0.1M diethylbarbiturate acetate buffer p H 8.2, on 80 mm x 80 mm glass plates. Antiserum (0.5"")was added to the aparose at SO C immediately prior to pouring. Five j t l of standard ferritin or unknown serum was added to each well with a microlitre syringe (SGE). Electrophoresis was performed immediately after sample application for 24 h at 6mA per plate with 0.1M diethyibarbiturate acetate buffer at pH 8.2. Gels were washed in distilled water. dried, stained with Coomassie blue
IRMA AND EIA OF FERRITIN
61
for 30 min at 56‘C and cleared with acid alcohol. Duplicate plates were stained with Perl’s stain to demonstrate the presence or absence of iron. The lengths of the immunoprecipitates (‘rockets’) were measured and unknown samples compared with a series of standards on the same plate.
Immunoelectrophoresis Immunoelectrophoresis was performed in 1.2%agarose in 0.1M veronal barbital buffer pH 8.6 on glass slides using an L.K.B. system. Rabbit anti-ferritin antiserum was used to form a ferritin precipitin band with purified liver ferritin and serum.
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RESULTS
Figure 1 shows immunoprecipitates from a ferritin electroimmuno assay. The height of each immunoprecipitate was measured and unknown samples were compared with a series of standards. Figure 2 illustrates a standard curve for this assay. Duplicate plates were stained for iron. Immunoprecipitates containing iron stained faintly yet discretely. The staining intensity of the immunoprecipitates which were positive for iron appeared to be similar. A 0.2% antibody concentration was used when the ferritin concentration was between 500 and 2000 pg/l in order to increase the height of the rocket. However, the immunoprecipitates were quite faint when antibody concentration was so low. Results of the reproducibility of the IRMA and the EIA are compared in Table 1. The ferritin content of serum from 5 different patients with increased serum ferritin levels was determined 5 times on different days by both IRMA and EIA. While the EIA appears to be the technique of choice for serum ferritin levels above the normal range, the reproducibility of the IRMA was better for the 5 additional normal patients shown in Table 1. Figure 3 illustrates the correlation between serum ferritin levels determined by IRMA and EIA (r = 0.99) of all sera studied. The figure also demonstrates that iron was detected in the immunoprecipitate of 9 out of 42 sera studied. These 9 samples were from 6 different patients (2 acute leukaemia, 1 myeloma, 1 eosinophilia, 1 chronic myeloid leukaemia and 1 Ca lung), 5 of whom had hepatomegaly and/or impaired liver function. However, hepatomegaly and impaired liver function were found in the same proportion among the patients whose immunoprecipitates did not stain for iron. Human liver ferritin was purified from 3 different liver samples after autopsy. All 3 ferritin
FIG. 1 EIA immunoprecipitate ‘rockets’. Standard ferritin 5 mg/l, 10 mg/l, 15 mg/l and 20 mg/l
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62
BROWN
ef al.
Pathology (1979), 11, January
FERRlTlN CONCENTRATlON (mgil)
FIG.2 EIA standard curve. The height of each 'rocket' is plotted against the ferritin concentration in each well
0 0 0 .
0
:
r = 0.99
00
10 50 FERRlTlN x 1o3[~g/i IMMUNORADIOMETRIC ASSAY
5
100
FIG.3 Correlation between EIA and IRMA serum ferritin estimations. Open circles represent sera where no iron was detected in EIA rockets. Closed circles represent sera where iron was detected in EIA rockets
IRMA AND EIA OF FERRITIN
TABLEI
1
~~
Immunoradiometric assay
SD Pgli
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
Pa tien t
I 2 3 4 5 6 7 8 9 10 SD CV
=
=
63
Reproducibility of serum ferritin estimations
3,300 4,600 6,200 12,200 14,700
22 45 149
850 850
900 3,100 4,200 2 4 3 3 10
~
Electroimmuno assay
cv
Mean
%
f d
26 18 15 25 28 40 23 14 1 1
4,100 5,900 6,300 12,200 15,700
190 300 580 290 1,100
5 5
9 3 7
Standard deviation Coefficient of variation
preparations formed a characteristic precipitin band during immunoelectrophoresis with rabbit anti-ferritin. All such precipitin lines stained with Perl’s stain, thus indicating a large content of iron in liver ferritin. Sera from patients with high serum ferritin levels also formed immunoelectrophoretic precipitin bands. While the different liver ferritin preparations formed the same precipitin band, there were small differences in the precipitin bands of ferritin from different sera. DISCUSSION
Immunological assays of ferritin measure both the iron-containing protein and apoferritin. The IRMA is the most sensitive technique available to measure serum ferritin concentration and the only satisfactory technique which can detect ferritin in normal serum. However, we have demonstrated that EIA is an alternative to the IRMA for accurate quantitation of ferritin protein in sera of patients with increasedserum ferritin levels. EIA is simple, accurate, reasonably rapid and can also give some indication of the average iron content of the molecule. Thus, for these patients, ETA has several advantages over the IRMA. Reissmann & Dietrich (1956) detected iron-rich ferritin in the plasma of 8 to 17 patients with infective hepatitis. Worwood et al. (1975) using sucrose density gradient centrifugation demonstrated that ferritin from the serum of a patient with haemochromatosis was more like apoferritin than the iron-rich protein found in liver cells. The techniques used in these previous studies are too complex to use as routine screening tests, so the number of patients studied was limited. The techniques used in our studies are simple enough to be used as screening tests for large numbers of patients if necessary. Circumstantial evidence suggests that ferritin circulating in serum has been released from reticuloendothelial cells (Jacobs & Worwood, 1975). Other studies (Worwood et al., 1975) indicate that of all the isoferritins found in different tissues, serum ferritin is most like the ferritin found in leucocytes. Increased ferritin concentrations in sera from patients with leukaemia are probably due to the increased concentration of ferritin in leukaemic cells (Worwood et al., 1974). The increased serum ferritin levels in patients with acute hepatocellular disorders (Reissmann & Dietrich, 1956) suggest that serum ferritin may be released from necrotic liver cells. All these observations suggest that the origin and perhaps
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64
BROWN el
af.
Pathology (1979), 11, January
the fate of ferritin in serum is manifold. Our observation that sera from different patients can form slightly different immunoelectrophoresis patterns against rabbit anti-ferritin antiserum suggests the heterogeneity of serum ferritin and thus also that ferritin in serum originates from many sources. As ferritin and apoferritin have the same electrophoretic mobilities (Mazur & Shorr, 1950) and different tissue ferritins have different electrophoretic mobilities (Allfrey et a/., 1967), the differences observed in the immunoelectrophoretic patterns of serum ferritin in our studies may indicate that the heterogeneity of ferritin in serum is due not only to the relative iron content of the molecule, but also to differences in the protein core itself. All sera studied were abnormal. Whether or not circulating ferritin protein in normal serum is in the form of apoferritin or iron-rich ferritin is still not known. Furthermore, it is not known which specific tissue isoferritins are found in normal serum. A clarification of these points may help our understanding not only of the origin and destiny of ferritin in serum, but also the role of serum ferritin in iron metabolism. We thank Dr R. Kimber, Dr B. Rudzki and Mr B. Blundell, ACKNOWLEDGEMENTS Institute of Medical and Veterinary Science, Adelaide, for their advice and assistance with the IRMA and Mrs S. Speers and Mr P. Power for their advice and assistance with the EIA. Address for reprint requests: R.D.B., Haematology Department. Royal Prince Alfred Hospital, Camperdown, New South Wales 2050. Australia
References ALLFREY, C. P.. LYNCH,E. C. & WHITELEY, C . E. (1967): Characteristics of ferritin isolated from human marrow, spleen. liver and reticulocytes. J . Lab. Clin. Med. 70, 419428. J . W. (1976): AROSIO,P., YOKOTA, M. & DRYSDALE, Characterization of serum ferritin in iron overload: possible identity to natural apoferritin. BF.J. Haemafol. 36, 199-207. M. R., LLEWELLIN, P. &JACOBS, A. (1971): BEAMISH. .4 rapid method for the detection of ferritin in serum. J . Clin. Pathol. 24, 581-582. I).P. &JACOBS. A. (1975): Accumulation of BEBTLEY, storage iron in patients treated for iron deficiency anaemia. Br. Med. J . 2, 64-66. D. P. & WILLIAMS, P. (1974): Serum ferritin BENTLEY, concentration as an index of storage iron in rheumatoid arthritis. J . Clin. Parho/. 27, 786-788. C . P. & BIEBER.M. M. (1973): Detection of BIEBER, ferritin as a circulating tumor-associated antigen in Hodgkin’s disease. Natl. Cancer Insr. Monogr. 36. 147-157. CRICHTON, R. R. (1971): Ferritin: structure, synthesis and function. N . Engl. J. Med. 284, 1413-1422. H. M. (1965): Small-scale DRYSDALE. J. W. & MUNRO. isolation of ferritin for the assay of the incorporation of “C-labelled amino acids. Biorheni. J . 95. 851-858.
HALLIDAY. J. W., GERA,K. L. & POWELL,L. W. (1975): Solid phase radioimmunoassay for serum ferritin. Clin. Chim. Acta. 58, 207-214. HUSSEIS,S.,hmo, J., OSHEA,M. et al. (1975): Serum ferritin assay and iron status in chronic renal failure and haemodialysis. Br. Med. J. 1,546-548. M. (1975): Ferritin in serum. JACOBS. A. & WORWOOD, N . Engl. J . Med. 292, 951-956. M. et al. (1972): JACOBS. A., MILLER,F., WORWOOD, Ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. Br. Med. J . 4, 206-208. C . B. (1966): Quantitative estimation of LAURELL, proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15, 45-52. C. B. (1972): Electroimmunoassay. Scund. LAURELL, J . Clin. Lab. Invest. 29, Suppl. 124, 21-37. M. & FLYNN, LETSKY,E. A,. MILLER,F., WORWOOD, D. M. (1974): Serum ferritin in children with thalassaemia regularly transfused. J. Clin. Puthol. 27, 652455. N. J., FARR,A. L. & LOWRY,0. H., ROSENBROUGH, RANDALL, R. M. (1951): Protein measurement with the fohn phenol reagent. J. B i d . Chem. 193, 265-275.
IRMA A N D EIA OF FERRITIN
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
MAZUR,A. & SHORR,E. (1950): A quantitative immunochemical study of ferritin and its relation tcv the hepatic vasodepressor material. J. Biol. Chem. 182, 607-627. MILES,L. E. M., LIPSCHITZ, D. A,, BIEBER,C. P. & COOK,J. D. (1974): Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal. Biochem. 61, 209-224. REISSMANN, K. R. & DIETRICH, M. R. (1956): On the presence of ferritin in the peripheral blood of patients with hepatocellular disease. J. Clin. Invest. 35. 588-595.
65
SIIMES,M. A,, ADDIEGO, J. E. & DALLMAN, P. R. (1974): Ferritin in serum: diagnosis of iron deficiency and iron overload in infants and children. Blood. 43, 581-590. WORWOOD, M., AHERNE, W., DAWKINS, S. &JACOBS, A. (1975): The characteristics of ferritin from human tissues, serum and blood cells. Clin. Sci. Mol. Med. 48, 441451. WORWOOD,M., SUMMERS,M., MILLER,F. et al. (1974): Ferritin in blood cells from normal subjects and patients with leukaemia. Br. J. Haematol. 28, 27-35.
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
IMMUNORADIOMETRIC AND ELECTROIMMUNO ASSAY OF INCREASED FERRlTlN AND APOFERRITIN LEVELS IN SERUM R. D. BROWN,G. C. MEEHAN,H. ROBINAND H. KRONENBERG Haematology Department, Royal Prince AEfred Hospital, Sydney
Summary Ferritin in serum from patients with increased serum ferritin levels has been studied both quantitatively and qualitatively. All techniques utilized in these studies are suitable to be used as routine screening tests for large numbers of patients. Electroimmuno assay (EIA) has been compared with the solid phase immunoradiometric (IRMA) assay as a technique to determine serum ferritin concentration (r=0.99) and is suggested as a useful. alternative when determining ferritin concentrations above 500 pg/l. Iron stained EIA gels have been used to indicate the iron content of the ferritin molecule in sera. This simple screening test has demonstrated that apoferritin is found more often than iron-rich ferritin in the serum of patients with elevated serum ferritin levels. lmmunoelectrophoresis precipitin bands suggest the heterogeneity of ferritin in serum from different patients.
Ferritin is the major iron storage protein of the body. Each ferritin molecule consists of a spherical protein shell, termed apoferritin and a central core containing a variable amount of ferric hydroxyphosphate. Apoferritin has a molecular weight of 450,000 daltons while ferritin molecules filled with iron may be up to 900,000 daltons. Ferritin is present in most tissues in the form of a characteristic tissue isoferritin. While high concentrations of ferritin are found in the liver and spleen, the concentration of ferritin in serum is normally very low (Crichton, 1971). Serum ferritin levels appear to indicate the size of the reticuloendothelial iron stores in most situations. Recently, a solid phase immunoradiometric assay (IRMA) has been introduced (Miles et al., 1974; Halliday et al., 1975) to determine serum ferritin concentrations. Previous methods could only detect ferritin in serum when the concentration was greatly increased (Reissmann & Dietrich, 1956; Beamish et al., 1971). The IRMA has been particularly useful for the evaluation of iron stores of patients with iron deficiency anaemia (Jacobs et al., 1972; Bentley & Jacobs, 1975), chronic renal failure (Hussein et al., 1975), thalassaemia (Letsky et al., 1974), idiopathic haemochromatosis (Jacobs et al., 1972), rheumatoid arthritis (Bentley & Williams, 1974) and in children (Siimes et al., 1974). However, because increased serum ferritin levels have been found in patients with a wide
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
60 BROWN C t 01. Pathology (19791, 11, January range of disorders, it is now realized that an increased serum ferritin level does not necessarily indicate elevated total iron stores. Bieber & Bieber (1973) have suggested that estimation of serum ferritin may be useful as a non-specific indicator of malignant disease. Recently, it has been demonstrated that the ferritin in serum from 2 patients with haemochromatosis and another with transfusion siderosis had a very low iron content (Arosio e t a / . . 1976: Worwood et a/., 1975). We report here some simple quantitative and qualitative studies of ferritin in sera from patients with increased serum ferritin levels. The immunoradiometric assay is compared with an electroimmuno assay (EIA) technique (Laurell, 1966, 1972) which has been adapted to determine not only the concentration of ferritin in serum, but also to indicate the iron content of the protein molecule.
MATERIALS AND METHODS Puricwr .selrcriorr Serum ferritin levels were determined by solid phase immunoradiometric assay as part of the haematological investipations of more than 800 patients. 30 of whom had serum ferritin levels above 1000 pgil. These 30 patients were the subjects of the studies reported below. All patients with serum ferritin levels above 1000 pg/l were selected for further investigation. This group of patients had a wide range of disorders ( 3 haemachromatosis, 12 acute leukaemia, 2 chronic myeloid leukaemia. 2 myelofibrosis. 4 alcoholic liver disease, 1 myeloma, 1 eosinophilia, 1 Ca lung, 1 lymphoma, 1 chronic lymphatic leukaemia. I Ca stomach and 1 refractory anaemia).
Hutnun liwr f i w i r i i i
Human liver ferritin was prepared from normal livers obtained at autopsy by a modification of the method of Drysdale & Munro (196s). Partially purified liver extract was chromatographed on a Sepharose 6 8 column using 0.02M phosphate buffer ( p H 7.5). Ferritin purity was confirmed by electrophoresis and protein was quantitated by the Lowry technique (Lowry er d.. 1951). .1nti-fcrririn uttti.rer.uni Antiserum was prepared b) injecting rabbits intramuscularly with 1 ml of alumina containing 1 mg of purified ferritin. This was repeated every 10 days for 40 days. Rabbits were then bled after a further 10 days. Purified ferritin was coupled to diazocellulose to provide a ferritin immunoadsorbant. Purified anti-ferritin antibody was prepared by incubation with ferritin immunoadsorbant. After iodination of the immunoadsorbant antibod) complex with I mCiiZ5lin the presence of chioralnine T. the purified labelled antibody was eluted off the immunoadsorbant at pH 2. Polystlrene tubes were coated with rabbit antiserum (500 pl) diluted 1 in 10,000in 0.2M N a H C 0 3 at pH 9.2 for 24 h at 4 C. The tubes were washed 3 times with 0.5M veronal buffered saline p H 8.0 containing 0.4",, bovine seruni albumin (BSA) and then 3 times writh distilled water. Inmn~icncira~i~~riIrrr.ic. us.sa:a?1 I R M A : The assay was performed b\ pipetting 200 pl of ferritin standard or I in 20 diluted serum into the bottom of the antibody coated tubes. Sera with high ferritin values were diluted I in 200. 1 in 2000 or 1 in 20,000 if necessary in BSA buffer. After 6 h incubation at 37 C the tubes were washed 3 times with ESA buffer and then 200 pl of '*'I labelled anti-ferritin antibody containing 10.000c.p.m. was added to each tube. The tubes were incubated at 4°C for 15 h and washed 3 times with 0.04M phosphate buffer prior to counting in a gamma counter (Packard). A logit-log transformation o f the dose-response curve was plotted for each assay. Elrc~rr.oirnniunoussuj' i EIA 1
Electroimmunodirfusion was performed according to the technique of Laurell (1966, 1972). Wells (3 mm) were punched in 0.9",, agarose gels (70 mm x 70 mm) in 0.1M diethylbarbiturate acetate buffer p H 8.2, on 80 mm x 80 mm glass plates. Antiserum (0.5"")was added to the aparose at SO C immediately prior to pouring. Five j t l of standard ferritin or unknown serum was added to each well with a microlitre syringe (SGE). Electrophoresis was performed immediately after sample application for 24 h at 6mA per plate with 0.1M diethyibarbiturate acetate buffer at pH 8.2. Gels were washed in distilled water. dried, stained with Coomassie blue
IRMA AND EIA OF FERRITIN
61
for 30 min at 56‘C and cleared with acid alcohol. Duplicate plates were stained with Perl’s stain to demonstrate the presence or absence of iron. The lengths of the immunoprecipitates (‘rockets’) were measured and unknown samples compared with a series of standards on the same plate.
Immunoelectrophoresis Immunoelectrophoresis was performed in 1.2%agarose in 0.1M veronal barbital buffer pH 8.6 on glass slides using an L.K.B. system. Rabbit anti-ferritin antiserum was used to form a ferritin precipitin band with purified liver ferritin and serum.
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
RESULTS
Figure 1 shows immunoprecipitates from a ferritin electroimmuno assay. The height of each immunoprecipitate was measured and unknown samples were compared with a series of standards. Figure 2 illustrates a standard curve for this assay. Duplicate plates were stained for iron. Immunoprecipitates containing iron stained faintly yet discretely. The staining intensity of the immunoprecipitates which were positive for iron appeared to be similar. A 0.2% antibody concentration was used when the ferritin concentration was between 500 and 2000 pg/l in order to increase the height of the rocket. However, the immunoprecipitates were quite faint when antibody concentration was so low. Results of the reproducibility of the IRMA and the EIA are compared in Table 1. The ferritin content of serum from 5 different patients with increased serum ferritin levels was determined 5 times on different days by both IRMA and EIA. While the EIA appears to be the technique of choice for serum ferritin levels above the normal range, the reproducibility of the IRMA was better for the 5 additional normal patients shown in Table 1. Figure 3 illustrates the correlation between serum ferritin levels determined by IRMA and EIA (r = 0.99) of all sera studied. The figure also demonstrates that iron was detected in the immunoprecipitate of 9 out of 42 sera studied. These 9 samples were from 6 different patients (2 acute leukaemia, 1 myeloma, 1 eosinophilia, 1 chronic myeloid leukaemia and 1 Ca lung), 5 of whom had hepatomegaly and/or impaired liver function. However, hepatomegaly and impaired liver function were found in the same proportion among the patients whose immunoprecipitates did not stain for iron. Human liver ferritin was purified from 3 different liver samples after autopsy. All 3 ferritin
FIG. 1 EIA immunoprecipitate ‘rockets’. Standard ferritin 5 mg/l, 10 mg/l, 15 mg/l and 20 mg/l
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
62
BROWN
ef al.
Pathology (1979), 11, January
FERRlTlN CONCENTRATlON (mgil)
FIG.2 EIA standard curve. The height of each 'rocket' is plotted against the ferritin concentration in each well
0 0 0 .
0
:
r = 0.99
00
10 50 FERRlTlN x 1o3[~g/i IMMUNORADIOMETRIC ASSAY
5
100
FIG.3 Correlation between EIA and IRMA serum ferritin estimations. Open circles represent sera where no iron was detected in EIA rockets. Closed circles represent sera where iron was detected in EIA rockets
IRMA AND EIA OF FERRITIN
TABLEI
1
~~
Immunoradiometric assay
SD Pgli
Pathology Downloaded from informahealthcare.com by Nyu Medical Center on 02/07/15 For personal use only.
Pa tien t
I 2 3 4 5 6 7 8 9 10 SD CV
=
=
63
Reproducibility of serum ferritin estimations
3,300 4,600 6,200 12,200 14,700
22 45 149
850 850
900 3,100 4,200 2 4 3 3 10
~
Electroimmuno assay
cv
Mean
%
f d
26 18 15 25 28 40 23 14 1 1
4,100 5,900 6,300 12,200 15,700
190 300 580 290 1,100
5 5
9 3 7
Standard deviation Coefficient of variation
preparations formed a characteristic precipitin band during immunoelectrophoresis with rabbit anti-ferritin. All such precipitin lines stained with Perl’s stain, thus indicating a large content of iron in liver ferritin. Sera from patients with high serum ferritin levels also formed immunoelectrophoretic precipitin bands. While the different liver ferritin preparations formed the same precipitin band, there were small differences in the precipitin bands of ferritin from different sera. DISCUSSION
Immunological assays of ferritin measure both the iron-containing protein and apoferritin. The IRMA is the most sensitive technique available to measure serum ferritin concentration and the only satisfactory technique which can detect ferritin in normal serum. However, we have demonstrated that EIA is an alternative to the IRMA for accurate quantitation of ferritin protein in sera of patients with increasedserum ferritin levels. EIA is simple, accurate, reasonably rapid and can also give some indication of the average iron content of the molecule. Thus, for these patients, ETA has several advantages over the IRMA. Reissmann & Dietrich (1956) detected iron-rich ferritin in the plasma of 8 to 17 patients with infective hepatitis. Worwood et al. (1975) using sucrose density gradient centrifugation demonstrated that ferritin from the serum of a patient with haemochromatosis was more like apoferritin than the iron-rich protein found in liver cells. The techniques used in these previous studies are too complex to use as routine screening tests, so the number of patients studied was limited. The techniques used in our studies are simple enough to be used as screening tests for large numbers of patients if necessary. Circumstantial evidence suggests that ferritin circulating in serum has been released from reticuloendothelial cells (Jacobs & Worwood, 1975). Other studies (Worwood et al., 1975) indicate that of all the isoferritins found in different tissues, serum ferritin is most like the ferritin found in leucocytes. Increased ferritin concentrations in sera from patients with leukaemia are probably due to the increased concentration of ferritin in leukaemic cells (Worwood et al., 1974). The increased serum ferritin levels in patients with acute hepatocellular disorders (Reissmann & Dietrich, 1956) suggest that serum ferritin may be released from necrotic liver cells. All these observations suggest that the origin and perhaps
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Pathology (1979), 11, January
the fate of ferritin in serum is manifold. Our observation that sera from different patients can form slightly different immunoelectrophoresis patterns against rabbit anti-ferritin antiserum suggests the heterogeneity of serum ferritin and thus also that ferritin in serum originates from many sources. As ferritin and apoferritin have the same electrophoretic mobilities (Mazur & Shorr, 1950) and different tissue ferritins have different electrophoretic mobilities (Allfrey et a/., 1967), the differences observed in the immunoelectrophoretic patterns of serum ferritin in our studies may indicate that the heterogeneity of ferritin in serum is due not only to the relative iron content of the molecule, but also to differences in the protein core itself. All sera studied were abnormal. Whether or not circulating ferritin protein in normal serum is in the form of apoferritin or iron-rich ferritin is still not known. Furthermore, it is not known which specific tissue isoferritins are found in normal serum. A clarification of these points may help our understanding not only of the origin and destiny of ferritin in serum, but also the role of serum ferritin in iron metabolism. We thank Dr R. Kimber, Dr B. Rudzki and Mr B. Blundell, ACKNOWLEDGEMENTS Institute of Medical and Veterinary Science, Adelaide, for their advice and assistance with the IRMA and Mrs S. Speers and Mr P. Power for their advice and assistance with the EIA. Address for reprint requests: R.D.B., Haematology Department. Royal Prince Alfred Hospital, Camperdown, New South Wales 2050. Australia
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IRMA A N D EIA OF FERRITIN
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SIIMES,M. A,, ADDIEGO, J. E. & DALLMAN, P. R. (1974): Ferritin in serum: diagnosis of iron deficiency and iron overload in infants and children. Blood. 43, 581-590. WORWOOD, M., AHERNE, W., DAWKINS, S. &JACOBS, A. (1975): The characteristics of ferritin from human tissues, serum and blood cells. Clin. Sci. Mol. Med. 48, 441451. WORWOOD,M., SUMMERS,M., MILLER,F. et al. (1974): Ferritin in blood cells from normal subjects and patients with leukaemia. Br. J. Haematol. 28, 27-35.