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Clinica Chimica Acta, 58 (1975) 207--214 © Elsevier Scicntific Publishing Company, Amsterdam -- Printed in The Netherlands

CCA 6713

SOLID PHASE RADIOIMMUNOASSAY FOR SERUM FERRITIN

JUNE W. HALLIDAY, K.L. GERA a'l c] L.W. POWEL[,

Department of Medicine, University of Queensland, Royal Brisbane ttospital, Brisbalze. 4029 (Australia) (Received July 1, i974)

Summary A solid-phase " d i r e c t " radio immunoassay which is highly sensitive (detecting less than 0.5 ng/ml) and reproducible is described for the assay of ferritin in h u m a n serum. The m e t h o d makes use of specific anti-ferritin antib o d y prepared using h u m a n liver ferritin. It has the advantage that tubes c o a t e d with specific antiserum m a y be stored for later use and lends itself readily to an a u t o m a t e d washing procedure. The c o n c e n t r a t i o n of ferritin present in the serum of normal males in the population was 56.9 ng/ml (geometric mean) with a 95% c o n f i d e n c e rarLge .~f 18 to 180 ng/ml. In normal females the c o n c e n t r a t i o n was 34.0 ng/ml (geometric mean) with a 95% confi( .~nce range of 10 Lo 143 ng/ml.

Introduction Ferritin has been considered until recently to i)e an iron storage protein found mainly in liver, spleen and b o n e marrow. However, using an i m m u n o radiometric m e t h o d Addison et al. [ ! ] den;onstrated ti~e presence of ferritin in the serum o f normal subjects. T h e y showed that i.he concentrat'.on of serum territin was low in iron deficient subjects and elevated in patients with increased iron stores. S u b s e q u e n t studies using this m e t h o d have confirmed that the serum ferritin c o n c e n t r a t i o n is an accurate inde:: of tc ,al b o d y iron stores [2]. ' The i m m u n o r a d i o m e t r i c m e t h o d makes u.¢., of a horse spleen ferritin imm u n o a b s o r b e n t for the isolation of antihuman ferritin a n t i b o d y . The p u r p o s e of the present r e p o r t is to describe a specific, highly sensitive, solid-phase radioin : , u n o a s s a y for use in the estimation of ferritin in h u m a n serum. The techni.iue is based on the principle first described by Catt and Tregear [3] and requires only very small a m o u n t s of ,25 I-labelled a n t i b o d y to h u m a n liver ferritin. It is sensitive to 0.3 ng ferritin protein per ml serum.

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Methods

Preparation c f human ferritin Ferritin was isolated and purified from normal human liver (obtained at autopsy from healthy young adult accident victims) by a modification of the method of Drysdale and Munro [4]. Tissue was homogenized in four volumes distilled water, heated to 70--75 ~ for 2 minutes, cooled rapidly to 4 ° and centrifuged at 1500 X g for 20 minutes. The pH was then adjusted to 4.6 with 0.2 M acetic acid, at 4 °. After further centrifugation at 1500 X g for 20 minutes the supernatant was adjusted to pH 5.0 and ammonium sulphate added (50% saturation). The precipitate formed was dialysed against 20 mM sodium phosphate buffer pH 7.5 and concentrated by Diaflo XM-100 membrane filtration. The resulting solution was subjected to gel chromatography on sepharose 6B using 20 mM sodium phosphate buffer. The purity of this preparation was confirmed by polyacrylamide gel electrophoresis, staining for both protein and iron.

Preparation of anti-ferritin antiserum Antiserum to human liver ferritin was prepared in rabbits by intramuscular injections of purified human liver ferritin. A 1 : 1 mixture of ferritin solution and Freund's adjuvant containing approximately 1.0 mg of ferritin protein as determined by a modified Lewis, method [5] was injected on three occasions at weekly intervals.

Preparation of purified an ti-ferritin an tibody Sepharose-bound ferritin was prepared according to the method of Perrotto (personal communication). Cyanogen-bromide activated Sepharose 4B (CNBr-sepharose) (Pharmacia, South Seas, Sydney, Australia} was hydrated in 1 mM sodium chloride. To 1.0 g of CNBr-sepharose was added 30 mg purified human liver ferritin dissolved in 0.1 M borate buffer pH 8.0 containing 0.5 M sodium chloride, and the mixture was shaken ovemi~,t,.~ at 4 °. After washing with 1 M ethanolamine p~! 8.4 m 1 mM sodium chloride, the absorbent was poured into a column and was ready for use in the preparation of anti-ferritin antibody. To 20 ml rabbit antiserum to human liver ferritin wa¢ added 20 ml of saturated ammonium sulphate. The precipitate formed by 50% saturation with ammonium sulphate was dialysed against 0.15 M sodium chloride followed by 0.1 M borate troffer ptI 8.0. This globulin fraction was passed through the sepharose-ferritin affinity column and washed with borate buffer. The purified anti-/etdtin antibody was eluted from the column using 0.01 M phosphate buffer pit 6.0 co~taining 3 M sodium thiocyanate, followed immediately by borate buffer pH 8.0. The column elua~e was immediately dialysed against borate buffer pH 8.0 las above). The titre of antibody in tile eluate was determined by gel diffusion against purified human liver ferritin.

lodinataon of anti-ferrilin antibody After removal from the affinity column, ~0--50 ml purified anti-ferritin antibody (protein content 3.0 mg/ml) was iodina;ed with 2 mCi 12sI' using

209

chloramine-T, by the m e t h o d o f - H u n t e r and G r e e n w o o d [ 6 ] . Free iodine was removed by passage through a sephadex G-100 (1 cm × 25 cm) column.

Assay of ferritin Flat b o t t o m e d p o l y s t y r e n e tubes (NC 1, Disposable Products, Adelaide, Australia) were coated by the addition of 500 pl of rabbit antiserum to h u m a n liver ferritin at dilution of 1 : 10 0 0 0 in p h o s p h a t e buffered saline {0.015 M) pH 7.4 containing 0.01% azide and incubated for 24 hours at 20 ° . The c o n t e n t s were then aspirated and the t u b e s washed 10 times with p h o s p h a t e b u f f e r e d saline and air dried. Standard curves were obtained by adding 100 pl o f standard ferritin solution in veronal b u f f e l e d saline (0.05 M veronal, 0.1 M sodium chloride pH 8.0) containing 4% bovine serum albumin to a series of c o a t e d tubes. T w o standard ranges have been used; 0.5 to 10 and 10 to 100 ng/ml, respectively. Quadruplicate standard t u b e s were used in each instance. Normal h u m a n serum (100 pl) diluted 1/10 and 1/20 in veronal buffered saline was added to duplicate tubes. Patients' sera were diluted routinely 1/20 and 1 / 2 0 0 to allow for high ferritin concentrations in some cases. After incubation for 24 hours at 20 ° the c o n t e n t s were aspirated and each t u b e washed 10 times with 2 ml p h o s p h a t e b u f f e r e d saline pH 7.4; 100 /~l 2 s I-labelled anti-ferritin a n t i b o d y containing 80 000 to 100 000 c o u n t s per m i n u t e was added carefully t o the b o t t o m of the tube. After further incubation for 2 hours at 20 ° the c o n t e n t s were again aspirated and the tubes washed 10 times with p h o s p h a t e buffered saline and c o u n t e d in a well-type a u t o m a t i c gamma c o u n t e r (Nuclear Chicago). Since the m e t h o d is a " d i r e c t " assay, the n u m b e r of c o u n t s per t u b e increased with i~creasing a m o u n t s of f e m t i n present. Results

Figs l a and l b show standard curves obtained for 0.5 to 10 ng/ml and 10 to 100.ng/ml, respectively. Individual points were the means of quadruplicate estimations and the standard deviation of the mean is shown for each point.

Non-specific binding In each assay the counts b o u n d in tb,'. ab,;ence o f ferritin i.e. d u e to non-specific binding of protein (bovine serum albumi:l) to the a n t i b o d y , have been subtracted to obtain the values plotted. The mean o f counts b o u n d in the absence of ferritin was 0.04% ± 0.001% (S.D.) of the tetal radioactivity.

Accuracy The recovery of added ferritin was determined by the addition of 0.5, 1.0, 1.5, 2.0 and 2.5 ng/ml, respectively, to the 5 ng/ml standard. Mean recoveries of ferritin were 93% +- 4.9% (S.D.) at the 0.5 ng level, 95% ± 3.7% at the 1.0 ng level, 89% +- 3.6% at the 1.5 ng level, 100% ± 4.1% at the 2.0 ng level and 93 ± 4.1% at the 2.5 ng level.

Sensitivity Sensitivity m a y be defined as the smallest single value which can be distin-

210

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[,'ig. I. S t a n d a r d c u r v e s for h u m a n ferr~tin a . s a y . (a) 1 to 10 n g / m l , ( b ) 10 to 1 0 0 n g / m l . R a b b i t a n t i h u m a n ferritin a n t i s e r t m , (1 in 10 0 0 0 in p h o s p h a t e b u f f e r e d s~dine, p H 7 . 4 ) w a s u s e d to c o a t t u b e s . S t a n d a r d d e v i a t i o n s are s h o w n l o t e a c h p o i n t .

guished from zero. With each set of standards in every assay five " b l a n k " tubes i.e. containing 4% bovine serum albvmin and no ferritin, were included. Sensitivity of the standard curve was then calculated from the 95% confidence limits at the zero point of the standard curve. T w e n t y such curJes allowed a mean sensitivity of 0.31 ng/ml to be calculated.

Specificity Samples were assayed at four dilutio1~s s,.'multaneeusly a~d the results were linear when plotted arithmetically. No significant difference was observed among results obtained at the four dilutions (Fig. 2). The addition of nonradioactive anti-ferritin a n t i b o d y prior to ' 2s I-anti-ferritin a n t i b o d y reduced the final radioactivity to the level of the blank, while the addition o f bovine serum albumin had no effect.

Reproducibility (a) Within batch variation: The ferritin c o n t e n t of 3 different normal sera was determined t w e n t y times in the one assay. The results are shown in Table I. (b) Between batch variation: Duplicate d e t e r m i n a t i o n s were carried o u t on several different, days, on each of 7 different sera (replicate determinations) and the results were highly reproducible {Table i ).

211

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in s u c c e s s i v e d : ! u t i o o s o f f o u r d i f f e r e n t s e r u m s a m p J c s .

Precision Precision was d e t e r m i n e d according to the m e t h o d of Virasoro et al. [ 7 ] . Duplicate d e t e r m i n a t i o n s were performed on 113 samples containing various a m o u n t s of ferritin. Standard deviations (S.D.) for different serum concentrations were calculated according to Snedecor [8] from the differences between duplicate determinations. S.D. = V~E--~/2N where d is the difference between duplicates and N is the n u m b e r of estimations. The precision over va;ious ranges of ferritin c o n c e n t r a t i o n s is shown in Table II. TABLE 1 REPLICATE

Number of deterrai . nations

20 20 20

DETERMINATIONS

.

Within batch . . . . Mean S.D. (ng/ml) (ng/ml) 40.4 202.0 251.0

4.1 15.3 32.3

OF SERUM FERRITIN

.

Number of Between batch . determiC.V. nations Mean S.D. C.V. (%) (ng/ml) (ng/ml) (%) ............................................. ......................... I0 7.5 12.8

5 5 4 6 4 20 12

78.5 73.5 220.0 47.9 67.3 19.8 14.0

14.5 13.5 20.5 7.9 5.3 1.7 1.95

18 18 9 1~~ 7 8.5 13.9

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T A B L E I1 PRECISION OF FERRITIN DETERMINATION Ferritin eoncen;.ration range (ng/ml)

Number pairs

Mcan ferritin concentration (ng/ml)

S.D.*

17 18 10 32 9 19 8

6.2 15.8 32.8 67.4 112.2 290.5 2972.0

0.5,i 2.24 2.9 6.7 5.3 9.5 56.0

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Effect o f variations in the conditions of the assay Incubation with standard ferritin solutions added to antiserum-coated tubes for 24 hours at 20 ° was compared with incubation for 4 hours at 37 °. There was no significant difference in tile final sensitivity of the assay when the

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213

different incubation m e t h o d s were used. Coated t u b e s have been stored for 6 weeks (a) at 4 ° {b) at 20 ° and {c) at --20 °. Assays carried o u t after storage showed that there was no significant variation in the results; in fact a l o w e r blank resulted when stored tubes were used. Likewise identical results were obtained when tubes c o a t e d with rabbit antiserum to h u m a n liver ferritin at a dilution of 1 : 10 000 were c o m p a r e d with those coated with specific antiferritin a n t i b o d y at a dilution of 1 : 5 0 0 0 ( Fig. 3). Assay o f sera o' tained from 40 male and 40 female normal controls {blood bank d o n o r s m:d l a b o r a t o r y staff} has shown that the distribution o f ferritin levels is skewed, as previously de:cribed by J a c o b s et al. [ 2 ] . The geometric mean of the ferritin values obtained in males was 56.9 ng/ml and the 95% confidence range 18 to 180 ng/ml and in females the geometric mean was 34.0 ng/ml with a 95% c o n f i d e n c e range of 10 to 143 ng/ml. Discussion

The m e t h o d described permits rapid and highly sensitive m e a s u r e m e n t of serum ferritin. It avoids the preparation of an i m m u n o a b s o r b e n t as used in previously described m e t h o d s . The use o f a specific a n t i b o d y to h u m a n liver ferritin, labelled with ~2 s I, resulted in a high specific activ ~y which increased the sensitivity of the m e t h o d and allowed e c o n o m i c use o f the a n t i b o u y . Precision, reproducibility, sensitivity, recovery and specificity, d e t e r m i n e d according to the m e t h o d s o f Virasoro et al. [ 7 ] , have been shown to c o m p a r e very favcurably with the previously described i m m u n o a b s o r b e n t m e t h o d [ 1 ] . As shown in Figs l a and l b an acceptable standard curve m a y be obtained using t w o ranges o f ferritir, c o n c e n t r a t i o n , 0.5 to 10 ng/ml and 10 to 100 ng/ml. We have f o u n d the 0.5 to 1(, ng/ml range to be m o s t suitable for routine assay purposes using initial dilutions o f 1 : 10, 1 : 20 and 1 : 200 of test sera. Since tubes stored for up to 6 weeks yielded reproducible results and low levels o f non-specific binding (blanks), large batches o f tubes m a y be c o a t e d with a single batch of antiserum for later use. Results m a y be obtained m o r e rapidly w i t h o u t loss o f sensitivity if incubation after addition of tests and standards is carried o u t at 37 ° for four hours. An a u t o m a t e d p r o c e d u r e for t u b e washing is a simple modification. A n t i b o d y m a y be stored frozen in batches of suitable size for iodination. Porter [9] has- reported ~hat ferritin c o n c e n t r a t i o n as d e t e r m i n e d by an inhibition-type radioimmunoassay was d e p e n d e n t on the iron c o n t e n t o f the ferritin. Using the solid-phase radioimmunoassay, purified apoferritin and ferritin of similar protein c o n t e n t were indistinguishable Thus the level o f ferritin determined by this m e t h o d doe.~ n o t appear to be d e p e n d e n t on the i~-on c o n t e n t of the protein. This i~ com~istent with the finding that horse apoferritin and horse ferritin p r o d u c e i(lentical precipitation curves w h e n rabbit a.ntiserum to either horse spleen ferritin e: apoferritin is used [ 1 0 ] . These data have been confirmed using h u m a n ferritin [ 9 ] . The mean concentrations reported here for normal subjects in o u r population are in agreement with those r e p o r t e d b y Addison et al. [ 1 ] . As in the former s t u d y [1] the values obtained in our s t u d y covered a wide range and the distribution o f these values was skewed.

214

Acknowledgements The au*~hors wish to thank Mr T. Rout for the preparation of purified anti-ferritin antibody. This work was supported in part by the National Health and Medical r,esearch Council and by Ciba-Geigy (Aust.). References 1 G . M . A d d i s o n , M . R . B e a m i s h , C . N . Holes, M. H o d g k i n s , A. J a c o b s a n d P. L l e w e l l i n , I. C l i n . P a t h o l . , 2 5 (1972) 326 2 A. J a c o b s , F. Miller, M. W o r w o o d , M . R B e a m i s h a n d C . A . W a r d r o p . Brit. Meal. J., 4 ( 1 9 7 2 ) 2 0 6 3 K.J. Catt and G.W. Tregear, Science, 158 (1967) 1580 4 J.W. Drysdale and H.N. Munro, Biochem. J.,95 (1965) 851 5 G . R . S e h a l t e r l e a n d R . L . P o l l a c k , A n a l . B i o c h e m . , 51 ( 1 9 7 3 ) 6 5 4 6 W.M. f l u n t e r a n d F . C . G r e e n w o o d , N a t u r e , 1 9 4 ( 1 9 6 3 ) 4 9 5 7 E. V i r a s o r o , G. C o p i n s c h i , O . D . B r u n o a n d R. L e c l e r c q , Clin. C h i m . A c t a , 31 ( 1 9 7 1 ) 2 9 4 8 G.W. S n e d e c o r , B i o m e t r i c s , 8 ( 1 9 5 2 ) 8 5 9 F.S. P o r t e r , J. L a b . Clin. Med., 8 3 ( 1 9 7 4 ) 1 4 7 I 0 A. M a z u r a n d E. S h o r t , J. Biol. C h e m . . 1 8 2 ( 1 9 5 0 ) 6 0 7