Upsala J Med Sci 82: 15-19, 1977 

A Solid Phase Radioimmunoassay Method for Ferritin 
in Serum using 1251-labelled Ferritin 

L E I F  WIDE and GUNNAR BIRGEGARD 

From the Departments of Clinical Chemistry and Internal Medicine, 
University Hospital, Uppsala, Sweden 

ABSTRACT 
A solid phase radioimmunoassay method for ferritin in 
serum was developed using ferritin conjugated with an 
1251-labelled ester. The method was based upon competitive 
inhibition utilizing the free antigen-binding sites of anti- 
bodies to ferritin bound to a ferritin which had been chemi- 
cally coupled to insoluble polysaccharide particles. The 
method was evaluated with regard to specificity, sensitivity, 
precision and practicability and was found to be a robust 
method suitable for the assay of ferritin in human serum in 
clinical studies. There was a significant (P<O.OOl) differ- 
ence between the ferritin levels in healthy men and women, 
mean values 100 and 66 arb U/I respectively. Male blood 
donors had significantly (P<0.002) lower ferritin levels in 
serum (mean 68.5 arb U/l) than other healthy men. Patients 
with iron overload or hepatic damage had high values, as 
expected, and the serum levels of ferritin reflected changes 
in iron deposits during phlebotomy and iron treatment in 
3 healthy men. 

INTRODUCTION 

In 1972 an immunoradiometric assay for ferritin in 
serum was described by Addison et al. (1). Since 
then several laboratories have used this method 
(8, 9, 1 1, 14) for which a production of an immuno- 
sorbent for ferritin antibody, labelling of the anti- 
body and subsequent elution of the labelled anti- 
body is necessary. Even if not all workers find the 
immunoradiometnc assay for ferntin as “exasperat- 
ingly difficult” t o  handle as Crosby (3, there is a 
need for a simpler method. Radioimmunoassays 
employing labelled antigens instead of labelled anti- 
bodies are commonly used in many laboratories 
for the assay of different antigens. Ferritin has been 
labelled with lZ5I by the chloramine-T technique but 
only to a very low specific activity (12). Attempts 
to label ferritin by that method to high specific ac- 
tivity were unsuccessful in our laboratory. The 
present work presents a method for measuring fer- 
ritin in serum by using ferntin labelled with a p -  

hydroxyphenylpropionic acid, N-hydroxysuccin- 
imid ester, which had been iodinated according to 
Bolton & Hunter (3), in a radioimmunoassay sys- 
tem with antibodies coupled to a solid phase. 

MATERIALS AND METHODS 
Ferritin preparation. Ferritin was purified from human 
spleen by a modification of the method described by Drys- 
dale & Munro (7), including a final repeated chromato- 
graphy on Sepharose 6B. The preparation was kindly 
supplied by Dr Tor Olofsson, University Hospital, Lund, 
Sweden. This preparation was used for immunization, 
for labelling with lZ5I and as a provisional reference stand- 
ard preparation. 

Antisera to ferritin. Three different antisera were com- 
pared. Two antisera were raised in two rabbits by weekly 
injections for 4 weeks with 0.25 mg of the ferritin prepara- 
tion in Freund‘s adjuvant. The animals were bled 3 weeks 
after the last injection. The third antiserum was a com- 
mercial product from Behringwerke, West Germany. 

Labelling of ferritin. To 400 mg of p-hydroxyphenyl- 
propionic acid N-hydroxysuccinimide ester was added 
2 mCi N a T  for labelling of the ester according to the 
technique of Bolton & Hunter (3). The iodinated ester 
was conjugated to 15 p g  of the ferritin preparation by in- 
cubation at 0°C for 40 min. The labelled product was puri- 
fied by chromatography on a Sephadex G 150 column. 
Two peaks with labelled material (apart from the free 
iodine) were obtained. The first peak was eluated with 
the void volume and contained almost all immunoreactive 
material. The second peak followed immediately after the 
first and had a very low immunoreactivity with the anti- 
ferritin antibodies. The radioactivity bound to immuno- 
reactive material comprised less than 30% of the total 
proteinbound radioactivity. 

Solidphase coupling of anti-ferrifin. For direct coupling 
of antibodies 5 to 10 p1 of the antisera were coupled to 
100 mg of CNBr-activated ultrafine Sephadex@ or micro- 
crystalline cellulose as previously described (17). Anti- 
bodies were also indirectly coupled (19), i.e. ferritin (200 
pg) was first coupled to the CNBr-activated particles 
(100 mg) and antifemtin-antibodies were then bound to 
the coupled ferritin. The particles were suspended in 
assay buffer solution. 

Upsulri J M e d  Sci 82 



L .  Wide and G. Birgegdrd 16 

95 

90 

80 

* 70 s 
.$ 60 
c' 50 
2 

- 
P LO 
Z 30 

20 

10 

5 

Ferritin o r b  U I I  

s 10 20 t o  I A O  200 500 

Fig. 1 .  Logit representation of the dilution curve for the 
reference standard and for serum sample with high ferritin 
content. 

Assay buffer solution. 0.05 M phosphate buffer, pH 7.4, 
containing 0.01 M EDTA, 0.2% bovine serum albumin, 
0.02 % NaN,, 0.08 M NaCl and 0.5 % Tweena 20. The pH 
of the final solution was adjusted to 7.4. 

Performance o f a s s a y .  The assays were technically per- 
formed a s  previously described for the assay of gonado- 
tropins (19). All samples were run in duplicate. 0.1 ml of 
test serum was incubated with 1 ml of the solid phase 
antibody suspension. The amount of polymer-coupled 
antibodies was chosen so that about 10% of added la- 
belled ferritin was bound after incubation for about 20 
hours. After 3 4  h of incubation at room temperature the 
labelled ferritin (40 000 cpm) was added in 0. I ml of assay 
buffer solution and the mixture was further incubated for 
20 hours with a slow vertical rotation of the test tubes. 
After washing three times with saline with O . S %  Tween 
20 the remaining radioactivity was measured in a gamma- 
counter. The assays were calibrated using the ferritin 
preparation diluted in 25% serum containing a small 
amount of ferritin as a provisional reference standard. 
The values given are expressed in arbitrary units per litre, 
obtained by giving the geometric mean for a population 
of 25 normal young men (no blood donors), age 26-30 
years, the value of 100. The results of the assay and dif- 
ferent quality control parameters were calculated using a 
Compucorp 445 desk computer with a logit-log program 

Gel chromatography. Fractionation of 3 ml of a serum 
sample from a healthy young man was performed by gel 
chromatography on a 2 6 x 9 3 0  mm Sephadex G 200 column 
at +4"C in a buffer solution of pH 7.5 containing 0.1 M 

Upsciln J M r d  S o  82 

(19). 

Tris-HCI, 0.2 M NaCI, 2 mM EDTA, 0.02% NaN,. Flow 
rate was 9 ml/h and 3 ml fractions were collected.' 

Subjects. Serum samples were obtained from the fol- 
lowing subjects: 25 male and 33 female medical students 
26-30 years old. Blood donors were excluded as well a s  
pregnant women. Thirty-seven male blood donors, ran- 
domly selected, were investigated. Two patients with ex- 
cess iron due to repeated transfusions, one with pure red 
cell aplasia and one with anaemia of chronic renal failure, 
as well a s  4 patients with well established alcoholic liver 
cirrhosis were selected. Three healthy young males were 
phlebotomized once weekly until they were anaemic and 
iron depleted, after which parenteral iron treatment was 
given in a dose not quite sufficient to restore the haemo- 
globin level to the initial values. From this experiment 
samples were selected before phlebotomy, at the time of 
lowest haemoglobin level and after iron treatment. All 
serum samples were kept at -20°C until analysed. 

R E S U L T S  

Using the conjugation method with iodinated ester, 
ferritin was labelled with '"1 to a specific activity 
of about 10 Ci/g. T h e  labelled ferritin was found to 
be sufficiently stable, when stored a t  + 4 T ,  to be 
used in the assays for at least 4 months. T h e  anti- 
sera were tested for ferritin-binding capacity both 
after direct and indirect coupling. The three anti- 
sera could b e  used for 250 0001100 000 assays per ml 
with both variants of the coupling method. T h e  esti- 
mated ferritin concentration in serum was not sig- 
nificantly different (p>0.05, paired t-test) when 
the three antisera were compared, testing serum 
samples from 10 different individuals. T h e  Sepha- 
dex@ particles gave a slightly better precision than 
microcrystalline cellulose. T h e  slope of the stand- 
ard line was steeper in assays with indirectly than 
with directly coupled antibodies (mean values: 1.22 
versus 0.97). T h e  ferritin concentration that gave a 
50% inhibition to the assay was 50 arb. U/I with the 
indirect and 110 arb. U/l with the direct coupling. 
One of the antisera raised in our laboratory and in- 
directly coupled to Sephadex@ particles was chosen 
for further experimental and clinical studies. 

Quality control data were obtained from nine as- 
says over a period of 4 months which included 
duplicate tests o n  a total of 793 serum samples. T h e  
average percentage of labelled ferritin bound to B" 
w a s  10.1k1.5 (S.D.). T h e  mean +_S.D. v a l u e s  for 
the slope was 1.22k0.07 and for the 50% inhibition 
50.2k5.8 arb. U/l. The intra-assay variation calcu- 
lated as the average percentage standard error of 
the mean of duplicates giving an inhibition between 
20 and 80% of total bound was 4.7. T h e  i n t e r f i n t r a  



Radioimmunoassay for ferritin in serum 17 

E280 
nm 

2.0 

1.5 

1.C 

0.5 

Ferritin 

arb U / I  

I0 

5 

, 

1 

Fig. 2. Ferritin concentra- 
tion in fractions from a Se- 
phadex G-200 gel chromato- 
graphy of a serum sample 
from a healthy young man. 

30 LO 50 so m 80 90 IOO 110 120 

assay variation calculated as the coefficient of var- 
iation from nine repeated assays of control samples 
on three levels of ferritin concentration was: 8% 
for 19 arb. U/l, 6.5% for 73 arb. U/l and 4 . 4 %  for 
180 arb. U/l. The average detection limit (p=0.0.5) 
of the nine assays was 4.6 arb. U/I of serum. 

The dose-response curve for serial dilutions of 
the reference standard, diluted in 2.5 % serum, par- 
alleled those for dilutions of sera with high ferritin 
content (Fig. 1). A fresh serum sample from a 
healthy young male (80 arb. U of ferritin per litre) 
was chromatographed on a Sephadex G 200 column 
and all fractions were assayed for ferritin content. 
The sensitivity was 1 ..5 arb. U/l. Two immunoreac- 
tive peaks eluted soon after the void volume were 
detected (Fig. 2). This elution pattern for immuno- 
reactive ferritin was almost identical with that ob- 
tained for the radioactivity when '251-labelled ferri- 
tin added to normal serum was gel chromato- 
graphed on the same Sephadex G-200 column. 

The levels of ferritin in serum in normal males 
and females and male blood donors is shown in 
Fig. 3. The geometric mean for males was given the 
value of 100 arb. U/l (range expressed as 9.5 7% lim- 
its: 40.3-246), which was significantly (p<O.001) 
higher than that for women, 66 arb. U/l (range 26- 
173). Male blood donors had significantly (p<0.002) 
lower levels than other normal men; geometric 
mean 68.5 arb. U/l. 

The 4 patients with liver cirrhosis had values of 

2 -771x54 

i j o  i i o  is0 160 170 
fraction number 

261, 292, 983 and 2496 arb. U/l. The highest value 
recorded, 3640 arb.U/1, was in a patient with 
chronic renal failure and iron overload due to re- 
peated transfusions and oral iron therapy. Her bone 
marrow was stained for hemosiderin and showed 
a strongly positive reaction. One patient with pure 
red cell aplasia, who had had several blood trans- 
fusions, rose from 433 arb.U/l to 1404 arb.U/l 
during a 3 month period of repeated transfusions. 

The ferritin levels in serum during induction of 
anaemia and after iron treatment of 3 healthy young 
men is shown in Fig. 4. Ferritin fell to a subnormal 
mean value when iron stores were depleted (no 
marrow hemosiderin could be detected). When iron 

Ferritin I 

Males Femoles Male blood donors 

F i g .  3. Serum ferritin levels in normal males, females 
and male blood donors. 

(Jp.su1u J Med Sci 82 



18 L .  Wide and G. Birgegdrd 

140’ 

IM- 

120. 

110. 

100. 

90i 
F e d i n  

so 

13 I& 15 16 19 w k s  

Fig. 4. Mean Hb concentrations and serum ferritin levels 
in 3 healthy young men during phlebotomy and iron treat- 
ment. 

was injected, ferritin levels rose, in one case almost 
10-fold. The iron dose was chosen so that iron 
stores would again be depleted before the initial 
haemoglobin level was reached, as also occurred: 
there was no stainable iron in the bone marrow and 
serum ferritin levels fell again to low levels. 

DISCUSSION 

One prerequisite for achieving high sensitivity in a 
competitive inhibition radioimmunoassay technique 
for ferritin using labelled ferritin is the labelling of 
this protein to a sufficiently high specific activity. 
Using the conjugation technique with ‘251-labelled 
ester, a specific activity of about 10 Cilg was ob- 
tained in the present study, which is about 1000 
times higher than that obtained by the chloramine-T 
technique (12). The labelled ferritin had to be 
further purified by gel chromatography, as less than 
30 % of the 1251-labelled protein was immunoreac- 
tive. It seems likely that this was due mainly to 
heterogeneity of the ferritin preparation used for 
labelling, and, to a lesser degree, a result of the 
labelling procedure, since this method is expected 
to damage the protein very little (3). Two variants 
were used for coupling of antibodies. The observa- 
tion of a higher value for the slope and a lower 
ferritin concentration at 50 76 inhibition using the 
indirect vs. the direct coupling method is in agree- 
ment with results from assay of gonadotropins (19). 

Ilprtrirt .I Mc.d Y( 182 

Support for a high specificity of the ferritin assay 
was obtained from several experiments with the 
method. On gel chromatography of serum, the im- 
munoreactivity was eluted in the same position as 
labelled ferritin. There was no difference in relative 
ferritin activity in serum when the three different 
antisera were compared in the assay. The relation 
between mean ferritin concentration in serum from 
healthy men and women was similar to that re- 
ported by other investigators ( 1 ,  4 ,  8, 9, 16). Male 
blood donors had significantly lower mean ferritin 
concentrations than other healthy males of the 
same age. The results of the clinical material and 
the variation in immunoreactive ferritin concentra- 
tion in serum for the three men participating in the 
phlebotomy study with iron therapy give further 
evidence for the high specificity of the ferritin 
assay. Several types of ferritin have been isolated 
from various tissues in man (6, 10) and some of 
them have been shown to cross-react immunologi- 
cally (2, 13). However, it is not known to what 
degree they cross-react in our radioimmunoassay 
system. The values for precision and sensitivity of 
the method were acceptable for the application of 
the method for the assay of ferritin in serum clini- 
cal studies. 

The method, which is technically simple to per- 
form in a clinical chemical laboratory, was robust 
and suffered from only a small intra- and inter-assay 
variation. There was no detectable change in the 
activity of control samples when different batches 
of labelled ferritin were compared. The labelled 
ferritin could be used for at least four months. All 
three antisera could be used for a large number of 
tests and with such high “titres” it is unlikely that 
this solid phase technique will be found wasteful of 
antibodies when compared with other radioim- 
munoassay systems ( I  8). 

For several reasons, we have chosen to express 
the activity in relation to the mean activity obtained 
in healthy men, no blood donors, 20-30 years of 
age. There is no international standard preparation 
of ferritin. We had no evidence for the homogeneity 
of the ferritin preparation which was available. 
Furthermore, Stauffer & Greenham (15) have re- 
cently shown that variations in iron content of the 
ferritin molecule constitute a serious source of error 
i n  estimations of ferritin protein by the method of 
Lowry et al. 

Expressing results in relation to a normal popula- 
tion simplifies calibration of new reference standard 



Rudioimmunoassuy f o r  ferritin in serum 19 

solutions in our own laboratory and facilitates com- 
parisons of our results with those from other labora- 
tories. 

In conclusion: a method is described for the as- 
say of ferritin in serum, which is based o n  the use 
of labelled ferritin in a solid phase system and which 
has been found to be a robust method suitable for 
clinical investigations. 

ACKNOWLEDGEMENTS 
The study was supported by a grant from the Swedish 
Medical Research Council (637). We are indebted to Dr 
Tor Olofsson, Lund, Sweden, for supply of the femtin 
preparation, and to Mrs Anna-Lena Barmark and Mr 
Christer Bengtsson for valuable technical assistance. 

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Received January 24,1977 

Address for reprints: 
Leif Wide, M.D. 
Departments of Clinical Chemistry 
University Hospital 
S-750 14 Uppsala 

Upsflln J M e d  Sci 82