High levels of neutralizing IL-6 autoantibodies in 0.1% of apparently healthy blood donors

High levels of neutralizing IL-6 autoantibodies in0.1% of apparently healthy blood donors

Pia Galle1,2, Morten Svenson1, Klaus Bendtzen1 and Morten B. Hansen2

1 Institute for Inflammation Research, Rigshospitalet, National University Hospital, Copenhagen,Denmark

2 Department of Clinical Immunology, Rigshospitalet, National University Hospital, Copenhagen,Denmark

IL-6-specific autoantibodies (aAb-IL-6) have been reported in diseased and healthy

individuals. We recently established a model for aAb-IL-6 in different mouse strains, based

on vaccination with immunogenic IL-6 analogues, in which titers of aAb-IL-6 above 1,000

resulted in an in vivo IL-6 deficiency. Here, we examined aAb-IL-6 in 4,230 blood donors.

Stable low titers of aAb-IL-6 were found in 9% of the donors, while 1% had titers ranging from

64 to greater than 10,000. Such aAb-IL-6-positive donors appeared normal with no overt

signs of pathology. Natural and recombinant forms of IL-6 bound avidly to their IgG, and their

plasma strongly neutralized IL-6 in vitro. Slightly elevated concentrations of IL-6 exclusively in

the form of IL-6-IgG complexes were present in their circulation. The complexes did not

contain soluble IL-6 receptors. Titers of 0.1% of the blood donors were as positive as the

vaccination-induced IL-6-deficient mice. Such donors might be IL-6 deficient, and if so, IL-6

seems be dispensable for several months in otherwise healthy individuals. Such highly

positive donors also explain why normal human IgG for pharmaceutical use may contain high

anti-IL-6 activity. Finally, transfusion of plasma with a high titer of aAb-IL-6 might, temporally,

render a recipient IL-6 deficient.

Key words: Cytokine autoantibodies / IL-6 deficiency / IVIg / Blood donors

1 Introduction

Autoantibodies (aAb) against cytokines may be induced

by exogenous administration of the cytokines in question

[1]. They may also arise spontaneously, as so-called

natural cytokine aAb [2, 3]. Natural IL-6-specific aAb

(aAb-IL-6) have been reported in diseased and healthy

individuals, and their significance continues to be

obscure [4]. High levels of aAb specific for some cytokine

or growth factors, such as anti-GM-CSF [5], anti-G-CSF

[6], anti-EPO [7] and anti-TPO-antibodies [8], have

pathologic potential. Other cytokine-specific aAb, such

as anti-IL-1 alpha and anti-IL-6, have primarily been

measured as low level aAb in apparently healthy blood

donors [4, 9], which suggests that they are harmless. The

antibodies are abundantly recovered in pharmaceutically

prepared human IgG for intravenous use (IVIg) [3]. The

natural anti-IL-6 antibodies are of potential interest

because the IL-6 system is involved in many aspects

of clinical medicine and is a drug discovery target [10].

A number of animal experiments and clinical trials with

passively administered monoclonal anti-IL-6 or anti-IL-6

receptor antibodies have provided important information

on the in vivo activity of anti-IL-6 antibodies [11–13]. For

antibodies to neutralize adequately in vivo, their binding

avidities and capacities are of central importance. In

addition, administration of a mixture of monoclonal

antibodies in relatively high amounts was shown to be

necessary to insure effective capturing of IL-6 in the form

of immune complexes that could be cleared from the

circulation [14]. In the absence of these requirements,

antibodies either failed to neutralize adequately or tended

to chaperone and hence increase systemic IL-6 [15].

In order to come closer to understanding the putative

physiological and clinical consequences of natural aAb-

IL-6, we have taken two initiatives. We first established a

model for aAb-IL-6 in different mouse strains based on

vaccination with immunogenic IL-6 analogues. In this

model, titers of aAb-IL-6 above 1,000 resulted in an in

vivo IL-6 deficiency with impaired systemic inflammatory

response [16]. Second, and in light of this model system,

we have now re-examined and further characterized the

aAb-IL-6 measurable in blood donor plasma with regard

to prevalence, concentration and binding avidity (titers),

[DOI 10.1002/eji.200425268]

Received 3/5/04Accepted 10/8/04

Abbreviations: aAb: Autoantibody aAb-IL-6: IL-6-specificaAb HS: High-sensitivity (ELISA) sIL-6R: Soluble IL-6receptor IVIg: Human IgG for intravenous use

Eur. J. Immunol. 2004. 34: 3267–3275 Neutralizing IL-6 autoantibodies in apparently healthy blood donors 3267

f 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

binding specificity and potential time-dependent fluctua-

tions in antibody binding activity. In parallel, we collected

accessible data on donor characteristics including

anamnestic health data, age, gender, race, donation

schedule and standard hematology parameters. We

tested the neutralizing capacity of plasma samples with

high titers of anti-IL-6 and examined the nature and level

of endogenous IL-6 and its corresponding soluble

receptors in antibody-positive samples. Finally, we did

mathematical calculations on antibody-binding data,

which, taken together with our animal data, suggest

that between 0.1 and 1% of apparently healthy blood

donors are more or less IL-6 deficient.

2 Results

2.1 Mini-pool screening for anti-IL-6

Six years ago we identified donors positive for anti-IL-6.

Plasma was collected from 4,230 donors, and mini-pools

containing plasma from 90 individuals were generated

and tested for binding of 125I-labeled IL-6 to IgG using

Protein G affinity chromatography. As shown in Fig. 1,

27–98% of the tracer bound to IgG in four pools (termed

8, 14, 20 and 26), and recovery of added natural IL-6 to

these pools was substantially decreased as judged by

the in-house IL-6 ELISA. Testing of single donors

confirmed saturable binding of IL-6 to IgG in 10% of

the plasma (judged by saturation binding of 125I-labeled

IL-6 to IgG) [17, 18]. The anti-IL-6 titers in the majority of

donors were below 64 (data not shown). However, 3 out

of 4,230 donors were highly positive (Fig. 2), and the 35

most positive donors harbored anti-IL-6 antibodies with

picomolar avidity at concentrations between 0.1 nM and

35 nM (Table 1). These donors were not different from the

entire donor population in terms of age, gender, race,

donation schedule, anamnestic health data or basic

hematology parameters (hemoglobin value, sedimenta-

tion rate, platelet count, total white blood cell count,

neutrophil count, monocyte and lymphocyte count).

Taken together, these donors had no obvious signs of

any pathologic processes such as an underlying chronic

inflammatory or autoimmune disease that should be

associated with altered activity in the IL-6 system.

Fig. 1. Mini-pool screening for anti-IL-6. A total of 47 mini-

pools, each containing plasma from 90 donors (1 ml/donor),

were tested for: (&) binding of 125I-labeled rIL-6 to IgG as

assessed by Protein G affinity chromatography and, con-

comitantly, (&) inhibition of IL-6 using the in-house human IL-

6 ELISA.

Fig. 2. Titration of donor plasma highly positive for anti-IL-6.

Dilutions of a highly positive plasma sample (isolated from

pool 14; see Fig. 1) were incubated with 125I-labeled IL-6

(3,500 cpm) in a final volume of 200 ll, and IgG-bound tracer

was assessed by Protein G affinity chromatography. Data are

shown as the mean of triplicates (SD less than 10%).

Table 1. IL-6 binding characteristics of plasma IgG from

blood donors highly positive for anti-IL-6a)

Single plasma

from pool no.

Binding avidity

(Kav)

Concentration

(1/2 Bmax)

8 9�2 pM 25�5 nM

14 7�3 pM 21�4 nM

20 5�1 pM 34�7 nM

a) Antibody binding characteristics were measured in

plasma samples (diluted from 25,000 to 80,000 times)

by saturation binding of varying concentrations of125I-labeled rIL-6 to IgG in soluble phase. Separation of

IgG-bound and free 125I-labeled rIL-6 was done by

secondary antibody precipitation. Average dissociation

constants (Kav) and maximal IL-6 IgG-binding capacities

(Bmax) were calculated from Scatchard plots. Results are

given as the mean � SEM of triplicate determinations.

3268 P. Galle et al. Eur. J. Immunol. 2004. 34: 3267–3275


2.2 Binding characteristics of anti-IL-6 fromhighlypositive donors

The three highly positive donors had anti-IL-6 titers of

several thousand, corresponding to concentrations in the

nanomolar range (Fig. 2 and Table 1). To examine

possible time-dependent fluctuations in the anti-IL-6

levels, five donors harboring different levels of anti-IL-6

were followed for 18–26 months. Positive donors re-

mained positive during the study period, and the

antibody-binding activities of their plasma did not

substantially fluctuate (Fig. 3).

2.3 The binding specificity of anti-IL-6 antibodies

Natural IL-6, recombinant IL-6 and 125I-labeled rIL-6

might differ slightly from each other with regard to epitope

recognition by antibodies. In contrast to natural IL-6,

recombinant IL-6 produced by E. coli is non-glycosy-

lated, and adding iodine to tyrosine residues further

modifies recombinant IL-6. We therefore investigated

antibody binding to these three forms using competition-

binding analysis. Anti-IL-6-positive plasma samples were

tested against 125I-labeled rIL-6 and natural or recombi-

nant IL-6. The proportion between bound unlabeled IL-6

and bound labeled IL-6 [(B/T) unlabeled / (B/T) labeled]

was assessed as shown in Fig. 4. A value of 1 is expected

if unlabeled and labeled IL-6 bind with the same avidity to

anti-IL-6. We observed, however, ratios between 1.8 and

3.8, an expression of higher avidity binding of unlabeled

rIL-6 and natural IL-6 as compared to radiolabeled rIL-6.

There was apparently no systematic difference between

antibody binding of recombinant and natural IL-6.

2.4 IgG-complexed IL-6 in blood fromanti-IL-6-positive individuals

IL-6 measurements in 157 randomly selected donor

plasma samples by a commercial high-sensitivity (HS) IL-

6 ELISA uncovered the following: the median concentra-

tion of IL-6 was 0.07 pM (1.6 pg/ml). The concentrations

did not fit a normal distribution because of a tail

representing approximately 5% of the plasma samples

with somewhat higher IL-6 concentrations. Interestingly,

these plasma samples were also anti-IL-6-positive. IL-6

levels were higher in randomly selected anti-IL-6-positive

samples compared to randomly selected anti-IL-6-

negative samples (Fig. 5). The IL-6 in anti-IL-6-positive

plasma was in complex with endogenous IgG, because it

was entirely absorbable to Protein G (Fig. 5).

2.5 Analysis of soluble IL-6 receptors (sIL-6R)

Since sIL-6R play an essential role in IL-6 physiology, we

examined the expression levels of sIL-6R and addressed

whether anti-IL-6 IgG binds potential IL-6/sIL-6R com-

plexes or prevents IL-6 from binding to sIL-6R. The 35

Fig. 3. Chronological follow-up of five blood donors with

different anti-IL-6 titers. Titrations were performed in

duplicate as described in the legend to Fig. 2. Anti-IL-6

titer: the dilution of plasma binding to 50% of 125I-labeled IL-

6 (3,500 cpm).

Fig. 4. Specificity of natural anti-IL-6 antibodies. Three

different plasma samples (symbolized by&,* and!) highly

positive for anti-IL-6 antibodies were incubated with 125I-

labeled rIL-6 and rIL-6 or one of four different sources of

natural IL-6. IgG-bound IL-6 was retained by affinity

chromatography on protein G. Unbound IL-6 (125I-labeled

rIL-6) was counted and measured by HS ELISA. Data were

plotted as the proportion between bound unlabeled IL-6 and

labeled IL-6 [(B/T) unlabeled / (B/T) labeled; B and T denote

bound and total, respectively]. The means of triplicate

determinations are shown (SD less than 20%).



most antibody-positive samples were not different from

45 negative sera regarding the levels of sIL-6R (ranging

from 0.2–1.5 nM with a median concentration of 0.6 nM

in each group), and there was no correlation between

anti-IL-6 titer and the concentration of sIL-6R. Further-

more, immune complexes isolated by chromatography

on Protein G contained undetectable levels of sIL-6R as

judged by ELISA (data not shown).

2.6 Dissociating IL-6 from IgG

We validated the HS IL-6 ELISA with regard to aAb-IL-6

by setting up standard curves in the presence of IVIg (final

concentration 5 g/l). There was a clear correlation

between the content of aAb-IL-6 and decreased recovery

of IL-6, just as observed with our in-house IL-6 ELISA; up

to 50% of the IL-6 disappeared upon mixture with certain

IVIg (data not shown). Dissociation of natural plasma IL-

6-IgG complexes followed by molecular size chromato-

graphy revealed that complexed IL-6 could be freed from

IgG (Fig. 6). The molar amounts of freed IL-6 were low in

comparison with the corresponding molar amounts of

high-avidity anti-IL-6 (between 0.1% and 1.6%). As

expected, the amounts of IL-6 in the dissociated and

non-dissociated conditions did not “add up”. We

conclude that different IL-6 ELISA systems inadequately

measure IL-6 bound to natural antibodies. In other words,

the assays measure free IL-6 but show varying respon-

siveness to IL-6 complexed to natural anti-IL-6 anti-

bodies. All the anti-IL-6-negative sera tested contained

low amounts of IL-6. Typically, a maximal 10% increase in

the amount of IL-6 could be freed by dissociation (Fig. 5

and data not shown).

2.7 Anti-IL-6 antibodies were neutralizing in vitro

As shown in Fig. 7, natural IL-6 antibodies competitively

inhibit IL-6 in vitro. Highly positive sera diluted 100 times

Fig. 5. Plasma IL-6 from anti-IL-6-positive individuals was

complexed to IgG. The concentration of IL-6 in 11 negative

(*) and 13 positive (*) donor plasma samples was

measured by HS IL-6 ELISA. Total IL-6 concentrations (the

horizontal axis) were slightly higher in anti-IL-6-positive

samples, and IL-6 was almost entirely associated with IgG as

assessed by its ability to absorb to Protein G (the vertical

axis). Data are shown as the mean of duplicate determina-

tions.

Fig. 6. IL-6 was freed from IgG in anti-IL-6-positive plasma

samples. Gray bars: Plasma subjected to dissociation

procedure. Black bars: Untreated plasma. Four anti-IL-6-

positive plasma samples (MBH 8, 18, 20 and E 32) were

subjected to the dissociation procedure. Data are presented

as the absolute amounts of IL-6 from 400 ll undiluted

plasma that were present in high molecular weight (HMW,

MW >30,000 g/mol) and low molecular weight (LMW, MW

<30,000 g/mol) fractions. Data are shown as the mean of

duplicate determinations.

Fig. 7. Anti-IL-6 antibodies potently inhibit IL-6 in vitro.

Typical IL-6 titration curves using B9 cells in the presence of

1% donor serum with (~ and !) or without (*) anti-IL-6

antibodies. Data are shown as the mean and SD of triplicate

determinations.



typically displaced the standard curve by one log. Taken

together with the data from Table 1, we conclude that

undiluted highly positive sera are able to neutralize

nanomolar concentrations of IL-6.

3 Discussion

The etiology of natural IL-6 autoimmunity is unclear, as are

its physiological and clinical consequences. This study

alludes to the latter aspects by carefully examining the

binding of IL-6 to IgG in a large number of donor samples.

In the circulation, the bioactive forms of IL-6 are mainly

free IL-6 or IL-6 in complex with sIL-6R. sIL-6R bind IL-6

with relatively low (nM) avidity until attached to cellular

gp130 [19]. The abundant presence of sIL-6R in normal

plasma (concentrations as high as 1.5 nM) may result in

binding of up to 50% of IL-6 [4].

In approximately 10% of blood donor plasma samples,

natural anti-IL-6 antibodies can be detected [17]. This

report describes substantial binding in a minority of these

samples and provides direct evidence that these

antibodies are true aAb avidly binding several exogenous

forms of natural and recombinant IL-6 as well as

endogenously (in vivo) produced IL-6. We have validated

the in vivo significance of different levels of anti-IL-6 in a

mouse model of aAb-IL-6 [16]. We can conclude that the

relatively low levels of aAb-IL-6 present in most of the

positive individuals do not significantly affect the

biological activity of IL-6. When present in high

concentrations, however, aAb-IL-6 are strong inhibitors

of IL-6 in vitro (Fig. 7), and their binding characteristics

(Fig. 3 and Table 1) favor an ability to strip and eliminate

systemic IL-6 by capturing the cytokine in immune

complexes. The presence of such complexes in the

majority of the aAb-IL-6-positive samples was expected,

and when compared to the molar concentrations of free

antibodies, the concentrations of the complexes were

indeed very low. The complexes seem to have no IL-6

bioactivity as judged by stimulation experiments on IL-6-

sensitive B9 cells (Fig. 7). In addition, they did not contain

detectable levels of sIL-6R. IL-6 complexed to aAb-IL-6

was measurable, albeit inadequately, by ELISA (Fig. 6),

and it could be released from IgG by a powerful

dissociation procedure (Fig. 6). The complexed IL-6

theoretically has biological potential, but practically we

believe that that this will not be the case. In this study the

35 most positive donors harbored antibodies with

picomolar avidity at concentrations ranging from

0.1 nM to 35 nM. By direct comparison with data from

our animal experiments [16], we conclude that between

0.1% and 1% of the donors are more or less IL-6

deficient.

In order to better interpret the physiological and clinical

impact of high titers of anti-IL-6 antibodies, we calculated

[20] the amounts of free (i.e. not antibody-bound and

hence biologically available) plasma IL-6 as a function of

total plasma IL-6 in three situations: 1) no antibodies are

present or 2) and 3) high-avidity anti-IL-6 antibodies are

present in high or low concentrations (25 nM and 0.2 nM,

respectively). Three concentrations of total IL-6 were

chosen: 1.6 pg/ml, which is the median concentration of

normal plasma IL-6, and concentrations 10 times and

100 times higher representing pathologic situations with

increased systemic levels of IL-6 (Fig. 8). These

calculations together with the results shown in Fig. 7

show that IL-6 is inactivated in highly positive blood

donors. The amounts of IL-6 that are measurable by

ELISA in dissociated antibody-positive plasma samples

will not exert any significant “IL-6 bioactivity” as long as

the high-avidity anti-IL-6 antibodies are concomitantly

present in high amounts.

The binding specificity studies showed that natural and

recombinant IL-6 bind with almost equal avidities to aAb-

IL-6, and iodinated recombinant IL-6 seems to bind with

lower avidity than natural IL-6. Hence, by using

radiolabeled recombinant IL-6, one may in fact under-

estimate the binding activity of anti-IL-6. Moreover, the

ability of the donor plasma to bind several sources of

natural IL-6 implies that the antibodies have the potential

to bind and influence IL-6 in recipients of aAb-IL-6-

containing blood products.

Fig. 8.Calculated free (bioavailable) IL-6 as a function of total

IL-6 in the presence or absence of high-avidity anti-IL-6

antibodies. [solid line: no antibodies; dotted line: anti-IL-6

antibodies (Kav =10 pM, Bmax =0.4 nM); dashed line: anti-

IL-6 antibodies (Kav = 10 pM, Bmax = 50 nM)]. The

calculations were done using a previously described formula

[20]: Free = T – 1/2(T + Bmax + Kav – [(T + Bmax + Kav)2 – 4�Bmax� T]1/2). T denotes total IL-6 (i.e. antibody-bound IL-6 +

free IL-6).



Three donors produced extremely high amounts of high-

avidity aAb-IL-6 (Table 1). Their plasma concentration of

anti-IL-6 antibodies was in the same range as our

vaccination-induced IL-6 “knock-out” animals [16] and

the pharmaceutically active concentrations of anti-TNF

antibodies in plasma from patients undergoing anti-TNF

treatment [21]. Hence, these apparently healthy blood

donors produced anti-IL-6 antibodies in concentrations

that were likely to “knock out” their own IL-6.

IL-6 is centrally involved in inflammation, immunity, bone

marrow function andmetabolism. It belongs to a group of

cytokines including leukemia inhibitory factor, oncostatin

M, ciliary neurotrophic factor and interleukin-11 that

display overlapping activities with IL-6 because of their

interaction with gp130. IL-6 gene-deficient mice develop

normally but display an immune deficiency characterized

by failure to efficiently control infections with vaccinia

virus or Listeria monocytogenes. Some Tcell-dependent

antibody responses are impaired as are aspects of

inflammatory acute-phase responses [22, 23]. IL-6 gene-

deficient animals may also be susceptible to other

pathologies such as obesity, with disturbed metabolism

and energy expenditure [24].

We followed individual aAb-IL-6-positive blood donors

for up to 3 years and observed that they continuously

produced steady levels anti-IL-6. While more studies are

needed to exclude a pathogenic role of anti-IL-6, the

presence of such highly positive donors indicates that IL-

6 was dispensable for longer periods of time in these

otherwise healthy individuals. This is not directly in

conflict with the above mentioned animal experiments on

IL-6 gene deficiency. Indeed, symptoms of IL-6 defi-

ciency may only show up when the system is profoundly

stressed. Again, this is in accordance with our observa-

tions in anti-IL-6 antibody-induced IL-6-deficient mice,

which despite their IL-6 deficiency behave like normal

mice until they are stressed. In spite of their healthy

appearance, however, we cannot rule out that anti-IL-6-

positive humans are susceptible to certain infections or

disorders as they may concomitantly be protected

against disorders in which IL-6 has a pathogenic role.

In this context it is interesting that aAb-IL-6 in patients

with alcoholic cirrhosis have been associated with

recurrent infections and increased mortality [25].

In Denmark 1% (corresponding to approximately 4,000

donations) of the blood units contain substantial amounts

of anti-IL-6 antibodies that are administered during

transfusion. In at least 0.1% of the transfused plasma

units (corresponding to 50 transfusions annually), mas-

sive amounts of anti-IL-6 antibodies are delivered to the

recipients. After transfusion of a plasma unit, the entered

IgG is typically diluted 25 times. Judged by the in vitro

neutralization experiments (Fig. 7) and the calculations

presented in Fig. 8, a highly positive plasma unit will

severely affect the IL-6 system in the recipient. Whether

or not this brings about an overlooked mechanism of

allogeneic blood transfusion-associated immune mod-

ulation [26] is under our investigation.

4 Materials and methods

4.1 Iodination of recombinant IL-6

Escherichia coli-derived recombinant hIL-6 (Amersham

Biosciences, Birkerød, Denmark) was iodinated by the

chloramine-T method and processed as previously de-

scribed [20]. The resulting 125I-labeled IL-6 was repeatedly

chromatographed on Sephadex G-75 (Amersham Bios-

ciences) to yield a tracer with more than 90% binding to

receptors on U-937 cells, with preserved bioactivity on B9

cells and with a specific activity of 100–250 cpm/pg.

4.2 Isolation and stimulation of mononuclear cells

Mononuclear cells (MNC) from four healthy blood donors

were obtained from the Blood Bank of Rigshospitalet

(National University Hospital, Copenhagen, Denmark). The

donors were negative for antibodies to IL-6. MNC were

purified from buffy coats by centrifugation on Lymphoprep

(Nycomed, Oslo, Norway). The cells were washed in RPMI

1640 containing 2 mM L-glutamine (Sigma, St. Louis, USA),

25 lg/ml gentamycin (Gibco BRL, Life Technologies, Paisley,

Scotland) and 5% normal human AB serum. Native IL-6 (nIL-

6) was generated by stimulating MNC at 37�C in 5% CO2

humidified air in the presence of 100 lg/ml E. coli LPS (Difco

Laboratories, Detroit, USA). After 12 h, the supernatant was

harvested and stored at –20�C until use.

4.3 Protein G affinity chromatography

Affinity chromatography of 100 ll plasma samples were

carried out at 4�C using columns containing 2000 ll ProteinG Sepharose 4 Fast Flow (Amersham Biosciences). Phos-

phate-buffered saline pH 7.4 (PBS) supplemented with 0.1%

(v/v) Triton X-100 and 0.1% (w/v) gelatin (Sigma) was used as

running buffer. Boundmaterial was elutedwith 0.1 M glycine/

HCl, pH 2.4.

4.4 Specificity analyses

Antibody specificity analyses were carried out with different

preparations of natural and recombinant IL-6 together with

radiolabeled rIL-6. Plasma samples that were positive for



anti-IL-6 were diluted to bind approximately 60% of a total of

15 ng/200 ll IL-6 including both unlabeled IL-6 and traced

IL-6 (15 pg/200 ll 125I-labeled IL-6). The mixture of plasma,

tracer and competitor was pre-incubated for 1 h at 37�C and

then subjected to affinity chromatography on Protein G.

Fractions corresponding to IgG-bound and free tracer were

counted in a gamma counter (1470 WizardTM gamma

counter, Wallac, Finland). In addition, free IL-6 wasmeasured

by in-house ELISA.

4.5 Screening plasma samples by RIA and ELISA

Plasma samples were collected from 4,230 individual blood

donors attending our department. All donors fulfilled the

criteria for blood donation stated by the Danish Society for

Clinical Immunology and the current edition of the Council of

Europe’s “Guide to the preparation and quality control of

blood components” (www.dski.suite.dk/TMS). Samples

were initially mini-pool screened for anti-IL-6. In brief,

mini-pools of 90 plasma samples were adjusted to 25%

(v/v) in PBS supplemented with 0.1% (v/v) Triton X-100

(Sigma), 0.1% (w/v) gelatin (Sigma) and 2 mM EDTA (Bie &

Berntsen, Rødovre, Denmark) (PBS+), and 3,500 cpm 125I-

labeled IL-6 was added; the final volume was 200 ll. Afterincubation for 20 h at 4�C, fractions representing IgG-bound

tracer and free tracer were separated by Protein G and

counted. In addition, anti-IL-6 binding activity was also

addressed with regard to natural IL-6. This was done by

measuring recovery of 1 ng/ml natural IL-6 in the presence of

25% plasma pools in an in-house IL-6 ELISA.

4.6 In-house IL-6 ELISA

This sandwich ELISA is based on specific polyclonal rabbit

anti-human IL-6 antibodies. It has been validated thoroughly

in terms of interference by natural human IL-6 antibodies [4,

17, 18, 27]. In brief, Immuno-Maxisorp plates (Nunc,

Roskilde, Denmark) were coated with Protein A affinity-

purified rabbit anti-human IL-6 IgG. Non-attached sites were

blocked with PBS containing 4% (w/v) skimmedmilk powder

(IRMA, Rødovre, Denmark), 1% (w/v) human serum albumin

(HSA) (SSI, Copenhagen, Denmark) and 0.005% (v/v) Tween

20 (Merck, Darmstadt, Germany). The wells were washed

with PBS/0.005% Tween 20 after each of the following steps:

1) 100 ll analyte incubated for 18 h at 4�C; 2) 100 llbiotinylated rabbit anti-human IL-6 IgG (2 lg/ml) in PBS/

0.005% (v/v) Tween 20/0.5% (w/v) HSA, incubated for 2 h at

20�C; 3) 100 ll streptavidin-peroxidase (0.1 lg/ml; Kirke-

gaard & Perry Laboratories, Gaithersburg, USA) in PBS/

0.005% (v/v) Tween 20/0.5% (w/v) HSA, incubated for

45 min at 20�C. Enzyme activities were quantitated using

1,2-phenylenediamine dihydrocloride (DakoCytomation,

Glostrup, Denmark). The working range of the ELISA was

from 150 pg/ml to 5,000 pg/ml. The inter- and intra-assay

coefficients of variation were below 15%.

4.7 HS IL-6 ELISA

Plasma IL-6 levels were quantified using the IL-6 Quantikine

High Sensitivity ELISA (R&D Systems, Minneapolis, MN).

According to the manufacturer’s validation and instructions,

this ELISA detects IL-6 bound to both sIL-6R and natural

anti-IL-6 antibodies.

4.8 sIL-6R ELISA

Plasma levels of sIL-6R were quantified by sIL-6R ELISA

(R&D Systems) according to the manufacturer’s instructions.

4.9 Secondary antibody precipitation and Scatchard

plots

The binding characteristics of aAb-IL-6 in selected antibody-

positive plasma samples were assessed as previously

described [20]. Appropriately diluted plasma samples were

mixed with 125I-labeled IL-6 ranging from 50,000 cpm to

700 cpm in PBS+ in a final volume of 100 ll. After incubationat 4�C for 20 h, 200 ll rabbit anti-human IgG (A424;

DakoCytomation) was added to precipitate more than

95% of the IgG. After incubation for 1 h at 4�C, three

volumes of PBS were added. The samples were centrifuged

immediately for 20 min (3000�g at 4�C), after which IL-6 in

the pellets (IgG-bound) and the supernatants (free) were

counted. The results from these saturation binding analyses

were subjected to Scatchard plots to estimate antibody

avidities (Kav) and binding capacities (Bmax).

4.10 Dissociation of IL-6/anti-IL-6 immune complexes

The immune complexes were separated in a two-step

procedure, as previously described. This dissociation

procedure separates more than 80% of nanomolar levels

of preformed recombinant IL-6/anti-IL-6 complexes with

preservation of both immune reactivity of IL-6 and antibody

avidity [28]. Briefly, plasma samples (33% v/v) were

incubated for 30 min in 67% (v/v) dissociation buffer [2 M

MgCl2 in 0.1 M acetic acid, pH 4.0 and 50% (v/v) ethylene

glycol]. The mixture was then subjected to molecular size

chromatography on Sephadex G-75 (Amersham Bios-

ciences). “Freed” IL-6 was measured by HS IL-6 ELISA

(R&D Systems).

4.11 Bioassay

The mouse B cell hybridoma cell line B9 (European

Collection of Cell Cultures, Wiltshire, UK) was cultured in

RPMI 1640 supplemented with 2 mM L-glutamine (Sigma),

50 lM 2-mercaptoethanol (Sigma), 5% fetal calf serum

(GibcoBRL), penicillin/streptomycin (Sigma) and 50 pg/ml



human rIL-6 at 37�C with 5% CO2. The neutralizing capacity

of anti-IL-6 antibody-positive samples was assessed by

standard titration of rIL-6 on B9 cells in the presence of 1%

heat-inactivated donor serum. B9 cells (10,000/well) were

incubated with serial dilutions of rIL-6 for 3 days (37�C, 5%

CO2). Cell viability was judged by an MTT assay [29].

4.12 Ethics

This study was approved by the local ethical authority under

the code number KF 01–110/98.

Acknowledgements: This investigation was supported

financially by The Danish Rheumatism Association, The

Danish Blood Donor Foundation, The Novo Nordisk Founda-

tion and The Danish Medical Research Council. The

excellent technical assistance of Søs Bonde is gratefully

acknowledged.

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Correspondence:Morten B. Hansen, Department of Clinical

Immunology, H:S Blood Bank, 2034, Rigshospitalet, National

University Hospital, Blegdamsvej 9, DK-2100 Ø Copenha-

gen, Denmark

Fax: +45-35390038

e-mail: [email protected]



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High levels of neutralizing IL-6 autoantibodies in 0.1% of apparently healthy blood donors