Growth Hormone & IGF Research 19 (2009) 357360

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Growth Hormone & IGF Research

journal homepage: www.elsevier.com/locate/ghir

Laboratory issues in the implementation of the marker method

David A. Cowan *, Christiaan Bartlett
Drug Control Centre, Department of Forensic Science and Drug Monitoring, Kings College London, London SE1 9NH, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history: Two important biomarkers for the identication of growth hormone or IGF-I administration are IGF-I and
Accepted 3 April 2009 P-III-P. These substances are determined in plasma or preferably in serum. There are a number of assays
Available online 1 July 2009 on the market for IGF-I but only two for P-III-P. The principles behind these assays and the choice of
assays for doping control purposes are discussed. The future possibility of quantication by mass spec-
Keywords: trometry is also briey discussed.
IGF-I 2009 Published by Elsevier Ltd.
P-III-P
Immunoassay
Mass spectrometry
Biomarkers

1. IGF-I placement with ethanol precipitation [5]. Binding proteins that

Most assays for insulin-like growth factor I (IGF-I) are calibrated
against the WHO International Reference Reagent for IGF-I for
immunoassay, 87/518. However, this is no longer readily available
and was initially replaced by the First International Standard for
IGF-I, 91/554, which has been prepared from DNA-derived human
insulin-like growth-factor-I expressed in yeast. It is intended to
serve as the primary reference material for IGF-I bioassay calibra-
tion. Each ampoule of the lyophilized preparation contains 150
International Units by denition and each Unit is equivalent to
1 lg. An international collaborative study compared the new stan-
dard 91/554 by immunoassay in terms of 87/518 and this gave a
geometric mean estimate (n = 10) of 191.9 (162.7226.1) lg per
ampoule. This standard has recently been replaced by the 1st
WHO International Standard for IGF-I, 02/254. This is a preparation
of recombinant material that has been established as the primary
reference material for IGF-I immunoassay by an international col-
laborative study [1]. Each ampoule contains 8.5 lg of IGF-1 by def-
inition with a stated expanded uncertainty of 7.739.23 lg.
In the circulation, 99% of IGF-I is bound to IGF binding proteins
(IGFBP-1 to IGFBP-6), of which 7580% is bound to IGFBP-3 and the
acid labile subunit (ALS) in a ternary complex with a molecular
weight of 150 kDa. The remaining 2025% is bound to lower
molecular weight binding proteins [24]. Due to this high degree
of binding protein interaction, immunoassays for IGF-I require a
sample pre-treatment step to free IGF-I from its bound form, thus
exposing the epitopes for antibody recognition.
The most common method of removing the binding proteins
from IGF-1, prior to assay of the freed IGF-I, is by using acid dis-
* Corresponding author.
E-mail address: David.cowan@kcl.ac.uk (D.A. Cowan).
are bound to ALS such as IGFBP-3 and IGFBP-5 are largely removed
during the precipitation step. However, multiple publications have
documented that smaller forms of IGFBP do not bind to ALS, such
as IGFBP-1 and IGFBP-4, and remain in the sample [6,7]. Some
IGF-I is also precipitated and could result in lower measured con-
centrations but, in the anti-doping context, this small bias will
err in favour of the competitor rather than producing a false posi-
tive result. Another claimed drawback of this approach is that less
than 50% of IGFBP-1 is removed by the ethanol precipitation meth-
od [6,7]. However, this conclusion is based on a study of poorly
controlled diabetes where IGFBP-1 may be much higher than ex-
pected in the healthy sports competitor.
An alternative to the acid displacement ethanol precipitation
approach is to use acid displacement followed by addition of IGF-
II in excess to ensure that all IGF-I has been displaced [8] and does
not rebind to the binding proteins. The authors claim that this
method is better when dealing with diabetic patients. This is unli-
kely to be of relevance to sport where, as already mentioned, any
bias will be in favour of the athlete. Of course, this method depends
on minimal cross-reactivity of IGF-II with the IGF-I antibodies.
Clemmons [9] provides a comprehensive review of current IGF-I
immunoassays and their limitations.
For the GH 2004 project to detect GH administration in sport,
the two IGF-I assays selected for use were the DSL-5600 and the
Immunotech A15729 assay. Both assays are of the 2-site IRMA
coated tube assay format using monoclonal antibodies. The DSL
assay uses acid displacement and ethanol precipitation sample
preparation whereas the Immunotech assay uses acid displace-
ment with IGF-II to prevent rebinding of the IGF-I. A summary of
the characteristics of each assay is presented in Table 1. Both assay
manufacturers state that the kit standards are calibrated
against the WHO International Reference Reagent for IGF-I for

1096-6374/$ - see front matter 2009 Published by Elsevier Ltd.

doi:10.1016/j.ghir.2009.04.005
358 D.A. Cowan, C. Bartlett / Growth Hormone & IGF Research 19 (2009) 357360

Table 1
Summary comparison of the two IGF-I assays used.

DSL Active with extraction DSL-5600 Immunotech (Beckman Coulter) A15729

Principle IRMA IRMA
Assay type 2-Site sandwich, mAb, CT 2-Site sandwich, mAb, CT
Serum pre-treatment Acid/ethanol precipitation, then neutralisation Acidication plus IGF-II
Serum volumea (lL) 50 50
Serum dilution 1:30 1:20
Incubation time 3 h, RT 1 h, RT
Calibration range 0500 ng/mL 01200 ng/mL
Signal detection Gamma counter, 125I Gamma counter, 125I
Manufacturers data
Sensitivity 0.8 ng/mL 2 ng/mL
Inter-assay CV (%) 1.58.2 6.8
Intra-assay CV (%) 1.53.4 6.3

IRMA = immunoradiometric assay.

CT = coated tube.
mAb = monoclonal antibody (Manufacturers claim the two assays use mAbs from different clones; personal communication).
ng/mL = nanograms per millilitre.
CV = coefcient of variation.
IGF-II = insulin-like growth factor II.
RT = room temperature.
a
In each assay, duplicate portions are taken from the treated serum extract.

immunoassay, 87/518. The use of one assay that relies on the eth- 1200 y = 0.7828x
anol precipitation method and the other relying on IGF-II to ensure 2
R = 0.826
that IGF-I is freed from binding proteins helps ensure the reliability
1000
of the data used in the growth hormone discriminant function de-
scribed elsewhere [10]. Although the use of radio-immunoassays
Immunotech IGF-I ng/mL

may present difculties for some laboratories since licensed facili-
ties are required, because only radio-labelled assays for P-III-P are
available, this minor difculty was not considered to be critical in
the roll-out of these assays to be able to detect growth hormone
administration.
The Immunotech A15729 IGF-I assay is an immunoradiometric
technique, using the 2-site sandwich principle. To release IGF-I
from its binding proteins, an acidic dissociation buffer is added
to each sample, which also contains an excess of IGF-II to prevent
rebinding. Once treated, duplicate portions of sample are pipetted
into tubes coated with mouse monoclonal anti-IGF-I antibody. A
second 125I-labelled anti-IGF-1 mouse monoclonal antibody is then
added, which forms a sandwich with the IGF-I and immobilised
rst antibody. After washing, the bound radioactivity is measured
by a gamma counter; the amount of radioactivity being directly
proportional to the amount of IGF-I in the original sample.
The DSL-5600 IGF-I assay, like the Immunotech assay, is an
immunoradiometric technique. However, this assay uses acid-eth-
anol extraction. Extraction solution is added to each serum sample,
which is then vortex mixed and incubated for 30 min. After centri-
fugation and removal of a portion of the supernatant the acid is
neutralised and duplicate aliquots are added to tubes coated with
anti-IGF-1 mouse monoclonal antibody. IGF-1 in the sample binds
to this antibody immobilised on the inner surface of the test tube.
The second 125I-labelled anti-IGF-1 antibody is then added in solu-
tion to form a sandwich complex. Decanting and washing the
tubes then removes any unbound material. The radioactivity in
the tube is measured by a gamma counter and compared to a cal-
ibration curve constructed under identical conditions; the amount
of radioactivity measured being directly proportional to the
amount of IGF-I present in the original sample.
Cross-correlation of the assays was performed using 124 serum
samples that had undergone only one freeze/thaw cycle. These
samples were obtained from adolescents because they have IGF-I
concentrations that cover the broadest range of normal, i.e. those
not administering growth hormone or IGF-I. The results are shown
in Fig. 1.
Using the simple proportionality model above is not strictly cor-
rect statistically. Plotting y/x against 1/x gives a slope that is signif-
800
600
400
200
0
0 500 1000 1500
DSL IGF-I ng/mL
Fig. 1. Cross-correlation of the Immunotech A15729 and DSL-5600 assays for IGF-I
(n = 124).
icant (p = 0.044). The alternative model of linearity gives a line of
slope of 0.714 and intercept of 47.1 with R2 of 0.835. This indicates
a possible small bias of one of the assays.
2. P-III-P
In contrast to the many assays on the market for IGF-I, there
are only two commercial assays available for the N-terminal pro-
peptide of type III procollagen (P-III-P or P-III-NP). These are the
RIA-gnost P-III-P (OCFK07-PIIIP) manufactured by CIS Biointerna-
tional (Gif-sur-Yvette Cedex, France) and UniQ P-III-NP RIA
(68570) made by Orion Diagnostica (Espoo, Finland).
Unlike IGF-I, P-III-P is not bound by binding proteins in the cir-
culation. Consequently there is no need for an extraction step in
the immunoassays. Importantly no International Standard exists
for P-III-P. Human P-III-P and bovine material are very similar with
about 97% sequence homology. The commercial kit manufacturers
do not provide information about the standards that they use and
it seems likely that bovine material is used. Importantly the two
assays employ different concentration units: Units/millilitre (CIS)
and nanograms/millilitre (Orion). A summary of the characteristics
of the two P-III-P assays is shown in Table 2.
 D.A. Cowan, C. Bartlett / Growth Hormone & IGF Research 19 (2009) 357360 359

Table 2
Summary comparison of the two P-III-P assays used.

CIS RIA-gnost P-III-P (OCFK07-P-III-P) Orion UniQ P-III-NP (68570)

Principle IRMA RIA
Assay type 2-Site sandwich, mAb, CT Competitive assay, pAb, 125I- labelled
P-III-NP, 2nd Ab bound to solid
particles
Units Units per millilitre (U/mL) Nanograms per millilitre (ng/mL)
Calibration range 0.54, 1.05, 2.18, 4.5, 8.8, 15.4 U/mL 0, 1, 2.5, 5, 10, 25, 50 ng/mL
Serum volume 2  20 lL 2  200 lL
Incubation time 2 h + 3 h, both @ RT 2 h @ 37 C + 0.5 h @ RT
Signal detection Gamma counter, 125Iodine Gamma counter, 125Iodine
Manufacturers data
Limit of detection 0.1 U/mL 0.3 ng/mL
Inter-assay CV (%) 7.811.3 4.57.2
Intra-assay CV (%) 2.94.0 3.07.0

IRMA = immunoradiometric assay.

RIA = radioimmunoassay.
mAb = monoclonal antibody.
pAb = polyclonal antibody.
RT = room temperature.
h = hour.

The CIS RIA-gnost P-III-P assay is an immunoradiometric assay
that enables the determination of P-III-P in serum using the princi-
ple of a 2-step sandwich assay. Standards, controls, and unknowns
are pipetted into tubes coated with mouse monoclonal anti-P-III-P
antibodies. During an incubation step, a complex is formed be-
tween the solid phase antibodies, P-III-P in the sample, and a sec-
ond 125I-labelled mouse monoclonal antibody. Free tracer is
removed by washing and the radioactivity specically bound is
measured using a gamma counter. The amount of radioactivity
measured is directly proportional to the amount of P-III-P present
in the original sample.
The CIS kit is designed for clinical use detecting elevated levels
of P-III-P typically found in patients with liver disease and the cal-
ibration curve ranges from approximately 0.515 U/mL. In the
healthy adult population, expected concentrations are typically in
the range of 0.3-0.8 U/mL (n = 451, RIA-gnost P-III-P kit instruc-
tion booklet). To better reect the normal population, a new cali-
bration curve was constructed by serial dilution of the highest
calibrant with 0.1% human serum albumin (protease-free) in 10%
phosphate buffered saline to give calibration points at 0.12, 0.24,
0.48, 0.96, 1.93, and 3.85 U/mL. Inter-assay precision CV has been
estimated at 9.5% by repeated analysis of the kit control (mean
2.08 U/mL, n = 18; see Table 3).
The Orion UniQ P-III-NP RIA is a competitive radioimmunoas-
say technique. A known amount of 125I-labelled P-III-NP and an
unknown amount of P-III-NP in the sample compete for a limited
number of high afnity rabbit polyclonal antibody binding sites. A
second antibody, covalently bound to solid particles, is added
which forms a complex with the P-III-NP bound to the rst anti-
body. After separation by centrifugation, the amount of 125I-la-
belled P-III-NP in each tube is measured by a gamma counter;
the amount of radioactivity measured being inversely propor-
tional to the amount of P-III-NP present in the original sample.
The calibration curve ranges from 0 to 50 ng/mL with a lowest cal-
ibrator of 1 ng/mL. The reference range for the normal healthy
adult population is 2.36.4 ng/mL (n = 232, UniQ P-III-NP RIA kit
instruction booklet). Compared to the other assays chosen, the
400 lL required by the Orion assay represents a relatively large
sample volume. In sports samples, in particular, serum volume
is often limited, and other anti-doping tests may be required. A
dilution experiment was therefore carried out in order to deter-
mine whether a smaller sample volume could be employed. Each
sample was analysed using duplicate serum volumes of 200 lL
(undiluted), 100 lL (1 in 2 dilution) and 50 lL (1 in 4 dilution).
For each dilution, the total sample volume was adjusted to
200 lL with 0.1% human serum albumin (protease-free) in 10%
phosphate buffered saline. The mean recovery from the 1 in 2
dilutions, across a concentration range of 514 ng/mL, was 101%
(range 91113%, n = 6). The 1 in 4 dilutions gave a mean recovery
of 118% (range 101133%, n = 6) with poorer performance at low-
er concentrations (less than 10 ng/mL). Although a small number
of samples were tested, these results are encouraging and suggest
duplicate volumes of 100 lL may be suitable for this assay at least
for screening purposes.
Correlation between the two P-III-P assays was demonstrated
by analysing the same 124 serum samples from adolescents used
for the cross-correlation of the IGF-I assays. These samples had also
undergone only one freeze/thaw cycle and were assayed for P-III-P

Table 3
Performance of commercial immunoassays for the quantication of IGF-I and P-III-P.

Analyte Assay name LODa LOQb Recoveries % (n = 3) Intra-assay precision (n = 8) Inter-assay precision
1/2 1/4 1/8 mean CV% mean SD CV%
IGF-I DSL-5600 36 ng/mL 95 ng/mL 92 90 94 74 5.5 89 3.9 4.3
278 ng/mL 3.9 242 ng/mL (n = 24) 14.2 5.9
A15729 1.4 ng/mL 4.3 ng/mL 92 94 98 138 1.6 285 ng/mL (n = 22) 19.5 6.9
455 ng/mL 2.2
P-III-NP RIA-gnost P-III-P 0.13 U/mL 0.33 U/mL 102 99 96 0.12 10.8 2.08 U/mL (n = 18) 0.2 9.5
3.46 U/ML 18.2
UniQ P-III-P 0.41 ng/mL 1.14 ng/mL 94 93 105 4.27 4.1 5.31 ng/mL (n = 20) 0.2 3.7
56.1 ng/mL 2.4

Ten individual measurements of zero calibrator.

a
Limit of detection, LOD = mean + 3*SD.
b
Limit of quantication, LOQ = mean + 10*SD.
360 D.A. Cowan, C. Bartlett / Growth Hormone & IGF Research 19 (2009) 357360
30
y = 9.2057x
R2 = 0.7676
25
Orion P-III-NP ng/mL
20
15
10
5 future. At least in the short-term, radioimmunoassay for P-III-P will
0 difculties associated with a mass spectrometric approach.
0.0 0.5 1.0 1.5 2.0 2.5
CIS P-III-P low calib U/mL
Fig. 2. Cross-correlation of the Orion and CIS assays for P-III-P (n = 124).
using both kits and the modied calibration curve for the CIS assay.
As in the case of IGF-I, adolescents also have P-III-P concentrations
that cover the broadest range of normal, i.e. those not administer-
ing growth hormone or IGF-I. The results are shown in Fig. 2.
These data show very good correlation and the slope of the line
is very similar to the slope of approximately 10 found by Abellan
et al. [11] suggesting the robustness of these two assays.
3. Mass spectrometry for the analysis of IGF-I
IGF-I is a protein with a molecular weight of around 7500 com-
prising 70 amino acids, six of which are cysteines that form three
intramolecular disulphide bonds. The typical concentration of
IGF-I in serum has been shown to be of the order of several hun-
dred nanograms per millilitre. Recent advances in mass spectrom-
etry have enabled the marketing of instruments that should have
sufcient capability to assay proteins of the size of IGF-I at the con-
centrations present in man using a reasonable sample volume.
Liquid chromatography coupled to 3D ion trap mass spectrom-
etry analysis of IGF-I was rst explored in the equine. Popot and
co-workers [1214] used immunoafnity purication after acid
displacement ethanol extraction and then analysis of the intact
protein using R3IGF-1 that has an arginine instead of glutamic acid
at position 3 as the internal standard. de Kock et al. [15] chose to
perform a protein digest using endoproteinase Asp-N, rather than
tryptic digest that they had found to be inefcient, followed by
reduction of the free thiols and LC tandem MS assay of the digest
using R3IGF-1 treated in the same way as the internal standard.
Kirsch et al. [16] claimed to have used tryptic digests effectively
to quantify both IGF-I and also IGFBP-3 in serum using heavy iso-
topes of suitable fragments as internal standards with a triple
quadrupole mass spectrometer. Bredehoft et al. [17] used electro-
spray ionisation tandem mass spectrometry and immunoafnity
sample preparation and achieved a limit of detection of between
20 and 50 ng/mL from a 60 lL sample of plasma and internal stan-
dard based on the homologous compounds R3IGF-1 and LONG-
TM 3
R IGF-1 that has an additional 13 amino-acids compared with
R3IGF-1. The future preparation of heavy labelled intact IGF-I
should improve the robustness of the technique and is likely to
make it the de facto reference method for IGF-I and help to resolve
much of the controversy surrounding IGF-I measurements in clin-
ical chemistry.
Unfortunately, because of the relatively large size of P-III-P
(approximately 40 kDa) and its relatively small concentration in
serum (about 5 ng/mL), a mass spectrometric assay for this protein
is rather more difcult and further advances in mass spectrometry
and sample preparation techniques are probably needed before
signicant progress can be made to quantify this substance.
4. Concluding remarks
A summary of the in-laboratory performance of the four chosen
assays is shown in Table 3.
These data indicate that these assays are t for purpose for the
detection of growth hormone administration as described in other
papers in this publication. The replacement of the immunoassays
for IGF-I by mass spectrometric methods seems likely in the near
continue to be the technique of choice for this protein due to the
3.0
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