www.elsevier.com/locate/jim

Journal of Immunological Methods 281 (2003) 9–15

Purification of bovine IGFBP-3 and the development of an

enzyme immunoassay for the protein

Mark Hennies, Helga Sauerwein*

Institut fur Physiologie, Biochemie und Hygiene der Tiere, Rheinische Friedrich Wilhelms-Universitat Bonn,

Katzenburgweg 7-9, 53115 Bonn, Germany

Received 10 May 2002; received in revised form 18 September 2002; accepted 8 April 2003

Abstract

Insulin-like growth factor binding protein-3 (IGFBP-3), the most prominent IGF-binding protein in serum, has been

demonstrated to modulate the effects of the IGFs but also to exert IGF-independent actions. Quantification of IGFBP-3 in

livestock species, in particular ruminants, is commonly limited to blotting methods in spite of the importance of these species.

Here we describe the development of a specific homologous enzyme-linked immunosorbent assay (ELISA) to measure bovine

IGFBP-3 in bovine plasma, serum and milk. IGFBP-3 purified from bovine serum was used both as standard and also for tracer

synthesis. A specific antiserum was raised in rabbits using a synthetic peptide based on the sequence of bovine IGFBP-3. The

measuring range of the assay was between 50 and 1000 ng IGFBP-3 per milliliter of plasma or milk. Mean recovery was 97.3%

for plasma and 100.1% for milk. Intra- and interassay coefficients of variation were 6.2% and 9.3%, respectively. For the

biological verification of the assay, IGFBP-3 was determined in plasma obtained from 12 dairy cows before and after being

injected with a depot-formulated growth hormone (GH) preparation. GH, a well-characterized stimulator of IGFBP-3, led to a

1.3-fold increase of basal IGFBP-3 concentrations during days 3 to 19 after the injection. The availability of an ELISA

procedure which permits precise and sufficiently sensitive quantification of bovine IGFBP-3 and which can be used on large

sample numbers thereby avoiding the need for radioactive labels, should facilitate further research studies.

D 2003 Elsevier B.V. All rights reserved.

Keywords: IGFBP-3 purification; ELISA; Growth hormone; Cattle

0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0022-1759(03)00195-9

Abbreviations: ALS, acid labile subunit; BSA, bovine serum

albumin; BXNHS, biotinamidocaproate N-hydroxysuccinimide

ester; DMSO, dimethylsulfoxide; EDTA, ethylenediaminetetra-

acetic acid; ELISA, enzyme-linked immunosorbent assay; Fc,

crystallizable region of an immunoglobulin; FPLC, fast protein

liquid chromatography; GH, growth hormone; IGF, insulin-like

growth factor; IGFBP, insulin-like growth factor binding protein;

PBS, phosphate-buffered saline; RPC, reversed-phase chromato-

graphy; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel

electrophoresis.

* Corresponding author. Tel.: +49-228-732804; fax: +49-228-

737938.

E-mail address: sauerwein@uni-bonn.de (H. Sauerwein).

1. Introduction

Growth factors such as insulin-like growth factor-1

(IGF-1) are essential for normal growth and develop-

ment. IGF-1 mediates many of the effects of growth

hormone (GH) and its biological activity is modulated

in vivo by the IGF binding proteins (IGFBPs). There

are six IGFBPs (IGFBP-1 to -6) with high affinities to

IGFs and nine low-affinity IGFBP-related proteins

(IGFBP-rP1 to 9) (Hwa et al., 1999). Besides interfer-

ing with the interaction between IGF-1 and its recep-

M. Hennies, H. Sauerwein / Journal of Immunological Methods 281 (2003) 9–1510

tor, intrinsic bioactivity has been reported for some

IGFBPs (Baxter, 2000). IGFBP-3, the predominant

IGFBP in the circulation, is regulated by various

genetic and environmental influences and is used as

a diagnostic parameter, for example, for GH disorders

(Blum et al., 1994; Kim et al., 2001). Analysis of

IGFBP-3 is not only relevant for human medicine, but

also for livestock species. Ruminant species provide

valuable models, for example, the sheep has been

studied with respect to GH neuroregulation (Dutour et

al., 1997) and also fetal growth, development and

disease (Gunn and Gluckman, 1995; Marks et al.,

1996; Gunn et al., 1998; Peters, 2001). Goats are used

in bone research (Yeung et al., 2001) and the cow is

of particular relevance for mammary gland physiol-

ogy (Baumrucker and Erondu, 2000). For meat pro-

ducers, circulating IGFBP-3 concentrations might be

useful indicators for growth and carcass composition

(McCann et al., 1997). However, specific ruminant

IGFBP-3 assays are not commercially available and,

so far, only one RIA has been published for sheep

IGFB-3 (Gallaher et al., 1998). Western ligand blot-

ting or Western immunoblotting are currently used for

quantitative measurements of this binding protein in

ruminants. Despite the various advantages these

methods have, their quantitative power is limited

(Rajaram et al., 1997). Due to the fact that bovine

IGFBP-3 is not available from commercial sources,

we aimed to purify the protein from serum to develop

an enzyme immunoassay for IGFBP-3 in bovine body

fluids, thereby circumventing the use of radiolabeled

ligands.

2. Materials and methods

2.1. Purification of IGFBP-3

For all chromatographic procedures, the Akta puri-

fier 10 liquid chromatography system (Amersham

Biosciences, Freiburg, Germany) was used. The indi-

vidual steps were carried out at room temperature

except that the column used for the third gel filtration

was cooled to 3 jC. Samples were filtered through a

0.22 Am syringe filter (Roth, Karlsruhe, Germany)

before application on the column. During the purifi-

cation procedure, IGFBP-3 containing fractions were

stored at � 20 jC until the next purification step.

For the first gel filtration, a 10� 2.5 cm column

containing SuperdexR 200 (Amersham Biosciences)

was equilibrated with 2 to 3 column volumes of

phosphate-buffered saline (PBS). Several column runs

were performed each with 5 ml normal bovine serum

obtained from an adult cow. The flow rate was 2 ml/

min using PBS as elution buffer. Fractions containing

proteins of approximately 150 kDa were pooled and

stored until the next purification step. Immediately

after thawing, the pH of the pool fractions from the

first gel filtration was adjusted to 2.5 with acetic acid

(100%) and incubated at room temperature for at least

1 h up to maximally 6 h. After this incubation, the

samples were subjected to a second gel filtration using

the same column as that used for the first purification

except that the buffer used for equilibration and

elution was the CIEX buffer (15 mM Na2HPO4, 15

mM sodium formate, 30 mM sodium acetate) adjusted

to pH 2.7 with 5 M HCl. Again, the IGFBP-3

containing fractions of several column runs were

collected and stored at � 20 jC. Under these buffer

conditions, IGFBP-3 eluted at a molecular weight of

approximately 40–50 kDa. For cation exchange chro-

matography, 250 ml of the prepurified IGFBP-3

solution (approximately 100 ml initial serum volume)

was used in one column run. Forty microliters of 2 M

NaCl was added per milliliter and the IGFBP-3

solution was then adjusted to pH 6.15 with 5 M

NaOH. After 1 h incubation and subsequent filtration,

the sample was loaded to an HR 5/10 column packed

with SourceR 15 S, 7� 0.5 cm (Amersham Bioscien-

ces) equilibrated with CIEX buffer of pH 6.15 con-

taining 0.77 mM NaCl and eluted with a flow rate of

1.5 ml/min. Unbound proteins were eluted by an

additional washing step with five column volumes

of the equilibration buffer. Bound proteins were eluted

by a gradual increase in NaCl concentration up to 0.5

M within 4 column volumes. IGFBP-3 containing

fractions were pooled and stored until required for

the reversed-phase chromatography (RPC). After

thawing and filtering, the pooled fractions were

loaded onto an RPC column (SourceR 15RPC ST

4.6/100; Amersham Biosciences). The column was

equilibrated with 0.065% trifluoro acetic acid (TFA),

and a gradient of 20 column volumes of 0.05% TFA

in acetonitrile was used for IGFBP-3 elution. After

identification of IGFBP-3 (see below), the pooled

fractions were lyophilized. This IGFBP-3 preparation

M. Hennies, H. Sauerwein / Journal of Immunological Methods 281 (2003) 9–15 11

was used for biotinylation or it was used as standard

after a further gel filtration: IGFBP-3 dissolved in

PBS was applied to a 1.6� 60 cm Superdex 200Rcolumn equilibrated with two column volumes of 0.2

M acetic acid, 1 M NaCl at a flow rate of 1 ml/min.

Immediately after elution of IGFBP-3, the peak frac-

tions were pooled and, after protein determination,

used for the calibration of a secondary standard of a

bovine serum pool. The concentration of purified

IGFBP-3 was determined photometrically at 280 nm

using the Schepartz Lab Biopolymer Calculator (Ver-

sion 4.1.1 10/4/98; Palmer, 1998) with a coefficient of

extinction of 0.6924 ml/mg� cm.

2.2. Electrophoresis, Western immunoblotting and

Western ligand blotting

Serum samples and purified IGFBP-3 were ana-

lyzed by non-reducing sodium dodecyl sulfate-

polyacrylamide gel electrophoresis (SDS-PAGE)

performed according to Laemmli (1970). Samples

were diluted in sample buffer (4.3% 1 M Tris/Cl,

pH 6.8, 40% glycerol, 2% SDS, 0.02% bromophenol

blue) and applied to the gel (5.6% stacking gel and a

12% resolving gel). Precision Protein Standardsk(BIO-RAD, Hercules, CA, USA) were used as molec-

ular weight markers. After electrophoresis, the gels

were used for Western blot analysis or were stained

with Coomassie Brilliant Blue R 250 (Roth) combined

with a standard silver staining method. After SDS-

PAGE, Western ligand and immunoblots were done

using a nitrocellulose membrane (Sartorius, Gottin-

gen, Germany), a semidry transfer apparatus (Amer-

sham Biosciences) and transfer buffer (50 mM Tris/

Cl, 0.38 M glycine, pH 8.3, 200 ml methanol/l).

Transfer was carried out at 68 mA and 3–4 V for 1

h. Non-specific binding was blocked with 1% bovine

serum albumin (BSA) and 0.02% Tween 20 in PBS.

The membrane was then incubated for 30 min with

biotinylated IGF-1 or primary antibodies (aBP-3-R15/

P4-14, or a well-characterized antibody against sheep

IGFB-3 (Gallaher et al., 1998)) in 1/4000 to 1/10,000

dilutions with blotting buffer (assay buffer as

described below containing 0.1% BSA). IGF-1 bio-

tinylation was done as described for IGFBP-3 below

using a 30-fold molar excess of biotinamidocaproate

N-hydroxysuccinimide ester (BXNHS, Sigma-Al-

drich, Taufkirchen, Germany). Bound ligand or anti-

bodies were incubated for 30 min with either a

streptavidin–peroxidase conjugate solution (10 ng/ml

blotting buffer; Sigma-Aldrich) or a second antibody–

peroxidase conjugate (anti-rabbit IgG, peroxidase con-

jugate, Sigma-Aldrich). After extensive washing

(PBS, 0.05% Tween 20), the peroxidase-containing

bands were visualized by chemiluminescence using

the ECL Plus Kit (Amersham Biosciences).

2.3. Generation of polyclonal antisera

Antisera were raised in crossbreed rabbits immu-

nized using 200 Ag of a synthetic peptide based on the

bovine IGFBP-3 amino acid sequence (STENQAGP-

STHRVPVSKY) bound to 400 Ag keyhole limpet

hemocyanin (Sigma-Aldrich) with glutardialdehyde.

The antigen was subsequently emulsified in complete

Freund’s adjuvant for the first immunization and in

incomplete Freund’s adjuvant for booster immuniza-

tions according to Breier et al. (1991). For booster

immunizations at 4-week intervals, half quantities of

the antigen were used. The rabbits were bled from an

ear vein 1 week after each booster injection. Blood was

allowed to clot at room temperature, and after centri-

fugation (2000� g), aliquots of antiserum were stored

at � 20 jC. For the IGFBP-3 enzyme-linked immu-

nosorbent assay (ELISA), the antisera of the rabbit

with the highest titer were pooled (aBP-3-R15/P4-14).

2.4. Development of ELISA procedure

2.4.1. Biotinylation of bovine IGFBP-3

For tracer synthesis, the purified IGFBP-3 was

biotinylated. A 50-fold molar excess of BXNHS in

dimethylsulfoxide (DMSO, 10 mg/ml) was added to

IGFBP-3 diluted in PBS (200 ng/ml) and the reaction

mixture incubated at room temperature for 3 h. The

reaction was stopped by the addition of 20 Al 1 M

NH4Cl. The biotinylated IGFBP-3 was separated

from free biotin with a PD-10 column (Amersham

Biosciences) equilibrated with 1% BSA solution in

PBS. The tracer containing fractions were pooled,

mixed with the same volume of glycerol and stored

at � 20 jC.

2.4.2. Assay procedure

Microtiter plates (EIA plate 9018, Corning Costar,

Cambridge, MA, USA) were coated with sheep IgG

Fig. 1. Purification of bovine IGFBP-3. Electrophoresis of bovine

serum and of the IGFBP-3 containing fractions after every

purification step. Proteins were stained with Coomassie Brilliant

Blue combined with a standard silver staining method. Lane 1:

Molecular weight markers; lane 2: bovine serum; lane 3: 150 kDa

eluate of the first gel filtration (PBS); lane 4: 40 kDa eluate of the

second gel filtration (pH 2.7); lane 5: cation exchange chromato-

graphy eluate; lane 6: reverse phase chromatography eluate; lane 7:

eluate obtained from the third gel filtration (purified IGFBP-3).

M. Hennies, H. Sauerwein / Journal of Immunological Methods 281 (2003) 9–1512

(100 Al/well in 50 mM sodium hydrogen carbonate,

pH 9.6, containing 150 ng anti-rabbit-crystallizable

region of an immunoglobulin (Fc) fragment antibod-

ies) at 4 jC for 20 h. After coating with 300 Al 2.5%casein in 0.05 M NaCl, pH 7.4 at room temperature

for 1.5 h, the plates were washed five times with

washing buffer. The plates were filled with assay

buffer and stored at 4 jC for up to several weeks

without appreciable loss of sensitivity. The assay

buffer contained 0.1% hydrolyzed gelatin, 0.12 M

NaCl, 0.02 M Na2HPO4, 0.01 M ethylenediaminete-

traacetic acid (EDTA), 0.005% chlorhexidine digluc-

onate (20%), 0.002% phenol red, 200 Al/l proteinaseinhibitor cocktail (completek, Boehringer Mannheim,

Germany) and 0.02% ProClin 150R (Supelco, Belle-

fonte, PA, USA).

IGFBP-3 standards, plasma or milk samples were

prediluted 1/10 to 1/100 in pooled goat serum

depending on IGFBP-3 content. Fifty microliters of

the standards or samples were pipetted into the wells

of the assay plates and 50 Al of antiserum diluted 1/

40,000 with assay buffer were added. After preincu-

bation at 4 jC for 20 h, 50 Al of biotinylated IGFBP-3

in pooled goat serum were added and the mixture was

incubated at 4 jC for another 24 h and then decanted.

One hundred microliters of a streptavidin–peroxidase

conjugate solution (200 ng/ml assay buffer; Sigma-

Aldrich) were added per well. The plates were incu-

bated at 4 jC for 30 min and, after five washes using

a microtiter plate washer (EL404, BIO-TEK

INSTRUMENTS, USA), the wells were filled with

150 Al of a freshly prepared substrate solution con-

taining 0.05 M citric acid, 0.055 M Na2HPO4, 0.05%

urea hydrogen peroxide and 2% of a tetramethylben-

zidine solution (12.5 mg/ml DMSO). The reaction

was stopped after 45 min by the addition of 50 Al 1 M

oxalic acid and the color development was deter-

mined photometrically at 450 nm (with 630 nm as

reference) on a microtiter plate reader (ELX800, BIO-

TEK INSTRUMENTS).

2.5. Animals and treatments

Blood plasma samples were obtained from an

animal experiment in which 12 Brown Swiss dairy

cows were treated once with bovine growth hormone

(POSILACR, Monsanto, USA) according to the man-

ufacturer’s directions for use. A detailed description of

the experimental design is given by Daxenberger et al.

(1998). Blood plasma samples were collected every

second day starting 2 weeks before until 4 weeks after

GH application. Blood samples collected from the

udder vein were stabilized using sodium citrate and

EDTA and, after centrifugation at 1900� g for 20 min

at 4 jC, the plasma samples obtained were stored at

� 20 jC until assayed.

3. Results

Fig. 1 shows SDS-PAGE analysis of the purifica-

tion of IGFBP-3 from bovine serum. From the first gel

filtration, the fractions around a molecular weight of

150 kDa containing a complex of IGFBP-3 with IGF-

1 or -2 and the acid labile subunit (ALS) were pooled

and thus most of the higher and lower molecular

weight proteins were excluded. After incubation at

pH 2.5 and the following gel filtration under acidic

conditions, free IGFBP-3 (molecular weight approx-

imately 40 kDa) was separated from the higher

molecular weight proteins (Fig. 1, lane 4). After the

Fig. 3. Standard curve and serial dilutions of three bovine serum

samples in the IGFBP-3 ELISA.

M. Hennies, H. Sauerwein / Journal of Immunological Methods 281 (2003) 9–15 13

cation exchange, the IGFBP-3 was visible (Fig. 1,

lane 5). Further purification and concentration was

done by RPC followed by an additional gel filtration

step (Fig. 1, lanes 6 and 7).

In Western ligand blot of bovine serum using

biotinylated IGF-1 as ligand, several bands of the

different IGFBPs were observed (Fig. 2, lane 4). The

doublet band at 39 and 43 kDa was identified as

IGFBP-3 by Western blot using a well-characterized

polyclonal antiserum against ovine IGFBP-3 (Fig. 2,

lane 7). The purified IGFBP-3 was identified by all

blotting methods and also by the antibody raised

against the peptide corresponding to the sequence of

the bovine protein (Fig. 2, lane 6). The latter anti-

serum was used to develop the bovine IGFBP-3

ELISA. Serial dilutions of bovine serum, plasma

and milk were parallel to the standard curve of

purified IGFBP-3 as shown for serum in Fig. 3. The

antiserum was highly specific for bovine IGFBP-3.

No major cross reaction could be detected with

IGFBP-3 from horse, pig, chicken and other rumi-

Fig. 2. Electrophoresis of a molecular weight marker (lane 1),

bovine serum (lanes 2 and 4) and purified bovine IGFBP-3 (lanes 3,

5, 6–8). Proteins were stained with Coomassie Brilliant Blue

combined with a standard silver staining method (A). Western

ligand blot (B); nitrocellulose membrane was incubated with

biotinylated IGF-1 followed by streptavidin–peroxidase conjugate

and chemiluminescence. Western immunoblots (C); nitrocellulose

membranes were incubated with the antiserum against a bovine

IGFBP-3-peptide (6), a well-characterized antiserum against ovine

IGFBP-3 (7) or normal rabbit serum (8) followed by a second

antibody–peroxidase conjugate and chemiluminescence.

nants, namely, goats and sheep. Neither human IGF-1,

human IGF-2 and bovine IGFBP-2 (GroPep, Ade-

laide, Australia) at concentrations up to 50 ng/well nor

bovine lactoferrin (Sigma-Aldrich) up to 50 Ag/wellshowed a cross reaction.

The measuring range for a sample diluted 1/10 (5

Al plasma per well) was between 50 and 1000 ng

IGFBP-3 per milliliter serum or milk. Recovery of

three different IGFBP-3 concentrations added to four

samples of bovine serum and milk, respectively,

ranged from 95.3% to 99.1% with an overall mean

recovery of 97.3% for serum and 84.1–109% with an

overall mean of 100.1% for milk. Intra- and interassay

Fig. 4. IGFBP-3 plasma concentrations in 12 lactating dairy cows

before and after treatment with depot-formulated bGH. For

statistical analysis, post-treatment time points were compared to

the pretreatment levels using a non-parametric test for two

dependent samples (Wilcoxon).

M. Hennies, H. Sauerwein / Journal of Immunological Methods 281 (2003) 9–1514

coefficients of variation for four control samples were

6.2% and 9.3% (n = 17), respectively.

To test whether the IGFBP-3 assay generated

biologically plausible results, it was first applied to

plasma samples from dairy cows that had been

treated with GH, a well-characterized stimulator of

IGFBP-3 secretion. As shown in Fig. 4, the mean

plasma levels were approximately 1.5 Ag/ml during

the 2-week pretreatment period. Three days after the

injections, IGFBP-3 plasma concentrations were

increased to about 2 Ag/ml, remained elevated until

day 19 ( p< 0.01) and reached pretreatment levels

thereafter.

4. Discussion

Here we describe the development of a homolo-

gous ELISA for bovine IGFBP-3. Since bovine

IGFBP-3 is not available from commercial sources

and due to the requirement for the protein as standard,

as tracer and for the characterization and validation of

the assay, purification of IGFBP-3 was the first step.

We chose an alternative purification procedure to that

of Gallaher et al. (1998). The use of IGF-1 or -2

linked to the solid phase in affinity chromatography

may be the faster and more efficient way to purify

IGFBPs but the requirement for large amounts of

these hormones is often limited by the high costs of

commercially available IGFs. Our purification proto-

col is based on standard chromatographic methods

such as gel filtration, cation exchange and RPC. The

limited capacity of gel filtration with a few milliliters

of serum per run can be overcome by using a

relatively short, wide column and several repeated

runs on a computer-controlled fast protein liquid

chromatography (FPLC) system. The purification

resulted in IGFBP-3 without visible contamination

as demonstrated by electrophoresis and silver staining.

The purified protein was identified as IGFBP-3 by

both Western ligand blot and by Western immunoblot

using the well-characterized antiserum developed by

Gallaher et al. (1998).

In contrast to a wide range of other hormonal

proteins and peptides such as human IGFBP-3 (Dia-

mandi et al., 2000) or IGF-I (Khosravi et al., 1996) for

which ELISA techniques are readily available, rumi-

nant IGFBP-3 has only previously been measured by

RIA (Gallaher et al., 1998). Most of the published

research literature available for livestock blood or

milk IGFBP-3 concentrations is based on radioactive

Western ligand blots. The ELISA developed in this

study will permit a sensitive and precise quantification

of IGFBP-3 in cattle which can also be used in high-

throughput systems.

An antiserum raised against a specific synthetic

peptide minimizes the risk of cross reactions with

other proteins, in particular with other closely related

IGFBPs. Our antiserum was specific for bovine

IGFBP-3 and comparable to the well-characterized

antiserum against ovine IGFBP-3. Plasma samples

exhibited parallel displacement to purified bovine

IGFBP-3 in the ELISA without interferences from

IGFs and IGFBP-2.

In cattle, the expression of IGFBP-3 mRNA and

the secretion of IGFBP-3 is well known to be stimu-

lated by GH (Cohick et al., 1992; Rausch et al., 2002).

An increase of IGF-1 in blood and in milk from GH-

treated cows has been consistently reported (Schams

et al., 1991; Zhao et al., 1994). For the particular cows

used in the present comparison, Daxenberger et al.

(1998) reported that IGF-1 concentrations in milk

were approximately doubled during GH treatment.

The increase we observed for IGFBP-3 was less

pronounced; on average, the concentrations were

1.3-fold higher during the treatment than before the

injections. These results were comparable to the 1.5-

fold increase in IGFBP-3 plasma concentrations after

GH treatment described by Rausch et al. (2002) but

lower than the 3.3-fold increase in the study of Cohick

et al. (1992) using ligand blot analyses. Nevertheless,

with the ELISA developed, we were able to pick up

these differences and could thus establish the expected

biological response. Since IGF-I alone is not a good

indicator of the galactopoietic potency of GH (Van-

derkooi et al., 1995), IGFBP-3 measurements might

be used in combination to distinguish between GH-

treated and non-treated animals.

In conclusion, the present ELISA technique per-

mits the quantitation of IGFBP-3 concentrations in

plasma, serum and milk of cattle; it therefore provides

a powerful alternative to the Western immunoblotting

and Western ligand blotting methods used to date.

Besides the improvements in terms of sensitivity,

precision and sample throughput, the omission of

radioactive labels offers a substantial advantage.

M. Hennies, H. Sauerwein / Journal of Immunological Methods 281 (2003) 9–15 15

Acknowledgements

We thank Dr. Bernhard Breier, Faculty of Medical

and Health Sciences, University of Auckland, New

Zealand for the antibody against ovine IGFBP-3.

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