development and validation of a biosensor-based immunoassay for progesterone in bovine milk

Download Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

Post on 13-Sep-2016




0 download

Embed Size (px)


  • Development and validation of a biosensor-based immunoassay

    for progesterone in bovine milk

    Els H. Gillis a, James P. Gosling b, Joseph M. Sreenan c, Marian Kane a,*

    aNational Diagnostic Centre, National University of Ireland, Galway, IrelandbDepartment of Biochemistry, National University of Ireland, Galway, IrelandcAnimal Reproduction Department, Teagasc, Athenry, Co. Galway, Ireland

    Received 30 January 2002; received in revised form 22 April 2002; accepted 2 May 2002


    We have developed a rapid automated immunoassay, using the BIACOREk surface plasmon resonance (SPR) biosensor, tomeasure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised

    to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7

    was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis

    (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the

    concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good

    with both intra-assay and inter-assay coefficients of variation < 5%. As results become available within minutes of injection and

    the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used

    in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and

    predict pregnancy.

    D 2002 Elsevier Science B.V. All rights reserved.

    Keywords: SPR; Biosensor assays; Progesterone; Heat detection

    1. Introduction

    Reproductive performance is a major factor affect-

    ing the production and economic efficiency of dairy and

    beef cow herds. According to Sreenan and Diskin

    (1992), a dairy herd is reproductively efficient when

    the calving interval is 365 days, when 90% of the cows

    calve in a 10-week period andwhen 5%or less cows are

    culled for reproductive failure. For herds using artificial

    insemination (AI), failure to detect heat is one of the

    major causes of a prolonged calving interval. Visual

    observation is currently the primary method for heat

    0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

    PII: S0022 -1759 (02 )00166 -7

    Abbreviations: AI, Artificial insemination; CV, Coefficient of

    variation; BIA, Biomolecular interaction analysis; CMO, Carbox-

    ymethyloxime; EDC, N-ethyl-NV(3-ethylaminopropyl) carbodiimide;ELISA, Enzyme-linked immunoassay; DMF, N,N-dimethylforma-

    mide; HBS-EP, HEPES-buffered saline; HEPES, N-[2-hydroxye-

    thyl]piperazine-NV-[2-ethanesulfonic acid]; LOD, Limit of detection;NHS, N-hydroxysuccinimide; SD, Standard deviation; SPR, Surface

    plasmon resonance.* Corresponding author. Tel.: +353-91-586559; fax: +353-91-


    E-mail address: (M. Kane).

    Journal of Immunological Methods 267 (2002) 131138

  • detection, but this is time consuming and repetitive.

    Farmers frequently achieve a heat detection rate of only

    3570% and up to 20% of cows presented for insemi-

    nation are not in heat (Sreenan andDiskin, 1992; Diskin

    and Sreenan, 2000).

    According to Senger (1994), an ideal system for

    detecting oestrous should have the following charac-

    teristics: (i) continuous surveillance of the cow; (ii)

    accurate and automatic identification of the cow in

    oestrous; (iii) operation for the productive life-time

    of the cow; (iv) minimal labour requirements; and

    (v) high accuracy and efficiency (95%) for identify-

    ing the appropriate physiological events that corre-

    late with oestrous or ovulation or both. Elevated

    milk progesterone concentrations indicate luteal

    dominance, while low amounts of progesterone are

    associated with oestrous. Progesterone measurement

    tests based on enzyme-linked immunoassays

    (ELISA) are available as kits for on-farm use (Nebel,

    1988). Most of these tests are, however, manual and

    designed as qualitative tests for the confirmation of

    oestrous and determination of pregnancy or non-

    pregnancy, rather than providing a precise concen-

    tration. Attempts at automation and in-line applica-

    tion of quantitative enzyme immunoassays for

    progesterone have been described (Koelsch et al.,

    1994; Claycomb et al., 1998; Pemberton et al.,

    2001), but, to date, such methods either suffer from

    poor sensitivity or are still too cumbersome for use

    in the milking parlour.

    Biomolecular interaction analysis (BIA) from Bia-

    core Ab uses the optical phenomenon of surface

    plasmon resonance (SPR) to monitor biomolecular

    interactions in real time without labelling. SPR meas-

    ures changes in refractive index of the solution close

    to the sensor surface, resulting from changes in the

    mass concentration of molecules in the solution

    (reviewed by Hashimoto, 2000; Markey, 2000). BIA-

    CORE instrumentation was developed primarily for

    use as a research tool, with a variety of applications

    such as screening biological samples for binding

    partners, the determination of kinetic and equilibrium

    constants for complex formation, epitope mapping

    and ligand fishing for known receptors. Recently, this

    principle has also gained popularity as an analytical

    tool for the determination of concentrations of analy-

    tes in biological fluids. In 1993, Minunni and Mascini

    described a BIACORE assay for the determination of

    the pesticide, atrazine, in drinking water, while Ster-

    nesjo et al. (1995) used a BIACORE to develop a

    biosensor-based immunoassay for the detection of

    sulfamethazine residues in milk. Since then, BIA

    assays have been described for other veterinary drug

    residues (Elliott et al., 1999; Gaudin and Maris,

    2001), toxins (Mullett et al., 1998; Daly et al.,

    2000) and vitamins (Bostrom Caselunghe and Linde-

    berg, 2000; Indyk et al., 2000).

    When compared to traditional techniques, BIA-

    CORE technology offers several significant advan-

    tages, such as high precision, speed, simplicity, and

    automation (Mellgren et al., 1996). This paper

    describes the development and validation, according

    to principles set out by Wong et al. (1997), of a BIA

    assay for real-time measurement of progesterone in

    bovine milk.

    2. Materials and methods

    2.1. Instrumentation

    A BIACORE 2000k biosensor instrument andCM5 sensor chips (research grade) were used (Biacore

    Ab, Uppsala, Sweden). The BIACORE 2000k wascontrolled by BIACORE Control Software version

    3.1.1 running underWindows 95. The software also in-

    cludes an interface to the separate BIAevalution soft-

    ware. The instrument running temperature was 25 jC.

    2.2. Reagents

    HEPES-buffered saline (HBS-EP) buffer pH7.4

    (10 mM N-[2-hydroxyethyl]piperazine-NV-[2-ethane-sulfonic acid] (HEPES), 0.15 M NaCl, 3.4 mM

    EDTA, 0.005% Surfactant P-20), and amine coupling

    kit (containing N-hydroxysuccinimide (NHS), N-

    ethyl-NV(3-ethylaminopropyl) carbodiimide (EDC),and ethanolamine hydrochloride) were obtained from

    Biacore Ab (Uppsala, Sweden). The Ridgeway milk

    progesterone ELISA was purchased from Ridgeway

    Science (Alvington, Gloucestershire, UK). Progester-

    one-3-carboxymethyloxime (CMO) was purchased

    from Steraloids (Wilton, NH, USA). Progesterone

    (MW 314.5) and N,N-dimethylformamide (DMF)

    were purchased from Sigma. All other reagents were

    of analytical grade.

    E.H. Gillis et al. / Journal of Immunological Methods 267 (2002) 131138132

  • 2.3. Monoclonal antibody

    The monoclonal antibody used was that described

    by ORorke et al. (1994). The antibody was raised

    against 11a-hydroxyprogesterone hemisuccinatebovine serum albumin conjugate, which was prepared

    by modification of the mixed anhydride method

    (Erlanger et al., 1957). Ascites fluid from mice

    infected with hybridoma cells secreting antibody

    39C5H7 was diluted 1/5000 in HBS-EP before use.

    2.4. Immobilisation of progesterone-3-CMO deriva-


    Simultaneous immobilisation of progesterone onto

    all four flowcells of a CM5 sensor chip was performed

    outside the biosensor system using conventional amine

    coupling (Sternesjo et al., 1995). For covalent immo-

    bilisation, carboxyl groups of the sensor chip surface

    are activated by derivatisation with N-hydroxysuccini-

    mide (NHS) mediated by N-ethyl-NV(3-ethylamino-propyl) carbodiimide (EDC). The resulting active

    ester groups will react spontaneously with amino

    groups (Johnsson et al., 1991). EDC and NHS were

    mixed (1:1) and 40 Al of the mixture were deposited onthe gold film for 10 min activation. An amine surface

    was then prepared by placing 40 Al of 1M ethylenediamine pH8.5 in contact with the activated surface for

    20 min. Capping any remaining active carboxyl groups

    was achieved by exposing the surface to 40 Al 1Methanolamine for 10 min. Progesterone-3-CMO (2 mg)

    was dissolved in 900 Al DMF and mixed with 450 Al of10 mM sodium acetate solution pH4.6 containing 5 mg

    EDC and 2 mg NHS. Progesterone was then immobi-

    lised on the surface by placing 40 Al of this solution incontact with the amine surface for approximately 15