Technical Report

Standardization and Validation of aSimple, Sensitive, Second AntibodyFormat Enzyme Immunoassay forGrowth Hormone Determination inMithun (Bos frontalis) PlasmaMohan Mondal,1* Chandan Rajkhowa,1 and B.S. Prakash2

1National Research Centre on Mithun (ICAR), Jharnapani, Medziphema, Nagaland,India2Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal,Haryana, India

To facilitate research into the action of growth hormone (GH) in mithun (Bosfrontalis), we standardized and validated a simple and highly sensitive enzymeimmunoassay (EIA) for GH determination in mithun blood plasma on microtiterplates using biotin-streptavidin amplification system and the second antibodycoating technique. Biotin was coupled to GH and used to bridge betweenstreptavidin-peroxidase and immobilized antiserum in competitive assay. TheEIA was carried out directly in 25 ml mithun plasma. The GH standards rangingfrom 0.25 ng/well/25 ml to 128 ng/well/25 ml were prepared in charcoal-treatedplasma collected from an aged (410 years) senile mithun. The sensitivity of EIAprocedure was 1.0 ng/ml plasma; the 50% relative binding sensitivity was seen at36 ng/ml plasma. Plasma volumes for the EIA, namely 12.5 and 25 ml, did notinfluence the shape of standard curve even though a drop in the optical density(OD)450 observed with higher plasma volumes was due to higher inherent GHcontent in mithun plasma collected from an aged (410 years) senile mithun.For the biological validation of assay, two mithuns were administered withsynthetic bovine GH-releasing factor (GRF; 10 mg/100 kg body weight;intravenous) and another two were administered sterile normal saline (controls).

*Correspondence to: Dr. Mohan Mondal, Scientist (Animal Physiology), National Research Centre on

Mithun (ICAR), Jharnapani, Medziphema, Nagaland-797 106, India.

E-mail: mohan_mondal@rediffmail.com

Received 09 March 2005; Accepted 10 May 2005

DOI 10.1002/zoo.20069

Published online 22 July 2005 in Wiley InterScience (www.interscience.wiley.com).

Zoo Biology 24:483–493 (2005)

�c 2005 Wiley-Liss, Inc.

Jugular blood samples were collected at �60, �45, �30, �15, �10, �5, 0, 5, 10,15, 30 min and thereafter at 15-min intervals beginning 1 hour before GRFinjection up to 8-hr post treatment, and samples were assayed for plasma GH. Intwo animals, a peak of GH was recorded at 15 min of GRF administration,which confirms the biological validation of the EIA. Plasma GH estimatedin blood samples collected for 6 consecutive weeks from two different agegroups of mithun (Group I, age 0–3 months; Group II, age 3–4 yr) showed higher(Po0.0001) mean plasma GH in younger than in adult mithuns and consequentlyhigher growth rates in the younger group. A parallelism test conducted between abuffer standard curve of bovine GH and GH measured from serial dilution ofmithun plasma containing high concentration of endogenous GH showed goodparallelism with a standard curve. In conclusion, the EIA developed for GHdetermination in mithun blood plasma is sufficiently reliable, economic, andsensitive enough to estimate mithun GH in all physiologic variations. Zoo Biol24:483–493, 2005. �c 2005 Wiley-Liss, Inc.

Key words: mithun endocrinology; EIA; GH; semi-wild; rare; growth rate

INTRODUCTION

Mithun (Bos frontalis) is a semi-wild ruminant species found mainly in theNorth-Eastern Hills region (NEHR) of India and in some locations of Bhutan,Myanmar, Bangladesh, China, and Malaysia. This ungulate of NEHR has animportant economical, social, and cultural role to the local population, once it actsas a potential source of meat and milk. Due to remoteness of their habitats and otherecological and sociopolitical factors, however, mithuns remain one of the leaststudied ungulates.

Growth hormone (GH) is necessary for most aspects of the postnatalsomatic growth [Nalbandov, 1963; Etherton and Kensinger, 1984]. Exogenous GHhas been found to stimulate a number of physiologic processes including bodygrowth and milk production [Machlin, 1973; Bauman and McCutcheon, 1986;McDowell et al., 1987; Burton et al., 1990]. Exogenous GH has also been reportedto improve fertilization of in vitro matured oocytes [Izadyar et al., 1997] as well as toimprove the superovulatory response [Gong et al., 1996] and to increase number offollicles in heifers [Tripp et al., 2000]. Estimation of GH in blood plasma of neonatalcalves to assess its genetic potential for milk production at an early stage hasalso been suggested [Woolliams and Lovendahl, 1991]. GH measurements inplasma of other species are currently being carried out either by sensitiveradioimmunoassay (RIA) procedures using 125I as the label [Petitclerc et al., 1987;Kazmer et al., 1992; Singh and Ludri, 1994; Jailkhani and Madan, 1999] orenzyme immunoassay (EIA) procedures [Hennies and Holtz, 1993; Prakash et al.,2003]. To facilitate further research into the action of GH in mithun, an efficientGH assay is needs to be standardized and validated in mithun blood plasma. NoRIA or EIA has been standardized or validated as yet for GH determinationin mithun blood plasma. We therefore decided to standardize and validate asensitive and convenient second antibody EIA for GH determination in mithunplasma using the second antibody technique and biotin-streptavidin peroxidaseamplification system.

484 Mondal et al.

MATERIALS AND METHODS

Preparation of Biotinyl-GH Conjugate

Biotinyl-GH conjugate was prepared following the procedures of Prakash et al.[2003].

Bovine GH Antibody

The bovine GH (bGH) antiserum (rabbit 3-anti-bGH; Pool 7–12) used in thepresent investigation was specific for estimation of GH. The details of the antiserumspecificity have been given by Hennies and Holtz [1993].

Preparation of Affinity-Purified Goat IgG Antirabbit IgG

The affinity-purified goat IgG antirabbit IgG was developed following theprocedure of Anandlaxmi and Prakash [2001].

EIA Procedure

First and second coating and washing of the microtiter plate were carried outas described in Mondal et al. [2005].

Assay Protocol

Duplicates of 25 ml of unknown plasma or bGH standards (USDA-bGH-B-1)prepared in charcoal-treated plasma ranging from 0.25 pg/25 ml/well to 128.00 ng/25ml/well were simultaneously pipetted into respective wells along with 100 ml of GHantibody diluted 1:40,000 in assay buffer (50 mM NaPO4, 0.15 M NaCl, and 0.02%thiomersal; pH 7.4) supplemented with 10% mithun plasma collected from old-aged(410 years) senile mithun with the aid of a dilutor-dispenser. Plates were incubatedovernight at room temperature after 30 min with constant agitation. They were thendecanted and washed twice with washing solution before addition of 100 ml ofbiotinyl-GH conjugate per well diluted 1:6,000 in assay buffer. The plates wereincubated further for 30 min with constant agitation, decanted, and washed fourtimes. Next, 20 ng streptavidin-peroxidase (Sigma, Munchen, Germany) in 100 ml ofassay buffer was added to all the wells and the plates were wrapped in aluminiumfoils and incubated for a further 30 min under constant agitation. All steps werecarried out at room temperature.

Substrate Reaction

The procedures followed for substrate reaction is similar to that described byMondal et al. [2005].

Biological Validation of the Mithun Plasma GH EIA

For the biological validation of the assay, two mithuns of 1–2 years of age wereadministered synthetic bovine GRF (10 mg/100 kg body weight, intravenously;Sigma-Aldrich Chemie, Munchen, Germany) and another two animals of similar agewere administered sterile saline (controls). Blood samples were collected at �60,�45, �30, �15, �10, �5, and 0 min before administration, then at 5, 10, 15, and30 min, and thereafter at an interval of 15 min post-administration up to 8 hr usingan indwelling jugular catheter. All experimental protocols and animal care met theregulations of the Institutional Animal Care and Utilization Committee (IACUC).

Standardization and Validation of Mithun GH EIA 485

In addition, blood samples collected weekly for a consecutive 6-week periodfrom 11 mithun calves (Group I; age range, 0–3 months) and 27 mithuns (Group II;age range, 3–4 years) were also analyzed for plasma GH.

Plasma was harvested from all collected blood samples and was stored at�201C until assayed for GH.

Animal Care and Management

All mithuns selected for the experiment were maintained at National ResearchCentre on Mithun, Medziphema Farm of Nagaland State of India. These animalswere maintained in semi-intensive system as detailed by Mondal et al. [2004]. Bodyweight of animals was recorded weekly.

Statistical Analysis

The results are presented as mean 7 standard error of the mean (SEM). Thedata on plasma GH levels and growth rates for consecutive 6 weeks for both groupswere analyzed using the analysis of variance (ANOVA) technique [Snedecor andCochran, 1980].

RESULTS

Standardization of EIA for Mithun Plasma GH Determination

Titration of biotinyl-GH antiserum.

A two-dimensional titer determination for the optimum dilution of GH labeland the antiserum was carried out. Antibody dilutions ranging from 1:10,000 to1:640,000 and the biotinyl-GH dilutions of 1:1,000 to 1:32,000 were tested. Theantibody titer of 1:40,000 and the biotinyl-GH conjugate titer of 1:6,000 were foundto be the most suitable and achieved an optical density (OD)450 of around 1.871.

Assay Validation

Assay interference and sensitivity.

To determine the possible interference of plasma with the assay sensitivity,bGH standards in various amounts of plasma (0, 12.5, 25, and 50 ml) were run in anassay (Fig. 1). There was significant plasma interference in the assay when comparedto buffer (0 ml plasma); however, no significant difference in the absolute bindingsensitivity was found between plasma volumes of 12.5 and 25 ml. Increasing plasmavolumes from 12.5 to 25 ml did not influence the shape of standard curve; however, adrop in the OD450 was seen with higher plasma volumes (Figs. 1 and 2). Keepingthese aspects in view, standards were subsequently prepared in charcoal-treatedplasma and run along with the unknowns in the assay. The nonspecific binding(OD450) using all these volumes of plasma was recorded to be within the range of0.091 to 0.99. All assays were therefore conducted by taking 25 ml of unknownplasma samples and standards per well just to increase assay sensitivity per milliliterof plasma in duplicates. The GH range that could be measured was between 1.0 to512 ng GH/ml and covered all possible physiologic variations. The lowest GHdetection limit significantly from zero concentration was 1.0 ng/ml plasma. The 50%relative binding (B/B0) sensitivity was 36.0 ng GH/ml plasma.

486 Mondal et al.

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Fig. 1. Influence of different volumes (12.5, 25, and 50 ml) of mithun plasma on opticaldensity displacement in growth hormone standard curve. Along with different volumesof plasma, the standards were also prepared in assay buffer. Optical density was measuredat 450 nm.

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Fig. 2. Influence of different volumes (12.5, 25 and 50 ml) of mithun plasma on percentagebinding in growth hormone standard curve. Along with different volumes of plasma, thestandards were also prepared in assay buffer. Optical density was measured at 450 nm.

Standardization and Validation of Mithun GH EIA 487

Intra- and interassay precision

Intra- and interassay coefficients of variation, determined using pooled plasmacontaining 2.0 and 256.0 ng/ml, were found to be 9.62%, 6.75%, and 8.83%, 7.12%,respectively.

Biological Validation

In two mithun calves administered with GRF, a mean peak value of 308.2 795.3 ng GH/ml was recorded at 15 min after GRF administration (Fig. 3). The meanGH level estimated in two control mithuns did not exhibit such a rise as thatrecorded in GRF-treated mithuns (Fig. 4).

GH Concentrations in Two Different Age Groups of Mithun and its Relationto Growth Rate

The GH concentrations between Group I and II differed significantly (P o0.0001; Fig. 5). The mean GH concentrations for 6 consecutive weeks for Group Iand II animals were found to be 86.63 7 3.76 and 33.17 7 0.82 ng/ml, respectively.Consequently, the growth rate in Group I (0.66 7 0.02 kg/day) was also foundto be significantly (P o 0.001) higher than that recorded in Group II mithuns(0.25 7 0.01 kg/day; Fig. 6).

Bovine GH Parallelism with Mithun Plasma

To conduct the parallelism test, a mithun plasma sample containing high levelof endogenous GH was serially diluted (containing 25, 12.5, 6.25, 3.13, 1.6, 0.8, 0.4,

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Fig. 3. Mean (7SEM) growth hormone profile of two growing mithuns before and afterGRF administration. Synthetic growth hormone releasing-factor (GRF [1–44]-NH2; 10 mg/100 kg body weight) was administered intravenously and blood samples were collected at�60, �45, �30, �15, �10, �5, and 0 min before administration, and 5, 10, 15, 30 min andthereafter at an 15-min intervals post-administration up to 8 hr.

488 Mondal et al.

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Fig. 4. Mean (7SEM) growth hormone profile of two growing mithuns before and afternormal saline administration. Normal saline was administered intravenously and bloodsamples were collected at �60, �45, �30, �15, �10, and �5 min before administration, and 5,10, 15, 30 min and thereafter at 15-min intervals post-administration up to 8 hr.

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Fig. 5. Changes in plasma growth hormone (mean7SEM) profiles in two different agegroups of mithun (Group I, age 0–3 months; Group II, age 3–4 yr) for a consecutive 6-weeksampling period. Blood samples were collected weekly.

Standardization and Validation of Mithun GH EIA 489

and 0.2 ml mithun plasma sample sizes) and run along with bGH standards (in assaybuffer) in an assay. It showed a good parallelism with the bovine standard curve,although a small deviation was found at very low plasma volumes (Fig. 7).

DISCUSSION

Many assay procedures, most of which are RIA, have been developed forestimation of GH in plasma or serum of other species, but to the best of ourknowledge the GH EIA standardized here is the first report of standardization ofdirect EIA in unextracted mithun plasma using the second antibody coatingtechnique and GH-biotin-streptavidin system. The use of a second antibody forcoating the microtiter plate wells instead of hormone-specific antibody is preferred asit reduces assay variabilities associated with uneven binding of the latter antibody tothe wells and further reduces the amount of hormone specific antibody needed in theEIA [Meyer, 1986].

In the direct EIA, less sample volume is desirable to reduce nonspecific bindingand plasma matrix effects and for a high degree of sensitivity [Mutayoba et al., 1990].This requires the use of highly specific antibody, a very efficient amplification system,and optimum ligand-antibody dilutions at a suitable incubation temperature. In ourGH EIA, there was a drop in the OD450 with increasing plasma volumes, althoughthe sensitivities per well and the relative binding percentages did not change when12.5 and 25 ml plasma were taken along with standards (Figs. 1 and 2). This effect ofplasma on optical densities can be compensated for by using the same plasmavolumes for standards and unknown samples. To compensate the interferenceof plasma, we supplemented 10% mithun plasma with diluted antisera in the assay.A high assay sensitivity of 1.0 ng/ml was obtained, whereas 25 ml plasma was taken

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490 Mondal et al.

for estimation. This was sufficient to estimate mithun GH for all physiologicvariations (Figs. 3 and 4).

In the present investigation, plasma GH levels of adult mithuns (Group II)were lower (P o 0.0001) than were those recorded in younger mithun calves(Group I). A similar trend has also been reported in other animal species [Mears andSchaalje, 1993; Mondal and Prakash 2003, 2004]. Higher basal plasma GH, whichmay be due to higher secretion rate or lower metabolic clearance rate of GH inyounger mithuns where growth potential is very high, is consistent with a role of GHfor body growth [Trenkle, 1971; Mondal and Prakash, 2003, 2004].

The GH EIA standardized here offers a reliable alternative to RIA, which isnot available for mithun GH determination. Apart from being nonradioactive innature, the EIA procedure has several other advantages over RIA, namely: (1) it maybe more sensitive than are RIA procedures; (2) it requires less plasma volumes ascompared to that needed for conventional RIA methods; (3) biotinylated GH has amuch longer shelf life (several years) as compared to iodinated GH (60 days); and(4) it is less labor intensive and is quick and relatively simple to carry out. Inaddition, because the procedure requires less capital investment in terms ofinstruments, the GH EIA provides a safe, cost-effective and less time-consumingalternative to conventional RIA procedures and can therefore be adopted inlaboratories of developing countries where financial constraints limit the adoption ofRIA. Highly purified GH preparations from cattle and other species of animals areavailable, and biotinylation of GH is not difficult as compared to iodinationprocedures. Good quality biotin and streptavidin peroxidase are also available

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Fig. 7. Parallelism for bovine growth hormone (bGH) standards with serially diluteddifferent volumes (25, 12.5, 6.25, 3.13, 1.6, 0.8, 0.4, and 0.2 ml) of mithun plasma.Standards for bGH ranged from 0.25 ng/25 ml/well to 128 ng/25 ml/well.

Standardization and Validation of Mithun GH EIA 491

commercially at rather cheaper costs compared to that of 125I preparation. Inconclusion, GH EIA as standardized here is an economic and sensitive enough toestimate mithun GH of all physiologic variations beginning with calves to adultmithuns.

ACKNOWLEDGMENTS

We thank Dr. M. Hennies, Institute of Tieranatomic, Bonn, Germany for thegenerous gift of highly specific bGH antibody. The supply of highly purifiedreference preparation of bovine GH by USDA Animal Hormone Program,Beltsville, USA is gratefully acknowledged. We also thank the Director, NationalDairy Research Institute, Karnal-132 001 (Haryana), India for providing facilitiesfor the present work.

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