Standardization and validation of a simple, sensitive, second antibody format enzyme immunoassay for growth hormone determination in mithun (Bos frontalis) plasma

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<ul><li><p>Technical Report</p><p>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</p><p>1National Research Centre on Mithun (ICAR), Jharnapani, Medziphema, Nagaland,India2Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal,Haryana, India</p><p>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 amplication 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 notinuence 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).</p><p>*Correspondence to: Dr. Mohan Mondal, Scientist (Animal Physiology), National Research Centre on</p><p>Mithun (ICAR), Jharnapani, Medziphema, Nagaland-797 106, India.</p><p>E-mail: mohan_mondal@rediffmail.com</p><p>Received 09 March 2005; Accepted 10 May 2005</p><p>DOI 10.1002/zoo.20069</p><p>Published online 22 July 2005 in Wiley InterScience (www.interscience.wiley.com).</p><p>Zoo Biology 24:483493 (2005)</p><p>c 2005 Wiley-Liss, Inc.</p></li><li><p>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 conrms 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 03 months; Group II, age 34 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 sufciently reliable, economic, andsensitive enough to estimate mithun GH in all physiologic variations. Zoo Biol24:483493, 2005. c 2005 Wiley-Liss, Inc.</p><p>Key words: mithun endocrinology; EIA; GH; semi-wild; rare; growth rate</p><p>INTRODUCTION</p><p>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.</p><p>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 efcientGH 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 peroxidaseamplication system.</p><p>484 Mondal et al.</p></li><li><p>MATERIALS AND METHODS</p><p>Preparation of Biotinyl-GH Conjugate</p><p>Biotinyl-GH conjugate was prepared following the procedures of Prakash et al.[2003].</p><p>Bovine GH Antibody</p><p>The bovine GH (bGH) antiserum (rabbit 3-anti-bGH; Pool 712) used in thepresent investigation was specic for estimation of GH. The details of the antiserumspecicity have been given by Hennies and Holtz [1993].</p><p>Preparation of Affinity-Purified Goat IgG Antirabbit IgG</p><p>The afnity-puried goat IgG antirabbit IgG was developed following theprocedure of Anandlaxmi and Prakash [2001].</p><p>EIA Procedure</p><p>First and second coating and washing of the microtiter plate were carried outas described in Mondal et al. [2005].</p><p>Assay Protocol</p><p>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.</p><p>Substrate Reaction</p><p>The procedures followed for substrate reaction is similar to that described byMondal et al. [2005].</p><p>Biological Validation of the Mithun Plasma GH EIA</p><p>For the biological validation of the assay, two mithuns of 12 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).</p><p>Standardization and Validation of Mithun GH EIA 485</p></li><li><p>In addition, blood samples collected weekly for a consecutive 6-week periodfrom 11 mithun calves (Group I; age range, 03 months) and 27 mithuns (Group II;age range, 34 years) were also analyzed for plasma GH.</p><p>Plasma was harvested from all collected blood samples and was stored at201C until assayed for GH.</p><p>Animal Care and Management</p><p>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.</p><p>Statistical Analysis</p><p>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].</p><p>RESULTS</p><p>Standardization of EIA for Mithun Plasma GH Determination</p><p>Titration of biotinyl-GH antiserum.</p><p>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.</p><p>Assay Validation</p><p>Assay interference and sensitivity.</p><p>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 signicant plasma interference in the assay when comparedto buffer (0 ml plasma); however, no signicant 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 inuence 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 nonspecic 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 signicantly from zero concentration was 1.0 ng/ml plasma. The 50%relative binding (B/B0) sensitivity was 36.0 ng GH/ml plasma.</p><p>486 Mondal et al.</p></li><li><p>10 100 1000 10000</p><p>0.00.10.20.30.40.50.60.70.80.91.01.11.21.31.41.51.6 20 l buffer</p><p>12.5l plasma25l plasma50l plasma</p><p>GH (pg/well/25l)</p><p>Abs</p><p>orb</p><p>anc</p><p>e a</p><p>t 450</p><p>nm</p><p>Fig. 1. Inuence 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.</p><p>100 101 102 103 104</p><p>0</p><p>10</p><p>20</p><p>30</p><p>40</p><p>50</p><p>60</p><p>70</p><p>80</p><p>90</p><p>100 20 l buffer12.5 l plasma25l plasma50l plasma</p><p>GH (pg/well/25l) </p><p>Perc</p><p>ent b</p><p>indi</p><p>ng</p><p>Fig. 2. Inuence 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.</p><p>Standardization and Validation of Mithun GH EIA 487</p></li><li><p>Intra- and interassay precision</p><p>Intra- and interassay coefcients 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.</p><p>Biological Validation</p><p>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).</p><p>GH Concentrations in Two Different Age Groups of Mithun and its Relationto Growth Rate</p><p>The GH concentrations between Group I and II differed signicantly (P o0.0001; Fig. 5). The mean GH concentrations for 6 consecutive weeks for Group Iand II animals were found to be 86.637 3.76 and 33.177 0.82 ng/ml, respectively.Consequently, the growth rate in Group I (0.66 7 0.02 kg/day) was also foundto be signicantly (P o 0.001) higher than that recorded in Group II mithuns(0.25 7 0.01 kg/day; Fig. 6).</p><p>Bovine GH Parallelism with Mithun Plasma</p><p>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,</p><p>-60 0 60 120 180 240 300 360 420 480</p><p>0</p><p>50</p><p>100</p><p>150</p><p>200</p><p>250</p><p>300</p><p>350</p><p>400</p><p>450</p><p>Time interval (minutes)</p><p>GH </p><p>conc</p><p>entra</p><p>tions</p><p> (ng/m</p><p>l) </p><p>Fig. 3. Mean (7SEM) growth hormone prole of two growing mithuns before and afterGRF administration. Synthetic growth hormone releasing-factor (GRF [144]-NH2; 10 mg/100 kg body weight) was administered intravenously and blood samples were collected at60, 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.</p><p>488 Mondal et al.</p></li><li><p>-60 0 60 120 180 240 300 360 420 480 540</p><p>10</p><p>20</p><p>30</p><p>40</p><p>50</p><p>60</p><p>70</p><p>80</p><p>90</p><p>Time interval (minutes)</p><p>GH c</p><p>once</p><p>ntr</p><p>atio</p><p>n (ng</p><p>/ml)</p><p>Fig. 4. Mean (7SEM) growth hormone prole 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.</p><p>1 2 3 4 5 6</p><p>15</p><p>25</p><p>35</p><p>45</p><p>55</p><p>65</p><p>75</p><p>85</p><p>95</p><p>105</p><p>115</p><p>125Group-IGroup- II</p><p>Experimental period (week)</p><p>GH</p><p> (n</p><p>g/m</p><p>l)</p><p>Fig. 5. Changes in plasma growth hormone (mean7SEM) proles in two different agegroups of mithun (Group I, age 03 months; Group II, age 34 yr) for a consecutive 6-weeksampling period. Blood samples were collected weekly.</p><p>Standardization and Validation of Mithun GH EIA 489</p></li><li><p>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).</p><p>DISCUSSION</p><p>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 rst 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-specic antibody is preferred asit reduces assay variabilities associated with uneven binding of the latter antibody tothe wells and further reduces the amount of hormone specic antibody needed in theEIA [Meyer, 1986].</p><p>In the direct EIA, less sample volu...</p></li></ul>

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