Small Ruminant Research 84 (2009) 22–27

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Small Ruminant Research

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Validation of a simple, sensitive enzyme immunoassay (EIA) for thedetermination of caprine plasma LH

A. Haldara,∗, R. Paula, S. Panb, A. Mitrac, C. Biswasd, D. Majumdard, S. Ghoshe,N.P. Singha, S.V. Ngachanf, K.M. Bajurbhorueag, B.S. Prakashh

a Animal Reproduction Discipline, ICAR Research Complex for NEH Region, Lembucherra-799210, West Tripura, Indiab Department of Animal Production & Management, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata, West Bengal, Indiac Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar-243122, Uttar Pradesh, Indiad Faculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, P.O. Krishi Viswavidyalaya, Mohanpur, Nadia-741252, West Bengal, Indiae Human Genetics Unit, Indian Statistical Institute, Kolkata, West Bengal, Indiaf ICAR Research Complex for NEH Region, Umiam-793103, Meghalaya, Indiag Division of Animal Science, Krishi Bhawan, New Delhi-110001, Indiah Reproductive Physiology Laboratory, National Dairy Research Institute, Karnal-132001, Haryana, India

a r t i c l e i n f o

Article history:Received 4 January 2009Received in revised form 21 April 2009Accepted 29 April 2009Available online 31 May 2009

Keywords:GoatEIAPlasma LH

a b s t r a c t

The study describes the development and validation of a simple and highly sensi-tive enzyme immunoassay (EIA) for the determination of goat plasma LH, utilizing thebiotin–streptavidin peroxidase amplification system in a competitive-binding assay. Micro-titer plates were coated with goat anti-rabbit globulin as the second antibody and biotinwas coupled to oLH and used as a bridge between streptavidin peroxidase and immobi-lized oLH beta antisera. A simple 4-step procedure was used at room temperature for thesample analysis: (1) overnight incubation of LH standards and plasma samples with the LHantibody in a 96-well micro-titer plate, pre-coated with a second antibody; (2) incubationin a biotinylated–LH conjugate for 30 min; (3) incubation with streptavidin peroxidase fora further 30 min and (4) lastly incubation in tetramethyl benzidine substrate for 40 minto develop colour. A two-dimensional titer determination test proved the antibody titer of1:100,00,000 and the biotinylated–LH conjugate titer of 1:4000 to be the most suitable.As the absolute binding sensitivity of different concentrations of oLH in 80 �l plasma wassimilar to that observed in buffer standards, all assays were conducted using 80 �l of theunknown plasma samples. A standard curve was obtained in the range of 25–12,800 pgLH/well in 80 �l hormone-free plasma. The sensitivity of the EIA procedure was 25 pg/wellLH, which corresponds to 0.31 ng/ml plasma. In a parallelism test, the relative percentagebinding curve for serially diluted goat plasma samples containing high levels of endogenous

LH and for the oLH standard ran parallel to each other, thereby confirming the actual LHestimation in goat plasma. The classical patterns of plasma LH during the estrous cycle andthe gradual increases in LH concentrations after gonadotrophin releasing hormone (GnRH)administration provided biological validation of the assay for the determination of plasma

es. Thissma LH

LH in caprine specianalysis of goat pla

∗ Corresponding author. Tel.: +91 9436464223; fax: +91 381 326846.E-mail address: haldar vet@yahoo.co.in (A. Haldar).

0921-4488/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.smallrumres.2009.04.007

EIA technique provides a simple and sensitive method for routine.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Hypophysial LH plays an important role in the ovu-lation and luteinization process in females. Its synthesisand secretion is regulated by hypothalamic factors,

A. Haldar et al. / Small Ruminant Research 84 (2009) 22–27 23

Table 1Different buffers and solutions used in the enzyme immunoassay.

Sl. no. Reagent Formula

1 Coating buffer 15 mM Na2CO3, 35 mM NaHCO3; pH 9.62 Phosphate buffer saline (PBS) 10 mM NaPO4, 0.5 M NaCl; pH 7.23 Blocking solution 1% bovine serum albumin (BSA) (Merck, Germany) in PBS buffer4 EIA assay buffer 0.05 M NaPO4, 0.15 M NaCl, 0.02% thiomersal (Sigma–Aldrich,

Germany); pH 7.45 Substrate buffer 0.05 M citric acid, 0.11 M Na2HPO4, 0.05% ureum peroxide (Merck,

Germany); pH 4.06

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Tetramethyl benzidene (TMB) solution

Substrate solutionWashing solution

ainly gonadotrophin releasing hormones (GnRH), andy circulating gonadal steroids, including estradiol androgesterone (Legan et al., 1977). The measurement ofirculating caprine LH is thus an important tool fornderstanding the hormonal mechanisms involved in theypothalamic–pituitary ovarian axis of the caprine species.

Radioimmunoassay (RIA) is currently the method mostommonly used for the quantification of oLH (Niswendert al., 1969; Scaramuzzi et al., 1970; Schanbacher et al.,991). Although the RIA method is reliable and accurate,he use of radio-isotopes has the disadvantages of a lim-ted half-life and licensing and safety considerations thatestrict the measurement of blood LH to specified labora-ories. The development of an enzyme immunoassay (EIA)

ethod provides a viable alternative to RIA.While EIA procedures have been developed for ovine LH

Valares et al., 2007), bovine LH (Mutayoba et al., 1990) anduffalo LH (Prakash et al., 2002), no EIA has thus far beenstablished for caprine plasma LH. Hence, it was decidedo develop a sensitive and convenient second antibodyIA technique on micro-titre plates for the quantificationf plasma LH in goats using the biotin–streptavidin per-xidase amplification system. The ability of this assay toonitor plasma LH levels during the estrous cycle and in

esponse to a dose of GnRH in the goat was included for thealidation of the method.

. Material and methods

.1. Experimental animals and management

The study was conducted on a goat farm of the Indian Council of Agri-ultural Research (ICAR) Centre for the North Eastern Hill (NEH) Region,embucherra, West Tripura, India. The area is located 12.8 m above meanea level in the north-eastern region at a 22◦56′N latitude and 90◦09′E lon-itude. Four Black Bengal does averaging 27 months of age, with a bodyeight ranging from 21.4 kg to 27.2 kg were selected for the biological val-

dation of the EIA technique. Routine deworming and vaccination of theoes were followed as per schedule. The does were fed according to theecommendations of ICAR (1998), grazing on natural pasture and receivingupplementary concentrate feeding. Fresh drinking water was availabled libitum to all the animals. The experimental protocol and animal careere met in accordance with the National guidelines for Care and Use ofgricultural Animals in Agricultural Research and Teaching.

.2. Preparation of hormone-free goat plasma

LH hormone free goat plasma was prepared for use in the assay stan-ard curve. Blood samples were collected in heparinized tubes (20 IUeparin/ml blood) from two goats at day 4 postpartum, in which the cir-ulating LH concentration was anticipated to be the bare minimum. The

12.5 mg of 3,3′ ,5,5′-tetramethyl benzidene (Merck, Germany) perml of dimethyl sulfoxide (Merck, Germany)340 �l TMB solution in 17 ml substrate buffer0.05% Tween 20 (Sigma–Aldrich, Germany) in distilled water

plasma was separated by centrifugation of the blood at 2500 × g for 10 minat 4 ◦C. To remove any trace of LH in the plasma, the plasma was treatedwith a charcoal and dextran mixture (Sarkar and Prakash, 2006) as follows:

The mixture of 14 g activated charcoal (Qualigens, India) and 1.4 g dex-tran T-70 (Sigma–Aldrich, Germany) per 100 ml plasma was washed withdistilled water by thorough mixing, using a magnetic stirrer overnight,at room temperature. After allowing the mixture to stand for 5 min, thesupernatant was discarded and the mixture again subjected to the wash-ing process three more times at 8 h intervals to remove all traces ofsuspended fine charcoal particles. Goat plasma was then added to thewashed charcoal–dextran mixture and mixed thoroughly for 2 h at 4 ◦C.The mixture was then further centrifuged (2500 × g) for 1 h at 4 ◦C and thesupernatant filtered using a disposable Acrodisc® syringe filter (Pall LifeSciences, India) to remove the suspended particles. The filtrate was againcentrifuged at 5000 × g for 1 h at 4 ◦C and the supernatant re-filtered. ThisLH-free plasma thus recovered, was stored at −20 ◦C for future use.

2.3. Stock solutions

The buffers and solutions used in the assay are presented in Table 1.

2.4. Hormonal antibody

Ovine LH beta antisera (NIDDK-anti-oLHbeta-IC-1 A.S. Lot# AFPP697071P) used in the assay was procured from Dr. A.F. Parlow, NationalHormone Peptide Program (NHPP), Harbor-UCLA Medical Centre, Carson,CA, USA. As per technical information supplied by Dr. A.F. Parlow, the speci-ficity of anti-oLHbeta-IC-1 for percentage binding with iodinated oBeta LH,oAlphaLH, oBetaFSH and oBetaTSH was 48.0, 0.0, 1.7 and 21.1, respectively.

2.5. Preparations of biotinylated LH conjugate

A biotinamidocaproate N-hydroxysuccinimide ester (Sigma–Aldrich,Germany) was coupled to ovine LH (oLH) to prepare the biotinylatedoLH conjugate. Here 40 �g oLH (NIDDK-oLH, Bio, Lot # AFP 5551B) wasdissolved in 200 �l 0.1 M carbonate buffer (coating buffer) and 12 �lbioptinamidocaproate-N-hydroxysuccinimideester (Sigma–Aldrich, Ger-many) dissolved in dimethyl sulfoxide (1 mg/ml) were added together andthe mixture immediately vortexed and incubated for 3 h at room tempera-ture, under constant agitation. The coupling reaction was then terminatedby the addition of 20 �l NH4Cl (1 M), and the reaction mixture incubatedfurther for 30 min, before the addition of 2 ml of a solution of 1% BSA in PBS(pH 7.4). The biotin–LH conjugate was isolated using a dialysis mixture in adialysis sac (D 6066-25 EA, 35 mm; Sigma–Aldrich, USA) overnight, at 4 ◦C,with 4 PBS replenishments. After dialysis, the conjugate was mixed withan equal volume of glycerol to prevent freezing and preserved at −20 ◦Cin 1 ml aliquots.

2.6. Preparation of plates: first coating with second antibody

Ninety-six-well micro-titer plates (Nunc, Genetix Biotech Asia Pvt.Ltd., India) were coated (first coating) with goat IgG anti-rabbit IgG (secondantibody), prepared as by Anandlaxmi and Prakash (2001), at a concen-tration of 1 �g/100 �l of coating buffer/well. The plates were covered withparafilm and incubated overnight at 4 ◦C.

inant R

A representative oLH standard curve with a range of0 and160 ng/ml is set out in Fig. 1. The optical density(OD450) of the 0 point was recorded as 1.4. The OD450for the non-specific binding using 80 �l plasma was low,

24 A. Haldar et al. / Small Rum

2.7. EIA procedure

2.7.1. Second coatingDuring the first day, 300 �l 1% BSA in the EIA assay buffer was added to

all the wells to block the remaining binding sites and then the plates wereincubated for 30 min at room temperature, under continuous shaking.

2.7.2. WashingThe coated plates were decanted and washed twice with 350 �l/well

of washing solution (0.05% Tween-20), using an automated micro-titerplate washer (Model: W-2002, Electronic Corporation of India Ltd., India).

2.7.3. Antigen–antibody reactionoLH standards (NIDDK-oLH, Bio, Lot# AFP 5551B) were prepared in

charcoal-dextran stripped goat plasma to provide a range of between 25and 12,800 pg/well/80 �l plasma corresponding to a range between 0.3and 160 ng/ml. The standard curve was constructed using 10 standardsolutions. Duplicates of each standard or unknown plasma sample (80 �l)were added to the respective wells. Thereafter, 100 �l of the LH antibody,diluted 1–10 million in EIA assay buffer was dispensed into each well,except the wells marked for non-specific binding (NSB). Plates were thencovered with parafilm and aluminum foil and subjected to constant gen-tle agitation for 30 min and then kept overnight, with incubation at roomtemperature.

2.7.4. WashingDuring the second day, the micro-titer plates were decanted and

washed twice with washing solution using the micro-titer plate washer.

2.7.5. Addition of biotinylated LH conjugateA volume of 100 �l biotinylated oLH conjugate, diluted 1:4000 in assay

buffer was added to each well. The micro-titer plates were further incu-bated for 30 min under continuous shaking at room temperature.

2.7.6. Addition of enzymeThe plates were decanted and washed four times. Thereafter, 20 ng

streptavidin peroxidase (Sigma–Aldrich, Germany) dissolved in 100 �l EIAassay buffer was added to all the wells. The plates were wrapped in alu-minum foil and incubated for a further 30 min under constant agitation atroom temperature.

2.7.7. Enzyme–substrate reactionThe plates were decanted and washed five times with the washing

solution and 150 �l of the substrate solution added to each well and incu-bated further in the dark for 40 min.

2.7.8. Stopping of the enzyme–substrate reactionThe enzyme–substrate reaction was terminated by the addition of

50 �l of 4 N H2SO4 (Prakash et al., 2002).

2.7.9. Measurement of optical densityAbsorbance by the yellow colour obtained after the enzyme–substrate

reaction was measured at 450 nm with the aid of an 8-channel automatic96 well micro-titer plate reader (Model: MS 5605A, Electronic Corporationof India Ltd., India).

2.8. Antigen–antibody titer test

A two-dimensional titer determination test for the optimum dilutionof biotinylated oLH and the antiserum was performed. In total 5 titer testswere run.

2.9. Plasma interference test

oLH standards in various volumes of hormone-free goat plasma (20,40 and 80 �l) were run in the assay to determine the possible interferenceof plasma with the accuracy of the assay sensitivity.

2.10. Test of parallelism between oLH standards and endogenous LH ingoat plasma

To determine whether parallelism exists between oLH standards andendogenous goat plasma LH, plasma samples containing high concentra-tions of endogenous LH were collected following gonadotrophin releasing

esearch 84 (2009) 22–27

hormone (GnRH) treatment of two goats and pooled. The pooled sam-ple was serially diluted with EIA assay buffer to obtain plasma volumesof 80, 40, 20, 10, 5 and 2.5 �l respectively. Parallelism was assessedbetween these serial dilutions and oLH standards (ranging from 25 to12,800 pg/80 �l/well) prepared in the EIA assay buffer.

2.11. Precision of the assay and detection limit

Plasma samples collected from the experimental does were usedto calculate the coefficients of variance. The intra-assay coefficient ofvariation was calculated from 4 pooled samples containing two LH con-centrations (40 and 0.6 ng/ml). Each sample was then analyzed three timesin the same assay. The inter-assay coefficient of variation was calculatedusing the same four samples containing the same LH concentration, andmeasured in 12 different assays. The detection limit of the assay wasrecorded from the standard curve of each assay.

2.12. Biological validation of the caprine LH EIA

To evaluate the suitability of the assay, the biological validation of theassay using Black Bengal does was performed under normal physiologicalconditions, as well as under hormone induced situations. (i) In Experiment1, blood samples were collected daily for a period of 38 days from two cyclicdoes into 4 ml heparinized (20 IU heparin/ml blood) polypropylene tubesby jugular vein puncture (08:00, prior to feeding); (ii) in Experiment 2, toguarantee a high and measurable plasma LH level, two does were adminis-tered 2 �g/10 kg body weight of a GnRH analogue (Receptal® vet, IntervetInt. GmbH, Germany) intramuscularly and subjected to serial blood sam-plings using an indwell 20 G intravenous (i.v.) catheter (Jelco®, MedexMedical Ltd., Great Britain) in the jugular vein at 15 min intervals – start-ing 1 h prior to GnRH administration – 8 h post-injection and thereafter at1 h intervals for another 10 h period.

Once collected, the blood samples were placed in an ice-box andreturned to the laboratory, where the blood samples were centrifuged(2500 × g for 10 min at 4 ◦C) and the plasma separated and collected inthe storage vial and stored (−20 ◦C), until the time of the hormone assay.

Plasma progesterone concentration determination was used as anindicator of ovulation during estrous cycle. Plasma progesterone concen-tration was estimated using a commercially available progesterone EIAtest kit supplied by Endocrine Technologies, Inc., Newark, CA, USA.

3. Results

3.1. Standard curve

Fig. 1. A representative standard curve for the enzyme immunoassay todetermine plasma LH in goats.

inant Research 84 (2009) 22–27 25

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anging from 0.072 to 0.125. The 50% relative bindingB/B0) sensitivity was 800 pg/80 �l plasma /well, whichorresponded to 10 ng/ml plasma.

.2. Titration of biotinyl–LH and LH antiserum

A two-dimensional titer determination test for theptimum dilution of biotinyl–LH and the oLH antiserumas performed. Initially, antibody dilutions ranging from:10,000 to 1:6.4 million and a biotinyl–LH dilution of:500 to 1:16,000 were tested. However, the optical den-ity (OD) for all the combinations was very high. The testas then run repeatedly, changing the antibody dilutions

ranging between 1:1.25 million and 1:80 million with theame biotinyl–LH dilutions between 1:500 and 1:16,000).he antibody titer of 1:10 million and the biotinyl–LH con-ugate titer of 1:4000 was found to be the most suitable andchieved an OD450 of approximately 1.4.

.3. Plasma interference test

oLH standards in the assay buffer and in various vol-mes of hormone-free goat plasma (20, 40 and 80 �l) wereun in the assay to determine the possible interferencef plasma with the accuracy of the assay. The percentageinding of different oLH standards in 80 �l goat plasmaas similar to that observed in 80 �l assay buffer (Fig. 2).

maller plasma volumes showed slight variations in theercentage binding from the buffer curve. Thus, there waslight decrease in the accuracy when measuring oLH stan-ards prepared in 20 and 40 �l plasma volumes. Hence,H standards ranging from 25 to 12,800 pg/well/80 �l wererepared in hormone-free plasma and subsequently allssays were conducted, using 80 �l of the unknown plasmaamples.

.4. oLH parallelism with goat plasma

The homology between oLH standards and endogenousH in goat plasma was assessed by conducting a parallelismest. To serve the purpose, a goat plasma sample containinghigh level of endogenous LH was serially diluted (contain-

ig. 2. Influence of different volumes (20, 40 and 80 �l) LH-free goatlasma on the percentage binding in the LH standard curve.

Fig. 3. Parallelism for ovine LH standards with serially diluted volumes(2.5, 5, 10, 20, 40 and 80 �l) of goat plasma.

ing 80, 40, 20, 10. 5 and 2.5 �l goat plasma) and run togetherwith the oLH standards (prepared in buffer) in an assay.The relative percentage binding for the increasing plasmavolumes and oLH standards were almost parallel to eachother, thereby confirming the actual LH determination ingoat plasma (Fig. 3).

3.5. Precision of the assay and detection limit

The precision of the assay was evaluated in term of twoparameters, namely repeatability (intra-assay coefficientof variation) and reproducibility (inter-assay coefficient ofvariation). The intra- and inter-assay coefficients of vari-ation, determined using the pooled plasma containing40 and 0.6 ng/ml in 12 assays were 7.71% and 14.3% and5.2% and 11.8%, respectively. The lowest plasma LH detec-tion level was 25 pg/80 �l plasma, which corresponded to0.31 ng/ml plasma.

3.6. Biological validation

The patterns of LH secretion obtained from the assayof blood samples collected from two cyclic goats are setout in Fig. 4a and b. The basal plasma LH concentrationsranged from 1.0 to 13.8 ng/ml during the follicular stageof the estrous cycle. The plasma LH surge reached a peakvalue of 66.4 ng/ml (Fig. 4a) or 57.4 ng/ml (Fig. 4b) in thetwo goats. This LH surge was further followed by ovula-tion that was confirmed by a rise in plasma progesteronelevel. During the luteal phase of the estrous cycle, plasmaLH concentrations fluctuated between 0.4 and 6.6 ng/ml inthe experimental goats.

The blood LH profiles for the two non-lactating cyclinggoats following GnRH treatment are set out in Fig. 5aand b. The mean basal LH concentration before the GnRHchallenge was 1.45 ± 0.19 ng/ml. The plasma LH concentra-tion increased gradually in both goats to a peak value of106.6 ng/ml (Fig. 5a) and 73.8 ng/ml (Fig. 5b) after 3.5 h

and 2.5 h following GnRH administration, respectively. Theplasma LH concentration remained high for several hoursand then gradually declined to values slightly higher thanthe basal level by 18 h.

26 A. Haldar et al. / Small Ruminant R

Fig. 4. (a, b) Plasma LH profiles during estrous cycle of cyclic goats.

Fig. 5. (a, b) Plasma LH profiles in goats after GnRH administration (i.m.,2 �g/10 kg body weight).

4. Discussion

To the best of current knowledge, the method describedhere is the first report using a double antibody techniqueand the LH–biotin–streptavidin system for goat plasma LH

esearch 84 (2009) 22–27

with the aid of EIA. The use of a second antibody in coatingthe wells instead of a hormone specific antibody is pre-ferred as it reduces assay variation associated with unevenbinding of the latter antibody to the wells. It further reducesthe amount of hormone specific antibody needed in the EIA(Meyer, 1986). The high degree of parallelism in the concen-tration of the hormone values plotted obtained followingserial dilutions of goat plasma containing high levels of LH,and the standard curve of oLH (Fig. 3) indicates considerablehomology between caprine and ovine LH used in the assay.

To obtain a high degree of sensitivity and accuracy in theEIA, 80 �l sample volumes are ideal in reducing the non-specific binding and plasma matrix effects (Mutayoba etal., 1990). This requires efficient LH biotination, the use ofa highly specific antibody and the optimum LH–biotin andLH antibody dilutions at suitable incubation temperatures.In this EIA there was a decrease in the optical density withplasma volumes of less of than 80 �l. The relative bindingpercentage decreased most when 20 �l LH-free plasma wasassayed along with the standards (Fig. 2). In order to com-pensate for this effect, it was necessary to use the sameplasma volumes for the standards and unknowns. Hence,80 �l plasma was recommended for the running of theassay. The minimum detection limit (25 pg/well plasma LH)was obtained when 80 �l of plasma was taken for the deter-mination. This volume was sufficient to determine a lowphysiological baseline plasma LH concentration in goats.Intra- and inter-assay coefficients of variation obtained inthe present study were also similar to those obtained bydifferent researchers using the EIA technique to determineplasma LH concentrations in other species (Mutayoba et al.,1990; Prakash et al., 2002; Valares et al., 2007).

The plasma samples assayed using the present EIA pro-cedure showed classical LH profiles in cyclic goats (Figs. 4aand b), as well as LH responses to a GnRH challenge (Fig. 5aand b). The detection of a LH surge in cyclic goats (Fig. 4aand b) and the gradual increase in plasma LH concentrationafter GnRH administration, with a subsequent and gradualdecrease of plasma LH concentrations following the chal-lenge (Fig. 5a and b), confirm the biological validation ofthe EIA technique for determination of plasma LH in goats.The plasma LH profiles during the estrous cycle have beenreported earlier in does (Chemineau et al., 1982; Leyva-Ocaritz et al., 1995) and ewes (Quirke et al., 1981; Bindonet al., 1984). The LH response after a GnRH challenge hasalso been reported in ewes (Yildiz et al., 2003; Valares etal., 2007), as well as in lambs (Recabarren et al., 2000).However, the plasma LH concentrations quoted are lowerthan obtained in the present trial. This could be due to thefact that several conditions were different in the presentstudy—including species, age of the animals (average 27months of age), the dose of GnRH (2 �g/10 kg body weight),number of GnRH administrations and the route of GnRHadministration.

The assay described here requires less expensive instru-mentation and reagents, compared to RIA and can be

adopted in developing countries where financial con-straints limit the adoption of RIA. Additionally, EIA canreduce licensing problems and health hazards when com-pared to the use of radio-isotopes. Highly purified LHpreparations of different species are currently available,

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nd the biotinylation of LH is not complicated, comparedo the iodination procedures. Biotin and streptavidin per-xidase of good quality are commercially available, cheaperhan the 125I preparations. The simplicity and the rapidityo complete the assay (four stages and 24 h to obtain theesults) provide an attractive alternative to other meth-ds. In conclusion, this enzyme immunoassay developedo determine circulating caprine plasma LH concentrationsas been demonstrated to be a simple and sensitive method

or the routine analysis of caprine LH.

cknowledgements

The authors wish to thank to Dr. A.F. Parlow, Nationalormone Peptide Program (NHPP), Harbor-UCLA Medi-al Centre, Carson, CA, USA, for providing the referencef oLH standards and oLH antiserum. The study wasnancially supported by the National Fund for Basic andtrategic Research in Agricultural Sciences, Indian Coun-il of Agricultural Research, New Delhi, India, Project no.FBSRA/PCN/AP-06/2006-07.

eferences

nandlaxmi, N., Prakash, B.S., 2001. Production and purification of goatantirabbit IgG. Ind. J. Dairy Sci. 54, 332–334.

indon, B.M., Piper, L.R., Thimonier, J., 1984. Preovulatory LH characteris-tics and time of ovulation in the prolific Booroola Merio ewe. J. Reprod.Fertil. 71, 519–523.

hemineau, P., Gauthier, D., Poirier, J.C., Saumande, J., 1982. Plasma levelsof LH, FSH, prolactin, oestradiol 17� and progesterone during natural

and induced oestrus in the dairy goat. Theriogenology 17, 313–323.

CAR, 1998. Nutrient requirements of livestock and poultry (Ranjhan, S.K.).Indian Council Agricultural Research (ICAR), Krishi Annusandhan Bha-van, New Delhi.

egan, S.J., Karsch, F.J., Foster, D.L., 1977. The endocrine control of sea-sonal reproductive function in the ewe: a marked change in response

esearch 84 (2009) 22–27 27

to the negative feedback action of estradiol on luteinizing hormonesecretion. Endocrinology 101, 818–824.

Leyva-Ocaritz, H., Munro, C., Stabenfeldt, G.H., 1995. Serum LH, FSH,estradiol-17� and progesterone profiles of native and crossbred goatsin a tropical semiarid zone of Venezuela during the estrous cycle.Anim. Reprod. Sci. 39, 49–55.

Meyer, H.H.D., 1986. Possibilities to improve enzyme immunoassay (EIA)techniques and their application in animal production. In: Proceed-ings of International Symposium on the Use of Nuclear Techniques inStudies of Animal Production and Health in Different Environments,IAEA, Vienna, pp. 255–262.

Mutayoba, B.H., Meyer, H.H.D., Schams, D., Schallenberger, E., 1990.Development of a sensitive enzyme immunoassay for LH determina-tion in bovine plasma using the streptavidin–biotin technique. ActaEndocrinol. (Copenh.) 122, 227–232.

Niswender, G.D., Reichert Jr., L.E., Midgley Jr., A.R., Nalbandov, A.V.,1969. Radioimmunoassay for bovine and ovine luteinizing hormone.Endocrinology 84, 1166–1173.

Prakash, B.S., Paul, V., Anandlaxmi, N., 2002. Development and validationof a simple, sensitive, second antibody format enzyme immunoassayfor LH determination in plasma. J. Immunol. Meth. 270, 281–290.

Quirke, J.F., Hanrahan, J.P., Gosling, J.P., 1981. Duration of oestrus, ovulationrate, time of ovulation and plasma LH, total oestrogen and proges-terone in Galway adult ewes and ewe lambs. J. Reprod. Fertil. 61,265–272.

Recabarren, S.E., Lobos, A., Cendoya, E., Correa, C., Rudolph, I., 2000.Pituitary responsiveness to diurnal and nocturnal GnRH pulses inmelatonin-treated ewe lambs. Reprod. Fertil. Dev. 12, 45–50.

Sarkar, M., Prakash, B.S., 2006. Application of sensitive enzymeimmunoas-says for oxytocin and prolactin determination in blood plasma of yaks(Poephagus grunniens L.) during milk let down and cyclicity. Theri-ogenology 65, 499–516.

Scaramuzzi, R.J., Caldwell, B.V., Moor, R.M., 1970. Radioimmunoassay of LHand estrogen during estrous cycle of the ewe. Biol. Reprod. 3, 110–119.

Schanbacher, B.D., Schemm, S.R., Rhind, S.M., 1991. Gonadotrophinconcentrations and ovulation rates in Suffolk ewes or passively immu-nized against inhibin alpha. J. Reprod. Fertil. 93, 133–139.

Valares, J.A., Abecia, J.A., Forcada, F., Palacin, I., Mata, L., Razquin, P., 2007.

Development of a simple enzyme immunoassay for the determinationof ovine luteinizing hormone. Vet. Res. Commun. 31, 427–436.

Yildiz, S., Saatci, M., Uzun, M., Guven, B., 2003. Effects of ram introductionafter the second prostaglandin F2 alpha injection on day 11 on theLH surge characteristics in fat-tailed ewes. Reprod. Dom. Anim. 38,54–57.