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J. Anim. Sci. 2003. 81:1294-1299
© 2003 American Society of Animal Science

Technical note: Time-resolved immunofluorometric assay for growth hormone in ruminants1

P. Løvendahl*,2, J. Adamsen*, R. Lund{dagger} and P. Lind{dagger}

* Danish Institute of Agricultural Sciences, Department of Animal Breeding and Genetics, DK 8830 Tjele, Denmark and and {dagger} Danish Veterinary Institute, Bülowsvej 27, DK 1790 Copenhagen V, Denmark

2 Correspondence:
P.O. Box 50, (phone: +45-8999-1338; fax: +45-8999-1300; E-mail:
Peter.Lovendahl{at}agrsci.dk).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
A noncompetitive, time-resolved immunofluorometric assay (TRIFMA) was developed using a selected pair of monoclonal antibodies (mab) raised against recombinant bovine GH, with the catching mab immobilized on microtiter plate wells and the detection mab labeled with Eu3+ as a tracer, arranged as a sandwich. Plates were coated with mab1.15 (680 ng/well) using a phosphate buffer (pH 4.9), and then blocked with assay buffer containing 1% (wt/vol) BSA. The assay procedure involved incubation of 50 µL of sample (plasma or serum) and 200 µL of assay buffer containing 25 ng of mab1.2-Eu3+ conjugate for 4 h at 25°C. Plates were then washed six times, incubated for 5 to 10 min with 250 µL of enhancement solution, and fluorescence read with a time-resolved fluorometer. The sensitivity of the assay was 0.1 ng/mL, and the working range was 0.2 to 200 ng/mL. Recovery of quantitative amounts of bovine GH added to plasma samples was close to 100%. Cross-reactivity with other bovine pituitary hormones or with GH from nonbovidae or cervidae species was not significant. Intra- and interassay CV during routine operation was 4.4 and 10.7%, respectively (mean = 3.54 ng/mL). Plasma concentrations of bovine GH determined by TRIFMA correlated closely (r2 >= 0.93) with RIA results, with a conversion ratio of 0.62 when the higher specificity of the monoclonal antibodies was taken into account. The TRIFMA is a reliable alternative to RIA methods because the assay employs no radiolabeled or hazardous chemicals, delivers results rapidly, and has little risk of down periods.

Key Words: Bovidae • Cervidae • Enzyme-Linked Immunosorbent Assay • Radioimmunoassay • Somatotropin


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
Measurement of GH in bovines is important to understanding the pulsatile secretion pattern and the metabolic role of this hormone. Assessing the pulsatile secretion pattern demands that several blood samples be collected and assayed in order to separate baseline concentrations from randomly occurring peaks in order to resolve their relation to feeding or other events (e.g., Breier et al., 1986; Woolliams et al., 1993). Traditionally, RIA has been the chosen and accepted method for GH assays. Although RIA is simple to perform, it also suffers from serious shortcomings, such as insufficient sensitivity (>=0.5 ng/mL), long processing time (>=20 h), handling of radioactive material and waste, and, most importantly, the short shelf life of radiolabelled GH.

Competitive ELISA-type assays for bovine GH (bGH) were developed by Hennies and Holtz (1993) using polyclonal antibodies. A noncompetitive ELISA, based on polyclonal antibodies, was described by Secchi et al. (1988), and one based on monoclonal antibodies (mab) was reported by Erhard et al. (1994). However, long incubation times were still required for these assays.

We have developed and validated a noncompetitive, time-resolved immunofluorometric assay (TRIFMA) for bGH based on two monoclonal antibodies. We use a time-resolved fluorescent label as the detection system because of the past success with this system in other assays (Løvendahl and Purup, 2002).


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Implications
 Literature Cited
 
Assay Development
Antigen and Reference Preparations.
Recombinantly-derived bGH (rbGH, lot AC5935-15; American Cyanamid Co., Princeton, NJ) was used to raise antibodies and to prepare calibrators. Purified pituitary bGH (bGH AFP-10325C; National Hormone and Pituitary Program [NHPP], Torrance, CA) was used as a reference in the validation steps. Cross-reactivity was studied with the following hormone preparations: (Monsanto-bGH, NHPP) and (Elanco-bGH, Eli Lilly, Copenhagen, Denmark), human pituitary GH (S 4776; Sigma Chemical Co., St. Louis, MO), horse pituitary GH (eGH AFP7112B; NHPP), synthetic porcine GH (SmithKline-Beecham Animal Health Inc., West Chester, PA), bovine prolactin (NIDDK AFP7170E; NHPP), bovine TSH (NIADDK-bTSH-13 AFP-9074C; NHPP), bovine LH (NHPP), and IGF-1 (1048058; Roche Diagnostics Inc., Indianapolis, IN).

Monoclonal Antibodies.
Three Balb/c mice were injected s.c. three times at 2-wk intervals with 10 µg of rbGH in PBS in incomplete Freund’s adjuvant. Five weeks after the third immunization, an i.p. booster injection of 10 µg of rbGH in PBS was given. Splenocytes were fused with myeloma cells (CRL 1580; American Type Culture Collection Inc., Rockville, MD) to form hybridomas (Jensen et al., 1993), which were screened by indirect ELISA. Four positive hybridoma clones showing the highest immunoreactivity and identified as IgG1 were then recloned and propagated to antibody production and purification on protein A-agarose columns (Kem-En-Tec A/S, Copenhagen, Denmark). A matching pair was selected, where mab1.15 was immobilized on microtiter plate wells and mab1.2 was used as the Eu3+-labelled detection antibody.

Assay Buffer.
The assay buffer contained 100 mmol/L of Tris-HCl, 150 mmol/L of NaCl, 7.7 mmol/L of NaN3, 20 µmol/L of diethylene triamine penta acetic acid, 0.5% BSA (A1662; Sigma), and 0.1% Tween-20 (Merck Schuchart GmbH, Hohenbrunn, Germany).

Coating of Microtiter Plates.
Microtiter plate wells (1244-550; Perkin-Elmer Life Sciences Wallac, Turku, Finland) were coated with 200 µL of the coating antibody solution (100 mM NaH2PO4, pH 4.90). The concentration of mab1.15 and the temperature and duration of coating were optimized to obtain a high signal to background ratio. Following incubation, plates were washed once with wash solution (1380-1088; Perkin-Elmer), and blocked with 250 µL of either assay buffer spiked with 0.5% BSA (A1662; Sigma) or with wash solution spiked with 0.5% Tween-20 (30 min, slow shaking). Blocking solution was aspirated and the wells were filled with 50 µL of blocking buffer. Sealed plates were stored at 4°C for up to 4 wk.

Labeling of Detection Antibody with Eu3+ or Biotin.
Labeling with europium was according to manufacturer instructions (1244-302; Perkin-Elmer), with purification on PD10 columns (Pharmacia, Uppsala, Sweden). Labeled antibody was characterized and stored at 4°C in aliquots, and the activity lasted at least 24 mo. Alternative labeling with biotin was according to instructions in the labeling kit (Immunoprobe Biotinylation kit BK-101, Sigma).

Heat-Inactivated Serum.
Serum for preparation of calibrators was inactivated by heating to 56 ± 1°C for 3 h, followed by removal of precipitates by filtration on a glass filter and centrifugation (4000 x g, 4°C, 30 min).

Assay Procedure, One-Step Method for Heparin Plasma or Serum.
Coated microtiter plates were washed once with wash solution (Perkin-Elmer). To each well, 50 µL of calibrator or sample was added, followed by 200 µL of assay buffer containing 25 ng/well of mab1.2-Eu3+ conjugate. Diluted antibody was passed through a sterile filter (0.2 µm) prior to use. Plates were incubated (25°C, slow shaking) for an optimized time interval. Plates were washed six times. Bound Eu3+ was dissociated by adding 250 µL of enhancement solution (1244-104; Perkin-Elmer), followed by 5 to 10 min of shaking. Time-resolved fluorescence was counted on a time-resolved fluorometer (DELFIA 1234 Research Fluorometer, or AutoDELFIA, Perkin-Elmer). Standard curves were fitted using a smoothing spline algorithm on log-log values (Multicalc program, Perkin-Elmer).

Modifications Necessary for Samples Containing Ethylenediaminetetraacetic Acid.
Two types of modifications were tested, but were not validated in detail. The first was a two-step split incubation. The samples (50 µL) were incubated with 100 µL of assay buffer in the wells for 1.5 h. After a single wash, a second incubation step followed with mab1.2-Eu3+ (25 ng/well) in 200 µL of assay buffer for 2.5 h, followed by four washes, enhancement, and reading as above. The other type of modification used biotin-labeled mab1.2 to replace the directly Eu3+-labeled mab1.2. Sample (50 µL) was incubated with 100 µL of assay buffer containing 25 ng of biotin-labeled mab1.2 (4 h). Then, 50 µL of assay buffer was added and the sample was incubated for another 4 h. Following six washes, 200 µL of assay buffer containing Eu3+-labeled streptavidin (1244-360, Perkin-Elmer) was added. The sample was again incubated (slow shaking, 30 min), followed by six washes, enhancement, and reading as above.

Validation and Application
The sensitivity and working range of the assay were estimated from an extended calibration curve. Linearity of dilution and recovery of rbGH in spiked samples were measured. Intra- and interassay CV were estimated during routine operation. The assay was validated for cross-reactivity with other hormones and GH from other species. TRIFMA and RIA results were compared using cattle plasma samples.

Quality-Control Plasma.
Plasma with a low bGH concentration was obtained from a lactating cow (QC-L). Plasma with medium bGH (QC-M) was obtained from a bull calf following i.v. stimulation with synthetic GHRH (hpGHRH29-NH2, Bachem Feinchemichalien AG, Bubendorf, Switzerland); plasma with high bGH (QC-H) was obtained from another calf following i.v. stimulation with GHRH in combination with thyrotropin-releasing hormone (Bachem). Aliquots of quality-control plasma were stored frozen (-20°C) in Cryo-vials (Nunc, Roskilde, Denmark).

Growth Hormone Release in Dairy Calves.
Twenty 9-mo-old Jersey calves and 12 9-mo-old Holstein calves were subjected to i.v. GHRH stimulation tests. Blood was sampled into heparinized tubes at -15, -5, 0, 5, 10, 15, 20, 30, 45, and 60 min relative to GHRH (0.5 µg/kg LW). Plasma was obtained by centrifugation (2,000 x g, 4°C, 20 min) and stored frozen (-20°C) until assayed by RIA (Løvendahl et al., 1994) and TRIFMA. Calves that were a subset of the animals previously used by Sørensen et al. (2002) were genotyped for the two bGH variants that possess the amino acids leucine (L) or valine (V) at position 127. Among the 20 Jersey calves, seven were of genotype LL, seven were LV, and six were of VV genotype, whereas the 12 Holsteins were of genotype LL (n = 11), and LV (n =1). Comparison of RIA and TRIFMA values were performed by linear regression within breed and genotype.

Application of the TRIFMA for serum or plasma samples from other species was studied by GH-release in four sheep, and in goat, kudu, reindeer, and giraffe without prior GHRH stimulation. Approval of all animal experiments and immunizations was obtained from the Danish Animal Experiments Inspectorate.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Implications
 Literature Cited
 
Selection of Antibodies and Optimal Coating Conditions
The highest counts and the ratio of signal to background (S:B) were obtained when plates were coated with mab1.15, with mab1.2 used as tracer at 25 ng/well (data not shown). Optimal conditions for coating of plates were chosen so that the S:B ratio was high and CV was low. Plate coating took place overnight at 4, 25, or 37°C (Table 1Go). Background counts were largely unaffected by the coating temperature. High counts and S:B ratio were found at the 680- and 1,360-ng/well coatings at 4°C, when the CV was also low. At 4°C the S:B ratio and CV were less affected by the concentration of the coating solution than at 25 or 37°C, where a high mab1.15 concentration was needed. Blocking by BSA was superior to the simpler Tween-20 blocking (data not shown). Hence, overnight coating at 4°C, with 680 ng/mL and blocking with BSA, was chosen for routine use.


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  		Table 1. The effects of coating and incubation conditions on counts, signal:background ratio (S:B), and coefficient of variation on calibrators at concentrations of 1 and 4 ng/mL
 
Incubation Conditions: One-Step Method.
Incubation was performed at 25°C with slow agitation of the plates, as is the standard for the automated equipment (AutoDELFIA, Perkin-Elmer). The counts increased with incubation time, but the S:B ratio did not increase above that at 4 h (Table 1Go). Results at 3 and 4 h were almost the same for both S:B ratio and CV, and four h was chosen for routine use. Automated running, including both pipetting and incubation steps, decreased background counts and thereby S:B ratio, and furthermore reduced CV. The volumes of plasma sample giving the highest S:B ratio and lowest CV were 50 and 75 µL; therefore, 50 µL was chosen for routine use. In its optimized version, this assay had a short incubation time, approaching that of commercially available hGH assays (e.g., hGH kit 1244-041, Perkin-Elmer). This is much shorter than the overnight incubations used in bGH ELISA (e.g., Secchi et al., 1988), or RIA (e.g., Løvendahl et al., 1994).

Calibration Curve, Sensitivity and Accuracy Profile.
The lowest concentration deviating from the zero calibrator by more than two standard deviation units was 0.1 ng/mL (see Figure 1Go). Counts increased to a plateau of approximately 300 ng/mL, but above 1,000 ng/mL counts declined. Seven calibrators were chosen for routine use: 0.2, 0.4, 1, 4, 40, 100, and 200 ng/mL. A zero calibrator was included as a quality-control sample. The average CV for each of the seven calibrators used in the routine assay was used to establish a precision profile (Figure 1Go). The lowest CV were found at the linear part of the curve, and values below 10% were found at concentrations above 0.3 ng/mL. The working range of the assay was between 0.2 and 200 ng/mL. By comparison, the bGH ELISA of Secchi et al. (1988) had a similar sensitivity, but the working range closed at 10 ng/mL. Our previously used RIA had a sensitivity of around 0.6 ng/mL (Løvendahl et al., 1994).



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  		Figure 1. Calibration curve and accuracy profile using an extended set of calibrators diluted in heat-treated bovine plasma. Time-resolved fluorescence is measured as counts per second x 1,000 (kcps). The horizontal bar indicates the working range of the assay.

 
Modified Assay Procedures.
Splitting the assay into more steps with intervening wash cycles was effective in removing interference from EDTA on the Eu3+-label without changing the shape of the calibration curve (data not shown). When the biotin modification was used on morning milk samples from 70 cows (pasteurized skim milk as matrix for calibrators), all milk samples had bGH concentrations below 0.1 ng/mL (data not shown). The modified assay versions appeared similar to the one-step method with respect to sensitivity and CV.

Dilution of Samples and Recovery of Recombinantly Derived Bovine Growth Hormone Added to Plasma Samples.
The linearity ratio, calculated as the ratio of measured:expected concentration, was close to unity when quality control sera were diluted serially (Table 2Go). However, due to the long working range, dilution is rarely needed, and then only when a high bGH concentration is brought about by administration of high doses of bGH secretagogue or treatment with exogenous bGH. Recovery ratio was likewise calculated as the ratio of measured:expected hormone concentration in spiked quality-control plasma and was found to be close to unity (Table 2Go). Recovery and dilution were studied in several other cattle samples, with results similar to those obtained with the quality-control plasma (data not shown). Thus, there was weak or no interference between plasma and added hormone.


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  		Table 2. Recovery of quantitative amounts of bovine GH added to three bovine quality-control (QC) plasma pools (QC-Low, QC-Medium and QC-High), and linearity of dilution in buffera
 
Cross-Reactivity with Other Reference Preparations and Hormones.
Pituitary bovine GH (NHPP) calibrators were measured as 0.47 to 0.60 times their expected concentration (Table 3Go). Likewise, synthetic analogs of bGH were measured as either lower (Monsanto) or equal to (Elanco) the preparation used as calibrator and antigen (Cyanamid). Similar differences in cross-reactivity with bGH analogs were found by Erhard et al. (1994). The cross-reactivity with other bovine hormones (bLH, bTSH, and b-PRL) was below the detection limit at concentrations up to 100 ng/mL (Table 3Go). Human, porcine, and equine GH preparations and IGF-I all showed negligible cross-reactivity.


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  		Table 3. Cross-reactivity (%) of various synthetic bovine GH preparations, GH from other species, and other pituitary hormones in units of bovine GH measured by time-resolved immuno fluorometric assay (TRIFMA)
 
Routine Performance Coefficient of Variation.
Routine assays, each with 2 to 3 plates, and each QC-H, QC-M, and QC-L determined in duplicate at both ends of each plate comprised approximately 700 determinations of each QC. Using mixed-model ANOVA, variance components were estimated for inter- and intraassay duplicates (i.e., usual intraassay) (PROC MIXED procedure of SAS, SAS Inst., Inc., Cary, NC). For duplicates, the CV was 3.4% in QC-M, and 4.4 to 4.6% in QC-H and QC-L. The intraassay variance included between-duplicate variance and drift from one end to the other of the assay. At this level, CV increased to 7.4, 6.0, and 5.5% for QC-L, QC-M, and QC-H, respectively. The interassay CV were 10.7, 9.1 and 15.5% for QC-L, QC-M, and QC-H respectively. The low variation between duplicates is comparable with other TRIFMA results (Løvendahl and Purup, 2002) and is a marked improvement compared with our former RIA (Løvendahl et al., 1994). The variation between assays is at least as low as our former RIA.

Growth Hormone Release in Dairy Calves and Comparisons with Radioimmunoassay Results.
Plasma bGH increased in Holstein and in Jersey calves following stimulation with GHRH (Figure 2Go). The highest release was seen in LL-Jersey calves, followed by Holsteins and then the heterozygote LV-Jerseys; the smallest release was seen in VV-Jersey calves. Although the values obtained using TRIFMA were numerically smaller than those found using RIA, the ranking of the breeds and genotypes was independent of the assay method used.



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  		Figure 2. Release of bGH in calves following stimulation with GHRH measured by RIA and time-resolved immunofluorometric assay (TRIFMA). Calves were Holsteins (HF) or Jerseys (Jer) of three GH (leucine/valine) genotypes (LL, LV, and VV).

 
Conversion of RIA to TRIFMA values was estimated by linear regression within breed and, for Jerseys, within genotype as: [A] y(Holsteins TRIFMA) = 0.616 ± 0.013 x (RIA) + 0.527 ± 0.1 R2 = 0.95 [B] y(Jersey pool TRIFMA) = 0.623 ± 0.013 x (RIA) + 0.498 ± 0.140 R2 = 0.93 [C1] y(Jersey-LL TRIFMA) = 0.584 ± 0.021 x (LL - RIA) + 1.022 ± 0.317 R2 = 0.93 [C2] y(Jersey-LV TRIFMA) = 0.671 ± 0.023 ± (LV - RIA) + 0.081 ± 0.219 R2 = 0.93 [C3] y(Jersey-VV TRIFMA) = 0.816 ± 0.021 x (VV - RIA) - 0.329 ± 0.166 R2 = 0.93. The slope of this regression was similar for Holstein and Jersey pool plasma samples. It differs from 1.00; as such, it may be seen as a difference in sensitivity between the two methods. The difference between genotypes in slopes for Jersey-LL, Jersey-LV, and Jersey-VV show that the RIA, the TRIFMA, or both have different affinities to the two types of bGH, but it does not reveal which of the assays are more or less sensitive to either type of bGH. When assays for hGH based on monoclonal vs. polyclonal antibodies were compared, a similar ratio of 0.63 between plasma concentrations was found and was interpreted as a difference in the antibody affinity to the different hGH forms (Andersen et al., 1998). However, the close correlation shows that animals and samples will be ranked similarly, independent of the assay method.

Samples from Bovidae, Cervidae, and Camelidae Species.
Stimulated release of GH by GHRH was performed in four sheep and three llamas (Figure 3Go), and acute samples without stimulation were obtained from goats, kudu, reindeer, and giraffe. The responses at 15 min following GHRH in sheep were similar to those obtained in cattle and were of comparable magnitude. Llamas had undetectable concentrations of bGH before GHRH and did not respond to GHRH. As the GHRH analog, hpGHRH29-NH2 is capable of stimulating a GH release in many mammalian species; this indicates that the present assay is not able to measure GH of llamas. Llamas, like alpaca, are of the Camelidae family, which has GH with an amino acid sequence similar to that of equine (Cascone et al., 1984). Purified eGH is not detectable in this TRIFMA (Table 3Go). Sample concentration (in ng/mL) of bGH varied between animals within each species. Close cross-reactivity among ovine, caprine, and bovine GH forms are expected because they are structurally similar and have almost identical amino acid sequences. This also applies to primitive ruminants, such as the chevrotain (Tragulus javanicus) (Wallis and Wallis, 2001). Based on these findings, we suggest that the assay may be applied to all Bovidae and Cervidae species, and measurements expressed in units of immunoreactive bGH.



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  		Figure 3. Concentrations of GH in plasma or serum from various species before ({circ}) or following GHRH stimulation ({diamondsuit}).

 

   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
Literature Cited
 
This time-resolved immuno fluoro-metric assay for bovine growth hormone has high sensitivity, is accurate, and shows no detectable cross reactivity with other bovine pituitary hormones. The assay has few and simple steps, provides results rapidly, and it presents no hazard to workers or the environment. It can be readily applied to samples from other species, and, with minor modifications, to samples containing chelating anticoagulants such as ethylenediaminetetracetic acid. Assaying milk is possible, but concentrations are expected to be less than the detection limit.


   Footnotes
 
1 Reference preparations of hormones were kindly provided by A. F. Parlow, National Hormone and Pituitary Program, Torrance, CA. Nonbovine plasma samples were kindly provided by K. Hove, Agricultural University, Norway (reindeer); B. Christoffersen, Egtved, Denmark (llama); J. Lilleør, Aalborg Zoo, Denmark (kudu, giraffe), C. Christensen, Gl. Estrup Museum, Denmark (goat). The authors are grateful to these people for their help and contributions and to M. Vestergaard, Danish Institute of Agricultural Sciences, for further samples and fruitful discussions. Back

Received for publication September 15, 2002. Accepted for publication January 20, 2003.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 


Andersen, M., P. H. Petersen, O. Blaabjerg, J. Hangaard, and C. Hagen. 1998. Evaluation of growth hormone assays using ration plots. Clin. Chem. 44:1032–1038.[Abstract/Free Full Text]

Breier, B. H., J. J. Bass, J. H. Butler, and P. D. Gluckman. 1986. The somatotropic axis in young steers: Influence of nutritional status on pulsatile growth hormone (GH) release and circulating insulin-like growth factor-I (IGF-I) concentrations. J. Endocrinol. 111:209–215.[Abstract]

Cascone, O., M. Biscoglio de Jimenez Bonino, C. Pena, J. A. Santome, A. I. Arnao de Nue, R. Ore, and M. Villavincencio. 1984. Acta Physiol. Pharmacol. Latinoam. 34:123–130.

Erhard, M. H., J. Kellner, S. Schmidhuber, D. Schams, and U. Lösch. 1994. Identification of antigenic differences of recombinant and pituitary bovine growth hormone using monoclonal antibodies. J. Immunoassay 15:1–19.[Medline]

Hennies, M., and W. Holtz. 1993. Enzyme immunoassay for determination of bovine growth hormone using avidin-biotin-peroxidase complexes. J. Immunol. Meth. 157:149–153.[Medline]

Jensen, H. E., B. Aalbæk, P. Lind, P. L. Frandsen, H. V. Krogh, and D. Stynen. 1993. Enzyme immunohistochemistry with mono- and polyclonal antibodies in the pathological diagnosis of systemic bovine mycoses. Acta Pathol. Microbiol. Immunol. Scand. 101:505–516.

Løvendahl, P., T. Liboriussen, J. Jensen, M. Vestergaard, and K. Sejrsen. 1994. Physiological predictors in calves of dairy breeds. Part 1. Genetic parameters of basal and induced growth hormone secretion. Acta Agric. Scand., Sect. A, Anim. Sci. 44:169–176.

Løvendahl, P., and H. M. Purup. 2002. Technical note: Time-resolved fluoro-immunometric assay for intact insulin in livestock species. J. Anim. Sci. 80:191–195.[Abstract/Free Full Text]

Secchi, C., P. A. Biondi, A. Berrini, T. Simonic, and S. Ronchi. 1988. A biotin-avidin sandwich enzyme-linked immunosorbent assay for bovine plasma. J. Immunol. Met. 110:123–128.[Medline]

Sørensen, P., R. Grochowska, L. Holm, M. Henryon, and P. Løvendahl. 2002. Polymorphism in the bovine growth hormone gene affects endocrine release in dairy calves. J. Dairy Sci. 85:1887–1993.[Abstract/Free Full Text]

Wallis O. C., and M. Wallis. 2001. Molecular evolution of growth hormone (GH) in Cetartiodactyla: Cloning and characterization of the gene encoding GH from a primitive ruminant, the Chevrotain (Tragulus javanicus). Gen. Comp. Endocrin. 123:62–72.[Medline]

Woolliams, J. A., K. D. Angus, and S. B. Wilson. 1993. Endogenous pulsing and stimulated release of growth hormone in dairy calves of high and low genetic merit. Anim. Prod. 56:1–8.


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