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Monoclonal antibody based sandwich enzyme‐linked immunosorbent assay for chicken growth hormone B. Houston , D. Peddie & C. Goddard To cite this article: B. Houston , D. Peddie & C. Goddard (1991) Monoclonal antibody based sandwich enzyme‐linked immunosorbent assay for chicken growth hormone, British Poultry Science, 32:3, 633-644, DOI: 10.1080/00071669108417388 To link to this article:

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British Poultry Science (1991) 32: 633-644


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B. HOUSTON, D. PEDDIE AND C. GODDARD Department of Cellular and Molecular Biology, AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian, EH25 9PS, Scotland Received for publication 18th July 1990

Abstract 1. Monoclonal antibodies which bind to different epitopes of chicken growth hormone (cGH) were used to develop a homologous sandwich enzyme-linked immunosorbent assay (ELISA). 2. The first antibody, which is species specific, was immobilised on microtitre plates and concentrations of cGH in biological fluids were estimated by revealing bound hormone using a second, biotinylated monoclonal antibody. 3. The sensitivity was 0.024 ng/ml, which is at least ten-fold greater than current radioimmunoassays (RIA) and there was no cross-reactivity to other chicken pituitary hormones or to growth hormone from other species. 4. The accuracy and precision of the assay were similar to RIA, and the growth hormone concentrations measured in plasma samples by both RIA and this new ELISA showed a high degree of correlation. 5. The assay takes only 4 h using pre-coated plates which can be stored at 4°C in sucrose. The advantages of being rapid and nonisotopic make this method attractive to both research and industrial laboratories.


Growth hormone (GH) has a number of well-defined functions including a central role in the control of growth and development, major effects on metabolism and the regulation of milk secretion (Heap et al, 1989). GH produced by recombinant DNA technology is used in the treatment of growth disorders in children and may soon be applied in agriculture to increase efficiency of milk production in dairy herds. Its use in the production of lean meat is a possibility because chronic treatment of pigs with porcine GH increases muscle growth at the expense of adipose tissue (Etherton, 1989) although commercial application of this may rely on transgenic biology. The manipulation of avian growth and efficiency of food utilisation has 633

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attractions to the poultry industry (Johnson, 1989) but the role of GH in birds is less well understood than in mammals. This was initially attributable to the unavailability of homologous GH but recently cDNAs encoding chicken GH (cGH) have been cloned and recombinant cGH has been produced (Souza et al, 1984; Zvirblis et al., 1987; Lamb et al., 1988). Unfortunately, this has not adequately resolved the role of cGH in controlling growth rate or food efficiency. Hypophysectomy reduces growth rate which can be restored by administration of exogenous cGH (Scanes, 1987) but in intact animals the effects of exogenous cGH are equivocal. Some studies have shown cGH to have no significant effects on growth rate (Burke et al., 1987; Peebles et al., 1988) whereas others have shown some effects, although these may be transitory (Leung et al., 1986) or require pulsatile administration at a particular age of animal (Vasilatos-Younken et al., 1988). A fundamental problem underlying studies using exogenous cGH is that the role of endogenous cGH in the regulation of growth is not fully understood because lines of chickens genetically selected for rapid growth rate have lower serum cGH concentrations than slow growing lines (Burke and Marks, 1982; Goddard et al., 1988). Before biotechnology can be applied to improve efficiency of growth in poultry using modified genetic selection programmes or by transgenic biology (Souza et al., 1984; Perry, 1988; Salter and Crittenden, 1989; Bosselman et al., 1989a, b; Petitte et al., 1989; Brazolot et al., 1989), the relationship between endogenous cGH, the GH receptor and insulin-like growth factors must be clearly understood. The measurement of cGH has invariably been by radioimmunoassay (RIA) but it is time consuming and has the inherent undesirability of isotopic methods. We report the development of an enzyme-linked immunosorbent assay (ELISA) for cGH based on two monoclonal antibodies, which has a broader range and greater sensitivity than RIA. The assay takes 4 h and has the potential to be used in research or industrial laboratories without requiring expensive radioisotope counting equipment. MATERIALS AND METHODS

Aluminium hydrogel (Superfos) was purchased from Speciality Chemicals, Vedbaek, Denmark. Polyethylene glycol 1500 was obtained from Boehringer Mannheim, Lewes, UK. Tissue culture media, foetal calf serum and 96 well tissue culture plates (Costar) were supplied by Northumbria Biologicals, Cramlington, UK. Microtitre plates were purchased from Dynatech Laboratories, Burgess Hill, UK. Ovine anti-mouse IgG and horseradish peroxidase ovine antimouse immunoglobulin G (IgG) were obtained from Scottish Antibody Production Unit, Carluke, UK. Freund's complete adjuvant, thyrotropin releasing hormone (TRH) and bovine serum albumin (BSA) (RIA grade) were obtained from Sigma, Poole, UK. Streptavidin peroxidase conjugate and Na 125I were purchased from Amersham International (Amersham, UK). All other chemicals were of analytical grade and were purchased from Sigma, BDH (Poole, UK) or Fisons (Loughborough, UK). Recombinant cDNA-derived cGH was a kind gift from Dr. L. Souza and bovine (b) GH was supplied by NIADDK. Monocomponent pituitary-derived cGH was prepared by the method of Houston et al.

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(1990). Chicken GH concentrations were calibrated by absorbence at 280 nm using an absorption coefficient of 7-5 for a 10 mg/ml solution. Antibodies were detected in serum and hybridoma culture conditioned medium by radiolabelled and enzyme-linked detection systems. The radiolabelled detection assay was based on an RIA for cGH as described by Goddard et al. (1988) except that ovine anti-mouse IgG was used as precipitating antibody at an initial dilution of 1:20 in assay buffer containing 1:200 normal mouse serum as carrier. In some assays 125I-labelled bGH was used instead of 125 I-labelled cGH. For the enzyme-linked detection system, microtitre plates were coated with cGH (0-1 /£g/ml in 50 mM Na2CO3, pH 9-6) overnight at 4°C. The GH solution was aspirated and 200 fi\ phosphate buffered saline (10 mM K2HPO4, 137 mM NaCl, 2-7 mM KC1, pH 7-4; Dulbecco's A phosphate buffered saline (PBS)) containing 2% w/v BSA was added and incubated for 30 min at room temperature. This was aspirated and the plates were washed 5 times with PBS containing 0-25% (v/v) Tween 20 (wash buffer). Serum samples or conditioned media (100 fil) were added, incubated for 2 h at room temperature and then washed again as before. Ovine anti-mouse IgG conjugated to horseradish peroxidase was added (1:1000 v/v in blocking buffer) and incubated for 2 h at room temperature. After a further 5 washes 100 /zl/well of freshly prepared substrate (3 mM o-phenylenediamine dihydrochloride, 0-01% v/v H2O2 in 100 mM phosphate-citrate buffer, pH 5-0) was added and the reaction stopped after 3 min by the addition of 50 fil 2M H2SO4. The optical density of each sample was read at 490 nm in in a MR700 plate reader (Dynatech Laboratories, Burgess Hill, UK) against a cGH free blank. Other positive and negative controls were included as appropriate. To prepare the monoclonal antibodies 8-week-old female BALB/c mice were immunised intraperitoneally (ip) with recombinant cDNA-derived cGH (100 fig in complete Freund's adjuvant) and then challenged one month later with 25 fig cGH in aluminium hydrogel. Two weeks later, 250 fil whole blood was removed by tail snip under anaesthesia, left overnight at 4°C to clot and the polyclonal titre was determined in serum. The highest responder mice were injected intravenously with 10 fig cGH in PBS and 3 d later splenocytes were fused with the NSO plasmacytoma cell line (Galfre and Milstein, 1981) at a ratio of 5:1 using 50% polyethylene glycol 1500. Cells were seeded into 96 well microtitre plates in 200 fil RPMI 1640 containing 10% (v/v) FCS, 25% (v/v) mixed thymocyte conditioned medium (Micklem et al., 1987), hypoxanthine, aminopterin and thymidine (HAT medium, Littlefield, 1964) and incubated at 37°C in 5% CO2. A further 100 fl\ of HAT medium was added 7 d later and wells were scored for macroscopically discernible colonies 14 d after the fusion. Conditioned medium from these wells was tested by radiolabelled or enzymelinked assay. Positive hybrids were cloned and stabilised by repeated limiting dilution (Micklem et al., 1987), removed from HAT medium into HT medium (without aminopterin) and then into RPMI 1640 containing 10% (v/v) FCS by dilution over a number of days. Ascitic fluids were produced in BALB/c mice (Hoogenraad and Wraight, 1986) and antibodies were purified by Sepharose-4B-protein A affinity chro-

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matography (Pharmacia LKB Biotechnology, Milton Keynes, UK) according to the manufacturers instructions. The antibodies were isotyped using a commercially available mouse monoclonal antibody isotyping kit (Biorad Ltd., Watford, UK). To prepare biotinylated antibody between 1-5 to 2 mg affinity purified antibody was dialysed overnight at 4°C against 4 1 100 m\t NaHCO3 buffer, pH 8*15). Biotin succinimide ester (5 mg) was dissolved in 2 ml dimethyl sulphoxide and was added to the antibody at a ratio of 50 /A per ml. It was gently mixed then incubated at room temperature for 2 h before being dialysed overnight at 4°C in PBS. The conjugate was stored at 4°C. Antibody coated microtitre plates were prepared in batches of 50. Purified antibody was diluted to a concentration of 1 /Ug/ml in 50 mM Na2CO3, pH 9-6 and 100 /A was added to each well and incubated overnight at 4°C. The plates were washed in wash buffer and blocked with 200 /zl/well blocking buffer for 1 h at 37°C. The plates were washed and 30% (v/v) sucrose (200 /zl/well) was added and incubated for 2 h at room temperature. The sucrose solution was aspirated and not washed but allowed to dry and stored at 4°C until required. For the ELISA procedure the antibody coated plates were washed as before. Chicken GH standard was diluted in blocking buffer so that concentrations of between 0-0625 to 8 ng/ml were added (in 100 jx\) to the appropriate wells for the standard curve. Samples (100 fA) of chicken serum or clarified chicken pituitary gland homogenate (Houston and Goddard, 1988) were added to other wells and plates were incubated at room temperature for 2 h. They were then washed as before and biotinylated antibody added (0-5 //g/well) and incubated for 1 h at room temperature. After a further 5 washes, 100 /^I/well of a 1:500 (v/v) solution of streptavidin peroxidase conjugate were added and the plates were incubated for 30 min at 37°C. The plates were developed using o-phenylenediamine as described for the detection assay. To measure the effect of TRH groups of 6 broiler-type chickens received a single subcutaneous injection of TRH (10 /^g/kg body weight) in 200 //I PBS buffer. Blood samples were taken from the right brachial vein immediately before and at 40, 80, 120 and 160 min after injection into heparinised tubes (Alpha Laboratories, Eastleigh, Hants, UK). Plasma was prepared by centrifugation at 2500 g for 15 min at 4°C and samples were stored at — 20°C until they were assayed. RESULTS

Two separate fusions yielded 1073 hybridomas of which 16 initially secreted antibody against cGH. A number of stable cell lines were established and two of these, monoclonal antibodies Mab B4E4 and Mab 6F5, were selected for the ELISA on the basis of their similar affinity for cGH but their difference in specificity for GH from other species. Antibody dilution curves using either 125I-labelled cGH or 125I-labelled bGH (Fig. 1) demonstrated that Mab B4E4 bound to GH from both species whereas Mab 6F5 only bound to cGH. Subsequent specificity studies demonstrated that bGH and rat GH were 10% and 1% as potent as cGH in their affinity for Mab B4E4 but did not bind



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to Mab 6F5. Neither chicken, turkey or ovine prolactin were able to displace 125 I-labelled cGH from either antibody (Goddard et al, 1987). The affinity constants determined by Scatchard analysis for binding to cGH were 1-8 ± 0-3 X 109 1/mole for Mab B4E4 and 2-5 ± 0-5 X 109 1/mole for Mab 6F5. Both antibodies were of the IgGt subtype and were able to identify cGH charge isomers by Western blotting which we have previously shown to be present in the chicken pituitary gland using a rabbit polyclonal antiserum against cGH (Houston and Goddard, 1988). In immunohistochemical studies both antibodies stained only somatotrophs in the anterior chicken pituitary gland. No staining was observed in the posterior pituitary gland (results not shown).




Antibody dilution

FIG. 1.—Dilution curves for monoclonal antibodies B4E4 and 6F5 binding to cGH(O) or 125I-labelled bGH (•).



Initial experiments were performed in which dose-response curves of A490 versus cGH concentration (1 pg/ml to 10 //g/ml) were constructed using different combinations of Mab B4E4 as the capture antibody (0-1 to 20 /Zg/well) and Mab 6F5 as the second (biotinylated) antibody (0-1 to 20 /zg/well). These experiments were repeated using Mab 6F5 as the capture antibody and Mab B4F4 as the second antibody. From the results of these experiments (not shown), the optimum combination was found to be Mab 6F5 (1-0 //g/well) as capture antibody and biotinylated Mab B4E4 (0-5 /ig/well) as second antibody. Using the protocol described in the materials and methods, this combination of antibodies gave a linear response to cGH over the range 0-0625 to 8 ng/ml. A typical standard curve is shown in Fig. 2. The linear regression of this curve was of the form y = 0-77x + 0-00016; r1 = 0-998. Also shown in Fig. 2



are dose-response curves obtained from serial dilutions of chicken serum and a chicken pituitary gland homogenate. The linear regressions of these curves were of the form y = 0-76x + 0-0015; r2 = 0-998 and y = 0-78* - 0-00044; r2 = 1-00 respectively. The gradient of the dose-response curve was therefore invariant irrespective of whether purified cGH, or cGH in serum or in a pituitary homogenate was being measured. The gradient was also unaffected when a dose-response curve was constructed by diluting purified cGH in chicken serum containing a known concentration of endogenous cGH (not shown).

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0-4 g c CD

8 o-2 a 64 8

4 2 Serum dilution (-fold)

64 8 4 2 1 Pituitary homogenate dilution (-fold) 0

2 4 6 8 Chicken growth hormone (ng/ml)

FIG. 2.—Standard curve for the ELISA of cGH compared with the dilution response curves for both chicken serum and chicken pituitary homogenate.

The cross-reactivity of other pituitary hormones in the assay was tested by comparing the response to bovine GH, chicken and ovine prolactin, chicken and ovine luteinising hormone and porcine follicle stimulating hormone over the concentration range 500 pg/ml to 1 ^g/ml (Fig. 3). There was no detectable cross-reactivity to any of the hormones tested. The specificity of the assay was further validated by demonstrating that the GH concentration in serum from hypophysectomised chickens was less than the minimum detectable dose of the assay (results not shown). The intra-assay coefficient of variation was assessed by repeated measurements in a single assay of pooled serum obtained from male and female layertype chickens at 4 and 12 weeks of age. Males of 4 weeks of age yielded a mean of 40-25 ng/ml with a coefficient of variation of 3-42%. Females exhibited a



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mean of 50-64 ng/ml with a coefficient of variation of 4-14%. In 12-week-old males the mean was 0-32 ng/ml with a coefficient of variation of 4-49% and in females was 12-36% with a pool size of 0-16 ng/ml (n = 20 for each sample).






Hormone concentration (ng/ml)

FIG. 3.—The effect of different concentrations of bovine GH, chicken and ovine prolactin, chicken and ovine luteinising hormone and porcine follicle stimulating hormone in the ELISA for chicken GH (•).

The inter-assay coefficient of variation was assessed by measuring the cGH concentration in two serum pools in 18 independent assays. In two separate pools with mean cGH concentrations of 0-385 ng/ml and 4-766 ng/ml the coefficients of variation were 14-2% and 9-65% respectively. The sensitivity of the assay, defined as the concentration of a given response three times greater than the standard deviation of the response at zero dose, was 0-024 ng/ml. The cGH concentration in 15 serum samples obtained from chickens between 6 and 21 weeks of age was measured by ELISA and also by RIA routinely used in this laboratory (Goddard et al., 1988). The concentrations measured by each method are plotted in Fig. 4, and ranged from 0-05 to 38-8 ng/ml. There was a good correlation between the values obtained with the two methods (r2 = 0-998). The linear regression of the curve obtained corresponded to the equation^ = 2-56 + l-24x. The effect of TRH (10 //g/kg body weight) on young broiler type chickens is shown in Fig. 5. TRH increased plasma GH concentrations from a baseline of 2-9 ng/ml to 16-1 ng/ml, a 5-5-fold increase. DISCUSSION

Knowledge of the relationship between GH, the GH receptor and the

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insulin-like growth factors in the regulation of growth in poultry is not sufficiently advanced to allow reliable progress in the regulation of growth to be made by the application of biotechnology. Transgenic chickens have been reported (Souza et al., 1984; Salter and Crittenden, 1989; Bosselman et al., 1989a; b) but their production by the use of retroviral vectors appears to be rather less 'routine' than the microinjection or stem cell approaches used in mammals (Palmiter et al., 1983; Thompson et al., 1989). Although a number of laboratories are active in this area (Perry, 1988; Petitte et al., 1989; Brazolot et al., 1989) the prospect of being able to test the effect of individual genes on growth regulation in transgenic poultry is still in the future. Current research relies on physiological experiments such as the effect of administration of hormones and growth factors in vivo, cell biology experiments studying responses of cells in vitro, study of the regulation of gene expression during development and the measurement of effects of genetic selection produced either experimentally or as a result of improvements by the poultry industry (see Scanes, 1987). For many of these experiments the analysis of cGH concentrations in plasma or in conditioned medium is an important step. As far as we are aware, RIA is universally employed despite the disadvantages of using radioisotopes and a 3-d period before results are available. We have therefore evaluated some of our monoclonal antibodies developed as research tools (Goddard et al., 1987) for use in an ELISA to measure cGH, because the introduction of biotin-streptavidin technology has enabled the design of rapid and sensitive non-isotopic immunoassays.









GH (ng/ml) by ELISA



FIG. 4.—Relationship between plasma concentrations of cGH measured by both ELISA and radioimmunoassay.





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Time (min) FIG. 5.—Plasma cGH concentrations measured by ELISA following a single subcutaneous injection of thyrotrophin releasing hormone (10 yUgAg body weight) to 6-week-oId broiler chickens.

The molecular heterogeneity of cGH (Houston and Goddard, 1988) could present a problem in the design of an immunoassay because the inability of the antibody to bind to any of the isomers would lead to an underestimate in the amount of cGH measured. However, monoclonal antibodies B4E4 and 6F5 have been used successfully for immunoaffinity chromatography, and in Western blotting studies which demonstrated that both antibodies recognise all cGH isomers identified (Houston et al., 1990; B. Houston, unpublished). Another important criterion in development of a sandwich immunoassay is that each antibody should in theory recognise a distinct epitope. The differential binding of the antibodies to bGH and cGH (Fig. 1) indicated that they recognise distinct epitopes, suggesting that they are suitable for an ELISA. The ELISA based on these two monoclonal antibodies was highly specific for cGH and showed no cross-reactivity with other pituitary hormones. Chicken prolactin, which has 26% structural homology to cGH at the amino acid level (Hanks et al., 1989) and has the greatest potential for cross-reactivity, was not detected by the assay. The ELISA reproducibly measured cGH concentrations in the range 0-0625 to 8 ng/ml and detected cGH concentrations as low as 0-024 ng/ml. The sensitivity was at least 10-fold greater than that obtained with RIA (Picaper et al., 1985; Goddard et al., 1988) and meant that cGH could be measured at higher dilutions, thus minimising potential interference from other proteins. The parallelism between cGH standards and dilutions of plasma and pituitary gland homogenate demonstrated that such

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interference was not a problem. The precision of the ELISA both between and within assay was similar to that achieved by RIA. The assay was further validated by estimation of GH concentrations in plasma samples. It reliably quantified the increase in cGH occurring in response to TRH administration to young broiler chickens (Fig. 5), which has been described on a number of occasions (Klandorf et al., 1985). There was also a strong correlation between cGH concentrations measured by the ELISA and those measured by the RIA used routinely in this laboratory. The ELISA has several advantages over RIA. If the plates are coated with capture antibody in advance, the assay can be completed within 4 h. This compares favourably with RIA protocols which take 2 to 4 d to complete. In addition, the ELISA does not use radionuclides and so avoids potentially hazardous labelling techniques and the problems associated with the disposal of radioactivity. It should therefore provide an attractive alternative to RIA in laboratories which do not have the facilities required to handle radioactivity. The accuracy and rapidity of this method makes genetic selection for a reduced plasma GH concentration a possibility in commercial laboratories, because previous work has suggested that a decreased plasma GH concentration is found in broilers genetically selected for an increased growth rate (Burke and Marks, 1982; Goddard et al., 1988). Furthermore, the assay will be of use when transgenic chickens become available, allowing assessment of the effects of incorporation of genes involved in the regulation of growth and development. Finally, because antibody B4E4 recognised a GH epitope common to a number of species it may provide a ubiquitous antibody for the basis of a GH ELISA in a number of domestic animals in conjunction with the development of other species-specific antibodies.


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Monoclonal antibody based sandwich enzyme-linked immunosorbent assay for chicken growth hormone.

1. Monoclonal antibodies which bind to different epitopes of chicken growth hormone (cGH) were used to develop a homologous sandwich enzyme-linked imm...
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