Development and characterization of polyclonal antibodies against chicken adipocytes

Comp. Biochem. Physiol. Vol. 99A, No. 1/2, pp. 195-198, 1991 Printed in Great Britain

0300-9629/91 $3.00 + 0.00 © 1991 Pergamon Press pie

DEVELOPMENT A N D C H A R A C T E R I Z A T I O N OF POLYCLONAL ANTIBODIES AGAINST C H I C K E N ADIPOCYTES* J. DONG,t D. P. FROMANt and C. Y. Hu:~ :~Department of Animal Science, tDepartment of Poultry Science, Withycombe Hall 112, Oregon State University, Corvallis, OR 97331-6702, U.S.A. Telephone: (503) 737-1915 (Received 22 August 1990)

Abstract--l. Antisera against chicken adipocytes were developed in sheep. These crude antisera showed a high degree of reactivity to adipocyte plasma membranes but also cross-reacted to a lesser extent with other tissues. 2. Antisera cross-reactivity was removed by adsorption of the antisera with various chicken tissue plasma membranes. 3. Antisera reacted with differing affinity to adipocyte plasma membranes from several species of animals.

INTRODUCTION Polyclonal antibodies against adipocyte plasma membrane have been developed in the rat (Pillion et al., 1979), cattle (Cryer et al., 1984), sheep (Nassar and Hu, 1991a) and chickens (Lee et al., 1986). These antibodies have been used to study adipocyte development, specific metabolic activities and to reduce body fat content (Flint et al., 1986; Nassar and Hu, 1991b). Polyclonal antibodies produced against adipocytes cross-react with other tissues which may be detrimental and consequently unsuitable for most in vivo studies. Recently, Butterwith et al. (1989) reported that in vivo injection of anti-chicken adipocyte cell membrane antisera may have caused red blood cell damage, underscoring the importance of characterizing polyclonal antibodies prior to use in in vivo studies. Therefore, the objectives of this study were to develop and characterize polyclonal antibodies to chicken adipocytes. MATERIALS

AND METHODS

Cell isolation Abdominal fat pads were obtained from market weight commercial broilers (Gallus domesticus) at the department slaughter facility. Adipocytes were isolated by collagenase digestion as described previously (Nassar and Hu, 1991a) except that digestion was carried out at 40-41°C instead of 37°C. Immunization procedure Freshly isolated adipocytes (~ 5 × 1 0 7 cells) were injected subcutaneously at various sites along the back of a ewe lamb (32 kg) and 1.2 × 1 0 7 cells were injected intramuscularly to the insides of the hind legs. This procedure was repeated four times at 14 day intervals, followed by a monthly

*Tech. paper no. 9339, Oregon Agric. Exp. Sta. This work was supported in part by the NIH Biomedical Research Suport Grant RR07079 and the Pacific Egg and Poultry Association.

booster injection. Blood samples were collected weekly to follow development of the antibody titer. Blood (I 50-250 ml) was collected regularly via jugular cannulation over the period of highest titer, two weeks after each booster injection and held at 4°C overnight. Serum was collected after centrifugation at 1000g for 15rain and sera were heated at 56°C for 30 min to inactivate complement. Isolation o f plasma membrane Plasma membrane (PM) of adipocytes (abdominal, inguinal and pericardial) was used as antigen in ELISA (Killefer and Hu, 1990) for determination of antisera titer and specificity. Adipocyte PM was obtained according to the method of Belsham et al. (1980) with slight modification. Isolated adipocytes were disrupted by vigorous inversion in the presence of two volumes of prewarmed (37°C) lysing medium (2.5raM ATP, 2.5raM MgC12, 0.1mM CaCI2, 1.0 mM KHCO3, 2.0 mM Tris base, pH 7.6), and PM were separated by centrifugation at 400g for 5 min. After aspirating the infranatant, the remaining contents were resuspended in lysing medium and the procedure repeated. Infranatant fractions were pooled, stored at 4°C, and processed as described by Belsham et al. (1980). PM of liver was obtained by the method of Loten and Redshaw-Loten (1985) and heart and kidney PM by the method of Lo et al. (1976). Sarcolemma was isolated from broiler breast and thigh muscle, essentially by the method of Danuta and Sarzala (1982). Fifty grams of muscle was minced and homogenized in a Waring blender in 500 ml of buffer containing 50raM CaC12, 5% sucrose, and 5 mM Tris-HC1, pH 7.2. After centrifuging at 14,000 g for 10 min, the pellets were washed with 5 volumes of buffer containing 1.0 mM CaC12, 35% sucrose and 5 mM Tris-HC1, pH 7.2 and homogenized using a Polytron at a setting of 6, and subsequently centrifuged at 12,000 g for I0 min to collect the supernatant. This washing was repeated twice. All supernatants were combined and the volume adjusted to give a final sucrose concentration of 1 5 ' . Suspensions were centrifuged again at 100,000g for 30 min. Pellets were resuspended in a small volume of 5.0 mM Tris-HCl, pH 7.2 and layered over a discontinuous sucrose gradient of 1545% and centrifuged at 100,000g for 2 hr. The membrane fraction at the interphase of the sucrose layer was removed and washed with 5 mM Tris-HC1 buffer, pH 7.2 and the preparation was centrifuged at 100,000 g for 30 min. Pellets were

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suspended in a small volume of the same solution and stored at - 20°C. Protein concentration of membrane preparations was assayed according to Bradford (1976) using bovine plasma albumin as a standard.

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Samples of the antisera (60 #g protein/ml) were adsorbed with double volumes of heart PM (650 #g protein/ml) or liver PM (700 #g protein/ml) for one hour at room temperature. Antisera were recovered in the supernatant after 2 rain of centrifugation in a Beckman microcentrifuge.

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ELISA was performed essentially according to Killefer and Hu (1990). Briefly, 100 #1 of 1 #g of PM protein in PBS (phosphate-buffered saline; 0.1M sodium phosphate, 0.15 M NaC1, pH 7.4) was coated onto each well of a 96-well polystyrene plate, either overnight at 4°C or 2 hr at 37°C in a humidified chamber. Wells were blocked with 350 #l/well of PBS containing 1% nonfat dried milk for 30 min at 37°C. PBS containing 0.05% Tween 20 and 0.01% sodium azide was used as washing medium. After washing three times, 50 #l/well of antiserum was added, and plates were incubated for 30 min at 37°C. The plate was washed three times and 50 #l/well of donkey antisheep serum (1/1000 dilution in PBS) was added, and plates were incubated as above. After washing three times, glycine buffer (0.1 M glycine, 1 mM ZnCI2, 1 mM MgC12, pH 10.4) containing P-Nitrophenyl phosphate at 1 mg/ml was added to each well in a volume of 50 #l/well, then incubated in darkness at 37°C for 30min. After terminating the reaction by addition of 50 #l/well of 0.5 N NaOH, the plate was read at 405 nm on an EIA spectrophotometer.

RESULTS AND DISCUSSION The titer of the antisera was assessed by ELISA using chicken abdominal adipocyte PM as antigen. The antisera titer reached a peak about two weeks after the first booster injection and started to decline at the end of the third week. Blood samples were collected two weeks after each booster injection, and the antiserum which had the highest titer was used in this study. The minimum detectable amount of antobody, defined as twice the optical density produced by normal serum, was 0.5 nl antiserum (100#1 of 1:204,800 dilution). This value is comparable to a previous study from this laboratory using sheep adipocyte PM as the antigen source (Nassar and Hu, 1991a). This demonstrates that the isolated adipocytes provide an adequate source of antigen for antibody production. With the exception of heart PM, the antiserum showed a relatively high degree of reactivity to chicken adipocyte PM compared to that of PM from liver, kidney and muscle (Fig. 1). This result is similar to that reported by Butterwith et al. (1989), although our data indicated that liver, muscle and kidney PM had a much lower reactivity than heart PM. This observation is quite different from studies in the rat (Flint et al., 1986) and sheep (Nassar and Hu, 1991a), which showed that liver had the highest cross-reactivity. Cryer et al. (1984) showed that cross-reactivity of antibodies developed against adipocyte PM with PM of various tissues was less than that of antibodies developed against whole fat cells. Since Butterwith et al. (1989) used chicken adipocyte PM and we used whole adipocytes as an antigen source, the

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Fig. l. The relative reactivity of antiserum to plasma membranes prepared from adipocyte (&), heart (V), liver (rq), kidney (O), sarcolemma (<>). Each point represents the mean of three experiments, each with three replications.

similar high cross-reactivity in both chicken studies cannot be attributed to use of whole adipocytes in this study. No site specific reactivity was observed. Reactivity of the developed antiserum with adipocyte PM appeared the same, regardless of location of the adipose tissue from which PM were isolated (Fig. 2). The antiserum was developed against adipocytes isolated from the abdominal fat pad. This observation also differs from a previous sheep study (Nassar and Hu, 1991a) which showed that antiserum raised against subcutaneous adipocyte PM reacted more strongly to adipocyte PM isolated from subcutaneous depot than it did with PM isolated from perirenal adipose tissue. Antiserum generated against chicken abdominal adipocytes reacted differently to adipocyte PM preparations from different species (Fig. 3) in correspondence with the evolutional relationship. PM of adipocytes from the turkey had a higher reactivity than from quail adipocyte PM. Both were higher than mammalian species PM. Adipocyte PM isolated from rat epididymal and inguinal sites had both similar and the lowest reactivity. This result is consistent

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Fig. 2. The relative reactivity of antiserum to adipocyt¢ plasma membranes isolated from abdominal (A), inguinal (O) and pericardial (V) fat pads. Each point represents the mean of three experiments, each with three replications.

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Fig. 3. The relative reactivity of antiserum to adipocyte plasma membranes from chicken (A), turkey (&), quail (l'q), pig (~7), rat epididymal (<>) and rat inguinal (O). Each point represents the mean of three experiments, each with three replications.

with previous observations (Lee et al., 1986; Nassar and Hu, 1991a). The degree of antiserum reactivity toward adipocyte PM from different species supports the hypothesis that antigenic determinant(s) on PM are species-specific. Reactivities of antisera adsorbed with heart and liver homogenates are shown in Figs 4 and 5, respectively. Compared to antisera adsorbed with liver homogenate, heart homogenate adsorbed antisera had a lower reactivity to all PM. This might be attributed to the heart having more cross-reactive proteins with adipocytes than that of liver or a wider variety of protein types than liver. Adsorption with heart homogenate removed most of the cross-reactivity with other tissues but conserved more than half of the non-adsorbed reactivity (Fig. 4). Liver homogenate was less effective in removing all the cross-reactivity (Fig. 5). These results indicate that adipocyte-specific antibodies can be obtained by adsorbing antisera with a homogenate of heart tissue. This implies the existence of specific antigens on

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Fig. 4. Effect of adsorption of antiserum with heart homogcnate on antiserum reactivity with plasma membranes prepared from adipocyte (V), heart ([[]), kidney (©), liver (~7), muscle (O). Adipocyte PM reacted with non-adsorbed antisera ( A ) and normal serum ( & ) were used as controls.

Each point represents the mean of three experiments, each with three replications.

Fig. 5. Effect of adsorption of antiserum with liver hom-

ogenate on antiserum reactivity with plasma membranes prepared from adipocyte (V), liver (~7), heart ([-7), kidney (O), muscle (O). Adipocyte PM reacted with non-adsorbed antisera (A) and normal serum (&) were used as controls. Each point represents the mean of three experiments, each with three replications.

chicken adipocytes which contradicts the conclusion of Butterwith et al. (1989). We have no explanation regarding the differing results of these two chicken studies. Further characterization of adipocyte PM, using gel electrophoresis and immunoblotting, are required to discern unequivocally the existence of chicken adipocyte-specific proteins.

REFERENCES

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Killefer J. and Hu C. Y. (1990) Production of a novel monoclonal antibody to porcine adipocyte plasma membrane. Proc. Soc. Exp. Biol. Med..194, 172-176. Lee S. R., Tume R. K. and Cryer A. (1986) Studies on the expression of adipocyte-specific cell surface antigens during the differentiation of adipocyte precursor cells in vitro. J. Develop. Physiol. 8, 207-226. Lo C., August T. R., Liberman U. A. and Edelman I. S. (1976) Dependence of renal (Na + + K+ )-adenosine

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triphosphatase activity on thyroid status. J. Biol. Chem. 251, 7826-7833. Loten E. G. and Redshaw-Loten J. C. (1985) Preparation of rat liver plasma membranes in a high yield. Analytical Biochem. 154, 183-185. Nassar A. H, and Hu C. Y. (1991a) Development and characterization of polyclonal antibodies specific to ovine adipocyte plasma membranes, lnt. J. Biochem. (in press).

Nassar A. H. and Hu C. Y. (1991b) Growth performance and carcass characteristics of Iambs treated with antiovine adipocyte plasma membrane antibodies. J. Anim. Sci. (in press). Pillion D. J., Grantham J. R. and Czech M. P. (1979) Biological properties of antibodies against rat adipocyte intrinsic membrane proteins. J. Biol. Chem. 254, 3211-3220.