Somatotropin Antibody Formation in Cows Treated with a Recombinant Bovine Somatotropin Over Two Lactations C. M. ZWICKL, H. W. SMITH, R. N. TAMURA, and P. H. BICK Toxicology DIvision Uily Research laboratories Eli Lilly and Company Greenfield, IN 46140 ABSTRACT

Blood plasma from cows treated with somidobove, a fonn of recombinant bovine somatotropin, was assayed for development of antibodies against the protein. Forty-three Holstein cows, selected from an animal safety study, were monitored. Cows were divided into four groups and treated with placebo, 960, 2880, or 4800 mg somidobove per dose at 28-d intervals during two successive lactation periods. Blood plasma was collected at intervals prior to and during the lactations, and levels of IgG antibody reactive with somidobove were determined in an enzyme-linked immunosorbent assay. Virtually all of the cows treated with somidobove developed low levels (less than 40 J.1g/ml) of antibody against somidobove. One or two cows from each group responded with somewhat higher levels, ranging from 40 to 200 J.1g/ml. Responses generally increased during the first 3 mo of treatment, then decreased, and remained constant with continued treatment. There was no sign of a memory response within or among the lactation periods, and no adverse health effects or decreases in lactational perfonnance were associated with antibody production. (Key words: antibody fonnation, somatotropin, cow) INTRODUCTION

Lactation in mammals is influenced by several honnones which exert direct or indirect

Received February 20, 1990. Accepted June 18, 1990. 1990 J Dairy Sci 73:2888-2895

effects on the growth, differentiation, and function of the mammary gland (8, 31). Whereas the minimal honnonal requirements for the maintenance of lactation vary among species, in ruminants somatotropin appears to play an essential role (13). This suggested that milk production in farm animals might be influenced by manipulating concentrations of somatotropin in plasma and prompted numerous studies to investigate the effects of supplemental somatotropin on lactating dairy cows. The studies consistently demonstrated that milk production could be increased by as much as 10 to 25% or greater in cows treated with somatotropin (10, 17, 19, 22, 26, 27, 28). Somatotropin supplementation offered an attractive strategy for the management of milk production in dairy herds because supplementation did not change the milk quality, caused no change or marginal changes in the composition of milk from treated cows (9, 10, 17), and had no reported side effects. Until recently, however, the practical consideration of this strategy was precluded by the limited amounts of somatotropin available from extraction of pituitary glands. The application of recombinant DNA methods to produce unlimited amounts of bovine somatotropin has eliminated this barrier and provides an opportunity to investigate the effectiveness and safety associated with somatotropin supplementation in greater detail (25, 32). As part of a target animal safety study using a recombinant bovine somatotropin, somidobove (16) (Eli Lilly and Co., Indianapolis, IN), we investigated the potential of the fonnulated somidobove preparation to elicit an antibody response when administered under conditions similar to those proposed for its commercial use.



MATERIALS AND METHODS Treatment with Somldobove

Forty-three Holstein cows, distributed among four groups and treated at 28-d intervals through two successive lactations with placebo or 960, 2880, and 4800 mg of somidobove, respectively, were monitored for somidobove antibody production. The 96O-mg dose originally was expected to approximate the dose that would be used in dairy herd management programs. Blood samples were collected at 1 and 3 wk prior to treatment and at regular intervals during the treatment periods. The length of each treatment period was 252 d from the day of initial injection. Plasma was separated from whole blood and assayed for antibodies against somidobove using an enzyme-linked immunosorbent (ELISA) assay. Production of Somldobove Antiserum

To produce somidobove antiserum to serve as a positive control in the antibody assay, two Holstein cows were immunized with a somidobove-adjuvant emulsion. Immunogen was injected intramuscularly in two sites on the back of each cow, in a total volume of 2 mI. Primary immunization consisted of 200 J.Lg of somidobove per cow in a 1:1 emulsion of complete adjuvant containing strain H37-Ra Mycoplasma (Difco Laboratories, Detroit, MI). Thereafter, cows were injected three additional times, at 2-wk intervals, with 1 mg of somidobove per cow in Freund's incomplete adjuvant (Difco Laboratories). This was followed by a final immunization, 20 d later, with 10 mg of somidobove in complete adjuvant. Blood samples were drawn intermittently during the immunization period to assess antibody titers, and a final sample was drawn 8 d following the last injection. Serum from the final bleedings contained 40 and 62 J.Lg/mI of somidobove antibody. Somldobove Antibody Enzyme-Linked Immunosorbent Assay

Wells of a 96-well microtiter plate (NUNC, Certified Irnrnuno-Plate I, Vangard International, Neptune, NJ), were coated with 100 J.LI of 2.5 J.Lg/mI goat antibovine IgO (H + L chain


specific, affInity purified, Kirkegaard and Perry Laboratories, Gaithersburg, MD), or with 100 J.Ll of 10 lJ.g/mI somidobove in .1 M bicarbonate buffer, pH 9.6. The plate was incubated at 24·C for 30 min, then rinsed three times with .02 M potassium phosphate-buffered saline (KPBS), pH 7.4, containing .01% porcine skin gelatin (60 Bloom, Sigma Chemical Co., St. Louis, MO), and .05% Tween 20 (Sigma Chemical Co.). Residual protein-binding sites on the plate were blocked with 150 J.Ll of .5% gelatin-KPBS for 30 min at 24·C. After the plate was blocked, it was rinsed as described, and 100 J.Ll of reference bovine IgG (chromatographically purified IgO, Cappel Laboratories, Malvern, PA), diluted twofold serially to concentrations from .977 to 250 ng/mI in .02 M KPBS, pH 7.4, containing .1% gelatin, were added, in duplicate, to the antibovine IgG-coated wells. Test plasma, as well as positive and negative control plasma, were diluted appropriately, and 100 J.Ll were added, in duplicate, to the somidobove-coated wells. Antisera from cows immunized with somidobove in adjuvant were used as positive assay controls. Following incubation for 30 min at 24·C, the plate was rinsed as described and 100 J.Ll of horseradish peroxidase-coupled goat antibovine IgO (Kirkegaard and Perry Laboratories) were added to the wells. The plate was incubated for 30 min at 24·C, then rinsed. One hundred microliters of 2,2'-azino-di[3ethylbenzthiazoline sulfonate] substrate solution (Kirkegaard and Perry Laboratories) were added to each well. Absorbances were read at 410 nrn, using a reference wavelength of 490 nrn (MR 600 Microplate Reader, Dynatech Laboratories, Chantilly, VA). When the absorbances of wells that contained the 250 ng/rnl reference IgG standard approached 2.0, the reaction was inhibited by adding 25 J.Ll of 2.5% sodium dodecyl sulfate, and the absorbance of each well was measured. A curve was fIt to the bovine IgG reference standards using a four-parameter logistic curve fitting program (l'iterCalc 2.1, HP Genenchem, Palo Alto, CA), and the concentrations of antisomidobove in test sample plasma were interpolated from the curve. Since the reference curve was produced by coating wells with antibovine IgO instead of somidobove, results are reported as relative IgO concentrations. AntiJournal of Dairy Science Vol. 73,

No. 10, 1990



TABLE 1. Incidence 1 of somidobove antibody responses classified as positive during the first lactation. Treatment group dose 2880 mg


4800 mg

Test day



0/11 (5.5, 8.0)2

0/12 (2.6, 1.6)

0/10 (3.5, 4.9)

0/10 (2.4, 1.5)


0/11 (3.7, 4.9)

0/12 (3.6, 2.4)

0/10 (4.6, 4.1)

0/10 (2.6, 1.5)


1/11 (4.2, 4.2)

8/12 (7.3, 4.2)

8/10 (19.5, 31.9)

10/10 (9.8, 3.0)


1/11 (3.0, 2.9)

11/12 (17.9, 18.7)

10/10 (31.6, 60.7)

10/10 (23.7, 17.1)


0/11 (2.3, 1.9)

10/12 (13.2, 9.4)

9/10 (17.1, 20.2)

10/10 (20.0, 12.7)


0/11 (2.6, 1.1)

11/12 (17.2, 11.2)

8/10 (19.8, 26.3)

10/10 (21.9, 18.5)

.9 - 16 2.5

3.0 - 66 8.5

1.9 - 203 10.2

5.3 - 69 14.5

Response3 Range Median

lIncidence is reported as the number of cows in the group that were classified as eliciting an antibody response according to the statistical decision rule described in the text over the total number of cows in the group. 2Yalues in parenlheses are the mean and SD somidobove antibody levels in micrograms per milliliter. 3Response ranges and median values include data from the treatment period only (d 28 to 252).

body levels were estimated from the 1:1000 dilution of plasma whenever the absorbances were low enough to warrant the use of that dilution. Antibody concentrations at each time point were analyzed statistically and classified as being positive if the difference between the concentration at the measured time point and the mean concentration determined in pretreatment samples from the same animal was greater than 2.4 J1g of IgG/ml plasma. This decision role resulted from classifying those responses that fell outside of a 9SnS nonparametric interval based on pretreatment antibody concentrations. The tolerance interval indicates a "likely" interval over which 9S% of the responses should fall if no antibody was produced. A nonparametric approach was used because of extreme skewness of the pretreatment data, which were not totally corrected after considering a logarithmic transfonnation. RESULTS

First Lactation Period

Twenty-eight days following the initial injection of somidobove, antibody responses were Journal of Dairy Science Yol. 73,

No. 10, 1990

detected in 67% of cows that received 960 mg, 80% of those that received 2880 mg, and 100% of those that received 4800 mg of somidobove (Table 1). By test d 84, all but one cow were positive. A large degree of variability among animals was seen, such that the presence of one or more high responding animals in a group biased the mean response for the group toward slightly higher antibody concentrations (Table 1). For this reason, when relationships between concentration and time were graphed, median response data were considered to be more representative of antibody concentrations produced by the majority of cows in a group. In groups treated with somidobove, the median antibody concentration increased with successive dosing through d 84, then remained constant at levels of 10 to IS J1g/ml by the 6th mo of treatment (Figure 1). The majority of responses ranged from 4.7 to 40 J1g/ml; however, one or two animals in each group responded with higher levels of antibody during a portion of the treatment period (Figure 2). The range of responses observed in four of the five higher responding cows was 40 to 70 J1g/ml, which was similar to those produced by the two positive control cows injected with somidobove in adjuvant. The fifth cow, from the 2880-mg







Somatotropin antibody formation in cows treated with a recombinant bovine somatotropin over two lactations.

Blood plasma from cows treated with somidobove, a form of recombinant bovine somatotropin, was assayed for development of antibodies against the prote...
704KB Sizes 0 Downloads 0 Views