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Use of the Capybara for Antisera Production

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Use of the Capybara for Antisera Production A n n a l s o f C linical and Laboratory Science, Vol. 4 , No. 1 Copyright © 1974, Institute for Clinical Science Use of the Capybara for Antisera Production ERNEST C. ADAMS, Ph .D., KATHLEEN M. LAYMAN, AND RALPH HARTNAGEL, B.S. Ames Research Laboratory, Ames Company and Toxicology Department, Research Division, Division Miles Laboratories, Inc., Elkhart, IN 46514 ABSTRACT Antisera to porcine insulin were produced in capybaras. The antisera were evaluated by radioimmuneassay, immune hemolysis and in vitro neutralization of insulin. The antisera were of high titer and useful for assay of insulin. Capybaras should be useful for any antisera for which guinea pigs are the preferred species. The serum proteins were shown to be similar by immuno- electrophoresis and immunodiffusion. Another similarity to guinea pigs is the requirement of an external source of ascorbic acid for capybaras. Introduction structure so that the guinea pig cells recog­ The production of antisera to insulin in nize pork, beef, or human insulin as being a number of species, such as human, rabbit, different from its endogenous insulin and goat, sheep, has been reported.11 However, thus produce good antibody. the guinea pig is preferred, particularly for Davidson and Haist4 reported that radioimmune assays. Part of the reason for guinea pig insulin is not neutralized by an the suitability of the guinea pig for im­ antiserum to beef insulin produced in the munization with insulin can be seen from guinea pig. Davidson, Zeigler and Haist the amino acid sequence of insulin shown reported that insulin isolated from the in figure 1.2,T The sequences for beef, pancreases of coypus5 and capybara6 were pork, and human insulin are very similar also not neutralized by antiserum to beef while that for guinea pig insulin is quite insulin produced in the guinea pig. This different. Some of these differences affect suggests that these animals would also be properties of guinea pig insulin without suitable for producing antisera to insulin. altering its biological activity. For instance, The capybara (Hydrocherus capybara) the B-10 histidine of beef insulin, which has is the largest rodent in the world (figure been reported to be responsible for zinc 2). It is found along the lakes and rivers of complexing, is changed to asparagine in South America. It is largely a water rodent guinea pig insulin and guinea pig insulin and in shape it resembles a guinea pig. It does not complex with zinc.13 These amino has webbed feet and its body is covered acid differences probably alter the tertiary with dark coarse hair. The adult weighs 19 2 0 ADAMS, LAYMAN AND HARTNAGEL F ig u r e 1. Amino acid sequence of beef insulin. approximately 70 to 80 pounds although pounds. The capybara also resembles the some capybara weigh as much as 150 guinea pig in that it requires an external source of ascorbic acid. Experimental A c q u i s i t i o n , C a r e , a n d F e e d in g Capybaras weighing approximately six kg are obtained from Charles P. Chase Co., Inc.® The animals are housed in rooms which are 150 feet square. A plastic box with straw is supplied as a bed and a plastic tub is filled with clean, warm water twice a day. The animals receive a diet of rolled oats, hay, dog pellets, and lettuce. They have access to a trace mineral salt block. 1 During the immunization schedules, an intramuscular injection of ascorbate is given five times weekly. I mmunization Eight mg pork insulin (Elanco, 25 units per mg) are dissolved in 0.5 ml 0.1 N hy- 0 7330 Northwest 66th Street, Miami, FL 33166. F i g u r e 2. Capybara. Scale reads in kilograms. f Harley Salt Company. USE OF THE CAPYBARA FOR ANTISERA PRODUCTION 2 1 drochloric acid and immediately neutralized If the insulin and adjuvant are well ho- with 0.5 ml 0.1 N sodium hydroxide to moginized and injected immediately, no which one ml Freund’s complete adjuvant hypoglycemic reactions develop. However, is added. The mixture is homogenized in it is well to be prepared at all injections the micro attachment for the Sorvall Omni­ with sterile 10 percent glucose in isotonic mixer whereby the vessel is cooled with ice saline. If hypoglycemic symptoms develop, water during the homogenization. Each the glucose solution is injected intraperi- footpad of the capybara is injected with toneally, 20 ml at a time, until the symp­ 0.25 ml of the homogenate. toms are alleviated. The glucose injections Subcutaneous boosters are given 21 and have to be repeated if hypoglycemia re­ 23 days after the primary immunization. develops. The animal must be watched, all For this booster, eight mg insulin are dis­ night if necessary, so that hypoglycemia can solved in 0.5 ml 0.1 N hydrochloric acid. be immediately counteracted. In general, if This is neutralized with 0.5 ml 0.1 N no symptoms of hypoglycemia have de­ sodium hydroxide and 1 ml Freund’s in­ veloped by six hours after injection, none complete adjuvant is added and homoge­ will appear. nized as before. The boosters (total volume To avoid anaphylactic reactions, the of one ml) are given in sites in each of the capybaras are injected intraperitoneally quarters of the animal. A test bleeding is with 0.5 ml phenindamine tartratef (6 mg made ten days after the second of the pair ml) one hour before the subcutaneous of boosters. boosters. On the 56th and 58th days after the Good antisera production after boosting primary immunization, another pair of is indicated by large swellings at the sites boosters are given. A test bleeding follows of boosting. on the tenth day. After this second pair of boosters, additional boosters are not B l e e d in g a n d P r o c e s s in g o f B lo o d given until test bleedings show the titer Test Bleedings. Volumes of 5 to 40 ml are has dropped to almost nothing. It takes at taken from a vein in the forepaw. Vacu- least twice as much time to reach this stage tainers® (ED TA ) of 5 or 10 ml capacity as the time between the other pairs of draw the blood at a rate which will not boosters. Additional boosters are given as collapse the vein. before. If, after an additional ten days, the While a capybara is not vicious, its teeth test bleeding indicates the desired titer, the are capable of inflicting a nasty wound. animal may be bled. Alternatively, boosters Thus, it is advisable to bind the jaws with may be used if, after waiting a period of gauze before bleeding. As the animal gets time, the titer drops. larger, it may be necessary to tranquilize it In some cases, for the primary immuniza­ with Sparine (promazine hydrochloride)$ tion, the insulin is mixed with Freund’s in­ before bleeding. Larger amounts of blood complete adjuvant and pertussis vaccine. may be obtained by surgical exposure of Eight mgs pork insulin (Elanco, 25 units the carotid under anesthesia. Good surgical per mg) are dissolved in 2.5 ml buffered technique is required so that the animal phenol (pH 2.6) to which are added 2.5 recovers. ml Freund’s incomplete adjuvant and 0.75 Bleedout. For bleedout, the animal is ml pertussis vaccine.* This is homogenized tranquilized with Sparine (promazine hy­ with an Omnimixer. A total of 2.9 ml is drochloride )t and anesthetized with Suri- given in footpads. t Roche. * Lilly. I Wyeth. 2 2 ADAMS, LAYMAN AND HARTNAGEL tal.§ The carotid is exposed and cannulated 10 ml 0.6 percent acetic acid. One tenth ml and the blood is collected in centrifuge of this solution is diluted to 10 ml in the bottles with 7.5 mg EDTA per 5 ml whole albumin buffer and 0.1 ml of this dilution blood as an anticoagulant. is further diluted to 5 ml in the albumin Processing. The blood is centrifuged and buffer. This gives a solution containing 500 the plasma separated. Calcium chloride so­ microunits insulin per ml. lution (52.5 mg per ml in saline), 0.1 ml is Labeled Insulin. The fractionated insulin added for every 7.5 mgs of EDTA to is diluted with the albumin buffer so that counteract the EDTA. After clotting, the 0.1 ml has approximately 30,000 counts per serum is removed and stored in a — 60° minute. freezer. Method. Two rows of silicone treated test tubes, each with 11 tubes, are set up. Five- E v a l u a t io n o f A n t i s e r a tenths ml of the albumin buffer is placed The method is based on the dextran- in tubes 2 through 11. One-one hundredth coated charcoal separation of Herbert.9 ml of antiserum is diluted with 5 ml of the Reagents. I125 labeled insulin is pur­ albumin buffer to give a 1/500 dilution. chased from Abbott Laboratories. As soon Five-tenths ml of this dilution is added to as the labeled insulin is received, it is tubes 1 and 2 of each row. Five-tenths ml diluted with an equal volume of 0.015 M, of tube 2 is transferred to tube 3 and the pH 7.4, phosphate 2 percent human serum serial dilution is continued through tube 10. albumin buffer and stored in a deep freeze. One-tenth ml of the 500 microunits per ml The labeled insulin (0.1 to 0.3 ml) is frac­ insulin standard is added to each tube in tionated on 1 X 20 cm columns of G-75 row 2.
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