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 immunoelectrophoresis 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. One-tenth ml of the albumin buffer Sephadex® equilibrated with the same buf is added to each tube in row 1. Finally, 0.1 fer. The first hot peak material is discarded ml of the diluted labeled insulin is added and the second peak material is used for to every tube. The tubes are mixed well, the assay. covered and placed in a 37° incubator Buffer. Sodium barbital, 14.714 g, and for two hours. Two ml of the dextran- sodium acetate, 9.714 g, are dissolved in coated charcoal suspension are added to distilled water and diluted to 500 ml. To each tube and mixed well. The tubes are 100 ml of this buffer base are added 1,800 centrifuged for 20 minutes. The super ml of sodium chloride (0.85 g per 100 ml) natants are decanted into plastic tubes and and 100 ml 0.1 N hydrochloric acid to yield counted in a well counter. a final pH of 7.4. Dose Response Curve. An antiserum dilu Albumin Buffer. Eight ml of 30 percent tion giving 30 to 50 percent binding is human serum albumin is added to 92 ml chosen. The 500 microunits per ml insulin of the buffer. standard is serially diluted in the albumin Dextran-Coated Charcoal. Five grams buffer for new standards. One-tenth ml of charcoal, Norit A, is suspended in 100 ml each of the standards is run with 0.5 ml of of the barbital buffer and 0.5 grams Dex- tran 80* is dissolved in 100 ml of the antiserum dilution and 0.1 ml labeled in barbital buffer. This is then mixed with the sulin. charcoal suspension. Hales-Randle Method. 8 Gamma globulin Insulin Standard. One mg pork insulin from the capybara anti-insulin is precipi (Elanco, 25 units per mg) is dissolved in tated with a rabbit anti-guinea pig gamma

§ Parke-Davis. globulin. To 20 ml of a 1/500 dilution of * Pharmacia—now T-70. the anti-insulin in isotonic saline are added USE OF THE CAPYBARA FOR ANTISERA PRODUCTION 2 3

4.5 ml of anti-guinea pig gamma globulin.* Immunodiffusion. This method was origi After being mixed well, the mixture is left nally described by Ouchterlony.10 One and standing overnight at 4°. The mixture is two-tenths grams Difco Bacto agar is dis centrifuged and the precipitate is washed solved in pH 7.4 phosphate-saline buffer twice with saline. The precipitate is re (6.8 g Na2HP04-2H20, 0.64 g KH2P04, suspended in 25 ml albumin-barbiturate and 0.2 g sodium azide diluted to one liter buffer, dispensed in 0.5 ml aliquots, and with saline). The suspension is boiled for stored at —60°. For use, a vial of this bind one hour until the agar is in solution. The ing reagent is made up to a volume of solution is filtered through glass wool and 16 ml. Insulin standards are prepared as the volume restored. The agar is poured for the dextran-coated charcoal method. into petri dishes to a depth of 3 mm and One-tenth ml of each standard is incubated allowed to gel for at least two hours. The at 4° with 0.1 ml of the binding reagent in agar is cut into the desired pattern with a plastic tubes for six hours. Then 0.1 ml Feinberg gel cutter. Protein solutions and labeled insulin (30,000 counts per minute) antisera are placed in the wells and allowed is added and the mixture incubated at 4° to diffuse for 8 to 24 hours. The precipitin for 18 hours. One ml of the albumin-bar lines are photographed with Polaroid black biturate buifer is added and mixed. The and white Polapan, Type 52, film. tube is centrifuged and the supernatant is decanted. The residue is then counted in a R esu lts well counter. In figure 3 is shown the immunoelectro- The immune hemolysis experiments were phoretic pattern of capybara and guinea carried out for us by Dr. Edward Arquilla pig sera against anti-guinea pig serum and of the University of California in Irvine, by anti-guinea pig gamma globulin. The pat his previously published methods.1 terns are very similar. The line for the Neutralization. This is based on the capybara serum against the anti-gamma method reported by Davidson.5’6 Carworth globulin is longer than that for the guinea C. F. white mice weighing approximately pig serum. 20 grams are injected subcutaneously with In figure 4 is shown the immunodiffusion mixtures of 0.5 ml of 100 milliunits of insu patterns for capybara and guinea pig sera lin and 0.5 ml of dilutions of antiserum. against anti-guinea pig serum and anti- The animals are observed for convulsions. Immunoelectrophoresis. Modifications of the Sheidegger microimmuno-electropho- retic technique as reviewed by Crowle3 are employed. Microscope slides are coated with 1 percent Noble Agar in barbital buf fer (0.025 n, pH 8.6) with 0.01 percent merthiolate added as a preservative. Elec trophoretic migration is carried out for two hours at 47 volts across the slides. The electrophoresed sera are diffused against the specific antisera for 16 to 48 hours. The F i g u r e 3. Immunoelectrophoretic patterns of unstained lines are photographed with capybara and guinea pig sera. Top: capybara Polaroid black and white Polapan, Type serum. Bottom: guinea pig serum. Outer slots 52, film. wells contain antiserum to guinea pig gamma globulin. Inner slot wells contain antiserum to * Pentex. whole guinea pig serum. 2 4 ADAMS, LAYMAN AND HABTNAGEL

m •t

F ig u r e 4. Immunodiffusion patterns of capybara and guinea pig sera. Top well: guinea pig serum. Bottom well: capybara serum. Left well: anti serum to whole guinea pig serum. Right well: antiserum to guinea pig gamma globulin. guinea pig gamma globulin. The patterns are similar. There may be some difference since one of the lines for capybara serum F ig u r e 6 . Immunodiffusion of guinea pig serum, does not appear to be blocked by the line capybara serum and guinea pig gamma globulin for guinea pig serum. against anti-capybara gamma globulin. Center well: anti-capybara gamma globulin. Upper right In figure 5 is shown the immunoelectro- well: guinea pig serum. Middle right well: capy phoresis of guinea pig serum against anti- bara serum. Lower right well: guinea pig gamma capybara gamma globulin and anti-guinea globulin. pig gamma globulin. The patterns appear identical. since only two capybaras were available at In figure 6 is shown the immunodiffusion that time. The titers and schedule are of guinea pig serum, capybara serum and shown in figure 7. The first capybara was guinea pig gamma globulin against anti- immunized in the footpads with insulin capybara gamma globulin. There is a line coupled to guinea pig gamma globulin or of identity, but the capybara serum con albumin with glutaraldehyde by the pro tains another antigen which is missing from cedure of Reichlin, Schnure, and Vance.12 the guinea pig serum. The second animal was immunized with The immunization schedule, given in the insulin. Materials for both injections were experimental section, is established as the homogenized with Freund’s complete adju best one. The first capybaras were taken vant. After three weeks, the animals were through a much more complicated scheme given intravenous boosters of insulin either alone or coupled to guinea pig albumin. The animal receiving insulin began to de velop a titer while the other did not. When the animal receiving insulin died, the first capybara was reimmunized in the footpads with insulin in Freund’s complete adjuvant and then boosted with insulin intravenously on schedule. Thus, the first capybara was F ig u r e 5 . Immunoelectrophoresis of guinea pig switched to the schedule of the second gamma globulin. Top slot: anti-capybara gamma globulin. Bottom slot: anti-guinea pig gamma capybara. The animal developed a titer. globulin. When the boosters were given subcutane- USE OF THE CAPYBARA FOR ANTISERA PRODUCTION 2 5

F i g u r e 7. Immuniza tion schedule, titers and growth curves for capybaras 1 and 2 .

ously in Freund’s incomplete adjuvant, the essary to skip a pair of boosters. After the titer increased further. Finally, the dose following booster, there was a greater in at each successive booster was increased. crease in titer. In retrospect, it was realized When the animal became sick, it was nec that the other capybara also had a titer

14 *

F i g u r e 8 . Immuniza «4 g tion schedule, titers and growth curves for capy- 12 5 baras 3 and 4. to

8 2 6 ADAMS, LAYMAN AND HARTNAGEL

approximately six kg and at first gained weight. After about 12 weeks, in spite of ascorbic acid in the diet, the animals de veloped typical symptoms of scurvy, loss of weight, bleeding gums, pain in joints, etc. The animals were hand-fed baby food rich in ascorbic acid, given tablets of ascor bic acid, and given ascorbic acid in the drinking water. The animals apparently re covered and resumed weight gain. How ever, the second capybara, after the first of a scheduled pair of boosters, developed neurological symptoms reported for scurvy and died despite treatment with ascorbic acid. It seems likely that the stress asso ciated with immunization increases the ascorbic acid requirement. The animals since then have been given intramuscular ascorbic acid. The surviving capybara reached a weight of 35 kg before being sacrificed. F ig u r e 9 . Evaluation of antiserum. The upper curve shows the percent binding of F 25 insulin In figure 8 are shown the titers and im with dilutions of antiserum. The lower curve shows munization schedule for the third and the percent binding of I125 insulin with the same fourth capybaras. Number 3 received in dilutions of antiserum and 50 microunits insulin. The difference between the two curves is a mea jections of Freund’s complete adjuvant in sure of the utility of the antiserum for radioim the footpad while number 4 received the munoassay. injections of pertussis adjuvant in the foot pad. Both animals received intramuscular increase when the interval between booster boosters in Freund’s incomplete adjuvant. injections was prolonged. These observa There appeared to be no difference be tions led to the schedule described in the tween using Freund’s complete adjuvant experimental part. and using the pertussis adjuvant. As can be In figure 7 are shown the growth curves seen, both animals developed good titer for the first two capybaras. Both started at and the titer increased when the booster pair was skipped. In figure 8 is also shown the growth curve for the two animals. In figure 9 is shown a typical serum eval uation study, using the dextran-coated char coal method. Usually, the 50 percent bind ing point is reported. In figure 10 is shown a dose response curve for one of the antisera with the dextran-coated charcoal method and with the Hales-Randle double antibody method. As shown in figure 11, the immune he molysis assay indicated a titer of about 1/ F ig u r e 1 0 . Dose response curve. 10,000. Hemolysis was inhibited completely USE OF THE CAPYBARA FOB ANTISERA PRODUCTION 2 7 by 0.1 ¡ig insulin. There was only 2 to 3 percent cross reaction with desalanine desasparagine insulin. In the neutralization experiments, three out of five mice had convulsions and died with 100 milliunits insulin and no anti serum; none convulsed with 100 milliunits insulin plus undiluted antiserum or 1/4, 1/8, 1/16, or 1/32 dilutions of antiserum while one of five convulsed and died with

100 milliunits insulin plus 1/64 dilution of F i g u r e 11. Immune hemolysis. The curve to antiserum. When other signs of hypogly the far right shows the percentage of cells lysed as a function of the amount of antiserum. The cemia such as marked salivation were used curve in the left lower corner shows the inhibition as criteria, five of five were hypoglycemic of this hemolysis by 0.1 microgram insulin. DAA in the control group, one of five was hypo insulin is desalanine desasparagine insulin pre pared by removing the B-30 alanine and the A-21 glycemic with the 1/64 dilution of anti asparagine with carboxypeptidase. DOP insulin serum and none was hypoglycemic in the is desoctapeptide insulin prepared by removing the other groups. C-terminal eight amino acids of the B chain with trypsin. In table I are given some values obtained for blood from one of the capybaras. more like the strain 13 guinea pigs than the strain 2 ones. D iscussion In addition to the use of the capybara for The similarity of capybaras to guinea producing antisera to insulin, it seems the pigs was confirmed by the patterns ob capybara would be a useful animal for any tained by immunodiffusion and immuno- antisera for which the guinea pig is the pre electrophoresis of the sera against anti ferred animal. This appears to be true for guinea pig serum, anti-guinea pig gamma a number of the viruses or parts of viruses, globulin and anti-capybara gamma globu such as hepatitis B. In the case of insulin, lin, as well as by the requirement for ascor bic acid. As originally postulated, immuni TA BLE I zation with insulin produced antisera that were useful for radioimmune assay. The Blood Values Obtained from One Capybara antisera were shown to have titer by radio- Hemoglobin gm per 100 ml 14.3 immune assay and immune hemolysis and Hematocrit percent 44.0 Erythrocyte ct. (millions) 3.56 to neutralize insulin. The dose response Leukocyte ct. (thousands) 7.0 curve was steeper for the capybara anti Differential Neutrophiles 52 serum than for a commercially obtained Lymphocytes 48 guinea pig antiserum. This may merely be Monocytes 0 the difference between using antiserum Eosinophiles 0 Basophiles 0 from one animal and a pooled antisera. The Cholesterol mg per 100 ml 45 2 to 3 percent cross reaction by desalanine Alkaline Phosphatase KA units 36.0 SGrOT Units 14 desasparagine insulin is approximately the SG PT Units 2 0 same as reported by Arquilla for strain 13 Total Bilirubin mg per 100 ml 0.65 Sodium mEq per liter 136 guinea pigs.2 The corresponding value for Potassium mEq per liter 4.9 strain 2 and mongrel guinea pigs was 10 Glucose mg per 100 ml 1 0 0 Urea Nitrogen mg per 100 ml 1 1 . 8 percent. This suggests that the capybara is 2 8 ADAMS, LAYMAN AND HARTNAGEL the failure of the endogenous insulin to R eferences combine with the antibodies has been sug 1. A rquilla, E. R ., O o m s , H., and M ercola, gested as a reason for the usefulness of the D.: Immunological and biological properties guinea pig. Possibly, there is also a similar of iodoinsulin labeled with one or less atoms of iodine per molecule. J. Clin. Invest. 47: failure of some of the viruses to combine 474-487, 1968. with antibodies of the guinea pig. 2. A r q u i l l a , E. R., B r o m e r , W. W., a n d The advantage of capybaras over guinea M e r c o l a , D.: Immunology conformation and biological activity of insulin. Diabetes 18: pigs for antisera production is the volume 193-205, 1969. of serum that can be obtained from a single 3. C r o w l e , A. J.: Immunodiffusion. New York, animal. A total bleed out of a capybara will Academic Press, p. 231, 1961. yield from 500 to 1000 ml serum, depend 4. D a v id s o n , J. K. a n d H a i s t , R. E .: Failure of guinea pig antibody to beef insulin to neutra ing on size, compared to 10 ml from a lize pancreatic and serum guinea pig insulin guinea pig. A large volume of antiserum in vitro. Can. J. Physiol. Pharmacol. 43:373— from one animal having the same titer, 378, 1965. 5. D a v id s o n , J. K., Z e ig l e r , M ., a n d H a i s t , R . specificity, and avidity is more useful than E.: Failure of guinea pig antibody to beef a serum pool obtained by mixing sera of insulin to neutralize coypu (nutria) insulin. varying titers, specificities and avidities Diabetes 17:8-12, 1968. from many animals. No more antigen is re 6 . D a v id s o n , J. K., Z e ig l e r , M., a n d H a i s t , R. E.: Failure of guinea pig antibodies to beef quired for the immunization of a capybara insulin, chicken insulin, and cod insulin to than for a guinea pig. Thus, there is econ neutralize capybara insulin. Diabetes 18:4, omy for scarce or expensive antigens. The 1969. 7. D a y h o f f , M. O. a n d Eck, R. V.: Atlas of ease of bleeding a capybara makes it pos Protein Sequence and Structure. The National sible to make repeated test bleedings, to Biomedical Research Foundation, Silver follow the rise of titer and to select an Springs, MD, pp. 262-264, 1968. 8 . H a l e s , C. N. a n d R a n d l e , P. J.: Immuno optimum time for collection of antiserum. assay of insulin with insulin antibody precipi For repeated test bleedings of a guinea pig, tate. Biochem. J. 88.137-146, 1963. it is necessary to make cardiac punctures 9. H e r b e r t , V., L a u , K., a n d G o t t l i e b , C. W.: and there is always the risk of losing a Coated charcoal immunoassay of insulin, J. Endocr. Metabol. 25:1375-1387, 1965. valuable animal. Capybaras are expensive. 10. O u c h t e r l o n y , O.: Diifusion-in-gel methods A very young one costs about sixty dollars of immunological analysis. Prog. Allergy 5: and older ones cost more; however, in pro 1-78, 1958. 11. P o p e , C. G. : The Immunology of Insulin. ducing antisera the initial cost of an animal Advances in Immunology, Dixon, F. J. and is trivial compared to the cost of preparing Humphrey, J. W., eds., Vol. 5, pp. 209-240, and purifying the antigen, the animal up 1966. keep as well as testing and quality control 12. R e i c h l i n , M., S c h n u r e , J. J., a n d V a n c e , V . K.: Introduction of antibodies to porcine of the antisera. The younger animals are ACTH in rabbits with nonsteroidogenic poly better for immunization and also easier to mers of BSA and ACTH. Proc. Soc. Exper. handle than older ones. When obtained Biol. Med. 28:347-350, 1968. 13. Zi m m e r m a n , A. E . a n d Y i p , C. C.: Physical young, they remain comparatively easy to and chemical properties of guinea pig insulin. handle, even as they grow older. Fed. Proc. 31:2, 243, 1972.