BIOCHEMICAL MEDICINE 29, 293-297 (1983)

Enzyme Immunoassay for Serum Ferritin of Pigs

JOSEPH E. SMITH. KATERI MOORE, AND DONNA BOYINGTON

Depurtment of Pathology. College of Veterinup Medicine, Kunscls State Uniljersity.

Manhattun. Kun.sus 66506

Received May 34. 1983

Total body iron stores are difficult to determine accurately. Serum iron and total iron binding capacities can be influenced by several diseases including chronic infection, renal failure, hypoproteinemia, and anemia (1). Hemoglobin and packed cell volume are relatively insensitive to lower iron stores because iron is preferentially shunted from other tissues to the erythron (2). In man, serum ferritin accurately reflects total body iron stores (3-7). It is low in iron deficiency and high in iron overload conditions. However, it is normal in precirrhotic familial hemochromatosis (8).

Neonatal pigs are susceptible to iron deficiency anemia. They do not receive adequate iron during fetal development (9), grow rapidly after birth, and may receive inadequate dietary iron under current management schemes (10). Here we describe an enzyme immunoassay to measure serum ferritin in baby pigs.

Apparatus MATERIALS AND METHODS

A miniwasher, microshaker, microtiter plates, and transfer plates (Dy- natech Laboratories, Alexandria, Va. 22314) were used in this study. Calorimetric measurements were made in a Gilford 300-N spectropho- tometer equipped with a EIA aspirating microcuvette and a thermal printer (Gilford Instrument Lab., Inc., Oberlin, Ohio 44074). Eppendorf pipettes were used for pipetting sera and standards and an Eppendorf Repeater (Brinkmann Instruments, Inc., Westbury, N.Y. 11590) was used for the remaining pipetting during the assay procedure.

Reagents

Washing solution. Polyoxyethylene sorbitan monooleate (0.05%) was dissolved in demineralized water.

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294 SMITH. MOORE. AND BOYINCXON

C’hrotnogenic~ reagrnc. Hydrogen peroxide. 0. I ml, (2%) and 0. I25 ml of 2.2’~azino-di-(3-ethyl benzthiazoline sulfonic acid) (40 mM) were diluted to 2.5 ml with citrate buffer (100 mM. pH 4.0).

Sfock sfopping .so/rrtion. Hydrofluoric acid (3.47 ml) and 6 ml of a I M sodium hydroxide solution were diluted to 1 liter of demineralized water.

dropping s~~~~ti~~l. EDTA solution (0.1 M, 0.25 ml) was diluted to 7.5 ml with stock stopping solution.

Antiferritin IgG-Peroxidase conjugate. IgG was conjugated with horse- radish peroxidase (Type VI: Sigma Chemical Co., St Louis, MO.) by the method of Nakane and Kawaoi (11) except that the sodium borohydride and column steps were omitted (12).

Purification ufferrih. Ferritin from pig spleen and liver was isolated by the method of Kitchen (I 3) which includes water solubility, stability at 7O”C, solubility at pH 4.8. insolubility in half-saturated ammonium sulfate, and gel chromatography on a column of Sephadex G-200 (Pharmacia Laboratories, Inc.. Piscataway, NJ. 08854). We modified the method by filtering the solubilized ammonium sulfate pellet through a 0.22-pm millipore filter (Millipore Corp., Bedford, Mass. 01730) and desalting on a Sephadex G-25 column ( 14 x 2.5 cm) that was equilibrated with a phosphate buffer (20 mM, pH 7). The ferritin obtained after Sephadex G-200 chromatography was concentrated by centrifuging at 95,OOOg for 4 hr. Polyacrylamide gel electrophoresis on 5%~ gels without sodium dodecyl sulfate gave one band when stained for iron with acid ferro~yanide or for protein with Coomassie blue dye. The protein concentration of the purified ferritin was determined by the method Lowry et ~1. (14) with bovine serum albumin as a standard.

F’erritin standurds. Ferritin (5. 10, 20, 30, 40. and SO ngiml) was diluted in phosphate-buffered saline containing 0.5% bovine serum albumin and 0. I% polyoxyethylene sorbitan monooleate.

A~~~errjr~n ZgG. New Zealand white rabbits were injected subcutaneously with 1 mg of ferritin emulsified in Freund’s complete adjuvant. A second injection was given 2 weeks later and the antisera collected 2 to 3 weeks following the second injection. The IgG was isolated with a Protein A- Sepharose CL4B column (15’). The IgG was eluted with 100 mM citrate buffer, pH 3.0, containing 0.05% sodium azide. One-milliliter fractions were collected in tubes containing 0.25 ml of Tris-WC1 buffer (1 M, pH 9.0). Fractions containing significant protein concentrations were pooled.

Prepcrration of untihody-coated plates. Antiferritin IgG was diluted to 10 pg/ml with a sodium carbonate buffer (IO mM, pH 9.6). Antiferritin IgG-carbonate (0. 2 ml) was incubated in each well of a microtiter plate that was covered with sealing tape. After incubating for 2 hr at 37°C. the microtiter plates were stored at -80°C until needed.

SERUM FERRlTIN 295

ASSAY PROCEDURE

Antibody-coated plates were washed with 0.25 ml/well of washing solution. The miniwasher was used to remove and add solutions from 12 wells simultaneously. After the third wash undiluted sera (0.1 ml) or ferritin standards (0.1 ml) were added to each well with a transfer plate. Both standards and samples were run in triplicate. During incubation at 37°C the plates were mixed on a microshaker. After 2 hr the plates were washed again with 0.25 ml/well of washing solution. After the third wash antiferritin IgG conjugate (0.1 ml) was added to each well and incubated at 37°C with mixing for 2 hr. After three washes, the chromogenic reagent (0.15 ml/well) was added and the plate incubated at 37°C for 15 min. The reaction was stopped by adding 0. I5 ml of the stopping solution. The absorbance of each well was measured at 414 nm. The spectropho- tometer was blanked with a well that contained an antibody coating but did not receive serum or standard and antiferritin IgG conjugate. A conjugate blank was run which contained human serum in lieu of either serum or standards.

RESULTS AND DISCUSSION

Specificity. Ferritin isolated from porcine liver and spleen did not crossreact with either antihuman ferritin antibody or antiequine ferritin antibody. Likewise, antiporcine ferritin antibody does not react with human ferritin or equine ferritin. Ferritin antibodies are apparently species specific.

Conjugate blank. Because of the low serum ferritin in pigs the selection of an appropriate blank is especially important. We tested blanks consisting of bovine serum albumin, polyoxyethylene sorbitan monooleate, equine sera, and human sera either singly or at various concentrations. Human sera was chosen as the most consistent and appropriate blank.

Standards. Ferritin standards were linear to 50 rig/ml (Fig. 1). Between 50 and 400 rig/ml the absorbance increased then began to level after 400 @ml. The enzyme reaction was linear for 20 min. Fifteen minutes was chosen as the reaction time because it allowed sufficient color development but avoided the nonlinearity that occurred after twenty minutes.

Sample dilution. Samples with ferritin concentrations greater than 50 rig/ml should be diluted with human sera and reassayed (Fig. 2).

Recovery. When known quantities of ferritin were added to porcine sera the recovery (Table 1) varied from 92.8 to 94.3% with a mean of 93.6%.

Precision. The coefficient of variation calculated from 25 samples done twice was 4.2%.

Norm& values. The ferritin levels of 20 serum samples from newborn pigs was 20.8 &ml with a standard deviation equal to 14.7. Those values

296 SMITH. MOORE. AND BOYINGTON

Ferritin (rig/ml)

FIG. I. Standard curve for porcine ferritin. The enzyme reaction was allowed to continue for IO (A) or 20 (0) min

06

04

02

Pig sera (%I

FIG. 2. Sample dilution and linearity. Swine serum was diluted with human sera.

TABLE I RECOVERY OF FERRITIN ADDED TO PORCINE SERUM

Added Estimated ferritin concentration (@ml) Wml)

0 - 5 10.6

IO 15.6 20 25.6 30 35.6 40 45.6

Observed concentration Recovery

@g/ml) (5%)

5.6 -

10.0 94.3 14.5 92.9 24.1 94.1 33.2 93.2 42.3 93.4

SERUM FERRITlN 297

are lower than the normal adult human values of 54.7 -+ 46.5 nglml (16). That may reflect the low transfer of iron across the placenta in pigs.

SUMMARY

An enzyme-linked immunosorptive assay for serum ferritin in pigs was developed. The assay uses rabbit antiporcine ferritin in a sandwich tech- nique. The normal value for newborn pigs was 20.8 ? 14.7 rig/ml.

ACKNOWLEDGMENTS

This work was supported in part by Section 1433C of Public Law PL-95-113. Public Health Service Grant HL-12972, and the Kansas Agricultural Experiment Station, and was published as contribution 82-381-j. Kansas Agricultural Experiment Station, Manhattan. Kansas 66506.

REFERENCES I. Kaneko, J. J., in “Clinical Biochemistry of Domestic Animals.” 3rd ed.. p. 649.

Academic Press, New York. 1980. 2. Beutler. E.. Annual Rev. Med. 12, I95 (1961). 3. Addison, G. M.. Beamish. M. R.. Hales. C. N.. Hodgkins. M., Jacobs. A.. and

Liewellin, P., J. C/in. Purh. 25, 326 (1972). 4. Jacobs, A., Miller, F., Worwood. M.. Beamish. M. R.. and Wardrop. C. A.. &it.

Med. .I. 4, 206 (1972). 5. Walters, G. 0.. Miller, F. W.. and Worwood. M.. J. C/in. Prrrh. 26, 770 (1973). 6. Siimes, M., Addiego. J. E.. Jr., and Dallman. P. R.. Blood 43, 581 (1974). 7. Lipschitz. D. A., Cook. J. D.. and Finch. C. A.. N. Eng. J. Med. 290, 1213 (1974). 8. Wands, J. R., Rowe, J. A.. Mezey, S. E.. Waterbury. L. A.. Wright, J. R.. Halliday.

J. W., Isselbacher, K. J., and Powell. L. W., N. Eng. J. Med. 294, 302 (1976). 9. Douglas, T. A., Renton. J. P.. Watts. C.. and Ducker. H. A., Camp. Bkxhem. Physiol.

43A, 665 (1972). IO. Parsons, M. J., Case, K. D.. Ku, P. K., and Miller. E. R. Michigun Sti,irw Res. Rep.

386, I4 (1979). II. Nakane, P.. and Kawaoi, J., J. Histochern. Cytochem. 22, 1084 (1974). 12. Saunders. G. C., Clinard. E. H.. Barlett. M. L.. and Sanders. W. M., J. Itcfect. Dis.

136, 5258 (1977). 13. Kitchen, H.. personal communication. 14. Lowry, 0. H., Rosenbrough. N. J.. Farr, A. L.. and Randall, R. J., .I. Biol. Chem.

193, 265 (1951). 15. Ey. P. L., Prowse, J. J.. and Jenkin. C. R.. Ifnmrrnoclrenr. 15, 429 (1978). 16. Lee, M.. and Burgett. M. W.. C/in. Chirn. Acftl 112, 241 (1981).