BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY 45, 188-196 (1991)

Sandwich Enzyme Immunoassay for Rat Transferrin with Two Monoclonal Antibodies and Its Application

TAKESHI MATSUMOTO,’ HIDEAKI SHIMA, TETSUYA KISHI, AND TADASHI SATO

Department of Biochemistry, Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Company Ltd., 16-89, Kashima-3-chome, Yodogawa-ku, Osaka, 532, Japan

Received August 24, 1990

The development of a sandwich enzyme immunoassay for rat transferrin with two mono- clonal antibodies is described. Microtiter plates coated with one monoclonal antibody (15C2H3) were used, and captured transferrin was estimated with a horseradish peroxidase- conjugated Fab’ fragment of another monoclonal antibody (22A06D2). In this assay, the measurable range is 5-150 rig/ml and the coefficients of variation within and between the assay series are 1.2-5.0 and 3.3-6.0%, respectively. Recovery was 101 + 9.7% when purified rat transferrin was added to rat plasma. No cross-reactivity with bovine, human, or mouse transferrin was shown. This assay for rat transferrin is a highly specific, sensitive, and expeditious method which may allow routine analysis of rat transferrin in blood or culture supematants of rat hepatocytes. 0 1991 Academic Press, Inc.

Transferrin is a carrier glycoprotein for iron which plays a central role in iron metabolism of vertebrate animals, transporting iron between its absorption, stor- age, and utilization sites (1). Interest in the study on transferrin has recently increased because of its profound effect on the stimulation of proliferation and differentiation of many cell populations (2-4) and because of its possible involve- ment as a neurotrophic factor (5).

For clinical use, the transferrin level in blood has also been widely used in the evaluation of both iron and protein nutrition (6-S). The rapid turnover plasma proteins, which include transferrin, may well reflect the host nutritional condition because they have shorter half-lives than other plasma proteins, such as albumin, which are usually used for nutritional assessment.

Roza et al. (9) reported that human transferrin was a poor measure of nutritional status. Further, many investigators reported that several conditions, for example, iron deficiency, affected transferrin level in blood (10-12). Therefore, we felt it was important to investigate in more detail the use of transferrin as a nutritional parameter. Experiments using animals would be very useful because we could easily change several conditions which might affect the transferrin level in blood.

’ To whom correspondence should be addressed.

188

0885-4505/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

IMMUNOASSAY FOR RAT TRANSFERRIN 189

The human transferrin level is usually measured in the hospital laboratory by radial immunodiffusion and turbidimetric assay. These immunological methods are very useful in the measurement of plasma transferrin because of the specificity. However, most of these assays are insensitive for measuring the small amount of transferrin in in vitro studies. And, in animal experiments a convenient method for measuring transferrin was not available. We needed a more precise and sen- sitive method for measuring animal transferrin.

In this report, we describe a sandwich enzyme immunoassay using two kinds of monoclonal antibodies against rat transferrin. This assay is specific, precise, highly sensitive, and suitable for the use in large population studies.

METHODS

Materials

2,6,10,14-Tetramethylpentadecane (Pristane) was obtained from Aldrich Chem- ical Company, Inc., Wisconsin. Horseradish peroxidase (grade I) was obtained from Boehringer-Mannheim GmbH, Manheim, Germany. N-(c-Maleimidoca- proyloxy) succinimide (EMCS)* was obtained from Dojindo Laboratory, Kuma- moto, Japan. Tween 20 was obtained from Kao Company Ltd., Tokyo, Japan. Collagen (type I) was obtained from Nitta Gelatin Company Ltd. Dexamethasone, insulin, pepsin, and trypsin inhibitor were obtained from Sigma Chemical Com- pany, St. Louis, Missouri. 2,2’-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and collagenase were obtained from Wako Pure Chemical Industries Ltd., Osaka, Japan.

BALB/c mice were obtained from Japan SLC, Inc., Shizuoka, Japan, and Wistar rats were obtained from Oriental Bioservice Company, Osaka, Japan.

Purification of Rat Transferrin

Rat transferrin was purified from normal Wistar rat serum by 50% ammonium sulfate precipitation, anion-exchange chromatography (Bakerbond ABx, J. T. Baker Chemical Co., Phillipsburg, NJ) and gel filtration (Superose 6, Pharmacia LKB Biotechnology AB., Uppsala, Sweden). The purity of rat transferrin was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (13) _

Preparation of Monoclonal Anti-Rat Transferrin Antibodies

Female BALB/c mice were twice immunized by an ip injection of rat transferrin (100 pg/body) emulsified in an equal volume of Freund’s complete or incomplete adjuvant. At 2 weeks after the second immunization, rat transferrin (50 pg/body) without adjuvant was injected ip into these mice. The spleen cells were harvested 3 or 4 days after the last immunization and were fused with SP-2/O-Ag14 murine myeloma cells with polyethylene glycol 1000 by the modified method of Kohler and Milstein (14). The fused cells were grown in HAT selection medium and were cloned by the limiting dilution method, more than two times. Anti-rat

* Abbreviations used: EMCS, N-(e-Maleimidocaproyloxy) succinimide; ABTS, 2,2’-Azinobis (3- ethylbenzothiazoline-6-sulfonic acid); ELISA, enzyme-linked immunosorbent assay; EDTA, ethylenediaminetetraacetic acid; GOT, glutamate oxalacetate transaminase; PBS, Dulbecco’s phos- phate-buffered saline.

190 MATSUMOTO ET AL.

TABLE 1 Monoclonal Antibodies against Rat Transferrin

Heavy Light Hybridoma chain type chain type

llB7 CL h llCl0 CL --a llE8 P K llH8 cc K 12F6 cc K 15A3 Yl K 15C2H3 Yl K 21AllD2 Yl K 21BO9B8 YI K 21BllE3 Yl K 22A06D2 Y1 K 22BlOB7 Yl K 22BllA3 P K 22DlOGlO YZ. K 24B3 P K

Note. The class and subclass of the heavy chain or light chain of these monoclonal antibodies were determined by the method using antibody capture on antigen-coated plates (15). Anti-mouse im- munoglobulins (types (Y, w, y,, yza, yZbr y,, K, y) were purchased from Zymed Lab. Inc.

y Not determined.

transferrin antibodies were assayed by an ELISA method (15). We obtained 15 hybridomas which produced monoclonal antibodies against rat transferrin (Table 1).

For the large preparation of monoclonal antibodies, cloned hybridoma cells (2 x lo6 cells) were injected into the peritoneum of a mouse pretreated with 0.5 ml/body pristane. One to two weeks after the injection, ascitic fluid was collected and centrifuged (15Og, 10 min) to remove cells and debris. Monoclonal anti-rat transferrin antibody was purified from the supernatants by 50% ammonium sulfate precipitation and anion-exchange chromatography (Mono Q, Pharmacia).

Preparation of Monoclonal Anti-Rat Transferrin Fab’-Peroxidase Conjugate

The Fab’-peroxidase conjugate was prepared by the method of Ishikawa et al. (16). Purified anti-transferrin antibody (15C2H3) was dialyzed overnight against 0.1 M citrate buffer (pH 3.5) and the dialyzate was digested to F(ab’), fragments by pepsin. After digestion, the F(ab’), fragments were isolated by gel filtration on Toyopearl HW-55F (Toso Co. Ltd., Tokyo, Japan). The F(ab’), fragments were concentrated by ultrafiltration using a PM-10 membrane (Amicon Div. W. R. Grace & Co., Danvers, MA) and then incubated with 10 mM dithiothreitol at 37°C for 30 min. The F(ab’), fragments were almost completely reduced to Fab’ fragments. Fab’ fragments were isolated by gel filtration (Sephadex G-25, Phar- macia) with 0.1 M phosphate buffer (pH 6.0) containing 5 mM EDTA. The Fab’ fragments were mixed with maleimide-peroxidase conjugate which was prepared by incubating peroxidase with EMCS (1:40 molar ratio of POD to EMCS) at

IMMUNOASSAY FOR RAT TRANSFERRIN 191

37°C for 7 hr. Fab’-peroxidase conjugate was isolated by gel filtration on Superose 12 (Pharmacia).

Assay Procedures

Microtiter plate wells (MaxiSorp F96, Nunc, Roskilde, Denmark) were coated by overnight incubation at 4°C with 50 ~1 monoclonal antibody (22A06D2) in PBS. The remaining protein binding sites were blocked with bovine serum albumin (5 mg/ml in PBS). The wells were washed three times with PBS containing 0.05% Tween 20 (PBS-T). The standard transferrin and samples were diluted with PBS- T containing BSA (1 mg/ml) (PBS-TB) appropriately and added to the wells. The wells were incubated for 2 hr at room temperature (25°C) and washed three times with PBS-T. Then, Fab’-peroxidase conjugates in PBS-TB were added to the wells and the wells were incubated at 25°C for 1 hr. After the incubation, the wells were washed six times with PBS-T. Finally, the peroxidase activity in each well was assayed with 100 ~1 substrate solution (2 mM ABTS, 0.7 mM hydrogen peroxide in 50 mM citrate buffer, pH 4.0). The enzyme reaction was stopped after 15 to 20 min with 100 ~1 of 1.5% oxalic acid. The optical density was read at 405 nm with a Biomek 1000 (Beckman Instruments Inc., Fullerton, CA). These data were analyzed with Immunofit EIA/RIA software (Beckman).

Applications of the Sandwich Enzyme Immunoassay to in Vivo and in Vitro Experiments

Male Wistar rats weighing 250-260 g were used. During the experimental period, rats were deprived of food, but were given free access to drinking water. Rats were anesthetized with sodium pentobarbital and blood was withdrawn from the inferior vena cava using a heparinized syringe at 0, 1, 3, 5, and 7 days after the beginning of fasting. Transferrin was measured with the sandwich enzyme im- munoassay, and albumin was measured by the bromcresol green method with a Tectron XA18 (Tectron Japan, Tokyo, Japan).

Rat hepatocytes were isolated from Wistar rats weighing 150-200 g by in situ perfusion of the liver with collagenase, essentially as described by Nakamura et al. (17). The isolated cells were suspended at 5 x 10’ cells/ml in William’s E medium containing 10% fetal calf serum, lo-” M dexamethasone and lo-’ M insulin, and 1 ml of the cell suspension was inoculated into a well of a plastic culture plate (24-well plate, Becton Dickinson, Rutherford, NJ), which had been coated with 0.03% collagen type I in 0.02 N acetic acid. These cells were pre- cultured at 37°C under 5% CO, in air for 19 hr. Then, the medium was changed and culture supernatants were collected at 0, 2, 4, 6, 8, and 9.5 hr. Transferrin was measured with the sandwich enzyme immunoassay and albumin was measured with an ELISA using anti-rat albumin antibody and perioxidase-conjugated anti- rat albumin antibody (Cooper Biomedical, Inc., Philadelphia, PA). GOT activity was measured by the Swedish standard clinical chemistry method with a Tectron XA18.

192 MATSUMOTO ET AL.

0.05 4 10 40 100 200

Transferrin (rig/ml)

FIG. 1. Typical standard curve for rat transferrin assayed by the sandwich enzyme immunoassay. Solid line indicates standard curve (four-parameter logistic-log curve fit, correlation coefficient >O.W).

RESULTS

Standardization of the Sandwich Enzyme Immunoassay

One (15C2H3) of the monoclonal antibodies obtained was labeled with horse- radish peroxidase and another one (22A06D2) was selected as coating antibody. To determine the optimal concentration of coating antibody, concentrations rang- ing from 3.5 to 13 pg/ml were tested. Binding of transferrin to the antibody- coated plate increased with the concentration of the antibody and no further increase in the binding was observed above 5 pg/ml. Therefore, a 5 pg/ml was chosen as the working concentration. Similarly, the working dilution of the mono- clonal Fab’-peroxidase conjugate, 1:200, was determined. Under these optimal conditions, a standard curve was routinely fitted with a four-parameter logistic curve to accommodate the assay with an asymmetric sigmoidal response (Fig. 1). The correlation between optical densities and concentrations of standard trans- fer-r-in from 5 to 150 rig/ml was highly significant (correlation coefficient > 0.999).

Specificity of the Sandwich Enzyme Zmmunoassay

We examined the specificity of this assay to evaluate its application to mea- surement of rat transferrin in a culture supematant of rat hepatocytes. The spec- ificity was determined with the competition between purified rat transferrin and the plasma of other animals (Table 2). Bovine, human, and mouse transferrin in the blood did not bind to the monoclonal antibodies (data not shown) and did

IMMUNOASSAY FOR RAT TRANSFERRIN 193

TABLE 2 Interference of the Sandwich Enzyme Immunoassay with Bovine, Human, and Mouse Serum

Species of plasma Measured transferrin added (wh1)

Inhibition (%)

None 48.9 k 1.8 Bovine 47.5 2 0.7 2.9 k 1.4 Human 47.8 ” 1.1 2.2 t 2.3 Mouse 48.3 k 1.4 1.2 k 2.0

Note. Rat transferrin added to bovine, human, or mouse plasma (final concentration, 1%) was measured by the immunoassay. Each value represents the mean t SD (n = 3).

not interfere with this assay. Therefore, we were able to measure specifically rat transferrin even if a sample contained large amounts of other animal transferrin(s).

Reproducibility and Recovery of the Sandwich Enzyme Immunoassay

Precision of this assay was defined over the range lo-80 rig/ml rat transferrin. Coefficients of variation within and between assays were 1.2-5.0 and 3.3-6.0%, respectively. For the recovery experiment, purified rat transferrin was added to rat plasma, and assay was performed. Recovery of rat transferrin was 101 k 9.7% (mean t SD, n = 6). These results indicated that this assay was very precise and reproducible.

Applications of the Sandwich Enzyme Zmmunoassay

With this immunoassay, we measured the plasma transferrin level of fasted rats (Fig. 2). The level decreased rapidly during the starvation period and stabilized after the third day, whereas the plasma albumin level did not change during this period.

This immunoassay was applied to measure the small amount of rat transferrin in culture supernatants of rat hepatocytes (Fig. 3). There was little damage to hepatocytes during the experimental period since GOT activities in the super- natants were only slightly increased. Transferrin and albumin concentrations in culture supernatants increased during the experimental period and the rate of transferrin production (109 ng/hr/well) was two-fifths that of albumin production (250 ng/hr/well), even though the usual plasma concentration of transferrin is one-tenth that of albumin in rats.

DISCUSSION

There have been many clinical studies of human transferrin. Results of these studies revealed that the transferrin level in blood is changed by protein-calorie malnutrition and several diseases (1,18,19). Radial immunodiffusion and immu- noturbidimetry are usually used for measuring transferrin, but these assays require a large amount of specific antiserum and sample, and the analyzer for immuno- turbidimetry is very expensive. Moreover, these methods are insufficiently sensitive to measure the small amount of transferrin produced by primary hepatocytes. On the other hand, enzyme immunoassay can be used for these purposes because of

194 MATSUMOTO ET AL.

h 300 3.0

‘0 \ r\

E” -0 \

w W V

c 200 2.0 -E c .-

z l. E Ic a ln P C m z

; 100 1.0

400 4.0

0 0

0 2 4 6 8

Days in fast

FIG. 2. Plasma transferrin and albumin levels of fasted rats. Opened and closed circles indicate albumin and transferrin concentrations, respectively. Each value represents the mean + SD of triplicates.

its high sensitivity and ease of handling. Recently, an enzyme immunoassay for human transferrin was reported (20). However, convenient methods for measuring animal transferrin specifically have not been available.

We describe here the sandwich enzyme immunoassay for rat transferrin, which is a simple, precise, and convenient method. The assay employs monoclonal antibodies as immunological reagents which can be supplied in unlimited quan- tities. And, microtiter plates are convenient to handle a large number of samples, especially with the use of instruments for measuring optical densities in situ.

Contamination with other iron-binding proteins does not interfere with this assay because it measures only rat transferrin protein with the use of the specific monoclonal antibodies.

We measured the plasma transferrin level of fasted rats (Fig. 2). The transferrin concentration of normal rat plasma was 359 f 41 mg/dl (n = 6). Fasting affected the transferrin level dramatically. After 3 days of fasting, the plasma level de- creased to 45.6% in comparison with that of nonfasted control rats. Gardiner et al. (11) reported a similar result.

Because of high sensitivity to rat transfer-tin and no cross-reactivity to bovine transferrin, we could measure the small amounts of transferrin which rat hepa- tocytes produced without using a radioisotope (Fig. 3). Rat hepatocytes produced

IMMUNOASSAY FOR RAT TRANSFERRIN 195

2.5

i \ z 2.0

Y

c .-

=, 1.5 Lc cn c m ; 1.0

.- L

: 0.5

2

I 0 0

0 2 4 6 8 10

Time in culture (hr)

FIG. 3. Transferrin and albumin production of rat hepatocytes in vitro. Opened and closed circles indicate albumin and transferrin concentrations, respectively. Closed triangles indicate GOT activities. Experimental conditions were described under Methods.

transferrin during the experimental period, and the rate of transferrin production was two-fifths that of albumin production under our conditions. Jeejeebhoy et al. (21) reported that the rate of transferrin production was one-third that of albumin with a system using hepatocyte suspensions in vitro. The plasma concentration of rat transferrin was one-eighth that of albumin under normal conditions (Fig. 2). This sandwich enzyme immunoassay will facilitate the in vivo and in vitro study of the synthesis and regulation of rat transferrin, in order to evaluate transferrin production.

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