Monoclonal antibodies against human hemopexin and their application

Journal oflmmunological Methods, 161 (1993) 265-268 © 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

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JIM06718

Short communication

Monoclonal antibodies against human hemopexin and their application Z. Hrkal, H. Cajthamlovfi, I. Kalousek and J. Barto~ovfi Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague-2, Czech Republic (Received 9 December 1992, revised received 25 January 1993, accepted 26 February 1993)

Six monoclonal antibodies raised against human serum hemopexin have been characterized. The antibodies reacted with serum hemopexin as well as with the isolated protein in apo-form and with heme-protein complexes on immunoblots obtained following both PAGE and SDS-PAGE. In the case of PAGE blots 1 ng of hemopexin could be detected using a streptavidin-AP detection system. ELISA procedures employing two different pairs of monoclonal antibodies gave working ranges of 0.3-3 mg/1 and 10-100/zg/1 respectively. Key words: Hemopexin; Blotting; ELISA

Introduction

Hemopexin is a glycoprotein (MW 58 kDa) that occurs in human plasma at concentrations of 0.5-1.0 g/1 (Miiller-Eberhard, 1970; Hrkal and Miiller-Eberhard, 1971; Miiller-Eberhard and Morgan, 1975). The physiological function of this protein is to bind heme released from the hemoglobin occurring in plasma in hemolytic states (Hrkal et al., 1980) and transport it to hepatocytes for degradation (Smith and Morgan, 1979, 1985). A possible role for hemopexin in preventing the oxidative damage caused by free

Correspondence to: Z. Hrkal, Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20 Prague-2, Czech Republic. Tel.: (00422) 295793; Fax: (00422) 292911. Abbreviations: Hpx, hemopexin; Hx, apohemopexin; HxX, heme-hemopexin complex; MAHpx, mouse monoclonal antibody raised against hemopexin; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; OPD, 1,2-phenylenediamine; HRP, horseradish peroxidase; AP, alkaline phosphatase.

heme has also been suggested (Vincent et al., 1988). Under pathological conditions of severe hemolysis when haptoglobin is depleted the level of hemopexin decreases. Thus the hemopexin level may serve as an indicator of the degree of hemolysis. Although several sophisticated methods of hemopexin assay have been developed (see Miiller-Eberhard, 1988), electroimmunoassay (EIA) employing polyclonal anti-hemopexin antibodies is still the most frequently used procedure in routine determinations. In this report we describe the production of mouse monoclonal anti-hemopexin antibodies and their use in the determination of serum hemopexin levels by an ELISA procedure.

Materials and methods

Isolation of hemopexin Hemopexin was isolated from fresh human serum by the chromatographic procedure previously described (Hrkal et al., 1992).

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Production of monoclonal antibodies

Hemopexin determination by ELISA

White B A L B / c mice were immunized by three injections with 100 /zg of highly purified hemopexin in complete Freund's adjuvant at 2 week intervals. 3 days after the last immunization a booster injection was given intravenously. 3-5 days later the mice were splenectomized and the lymphocyte suspension (5 x 107 cells) was mixed with 2 x 107 cells of the SP-2/0-Ag 14 myeloma cell line. Fusion was performed according to K6hler and Milstein (1975). The cell suspension was cultivated in HAT medium and the individual cell colonies were screened for antibody binding to hemopexin using an ELISA technique. The recloned hybridomas were inoculated into the peritoneum of the B A L B / c mice and the ascitic fluid collected within 7-10 days. Isolation of immunoglobulins was performed on Protein G-Sepharose 4 Fast Flow as described by the manufacturer (Pharmacia LKB Biotechnology, Handbook). The anti-Hpx antibodies were dialysed against TBS pH 8.0, concentrated by ultrafiltration to give a 1 mg/ml solution and stored frozen at -50°C in 0.5 ml aliquots.

Hemopexin levels were determined using a sandwich ELISA procedure with monoclonal antibodies. The microtiter plates were coated with 100 /zl of 10 /zg/ml anti-hemopexin monoclonal antibody at 4°C overnight. After three washings with PBS-Tween 20 and after blocking unoccupied sites with 2% dried non-fat milk, the hemopexin standards were added to individual wells at concentrations of 0.12-10 /xg/ml and human sera at dilutions of 1/50 ~ 1/3200 (six replicates at each concentration) were added to other wells. Incubation proceeded at 37°C for 90 min. After three washes the plate was incubated with the second monoclonal antibody labeled with HRP for 90 min at 37°C. Color was developed and the absorbance recorded as above.

Determination of the binding characteristics of the monoclonal antibodies by ELISA Individual wells of a 96 well microtiter plates were coated with 100 /zl hemopexin solution (4 /~g/ml) by overnight incubation at room temperature. After washing and blocking the unoccupied binding sites with 0.02% gelatin solution the plate was incubated at 37°C for 90 min with anti-hemopexin monoclonal antibody (1 mg/ml) diluted with 50 mM phosphate pH 7.4 containing 1% BSA, was added over a range of concentrations (1/100-1/107). Six replicates were assayed at each concentration. After washing with PBSTween 20 solution, the peroxidase-labeled swine antibody against mouse immunoglobulins diluted 1/1000 was added and the plate incubated at 37°C for 90 min. The color reaction was then developed with OPD and H 2 0 2. The reaction was stopped with 4 N H 2 S O 4 and the absorbance at 490 nm was recorded with a Dynatech photometer. The readings were statistically evaluated and the binding curves analysed by nonlinear regression employing Hill's equation.

Results and discussion

Six monoclonal antibodies against human hemopexin were produced and their binding profiles with isolated hemopexin were studied using the ELISA technique. Their basic characteristics are given in Table I. The panel of monoclonal antibodies was further used for the identification of hemopexin in immunoblots obtained following both PAGE and SDS-PAGE of both isolated hemopexin, heme-hemopexin complexes and human serum. All the antibodies reacted almost equally well with apohemopexin as with the equimolar heme-hemopexin complex. The monoclonal antibodies appeared to react better with hemopexin separated by PAGE than by SDSPAGE. With the streptavidin-AP detection sysTABLE I CHARACTERISTICS OF THE ANTI-HUMAN HEMOPEXIN MONOCLONAL ANTIBODIES Antibody

lgG subclass

Ka a m o l - l

MAHpx(1) MAHpx(2) MAHpx(3) MAHpx(4) MAHpx(5) MAHpx(6)

IgG 1 IgG1 IgG 1 IgG1 IgG1 IgG1

2.5110 s 1.28 × 108 2.66 x 109 7.94 x 10 s 1.03 x 109 5.08 X 108

a Ka = apparent binding constant.

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tem a detection limit of 1 ng of hemopexin was achieved by PAGE blots employing MAHpx(3). Reactivity with reduced hemopexin on SDS gels was poor for all antibodies. Finally, an ELISA procedure for hemopexin determination employing two monoclonal antibodies in a sandwich format was worked out. The anti-hemopexin monoclonal antibodies MAHpx(2) and MAHPx(6) were labelled with HRP for detection while the remaining antibod-

ies served as the first (coating) antibody. Of the five antibodies tested the combinations of MAHpx(5) with HRP-labeled MAHpx(6) and unlabelled MAHpx(6) with HRP-labeled MAHpx(2) gave satisfactory results as far as sensitivity and accuracy were concerned. Fig. 1 shows the doseresponse curves obtained with isolated hemopexin (Fig. 1A) and with human serum (Fig. 1B). The midpoint of the dose-response curve corresponded to a hemopexin concentration of 40/xg/1 for the combination of MAHpx(5) with MAHpx(6)-HRP (curve a) and 1.7 mg/1 for the combination of MAHpx(6) with MAHpx(2)-HRP (curve b).

^ Acknowledgements E t-

The authors wish to thank Dr. Suttnar for kindly performing the HRP labeling and Ms. Nfimcovfi, Ms. Nouzovfi, Ms. Sedlmaierovfi, and Ms. Velebnfi for their skillful technical assistance.

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Fig. 1. Dose-response curves for hemopexin (Hpx) determination by an ELISA procedure employing (a) MaHpx(5) and MAHpx(6)-HRP and (b) MAHpx(6) and MAHpx(2)-HRp anti-hemopexin monoclonal antibodies. A shows the curves obtained with isolated hemopexin, B those obtained with pooled human serum.

References Bjerum, O.J. and Schafer-Nielsen, C. (1986) Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus. In: M.J. Dunn (Ed.), Electrophoresis 86. Verlag Chemie, Weinheim, p. 315. Hrkal, Z. and Muller-Eberhard, U. (1971) Partial characterization of the heme binding serum glycoproteins rabbit and human hemopexin. Biochemistry 10, 1746. Hrkal, Z., Kalousek, I. and Vodrfi2ka Z. (1980) Haeme binding to albumin and equilibria in the albumin-ferrihaemoglobin and albumin-haemopexin system. Int. J. Biochem. 12, 619. Hrkal, Z., (~abart, P. and Kalousek, I. (1992) Isolation of human haemopexin in apo-form by chromatography on S-Sepharose Fast Flow and Blue Sepharose CL-6B. Biomed. Chromatogr. 6, 212. K6hler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Mfiller-Eberhard, U. (1970) Hemopexin. New Engl. J. Med. 283, 1090. Miiller-Eberhard, U. (1988) Hemopexin. Methods Enzymol. 163, 536. Miiller-Eberhard, U. and Morgan, W.T. (1975) Porphyrinbinding proteins in serum. Ann. N.Y. Acad. Sci. 244, 624. Pharmacia LKB Technology. Monoclonal Antibody Purification (A Handbook), p. 41.

268 Smith, A. and Morgan W.T. (1979) Haeme transport to the liver by haemopexin. Biochem. J. 182, 47. Smith, A. and Morgan, W.T. (1985) Hemopexin-mediated heme transport to the liver. J. Biol. Chem. 260, 8325.

Vincent, S.H., Grady, R.W., Shakai, N., Snider, J.M. and Miiller-Eberhard, U. (1988) The influence of heme binding proteins in heme-catalyzed oxidations. Arch. Biochem. Biophys. 265, 539.