A Monoclonal Antibody to a-Human AtrialNatriuretic Polypeptide

MASASHI MUKOYAMA, KAZUWA NAKAO, HIDEO SUGAWA,

NARITO MORII, AKIRA SUGAWARA, TAKAYUKI YAMADA,

HIROSHI ITOH, SHOZO SHIONO, YOSHIHIKO SAITO, HIROSHI ARAI,

TOHRU MORI, HISAO YAMADA, YUTAKA SANO, AND HIROO IMURA

SUMMARY A monoclonal antibody to o-human atrial natriuretic polypeptide (a-hANP), KY-ANP-I, hasbeen produced by fusion of a nonproducmg mouse myeloma cell tine, X63-Ag8.653, with spleen ceDs fromBALB/c mice immunized with synthetic a-hANP conjugated to bovine thyrogtobuhn using the carbodiimidecoupling procedure. Hybrklomas were screened for antibody production by rarJiolmmunoassay using culturemedia and l2SI-a-hANP. They were cloned by the llmtHng dilution technique, expanded hi culture, and injectedmtraperHoneaDy into BALB/c mice. The obtained antibody belonged to the immunogiobunn G, subclass.Analysis by a Scatchard plot revealed a high affinity for a-hANP, with an association constant of 3.1 x 10"M~'. With this monoclonal antibody, a specific radioimmunoassay for a-hANP has been established. Theantibody in mouse ascites was available for radioimmunoassay at a final dilution of 1:10*. Values of IC|0 andIC<, in this radioimmunoassay were 3 and 30 fmol/tube, respectively. The radioimmunoassay showed across-reactivity of 0.9% with a-rat ANP. a-hANP-<8-22) and a-ANP-(l-6) exhibited less cross-reactivity thana-rat ANP on a molar basis. There was no cross-reaction with a-ANP-(17-28). Thus, the recognized epitopemust be located in the N-terminal half of the ring structure of a-hANP including Met12 residue. Thisradioimmunoassay could detect y-hANP and 0-hANP as well as a-hANP. The monoclonal antibody was alsouseful for unmunohistochemical studies. ANP-positive cells were finely stained hi the human atrium using theavidin-btotin-peroxidase complex technique. These results indicate that this monoclonal antibody to a-hANPwill become a powerful tool for investigating the physiological and pathophvsiok>gkal significance of ANP.(Hypertension 12: 117-121,1988)

KEY WORDS • atrial natriuretic factorimmunohistochemistry

monoclonal antibody • radioimmunoassay

FOLLOWING the discovery of potent diuretic,natriuretic, and vasorelaxant properties inextracts from the rat atrium by de Bold et

al.,1 a family of cardiac peptides named atrial natri-

From the Second Division, Department of Medicine (M. Mukoyama,K. Nakao, H. Sugawa, N. Morii, A. Sugawara, T. Yamada, H. Itoh, S.Shiono, Y. Saito, H. Arai, T. Mori, H. Imura), and the Department ofClinical Molecular Biology (T. Mori), Kyoto University School ofMedicine, the Radioisotope Research Center (K. Nakao), Kyoto Uni-versity, and the Department of Anatomy (H. Yamada, Y. Sano), KyotoPrefecture! University of Medicine, Kyoto, Japan.

Supported in part by research grants from the Japanese Ministry ofEducation, Science and Culture; the Japanese Ministry of Health andWelfare "Disorders of Adrenal Hormone" Research Committee,Japan, 1987; Japan Tobacco Inc.; and Yamanouchi Foundation forResearch on Metabolic Disorders; and by grants for cardiovasculardisease research (6OA-3 and 62A'-1) from the Japanese Ministry ofHealth and Welfare.

Address for reprints: Kazuwa Nakao, M.D., Ph.D., Second Divi-sion, Department of Medicine, Kyoto University School of Medi-cine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan.

Received May II, 1987; accepted April 8, 1988.

uretic polypeptides (ANPs), also known as atrialnatriuretic factor (ANF), was isolated from humanand rat atrial tissues and implicated in the control ofwater and electrolyte balance and blood pressure.2-5

Using a polyclonal rabbit antiserum against a-ANP-(17-28) (ANF-[115-126]), we have established aspecific radioimmunoassay (RIA) for ANP that rec-ognizes a-human ANP (a-hANP or human ANF-[99-126]) and a-rat ANP (a-rANP or rat ANF-[99-126]) equally.6 With the aid of this RIA, we havedemonstrated that ANP is released from the heartand circulates in the body as a hormone7-8 and thatANP exists not only in the atrium but in the extra-atrial tissues, including the brain,9 spinal cord,10

kidney," and lung.12 This antiserum against a-ANPwas also used for immunohistochemical studies.13- M

Furthermore, intracerebroventricular injection of thisantiserum blocked the endogenous ANP action inthe brain and potentiated water intake in rats.13

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Polyclonal antibodies, however, have several draw-backs in their use, such as the limited supply and thecontamination with irrelevant antibodies not directedagainst ANP.

The monoclonal antibody technique introducedby Kohler and Milstein16 has now come into wide-spread use for the study of various substances,including biologically active peptides.l7-18 The pres-ent article describes the preparation and character-ization of a monoclonal antibody to a-hANP and itsuseful applications.

Materials and MethodsPreparation of Monoclonal Antibody

Synthetic a-hANP was conjugated to bovine thy-roglobulin (Sigma Chemical, St. Louis, MO, USA)using the carbodiimide (Nakarai Chemicals, Kyoto,Japan) coupling procedure.6 Adult female BALB/cmice (Shizuoka Animal Center, Hamamatsu, Japan)were initially immunized by combined subcutane-ous and intraperitoneal injections with the conju-gate containing 30 /Ag of a-hANP emulsified incomplete Freund's adjuvant (Difco Laboratories,Detroit, MI, USA). They were boosted in the samemanner 3 weeks later and then bled to test for thepresence of antibody by RIA with 125I-a-hANP. Fiveout of 10 mice gave a positive antibody response, andthe two mice giving the greatest response were furtherboosted intravenously with the conjugate containing50 /xg of the antigen 5 weeks after the second injec-tion. Four days later the spleens were harvested fromthe mice for cell fusion.

Fusion of spleen cells with a nonproducing mousemyeloma cell line, X63-Ag8.653,19 was performedin the ratio of 6: 1 with 50% polyethylene glycol4000 (Merck, Darmstadt, West Germany) accordingto the method described by Galfre et al.20 with slightmodifications. After the fusion, cells were distrib-uted into microtiter plates and cultured in hypo-xanthine-aminopterin-thymidine medium contain-ing 15% fetal calf serum for hybridoma selection.As cells proliferated, culture media were periodi-cally screened for their ability to bind l25I-a-hANP.Cells from the well that gave the highest antibodytiter were cloned twice by the limiting dilutiontechnique in the presence of a mouse thymocytefeeder layer. Positive clones were expanded inculture and injected intraperitoneally into BALB/cmice to produce ascitic fluid with highly concen-trated antibody.

Characterization of Monoclonal AntibodyIsotyping of the monoclonal antibody was per-

formed by the Ouchterlony technique (Mouse Mono-clonal Typing Kit, Miles Laboratories, Elkhart, IN,USA). Binding affinity was analyzed by construct-ing a Scatchard plot of a-hANP with the RIAdescribed in the next section. Epitope analysis wasperformed by checking cross-reactivities with sev-eral ANP-related peptides in the RIA for a-hANP.

RadioimmunoassayRIA with the monoclonal antibody was per-

formed following the polyclonal antiserum methodas previously described in detail.6 In brief, a-hANPwas radioiodinated by the chloramine-T method.The specific activity of l25I-a-hANP ranged from 400to 800 pCi/fig. The RIA incubation mixture (500 fi\Jtube) consisted of 100 /xl of standard a-hANP (0.8-1600 fmol/tube) or sample, 100 .̂1 of a dilution ofantibody (antisera, culture media, or ascites), 100 n\of l25I-a hANP (approximately 10,000 cpm), and 200^1 of the standard buffer (0.1 M phosphate buffer,pH 7.0, containing 0.5% gelatin [Merck], 1 mMNa2EDTA, 0.2 mM cystine, 0.1% Triton X-100, and0.01% merthiolate). The mixture was incubated for24 to 48 hours at 4 °C. Bound and free ligands wereseparated by adding 1.0 ml of a suspension ofdextran-coated charcoal consisting of 250 mg ofactivated charcoal (Norit SX Plus, Norit-VereengingN.V., Netherlands) and 25 mg of Dextran T70(Pharmacia Fine Chemicals, Uppsala, Sweden) in100 ml of 0.05 M phosphate buffer, pH 7.4, contain-ing 0.01% merthiolate, and the supernatant wascounted by a gamma counter (Aloka Autowellgamma system ARC-600, Tokyo, Japan). Mouseascitic fluid was available at a final dilution of 1:106

to yield a total tracer binding of about 30%.

Measurement of a-hANP-Like Immunoreactiviry inHuman Atria

The RIA with the monoclonal antibody was usedto measure levels of a-hANP-like immunoreactivity(LI) in extracts from human atria. Five human rightauricular tissues were obtained at cardiac surgeryand stored at —70 °C until extraction. Tissues wereboiled for 5 minutes in 10 volumes (2-5 ml) of 1 Macetic acid containing 20 mM HC1 and then homog-enized with a Polytron homogenizer (KinematicaGmbH, Lucerne, Switzerland).2-6 The homogenatewas centrifuged at 18,000 g for 30 minutes at 4 °C,and the supernatant was stored at - 20 °C until RIA.

ImmunohistochemistryA tissue sample from the human right auricle was

obtained at coronary bypass surgery. The tissue blockwas immersed in the buffered formaldehyde fixatives(4% paraformaldehyde in 0.1 M phosphate buffer, pH7.0) for 2 days and then rinsed with 20% sucrosesolution for another 2 days. The free-floating sections(16 pm thick) were obtained by cutting with a cryostat(Histostat, AO Scientific Instruments, Buffalo, NY,USA) after the tissue block was frozen with CO2 gas.The sections were sufficiently washed with 0.1 Mphosphate buffer, pH 7.4, containing 0.5% TritonX-100 and were incubated in 1% H2O2 solution for 1hour to suppress endogenous peroxidase activity. Thesections were successively incubated in 1) the mono-clonal antibody (ascites diluted in 1: IQ* with 0.1 Mphosphate buffer, pH 7.4, containing 0.5% TritonX-100) at 4 °C for 36 hours, 2) biotinyl anti-mouse

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MONOCLONAL ANTIBODY TO ANP/Mukoyama et al. 119

immunoglobulin G solution at 20 °C for 120 minutes,and 3) avidin-biotin-peroxidase complex solution at20 °C for 90 minutes. These reagents for the avidin-biotin-peroxidase complex method were purchasedfrom Vector Laboratories, Burlingame, CA, USA(PK 4002 kit); 3,3'-diaminobenzidine was employedfor coloration of peroxidase. The principle of immu-nohistological technique has been given elsewhere.21

PeptidesSynthetic a-hANP was kindly donated by Profes-

sor Hisayuki Matsuo (Miyazaki Medical College,Miyazaki, Japan); -^hANP (human ANF-[1-126])purified from human atria was also donated byProfessor Hisayuki Matsuo. Synthetic a-rANP andthree fragment peptides, a-ANP-(l-6) (ANF-[99-104]), a-hANP-(8-22) (human ANF-[106-120]), anda-ANP-( 17-28), were generously supplied by Pro-fessor Yoshiaki Kiso (Kyoto Pharmaceutical Uni-versity, Kyoto, Japan). Synthetic /3-hANP (an anti-parallel dimer of a-hANP) was provided by Dr. KenInouye (Shionogi Research Laboratories, Osaka,Japan). The homogeneity of peptides was confirmedby reverse-phase high performance liquid chroma-tography and amino acid analysis.

ResultsAntibody Preparation and Characterization

After the fusion, hybridomas grew in nearly 30%(212 of 768) of the wells. ANP antibody-producingcells were recognized in about 4% (8 of 212) of thesewells. After further culture and cloning, one clonethat produced antibody with the strongest responsewas selected for expansion and characterization. Theobtained monoclonal antibody belonged to the immu-noglobulin Gi subclass, as determined by the Ouch-terlony technique. Analysis by a Scatchard plotrevealed a high affinity for a-hANP, with an associa-tion constant (KB) of 3.1 x 1010 M~' (Figure 1).

A standard curve of a-hANP and cross-reactivitiesof its related peptides in the RIA with the monoclo-nal antibody are shown in Figure 2. The RIAshowed a cross-reactivity of 0.9% with a-rANP ona molar basis. a-hANP-(8-22) and a-ANP-(l-6)exhibited less molar cross-reactivity than did a-rANP (0.2 and 0.04%, respectively). There was nocross-reaction (<0.01%) with the C-terminal frag-ment, a-ANP-(17-28).

Radloimmunoassay and Measurement ofa-hANP-Like Immunoreactivity inHuman Atria

In the standard curve of a-hANP shown in Figure2, the IC50 value was 30 fmol/tube and the detectionlimit, in which 10% of total tracer binding would beinhibited (IC10), was 3 fmol/tube. The intra-assayand interassay coefficients of variation (for 6 fmol/tube of a-hANP) were 5.2% (n = 6) and 9.2% (n =6), respectively. The mean recoveries for 15 and 30fmol/tube of unlabeled a-hANP added to human

B/F

0.4

0.3

0.2

0.1

0.5 1.0

[BKx1<r1lM)1.5

FIGURE 1. Scatchard plot of the binding of a-hANP tothe monoclonal antibody KY-ANP-I. Diluted culture super-natant containing KY-ANP-I was incubated with t25I-a-hANP (approximately 10 pM) and increasing concentra-tions of standard a-hANP (0-160 pM) for 48 hours at4 °C, and the specific binding was determined as describedin Materials and Methods. [B] = concentration of specif-ically bound a-hANP; B/F = bound/free ratio of theligand.

atrial extracts were 104% (n = 4) and 107% (n = 4),respectively.

Serial dilutions of extracts from human atria gavecompetition curves parallel to the standard curvefor a-hANP. The level of a-hANP-LI in humanatria obtained with this RIA was 169 ± 71.5 /ng/g(mean ± SE; n = 5), which was quite comparableto that assayed with the polyclonal antiserum.6 Ahighly significant correlation was observed betweenthe results of the two RIAs (r = 0.9%, p < 0.001).The RIA detected not only a-hANP but ^hANPprepared from human atria on an equimolar basis.

B/Bo

102 103

Peptide (fmol/Tube)

10s

FIGURE 2. Typical standard curve of a-hANP and cross-reactivity profiles of its related peptides in the RIA withthe monoclonal antibody KY-ANP-I. mI-a-hANP andvarious concentrations of standard a-hANP or relatedpeptides were incubated with KY-ANP-I for 24 hours at4 °C, as described in Materials and Methods.

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120 HYPERTENSION VOL 12, No 2, AUGUST 1988

'^^fc

«*t .

FIGURE 3. Immunohistochemical staining ofANP-containing cardiocytes in the human rightauricle obtained at cardiovascular surgery withthe aid of the monoclonal antibody KY-ANP-1.(X150.)

I - *

In addition, the RIA recognized synthetic /3-hANP,with a cross-reactivity of 80% on a molar basis.

ImmunohistochemistryANP was shown on light microscopy to be a finely

granular immunoreactive substance (Figure 3). Theseimmunoreactivities were observed in the periphery ofthe sarcoplasm in the perinuclear region of atrialmyocytes. However, no immunopositive substanceswere detected in the epicardium, endocardium, or anyconnective tissues in the human atrium. The speci-ficity of antibody binding to atrial myocytes wasverified by the inhibition of staining in the presence ofexcess synthetic a-hANP. Furthermore, human ven-tricular tissues obtained at autopsy from patientswithout cardiac complications were hardly stainedwith this monoclonal antibody.

DiscussionIn the present study we have established and

characterized a monoclonal antibody to a-hANP(KY-ANP-I). The antibody obtained in this studyhas a high affinity for a-hANP, with a Ke of 3.1 x1010 M"1. The K, value is comparable to those ofmonoclonal antibodies to human renin previouslyreported.22 The epitope of the monoclonal antibodyis presumed to be located in the N-terminal half ofthe ring structure of a-hANP including Met12 res-idue. Therefore, this monoclonal antibody reactslittle with a-ANP of rabbits, rats, and mice, whichcontains isoleucine at the twelfth amino acid resi-due instead of methionine. Recently, amino acidsequences of a-ANP from dogs, pigs, and cattlehave been demonstrated to be identical to a-hANP.23-24 Thus, the monoclonal antibody highlyspecific for a-hANP could be useful for experimentsin these animals and, especially, for application tostudies in humans.

Monoclonal antibodies to biologically active pep-tides are now commonly prepared and applied invarious fields to investigate the finely organizedsystem of the peptide concerned. Since they arehomogeneous in chemical properties, including

affinity and epitope specificity, and available on anunlimited scale, they are particularly useful forantigen purification, neutralization experiments,immunohistochemistry, and highly sensitive immu-noassay techniques, including immunoradiometricassay and enzyme immunoassay. In the presentstudy, the monoclonal antibody to a-hANP wasapplied to RIA and immunohistochemistry. In addi-tion, applications of this specific and high affinityantibody to the neutralization experiment and thesensitive enzyme immunoassay for ANP are now inprogress in our laboratory. Recently, John et al.25

reported a monoclonal antibody to atriopeptin, andthe administration of this antibody blocked theelevation of cyclic guanosine 3',5'-monophosphatelevels induced by volume expansion in rats.26

In conclusion, these results indicate that themonoclonal antibody to a-hANP (KY-ANP-I) willbecome a powerful tool for investigating the phys-iological and pathophysiological significance of ANP.

AcknowledgmentsWe thank Mrs. Hiroko Tabata, Miss Atsuko Furu, and Miss

Kazuko Horii for their secretarial assistance.

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and H AraiM Mukoyama, K Nakao, H Sugawa, N Morii, A Sugawara, T Yamada, H Itoh, S Shiono, Y Saito

A monoclonal antibody to alpha-human atrial natriuretic polypeptide.

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1988 American Heart Association, Inc. All rights reserved.

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