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Biochimica et Biv12hysicaAttn. 1073(1991)538-542 ~5199] ElsevierSciencePublishersBN. 0304-4165/91/$03,50 ADONIS 030441659100]30E

BBAGEN 2348,~

Anti-bilirubin monoclonal antibody IlI. Preparation and properties of monoclonal antibodies to unconjugated bilirubin-IXa Yuiraa Okamura 1, Masahiko Yamazaki t, Tokio Yamaguchi L, Yasuo Komoda 2, Akiko Sugimoto 3 and Hiroshi Nakajima J Department o/Biochemical Genetics. Medical Research Institute, Tokyo Medical and Dental University. Tokyo (da.~an), : Division of Molecular BDIo~,, Tokyo Medicalnnd Dental University, Tokj~o(Japan) and "¢Division of Medicinal Ckemtctry. Institute of Medical and Detlt~tl Engineering, Tokyo Medical and Dental Unieersity, Tokyo (Japan)

(Received 20 November1990)

Key words: Bilirubin-albumin;Monoclonalantibody Anti-bBirubin-lXa monoclonal antibodies exclusively specific for unconjugated hilirubia-IXo are prepared and characterized. Using modified M B S (metamaleimidobenzoyI-N-hydroxysuccinimide ester) method, bilirebin-lXa was covalently coupled to bovine senna albumin (liSA) retaining its intramoleeular hydrogen bonds as well as three-dimenslonal structure. Somatic cell fusion was performed between meriae spleen cells immunized with the bilimhin-IXtt-BSA and murine myeloma P3-X63-Ag8-UI ceils. Alter screening assay, 58 clones were identified which were producing antibodies not to albumin nor MBS, but to haptenie bilirubin-IXa. In Inhlbitlon analysis, the antibodies in the culture supernatant reacted only with bBirubin-lXa to a varying degree, but did not react with conjugated bilirubin-lX(t, including hilirub~a-IXa monoglucuronide, bilirubin-lXa digtucuronide, and ditaarenic bilirubin-lXa.

Introduction The conventional method for determination of bilirubin in biological specimens is based on the diazo-couvling reaction of Ehrlich. With the method, however, it is theoretically impossible to determine individual bilirubin conjugates, since bilirubin undergoes splitting into dipyrrolic compounds by diazo-coupling. HPLCanalysis of bilirubin, which is developed in our laboratory in 1979 [9], is the first procedure able to determine individual bilirubin conjugates in biological specimens. The data on the separation and determination of the bilicubin conjugates in serum or bile by H P L C have

Abbrevialions: BSA. bovine serum albumin; MBS, metamal¢iraidobenzoyI-N.hydroxysucelnimide ester; HPLC, kigh.pe:formaa¢¢liquid chromatography; HSA, human serum albumin; PSB, phosphatebuffmed saline; UCB, unconjugated bilirubin-lXw, p-TSA, p. tolueaesul[onlcacid; BDT, ditauronic hifirubln.lXeq BMG, bilirubinIXc¢ monoglucuronldc;BDG. bilirubin-IXa di~,lucuronJd¢. Correspondence: H. Nakajima, Department of BiochemicalGenetics, Medical Research Institute, Tokyo Medical and Denial University, 1-5-~,5Yushima, Burtkyo.ku,Tokyo 113, Japan.

been shown to provide valuable information for understanding the pathogenesis of disease; The H P L C analysis, however, is time consuming a n d appears unsuitable for clinical use. These observations have prompted as to prepare monoclonal antibodies specific for bilirubin a n d its conjugates. In the previous papers of this series [1,2], preparation of anti-bilirubin monoclonal antibodies was reported, a n d these antibodies have reacted with unconjugated hilirubin-lXn as well as bilirubin-lXa monoglucuronide a n d bilirubin-IXa diglucuronide to a similar degree. The present communication describes preparation and characterization of anti-bilirubin m o n o d o n a l antibody which is exclusively specific for unconjugated bifirubin-lXa. Materials and Methods Chemicals a n d reagents

Bilirubin-IXa was purchased from Sigma, ditauronic bilirubin-lX~ was from Funakoshi, Tokyo, a n d their purity was checked by thin-layer chromatography on silica gel plate. Bovine serum albumin (BSA) and human serum albumin (HSA) were also from Sigma, and

539 metamaleimidobenzoyl-N-hydroxysuecinimide ester (MBS) was from Pier~ • Chemicals, Rockford. IL, U.S.A. Other chemicals were of analytical ~ a d e .

Buffers Phosphate-buffered saline (PBS): 9.15 M NaCI in 0.01 M sodium phosphate buffer (pH 7,4). Wash buffer (T-PBS): PBS containing 500/~I/I of Tween-20, Blocking solution: 200 ml of B:ackace (Dainihon-Seiyaku, Osaka, Japan} in 600 ml ~;l distilled water. HSA-PBS: PBS containing 10 m g / m l HSA. Urease-conjugated rabbit anti-mouse F ( a b ' ) , immunoglobufin was from Nakarai ChemicaL, Kyoto, Japan, and was diluted 100-fold with 0.25% HSA-PBS. The substrate solution for urease was also from Nakarai. Peroxidase-conjugated rabbit anti-mouse F(ab')2 immunoglobulin was from Nakarai, and was diluted 100-fold with 0.2% HSA-PBS.

Media for cell culture HT: Dulbccco's modified Eagle's medium wi~h 20% ( v / v ) ~etal calf serum containing 13.6 mg of hypoxanthine and 3.9 mg of thyrnidine per liter. HAT: H T containing 1.76 mg of aminoptedn per liter. Protein was determined as de.scribed ~reviously [1],

Coupling of bilirubin-lXrt to carrier proteins Unconjugated bi]itubin-IXa (UBC) was covalently coupled to BSA in order to prepare the immunogcn. Special care was taken to preserve the tertiary structure of UBC during the coupling reaction. For this purpose, MBS was selected as a cross-linking reagent, and the reaction was proceeded through four steps as described below [3-5]. Step 1: Acid-catalyzed addition of nueleophiles to UCB [6]. UCB (25 rag) was dissolved in 50 ml of chloroform with 100 mg of cysteamineoHC] and a few drops of p-toluenesulfonic acid (p-TSA) was added as a catalyst. The mixture was gently stirred overnight at room temperature, it was then filtered with glass filter, and the residue was lyophilized. In order to remove a n y excess cysteamine-HCI, the lyophilized residue (30 me) was dissolved in 4 to 5 ml of methanol/ehlorofrm (1 : L v/v), and the solution was placed on a tapered preparative silica gel plate (20 × 20 cm) with a preabsorbcnt zone, and developed for 30 rain in chloroform/ methanol/triethylamine (510 : 90 : 20, v/v). Bands were scraped and immediately dissolved in 20 ml of methanol and 50 ml of chloroform. The sample was completely evaporated and lyophillzed, dissolved in 1 ml of methanol and 9 ml of chloroform, and then evaporated and lyoplfilized. Approx. 6 mg of UCB-eysteamine-HCI was obtained. Step 2: MBS-acylated UCB. UCB-cysteamine-HCl (6 me) was dissolved in 1 ml of dimethylsulfoxide and 1 ml of 0.4 M potassium phosphate buffer ( p H 7.4) (a).

MBS (4.6 me) was dissolved in I ml of tetrahydrofaran (b). 0.5 ml of (b) was then added to (a) and incubated for 30 aria at room temperature. In order to remove excess tetrahydrofuran, the mixture was flushed with nitrogen and then immediately processed to step 4. Step 3: Sodium borohydride reduction of disulfide bonds in BSA. A total of 20 m g of N a B H 4 and 0.2 ml of n-butanol were alternately added portionwise to a solution nf gSA (10 rag) in 6 M urea/0.1 M EDTA. The maxture was incubated for 20 rain at room temperature and then excess N a B H 4 was decomposed by adding 1 ml of 0.I M sodium phosphate (monobasie) and 0.4 ml of acetone. Thus, introduction of free thiol group into BSA was achieved, and tb.e solution was immediately processed to step 4. S~ep 4: Conjugation of MBS-acylated UCB to reduced BSA. The solution of reduced BSA (step 3) was incubated at 25~C for 3 h with the MBS-aeylated UCB solution (step 2) and in order to remove unreacted BSA, the mixture was subjected on a Sephadex 0 - 7 5 column equilibrated and ehited with PBS.

Immunization and cell fusion Female B a l b / c mice were immunized by intraperituneal injection of 1120pA (100/zg) of covalentiy coupled UCB-BS,.~ emulsified with art equal volume of Freund's Complete adjuvant. Booster injections without adjuvant, 200 /~l (400 /zg), respectively, were given intraperitoneaIly 9 weeks later on the last 4 days before cell fusion according to St~ihliet el. [7]. Procedure for cell fusion and subsequem selection of hybridomas were described previously [I].

Screening assay 100 ~l of the four kinds of solutions listed below were applied respectively to the wells of 96-well immunotitration plates (Nunc, Roskilde, Denmark) as immunosorbent, (i) Cnvalenfly coupled UCB-MBS-BSA in PBS (10 p g BSA/ml). (it) BSA in PBS (10 .ag/ml). (iii) Reduced BSA which has free thiol 'cToups in PBS (10 ~tg/ml). (iv) BSA-MBS-glyeine in PBS 0 0 #g/ml). After incubation overnight at 4 ° C the wells were first washed three times with T-TBS to remove any non-adherent immunosorbents, then filled with Blockaee and kept at room temperature for 1 h in order to block the remaining binding sites on the well surfaces. The unbound Blockace was removed by washing three times with T-PBS. CuItured supematant (100 p,I) of growing hybridomas, d i h t e d 10-fold with 0.1~ HSA, were added to the immunosorbent-eoated wells, Prt~cedures for screening assay were as described previously [1], Supernatants which showed high absorbance only with immanosotbent (i) but showed low ahsorbance with imnmnosorbents (it), (iii) and (iv) were selected as positive and used for further study.

5~.0

Inhibition assay Detailed procedures for the inhibition assay were described previously [1]. The diluted supernatants were mixed with serially diluted inhibitors in PBS or HSAPBS. After 2 h at 4=C, the inhibitor-antibody mixtures were transferred to 96-well immunotiter plates which had been coated with 1 # g / 1 0 0 v.l of UCB-MBS-BSA and blocked with B10ckaee. After 30 rain at 3 7 ° C , unbound fractions of antibodies were removed with three washes of T-PBS. Consumption of the binding sites of the monoclonal antibodies was determined by a decrease in absorbanee at 590 rim, and expressed as B/B o (B, absorbance with inhibitor; B0, absorbance without inhibitor) against the inhibitor concentrations expressed as log (l) (i, inhibitor concentration).

Preparation of inhibitors lnhibitors were prepared as described previously [1]. In the present experiments, ditanronic bilirubin-IXa (BDT) was dissolved in PBS to make a 1 . 1 0 -4 M solution.

~bo ,~DD ~.bO blJO "~bo WAVELENGYH(mr) Fig. 2. Absorption spectra of mesobilirubin-lX= IA), UCB (B), and the cysteamiae-adduc~,of UBC (C)-

Results and D|scussion

Coupling of UCB to carrier protein Covalently coupled UCB-BSA was prepared as an immunogen using MBS as a cross-linker, and the reaclieu proceeded in four steps (Fig. 1) [3-5]. Special care was taken to preserve intramolecular hydrogen bonds of UCB, and acid-catalyzed addition of cysteamine to UCB via vinyl group in tetrapyrrole was utilized in the present experimc=Jts. The procedure used was a modification of the method reported b y Manitto and Monti [6], and after step 1, addition of cysteamine to exo-vinyl group of UCB was explored as follows. (i) Absorption

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spectra: Absorption maximum of UCB at 448 nm was shifted to 434 nm b y the addition of cysteamine (Fig. 2). (it) Ninhydrin reaction: The reaction product was positive in ninhydrin reaction indicating the presence of amino group in the molecule. (iii) Diazo reaction: The product was negative in diazo reaction in the absence of ethanol, and turned positive after addition of ethanol. This indicates the reaction product preserved intramolecular hydrogen bonds in the bilirubin moiety. The azo-derivatives of the reaction product were separ~:ted by means of H P L C (Fig, 4), a n d the retention time of endo-vinyl type azo-pigments was unchanged, while those of exo-vinyl type azo-pigraeuts was shifted. These results indicated that eysteamine was coupled with exovinyl group of the bilirubin moiety. (iv) FAB mass spectrometry: The product represented a molecular ion peak at m / z 662 (M + H ÷) in FAI~ mass s ~ t r u m a n d the value is in agreement with the molecular formula C3sH43NsO6S, indicating the presence of an additional cysteamino group in UCB molecule (Fig. 3). (v) NMR: In 1H - N M R spectrum (270 MHz), three olefinic protons at 5.30 p p m (1H, dd (double doublet), or = 11..4, 3.0 Hz), 6.21 ppm (1H, dd, J - 17.6, 3.0 Hz), 6.58 ppm (IH, dd, J = 17.6, 11,4 Hz) derived from an exo-vinyi group of bilirubin-lXa were not observed, white endovinyfic protons of bilJrubin lX0t, 5.63 ppm (1H, d (doublet), J = i7~2 Hz), 5.64 pm (IH, d, J = 1.22 Hz), 6.82 p p m (1H, rid, d = 17.2, 12.2 Hz) still remained. New signals due to cysteamino group, 1.50 p p m (3H, d. J = 7.2 Hz, -CH3) , 2.76 ppm (2H, t (triplet), J - 7.0 Hz, -CH2-NH2) , 2.89 p p m (2H. t, d = 7.0 Hz, -S-CHz- ) were observed. These results indicated that the addition

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of eysteamine occurred at the exo-vinyl group of billruhin-IXa. Steps 2 - 4 were then performed in accordance with Kitagawa's method, and covalent coupling between bilirubin-lX~ and BSA was achieved. The resulting complex bound an average 5 - 7 bilirubin-IXa per molecule of BgA. Cell fusion and screening assay Approx. 2 - 3 weeks after cell fusion, the supernatants of hybridomas were tested, Prior to assay, in order to

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exclud~ clones producing antibodies against unwanted epitopes, the fonowing three immunosorbents were prepared for negative control. (i) BSA in PBS: (ii) reduced BSA having free t h i n groups; and (iii) BSA-MBSgtycine: another site of MBS which coupled with BSA was blocked by glyeine in order to prevent non-specific reaction with immunoglobulins. In 3893 supernatants of the wells in which hybfidomas were growing, 58 clones produced antibodies that bound to haptenic UCB but did not bind any of the above three immunosorbents. Among them, 22 clones with relatively high reactivity were selected. Inhibition assay with UCB In 18 out of 22 clones, binding activity of monoelonal antibodies to UCB was inhibited by addition of dissolved UCB in the range of 10-7 to 10 -4 M. One of the clones, designated 5M2 was inhibited at 10 -7 M. 3 C I I , 4829, 588, and 516 were inhibited at 10 -5 M,

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Fig. 4. Separationof azo-pigraea~sof UCB (A) and of the cysteamine adducts of UCB (B) by meansof HPLC. t: endc-vi~yltype azn-piBmerits; II: exo-vinyltypeazo-pig~ent~.

Fig. 5, Inhibition 3~ay with UCII. Culture supemalants were assayed

with UCB in PBS (O). UCB in U.]%HSA (O). and 0.1% HSA alone (ta). B: absorbancewith inhibitor; Bo: ahsurbancewithout inhibitor; and i: cozaceatrationof

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Fig. 6. Inhibition assay with UCB, BMG, BDG, ~ d BDTr PuIifiod [gG of 5M2 was agsayM ~vith UCB CoL BMG (I), BDG (I), a~d BDT (12). B: absorhane* with inhibitor; no: absorbance without inhibitor; and i: concentrationof inhibitor. aA17, 4A32, 4A57, 4Bl6, 4B25, 4C9, 4C15, and 5F13 at 10 -4"~ M, and 4A13, 4A54, 5A1, 5J5, a n d 5L2 at 10 -4 M. AI1 antibodies reacted with UCB in HSA-PBS with a sensitivity one or two orders of magnitude smaller than with UCB in the anionic form in protein-free solution (PBS), presumably due to sterie hindrance b y HSA b o u n d to UCB (Fig. 5).

Inhibition assay with ditauronic bilirubin.lXa (BDT), bilirubin-lXo monogluculonide (BMG), and Mlffubin-IXa diglucuranide (BDG) Almost all antibodies were inhibited weakly b y BDT, BMG, or B D G at concentrations as high as 10 -4's M. In particular, 5M2 was inhibited exelusive/y by UCB (Fig. 6). These results support the assumption that UCB in the immunosorbent preserved its tertiary structure.

Inh~biaon assay with ~iliverdm, heroin, azodipyrrole, and cholw acid Clone 5M2 was selected, a n d the l g O was purified from routine ascltes which has been injected intraperitoneally with the hybridoma using Protein-A MAPS-II Kit (Bin-Rod). 5M2 was not inklbited by biliv:rdin or hernia, though these compounds have equally tetrapyrrolic structure. Furthermore, azodipyrrole did not react with the antibody (Fig. 7). Preparation of monoclonal antibodies each specific for other hilirubin derivatives incleding B M G a n d B D G is now under way a n d will be presented elsewhere. lmmunochemical determination of individual bilirubin derivatives using monoelonal antibodies thus obtained enables us to perform analysis of exact amounts of the bilirubins in biological specimens with a high sensitivity, accuracy, and reproducibility. The methods represents a similar order of sensitivity with H P L C which has been first developed in our laboratory in 1979 [9]. The

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I ~ ' -7 -6 -5 - 4 I.~(i l Fig. 7. Ia~hibition essay with biliverdin, he,ran, azodipyrrole, and UCB. Purified IgG of 5M2 was assayedwith biliverdin(11).heroin(I). ch01ivacid (0), azodipyrro|e (t~), and UCB (0). B: absorbane¢ '.~th inhibiter; 80: absorbance without inhibitor; and ~: concentration ot inhibitor.

disadvantage of the H P L C is the inability to assure the homogeneity of each fraction dutcd, and further it is unsuitable for clinical use since the analytical procedure is time consuming and takes approx. 1 h for the analysis of a single sample. These monoclonal antibodies are producible in large amounts a n d are commercially available. These antibodies arc also useful to identify intracellular localization of bilirubin and its derivatives immunohistochenMeally, and to trace in viva bilirubin metabolism morphologically. Acknowledgements We wish to thank Professor K. lshida, the Second Department of Surgery, Saitama Medical College, Saitama, Japan, for reading a n d commenting critically on the manuscript. Y.O. a n d M.Y. are visitit~ research follows from the Second Department of Surgery, Saltama Medical College, Saitama, Japan. References 1 Shimizu,S,, Izurni, Y.. YamazakLM. Shimizu,K. Yalnaguehi, T. and Nakaiima~H. 0988) Bicchim. Biophys.A¢ta 967.255-260, 2 Izuml. Y,, Yamazakl, M, Shimizu. S., Shimizu, K, Yamaguchi, T. and Nakajima, H. (1988) Biochim, Biophys,Acta 967, 261-265. 3 Kitagawa, T., Kawa~aki,T. and Muncchik~, H. (1982)J. o,i(x:h~a'n. 92. 585-590. 4 Kitagawa. T. and Aikawa, T. (1976) J. Bioeheal. 79. 233-236. 5 Kitagawa, T, Kanaraura. T., Wakamatsu, H., Kate, H,. Yaao, $. and Asanuma, Y. (1978) $. Bioohem, 84. 491-494. 6 Manitto. P. and MvatL D. (1973) Experiantia 29, 137-139. 7 $ti~tli,C.T., Staehelin, T., Miggaamo,V.. 8chmidt, J. and Hgtrlng,P. (1980) J, lramunol, Methods 32, 298-304. S Blaakaert. N.. Gullan, J.l, and Schmid.R. (1979) Proe. Nail. Aead. Sci. UgA 76. 203"L2041. 9 Yamaguchi, T., Yamaguchi, N., Nakajima, H., Komoda, Y. and ]s~tawa, M, (1979) Par=, Jpn. Acad. ~Sa, 89-93.