44(1)-11.fm

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1 Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006, pp 자성미세입자에의항체고정화방법이면역결합반응에미치는영향 s**ç içm Ço Ç o Ç *Çqm *Çml k rl e, *pn k e ( o 5p r, o 3p }ˆ) Effect of Antibody Immobilization Method to Magnetic Micro Beads on its Immunobinding Characteristics Hyo Jin Choi**, Sang Youn Hwang, Dae Ho Jang, Hyung Min Cho, Jung Hye Kang, Gi Hun Seong*, Jae Bum Choo* and Eun Kyu Lee Bioprocessing Research Laboratory, Department of Chemical Engineering, *Department of Applied Chemistry, Hanyang University, 1271, Sa 1-dong, Sangnok-gu, Ansan , Korea (Received 5 December 2005; accepted 3 February 2006) k q p pn p qrp pn l l v p l p ˆ nkp q pq pn l r v p ll p p l l kk q m. q pq pn nl (1) q pq l r, (2) ligand( )m r v p pr, (3) q p pn q pqp, (4) r vp ˆ (5) q pqp q n v l l p rp eˆ p. p q pq pn p p,, v kp s, q p np, np r p p ep v p. l l p e } q pql IgG ~ r eˆ IgG r v l p q m. p o l q pq l v p pr p pl v kp s l l p kp, IgG ~ r e q pq r v rp l r vp IgG p ˆrp p vl l kk k. p ph n n pr p tp plp, IgG ~ r q pq n n r vp IgGm ˆrp p p k pl. IgG ~p r l Fcvl C-terminusl pr p ˆ p p n l p t n, IgG ~ p k m r eˆ r o p p k 2 k. h Abstract Recent technical advances in the biorecognition engineering and the microparticle fabrication may enable us to develop the single step purification using magnetic particle, because of its simplicity, efficacy, ease of automation, and process economics. In this study, we used commercial magnetic particles from Seradyn, Inc. (Indianapolis, USA). It was ca. 2.8 micron in diameter, consisted of polystyrene core and magnetite coating, and its surface had carboxyl groups. The model, capture protein was IgG and anti-igg was used as the ligand molecule. We studied the different surfaces ( nude, ester-activated, and anti-igg coated) for their biorecognition of IgG. At a high ph condition, we could reduce non-specific binding. Also anti-igg immobilized magnetic particle could capture IgG more selectively. We attempted oriented immobilization of anti-igg, in which the polysaccharides moiety near the C-terminus was selectively oxidized and linked to the hydrazine-coated MP, to improve the efficacy of biorecognitive binding. Using this method, the IgG capturing ability was improved by ca. 2 fold. From the binary mixture of the IgG-insulin, IgG could be more selectively captured. In summary, the oriented immobilization of oxidized anti-igg proved to be as effective as the streptavidin-biotin system and yet simpler and cost-effective. This immobilization method can find its applications in protein biochips and biotargeting. Key words: Magnetic Particles, Biorecognition of IgG, Oriented Immobilization, Surface Modification To whom correspondence should be addressed. eklee@hanyang.ac.kr **Current address: Celltrion Quality Control Group, Incheon, Korea. 65

2 66 vë lëq Ës Ë r Ë Ëtq Ëpp 1. rp l p rp eˆ o r n q p pn q (magnetic separation) p. q p tn pr n r nkp ˆ kl vr r vp qlp p. q p ~ pq l v v p p ˆk l m s p r vp p qrp v p [1]. pnl d pl (scalability), p,, q p np, np r p l v p. q pq v, ˆp, p r,, immunoassay, k r, pm l n p. q pq pn vp ˆr pp rl v. (1) r v p p vp q p q l o eˆ, (2) nk l vp r v p pe l, (3) q p pn l q pq p, (4) q pql r vp v ˆ eˆ. p, r v p pr(specific)p o l r v prp p p vp q pq l eˆ, q pq p eˆ p v kp q ktp f vp q pq l o eˆ p (Fig. 1). p r ~ l p p m l l rrp p. ~ r p n o l p r p p ss o p p n p. p rrp o l ~ r eˆ. r vp ol lr p q k v o l r eˆ p. ~p r n vp ˆ p pn l r eˆ p. ~p n p H-chain(Heavy chain)l r l p p N-linked ˆ p v pp IgGp n CH2 domainp Asn297 vll p N-glycosylation p v p [2]. ~p ˆ p pn l r eˆ n v sp kr k o p r np p k p [3, 4]. l l q pq pn l r vp rr r p q m. r vp IgG ˆrp o l q pql IgG ~ r eˆ, r rl p rp n p k p pn r ˆ p pn p pn l p n IgGmp p k. Surface plasmon resonance biosensor(spr) pn l r m r l p p p m., IgG ~ r eˆ q pq n l IgG p ˆrp o s p }kp q pq pn l rp tp p l l p m m e l n q pq CM-MP(carboxyl modified magnetic particle) Seradyn, Inc.(Indianapolis, USA)l p m. v p 2.8 µmp, dž ll q~ p Žeˆp f q p ˆ p e v p. v n mouse IgG polyclonal ~m mouse IgG, BSA(Bovine Fig. 1. Schematics of IgG purification by using anti-igg coated magnetic particles. o44 o k

3 serum albumin) Sigma-Aldrich(St. Lois, USA) l p m. r p o Bradford dyem SDS-PAGE ekp Bio-Rad (Hercules, USA) l p m. r o l n streptavidin biotinp Pierce(Rockford, USA) l p m p p v hydrazide v p. p pd p (t)s pml k NHS-EDC mk m mmi mouse-igg zm o p e q pqp l IgG ~ r e. q pq 1mgp 50 mm MES(2-(N-morpholino ethanesulfonic acid, ph 6.1) 3 }, 230 mlp 50 mg/mlp NHS (N-hydroxysuccinimide)m p p 10 mg/ml EDC(1-ethyl-3- (3-dimethylamino-propyl) carbodiimide) l 30 kp pe tp f NHS e. IgG ~ l ph m p l o l 1 mg/mlp IgG ~ 10 mm sodium acetate(ph 3.5, 4.5, 5.5), 10 mm MES(pH 6.5), 10 mm PBS(phosphate buffered saline, ph 7.5) pn l IgG ~ 100 µg/ml 1 mlp m. q pq lp p l eˆ IgG ~ eˆ ml pe. p oe pn l k p kp IgG ~p kp UV r l r IgG ~p kp r m. q pql kp p ester t ~ kt o l 1Mp ethanolamine(ph 8.0)p 15 k peˆ p } m IgG zm l mk o q pq 1mgp l 0.1 M MES(pH 4.8) pn l 3 } m. 0.1 M MES(pH 4.8)l 0.5 M acipic dihydrazide peˆ q pq lp p l 1 mlp lt. 3 mgp EDC 3e k e ˆ peˆ 3 v, 1 M NaCl, 3 v p } m. IgG ~p ˆ p eˆ o l 1 ml p 0.1 M sodium phosphate(ph 7.0) l 1 mgp IgG ~ n eˆ 10 mgp sodium m-periodate 30 ml pe [5]. p desalting column(sephadex G- 25) pn l kp sodium m-periodatem IgG ~ [6]. srp 0.1 M sodium phosphate(ph 7.0) pn l q pq } eˆ IgG ~ 50 µg ~ l ml k m. p 0.1 M sodium phosphate(ph 7.0), 3 v, 1 M NaCl, 3 v p } m Biotin-streptavidinl mk IgG z o q pql streptavidinp r eˆ o l 1 mgp q p q 50 mm MES(pH 6.1) 3 } 2 mg/mlp streptavidin hydrazide nk 200 µlm 10 mgp EDC l k pe [7]. Biotin e rp 1 mg/mlp IgG ~ eˆ 1 mg/mlp biotin hydrazide nkp 1e k ml pe. pp o l 0.01 M sodium phosphate(ph 7.4) pn l 4 o Cl k Œ e [7]. k r ~ pn l p o IgG zj IgGm m r eˆ IgG ~m IgG p p p o l IgG ~ r l p 1 mgp q pql 100 µgp IgG l eˆ ml pe. p 30, 60, 1e, anti- IgGm IgG p pp m IgG yl l no r IgG ~m IgG o l ˆ s p }k k. r IgG ~m IgGl p ˆ 1 mlj 2 k pe. ˆ p o n 1 M sodium chloride, 200 mm sodium carbonate(ph 11.5), 100 mm sodium bicarbonate(ph 9.2), alkaline SDS(0.2 M Tris base/1.0 Í SDS), 100 mm sodium citrate(ph 3.0), 100 mm glycine(ph 2.0), 250 mm sodium hydroxide, 100 mm hydrochloric acid, 100 mm glycine(ph 2.3)+1Í DMSO p. oe pn l k p l ˆ IgGkp p m phi Šmn y (1) l k } v kp e ( nude ) (2) NHS/EDC pn l p eˆ 1 Mp ethanolamine (ph 8.0)p pn l blockinge p e ˆ l pr p IgGm BSAp kp p k. 1mgp q pql 25, 50, 100, 200, 400 µgp IgGm p kp BSA 1mLp m m. php m p p o l 50 mm sodium acetate(ph 3.5, ph 5.5), 50 mm sodium phosphate(ph 7.5), 50 mm sodium bicarbonate(ph 9.5) pn m. 1e k eˆ peˆ k p l r~p kl UV r k l kp IgGm BSAp kp l IgGm BSAp k p r m IgG z m mmm m k q pql r IgG ~m IgG ˆ e, 100 µg/mlp IgG 1 ml ltl e ˆ pe. p oe l k p l kp IgGp kp r p f p IgG kp r m. ˆ r rp l v q n q pq p IgG ~m p IgGp kp m m (binary mixture)i m n Amine-couplingp pn r, r, biotinstreptavidinp pn l 30~35 µgp IgG ~ r q pq l p p l 50 mm sodium bicarbonate(ph 9.5) l kp 60 µgp IgGm pd p 1e k ml eˆ pe. oe q pq p p n l } m. r IgG ~m p p non-denaturing SDS-PAGE pn l p m. p, 20Í acrylamide gelp n mp gelp coomassie brilliant blue m p pn m. SDS-PAGE p v r p o densitometer(total Lab, Durham, USA) pn m. Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

4 68 vë lëq Ës Ë r Ë Ëtq Ëpp Surface plasmon resonance mk IgG z o SPR biosensor Biacore AB (Uppsala, Sweden)p Biacore 3000 edšp pn m. p CM5 p pn m. p p carboxymethyl dextran p l pp 4 p flow cellp p. pr p dextran l p r pp eˆ o kr p coupling buffer pn l rr p o (preconcentration test). ph 3.5, 4.5, 5.5p 10 mm sodium acetate m l tp l rp ph s p r m. Dextran p carboxyl e ~ p amine m o p veˆ o l, 0.2 M EDC(N-ethyl-N'-dimethylaminopropyl carbodiimide)m 0.05 M NHS (N-hydroxysuccinimide) 7 k tl p p sp NHS ester e. Preconcentration test r php 10 mm sodium acetatel (IgG ~ ~) s 20 µg/mlp ~ l flow cell 2(fc 2) o 5 µl/minp o p r m. IgG ~ v kp NHS ester 1M ethanolaminep 7 k tl blocking e Surface plasmon resonance mk IgG z o CM5 p e 0.2 M EDCm 0.05 M NHS 5µL/min p 3 k tl NHS ester e. k e o l 5 mm adipic acid hydrizide 5µL/minp 7 tl. k v kp NHS ester 1M ethanolaminep 5µL/minp o p 7 tl blockinge. q pql jp IgG ~m p p eˆ ~ s 20 µg/ml 10 mm sodium acetate(ph 3.5)l l 5µL/minp ~m p kp flow cell 4l r m. ~p (Schiff base)p kr o l 0.1 M sodium cyanoborohydride 2µl/min 20 tl SPRl mk IgG zm o i 10.8 mg/ml IgG s 321.3, 160.7, 80.3, 40.2, 20.1, 10.0, 5.0, 2.5 µg/mlp 0.01 M PBS(pH 7.2)l l 30 µl/minp fc(flow cell)1l 4 v tl. e p 2 (association time), 3 (dissociation time)p tl 5 e m. e p e tp o 250 mm NaCl+12.5 mm NaOH 2 30 µl/minp tl q (regeneration) m. 3. y 3-1. IgG z o i nm ph o e q pq NHSm EDC pn l eˆ l IgG ~ r o r ph } o l ph v e k. ph v IgG ~m q pq p r lp v p p ph 3.5 p nl p pp pl v kk. p l l v IgG ~p amine-coupling r ph 4.5l v m. o44 o k Fig. 2. Adsorption isotherm of anti-igg immobilized on magnetic particle (N=3) m mmi o IgG z h NHSm EDC p eˆ 1 mgp q pql 10 mm sodium acetate(ph 4.5) l kp IgG ~p kp v eˆ ltp f r p p k. Fig. 2l ˆ p 1mgp q pql k 70 µgp IgG ~. p Langmuir modell rn p. q/q m =(K d C s )/(1 + K d C s ) l q p vp k(g/mg-mp), q max v p (µg/mg-mp), C s nk p vp (µg/ml) K d (ml/µg)p [8]. op el p l q max 105 µg/mg, K d ml/µg ˆ IgG zm o l m q pq l IgG ~ r eˆ n e p p k. NHSm EDC pn l eˆ q pql 10 mm sodium acetate(ph 4.5) l lp IgG ~ 80 µgp l 30, 1e, 2e, 4e, 6e q pql r IgG ~p k p p m. 1e v p pp pl p pp p pl v kk Šmn ml qm l ph o q pq l prp vp kp tp o l pl r ph s p p k. r vp IgGm vp BSAp p 50 mm sodium bicarbonate(ph 3.5~9.5)p l n eˆ pq l kp m. n q pq (1) p k } v kk e ˆ (2) NHSm EDC pn l p eˆ ql e 1 M ethanolamine(ph 8.0)p pn l blockinge p e ˆ p pn l e m. Fig. 3l ˆ p ph kv q pq l IgGm BSA p kp tl p p p. p php m p q pq v p r l p m p p. ph IgGp pi p 7.4 o kv IgGp p p

5 k r ~ pn l p 69 Fig. 3. Amount of protein adsorbed on magnetic particle surfaces. : nude MP with BSA, : nude MP with IgG, : NHSactivated (after ethanolamine blocking) MP with BSA, : NHS-activated (after ethanolamine blocking) MP with IgG. Fig. 4. Time course of IgG adsorption to anti-igg-coated magnetic particle. k is min -1 (N=3). ˆ l le p ˆ q pqmp rr r p p f kp tl, p po pi p 4.7p BSA ph 4.7 kvp f q pq l kp tl. p vp l rr r p tn n m p p k p [9] IgGm n m IgG ~ r eˆ q pql IgG p ˆrp p vl l p k. pr pp t p o l 50 mm sodium bicarbonate(ph 9.5) pn m. q pq l (1) k } v kp n, (2) NHSm EDC pn l eˆ 1 M ethanolamine(ph 8.0)p pn l blockingeˆ n, (3) IgG ~ amine-coupling p r eˆ nl IgGm BSAp p p k. Table 1l ˆ p IgG ~ r eˆ q pq pn n e, p e p n r vp IgGmp ˆr p 6~8 kv p p pl. v IgG ~ l r eˆ n IgGmp ˆr pp p p p p m o IgG zj IgG m m IgG ~ amine-coupling p r q pql IgG n e p r m. IgG ~ aminecoupling p r l p q pql 100 µgp IgG lt v IgGp kp p, IgG Table 2. Buffers used for elution of IgG adsorbed on anti-igg-coated magnetic particle No. Elution buffer Elution yield ( ) 1 1 M sodium chloride mm sodium carbonate (ph11.5) mm sodium bicarbonate (ph 9.2) Alkaline SDS (0.2 M Tris base/1.0 SDS) mm sodium citrate (ph3.0) mm glycine (ph 2.0) GmM sodium hydroxide mm hydrochloric acid mm glycine (ph 2.3)+1 DMSO 86.0 ~m IgG pp p 1e p p p m (Fig. 4) IgG yo r IgG ~m IgGp pr ˆ p o l Table 2 l re p ph, mp s p m p k. p tl 100 mm glycine(ph 2.0) 100 mm glycine (ph 2.3)+1Í DMSO pn n pp 86Í q k. p p IgG ~m IgGp ˆ l m s s p n rp p k pl., glycine p n n DMSO l n n p ˆ l p acetonitrilep DMSOm p o v p ˆ pp lt m p m [10]. Table 1. Effect of surface on adsorption capacity of BSA and IgG Relative adsorption capacity BSA IgG * Surface with carboxyl group(nude) Surface with hydroxyl group(nhs-activated and ethanolamine-blocked) Anti-IgG immobilized surface(ligand-coated) (*) Relative adsorption capacity of IgG as compound to BSA s (1.0). Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

6 70 vë lëq Ës Ë r Ë Ëtq Ëpp 3-8. o IgG zm m k r vp IgGp rp ˆ q pql r IgG ~p q np q pq pn p l l tn p. q sp ˆ pp p 100 mm glycine (ph 2.3)+1Í DMSO pn l IgG ˆ eˆ, n q pq pn l IgGp ˆ p m. q p q 8 n, p p 70Í r ov p k pl. Kandimalla [10]p Sepharosel r EP(ethyl parathion) ~ pn l r ~p q n p e m. 100 mm glycine(ph 2.3)+1Í DMSO pn mp 14~15 v p 70Í p p o v m. p p k q pql r ~p q n p p n o l q pq l ~p k lp r ~p ˆ p e k p eˆ p q p k m r eˆ, streptavidinp r eˆ q pql biotinylationeˆ ~ r eˆ p p n m. p rp p ~l p p op 2 p, Table 3p k p p r p p n n, ~ l l p p op k 0.9 p ~p ˆ p pn l r eˆ nl ~ l k 1.5 o p p k 1.6 p k pl. p r p nl ~ pep ε-aminep pn ~l pep p sq l t p r p l, r ~p l p ~p p l m p m [4]. ~p ˆ p e r eˆ nl ~ r p r l o p p kv., ~p ˆ o psp spacer armp qn l pq p p l tl o p r p np o p [11]. p streptavidin biotinp pn p e q pq l streptavidinp o eˆ, IgG ~p ˆ p e biotinylationeˆ v peˆ p. p IgG ~l IgGp ~ l k 1.5 ~ e r eˆ p pn nm p k (Table 3). o p l, ~ e r eˆ biotin-streptavidinp pn p d ˆ m (Binary mixture)i m n r pn nm IgG ~p ˆ p e r eˆ n, streptavidin biotin r p pn l IgG ~ r eˆ q pq IgGm pd p kl ~ m. Table 4m p r n IgGp k 30Í p Ž, r m biotin-streptavidinp pn nl 94Í, 97Íp IgG p k p., r IgG ~kl l p p t nm biotin-streptavidinp pn n p ~l k 1.7 p op m. p Table 3l re m o p k p. p pd p sq l pr p ov p l l p sq nkl r vp ˆrp on p., p vp r r omp r p p p l p p lt sp l m p p k p [3]. l t pd p kp p r p kl p p l p k, r IgG ~m IgG ˆrp p p m (Fig. 5). biotin-streptavidinp pn n pr p ˆ k IgG ~p ˆ p e pn r p n rp Ž SPRl mk IgG z o i m CM5 l amine couplingp r IgG ~ 1,076 RU(428 nm)p fc 2l, ˆ k p e CM 5 p k m pe p t n 1,047 RU (418 nm) r l [12]. pm p r IgG ~l 321.3, Table 3. Comparison of IgG binding affinity by ligand coupling methods (N=3) ImmobilizationGMethods Anti-IgGGImmobilization IgG adsorbed Binding affinityg(the number of IgG/the G(µg/mg MP) (µg/mg MP) number of anti-igg) Random(amine-coupling) 31.9 ± ± Oriented(oxidized carbohydrate coupling) 35.7 ± ± Biotin-streptavidin 43.8 ± ± Table 4. Comparison of IgG binding affinity from IgG-insulin mixture by ligand coupling methods (N=3) ImmobilizedG Adsorbed IgG Ratio of immobilized antibody anti-iggg(µg) Percentage ( ) Mass (µg) to bound antigen Random(amine-coupling) Oriented(oxidized carbohydrate coupling) Biotin-streptavidin o44 o k

7 k r ~ pn l p Fig. 5. SDS-PAGE (20 acrylamide gel) analysis. Lane 1: binary mixture sample (0.6 mg/ml IgG and 0.6 mg/ml insulin), lane 2: after reaction by random immobilized anti-igg, lane 3: after reaction by oriented immobilized anti-igg, lane 4: after reaction by biotinylated anti-igg. e l q pq pn rl ˆ p p m. pn IgG ~ r n ph 4.5l r pp q k 1mg k 70 µgp IgG ~ r l. vp amine-coupling r IgG ~p ˆ p eˆ r p pn l, o om lr p ~p ˆ p pn l r eˆ n IgG r o p k 2 k. p p pd p lp nk l IgG ~ r eˆ q pq l pe p le p r eˆ n IgGm n p p m. ~p ˆ p e r eˆ n o p, rp l rpl. p SPRp pn l o ~ p l p m. pl p ~ r e o p eˆ p rp p pn k k rr, pm k p pnl p Ž. r( ) oprqo, lqo, p vop tp n e e vo l( pmpk p o pe )p l p. pl. y Fig. 6. IgG isotherm to immobilized anti-igg antibody. Open circle: orientedly immobilized anti-igg antibody, closed circle: randomly immobilized anti-igg antibody , 80.3, 40.2, 20.1, 10.0, 5.0, 2.5 µg/mlp IgG 30 µl/minp tl p p m. Fig. 6l lt p r IgG ~p n r IgG ~ k 6 p IgGm p p p ˆ. p p pp p l o p o p p l p o p p k p., r IgG ~m p IgGp, p n IgG ~ 0.04 p IgGm p, p n 0.27 p o p p k p. p q pq e rp p p p. p dextran l r lp ~ p o l o rp rp kp o p. v, r eˆ IgG ~p n r IgG ~ 6~7 p p p p p m. 1. Hubbuch, J. J. and Owen, R. T. T., High-gradient magnetic Affinity Separation of Trypsin from Porcine Pancreatin, Biotechnol. Bioeng., 79, (2002). 2. Yoo, E. M., Chintalacharuvu, K. R. and Penichet, M. L., Myeloma Expression System, J. Immunol. Methods, 261, 1-20(2002). 3. Turkova, J., Orient Immobilization of Biologically Active Proteins as a Tool for Revealing Protein Interactions and Function, J. Chromatogra. B, 722, 11-31(1999). 4. Wimalasena, R. L. and Wilson G. S., Factor Affecting the Specific Activity of Immobilized Antibodies and Their Biological Active Fragments, J. Chromatogra., 572, (1991). 5. Hermanson, G. T., Bioconjugate Techniques, pp.115, Academic press, New York, USA(1996). 6. Bilkoà, Z., Mazurovà, J. C., Horàk, D. and Turkovà, J., Oriented Immobilization of Chymotrypsin by Use for Suitable Antibodies Coupled to a Nonporous Solid Support, J. Chromatogra. A, 852, (1999). 7. Babashak, J. V. and Phillips, T. M., Use of Avidin-coated Glass Beads as a Support for High-performance Immunoaffinity Chromatography, J. Chromatogra., 444, 21-28(1998). 8. Abudiab, T. and Jr. Beitle, R. R., Preparation of Magnetic Immobilized Metal Affinity Separation Media and its Use in the Isolation of Protein, J. Chromatogra. A., 795, (1998). 9. Peng, Z. G., Hidajat, K. and Uddin, M. S., Adsorption of Bovine Serum Albumin on Nanosized Magneticparticles, J. Colloid Interface Sci., 271, (2004). Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

8 72 vë lëq Ës Ë r Ë Ëtq Ëpp 10. Kandimalla, V. B., Neeta, N. S., Karanth, N. G., Thakur, M. S., Roshini, K. R., Rani, B. E. A., Pasha, A.Gand Karanth, N. G. K., Regeneration of Ethyl Parathion Antibodies for Repeated use in Immnosensor: a Study on Dissociatin of Antigens from Antibodies, Biosens. Bioelectron., 20, (2004). 11. Orthner, C. L., Highsmith, F. A. and Tharakan, J., Comparison of the Performance of Immunosorbents Prepared by Site-directed of Random Coupling of Monoclonal Antibodies, J. Chromatogra., 558, 55-70(1991). 12. Nelson, R. W., Nedelkov, D. and Tubbs, K. A., BIACORE and Mass Spectrometry: Identification of Epitope-tagged Proteins in E. coli Lysates, BIAjournal, 7, 25-26(2000). o44 o k