Journal of Biochemical and Biophysical Methods, 21 (1990) 75-80 Elsevier


JBBM 00820

Short N o t e

Purification of human C-reactive protein by immunoaffinity chromatography using mouse monoclonal antibody W a t a r u N u n o m u r a 1,2, M a s a h i k o H a t a k e y a m a a,a a n d H i d e m a t s u Hirai 1 1 Turnout Laboratory and 2 Nippon Bio-Test Laboratories Inc., Tokyo, Japan

(Received 5 November 1989) (Accepted 12 February 1990)

Summa~ Human C-reactive protein (CRP) was purified from sera with a high concentration of CRP by immunoaffinity chromatography using mouse monoclonal antibody (mAb) to CRP (No. 18) and ion exchange on DEAE-Sephacel. CRP bound to the immunoaffinity column was eluted by 0.5 M acetate buffer, pH 5.0 containing 1.5 M NaC1. Final recovery of CRP was 90%. No significant immunochemical differences were observed between CRP obtained by the immunoaffinity chromatography and CRP purified by the method of Hokama and Riley [1]. Key words: C-reactive protein; Monoclonal antibody; Purification; Immunoaffinity chromatography; ELISA; Double diffusion test


For the determination of CRP, radioimmunoassay (RIA) [2] a n d / o r enzyme immunoassay [3] are available. Accurate measurement of CRP can be achieved by the use of polyclonal antibodies with a high specificity prepared from hyperimmune antisera [2,3]. To obtain such antibodies, highly purified CRP is needed. So far, CRP has been purified by lecithin-Ca 2+ precipitation [4], C-polysaccharide (CPS) affinity chromatography [5] or phosphorylcholine (PC) affinity chromatography [6]. However, the lecithin-Ca 2+ precipitation method is rather tedious and the recovery is low. Furthermore, isolation of CPS in sufficient amount from Streptococcus pneumoniae is not easy [7]. Correspondence address: Wataru Nunomura, Tumour Laboratory, Higashitokura 1-15-3, Kokubunji, Tokyo 185, Japan. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

76 Compared to these methods, immunoaffinity chromatography using monoclonai antibody to CRP is more efficient. Immunoaffinity columns based on polyclonat antibodies are not always applicable in this context because it is quite difficult to eIute the CRP in good yield. In this paper, an attempt to purify CRP from sera with a high serum CRP concentration by an immunoaffinity chromatography employing a m A b is reported.

Materials and Methods

Preparation of CRP and antibodies against CRP CRP purified from sera with a CRP concentration over 50 mg/1 by the method of H o k a m a and Riley [11, is designated as H - C R P in this paper. Using this CRP, 5 mAbs (Nos. 17, 18, 19, 20 and 23) were established [8]. Polyclonal antibodies to CRP (1 m g / a n i m a l ) and to normal human serum with a CRP level less than 1000 n g / m l , were raised in rabbits by immunization with the proteins emulsified in Freund's complete adjuvant (Wako Pure Chemical Industries, Japan). Booster injections were made 1 and 2 weeks after the first immunization. G o a t antibody (IgG fraction) tO human CRP was purchased from Nippon Bio-Test Laboratories Inc., Japan. ELISA for CRP A 96-well microplate was coated with 0.1 ml of purified mAbs ( 2 0 / x g / m l ) or the goat antibody to CRP (20 ~ g / m l ) , and then incubated with 1% bovine serum albumin (BSA) in a 0.01 M phosphate buffer, p H 7.2, containing 0.15 M NaC1 (PBS) for 2 h and 20/~1 of CRP (10-100 n g / m l ) dissolved in a 0.01 M Tris-HC1 buffer, p H 8.0; containing 0.15 M NaC1 (TBS). After the contents were removed, the wells were washed three times with TBS. Then, 20 /~1 of alkaline phosphatase (ALP)-conjugated mAbs, prepared by a glutaraldehyde method [9], were added. The incubation was performed for 1 h, and A L P activity was measured using a commercial kit (Shino-Test Laboratories, Japan). BSA (1%) dissolved in T B S was used as blank. All procedures were performed at room temperature. Dissociation condition of mAb-CRP immune complex The condition for the dissociation of immune complexes was examined using ELISA as described above. A 96-well microplate was coated with 0.1 ml of C R P (20 ~ g / m l ) dissolved in TBS and then incubated with 1% BSA. m A b s dissolved in TBS were a d d e d and incubated for 2 h. After the mAbs were removed, the plate was washed three times with each of the following buffers: (a) 0.01 M Tris-HC1, p H 8.0; (b) 1.0 M Tris-HC1, p H 7.0: (c) 0.5 M acetate buffers, p H 6.0, p H 5.0 and p H 4.5 containing 1.5 M NaCI: and (d) 8 M urea dissolved in PBS as control. After a further wash by TBS. a solution of ALP-conjugated goat I g G antibody to mouse [gG was added. ALP activity was measured according to the method described above.


Purification of CRP by immunoaffinity chromatography using mAb (No. 18) 50 mg of purified No. 18 (as IgG) was coupled to 10 ml of cyanogen bromide activated Sepharose 4B (3 g). The following procedures were carried out at 4 ° C. The sera with high CRP concentration were centrifuged at 15 000 rpm for 30 min to remove insoluble materials and then were applied to No. 18-coupled Sepharose 4B. After the column was washed with TBS, bound protein was eluted by 0.5 M acetate buffer, p H 5.0, containing 1,5 M NaC1 (buffer A): Eluted fractions were immediately neutralized by adding 2-amino-2-hydroxymethyl-l,3-propanediol (Tris), and dialyzed against 0.05 M citrate buffer, p H 7.0, containing 0.1 M NaC1. Further purification was performed by a DEAE-Sephacel column (1.0 × 10 cm, Pharmacia, Sweden) equilibrated with the same buffer. CRP absorbed to the column was eluted with 0.05 M citrate buffer, p H 7.0, containing 0.5 M NaC1. The immunoaffinity purified CRP is named IP-CRP in this paper. The purity was assayed by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) [10].

Immunodiffusion analysis Double immunodiffusion test in 1.2% agarose gel was performed according to the method of Ouchterlony [11]. The purified mAbs and CRP were applied to each well at a concentration of 5 m g / m l and 1 m g / m l , respectively. The incubation was made at room temperature for 18 h. After the precipitin reaction was completed, the agarose plate was incubated with buffer A.


Fig. 1 shows the dissociation of the complex of CRP and No. 17 or No. 18 at various p H values. CRP bound to No. 18 was completely dissociated by 0.5 M acetate buffer, p H 5.0 and pH 4.5, containing 1.5 M NaC1. On the other hand, CRP bound to No. 17 was not dissociated by these buffers. Urea (8 M) dissociated the immune complex with both No. 17 and No. 18. Fig. 2 further demonstrates the dissociation of the immune complex by buffer A. When the plate was incubated with buffer A, the precipitin line formed with No. 18 disappeared, .whereas that with No. 17 remained. 15 mg of CRP could be bound to 50 mg of No. 18 coupled to Sepharose 4B, and eluted from the column with buffer A: S o m e contaminants in the eluted fractions could easily be removed by means of DEAE-Sephacel chromatography. Rerunning three times, the mean final recovery of C R P was 90.2 + 2.2% ( m e a n + SD). So far, the column has been found to be used more than 10 times. On SDS-PAGE, IP-CRP migrated as a single band at 24 k D a (Fig. 3) corresponding to that of H-CRP [8]. The IP-CRP gave a single precipitin line in the y-globulin region in immunoelectrophoresis against antiserum to CRP, while it did not react with antiserum to normat human serum. Hyperimmunized antiserum to IP-CRP also did not react with normal human serum (data not shown).

78 A 510/~20


0.2 I


E ~Eu 0 ,I


pH B.O 7.0 7.0 M 0.01 0-5 1.0 Tris-HCI


5,0 4.5 / Urea 0.5 B,O Aceta'~e PBS

1.5M NaCl

Fig. 1. Effect of vanpus pHs on the dissociation of CRP-mAb complexes. 17 and 18 represent the complex of CRP with mAb No. 17 and No. 18, respectively. Figures in the middle row (M) indicate molar concentrations. All buffers contained 1.5 M NaC1. Urea was dissolved in PBS at a concentration of 8M.

Immunochemical characteristics of IP-CRP were compared with those of H-CRP. No difference between IP-CRP and H-CRP was detected by Ouchterlony's test using antisera to IP-CRP or H-CRP (data not shown).

Fig. 2. Dissociation of CRP-mAb complexes with buffer A (0.5 M acetate buffer, pH 5.0, containing 1.5 M NaC1) in agarose double immunodiffusion. C, P, 17 and 18 represent CRP (1 mg/ml), rabbit polyclonal antibody to CRP, mAb No. 17 and No. 18 (each concentration was 5 m g / m i as lgG), respectively, (A) before and (B) after the treatment with buffer A.


Fig. 3. SDS-PAGEof IP-CRP (lane 1) and H-CRP (lane 2): M represents marker proteins: rabbit muscle phosphorylase b (94 kDa), BSA (68 kDa), ovalbumin (40 kDa), bovine erythrocytescarbonic anhydrase (30 kDa), soy bean trypsin inhibitor (2011kDa), ~ d bovine milk c~-lactalbumin(14.4 kDa). Discussion

Human CRP could be isolated in high purity and in sufficient yield (more than 85%) from sera with a high concentration of CRP by using mAbs coupled to Sepharose 4B. The amount of CRP bound to No. 18-coupled Sepharose 4B was 0.3 m g / m g - N o . 18. Pepys et al. [5] reported that 7 ml of Sepharose 4B bearing 16.7 mg CPS bound 1.6 mg CRP/mg-CPS. The results indicate that the CRP-binding capacity of CPS-coupled Sepharose is larger than that coupled with No. 18. The CRP molecule has plural numbers of antigenic determinants. The CRP has been demonstrated to be a pentameric molecule by structural study and electron-microscopic observation [12]. Therefore, one CRP molecule binds several molecules of mAb. The mAb from mouse ascites fluid can easily be purified by protein A-affinity chromatography or salting-out and DEAE-cellulose chromatography. The contaminating protein in fractions eluted from the No. 18 coupled column was demonstrated to be nonspecific IgG by immunoelectrophoresis using a specific antibody to IgG (DAKO). Preliminary fractionation with ammonium sulfate of sample sera faciliates the further purification. The nonspecific IgG is precipitated with 50% saturation of ammonium sulfate at 20 ° C, while CRP remains in the supernatant [8]. Actually, when the fraction obtained by the treatment with ammonium sulfate (50-75% saturation) was applied to No. 18-Sepharose 4B, the CRP eluted by buffer A was practically uncontaminated with other proteins.


References 1 Hokama, Y. and Riley, R.F. (1963) Purification of C-reactive protein, an acute phase protein of human serum. Biochim. Biophys. Acta 74, 305-308. 2 Claus, D.R., Osmand, A.P. and Gewurz, H. (1976) Radioimmunoassay of human C-reactive protein and levels in normal sera. J. Lab. Clin. Med. 87, 120-128. 3 Unten, S.K. and Hokama, Y. (1987) Enzyme immunoassay for C-reactive protein analysis. J. Clin. Lab. Anal. 1, 136-139. 4 Hokama, Y., Tam, R., Hirano, W. and Kimura, L. (1974) Significance of C-reactive protein binding by lecithin: a simplified procedure for CRP isolation. Clin. Chim. Acta 50, 53-62. 5 Pepys, M.B., Dash, A.C. and Ashley, M.J. (1977) Isolation of C-reactive protein by affinity chromatography. Clin. Exp. Immunol. 30, 32-37. 6 Volanalds, J.E., Clements, W.L. and Schrohenloher, R.E. (1978) C-reactive protein: purification by affinity chromatography and physicochemical characterization. J. Immunol. Methods 23, 285 295. 7 Gotschlich, E.C. and Lia, Teh-Yung (1967) Structural and immunological studies on the pneumococcal C polysaccharide. J. Biol. Chem. 242; 463-470. 8 Hirai, H., Nunomura, W. and Hatakeyama, M. (1986) Monoclonal antibodies to human C-reactive protein (CRP) and some characterization of rat serum protein retated to CRP. Protides Biol. Fluid. 34, 283-286. 9 Avrameas, S. (1969) Coupling of enzyme to proteins with glutaraldehyde. Use of conjugates for the detection of antigen and antibody. Immunochem. 6, 43-52. 10 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. 11 Ouchterlony, O. (1948) Antigen-antibody reaction in gels. Arkiv. Kern. Mineral Geol. 26B, 14. 12 Osmand, A.P:, Friedenson, B., Gewurz, H., Painter, R.H., Hofmann, T. and Shelton, E. (1977) Characterization of C-reactive protein and complement subcomponent Clt as homologous proteins displaying cyclic pentameric symmetry (pentraxins). Proc. Natl. Acad. Sci. USA 74, 739-743.

Purification of human C-reactive protein by immunoaffinity chromatography using mouse monoclonal antibody.

Human C-reactive protein (CRP) was purified from sera with a high concentration of CRP by immunoaffinity chromatography using mouse monoclonal antibod...
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