265

Journal of Immunological Methods, 138 (1991) 265-271 ~9 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 002217599100149D JIM05896

Purification of human complement factor D from the peritoneal fluid of patients on chronic ambulatory peritoneal dialysis E m m a n u e l l e C a t a n a a n d Jiirg A. Schifferli Dwtsion o/Nephrology, Department of Medicine, H~pital Cantonal Uni~ersitatre, Genes'a, Switzerland (Received 19 November 1990, revised received 17 December 1990. accepted 14 January 1991 )

We describe a simple procedure for the purification of human factor D from the peritoneal fluid (PF) of patients with end stage renal failure (ESRF) on chronic ambulatory peritoneal dialysis (CAPD). The main advantages of this method are: (1) a relative enrichment of factor D in PF versus plasma (factor D / t o t a l protein enriched 3.8-fold);thus, added to the elevated concentration of factor D in ESRF, the enrichment compared to normal human serum is approximately 50 fold. (2) This biological source of factor D is almost unlimited, since around 10 liters of PF are removed per day from each patient. The purification is performed in three simple steps - Bio Rex 70, Heparin Sepharose CL 6B and Mono S FPLC - and milligrams of highly purified factor D are obtained. Peritoneal fluid might be a valuable source for the purification of other low MW proteins which accumulate in renal failure. Key words: Complement factor D; Purification; Peritoneal fluid; Chronic ambulatory peritoneal dialysis; Renal failure; (Human)

Introduction

Factor D, the serine esterase responsible for activating the C3 convertase of the alternative pathway is known to be present in plasma only in its activated form (Lesavre and Miiller-Eberhard, 1978; Schreiber et al., 1978; Lesavre et al., 1979). It consists of a single polypeptide chain with a molecular weight of 24 kDa (Fearon et al., 1974; Fearon and Austen, 1975; Volanakis et al., 1977). The low plasma concentration of this enzyme, 2

Correspondence to: J.A. Schifferli, Division de N6phrologie, H6pital Cantonal Universitaire, 1211 Gen~ve 4, Switzerland. Abbreoiations: CAPD, chronic ambulatory peritoneal dialysis: ESRF, end stage renal failure; NHS, normal human serum; PBS, phosphate-buffered saline; PF, peritoneal fluid; SDSPAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

~ g / m l (Lesavre and Miiller-Eberhard, 1978) is known to increase ten-fold in end stage renal failure (Sturfelt et al., 1985; Volanakis et al., 1985; Pascual et al., 1988, 1989). However, for ethical reasons, it is not possible to remove large quantities of plasma from patients with ESRF in order to purify factor D. Volanakis and Macon (1987) have noticed that factor D was present in the urine of patients with severe tubular dysfunction, and have purified factor D from the urine of a patient with Fanconi's syndrome. However, such patients are rare and urine usually contains only very small amounts of factor D (Pascual et al., 1988). Another unlimited biological source of factor D may be the peritoneal fluid (PF) of patients undergoing chronic ambulatory peritoneal dialysis (CAPD), since around 10 liters are drained every day, and this fluid has been shown to contain

266 proteins of small MW such as /3~ microglobulin (Acchiardo et al., 1989). Thus, we measured factor D in PF in order to evaluate whether it might be used as starting material for factor D purification.

Materials and methods

Bio-Rex 70 was obtained from Bio-Rad (Richmond, CA, U.S.A.). Heparin Sepharose CL 6B, Superose 12, Mono S HR 5 / 5 and the fast liquid chromatography system (FPLC) were obtained from Pharmacia (Uppsala. Sweden). The Milex-GV filter (0.22 ~m) was purchased from Millipore (Molheim, France). (2-morpholino)ethane sulfonic acid monohydrate (MES) was obtained from Fluka (Chemie, Buchs, Switzerland). Chemicals were purchased from Merck, Darmstadt, F.R.G., if not otherwise specified.

was standardized using dilutions of pooled normal serum from 25 donors. Antigenic assa)' for factor D by radioimmunoassay (RIA). The purified factor I) obtained by the present method was radiolabelled with ~25iodineBolton-Hunter reagent (Amersham, Buckinghamshire, U.K.) to a specific activity of 4.21 ~tC//~g. The RIA was standardized using the same pooled normal serum (estimated factor I) concentration: 2 ktg/ml) and a fixed amount of radiolabelled factor D (0.32 ng). Optimal results were obtained by adding in sequence 667 ng of monoclonal antifactor D Ab (incubation 30 min at 37°C). followed by an excess of goat anti-mouse Ab (5 p,I in a final volume 120 p.l PBS-I% polyethylene glycol for 16 h at 4°(').

A ntisera, pol.vclonal and monoclonal antibodies Goat antiserum to mouse IgG was kindly provided by Prof. S. Izui. Rabbit polyclonal (L71) and mouse monoclonal antibodies (50-106) to human factor D have been described previously (Pascual et al., 1990).

Electrophoresis and immunoblot SDS-PAGE using 12% or a linear gradient (5 20%) polyacrylamide were performed according to Laemmli (1970) and stained with silver nitrate (Wray et al.. 1981) or Coomassic Brilliant Blue. The samples were diluted 1/2 (v/v) with the SDS-PAGE sample buffer. Molecular weights were determined using non-reduced marker proteins from Pharmacia (phosphorylase h (94,000), alburnin (67,000), ovalbumin (43,0(X)), carbonic anhydrase (30,000), trypsin inhibitor (20,100) and a-lactalbumin (14,000)). Immunoblots of factor D were performed after transfer to a 0.45/~m pore size nitrocellulose filter (Schleicher and Schull, Feldbach, Switzerland). Binding sites on the nitrocellulose were blocked with 3% Rapilait (Migros, Geneva, Switzerland) in phosphate-buffered saline. After extensive washing with 1% Rapilait-PBS, the nitrocellulose sheets were incubated with rabbit polyclonai anti factor D-antiserum followed by 12~I-labelled protein G (Amersham, Buckinghamshire. U.K.), and revealed by autoradiography.

Haemolytic and antigenic assays"for factor D Haemolvtic assay. Factor D activity was monitored by the lysis of guinea pig erythrocytes in agarose gels using serum immunochemically depleted of factor D (RD) (Martin et al., 1976; Pascual et al., 1988). 1 U of factor D activity was defined as the haemolytic area obtained using 10 ~1 of human serum (i.e., 100 U/ml). The assay

Factor D preparation The pH of PF removed from patients with ESRF on CAPD was adjusted to 6.5 with glycineHCI. The conductivity was adjusted to 14 mS (if above 14 mS) and the PF was then loaded onto a Bio-Rex 70 column (5.6 cm × 30 cm) equilibrated with the buffer described above. Elution was performed at a flow rate of 50 ml/h. 4 liters of PF

Buffers Superose 12 buffer: NaC1 0.15 M, N a / K phosphate 0.01 M, pH 7.4 (PBS). Bio-Rex 70 buffer: 20 mM sodium phosphate. 100 mM NaCI, 2 mM ethylenediaminotetraacetate (EDTA), pH 6.5. Heparin Sepharose CL 6B buffer: 10 mM sodium citrate, 50 mM Tris (hydroxy methyl)aminomethanc, conductivity adjusted to 14 mS with NaCI (room temperature), pH 6. Mono S buffer: 50 mM (2-morpholino)ethane sulfonic acid monohydrate (MES), 30 mM NaC1, pH 6. SDS-PAGE sample buffer: 125 mM Tris-HCl pH 6.8, 8% SDS, 17% glycerol, 0.02% bromophenol blue.

267 were loaded, although later studies indicated that up to 20 liters could be added to the Bio-Rex 70 without loss of factor D in the break-through peak. Factor D was eluted with a 1.5 liters linear concentration gradient to 1 M NaC! in the same buffer and absorbance was monitored at 280 nm. The fractions containing factor D haemolytic activity were pooled (90 ml), concentrated to 20 ml using an Amicon cell with a YM5 membrane (cut off MW: 5000) (Lausanne, Switzerland), and dialysed overnight at 4°C against the Heparin Sepharose buffer, before being loaded onto the heparin Sepharose CL 6B column (2.5 cm × 16 cm) operated at a flow rate of 20 m l / h . Elution was performed with a linear concentration gradient to 1.5 M NaCI. The fractions containing haemolytic factor D were pooled and further purified by chromatography on a strong cation exchanger Mono S H R 5 / 5 column (0.5 cm x 5 cm) by fast protein liquid chromatography (FPLC). All samples and buffers were filtered through Milex GV 0.22 /tin filters prior to use. The column was loaded at a flow rate of 2 m l / m i n and eluted at the same rate with a discontinuous NaCI gradient ranging from 30 mM to 500 mM. The fractions containing the haemolytic factor D activity peak were then immediately pooled and dialysed extensively at 4°C against 0.1 M Tris-HCl, 0.2 M NaCI, 0.002 M EDTA buffer pH 8. The concentration of

the purified factor D was determined by the Folin assay (Lowry et al,, 1951).

Results Because peritoneal fluid in patients on C A P D is known to contain proteins of small MW, we assayed factor D activity in ten such fluids. All PF were positive, although to a variable degree. The mean factor D activity was approximately 2 / 3 of that of pooled N H S (68.9 U / m l (range: 10-223 U / m l ) , versus 100 U / m i ) . Thus, the factor D concentration was much lower than in the patients' sera, which contained as expected between 10- and 15-fold more factor D than N H S (1269 U / m l (725-2000 U / m l ) ) . However, when the protein content was taken into account (serum protein concentration 64.9 g / l (55-73 g/l)), PF protein concentration 1.18 g / l (0.72--2.27 g/l)), most of the PF were enriched in factor D (3.82 fold (0.5-13.5-fold)), indicating that this small MW protein was lost preferentially to larger proteins in PF, as illustrated in Fig. 1. Thus, compared to normal serum, there was an approximate 50-fold enrichment of factor D in PF, i.e., the peritoneal membrane can be considered as being a useful first step in the purification procedure of factor D. To see whether factor D in PF was functionally

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Fig. 1. Gel filtration chromatography (Superose 12) by FPI.C of a patient's serum and the corresponding peritoneal fluid (PF), which shows that PF is poor in high MW proteins. 100 ~.l of 1/2 diluted samples were applied. Markers: IgM in fraction 33, IgG in fraction 50, albumin in fraction 55, and factor D haemolytic activity in fraction 74.

268

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active, we compared the haemolytic/antigenic ratio of factor D from pooled normal serum, and from the PF and sera of the ten patients with ESRD. As previously reported (Volanakis et al., 1985; Pascual et al., 1988), the h a e m o l y t i c / antigenic ratio was similar in normal serum and sera of patients with ESRD (maximum 10% difference). The results for factor D in PF were identical, indicating that it had not lost any activity.

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Factor D was purified in three steps at 4°C. The 4 liters of PF were loaded on the Bio-Rex column, which was washed extensively before being eluted with a salt gradient. This was the most effective purification step removing 99.6% of the loaded proteins (see Table I). The eluted fractions containing factor D were dialysed, applied to Heparin Sepharose, and eluted with a salt gradient (see Fig. 2). The last step was chromatography on M o n o S. This final purification step was conveni-

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