- Email: [email protected]
Dissociation and isolation of antigen and antibody from immune complexes
Journal of Immunological Methods, 53 (1982) 51-59
5l
Elsevier Biomedical Press
Dissociation and Isolation of Antigen and Antibody from Immune Complexes Klaus H6ffken, Friederike Bosse, Ulrike Steih and Carl G. Schmidt Department of Internal Medicine (Cancer Research), West German Tumour Centre, University of Essen, Hufelandstrasse 55, D-4300 Essen 1, F. R. G.
(Received 14 October 1981, accepted 3 February 1982)
Ion-exchange chromatography in dissociating buffers or isoelectric focusing were used to isolate antigen or antibody from HSA-anti-HSA immune complexes (ICs). Both methods permitted dissociation of ICs and isolation of antigen and antibody components, thus providing a reliable means for a more accurate definition of the various antigens involved in immune complex diseases.
Key words: immune complexes - - isolation of immune complexes - - isoelectric focusing - - ion-exchange chromatography
Introduction A n t i g e n - a n t i b o d y c o m p l e x e s are k n o w n to be involved in the p a t h o g e n e s i s of a v a r i e t y of diseases ( M a i n i a n d H o l b o r o w , 1977; T h e o f i l o p o u l o s a n d Dixon, 1979; .H/3ffken a n d Schmidt, 1980). Evidence for this has been o b t a i n e d b y using several r e p r o d u c i b l e assays a i m e d at d e t e r m i n i n g i m m u n e c o m p l e x e s (ICs) in b o d y fluids, a l t h o u g h at p r e s e n t these assays d o not c o n f o r m to agreed s t a n d a r d s . W h i l e a c o m p a r i s o n of various m e t h o d s q u a n t i f y i n g reference p r e p a r a t i o n s of aggregated i m m u n o g l o b u l i n s showed g o o d correlation b e t w e e n some of the tests, conflicting results were o b t a i n e d when specific sera were assayed. Thus, for example, sera collected f r o m p a t i e n t s with different diseases were f o u n d to be positive for ICs in o n e assay b u t negative in a n o t h e r ( L a m b e r t et al., 1978). This is not surprising c o n s i d e r i n g the different principles u n d e r l y i n g the assays as well as the different c o m p o s i t i o n of the ICs e n c o u n t e r e d in different diseases. These limitations could be Abbreviations: HSA, human serum-albumin; anti-HSA, anti-HSA antiserum: HSA-anti-HSA, HSA-anti-
HSA immune complexes; ICs, immune complexes; ID, immunodiffusion; IgG, immunoglobulin G; anti-hum IgG, anti-human IgG antiserum; anti-rab IgG, anti-rabbit IgG antiserum; PBS, phosphatebuffered saline; SpA, Staphylococcus protein A. 0022-1759/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
52 overcome by specifically measuring the antigen moiety involved in the formation of ICs, but most methods currently in use are antigen-non-specific quantifying either the antibody or the complement components attached to the ICs. In order to develop antigen-specific IC assays, several attempts have been made to isolate and characterize ICs (reviewed by Jones and Orlans, 1981) including: (a) the dissociation of antigen and antibody by acid, alkaline or high-molar salt solutions followed by filtration methods (Kabat and Mayer, 1961: Givol et al., 1962: Slobin and Sela, 1965); (b) the generation in immunoglobulin-tolerant rabbits of antibodies against the antigen moiety of ICs bound to Raji cells (Theofilopoulos et al., 1978; Koestler et al., 1981); and (c) the separation of antigens from antibodies by isoelectric focusing (Maidment et al., 1980, 1981). The experiments described here were undertaken to evaluate the applicability of ion-exchange chromatography or isoelectric focusing to the isolation of antigens involved in IC formation. It was found that ICs were dissociated readily by high-molar solutions of urea at neutral pH or by acid citrate buffer as well as by electrofocusing. In addition, the antigen component could be recovered by preparative isoelectric focusing or ion-exchange chromatography and the antibody moiety isolated by ion-exchange chromatography.
Material and Methods
Preparation of immune complexes in vitro Equal volumes of human serum albumin (HSA; Sigma, Munich) at 25 mg/ml of phosphate-buffered saline (PBS, pH 7.4; Oxoid, Wesel) and the immunoglobulin fraction of rabbit anti-HSA antiserum (anti-HSA; Dako, Boehringer-Ingelheim, Garching) were incubated at 37°C for 1 h and at 4°C for 2 days before being applied to a staphylococcus protein A-Sepharose 4B column (SPA; Pharmacia, Freiburg) equilibrated in PBS containing 0.01% sodium azide (NAN3). Elution of unbound material was continued until optical density returned to the background level in an LKB chromatography unit. Thereafter, material bound to protein A was displaced by 2.5 M sodium thiocyanate (NaSCN). Salt was separated from protein by filtration on a column of Sephadex G-25. The binding and non-binding fractions, respectively, were pooled, concentrated by dialysis against Aquacide II (Calibiochem, La Jolla, CA), dialyzed against PBS at 4°C for 18h and stored at - 2 0 ° C until use. The composition of the ICs was analyzed by double immunodiffusion against anti-HSA or anti-rabbit immunoglobulin antiserum, demonstrating the presence of both HSA and rabbit immunoglobulin in the fraction bound to protein A (data not shown). Isoelectric focusing of the unbound material revealed that excess HSA antigen had been removed from the IC preparation (Fig. 3a). To obtain a sufficient amount of ICs for further analysis by ion-exchange chromatography or preparative isoelectric focusing, binding material from 5 consecutive SpA elutions was pooled at a final protein concentration of 20 mg/ml as estimated by Lowry's method (1951) with crystalline bovine serum albumin as standard.
53
Buffers Phosphate buffer was prepared by adding 0.01 M N a H z P O 4 to 0.01 M N a 2 H P O 4 to give a final pH of 7.4. Citrate buffer was prepared as outlined in the Geigy Scientific Tables. Solutions A consisted of 0.1 M or 1.0 M citric acid monohydrate, containing 200 ml/1 of 1 N or 10 N NaOH. Solutions B consisted of 0.1 N or 1.0 N HC1. Mixing 399 ml of solution A with 601 ml of solution B gave 0.1 M or 1.0 M citrate buffers, pH 3.0.
Ion-exchange chromatography A 2.5 × 37 cm column was prepared with DEAE 52 cellulose (Whatman, Ferri6res) equilibrated in 0.01 M phosphate buffer containing 8 M urea, pH 7.3, and kept at room temperature. HSA and human IgG dissolved in the starting buffer were used as standards as shown in Fig. 1. After recovery of the non-binding fraction, material bound to the anion exchanger was eluted with 0.01 M phosphate buffer containing 0.5 M NaC1 and 8 M urea, pH 7.4. Fractions of 150 drops were collected, pooled, concentrated to the original volumes and dialyzed as described. A 2.5 × 42 cm CM-Sepharose CL-6B column (Pharmacia) was equilibrated in 0.1 M citrate buffer, p H 3.0, and kept at 4°C. Samples applied to the column were dissolved in (or dialyzed for 24 h against) the equilibrating buffer. Elution was performed with a linear salt gradient of increasing ionic strength. The gradient was prepared in a gradient former by mixing 1 liter 0.1 M citrate buffer with 1 liter 1.0 M citrate buffer, both at pH 3.0.
Analytical isoelectricfocusing Analytical flat bed electrofocusing was performed according to the method outlined in LKB Application Note 250, using LKB Ampholine PAG plate gels, pH 3.5-9.5. Samples were applied to filter papers or directly to the gel surface and focused at 10 W constant power, 600 V and 25 mA at 10°C for 3 h. Fixing, staining and destaining was performed according to the Application Note mentioned.
Preparative isoelectricfocusing Preparative electrofocusing was performed according to the method outlined in LKB Application Note 198. Briefly, the granular bed was prepared by mixing 5 g Ultrodex (LKB) with 100 ml of 2% ampholytes (pH 3.5-10; LKB) and with 2 ml of the test sample. After evaporation of 27% of the original gel weight, electrofocusing was performed at 8 W constant power, 500V and 15 mA at 10°C for 16-18 h. Anode buffer was 1 M H3PO 4 and cathode buffer was 1 M NaOH. At the end of the run an imprint was prepared by applying a Whatman filter paper no. 1 to the gel surface for 2 min before pressing a 30-zone fractionating grid into the gel bed. The p H gradient was determined with a surface p H electrode (LKB) and fractions of 3 - 4 zones were collected and eluted with PBS through P E G G elution columns (LKB). Fractions were concentrated to the original sample volume before tested by immunodiffusion.
Immunodiffusion Double immunodiffusion was performed by the method of Ouchterlony using 1% agar (Difco Labs., Detroit, MI) in PBS containing 0.01% NaN 3. Antisera were rabbit
54
immunoglobulins against HSA (anti-HSA; Dako, Boehringer-lngelheim, Garching), rabbit immunoglobulin against human IgG (anti-hum IgG, Dako, Boehringer-lngelheim), and goat antiserum against rabbit IgG (anti-rab IgG; Hyland, Munich).
Results
A nion-exchange chromatography As shown in Fig. l, chromatography of a mixture of HSA and IgG on DEAE 52 cellulose equilibrated in 0.01 M phosphate buffer containing 8 M urea, pH 7.4,
oasj DEAE 52
a HSA",'IgG
0,01M PO4 0,5 M NaCL IgG 8 M UREA
o 0.4 0.3 o 0.2 O 0.1
L
1'0 I._D~.HSA -~lgG 4-
HSA(IgG)
2'0
3~0 4-
(4-)
4'0 5~0 FRACTIONNUMBER (x 150 drops)
DEAE 52
b HSA-~HSA
0.4
Ec 0"5l 0.3 ~ 0.10"2 O
o.s
0.01M PO M No~:L 8 M UREA 1 ~HSA(zHSA)
~
I
1'0
2~0
L~ d.HSA --
3~0 4-
(4-)
SpA-SEPHAROSE 4B
E
c 2nd
0.~
~ .
4'0 5'0 FRACTIONNUMBER (x 150 dropsl peak from_b_
HSA2.5M NaSCN
O0".~ t0
I_D~HSA &rablgG
4--
1'0
4(4-)
2~0 3'0 FRACTIONNUMBER (x 150 drops/
Fig. 1. Isolation of antigen and antibody by anion-exchange chromatography followed by affinity chromatography, a: stepwise elution of a mixture of HSA and human IgG dissolved in equilibrating buffer (8 M urea in 0.01 M phosphate buffer, pH 7.4) by adding 0.5 M NaC1 to the starting buffer. lmmunodiffusion analysis (ID) showed the content of the 2 fractions obtained, b: stepwise elution of HSA-anti-HSA immune complexes [HSA-aHSA] previously dialyzed against the equilibrating buffer. Immunodiffusion analysis (ID) shows that the first peak contained rabbit anti-HSA immunoglobulin only and the second peak, developed by adding 0.5 M NaCI to the equilibration buffer, contained HSA and trace amounts of immunoglobulin, c: protein A-Sepharose 4B affinity chromatography of the second peak recovered from ion-exchange chromatography (Fig. lb). The first peak contained HSA but no immunoglobulin whereas the second peak contained the remaining ICs as determined by immunodiffusion (ID).
55 resulted in a first peak of IgG. After increasing the ionic strength of the eluting buffer by adding 0.5. M NaCI, the fraction that had bound to the exchanger eluted from the column which contained HSA and trace amounts of IgG (Fig. la). Following dialysis of HSA-anti-HSA immune complexes against the starting buffer at 4°C for 24 h, a first peak of anti-HSA and a second peak containing a mixture of antigen and antibody was obtained (Fig. lb). After dialysis against PBS and concentration to the original sample volume, the second peak was re-chromatographed on a column of protein A-Sepharose (Fig. lc). Subsequent analysis for antigen and antibody by radial immunodiffusion indicated that the non-binding fraction contained only HSA whereas the binding fraction eluted with 2.5 M NaSCN contained both antigen and antibody.
Cation-exchange chromatography CM-Sepharose cation-exchange chromatography of HSA-anti-HSA immune complexes previously dissociated in acid citrate buffer, gave a first peak containing the anti-HSA antibody and a second peak containing a mixture of HSA and anti-HSA antibody (Fig. 2). Unlike anion-exchange chromatography, both proteins were positively charged at the pH of the buffer, and bound to the exchanger. Subsequent elution was by a linear salt gradient of constant low pH which precluded re-association of antigen and antibody.
C M - S E P H A R O S E in 0.1 M CITRATE BUFFER 0.50.4E
o 0.3-
Go
04 a
00.20.1-
,6o
I
I
I
I ii I lii iv FRACTION NUMBER (x150 drops)
I_.D d. H S A
aCrab IgG
I
v
--
--
--
+
+
+
+ +
+
{+)
(+)
I
Fig. 2. Elution profile of HSA-anti-HSA immune complexes on CM-Sepharose cation-exchange chromatography. ICs were dialyzed against the equilibration buffer at 4°C for 24 h and elution was performed with a linear gradient of increasing ionic strength at constant pH. Immunodiffusion analysis (ID) shows localization of HSA and rabbit anti-HSA immunoglobulin.
56
Isoelectric focusing I n accordance with the f i n d i n g previously reported by M a i d m e n t et al. (1980), Fig. 3 shows that i m m u n e complexes can be dissociated by analytical isoelectric focusing. Subsequently, preparative electrofocusing was used to investigate to what extent separation of antigen from a n t i b o d y was possible. As shown in Fig. 4, fractions 3 - 5 c o n t a i n e d both H S A a n d a n t i - H S A a n t i b o d y whereas the antigen was c o n f i n e d to fraction 2 a n d the a n t i b o d y to fractions 6 a n d 7. Z o n e 8 was omitted from fraction 2 since it already c o n t a i n e d i m m u n o g l o b u l i n .
a
b
c
d
Fig. 3. Separation of I-ISA-anti-HSA immune complexes by analytical isoelectric focusing, a: HSA obtained as non-bindingfraction from protein A chromatography of HSA-anti~HSAICs indicating that the ICs generated in vitro were in antigen excess before affinity chromatography on protein A. b: HSA-anti-HSA ICs bound to and eluted from protein A-Sepharose 4B. c: rabbit anti-HSA immunoglobulins bound to and eluted from protein A-Sepharose 4B. d: HSA (30/~g dissolved in 5 ~1 PBS).
57
115
~,ob ~
:
(¥)
-~
+
+ +
-
-
Fig. 4. Imprint showing localization of the major protein bands obtained by preparative isoelectric focusing of 40 mg HSA-anti-HSA immune complexes. Immunodiffusion analysis (ID) indicates the composition of the 8 fractions collected on termination of the procedure. The pH gradient was measured with a surface pH electrode.
Discussion
These experiments show that ion-exchange chromatography at neutral or acid p H is capable of isolating antigen and antibody from previously dissociated ICs. By a modification of the method described by Slobin and Sela (1965) dissociation was achieved by the use of 8 M urea in neutral phosphate buffer or by acid citrate buffer. It m a y be noted that the choice of buffer appears important for maintaining the immunological and biochemical properties of both the antigen and the antibody which may suffer alteration during dissociation, when acid or alkaline buffers are used (Slobin and Sela, 1965). In contrast the effect of urea or an electric field on protein structure is thought to be negligible. With the model ICs used in the present experiments, however, reconstitution of antigen-antibody complexes by immunodiffusion was successful only with fractions derived from cation-exchange chromatography at acid pH. The other procedures used apparently altered the properties of the isolated antigen and antibody so that no precipitation occurred upon immunodiffusion analysis (data not shown). In accordance with the findings of Maidment et al. (1980, 1981), dissociation of ICs was also achieved by analytical and preparative isoelectric focusing. However, the results of immunodiffusion analysis of the preparative electrofocusing fractions showed that only the antigen component can be isolated in sufficient amounts, the antibody fractions (pH > 5.0) being heavily contaminated with antigen. The essential steps suggested for the isolation of antigen and antibody moieties
58
from ICs are schematically outlined in Fig. 5. ICs from sera or other body fluids can be initially separated by gel filtration followed by affinity chromatography on agents with known affinity for ICs such as staphylococcal protein A, conglutinin or Clq. The antigen component of the ICs may then be recovered by preparative isoelectric focusing, provided the antigen has an isoelectric point similar to albumin (pl 4.7). Alternati,~ely, after dissociation of ICs with high-molar urea or acid citrate the antigen can be isolated by ion-exchange chromatography followed by affinity chromatography on protein A. The antibody moiety may be simultaneously isolated by ion-exchange chromatography in dissociating buffers. It should be noted, however, that the procedure employed for preparation and partial purification of the model ICs does not exclude the presence of non-specific rabbit IgG contaminants in the antibody fractions. No attempt was made in the present investigation to analyze this possibility further, mainly because the separation of immunoglobulin (both non-antibody and antibody IgG) from albumin suggested that the ICs present in the original preparation had been dissociated. Several attempts have been reported to isolate antigen from ICs present in the sera of patients with a variety of benign and malignant diseases. In most of these studies gel filtration in dissociating buffers has been used to obtain information about the properties of eluants and gel matrices. However, these attempts have encountered difficulties in developing standard procedures, in the absence of defined antigen-antibody systems. This accords with findings from similar studies in this laboratory. Using methods described for successful isolation of antigen from ICs by gel filtration (e.g., Bowen and Baldwin, 1976; Papsidero et al., 1978; Kilpatrick and Virella, 1980) we were unable to dissociate and separate model ICs of high affinity or ICs present in sera from breast cancer patients (unpublished). Thus further investigations were warranted in the attempt to establish reproducible methods for
pH
JC
~ GELFILTRATION
19 S
eL
PROTEIN-A
PREPARATIVE IEF
3-4.7
~ ANTIGEN
2ndpeok
SEPHAROSE ANTIBOOY
ION-EXCHANGE /st peak 2ndpeak
CHROMATOGRAPHY
_
~PROTEIN A SEPHAROSE 1
stlpeak
ANTIGEN
Fig. 5. Schematic outline of the major steps for partial purification and dissociation of immune complexes from body fluids and subsequent isolation of antigen and antibody moieties.
59
the isolation of antigen and antibody from preformed ICs of high affinity. From the results obtained it can be concluded that ion-exchange chromatography in dissociating buffers and isoelectric focusing permit the isolation~of antigen and antibody moieties from such ICs. Although only artificial ICs were analyzed here, the separation procedures described may prove of value in the detailed analysis of other ICs. Investigations are now in progress to define the antigens involved in the formation of ICs present in pleural or peritoneal effusions from various cancer patients.
Acknowledgements This study was supported in part by grants from the Landesamt for Forschung, Ministerium ftir Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Dtisseldorf, and the Deutsche Krebshilfe. We are indebted to Miss I. Rademacher for technical and to Mrs. G. B6ttcher for secretarial assistance. Dr. Valerie E. Moore and Dr. H. Von Melchner are thanked for critical review of the manuscript.
References Bowen, J.G. and R.W. Baldwin, 1976, Transplantation 21,213. Bryce, C.F.A. and R.R. Chrichton, 1971, J. Chromatogr. 63, 267. Givol, D., S. Fuchs and M. Sela, 1962, Biochim. Biophys. Acta 63, 222. H6ffken, K. and C.G. Schmidt, 1980, Dtsch. Med. Wschr. 105, 1697. Jones, V.E. and E. Orlans, 1981, J. lmmunol. Methods 44, 249. Kabat, E.A. and M.M. Mayer, 1961, Experimental Immunochemistry (Thomas, Springfield, IL) Ch. 5i. Kilpatrick, J.M. and G. Virella, 1980, Clin. Exp. Immunol. 40, 445. Koestler, T.P., L.D. Papsidero, T. Nemoto and T.M. Chu, 1981, Cancer Res. 41, 2900. Lambert, P.H., F.J. Dixon, R.H. Zubler, V. AgneUo, C. Cambiaso, P. Casali, J. Clarke, J.S. Cowdery, F.C. McDuffie, F.C. Hay, I.C.M. MacLennan, P. Masson, H.J. Maller-Eberhard, K. Pettinen, M. Smith, G. Tappeiner, A.N. Theofilopoulos and P. Verroust, 1978, J. Clin. Lab. lmmunol. 1, 1. Lowry, O.H., N.L. Rosebrough, R.J. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Maidment, B.W., L.D. Papsidero and T.M. Chu, 1980, J. lmmunol. Meth. 35, 297. Maidment, B.W., L.D. Papsidero, T. Nemoto and T.M. Chu, 1981, Cancer Res. 41,795. Maini, R.N. and E.J. Holborow, 1977, Ann. Rheum. Dis. 36 (Suppl. 1). Papsidero, L.D., S.R. Harvey, M.C. Snyderman, T. Nemoto, L. Valenzuela and T.M. Chu, 1978, Int. J. Cancer 21,675. Slobin, L.I. and M. Sela, 1965, Biochim. Biophys. Acta 107, 593. Theofilopoulos, A.N. and F.J. Dixon, 1979, Adv. Immunol. 28, 89. Theofilopoulos, A.N., R.A. Eisenberg and F.J. Dixon, 1978, J. Clin. Invest. 61, 1570.
5l
Elsevier Biomedical Press
Dissociation and Isolation of Antigen and Antibody from Immune Complexes Klaus H6ffken, Friederike Bosse, Ulrike Steih and Carl G. Schmidt Department of Internal Medicine (Cancer Research), West German Tumour Centre, University of Essen, Hufelandstrasse 55, D-4300 Essen 1, F. R. G.
(Received 14 October 1981, accepted 3 February 1982)
Ion-exchange chromatography in dissociating buffers or isoelectric focusing were used to isolate antigen or antibody from HSA-anti-HSA immune complexes (ICs). Both methods permitted dissociation of ICs and isolation of antigen and antibody components, thus providing a reliable means for a more accurate definition of the various antigens involved in immune complex diseases.
Key words: immune complexes - - isolation of immune complexes - - isoelectric focusing - - ion-exchange chromatography
Introduction A n t i g e n - a n t i b o d y c o m p l e x e s are k n o w n to be involved in the p a t h o g e n e s i s of a v a r i e t y of diseases ( M a i n i a n d H o l b o r o w , 1977; T h e o f i l o p o u l o s a n d Dixon, 1979; .H/3ffken a n d Schmidt, 1980). Evidence for this has been o b t a i n e d b y using several r e p r o d u c i b l e assays a i m e d at d e t e r m i n i n g i m m u n e c o m p l e x e s (ICs) in b o d y fluids, a l t h o u g h at p r e s e n t these assays d o not c o n f o r m to agreed s t a n d a r d s . W h i l e a c o m p a r i s o n of various m e t h o d s q u a n t i f y i n g reference p r e p a r a t i o n s of aggregated i m m u n o g l o b u l i n s showed g o o d correlation b e t w e e n some of the tests, conflicting results were o b t a i n e d when specific sera were assayed. Thus, for example, sera collected f r o m p a t i e n t s with different diseases were f o u n d to be positive for ICs in o n e assay b u t negative in a n o t h e r ( L a m b e r t et al., 1978). This is not surprising c o n s i d e r i n g the different principles u n d e r l y i n g the assays as well as the different c o m p o s i t i o n of the ICs e n c o u n t e r e d in different diseases. These limitations could be Abbreviations: HSA, human serum-albumin; anti-HSA, anti-HSA antiserum: HSA-anti-HSA, HSA-anti-
HSA immune complexes; ICs, immune complexes; ID, immunodiffusion; IgG, immunoglobulin G; anti-hum IgG, anti-human IgG antiserum; anti-rab IgG, anti-rabbit IgG antiserum; PBS, phosphatebuffered saline; SpA, Staphylococcus protein A. 0022-1759/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
52 overcome by specifically measuring the antigen moiety involved in the formation of ICs, but most methods currently in use are antigen-non-specific quantifying either the antibody or the complement components attached to the ICs. In order to develop antigen-specific IC assays, several attempts have been made to isolate and characterize ICs (reviewed by Jones and Orlans, 1981) including: (a) the dissociation of antigen and antibody by acid, alkaline or high-molar salt solutions followed by filtration methods (Kabat and Mayer, 1961: Givol et al., 1962: Slobin and Sela, 1965); (b) the generation in immunoglobulin-tolerant rabbits of antibodies against the antigen moiety of ICs bound to Raji cells (Theofilopoulos et al., 1978; Koestler et al., 1981); and (c) the separation of antigens from antibodies by isoelectric focusing (Maidment et al., 1980, 1981). The experiments described here were undertaken to evaluate the applicability of ion-exchange chromatography or isoelectric focusing to the isolation of antigens involved in IC formation. It was found that ICs were dissociated readily by high-molar solutions of urea at neutral pH or by acid citrate buffer as well as by electrofocusing. In addition, the antigen component could be recovered by preparative isoelectric focusing or ion-exchange chromatography and the antibody moiety isolated by ion-exchange chromatography.
Material and Methods
Preparation of immune complexes in vitro Equal volumes of human serum albumin (HSA; Sigma, Munich) at 25 mg/ml of phosphate-buffered saline (PBS, pH 7.4; Oxoid, Wesel) and the immunoglobulin fraction of rabbit anti-HSA antiserum (anti-HSA; Dako, Boehringer-Ingelheim, Garching) were incubated at 37°C for 1 h and at 4°C for 2 days before being applied to a staphylococcus protein A-Sepharose 4B column (SPA; Pharmacia, Freiburg) equilibrated in PBS containing 0.01% sodium azide (NAN3). Elution of unbound material was continued until optical density returned to the background level in an LKB chromatography unit. Thereafter, material bound to protein A was displaced by 2.5 M sodium thiocyanate (NaSCN). Salt was separated from protein by filtration on a column of Sephadex G-25. The binding and non-binding fractions, respectively, were pooled, concentrated by dialysis against Aquacide II (Calibiochem, La Jolla, CA), dialyzed against PBS at 4°C for 18h and stored at - 2 0 ° C until use. The composition of the ICs was analyzed by double immunodiffusion against anti-HSA or anti-rabbit immunoglobulin antiserum, demonstrating the presence of both HSA and rabbit immunoglobulin in the fraction bound to protein A (data not shown). Isoelectric focusing of the unbound material revealed that excess HSA antigen had been removed from the IC preparation (Fig. 3a). To obtain a sufficient amount of ICs for further analysis by ion-exchange chromatography or preparative isoelectric focusing, binding material from 5 consecutive SpA elutions was pooled at a final protein concentration of 20 mg/ml as estimated by Lowry's method (1951) with crystalline bovine serum albumin as standard.
53
Buffers Phosphate buffer was prepared by adding 0.01 M N a H z P O 4 to 0.01 M N a 2 H P O 4 to give a final pH of 7.4. Citrate buffer was prepared as outlined in the Geigy Scientific Tables. Solutions A consisted of 0.1 M or 1.0 M citric acid monohydrate, containing 200 ml/1 of 1 N or 10 N NaOH. Solutions B consisted of 0.1 N or 1.0 N HC1. Mixing 399 ml of solution A with 601 ml of solution B gave 0.1 M or 1.0 M citrate buffers, pH 3.0.
Ion-exchange chromatography A 2.5 × 37 cm column was prepared with DEAE 52 cellulose (Whatman, Ferri6res) equilibrated in 0.01 M phosphate buffer containing 8 M urea, pH 7.3, and kept at room temperature. HSA and human IgG dissolved in the starting buffer were used as standards as shown in Fig. 1. After recovery of the non-binding fraction, material bound to the anion exchanger was eluted with 0.01 M phosphate buffer containing 0.5 M NaC1 and 8 M urea, pH 7.4. Fractions of 150 drops were collected, pooled, concentrated to the original volumes and dialyzed as described. A 2.5 × 42 cm CM-Sepharose CL-6B column (Pharmacia) was equilibrated in 0.1 M citrate buffer, p H 3.0, and kept at 4°C. Samples applied to the column were dissolved in (or dialyzed for 24 h against) the equilibrating buffer. Elution was performed with a linear salt gradient of increasing ionic strength. The gradient was prepared in a gradient former by mixing 1 liter 0.1 M citrate buffer with 1 liter 1.0 M citrate buffer, both at pH 3.0.
Analytical isoelectricfocusing Analytical flat bed electrofocusing was performed according to the method outlined in LKB Application Note 250, using LKB Ampholine PAG plate gels, pH 3.5-9.5. Samples were applied to filter papers or directly to the gel surface and focused at 10 W constant power, 600 V and 25 mA at 10°C for 3 h. Fixing, staining and destaining was performed according to the Application Note mentioned.
Preparative isoelectricfocusing Preparative electrofocusing was performed according to the method outlined in LKB Application Note 198. Briefly, the granular bed was prepared by mixing 5 g Ultrodex (LKB) with 100 ml of 2% ampholytes (pH 3.5-10; LKB) and with 2 ml of the test sample. After evaporation of 27% of the original gel weight, electrofocusing was performed at 8 W constant power, 500V and 15 mA at 10°C for 16-18 h. Anode buffer was 1 M H3PO 4 and cathode buffer was 1 M NaOH. At the end of the run an imprint was prepared by applying a Whatman filter paper no. 1 to the gel surface for 2 min before pressing a 30-zone fractionating grid into the gel bed. The p H gradient was determined with a surface p H electrode (LKB) and fractions of 3 - 4 zones were collected and eluted with PBS through P E G G elution columns (LKB). Fractions were concentrated to the original sample volume before tested by immunodiffusion.
Immunodiffusion Double immunodiffusion was performed by the method of Ouchterlony using 1% agar (Difco Labs., Detroit, MI) in PBS containing 0.01% NaN 3. Antisera were rabbit
54
immunoglobulins against HSA (anti-HSA; Dako, Boehringer-lngelheim, Garching), rabbit immunoglobulin against human IgG (anti-hum IgG, Dako, Boehringer-lngelheim), and goat antiserum against rabbit IgG (anti-rab IgG; Hyland, Munich).
Results
A nion-exchange chromatography As shown in Fig. l, chromatography of a mixture of HSA and IgG on DEAE 52 cellulose equilibrated in 0.01 M phosphate buffer containing 8 M urea, pH 7.4,
oasj DEAE 52
a HSA",'IgG
0,01M PO4 0,5 M NaCL IgG 8 M UREA
o 0.4 0.3 o 0.2 O 0.1
L
1'0 I._D~.HSA -~lgG 4-
HSA(IgG)
2'0
3~0 4-
(4-)
4'0 5~0 FRACTIONNUMBER (x 150 drops)
DEAE 52
b HSA-~HSA
0.4
Ec 0"5l 0.3 ~ 0.10"2 O
o.s
0.01M PO M No~:L 8 M UREA 1 ~HSA(zHSA)
~
I
1'0
2~0
L~ d.HSA --
3~0 4-
(4-)
SpA-SEPHAROSE 4B
E
c 2nd
0.~
~ .
4'0 5'0 FRACTIONNUMBER (x 150 dropsl peak from_b_
HSA2.5M NaSCN
O0".~ t0
I_D~HSA &rablgG
4--
1'0
4(4-)
2~0 3'0 FRACTIONNUMBER (x 150 drops/
Fig. 1. Isolation of antigen and antibody by anion-exchange chromatography followed by affinity chromatography, a: stepwise elution of a mixture of HSA and human IgG dissolved in equilibrating buffer (8 M urea in 0.01 M phosphate buffer, pH 7.4) by adding 0.5 M NaC1 to the starting buffer. lmmunodiffusion analysis (ID) showed the content of the 2 fractions obtained, b: stepwise elution of HSA-anti-HSA immune complexes [HSA-aHSA] previously dialyzed against the equilibrating buffer. Immunodiffusion analysis (ID) shows that the first peak contained rabbit anti-HSA immunoglobulin only and the second peak, developed by adding 0.5 M NaCI to the equilibration buffer, contained HSA and trace amounts of immunoglobulin, c: protein A-Sepharose 4B affinity chromatography of the second peak recovered from ion-exchange chromatography (Fig. lb). The first peak contained HSA but no immunoglobulin whereas the second peak contained the remaining ICs as determined by immunodiffusion (ID).
55 resulted in a first peak of IgG. After increasing the ionic strength of the eluting buffer by adding 0.5. M NaCI, the fraction that had bound to the exchanger eluted from the column which contained HSA and trace amounts of IgG (Fig. la). Following dialysis of HSA-anti-HSA immune complexes against the starting buffer at 4°C for 24 h, a first peak of anti-HSA and a second peak containing a mixture of antigen and antibody was obtained (Fig. lb). After dialysis against PBS and concentration to the original sample volume, the second peak was re-chromatographed on a column of protein A-Sepharose (Fig. lc). Subsequent analysis for antigen and antibody by radial immunodiffusion indicated that the non-binding fraction contained only HSA whereas the binding fraction eluted with 2.5 M NaSCN contained both antigen and antibody.
Cation-exchange chromatography CM-Sepharose cation-exchange chromatography of HSA-anti-HSA immune complexes previously dissociated in acid citrate buffer, gave a first peak containing the anti-HSA antibody and a second peak containing a mixture of HSA and anti-HSA antibody (Fig. 2). Unlike anion-exchange chromatography, both proteins were positively charged at the pH of the buffer, and bound to the exchanger. Subsequent elution was by a linear salt gradient of constant low pH which precluded re-association of antigen and antibody.
C M - S E P H A R O S E in 0.1 M CITRATE BUFFER 0.50.4E
o 0.3-
Go
04 a
00.20.1-
,6o
I
I
I
I ii I lii iv FRACTION NUMBER (x150 drops)
I_.D d. H S A
aCrab IgG
I
v
--
--
--
+
+
+
+ +
+
{+)
(+)
I
Fig. 2. Elution profile of HSA-anti-HSA immune complexes on CM-Sepharose cation-exchange chromatography. ICs were dialyzed against the equilibration buffer at 4°C for 24 h and elution was performed with a linear gradient of increasing ionic strength at constant pH. Immunodiffusion analysis (ID) shows localization of HSA and rabbit anti-HSA immunoglobulin.
56
Isoelectric focusing I n accordance with the f i n d i n g previously reported by M a i d m e n t et al. (1980), Fig. 3 shows that i m m u n e complexes can be dissociated by analytical isoelectric focusing. Subsequently, preparative electrofocusing was used to investigate to what extent separation of antigen from a n t i b o d y was possible. As shown in Fig. 4, fractions 3 - 5 c o n t a i n e d both H S A a n d a n t i - H S A a n t i b o d y whereas the antigen was c o n f i n e d to fraction 2 a n d the a n t i b o d y to fractions 6 a n d 7. Z o n e 8 was omitted from fraction 2 since it already c o n t a i n e d i m m u n o g l o b u l i n .
a
b
c
d
Fig. 3. Separation of I-ISA-anti-HSA immune complexes by analytical isoelectric focusing, a: HSA obtained as non-bindingfraction from protein A chromatography of HSA-anti~HSAICs indicating that the ICs generated in vitro were in antigen excess before affinity chromatography on protein A. b: HSA-anti-HSA ICs bound to and eluted from protein A-Sepharose 4B. c: rabbit anti-HSA immunoglobulins bound to and eluted from protein A-Sepharose 4B. d: HSA (30/~g dissolved in 5 ~1 PBS).
57
115
~,ob ~
:
(¥)
-~
+
+ +
-
-
Fig. 4. Imprint showing localization of the major protein bands obtained by preparative isoelectric focusing of 40 mg HSA-anti-HSA immune complexes. Immunodiffusion analysis (ID) indicates the composition of the 8 fractions collected on termination of the procedure. The pH gradient was measured with a surface pH electrode.
Discussion
These experiments show that ion-exchange chromatography at neutral or acid p H is capable of isolating antigen and antibody from previously dissociated ICs. By a modification of the method described by Slobin and Sela (1965) dissociation was achieved by the use of 8 M urea in neutral phosphate buffer or by acid citrate buffer. It m a y be noted that the choice of buffer appears important for maintaining the immunological and biochemical properties of both the antigen and the antibody which may suffer alteration during dissociation, when acid or alkaline buffers are used (Slobin and Sela, 1965). In contrast the effect of urea or an electric field on protein structure is thought to be negligible. With the model ICs used in the present experiments, however, reconstitution of antigen-antibody complexes by immunodiffusion was successful only with fractions derived from cation-exchange chromatography at acid pH. The other procedures used apparently altered the properties of the isolated antigen and antibody so that no precipitation occurred upon immunodiffusion analysis (data not shown). In accordance with the findings of Maidment et al. (1980, 1981), dissociation of ICs was also achieved by analytical and preparative isoelectric focusing. However, the results of immunodiffusion analysis of the preparative electrofocusing fractions showed that only the antigen component can be isolated in sufficient amounts, the antibody fractions (pH > 5.0) being heavily contaminated with antigen. The essential steps suggested for the isolation of antigen and antibody moieties
58
from ICs are schematically outlined in Fig. 5. ICs from sera or other body fluids can be initially separated by gel filtration followed by affinity chromatography on agents with known affinity for ICs such as staphylococcal protein A, conglutinin or Clq. The antigen component of the ICs may then be recovered by preparative isoelectric focusing, provided the antigen has an isoelectric point similar to albumin (pl 4.7). Alternati,~ely, after dissociation of ICs with high-molar urea or acid citrate the antigen can be isolated by ion-exchange chromatography followed by affinity chromatography on protein A. The antibody moiety may be simultaneously isolated by ion-exchange chromatography in dissociating buffers. It should be noted, however, that the procedure employed for preparation and partial purification of the model ICs does not exclude the presence of non-specific rabbit IgG contaminants in the antibody fractions. No attempt was made in the present investigation to analyze this possibility further, mainly because the separation of immunoglobulin (both non-antibody and antibody IgG) from albumin suggested that the ICs present in the original preparation had been dissociated. Several attempts have been reported to isolate antigen from ICs present in the sera of patients with a variety of benign and malignant diseases. In most of these studies gel filtration in dissociating buffers has been used to obtain information about the properties of eluants and gel matrices. However, these attempts have encountered difficulties in developing standard procedures, in the absence of defined antigen-antibody systems. This accords with findings from similar studies in this laboratory. Using methods described for successful isolation of antigen from ICs by gel filtration (e.g., Bowen and Baldwin, 1976; Papsidero et al., 1978; Kilpatrick and Virella, 1980) we were unable to dissociate and separate model ICs of high affinity or ICs present in sera from breast cancer patients (unpublished). Thus further investigations were warranted in the attempt to establish reproducible methods for
pH
JC
~ GELFILTRATION
19 S
eL
PROTEIN-A
PREPARATIVE IEF
3-4.7
~ ANTIGEN
2ndpeok
SEPHAROSE ANTIBOOY
ION-EXCHANGE /st peak 2ndpeak
CHROMATOGRAPHY
_
~PROTEIN A SEPHAROSE 1
stlpeak
ANTIGEN
Fig. 5. Schematic outline of the major steps for partial purification and dissociation of immune complexes from body fluids and subsequent isolation of antigen and antibody moieties.
59
the isolation of antigen and antibody from preformed ICs of high affinity. From the results obtained it can be concluded that ion-exchange chromatography in dissociating buffers and isoelectric focusing permit the isolation~of antigen and antibody moieties from such ICs. Although only artificial ICs were analyzed here, the separation procedures described may prove of value in the detailed analysis of other ICs. Investigations are now in progress to define the antigens involved in the formation of ICs present in pleural or peritoneal effusions from various cancer patients.
Acknowledgements This study was supported in part by grants from the Landesamt for Forschung, Ministerium ftir Wissenschaft und Forschung des Landes Nordrhein-Westfalen, Dtisseldorf, and the Deutsche Krebshilfe. We are indebted to Miss I. Rademacher for technical and to Mrs. G. B6ttcher for secretarial assistance. Dr. Valerie E. Moore and Dr. H. Von Melchner are thanked for critical review of the manuscript.
References Bowen, J.G. and R.W. Baldwin, 1976, Transplantation 21,213. Bryce, C.F.A. and R.R. Chrichton, 1971, J. Chromatogr. 63, 267. Givol, D., S. Fuchs and M. Sela, 1962, Biochim. Biophys. Acta 63, 222. H6ffken, K. and C.G. Schmidt, 1980, Dtsch. Med. Wschr. 105, 1697. Jones, V.E. and E. Orlans, 1981, J. lmmunol. Methods 44, 249. Kabat, E.A. and M.M. Mayer, 1961, Experimental Immunochemistry (Thomas, Springfield, IL) Ch. 5i. Kilpatrick, J.M. and G. Virella, 1980, Clin. Exp. Immunol. 40, 445. Koestler, T.P., L.D. Papsidero, T. Nemoto and T.M. Chu, 1981, Cancer Res. 41, 2900. Lambert, P.H., F.J. Dixon, R.H. Zubler, V. AgneUo, C. Cambiaso, P. Casali, J. Clarke, J.S. Cowdery, F.C. McDuffie, F.C. Hay, I.C.M. MacLennan, P. Masson, H.J. Maller-Eberhard, K. Pettinen, M. Smith, G. Tappeiner, A.N. Theofilopoulos and P. Verroust, 1978, J. Clin. Lab. lmmunol. 1, 1. Lowry, O.H., N.L. Rosebrough, R.J. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Maidment, B.W., L.D. Papsidero and T.M. Chu, 1980, J. lmmunol. Meth. 35, 297. Maidment, B.W., L.D. Papsidero, T. Nemoto and T.M. Chu, 1981, Cancer Res. 41,795. Maini, R.N. and E.J. Holborow, 1977, Ann. Rheum. Dis. 36 (Suppl. 1). Papsidero, L.D., S.R. Harvey, M.C. Snyderman, T. Nemoto, L. Valenzuela and T.M. Chu, 1978, Int. J. Cancer 21,675. Slobin, L.I. and M. Sela, 1965, Biochim. Biophys. Acta 107, 593. Theofilopoulos, A.N. and F.J. Dixon, 1979, Adv. Immunol. 28, 89. Theofilopoulos, A.N., R.A. Eisenberg and F.J. Dixon, 1978, J. Clin. Invest. 61, 1570.