Journal of Immunological Methods, 132 (1990) 103-110
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Elsevier JIM05662
Characterization of immune complexes by isoelectric focusing in agarose gels Wayne L. Hoffman, Patrick J. Kelly and Margaret Larrumbide Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A.
(Received 13 February 1990, revisedreceived27 April 1990, accepted 1 May 1990)
A method is described for the characterization of immune complexes on thin-layer agarose isoelectric focusing (IEF) gels. This method involves dissociating immune complexes and then maintaining this dissociation during IEF in agarose gels containing 9 M urea. After IEF, the immune complex components can be quantitatively transferred to nitrocellulose in less than 15 rain, and a variety of immunostaining procedures can be used to probe these blotted components. No loss of biological activity was detected in any of the blotted components. Key words: Immunecomplex; Isoelectricfocusing;Agarosegel; Urea; (Antigen); (Antibody)
Introduction
Analysis of immune complex (IC) components usually requires an initial dissociation of the immune complexes, followed by the characterization of individual components. Several methods have been used to characterize these components including: ion exchange chromatography (Hoffken et al., 1982), chromatofocusing (Kneba et al., 1983), SDS-polyacrylamide gel electrophoresis (Casali and Lambert, 1979; Male and Roitt, 1979; Falus et al., 1981), and isoelectric focusing (Maidment et al., 1980; Hoffken et al., 1982).
Correspondence to: W.L. Hoffman, Arthritis Consultation Center, PresbyterianHospital, 8200 Walnut Hill Lane, Dallas,
TX 75231, U.S.A. (Tel.: 214-696-7465). Abbreviations: BSA, bovine serum albumin; IC, immune complex(es); IEF, isoelectrie focusing; NFDM, non-fat dried milk; pl, isoelectric point; SA-HRP, streptavidin-horseradish peroxidase conjugate; STI, soybean trypsin inhibitor; TBS, Tris-buffered saline.
IEF is an ideal method to characterize immune complex components because it is rapid, requires small amounts of IC to study and normally does not denature protein components. Although there are several reports discussing the use of IEF to characterize immune complex components (Maidment et al., 1980, 1981; Hoffken et al., 1982), the ability of this method to characterize immune complex components under non-denaturing conditions has recently been questioned by Haas and Schlaak (1987). The data presented in this report confirms the work of Haas and Schlaak (1987) and demonstrates that agarose IEF gels will not dissociate insoluble immune complexes in the absence of denaturing solvents. However, the data presented in this paper shows that if denaturing solvents were present during both sample preparation and IEF analysis, then the IC could be dissociated and their individual components analyzed by IEF. The incorporation of 9 M urea into rehydratable IEF gels prevents the reassociation of the immune complex components during IEF, but the 9 M urea does not interfere with the transfer of
0022-1759/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
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the immune complex components to nitrocellulose. After binding to the nitrocellulose membrane, the IC components retain biological activity.
Materials and methods
Reagents Urea (Sequanal grade) was obtained from Pierce Chemical Co. (Rockford, IL). Bovine serum albumin (BSA) was obtained from ICN Biomedicals (Costa Mesa, CA). Ovalbumin (5 × crystallized) was obtained from Calbiochem (La Jolla, CA). Soybean trypsin inhibitor (STI), papain and biotin-amidocaproate N-hydroxysuccinimide ester were obtained from Sigma Chemical Co. (St. Louis, MO). Streptavidin-horseradish peroxidase conjugate (SA-HRP) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Affinity-purified rabbit anti-BSA, affinity-purified rabbit anti-ovalbumin, and affinity-purified rabbit anti-STI were prepared in this laboratory.
Isolation of human IC Human IC were isolated from the serum of a patient with vasculitis by gel filtration on an A-5m column (Bio-Rad, Richmond, CA). The high molecular weight fractions were pooled to exclude monomeric IgG, and then precipitated with 15% polyethylene glycol ( M r 20,000). The precipitated material was resuspended in 0.1 M phosphate-pH 7 and then passed through a rabbit anti-human IgG affinity column. After extensive washing with 0.1 M phosphate buffer, p H 7, the bound IC material containing IgG was eluted with 0.1 M glycine-HC1, p H 3. The isolated human IC were immediately neutralized, and then concentrated and equilibrated for IEF in a Centricon-10 microconcentrator.
Preparation of Fc fragments H u m a n IgG Fc fragments were prepared from Cohn fraction II according to the method of Porter (1958). The Fc fragments were affinity-purified on Protein A-Sepharose CL-4B (Pharmacia, Piscataway, N J).
Preparation of insoluble IC
Biotinylation of probes
The antigen-antibody systems used in this study were BSA : rabbit anti-BSA, ovalbumin : rabbit anti-ovalbumin and STI :rabbit anti-STI. The ratios of antigen: antibody were varied to determine the three different equivalence points and the in vitro formation of insoluble immune complexes was quantitated by dissolving the IC precipitates in 1 M N a O H and reading the absorbance at 280 nm. Insoluble IC were washed two times in 0.1 M phosphate buffer (pH 7) and then resuspended in the appropriate IEF application buffer as stated in the text. Unless noted otherwise, a 3 : 1 molar ratio of antibody : antigen was used for the preparation of insoluble IC.
Protein probes were biotinylated according to a modification of the procedure of Updyke and Nicolson (1984). Each probe was dialyzed in 0.1 M N a H C O 3, p H 8.5, and concentrated to 10 m g / m l . A 12:1 molar ratio of biotin-amidocaproate N-hydroxysuccinimide ester : probe protein was used. The biotin ester was dissolved in dimethyl sulfoxide (20 mg/ml), then added to the protein solution and reacted for 4 h at 20 o C. The unreacted and hydrolyzed biotin ester was removed by gel filtration on a Pharmacia PD-10 (G-25) column equilibrated with 0.1 M phosphatep H 7.
Isoelectric focusing Preparation of soluble IC Soluble ovalbumin:rabbit anti-ovalbumin IC were made in both 1 : 3 and 1 : 10 molar ratios Of antibody:antigen. The IC were purified by gel filtration on a Pharmacia Superose-12 FPLC column (Pharmacia, Piscataway, N J). The isolated IC were concentrated and equilibrated for IEF in a Centricon-10 microconcentrator (Amicon, Danvers, MA).
IEF in agarose gels without urea was as previously described (Hoffman and Jump, 1985). IEF in agarose gels containing 9 M urea was done in rehydratable agarose gels as recently described (Hoffman et al., 1989b). The rehydratable agarose gels used in this study contained 2.5% linear polyacrylamide, 1% agarose IEF (Pharmacia, Piscataway, NJ) and 3% preblended LKB Ampholine carrier ampholytes (pH 3.5-9.5) (Pharmacia,
105 Piscataway, N J). The rehydratable agarose gels were stored 2-10 weeks at 4 ° C prior to drying and rehydration on the same day in the 9 M urea-ampholyte solution. IC samples were resuspended or diluted at least ten-fold either in 0.1 N H 4 H C O 3 (pH unadjusted) or in freshly made 10 M urea adjusted to p H 3 with HC1. A final concentration of at least 9 M urea, pH 3 was needed to dissociated the IC in the sample buffer, and samples were applied to the IEF gel surface within 15 min after dilution in the sample buffers. After IEF, the agarose gels were either acid fixed and stained with Coomassie blue R-250 (Hoffman and Jump, 1985) or the focused IC components were transferred to nitrocellulose and probed with biotinylated reagents.
Transfer of focused proteins to nitrocellulose Transfer of proteins to nitrocellulose from both conventional and rehydratable gels was as previously described (Hoffman and Jump, 1985), except that all the gels were routinely rinsed for 15-20 s before blotting. The longer rinse time eliminated detectable non-specific background. TBS buffer (0.02 M Tris, 0.5 M NaC1, 0.01% thimerosal, p H 7.4) was used to soak the membrane and rinse the gel.
Immunofixation and staining of nitrocellulose blots After transfer of the proteins to nitrocellulose, the unoccupied sites on the membrane were blocked for 1 h at 2 0 ° C with 5% non-fat dried milk ( N F D M ) in TBS. After blocking, the membrane was incubated for 5 h at 2 0 ° C in 5% N F D M - T B S containing an appropriate biotinylated probe. The blots were then washed for 20 min in 0.5% NFDM-TBS, with three changes in that time period. The membrane was then incubated for 20 min in streptavidin-HRP diluted 1/2000 in 1% BSA (Hoffman and Jump, 1989a), washed in 0.5% N F D M - T B S as before, given a final rinse in TBS alone, and developed with 4chloro-l-naphthol as previously described (Hoffman and Jump, 1989a). When human IC were studied, an extra blocking step was added after the milk block. The bound human IC components on the nitrocellulose blot were equilibrated for 1 h with human Fc fragments (0.5 m g / m l in 1% N F D M ) to block
potential rheumatoid factors in the dissociated IC. Human Fc fragments (0.5 m g / m l in 1% N F D M ) were also added to the biotinylated F(ab')2 probes to maintain the blocking of the potential rheumatoid factors on the blot and prevent these rheumatoid factors from binding to any Fc contaminants in the probes. The ability of 0.5 m g / m l human Fc fragments to effectively block rheumatoid factor binding has been previously demonstrated (Hoffman et al., 1990).
Results
To demonstrate that IEF gels containing 9 M urea do not permit formation of IC, ovalbumin and anti-ovalbumin standards were layered on IEF gels with and without 9 M urea. Similar application sites on both gels were chosen to permit IC formation during electrophoresis. As seen in Fig. 1, anti-ovalbumin (Ab), with an isoelectric point (pI) more basic than ovalbumin (Ag), was layered on the gel closer to the acidic wick than ovalbumin. When the pH gradient began to form, the antibody was forced to pass through the region of the IEF gel containing the antigen in order to reach its more basic pl. Fig. 1 clearly demonstrates that when the IEF gel did not contain urea (Fig. 1A), IC formed and the antigen and antibody were not focused. However, when 9 M urea was included in the IEF gel (Fig. 1B), IC formation was prevented and both antigen and antibody focused. Lower concentrations of urea in the IEF gel permitted the formation of some IC (data not shown). Fig. 2 confirms the importance of including 9 M urea in the IEF gel to maintain dissociation of the IC, but it also emphasizes the importance of utilizing the proper sample buffer to ensure that the IC are completely dissociated before application to the surface of the IEF gel. Fig. 2 shows a comparison of antigen (ovalbumin), antibody (rabbit anti-ovalbumin) and ovalbumin:rabbit anti-ovalbumin IC (ovalbumin IC), diluted in four different buffers, analyzed on an IEF gel without (Fig. 2A) and with (Fig. 2B) 9 M urea. The ovalbumin antigen (lane 1) and the anti-ovalbumin antibody (lane 2) focused on both gels. When the o v a l b u m i n : a n t i - o v a l b u m i n immune com-
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U l-'ecl
(-) A
(÷) B
pH8
Ab
Ag
Ag opplic. Ab
applic.
pH4
Fig. 1. Immune complex components maintain dissociation during IEF in gels containing 9 M urea. On an IEF gel without urea (A) and one containing 9 M urea (B), 5 Fg of ovalbumin (Ag) and 25 #g of rabbit anti-ovalbumin (Ab) were layered onto the gel surfaces in 10 M urea, pH 3. The individual application sites of the ovalbumin (Ag Applic.) and rabbit anti-ovalbumin (Ab Applic.) are indicated on the figure and these sites were chosen to permit immune complex formation during electrophoresis. After electrophoresis, the gels were acid-fixed and stained as described in the materials and methods section. The focused antibody (Ab) and antigen (Ag) are indicated by the arrows. plexes were dissolved in 10 M urea, p H 3 and analyzed by I E F (lane 3), only the gel containing 9 M urea (Fig. 2B, lane 3) maintained complete dissociation of the ovalbumin IC, allowing the corresponding antigen and antibody to focus. The gel lacking 9 M urea (see Fig. 2A, lane 3) allowed the reassociation of the IC, resulting in precipitation of most of the reformed ovalbumin IC at the sample application site. Lanes 4, 5, and 6 of Fig. 2 demonstrated that preparation in 10 M urea alone (lane 4), p H 3 buffer alone (lane 5), or 0.1 M N H 4 H C O 3 alone were not sufficient to completely dissociate the ovalbumin IC before application on either the normal I E F gel or the gel containing 9 M urea. Insoluble IC that never entered the gel were mostly washed off the gel surface during the fixing and staining process and
were detected as irregular blotches at the application site (see lanes 4-6). Only 10 M urea, p H 3 was able to completely dissociate the ovalbumin IC before analysis on IEF, and 9 M urea in the gel ~,as needed to maintain this dissociation. Comparison of the p I of the ovalbumin and anti)valbumin standards on the two gels in Fig. 2 demonstrated that the 9 M urea changed the p I of both proteins. Since 10 M urea, p H 3 was used to dissociate he ovalbumin IC and 9 M urea was used in the EF gel to maintain dissociation, it was not known o what extent the dissociated IC component proeins would maintain biological activity and anti;enicity under these conditions. Fig. 3 demontrates that the antibodies retained biological acivity and the antigens retained reactive epitopes fter dissociation in 10 M urea, p H 3 and analysis n an I E F gel containing 9 M urea. In lane 1, valbumin IC were dissociated, acid fixed and ~:ained to show the I E F profiles of the IC samples that were blotted to a nitrocellulose membrane in lanes 2 and 4. The ovalbumin from the dissociated ovalbumin IC (lane 2) and the ovalbumin standard (lane 3) were both reactive when the nitrocellulose blot was probed with biotinylated antibody (affinity-purified rabbit anti-ovalbumin). The rabbit anti-ovalbumin antibody from the dissociated ovalbumin IC (lane 4) and the rabbit antiovalbumin standard (lane 5) both retained the capacity to bind antigen when the nitrocellulose blot was probed with biotinylated ovalbumin (5 × crystallized). For comparison, the identical amount of anti-ovalbumin antibody standard was resuspended in 0.1 M N H 4 H C O 3 and analyzed on a parallel urea-free I E F gel, followed by blotting to nitrocellulose and probing with biotinylated ovalbumin (data not shown). The anti-ovalbumin antibody analyzed in the presence of urea (Fig. 3, lane 5) bound as much antigen as the antiovalbumin antibody analyzed in the absence of urea, suggesting that the anti-ovalbumin antibody was not permanently inactivated by dilution in 10 M urea, p H 3 or by I E F in gels containing 9 M urea. Control studies demonstrated that both biotinylated probes were specific for the respective antigen or antibody and did not bind to other proteins either in the presence or absence of 9 M urea.
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pH 8
pH 4 1
2
5
4
5
6
t
2
3
4
5
6
Fig. 2. Importance of the sample buffer and urea in the dissociation and isoelectric focusing analysis of i m m u n e complexes. The following samples were analyzed on IEF gels without urea (A) or IEF gels containing 9 M urea (B): lane 1, 5 lag ovalbumin; lane 2, 20 lag rabbit anti-ovalbumin; lane 3, 40 lag of ovalbumin : anti-ovalbumin i m m u n e complexes (ovalbumin IC) in 10 M urea, p H 3; lane 4, 40 #g ovalbumin IC in 10 M urea, p H 8; lane 5, 40 lag ovalbumin IC in 0.1 M glycine, p H 3; and lane 6, 40 # g ovalbumin IC in 0.1 M N H 4 H C O 3. Both gels were acid-fixed and stained as described in the materials and methods section. The arrow indicates the sample application site.
To assess whether the a n t i b o d y : a n t i g e n ratio of the IC would effect dissociation or I E F separation of IC, both soluble and insoluble IC were compared. Fig. 4 demonstrates that whether the
pH 8 ~i~~ ~i!~i
IC were formed in antibody excess (lanes 3 and 4) or antigen excess (lanes 5 and 6), the IC dissociated completely. The antibody excess IC analyzed in lanes 3 and 4 were insoluble complexes and the precipitates were washed in 0.1 M phosphate, p H 7 before dissolving in the 10 M urea, p H 3 sample buffer. The antigen excess IC analyzed in lanes 5 and 6 were soluble and were isolated by gel filtration chromatography. The amount of the soluble
pH 8
pH 4 1
2
3
4
5 4
Fig. 3. Retention of antibody activity and antigen immunogenicity following IC dissociation, separation by IEF, and blotting to nitrocellulose. O v a l b u m i n : a n t i - o v a l b u m i n i m m u n e complexes were dissociated in 10 M urea, p H 3 and analyzed on an IEF gel containing 9 M urea. Antibody and antigen standards were also diluted in 10 M urea, p H 3 before analysis on the same IEF gel. Lane 1 contained 40 lag of ovalbumin IC and was acid-fixed and stained as described in the materials and methods section. Lane 2, containing 0.5 lag of ovalbumin IC, and lane 3, containing 0.1 lag of the ovalbumin standard, were blotted to nitrocellulose, probed with biotinylated rabbit antiovalbumin (5 lag/ml), and developed with streptavidinhorseradish peroxidase (SA-HRP) as described in the materials and methods section. Lane 4, containing 0.5 lag of ovalbumin IC, and lane 5, containing 0.5 lag of the rabbit anti-ovalbumin standard, were blotted to nitrocellulose, probed with biotinylated ovalbumin (5 lag/ml), and developed with S A - H R P as described in materials and methods section. The arrow indicates the sample application site.
pH 4 1
2
3
4
5
6
Fig. 4. Dissociation and IEF separation of immune complexes are u n a f f e c t e d b y the a n t i b o d y : a n t i g e n ratio. O v a l b u m i n : a n t i - o v a l b u m i n i m m u n e complexes were dissociated in 10 M urea, p H 3 and analyzed on an IEF gel containing 9 M urea. Antibody and antigen standards were analyzed under identical conditions. The following samples were analyzed: lane 1, 5 lag of the ovalbumin standard; lane 2, 10 lag of the rabbit anti-ovalbumin standard; lane 3, 40 lag of insoluble ovalbumin IC prepared using a 5 : 1 ratio of antibody to antigen; lane 4, 40 lag of insoluble ovalbumin IC prepared using a 3:1 ratio; lane 5, 10 lag of soluble ovalbumin IC prepared using a 1 : 3 ratio; and lane 6, 10 lag of soluble ovalburnin IC prepared using a 1 : 10 ratio. The gel was acidfixed and stained as described in the materials and methods section. The arrow indicates the sample application site.
108 IC analyzed in lanes 5 and 6 was reduced in an attempt to match the amount of antigen in lanes 3 and 4. This reduction in sample size emphasized that the antibody component was significantly reduced in these IC and confirmed that the IC in lanes 5 and 6 were formed in antigen excess. BSA : anti-BSA IC and STI : anti-STI IC at different a n t i g e n : a n t i b o d y ratios were also completely dissociated by the 10 M urea, p H 3 sample buffer, and the 9 M urea in the gel maintained complete dissociation of these IC (data not shown). The anti-BSA and anti-STI antibody standards and those dissociated from the corresponding IC retained biological activity when compared to antibody standards on gels lacking 9 M urea. The BSA and STI antigens also retained antigenicity under the same conditions. The ovalbumin IC data is representative of these other two IC sampies. Although the ovalbumin IC were representative of the other IC systems tested, the three IC differed in the p I of the antigen and antibody components and in the observed avidity of the antibodies for their respective antigens. For example, the anti-BSA antibody had the lowest observed avidity of the three antibodies tested and the BSA IC were dissociated in a 6 M urea, p H 3 sample buffer. This sample buffer did not dissociate either the ovalbumin IC or the STI IC. In contrast, the anti-STI antibody had the highest observed avidity of the three antibodies tested, but STI IC were completely dissociated in the 10 M urea, p H 3 sample buffer and were focused in the I E F gels containing 9 M urea. Lower urea concentrations in either the sample buffer or in the I E F gel did not permit complete dissociation of the STI IC. Since IC components retained biological activity under these conditions (see Fig. 3), they are recommended for the analysis of all IC. I m m u n e complexes from a patient with vasculitis were dissociated in 10 M urea, p H 3 and focused in an I E F gel containing 9 M urea. Human fibronectin was identified in the dissociated IC (Fig. 5, lane 1) by biotinylated F(ab')2 fragments of rabbit anti-fibronectin, and human C 3 was identified in the dissociated IC by biotinylated F(ab')2 fragment of rabbit anti-C 3 (Fig. 5, lane 2). Probing of the same IC sample with biotinylated F(ab')2 fragments of normal rabbit
pH8
pH4 1
2
3
Fig. 5. Analysis of components in human immune complexes by IEF in 9 M urea. Isolated soluble immune complexes from a vasculitis patient were dissociated in 10 M urea, pH 3 and separated on a rehydratable IEF gel containing 9 M urea. Three lanes, each containing 1 /~g of the dissociated IC, were probed with the following reagents: lane 1, biotinylated F(ab')2 fragments of rabbit anti-fibronectin (5 /xg/ml); lane 2, biotinylated F(ab')2 fragments of rabbit anti-C3 (5/~g/ml); and lane 3, biotinylated F(ab')2 fragments of normal rabbit IgG (5 t~g/ml). The blots were developed with SA-HRP as described in the materials and methods section.
I g G (Fig. 5, lane 3) did not detect any reactivity, demonstrating that there was no detectable rheumatoid factor activity or other anti-globulin activity between the IC components and the rabbit F(ab')2 fragments. Therefore, the use of hum a n Fc fragments to block both the IC components and the biotinylated probes (see materials and methods section) permitted the detection of only the specific antigens.
Discussion S D S - P A G E is a commonly employed technique for the analysis of IC (Casali and Lambert, 1979; Male and Roitt, 1979; Falus et al., 1981). Unfortunately, some antigenic determinants may be irreversibly denatured by SDS. Reducing agents
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like fl-mercaptoethanol will dissociated the proteins into their respective subunits and after transfer to nitrocellulose the components may lack biological activity and antigenicity (Tovey et al., 1987). For example, denatured IC protein components may not be detected by monoclonal antibodies (Braun et al., 1983; De Blas and Cherwinski, 1983). Since IEF does not contain dissociating or reducing agents, IEF would be the method of choice to analyze IC components, because it might allow the maintenance of both biological activity and antigenicity of these separated components. Although there are several reports describing the use of IEF for the analysis of IC in the absence of denaturing reagents (Maidment et al., 1980, 1981; Hoffken et al., 1982), the ability of these IEF methods to dissociate and analyze IC components has recently been questioned (Haas and Schlaak, 1987). Disagreements over whether IEF is an effective method to analyze IC might be explained by differences in antibody avidity, sample preparation, or IC analysis methods, such as tube gels versus slab gels. Like Haas and Schlaak (1987), we were unable to separate IC components in slab gels unless the dissociating conditions described in this paper were used in both sample preparation and IEF analysis. The data in this paper demonstrate that 10 M urea, pH 3 is needed to dissociate the three IC tested and 9 M urea is needed in the IEF gel to maintain this dissociation (Figs. 1 and 2). These dissociating conditions permitted IC component separation during IEF and were effective in dissociating both soluble and insoluble IC (Fig. 4). Although the IC were both dissociated and analyzed in denaturing buffers, they retained their biological activity and antigenicity when transferred to nitrocellulose (Fig. 3). It was previously shown that IgG rheumatoid factors also retained biological activity when analyzed and blotted under these identical conditions (Hoffman et al., 1989b). Therefore, the separated IC components appeared to be stabilized and in a native conformation after being bound to nitrocellulose. This retention of biological activity by IC components following IEF separation is a major advantage over the SDS-PAGE analysis of IC. The time of analysis is another advantage that this method has
over SDS-PAGE. Running the IEF gel takes about 30 min, and the proteins can be blotted to nitrocellulose in 15 min with a 95% transfer efficiency (I4offman et al., 1989b). Although three IC systems were studied, only one representative IC system was presented. All of the IC were dissociated and focused in this gel system (data not shown). All of the IC components also displayed pI changes when analyzed in IEF gels containing 9 M urea. Urea causes an unfolding of proteins, and the unfolded proteins frequently display a change in pI as previously noted (Hoffman et al., 1989b). In summary, a new technique is described which allows the rapid analysis of IC components using IEF. The focused components can be transferred to nitrocellulose in 15 min, and the bound proteins retain biological activity and antigenicity.
Acknowledgements This work was supported by a grant from the National Institutes of Health (AR-36323) and by the John and Katie Jackson Foundation.
References Braun, D.K., Pereira, L., Norrild, B. and Roizman, B. (1983) Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for detection of monoclonal antibodies and for studies of the properties of viral proteins. J. Virol. 46, 103. Casali, P. and Lambert, P.H. (1979) Purification of soluble immune complexes from serum using polymethylmetacrylate beads coated with conglutinin or Clq. Clin. Exp. Immunol. 37, 295. De Bias, A.L. and Cherwinski, H.M. (1983) Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies. Anal. Biochem. 133, 214. Falus, A., Meretey, K., Glikmann, G., Svehag, S.E., Fabian, F., Bohm, U. and Bozsoky, S. (1981) fl2-Microglobulin-containing IgG complexes in sera and synovial fluid of rheumatoid arthritis and systemic lupus erythematosus patients. Scand. J. Immunol. 13, 25. Haas, H. and Sctdaak, M. (1987) Conventional isoelectric focusing does not dissociate immune complexes. J. Immunol. Methods 103, 79. Hoffken, K., Bosse, F., Steih, U. and Schmidt, C.G. (1982) Dissociation and isolation of antigen and antibody from immune complexes. J. Immunol. Methods 53, 51.
110 Hoffman, W.L. and Jump, A.A. (1985) Rapid blotting of IgG to nitrocellulose with minimal IgM contamination. J. Immunol. Methods 76, 263. Hoffman, W.L. and Jump, A.A. (1989) Inhibition of the streptavidin-biotin interaction by milk. Anal. Biochem. 181,318. Hoffman, W.L., Jump, A.A., Kelly, P.J. and Elangovan, N. (1989) Rehydratable agarose gels: Application to isoelectric focusing in 9 molar urea. Electrophoresis 10, 741. Hoffman, W.L., Jump, A.A. and Smiley, J.D. (1990) Synthesis of specific idiotypes by rheumatoid synovium. Arthritis Rheum., in press. Kneba, M., Krieger, G., Kehl, A., Bause, I. and Nagel, G.A. (1983) Chromatofocusing combined with the ELISA technique. A sensitive method for the analysis of immune complexes. J. Immunol. Methods 61, 233. Maidment, Jr., B.W., Papsidero, L.D. and Chu, T.M. (1980) Isoelectric focusing-a new approach to the study of immune complexes. J. Immunol. Methods 35, 297.
Maidment, Jr., B.W., Papsidero, L.D., Gamarra, M., Nemoto, T. and Chu, T.M. (1981) Isoelectric focusing analysis of soluble immune complexes bound to protein A-Sepharose. Anal. Biochem. 111, 336. Male, D. and Roitt, I.M. (1979) Analysis of the components of immune complexes. Mol. Immunol. 16, 197. Porter, R.R. (1958) Separation and isolation of fractions of rabbit gamma-globulin containing the antibody and antigenic combining sites. Nature 182, 670. Tovey, E.R., Ford, S.A. and Baldo, B.A. (1987) Protein blotting on nitrocellulose: Some important aspects of the resolution and detection of antigens in complex extracts. J. Biochem. Biophys. Methods 14, 1. Updyke, T.V. and Nicolson, G.L. (1984) Immunoaffinity isolation of membrane antigens with biotinylated monoclonal antibodies and immobilized streptavidin matrices. J. Immunol. Methods 73, 83.
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Elsevier JIM05662
Characterization of immune complexes by isoelectric focusing in agarose gels Wayne L. Hoffman, Patrick J. Kelly and Margaret Larrumbide Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, U.S.A.
(Received 13 February 1990, revisedreceived27 April 1990, accepted 1 May 1990)
A method is described for the characterization of immune complexes on thin-layer agarose isoelectric focusing (IEF) gels. This method involves dissociating immune complexes and then maintaining this dissociation during IEF in agarose gels containing 9 M urea. After IEF, the immune complex components can be quantitatively transferred to nitrocellulose in less than 15 rain, and a variety of immunostaining procedures can be used to probe these blotted components. No loss of biological activity was detected in any of the blotted components. Key words: Immunecomplex; Isoelectricfocusing;Agarosegel; Urea; (Antigen); (Antibody)
Introduction
Analysis of immune complex (IC) components usually requires an initial dissociation of the immune complexes, followed by the characterization of individual components. Several methods have been used to characterize these components including: ion exchange chromatography (Hoffken et al., 1982), chromatofocusing (Kneba et al., 1983), SDS-polyacrylamide gel electrophoresis (Casali and Lambert, 1979; Male and Roitt, 1979; Falus et al., 1981), and isoelectric focusing (Maidment et al., 1980; Hoffken et al., 1982).
Correspondence to: W.L. Hoffman, Arthritis Consultation Center, PresbyterianHospital, 8200 Walnut Hill Lane, Dallas,
TX 75231, U.S.A. (Tel.: 214-696-7465). Abbreviations: BSA, bovine serum albumin; IC, immune complex(es); IEF, isoelectrie focusing; NFDM, non-fat dried milk; pl, isoelectric point; SA-HRP, streptavidin-horseradish peroxidase conjugate; STI, soybean trypsin inhibitor; TBS, Tris-buffered saline.
IEF is an ideal method to characterize immune complex components because it is rapid, requires small amounts of IC to study and normally does not denature protein components. Although there are several reports discussing the use of IEF to characterize immune complex components (Maidment et al., 1980, 1981; Hoffken et al., 1982), the ability of this method to characterize immune complex components under non-denaturing conditions has recently been questioned by Haas and Schlaak (1987). The data presented in this report confirms the work of Haas and Schlaak (1987) and demonstrates that agarose IEF gels will not dissociate insoluble immune complexes in the absence of denaturing solvents. However, the data presented in this paper shows that if denaturing solvents were present during both sample preparation and IEF analysis, then the IC could be dissociated and their individual components analyzed by IEF. The incorporation of 9 M urea into rehydratable IEF gels prevents the reassociation of the immune complex components during IEF, but the 9 M urea does not interfere with the transfer of
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the immune complex components to nitrocellulose. After binding to the nitrocellulose membrane, the IC components retain biological activity.
Materials and methods
Reagents Urea (Sequanal grade) was obtained from Pierce Chemical Co. (Rockford, IL). Bovine serum albumin (BSA) was obtained from ICN Biomedicals (Costa Mesa, CA). Ovalbumin (5 × crystallized) was obtained from Calbiochem (La Jolla, CA). Soybean trypsin inhibitor (STI), papain and biotin-amidocaproate N-hydroxysuccinimide ester were obtained from Sigma Chemical Co. (St. Louis, MO). Streptavidin-horseradish peroxidase conjugate (SA-HRP) was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Affinity-purified rabbit anti-BSA, affinity-purified rabbit anti-ovalbumin, and affinity-purified rabbit anti-STI were prepared in this laboratory.
Isolation of human IC Human IC were isolated from the serum of a patient with vasculitis by gel filtration on an A-5m column (Bio-Rad, Richmond, CA). The high molecular weight fractions were pooled to exclude monomeric IgG, and then precipitated with 15% polyethylene glycol ( M r 20,000). The precipitated material was resuspended in 0.1 M phosphate-pH 7 and then passed through a rabbit anti-human IgG affinity column. After extensive washing with 0.1 M phosphate buffer, p H 7, the bound IC material containing IgG was eluted with 0.1 M glycine-HC1, p H 3. The isolated human IC were immediately neutralized, and then concentrated and equilibrated for IEF in a Centricon-10 microconcentrator.
Preparation of Fc fragments H u m a n IgG Fc fragments were prepared from Cohn fraction II according to the method of Porter (1958). The Fc fragments were affinity-purified on Protein A-Sepharose CL-4B (Pharmacia, Piscataway, N J).
Preparation of insoluble IC
Biotinylation of probes
The antigen-antibody systems used in this study were BSA : rabbit anti-BSA, ovalbumin : rabbit anti-ovalbumin and STI :rabbit anti-STI. The ratios of antigen: antibody were varied to determine the three different equivalence points and the in vitro formation of insoluble immune complexes was quantitated by dissolving the IC precipitates in 1 M N a O H and reading the absorbance at 280 nm. Insoluble IC were washed two times in 0.1 M phosphate buffer (pH 7) and then resuspended in the appropriate IEF application buffer as stated in the text. Unless noted otherwise, a 3 : 1 molar ratio of antibody : antigen was used for the preparation of insoluble IC.
Protein probes were biotinylated according to a modification of the procedure of Updyke and Nicolson (1984). Each probe was dialyzed in 0.1 M N a H C O 3, p H 8.5, and concentrated to 10 m g / m l . A 12:1 molar ratio of biotin-amidocaproate N-hydroxysuccinimide ester : probe protein was used. The biotin ester was dissolved in dimethyl sulfoxide (20 mg/ml), then added to the protein solution and reacted for 4 h at 20 o C. The unreacted and hydrolyzed biotin ester was removed by gel filtration on a Pharmacia PD-10 (G-25) column equilibrated with 0.1 M phosphatep H 7.
Isoelectric focusing Preparation of soluble IC Soluble ovalbumin:rabbit anti-ovalbumin IC were made in both 1 : 3 and 1 : 10 molar ratios Of antibody:antigen. The IC were purified by gel filtration on a Pharmacia Superose-12 FPLC column (Pharmacia, Piscataway, N J). The isolated IC were concentrated and equilibrated for IEF in a Centricon-10 microconcentrator (Amicon, Danvers, MA).
IEF in agarose gels without urea was as previously described (Hoffman and Jump, 1985). IEF in agarose gels containing 9 M urea was done in rehydratable agarose gels as recently described (Hoffman et al., 1989b). The rehydratable agarose gels used in this study contained 2.5% linear polyacrylamide, 1% agarose IEF (Pharmacia, Piscataway, NJ) and 3% preblended LKB Ampholine carrier ampholytes (pH 3.5-9.5) (Pharmacia,
105 Piscataway, N J). The rehydratable agarose gels were stored 2-10 weeks at 4 ° C prior to drying and rehydration on the same day in the 9 M urea-ampholyte solution. IC samples were resuspended or diluted at least ten-fold either in 0.1 N H 4 H C O 3 (pH unadjusted) or in freshly made 10 M urea adjusted to p H 3 with HC1. A final concentration of at least 9 M urea, pH 3 was needed to dissociated the IC in the sample buffer, and samples were applied to the IEF gel surface within 15 min after dilution in the sample buffers. After IEF, the agarose gels were either acid fixed and stained with Coomassie blue R-250 (Hoffman and Jump, 1985) or the focused IC components were transferred to nitrocellulose and probed with biotinylated reagents.
Transfer of focused proteins to nitrocellulose Transfer of proteins to nitrocellulose from both conventional and rehydratable gels was as previously described (Hoffman and Jump, 1985), except that all the gels were routinely rinsed for 15-20 s before blotting. The longer rinse time eliminated detectable non-specific background. TBS buffer (0.02 M Tris, 0.5 M NaC1, 0.01% thimerosal, p H 7.4) was used to soak the membrane and rinse the gel.
Immunofixation and staining of nitrocellulose blots After transfer of the proteins to nitrocellulose, the unoccupied sites on the membrane were blocked for 1 h at 2 0 ° C with 5% non-fat dried milk ( N F D M ) in TBS. After blocking, the membrane was incubated for 5 h at 2 0 ° C in 5% N F D M - T B S containing an appropriate biotinylated probe. The blots were then washed for 20 min in 0.5% NFDM-TBS, with three changes in that time period. The membrane was then incubated for 20 min in streptavidin-HRP diluted 1/2000 in 1% BSA (Hoffman and Jump, 1989a), washed in 0.5% N F D M - T B S as before, given a final rinse in TBS alone, and developed with 4chloro-l-naphthol as previously described (Hoffman and Jump, 1989a). When human IC were studied, an extra blocking step was added after the milk block. The bound human IC components on the nitrocellulose blot were equilibrated for 1 h with human Fc fragments (0.5 m g / m l in 1% N F D M ) to block
potential rheumatoid factors in the dissociated IC. Human Fc fragments (0.5 m g / m l in 1% N F D M ) were also added to the biotinylated F(ab')2 probes to maintain the blocking of the potential rheumatoid factors on the blot and prevent these rheumatoid factors from binding to any Fc contaminants in the probes. The ability of 0.5 m g / m l human Fc fragments to effectively block rheumatoid factor binding has been previously demonstrated (Hoffman et al., 1990).
Results
To demonstrate that IEF gels containing 9 M urea do not permit formation of IC, ovalbumin and anti-ovalbumin standards were layered on IEF gels with and without 9 M urea. Similar application sites on both gels were chosen to permit IC formation during electrophoresis. As seen in Fig. 1, anti-ovalbumin (Ab), with an isoelectric point (pI) more basic than ovalbumin (Ag), was layered on the gel closer to the acidic wick than ovalbumin. When the pH gradient began to form, the antibody was forced to pass through the region of the IEF gel containing the antigen in order to reach its more basic pl. Fig. 1 clearly demonstrates that when the IEF gel did not contain urea (Fig. 1A), IC formed and the antigen and antibody were not focused. However, when 9 M urea was included in the IEF gel (Fig. 1B), IC formation was prevented and both antigen and antibody focused. Lower concentrations of urea in the IEF gel permitted the formation of some IC (data not shown). Fig. 2 confirms the importance of including 9 M urea in the IEF gel to maintain dissociation of the IC, but it also emphasizes the importance of utilizing the proper sample buffer to ensure that the IC are completely dissociated before application to the surface of the IEF gel. Fig. 2 shows a comparison of antigen (ovalbumin), antibody (rabbit anti-ovalbumin) and ovalbumin:rabbit anti-ovalbumin IC (ovalbumin IC), diluted in four different buffers, analyzed on an IEF gel without (Fig. 2A) and with (Fig. 2B) 9 M urea. The ovalbumin antigen (lane 1) and the anti-ovalbumin antibody (lane 2) focused on both gels. When the o v a l b u m i n : a n t i - o v a l b u m i n immune com-
106
U l-'ecl
(-) A
(÷) B
pH8
Ab
Ag
Ag opplic. Ab
applic.
pH4
Fig. 1. Immune complex components maintain dissociation during IEF in gels containing 9 M urea. On an IEF gel without urea (A) and one containing 9 M urea (B), 5 Fg of ovalbumin (Ag) and 25 #g of rabbit anti-ovalbumin (Ab) were layered onto the gel surfaces in 10 M urea, pH 3. The individual application sites of the ovalbumin (Ag Applic.) and rabbit anti-ovalbumin (Ab Applic.) are indicated on the figure and these sites were chosen to permit immune complex formation during electrophoresis. After electrophoresis, the gels were acid-fixed and stained as described in the materials and methods section. The focused antibody (Ab) and antigen (Ag) are indicated by the arrows. plexes were dissolved in 10 M urea, p H 3 and analyzed by I E F (lane 3), only the gel containing 9 M urea (Fig. 2B, lane 3) maintained complete dissociation of the ovalbumin IC, allowing the corresponding antigen and antibody to focus. The gel lacking 9 M urea (see Fig. 2A, lane 3) allowed the reassociation of the IC, resulting in precipitation of most of the reformed ovalbumin IC at the sample application site. Lanes 4, 5, and 6 of Fig. 2 demonstrated that preparation in 10 M urea alone (lane 4), p H 3 buffer alone (lane 5), or 0.1 M N H 4 H C O 3 alone were not sufficient to completely dissociate the ovalbumin IC before application on either the normal I E F gel or the gel containing 9 M urea. Insoluble IC that never entered the gel were mostly washed off the gel surface during the fixing and staining process and
were detected as irregular blotches at the application site (see lanes 4-6). Only 10 M urea, p H 3 was able to completely dissociate the ovalbumin IC before analysis on IEF, and 9 M urea in the gel ~,as needed to maintain this dissociation. Comparison of the p I of the ovalbumin and anti)valbumin standards on the two gels in Fig. 2 demonstrated that the 9 M urea changed the p I of both proteins. Since 10 M urea, p H 3 was used to dissociate he ovalbumin IC and 9 M urea was used in the EF gel to maintain dissociation, it was not known o what extent the dissociated IC component proeins would maintain biological activity and anti;enicity under these conditions. Fig. 3 demontrates that the antibodies retained biological acivity and the antigens retained reactive epitopes fter dissociation in 10 M urea, p H 3 and analysis n an I E F gel containing 9 M urea. In lane 1, valbumin IC were dissociated, acid fixed and ~:ained to show the I E F profiles of the IC samples that were blotted to a nitrocellulose membrane in lanes 2 and 4. The ovalbumin from the dissociated ovalbumin IC (lane 2) and the ovalbumin standard (lane 3) were both reactive when the nitrocellulose blot was probed with biotinylated antibody (affinity-purified rabbit anti-ovalbumin). The rabbit anti-ovalbumin antibody from the dissociated ovalbumin IC (lane 4) and the rabbit antiovalbumin standard (lane 5) both retained the capacity to bind antigen when the nitrocellulose blot was probed with biotinylated ovalbumin (5 × crystallized). For comparison, the identical amount of anti-ovalbumin antibody standard was resuspended in 0.1 M N H 4 H C O 3 and analyzed on a parallel urea-free I E F gel, followed by blotting to nitrocellulose and probing with biotinylated ovalbumin (data not shown). The anti-ovalbumin antibody analyzed in the presence of urea (Fig. 3, lane 5) bound as much antigen as the antiovalbumin antibody analyzed in the absence of urea, suggesting that the anti-ovalbumin antibody was not permanently inactivated by dilution in 10 M urea, p H 3 or by I E F in gels containing 9 M urea. Control studies demonstrated that both biotinylated probes were specific for the respective antigen or antibody and did not bind to other proteins either in the presence or absence of 9 M urea.
107
pH 8
pH 4 1
2
5
4
5
6
t
2
3
4
5
6
Fig. 2. Importance of the sample buffer and urea in the dissociation and isoelectric focusing analysis of i m m u n e complexes. The following samples were analyzed on IEF gels without urea (A) or IEF gels containing 9 M urea (B): lane 1, 5 lag ovalbumin; lane 2, 20 lag rabbit anti-ovalbumin; lane 3, 40 lag of ovalbumin : anti-ovalbumin i m m u n e complexes (ovalbumin IC) in 10 M urea, p H 3; lane 4, 40 #g ovalbumin IC in 10 M urea, p H 8; lane 5, 40 lag ovalbumin IC in 0.1 M glycine, p H 3; and lane 6, 40 # g ovalbumin IC in 0.1 M N H 4 H C O 3. Both gels were acid-fixed and stained as described in the materials and methods section. The arrow indicates the sample application site.
To assess whether the a n t i b o d y : a n t i g e n ratio of the IC would effect dissociation or I E F separation of IC, both soluble and insoluble IC were compared. Fig. 4 demonstrates that whether the
pH 8 ~i~~ ~i!~i
IC were formed in antibody excess (lanes 3 and 4) or antigen excess (lanes 5 and 6), the IC dissociated completely. The antibody excess IC analyzed in lanes 3 and 4 were insoluble complexes and the precipitates were washed in 0.1 M phosphate, p H 7 before dissolving in the 10 M urea, p H 3 sample buffer. The antigen excess IC analyzed in lanes 5 and 6 were soluble and were isolated by gel filtration chromatography. The amount of the soluble
pH 8
pH 4 1
2
3
4
5 4
Fig. 3. Retention of antibody activity and antigen immunogenicity following IC dissociation, separation by IEF, and blotting to nitrocellulose. O v a l b u m i n : a n t i - o v a l b u m i n i m m u n e complexes were dissociated in 10 M urea, p H 3 and analyzed on an IEF gel containing 9 M urea. Antibody and antigen standards were also diluted in 10 M urea, p H 3 before analysis on the same IEF gel. Lane 1 contained 40 lag of ovalbumin IC and was acid-fixed and stained as described in the materials and methods section. Lane 2, containing 0.5 lag of ovalbumin IC, and lane 3, containing 0.1 lag of the ovalbumin standard, were blotted to nitrocellulose, probed with biotinylated rabbit antiovalbumin (5 lag/ml), and developed with streptavidinhorseradish peroxidase (SA-HRP) as described in the materials and methods section. Lane 4, containing 0.5 lag of ovalbumin IC, and lane 5, containing 0.5 lag of the rabbit anti-ovalbumin standard, were blotted to nitrocellulose, probed with biotinylated ovalbumin (5 lag/ml), and developed with S A - H R P as described in materials and methods section. The arrow indicates the sample application site.
pH 4 1
2
3
4
5
6
Fig. 4. Dissociation and IEF separation of immune complexes are u n a f f e c t e d b y the a n t i b o d y : a n t i g e n ratio. O v a l b u m i n : a n t i - o v a l b u m i n i m m u n e complexes were dissociated in 10 M urea, p H 3 and analyzed on an IEF gel containing 9 M urea. Antibody and antigen standards were analyzed under identical conditions. The following samples were analyzed: lane 1, 5 lag of the ovalbumin standard; lane 2, 10 lag of the rabbit anti-ovalbumin standard; lane 3, 40 lag of insoluble ovalbumin IC prepared using a 5 : 1 ratio of antibody to antigen; lane 4, 40 lag of insoluble ovalbumin IC prepared using a 3:1 ratio; lane 5, 10 lag of soluble ovalbumin IC prepared using a 1 : 3 ratio; and lane 6, 10 lag of soluble ovalburnin IC prepared using a 1 : 10 ratio. The gel was acidfixed and stained as described in the materials and methods section. The arrow indicates the sample application site.
108 IC analyzed in lanes 5 and 6 was reduced in an attempt to match the amount of antigen in lanes 3 and 4. This reduction in sample size emphasized that the antibody component was significantly reduced in these IC and confirmed that the IC in lanes 5 and 6 were formed in antigen excess. BSA : anti-BSA IC and STI : anti-STI IC at different a n t i g e n : a n t i b o d y ratios were also completely dissociated by the 10 M urea, p H 3 sample buffer, and the 9 M urea in the gel maintained complete dissociation of these IC (data not shown). The anti-BSA and anti-STI antibody standards and those dissociated from the corresponding IC retained biological activity when compared to antibody standards on gels lacking 9 M urea. The BSA and STI antigens also retained antigenicity under the same conditions. The ovalbumin IC data is representative of these other two IC sampies. Although the ovalbumin IC were representative of the other IC systems tested, the three IC differed in the p I of the antigen and antibody components and in the observed avidity of the antibodies for their respective antigens. For example, the anti-BSA antibody had the lowest observed avidity of the three antibodies tested and the BSA IC were dissociated in a 6 M urea, p H 3 sample buffer. This sample buffer did not dissociate either the ovalbumin IC or the STI IC. In contrast, the anti-STI antibody had the highest observed avidity of the three antibodies tested, but STI IC were completely dissociated in the 10 M urea, p H 3 sample buffer and were focused in the I E F gels containing 9 M urea. Lower urea concentrations in either the sample buffer or in the I E F gel did not permit complete dissociation of the STI IC. Since IC components retained biological activity under these conditions (see Fig. 3), they are recommended for the analysis of all IC. I m m u n e complexes from a patient with vasculitis were dissociated in 10 M urea, p H 3 and focused in an I E F gel containing 9 M urea. Human fibronectin was identified in the dissociated IC (Fig. 5, lane 1) by biotinylated F(ab')2 fragments of rabbit anti-fibronectin, and human C 3 was identified in the dissociated IC by biotinylated F(ab')2 fragment of rabbit anti-C 3 (Fig. 5, lane 2). Probing of the same IC sample with biotinylated F(ab')2 fragments of normal rabbit
pH8
pH4 1
2
3
Fig. 5. Analysis of components in human immune complexes by IEF in 9 M urea. Isolated soluble immune complexes from a vasculitis patient were dissociated in 10 M urea, pH 3 and separated on a rehydratable IEF gel containing 9 M urea. Three lanes, each containing 1 /~g of the dissociated IC, were probed with the following reagents: lane 1, biotinylated F(ab')2 fragments of rabbit anti-fibronectin (5 /xg/ml); lane 2, biotinylated F(ab')2 fragments of rabbit anti-C3 (5/~g/ml); and lane 3, biotinylated F(ab')2 fragments of normal rabbit IgG (5 t~g/ml). The blots were developed with SA-HRP as described in the materials and methods section.
I g G (Fig. 5, lane 3) did not detect any reactivity, demonstrating that there was no detectable rheumatoid factor activity or other anti-globulin activity between the IC components and the rabbit F(ab')2 fragments. Therefore, the use of hum a n Fc fragments to block both the IC components and the biotinylated probes (see materials and methods section) permitted the detection of only the specific antigens.
Discussion S D S - P A G E is a commonly employed technique for the analysis of IC (Casali and Lambert, 1979; Male and Roitt, 1979; Falus et al., 1981). Unfortunately, some antigenic determinants may be irreversibly denatured by SDS. Reducing agents
109
like fl-mercaptoethanol will dissociated the proteins into their respective subunits and after transfer to nitrocellulose the components may lack biological activity and antigenicity (Tovey et al., 1987). For example, denatured IC protein components may not be detected by monoclonal antibodies (Braun et al., 1983; De Blas and Cherwinski, 1983). Since IEF does not contain dissociating or reducing agents, IEF would be the method of choice to analyze IC components, because it might allow the maintenance of both biological activity and antigenicity of these separated components. Although there are several reports describing the use of IEF for the analysis of IC in the absence of denaturing reagents (Maidment et al., 1980, 1981; Hoffken et al., 1982), the ability of these IEF methods to dissociate and analyze IC components has recently been questioned (Haas and Schlaak, 1987). Disagreements over whether IEF is an effective method to analyze IC might be explained by differences in antibody avidity, sample preparation, or IC analysis methods, such as tube gels versus slab gels. Like Haas and Schlaak (1987), we were unable to separate IC components in slab gels unless the dissociating conditions described in this paper were used in both sample preparation and IEF analysis. The data in this paper demonstrate that 10 M urea, pH 3 is needed to dissociate the three IC tested and 9 M urea is needed in the IEF gel to maintain this dissociation (Figs. 1 and 2). These dissociating conditions permitted IC component separation during IEF and were effective in dissociating both soluble and insoluble IC (Fig. 4). Although the IC were both dissociated and analyzed in denaturing buffers, they retained their biological activity and antigenicity when transferred to nitrocellulose (Fig. 3). It was previously shown that IgG rheumatoid factors also retained biological activity when analyzed and blotted under these identical conditions (Hoffman et al., 1989b). Therefore, the separated IC components appeared to be stabilized and in a native conformation after being bound to nitrocellulose. This retention of biological activity by IC components following IEF separation is a major advantage over the SDS-PAGE analysis of IC. The time of analysis is another advantage that this method has
over SDS-PAGE. Running the IEF gel takes about 30 min, and the proteins can be blotted to nitrocellulose in 15 min with a 95% transfer efficiency (I4offman et al., 1989b). Although three IC systems were studied, only one representative IC system was presented. All of the IC were dissociated and focused in this gel system (data not shown). All of the IC components also displayed pI changes when analyzed in IEF gels containing 9 M urea. Urea causes an unfolding of proteins, and the unfolded proteins frequently display a change in pI as previously noted (Hoffman et al., 1989b). In summary, a new technique is described which allows the rapid analysis of IC components using IEF. The focused components can be transferred to nitrocellulose in 15 min, and the bound proteins retain biological activity and antigenicity.
Acknowledgements This work was supported by a grant from the National Institutes of Health (AR-36323) and by the John and Katie Jackson Foundation.
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110 Hoffman, W.L. and Jump, A.A. (1985) Rapid blotting of IgG to nitrocellulose with minimal IgM contamination. J. Immunol. Methods 76, 263. Hoffman, W.L. and Jump, A.A. (1989) Inhibition of the streptavidin-biotin interaction by milk. Anal. Biochem. 181,318. Hoffman, W.L., Jump, A.A., Kelly, P.J. and Elangovan, N. (1989) Rehydratable agarose gels: Application to isoelectric focusing in 9 molar urea. Electrophoresis 10, 741. Hoffman, W.L., Jump, A.A. and Smiley, J.D. (1990) Synthesis of specific idiotypes by rheumatoid synovium. Arthritis Rheum., in press. Kneba, M., Krieger, G., Kehl, A., Bause, I. and Nagel, G.A. (1983) Chromatofocusing combined with the ELISA technique. A sensitive method for the analysis of immune complexes. J. Immunol. Methods 61, 233. Maidment, Jr., B.W., Papsidero, L.D. and Chu, T.M. (1980) Isoelectric focusing-a new approach to the study of immune complexes. J. Immunol. Methods 35, 297.
Maidment, Jr., B.W., Papsidero, L.D., Gamarra, M., Nemoto, T. and Chu, T.M. (1981) Isoelectric focusing analysis of soluble immune complexes bound to protein A-Sepharose. Anal. Biochem. 111, 336. Male, D. and Roitt, I.M. (1979) Analysis of the components of immune complexes. Mol. Immunol. 16, 197. Porter, R.R. (1958) Separation and isolation of fractions of rabbit gamma-globulin containing the antibody and antigenic combining sites. Nature 182, 670. Tovey, E.R., Ford, S.A. and Baldo, B.A. (1987) Protein blotting on nitrocellulose: Some important aspects of the resolution and detection of antigens in complex extracts. J. Biochem. Biophys. Methods 14, 1. Updyke, T.V. and Nicolson, G.L. (1984) Immunoaffinity isolation of membrane antigens with biotinylated monoclonal antibodies and immobilized streptavidin matrices. J. Immunol. Methods 73, 83.
