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Cascade immunoelectrophoresis: Combined electrophoretic and solid-phase processing of immunoreactive protein by zonal immobilization
Journal of Immunological @ Elsevier/North-Holland
Methods, 42 (1981) Biomedical Press
229-241
229
CASCADE IMMUNOELECTROPHORESIS: COMBINED ELECTROPHORETIC AND SOLID-PHASE PROCESSING OF IMMUNOREACTIVE PROTEIN BY ZONAL IMMOBILIZATION
JOHN R. SHAINOFF
and BEATRIZ
N. DARDIK
Thrombosis Research Section, The Cleveland Clinic Foundation, U.S.A.
Cleveland, OH 44106,
(Received 1’7 October 1980, accepted 26 November 1980)
A new approach to immunochemical analysis of complex mixtures of proteins has been devised through development of a system for (1) sequentially absorbing, desorbing, and cascading antibodies in a profile indicative of both the quantity and electrophoretic characteristics of the antigen, and (2) for carrying out multiple tests of antigenic determinants at sub-picomole levels regardless of the solubility or precipitin forming characteristics of the immunoreactive protein. The procedure employs zonal immobilization in which a novel, aldehyde-rich gel (glyoxyl agarose) is used interchangeably as an inert support for electrophoresis and as an immobilizing matrix to fix the protein for analysis by solid-phase techniques. Proteins may be separated on the gel as with ordinary agarose, and then driven to combine covalently with the gel upon exposure to NaCNBHs. After removing NaCNBHs, the distribution of specified antigens is then established by exposing the gel to antibody and profiling the pattern of antibody uptake by a cross-electrophoretic technique in which the absorbed antibody is (1) desorbed with dodecyl sulfate, then (2) transferred through a gel containing potassium ion to halt migration of the detergent, and (3) displayed by immunoprecipitation with anti-IgG antibodies. To cascade the process further, the IgG may be immobilized and used as a surrogate antigen to bind additional antibody protein. In addition to enhancing sensitivity, the cross-electrophoretic measurement of uptake of antibody by immobilized antigen eliminates dependence on direct immunoprecipitation of the antigen, and may accordingly be of particular advantage with non-precipitin forming antigens and monoclonal antibodies.
INTRODUCTION
Conventional immunoelectrophoretic methods depend on direct precipitation for the detection and measurement of electrophoretically mobilized immunoreactive substances. The dependence on direct immunoprecipitation prohibits application to analysis of proteins which either undergo selfprecipitation or fail to form insoluble complexes. These limitations became evident to us in attempts to detect fibrinogen-related antigens in blood after separating them by electrophoresis in sodium dodecyl sulfate (SDS). Existing methods for removing SDS to allow precipitation of antigens by antibody (Converse and Papermaster, 1975; Schafer Nielsen and Bjerrum, 1975)
230
caused non-specific precipitation of plasma fibrinogen and its polymeric forms, while many degraded forms of fibrinogen failed to produce a distinct reaction. Since we could not depend on full mobilization and direct immunoprecipitation, we explored the opposite approach of immobilizing the antigens after electrophoresis and detecting them with mobilized antibody. With prospect that direct attachment of antigens to the gel matrix would offer important advantages over the usual methods for post-electrophoretic fixation, we sought to develop a gel matrix that could be utilized as a separation medium and then activated to bind the separated protein. An aldehydic derivative of agarose described as ‘glyoxyl agarose’ (Shainoff, 1980) enabled us to achieve that goal. We now describe a novel antibody absorption procedure for profiling the distribution of the immobilized antigen. The approach as conceived (Fig. 1) and applied here to profile mobilities of fibrinogen in SDS involves 4 steps in sequence to (1) prepare the specimen by electrophoretically arraying and then immobilizing the protein on glyoxyl agarose, (2) expose the immobilized protein to specific antibody by electrophoretically transporting anionically modified antibody-IgG into the sample gel and then back out, (3) displace the absorbed antibody-IgG from the immobilized antigen by electrophoretically transporting SDS through the sample gel to desorb the IgG and transfer it into an abutting spacer gel, and (4) display the desorbed antibody by electrophoretically transporting K’ cathodahy into the spacer to halt migration of the detergent while concomitantly transferring the IgG anodally into a contiguously placed gel for immunoprecipitation with anti-IgG antibody. The first steps are analogous to an immunologic staining procedure and have been employed as such in studies of factor VIII-R antigen (Hoyer and Shainoff, 1980). The second two steps serve as a device for quantification and for cascading sensitivity through use of a second antibody. MATERIALS
AND METHODS
Primary antibody
Rabbit anti-rat fibrinogen antibodies were purified (Shainoff and Braun, 1973) to yield fully immunoreactive protein and were processed further by chromatography (Levy and Sober, 1960) to remove IgM. They were then modified by carbamylation to promote anodic migration in electrophoresis according to Bjerrum et al. (1973) and were also labeled with lissaminerhodamine B to facilitate tracking, the two modifications being carried out together. The antibody (1 ml at 20-30 mg/ml) was diluted at 0°C with 0.015 M Na borate (2 ml) adjusted with HCl to pH 9, and 0.1 ml of lissamine-rhodamine B sulfonyl chloride (Eastman Chemical Co.) at 12.5 mg/ml in acetone was admixed drop-wise and stirred while maintaining pH 9 with 0.02 M NaOH for 2 h. The solution was then adjusted to pH 8.0 and brought to 45”C, whereupon 102 mg of dry KCNO was added and incubated
--I--
f
NaCNBH3,
then
Immobilization immersion in
of specimen
Fig. 1. Schematic
a4
-
t
0
Spacer Antibody loading gel
-
excess antibody
Electrophoretic retrieval of
Electrophoresit of
contact gel
Specimen gel
Contact gel
_
-
2. Exposure to antibody
of the procedure,
by
Electrophoresis
Application well
Y!
0
n
H
Glyoxyl agarose (pH
1. Arraying of antigen
ii
1
with SDS
boundary
ahead of antibody
Excission of SDS
Aantibody
transfer of
Desorption and
Contact/spacar gel
3. Desorption of antibody
I
0
with anti.lgG
of primaty antibody
Immunopmcipitatum
precipitation gel
_
and IgG to immuno-
precipitation of SDS,
of K+ to spacer for
concurrent transfer
in sodium buffer
Desorbed antibody
in potassium buffer
Ab K+ I I
0
0
-
gel with anti-IgG
lmmunoprecipitation
4. Measurement of antibody
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for 100 min while maintaining pH 8 with a solution of 0.2 M HCl and 0.2 M KCl. The modified antibody was then separated from the reactants on Sephadex G-25 equilibrated with 0.03 M Na phosphate at pH 6.85 for use in electrophoresis. Incorporation of dye intc the protein was estimated to range from 1.5 to 2 moles/mole protein, the calculation being based on absorbancies (A) of protein and dye at 280 and 575 nm [dye/protein = 3.73 AsT5/ (Azso-0.22 AsT5)]. The degree of carbamylation was assessed only by verifying expected (Bjerrum et al., 1973) shifts in electrophoretic mobility of the protein. Second antibody Swine anti-rabbit
immunoglobulin antibody (340 Sewell titer, BioRad Laboratories) at l/250 dilution in 0.03 M K phosphate (pH 6.85) buffered 1% agarose was used for electroimmunoassay of the primary antibody.
Electrophoresis
Gels from ordinary agarose were prepared for electrophoresis as described by Laurel1 (1965) and detailed by Weeke (1973) except for composition. Phosphate (0.03 M) was selected for buffering by anion alone to avoid large pH shifts on displacing Na’ with K’ in step 4, and neutral pH 6.85 was required instead of the usual alkaline conditions for immunoelectrophoresis because the immunoglobulins used as secondary antibody had zero mobility at that pH with K’ as counter-ion. Neutral pH was maintained throughout electrophoresis by recirculating buffer between the electrode chambers through a peristaltic pump. All gels were cast at 1.5 mm depth in an 8 cm X 8 cm format on glass plates, and electrophoresis was carried out with cooling at 14°C. The agarose content of spacer and contact gels amounted to 1.5% except for the 2% specified for step 3. Glyoxyl agarose was used only in the specimen gels. Specimen
preparation
(step 1)
Specimen gels containing electrophoretically separated antigens were prepared according to earlier (Shainoff, 1980) guidelines for zonal immobilization. Specifically, 12.5 ml of a 2% stock suspension of glyoxyl agarose (1.0-1.2 mEq aldehyde/g agarose matrix) was melted together with 1 g of regular agarose plus 37 ml of 0.03 M Na phosphate at pH 6.85, plus 0.5 ml of 10% SDS to obtain gel with 0.5% glyoxyl and 2% regular agarose. Amounts of protein applied to the gels were gauged so that zonal concentrations after separation would not greatly exceed 1 mg/ml. For plasma, 2 ~1 at l/6 dilution in SDS-phosphate buffer was applied across 1 mm X 5 mm slots in the gel, and voltage was applied (150 V/8 cm) until a marker consisting of ribonuclease labeled with lissamine-rhodamine traversed 7 cm. Immobilization of the separated protein was then achieved by immersing the gel with gentle agitation in a solution (200 ml) of 0.2 M Na carbonate (pH lo), 1% Lubrol, and 0.02 M NaCNBH, for 2 h, whereafter the gel was washed (3X)
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by immersion in 500 ml of 0.03 M Na phosphate at pH 6.85 overnight in preparation for loading of the primary antibody (step 2). For storage beyond a week, NaN3 was added (0.2 mg/ml). As indicated before (Shainoff, 1980), good fixation could be achieved without resorting to alkaline pH, but required higher (0.2 M) concentrations of NaCNBH3. Loading
of the primary
antibody
(step 2)
Antibody was applied and retrieved by electrophoresing it through a spacer gel into the specimen and then back out, the spacer being interfaced to prevent slow migrating immunoglobulin from contacting the specimen. A 5-7 mm wide strip of gel containing the antibody, carbamylated and labeled as described, was laid 2-4 mm from the specimen, and intervening space filled with melted gel (<6O”C) to form the spacer. Fairly high concentrations of antibody were used. For specimens containing zonal concentrations of fibrinogen on the order of 0.1 mg/ml (concentrations which at equivalence would titrate 0.17 mg antibody per ml), the antibody in the loading strip was set at 1 mg/ml to provide a g-fold excess. Transport of the antibody from the loading strip into the specimen gel required approximately 1 h at 75 mA and 150 V. As a refinement (not illustrated in the diagram) to prevent transport of antibody beyond the specimen, a strip of dialysis membrane and a thick slab of gel were laid across the anodal edge of the specimen to provide a removable, protein-excluding shunt to the anodal contact gel, Specifically, the membrane (1.5 cm wide) was laid to cover much of the specimen and extend beyond it in the anodal direction, and the overlaid slab (6 mm thick X 2 cm wide) was placed in position overlapping the anodal edge of the specimen by only 2-3 mm and extending beyond the membrane in the anodal direction.’ On completion of antibody transfer the current was reversed briefly (10 min) to carry accumulated portions of antibody away from the membrane and back into the specimen. The antibody was then allowed to remain in the specimen for 3 h before resuming electrophoresis to remove non-absorbed portions. More conveniently, the specimen with antibody loaded was excised from the plate, stored at 4°C overnight in a wrapped Petri dish, and then replaced with fresh contact gel poured around it on a clean plate. To remove excess antibody from the specimen, current was applied for 3 h in direction opposite to that used for loading. Otherwise, unloading took longer due to unequal distribution of slow and fast migrating immunoglobulins in the gel. To recover the antibody in concentrated form for use on other specimens, a thick strip of gel and a membrane barrier were laid over the anodal contact gel to impose a voltage drop at a position l-l.2 cm from the specimen as used to limit migration in the loading procedure. The recovered antibody could be seen by fluorescence concentrated in a tight (3 mm) zone at the barrier, and immunoabsorbed portions could be seen in weakly fluorescent bands coinciding with position(s) of the immobilized antigen within the specimen. The zone containing the retrieved antibody was
234
excised, and the recovered antibody was stored in the gel for loading the next specimen as in step 2. For prolonged storage, the excised gel was coated with 20 ~1 of 30 mM NaNa, and kept at 4°C in a wrapped dish. The immunoabsorbed portions of antibody in the specimen gel were analyzed by desorption and immunoprecipitation (steps 3 and 4). Desorption
of antibody
(step 3)
Displacement of immunoadsorbed antibody out of the specimen gel with SDS was commenced only after careful inspection with long-wave ultraviolet light and with safety glasses for ‘hot spots’ of non-specifically entrapped or precipitated antibody which could be confused with specifically bound antibody after displacement. Such flaws, when occasionally found, occurred at edges where heat from poor junctions or bubbles caused denaturative deposition of immunoglobulins. For added precaution, the edges of the specimen were regularly trimmed by 1 mm to eliminate undetected artefacts. At this point, standards consisting of known quantities (1.0-0.4 pg) of the antibody preparation were added to 1 mm holes punched near the ends away from immobilized protein in the specimen gel. In addition to serving as standards for measurement of antibody retained by protein in the specimen, the applied antibody also aided tracking because of high fluorescence. To free the antibody, contact gel of 2% agarose was poured (<9O”C) to surround the trimmed specimen, then a strip 4 mm wide was excised at the cathodal side of the specimen for adding gel containing 0.2% SDS in buffer, and current was applied (150 V) to transport the SDS into the specimen. The location of the SDS was easily followed from the sharp refractile band at its leading edge. Alternatively, cytochrome-C could be applied (2 ~1 at 6 mg/ml) to the specimen to serve as an indicator which would not move until overtaken by the SDS, and would then migrate near the detergent front. The SDS was allowed to stand in the specimen for 0.5 h to complete desorption of antibody, and then electrophoresis was resumed until the refractile band migrated to 4 cm beyond the specimen, whereupon desorbed antibody could be seen at position 3 cm from the specimen, a spacing which allowed removal of a major portion of the SDS by excising a 1.5 cm strip of gel in the region just to the front of the fluorescent antibody. When the SDSrich gel was not removed a heavy precipitation of the detergent would ensue on adding K’ in step 4, and cause a partial co-precipitational loss of antibody. After excising the strip of SDS-rich gel, the gap was closed by repositioning to abut the residual contact gel, and current was reversed for about 20 min to transport the antibody back to within a few millimeters from the specimen. The back transport reversed spread and sharpened the antibody protein into a tight zone, which sharpened subsequent precipitin formation with the second antibody (step 4). Immunoassay
of desorbed
antibody
(step 4)
Quantification of desorbed antibody with swine anti-immunoglobulin as
second antibody was patterned after the technique of Bjerrum et al. (1973) who first described measurement of carbamylated IgG with unmodified IgG, the principal difference being use of potassium buffer to prevent dodecyl sulfate from interfering. Gel containing unmodified second antibody in potassium buffer was poured at the anodal side of the desorbed primary antibody, and, after changing to potassium buffer in the anode reservoir, current was applied at 60 V overnight to transport the primary antibody into the immunoprecipitation gel. Rapid precipitation of residual detergent, ensuing almost immediately on applying current, prevented it from permeating the immunoprecipitation gel provided low amperage (<80 mA/ cm2cross-section) was maintained as a precaution against melting the precipitate. RESULTS
Analyses of purified fibrinogen and normal rat plasma by the procedure as detailed showed a single peak (Fig. 2A) at the position expected for fibrinogen, and with area corresponding to retention of 2.3 -t 0.05 (S.E.M., N = 10) 1.18of primary antibody per pg of fibrinogen, which corresponded to 4.9 moles of IgG absorbed per mole of fibrinogen. These combining proportions were greater than observed in immunoprecipitational titration (Shainoff and Braun, 1973) where 1.73 pg of antibody were bound per pg of fibrinogen at equivalence, but were 66% less than one would calculate on basis that twice the equivalent combining proportion (or 3.46 I-(gper pg fibrinogen) might be expected to be absorbed with exposure to excess antibody. Thus, retention of antibody appeared to be consistent, but somewhat below the maximum that might be anticipated from the observed (Shainoff and Braun, 1973) and usual (Nussenzweig et al., 1961) content of antigenic determinants in fibrinogen. Higher retention, corresponding to 2.9 pg antibody per I.rg fibrinogen, was obtained when the procedure in step 2 was changed to remove the excess antibody from the specimen by washing instead of electrophoresis. When washed specimens were subjected to electrophoresis, a small portion of antibody leached away from the fibrinogen, as evidenced by the appearance of a faintly fluorescent streak emerging from the band of absorbed antibody. Accordingly, much of the incomplete retention suggested by the preceding calculations arose from removal of weakly bound antibody in the course of removing unbound antibody by the electrical field. Yet, enormous wastes of unbound antibody were incurred by washing. Since retrieval of antibody from washings proved troublesome by comparison to the speed and convenience of the electrophoretic method, and since results of comparable quality were obtained by either procedure we did not pursue the question of significance of the weakly bound antibody. A question of the extent to which antigenic determinants became masked by attachment of the fibrinogen to the gel was examined operationally by
237
increasing the content of glyoxyl agarose in the gel to determine whether the enriched immobilizing matrix would adversely affect antibody uptake. On changing composition of the specimen gel from 0.5% glyoxyl agarose and 2% regular agarose to a composition of 1.25% glyoxyl and 1.25% regular, retention of antibody fell to 1.9 and 1.7 pg per pg fibrinogen in two trials. With gel composed of 1.25% glyoxyl agarose alone, retention fell further to 1.5 pg per pg. These measurements led us to conclude that the immobilization altered accessibility of the fibrinogen to antibody to a moderate degree that was dependent in part upon the actual glyoxyl agarose content, but tended to diminish with regular agarose added to the glyoxyl agarose. Uptake of antibody by plasma protein other than fibrinogen was not observed except with one rabbit anti-human fibrinogen preparation which, despite purification contained some antibody that was retained by IgG in the specimen gel. The contaminant was not absorbed by undenatured IgG, but could be removed by absorption with SDS-treated IgG immobilized (Shainoff, 1980) on beaded glyoxyl agarose, and accordingly appeared to correspond to rheumatoid factor. The experience demonstrated that antibody specificity should be tested with SDS-treated protein when employed for measurement of SDS-treated antigens. Desorption of retained antibody with SDS in step 3 proceeded to completion as judged by full mobilization of the fluorescent antibody out of the specimen. However, full removal of fluorescence was not-obtained in early trials which employed labeling of the antibody with fluorescein isothiocyanate instead of the adopted lissamine-rhodamine sulfonylchloride. The fluorescein initially appeared desirable because labeling with 5 moles per mole antibody induced the same alteration of mobility as achieved by the carbamylation procedure, but despite numerous attempts we were unable to refine the fluoresceinated antibody to abolish irreversible staining of the Fig. 2. Results of the procedure as applied to analysis of the distribution of fibrinogenrelated antigens in SDS/glyoxyl agarose electrophoregrams of plasma from normal (A) and malignant hypertensive (B) rats. The electrophoretically arrayed and immobilized protein in the specimen gels shown lying across the bottom of the illustrations were exposed to rabbit anti-fibrinogen antibody, and absorbed antibody was desorbed and transported (upwards) for measurement by immunoprecipitation with swine anti-rabbit immunoglobulin antibody. The photographs were taken after staining with Coomassie blue. The peaks at the ends of the precipitin lines portray rabbit antibody applied directly at the edges of the specimens for calibration. The occurrence of only one peak apart from that of the standard for the normal specimen (A) corroborated specificity of the antibody. As calculated from comparison with area under the peak for the standard (0.75 pg applied), and from the predetermined amount of thrombin coagulable protein in the specimen (0.89 pg from 2 ~1 plasma at l/6 dilution), retention of antibody corresponded to 2.2 pg/pg fibrinogen. Many additional peaks arose from antibody displaced from polymers and degraded forms of fibrinogen in the abnormal specimen (B) shown for illustration of the resolving power of the procedure. The standards in B consisted of 1.0 and 0.5 fig antibody, and content of thrombin coagulable protein in the applied plasma (2 /.d/6) was 1.9 pg.
238
antigen and occasionally other proteins. Similar problems were encountered with several other fluorescent isothiocyanates for reasons as yet unknown, but which we suspect involved a slow exchange of the label which did not occur with lissamine-rhodamine sulfonylchloride. In preparation for immunoprecipitational assay of the desorbed antibody, attempts were made to use Lubrol@ to retard transport of the SDS (Converse and Papermaster, 1975), but the degree of retardation achieved did not suffice to prevent the antibody from being swept away before completion of the immunoprecipitation. With the new approach of precipitating the dodecyl sulfate by electrophoretic addition of K’, some experience was needed to carry out the recommended positioning of the detergent for precipitation. In practice, the positions of the precipitated detergent and the fluorescent antibody were discernible within minutes after applying current for adding K’, so that regions of gel rich in detergent could be excised without risk of removing antibody. Further, the detergent could be redissolved and repositioned at will by simply reversing current to return Na’ to the buffer. Immunoassay of the desorbed primary antibody in step 4 proceeded as anticipated from studies by Bjerrum et al. (1973), except for need to maintain neutral pH with which the second antibody (swine anti-rabbit immunoglobulin) had zero mobility in the K’ buffer. This pH also proved suitable in studies using secondary antibody derived from rabbits, but not from goats. Detection of minor components of fibrinogen related antigens in specimens, as illustrated in Fig. ZB, depended on near complete removal of nonabsorbed portions of primary antibody. Portions left in the specimen contributed to the base of the precipitin line used for measurement of specifically retained antibody. When large portions were left a correspondingly large displacement of the baseline resulted, and irregularities in the distribution of antibody attained in loading would cause correspondingly irregular baselines. The distinct peaks observed in the illustrated results were presumed to be produced by antibody displaced from fibrinogen-related antigens in the specimens, because they could be seen by fluorescence to arise from appropriately shaped bands resembling immobilized protein in the specimen, were found in duplicate analyses, and were not observed in specimens of sera devoid of immunoreactive fibrinogen. Some minor components resolved in Fig. 2B corresponded to as little as 1% of the total fibrinogen, the equivalent of 20 ng or 6 X lo-i4 mole of fibrinogen in the specimen. DISCUSSION
The overall procedure differs from direct immunoelectrophoresis mainly in the use of zonal immobilization, made possible by the development of a medium that can be used interchangeably and sequentially either for separating or immobilizing proteins. We view the ability to array proteins on an immobilizing substrate in position indicative of physical properties of the protein as adding a new dimension to solid-phase technology that is anal-
239
ogous to the advance gained by applying electrophoresis to analysis of proteins in fluid phase. Although protein immobilization is widely used in immunoabsorption procedures (Robbins and Schneerson, 1974), reactivities of the immobilizing ligands have been such as to allow only random distribution of protein on the substrate. Thus, the substrates could be used only for immobilization of highly purified (monospecific) protein to obtain defined composition. Electrophoretic arraying of protein on glyoxyl agarose and subsequent immobilization with NaCNBH3 yield a dimensionally defined immunoabsorbent, step 1 of the procedure. As indicated before (Shainoff, 1980), the NaCNBH3 that is used to link the protein to the gel has no effect on either the protein or the gel separately, but acts in a highly specific manner (Borch et al., 1971) to drive reduction of the extremely weak and highly dissociable Schiff-base linkages that are formed between amines and simple alkanals. Thus, the protein and gel are left unaltered except at points of linkage catalyzed by the reducing agent. Removal of the NaCNBH3 returns the system to its non-reactive state, except for the immunoreactive or other specific epitopes imparted by the immobilized protein, the distribution of which is established by the pattern of antibody uptake (steps Z-4). As evidenced by the results obtained, negligible uptake of antibody by the gel occurs except through the imparted epitope even though the aldehyde groups of the gel are left unquenched. The ability to continue using the gel as a separation medium without quenching is an important advantage, because quenching reactions usually impart complications through added electrical charge. Since the immobilized state of the antigen preserves its distribution in the specimen, a battery of specific antibodies can be applied either to obtain consecutive details of composition, or to amplify sensitivity. Studies in progress are profiling not only the molecular weight distribution of fibrinogen-related antigens as illustrated here, but also the content of fibrinopeptides and neoantigenic determinants of the variant forms of the protein through sequential use of specific antibody (Plow et al. 1971; Nossel et al., 1976) to these epitopes. Sensitivity of the procedure is quite high. Based on the estimated retention of an average of two-thirds mole equivalent of primary antibody per antigenic determinant in fibrinogen examined in the present study, and the use of the retained IgG as a surrogate antigen having an effective valence of 8 for precipitation of the secondary antibody, we calculate that the method has about 5 times (2/3 X 8) greater sensitivity than direct immunoassay. If warranted to amplify sensitivity further the retained IgG could be immobilized with NaCNBH3 instead of measured directly, and then used as a second surrogate to cascade antibody uptake by another order of magnitude. It is important to emphasize that the procedure was not devised as a substitute for direct immunoelectrophoresis, but as an alternative when direct immunoprecipitation cannot be relied upon, a situation that arises when
240
studying either self-precipitating or non-immunoprecipitable antigens, and one that also arises in studies depending on use of monoclonal antibodies which usually do not yield precipitins. The 4 steps in the procedure entail twice the work of direct cross-immunoelectrophoresis. Additional effort may be required to ensure specificity of the antibody. The method depends critically on the use of purified antibody. Unlike direct immunoprecipitation methods with which unrelated antigens form separate precipitin lines, only one line is formed here - that produced by retained immunoglobulin. The need to purify antibody adds to cost of preparing it for use in the procedure, but is compensated manifold by the ability to recover all but specifically retained portion of the antibody applied in carrying out the procedure. By contrast, excess antibody spent in carrying out direct immunoprecipitation is usually lost, a consequence that adds considerably to the cost of the direct method. Several of the techniques developed for the procedure can be used to advantage in conjunction with other procedures. These include covalent immobilization to fix proteins for immunofluorescent staining or autoradiography, electrophoretic addition and removal of antibody to minimize losses in immunologic staining, and use of K’ instead of Lubrol@ (Converse and Papermaster, 1975) to limit migration of SDS. In addition, glyoxyl agarose provides an efficient protein immobilizing substrate for affinity purification of antibody, and for screening cell cultures for production of monoclonal antibodies. We view the techniques presented here as examples of ways in which zonal immobilization can provide a basis for the design of new immunologic procedures. ACKNOWLEDGEMENTS
Dolores Andrasic provided technical assistance. The work was supported in part by Grants HL-16361 and HL-19767 from the National Heart, Lung and Blood Institutes of the U.S. Public Health Service, and by an advanced research fellowship from the American Heart Association, North East Ohio Affiliate. REFERENCES Bjerrum, O.J., A. Ingold, H. Lowenstein and B. Weeke, 1973, Stand. J. Immunol. 2 (Suppl. l), 145. Borch, R.F., M.D. Bernstein and H.D. Durst, 1971, J. Am. Chem. Sot. 93, 2897. Converse, C. and D.S. Papermaster, 1975, Science 189, 469. Hoyer, L. and J.R. Shainoff, 1980, Blood 55, 1056. Laurell, C.B., 1965, Anal. Biochem. 10, 358. Levy, H.B. and H.A. Sober, 1960, Proc. Sot. Exp. Biol. Med. 103, 250. Nossel, H.L., V.P. Butler, Jr., G.D. Wilner, R.E. Canfield and E.J. Harfenist, 1976, Thromb. Haemostas. 35, 101.
241 Nussenzweig, V., M. Seligman, P. Grabar and M. Boucart, 1961, Ann. Inst. Pasteur 100, 490. Plow, E.F., C. Houghie and T.S. Edgington, 1971, J. Immunol. 107, 1496. Robbins, J.B. and R. Schneerson, 1974, in: Methods in Enzymology, Vol. 44, eds. W.B. Jackoby and M. Wilchak (Academic Press, NY) p. 703. Schafer Nielsen, C. and O.J. Bjerrum, 1975, Stand. J. Immunol. 4 (Suppl. 2), 73. Shainoff, J.R., 1980, Biochem. Biophys. Res. Commun. 95, 690. Shainoff, J.R. and W.E. Braun, 1973, Anal. Biochem. 55, 206. Weeke, B., 1973, Stand. J. Immunol. 2 (Suppl. l), 15.
Methods, 42 (1981) Biomedical Press
229-241
229
CASCADE IMMUNOELECTROPHORESIS: COMBINED ELECTROPHORETIC AND SOLID-PHASE PROCESSING OF IMMUNOREACTIVE PROTEIN BY ZONAL IMMOBILIZATION
JOHN R. SHAINOFF
and BEATRIZ
N. DARDIK
Thrombosis Research Section, The Cleveland Clinic Foundation, U.S.A.
Cleveland, OH 44106,
(Received 1’7 October 1980, accepted 26 November 1980)
A new approach to immunochemical analysis of complex mixtures of proteins has been devised through development of a system for (1) sequentially absorbing, desorbing, and cascading antibodies in a profile indicative of both the quantity and electrophoretic characteristics of the antigen, and (2) for carrying out multiple tests of antigenic determinants at sub-picomole levels regardless of the solubility or precipitin forming characteristics of the immunoreactive protein. The procedure employs zonal immobilization in which a novel, aldehyde-rich gel (glyoxyl agarose) is used interchangeably as an inert support for electrophoresis and as an immobilizing matrix to fix the protein for analysis by solid-phase techniques. Proteins may be separated on the gel as with ordinary agarose, and then driven to combine covalently with the gel upon exposure to NaCNBHs. After removing NaCNBHs, the distribution of specified antigens is then established by exposing the gel to antibody and profiling the pattern of antibody uptake by a cross-electrophoretic technique in which the absorbed antibody is (1) desorbed with dodecyl sulfate, then (2) transferred through a gel containing potassium ion to halt migration of the detergent, and (3) displayed by immunoprecipitation with anti-IgG antibodies. To cascade the process further, the IgG may be immobilized and used as a surrogate antigen to bind additional antibody protein. In addition to enhancing sensitivity, the cross-electrophoretic measurement of uptake of antibody by immobilized antigen eliminates dependence on direct immunoprecipitation of the antigen, and may accordingly be of particular advantage with non-precipitin forming antigens and monoclonal antibodies.
INTRODUCTION
Conventional immunoelectrophoretic methods depend on direct precipitation for the detection and measurement of electrophoretically mobilized immunoreactive substances. The dependence on direct immunoprecipitation prohibits application to analysis of proteins which either undergo selfprecipitation or fail to form insoluble complexes. These limitations became evident to us in attempts to detect fibrinogen-related antigens in blood after separating them by electrophoresis in sodium dodecyl sulfate (SDS). Existing methods for removing SDS to allow precipitation of antigens by antibody (Converse and Papermaster, 1975; Schafer Nielsen and Bjerrum, 1975)
230
caused non-specific precipitation of plasma fibrinogen and its polymeric forms, while many degraded forms of fibrinogen failed to produce a distinct reaction. Since we could not depend on full mobilization and direct immunoprecipitation, we explored the opposite approach of immobilizing the antigens after electrophoresis and detecting them with mobilized antibody. With prospect that direct attachment of antigens to the gel matrix would offer important advantages over the usual methods for post-electrophoretic fixation, we sought to develop a gel matrix that could be utilized as a separation medium and then activated to bind the separated protein. An aldehydic derivative of agarose described as ‘glyoxyl agarose’ (Shainoff, 1980) enabled us to achieve that goal. We now describe a novel antibody absorption procedure for profiling the distribution of the immobilized antigen. The approach as conceived (Fig. 1) and applied here to profile mobilities of fibrinogen in SDS involves 4 steps in sequence to (1) prepare the specimen by electrophoretically arraying and then immobilizing the protein on glyoxyl agarose, (2) expose the immobilized protein to specific antibody by electrophoretically transporting anionically modified antibody-IgG into the sample gel and then back out, (3) displace the absorbed antibody-IgG from the immobilized antigen by electrophoretically transporting SDS through the sample gel to desorb the IgG and transfer it into an abutting spacer gel, and (4) display the desorbed antibody by electrophoretically transporting K’ cathodahy into the spacer to halt migration of the detergent while concomitantly transferring the IgG anodally into a contiguously placed gel for immunoprecipitation with anti-IgG antibody. The first steps are analogous to an immunologic staining procedure and have been employed as such in studies of factor VIII-R antigen (Hoyer and Shainoff, 1980). The second two steps serve as a device for quantification and for cascading sensitivity through use of a second antibody. MATERIALS
AND METHODS
Primary antibody
Rabbit anti-rat fibrinogen antibodies were purified (Shainoff and Braun, 1973) to yield fully immunoreactive protein and were processed further by chromatography (Levy and Sober, 1960) to remove IgM. They were then modified by carbamylation to promote anodic migration in electrophoresis according to Bjerrum et al. (1973) and were also labeled with lissaminerhodamine B to facilitate tracking, the two modifications being carried out together. The antibody (1 ml at 20-30 mg/ml) was diluted at 0°C with 0.015 M Na borate (2 ml) adjusted with HCl to pH 9, and 0.1 ml of lissamine-rhodamine B sulfonyl chloride (Eastman Chemical Co.) at 12.5 mg/ml in acetone was admixed drop-wise and stirred while maintaining pH 9 with 0.02 M NaOH for 2 h. The solution was then adjusted to pH 8.0 and brought to 45”C, whereupon 102 mg of dry KCNO was added and incubated
--I--
f
NaCNBH3,
then
Immobilization immersion in
of specimen
Fig. 1. Schematic
a4
-
t
0
Spacer Antibody loading gel
-
excess antibody
Electrophoretic retrieval of
Electrophoresit of
contact gel
Specimen gel
Contact gel
_
-
2. Exposure to antibody
of the procedure,
by
Electrophoresis
Application well
Y!
0
n
H
Glyoxyl agarose (pH
1. Arraying of antigen
ii
1
with SDS
boundary
ahead of antibody
Excission of SDS
Aantibody
transfer of
Desorption and
Contact/spacar gel
3. Desorption of antibody
I
0
with anti.lgG
of primaty antibody
Immunopmcipitatum
precipitation gel
_
and IgG to immuno-
precipitation of SDS,
of K+ to spacer for
concurrent transfer
in sodium buffer
Desorbed antibody
in potassium buffer
Ab K+ I I
0
0
-
gel with anti-IgG
lmmunoprecipitation
4. Measurement of antibody
232
for 100 min while maintaining pH 8 with a solution of 0.2 M HCl and 0.2 M KCl. The modified antibody was then separated from the reactants on Sephadex G-25 equilibrated with 0.03 M Na phosphate at pH 6.85 for use in electrophoresis. Incorporation of dye intc the protein was estimated to range from 1.5 to 2 moles/mole protein, the calculation being based on absorbancies (A) of protein and dye at 280 and 575 nm [dye/protein = 3.73 AsT5/ (Azso-0.22 AsT5)]. The degree of carbamylation was assessed only by verifying expected (Bjerrum et al., 1973) shifts in electrophoretic mobility of the protein. Second antibody Swine anti-rabbit
immunoglobulin antibody (340 Sewell titer, BioRad Laboratories) at l/250 dilution in 0.03 M K phosphate (pH 6.85) buffered 1% agarose was used for electroimmunoassay of the primary antibody.
Electrophoresis
Gels from ordinary agarose were prepared for electrophoresis as described by Laurel1 (1965) and detailed by Weeke (1973) except for composition. Phosphate (0.03 M) was selected for buffering by anion alone to avoid large pH shifts on displacing Na’ with K’ in step 4, and neutral pH 6.85 was required instead of the usual alkaline conditions for immunoelectrophoresis because the immunoglobulins used as secondary antibody had zero mobility at that pH with K’ as counter-ion. Neutral pH was maintained throughout electrophoresis by recirculating buffer between the electrode chambers through a peristaltic pump. All gels were cast at 1.5 mm depth in an 8 cm X 8 cm format on glass plates, and electrophoresis was carried out with cooling at 14°C. The agarose content of spacer and contact gels amounted to 1.5% except for the 2% specified for step 3. Glyoxyl agarose was used only in the specimen gels. Specimen
preparation
(step 1)
Specimen gels containing electrophoretically separated antigens were prepared according to earlier (Shainoff, 1980) guidelines for zonal immobilization. Specifically, 12.5 ml of a 2% stock suspension of glyoxyl agarose (1.0-1.2 mEq aldehyde/g agarose matrix) was melted together with 1 g of regular agarose plus 37 ml of 0.03 M Na phosphate at pH 6.85, plus 0.5 ml of 10% SDS to obtain gel with 0.5% glyoxyl and 2% regular agarose. Amounts of protein applied to the gels were gauged so that zonal concentrations after separation would not greatly exceed 1 mg/ml. For plasma, 2 ~1 at l/6 dilution in SDS-phosphate buffer was applied across 1 mm X 5 mm slots in the gel, and voltage was applied (150 V/8 cm) until a marker consisting of ribonuclease labeled with lissamine-rhodamine traversed 7 cm. Immobilization of the separated protein was then achieved by immersing the gel with gentle agitation in a solution (200 ml) of 0.2 M Na carbonate (pH lo), 1% Lubrol, and 0.02 M NaCNBH, for 2 h, whereafter the gel was washed (3X)
233
by immersion in 500 ml of 0.03 M Na phosphate at pH 6.85 overnight in preparation for loading of the primary antibody (step 2). For storage beyond a week, NaN3 was added (0.2 mg/ml). As indicated before (Shainoff, 1980), good fixation could be achieved without resorting to alkaline pH, but required higher (0.2 M) concentrations of NaCNBH3. Loading
of the primary
antibody
(step 2)
Antibody was applied and retrieved by electrophoresing it through a spacer gel into the specimen and then back out, the spacer being interfaced to prevent slow migrating immunoglobulin from contacting the specimen. A 5-7 mm wide strip of gel containing the antibody, carbamylated and labeled as described, was laid 2-4 mm from the specimen, and intervening space filled with melted gel (<6O”C) to form the spacer. Fairly high concentrations of antibody were used. For specimens containing zonal concentrations of fibrinogen on the order of 0.1 mg/ml (concentrations which at equivalence would titrate 0.17 mg antibody per ml), the antibody in the loading strip was set at 1 mg/ml to provide a g-fold excess. Transport of the antibody from the loading strip into the specimen gel required approximately 1 h at 75 mA and 150 V. As a refinement (not illustrated in the diagram) to prevent transport of antibody beyond the specimen, a strip of dialysis membrane and a thick slab of gel were laid across the anodal edge of the specimen to provide a removable, protein-excluding shunt to the anodal contact gel, Specifically, the membrane (1.5 cm wide) was laid to cover much of the specimen and extend beyond it in the anodal direction, and the overlaid slab (6 mm thick X 2 cm wide) was placed in position overlapping the anodal edge of the specimen by only 2-3 mm and extending beyond the membrane in the anodal direction.’ On completion of antibody transfer the current was reversed briefly (10 min) to carry accumulated portions of antibody away from the membrane and back into the specimen. The antibody was then allowed to remain in the specimen for 3 h before resuming electrophoresis to remove non-absorbed portions. More conveniently, the specimen with antibody loaded was excised from the plate, stored at 4°C overnight in a wrapped Petri dish, and then replaced with fresh contact gel poured around it on a clean plate. To remove excess antibody from the specimen, current was applied for 3 h in direction opposite to that used for loading. Otherwise, unloading took longer due to unequal distribution of slow and fast migrating immunoglobulins in the gel. To recover the antibody in concentrated form for use on other specimens, a thick strip of gel and a membrane barrier were laid over the anodal contact gel to impose a voltage drop at a position l-l.2 cm from the specimen as used to limit migration in the loading procedure. The recovered antibody could be seen by fluorescence concentrated in a tight (3 mm) zone at the barrier, and immunoabsorbed portions could be seen in weakly fluorescent bands coinciding with position(s) of the immobilized antigen within the specimen. The zone containing the retrieved antibody was
234
excised, and the recovered antibody was stored in the gel for loading the next specimen as in step 2. For prolonged storage, the excised gel was coated with 20 ~1 of 30 mM NaNa, and kept at 4°C in a wrapped dish. The immunoabsorbed portions of antibody in the specimen gel were analyzed by desorption and immunoprecipitation (steps 3 and 4). Desorption
of antibody
(step 3)
Displacement of immunoadsorbed antibody out of the specimen gel with SDS was commenced only after careful inspection with long-wave ultraviolet light and with safety glasses for ‘hot spots’ of non-specifically entrapped or precipitated antibody which could be confused with specifically bound antibody after displacement. Such flaws, when occasionally found, occurred at edges where heat from poor junctions or bubbles caused denaturative deposition of immunoglobulins. For added precaution, the edges of the specimen were regularly trimmed by 1 mm to eliminate undetected artefacts. At this point, standards consisting of known quantities (1.0-0.4 pg) of the antibody preparation were added to 1 mm holes punched near the ends away from immobilized protein in the specimen gel. In addition to serving as standards for measurement of antibody retained by protein in the specimen, the applied antibody also aided tracking because of high fluorescence. To free the antibody, contact gel of 2% agarose was poured (<9O”C) to surround the trimmed specimen, then a strip 4 mm wide was excised at the cathodal side of the specimen for adding gel containing 0.2% SDS in buffer, and current was applied (150 V) to transport the SDS into the specimen. The location of the SDS was easily followed from the sharp refractile band at its leading edge. Alternatively, cytochrome-C could be applied (2 ~1 at 6 mg/ml) to the specimen to serve as an indicator which would not move until overtaken by the SDS, and would then migrate near the detergent front. The SDS was allowed to stand in the specimen for 0.5 h to complete desorption of antibody, and then electrophoresis was resumed until the refractile band migrated to 4 cm beyond the specimen, whereupon desorbed antibody could be seen at position 3 cm from the specimen, a spacing which allowed removal of a major portion of the SDS by excising a 1.5 cm strip of gel in the region just to the front of the fluorescent antibody. When the SDSrich gel was not removed a heavy precipitation of the detergent would ensue on adding K’ in step 4, and cause a partial co-precipitational loss of antibody. After excising the strip of SDS-rich gel, the gap was closed by repositioning to abut the residual contact gel, and current was reversed for about 20 min to transport the antibody back to within a few millimeters from the specimen. The back transport reversed spread and sharpened the antibody protein into a tight zone, which sharpened subsequent precipitin formation with the second antibody (step 4). Immunoassay
of desorbed
antibody
(step 4)
Quantification of desorbed antibody with swine anti-immunoglobulin as
second antibody was patterned after the technique of Bjerrum et al. (1973) who first described measurement of carbamylated IgG with unmodified IgG, the principal difference being use of potassium buffer to prevent dodecyl sulfate from interfering. Gel containing unmodified second antibody in potassium buffer was poured at the anodal side of the desorbed primary antibody, and, after changing to potassium buffer in the anode reservoir, current was applied at 60 V overnight to transport the primary antibody into the immunoprecipitation gel. Rapid precipitation of residual detergent, ensuing almost immediately on applying current, prevented it from permeating the immunoprecipitation gel provided low amperage (<80 mA/ cm2cross-section) was maintained as a precaution against melting the precipitate. RESULTS
Analyses of purified fibrinogen and normal rat plasma by the procedure as detailed showed a single peak (Fig. 2A) at the position expected for fibrinogen, and with area corresponding to retention of 2.3 -t 0.05 (S.E.M., N = 10) 1.18of primary antibody per pg of fibrinogen, which corresponded to 4.9 moles of IgG absorbed per mole of fibrinogen. These combining proportions were greater than observed in immunoprecipitational titration (Shainoff and Braun, 1973) where 1.73 pg of antibody were bound per pg of fibrinogen at equivalence, but were 66% less than one would calculate on basis that twice the equivalent combining proportion (or 3.46 I-(gper pg fibrinogen) might be expected to be absorbed with exposure to excess antibody. Thus, retention of antibody appeared to be consistent, but somewhat below the maximum that might be anticipated from the observed (Shainoff and Braun, 1973) and usual (Nussenzweig et al., 1961) content of antigenic determinants in fibrinogen. Higher retention, corresponding to 2.9 pg antibody per I.rg fibrinogen, was obtained when the procedure in step 2 was changed to remove the excess antibody from the specimen by washing instead of electrophoresis. When washed specimens were subjected to electrophoresis, a small portion of antibody leached away from the fibrinogen, as evidenced by the appearance of a faintly fluorescent streak emerging from the band of absorbed antibody. Accordingly, much of the incomplete retention suggested by the preceding calculations arose from removal of weakly bound antibody in the course of removing unbound antibody by the electrical field. Yet, enormous wastes of unbound antibody were incurred by washing. Since retrieval of antibody from washings proved troublesome by comparison to the speed and convenience of the electrophoretic method, and since results of comparable quality were obtained by either procedure we did not pursue the question of significance of the weakly bound antibody. A question of the extent to which antigenic determinants became masked by attachment of the fibrinogen to the gel was examined operationally by
237
increasing the content of glyoxyl agarose in the gel to determine whether the enriched immobilizing matrix would adversely affect antibody uptake. On changing composition of the specimen gel from 0.5% glyoxyl agarose and 2% regular agarose to a composition of 1.25% glyoxyl and 1.25% regular, retention of antibody fell to 1.9 and 1.7 pg per pg fibrinogen in two trials. With gel composed of 1.25% glyoxyl agarose alone, retention fell further to 1.5 pg per pg. These measurements led us to conclude that the immobilization altered accessibility of the fibrinogen to antibody to a moderate degree that was dependent in part upon the actual glyoxyl agarose content, but tended to diminish with regular agarose added to the glyoxyl agarose. Uptake of antibody by plasma protein other than fibrinogen was not observed except with one rabbit anti-human fibrinogen preparation which, despite purification contained some antibody that was retained by IgG in the specimen gel. The contaminant was not absorbed by undenatured IgG, but could be removed by absorption with SDS-treated IgG immobilized (Shainoff, 1980) on beaded glyoxyl agarose, and accordingly appeared to correspond to rheumatoid factor. The experience demonstrated that antibody specificity should be tested with SDS-treated protein when employed for measurement of SDS-treated antigens. Desorption of retained antibody with SDS in step 3 proceeded to completion as judged by full mobilization of the fluorescent antibody out of the specimen. However, full removal of fluorescence was not-obtained in early trials which employed labeling of the antibody with fluorescein isothiocyanate instead of the adopted lissamine-rhodamine sulfonylchloride. The fluorescein initially appeared desirable because labeling with 5 moles per mole antibody induced the same alteration of mobility as achieved by the carbamylation procedure, but despite numerous attempts we were unable to refine the fluoresceinated antibody to abolish irreversible staining of the Fig. 2. Results of the procedure as applied to analysis of the distribution of fibrinogenrelated antigens in SDS/glyoxyl agarose electrophoregrams of plasma from normal (A) and malignant hypertensive (B) rats. The electrophoretically arrayed and immobilized protein in the specimen gels shown lying across the bottom of the illustrations were exposed to rabbit anti-fibrinogen antibody, and absorbed antibody was desorbed and transported (upwards) for measurement by immunoprecipitation with swine anti-rabbit immunoglobulin antibody. The photographs were taken after staining with Coomassie blue. The peaks at the ends of the precipitin lines portray rabbit antibody applied directly at the edges of the specimens for calibration. The occurrence of only one peak apart from that of the standard for the normal specimen (A) corroborated specificity of the antibody. As calculated from comparison with area under the peak for the standard (0.75 pg applied), and from the predetermined amount of thrombin coagulable protein in the specimen (0.89 pg from 2 ~1 plasma at l/6 dilution), retention of antibody corresponded to 2.2 pg/pg fibrinogen. Many additional peaks arose from antibody displaced from polymers and degraded forms of fibrinogen in the abnormal specimen (B) shown for illustration of the resolving power of the procedure. The standards in B consisted of 1.0 and 0.5 fig antibody, and content of thrombin coagulable protein in the applied plasma (2 /.d/6) was 1.9 pg.
238
antigen and occasionally other proteins. Similar problems were encountered with several other fluorescent isothiocyanates for reasons as yet unknown, but which we suspect involved a slow exchange of the label which did not occur with lissamine-rhodamine sulfonylchloride. In preparation for immunoprecipitational assay of the desorbed antibody, attempts were made to use Lubrol@ to retard transport of the SDS (Converse and Papermaster, 1975), but the degree of retardation achieved did not suffice to prevent the antibody from being swept away before completion of the immunoprecipitation. With the new approach of precipitating the dodecyl sulfate by electrophoretic addition of K’, some experience was needed to carry out the recommended positioning of the detergent for precipitation. In practice, the positions of the precipitated detergent and the fluorescent antibody were discernible within minutes after applying current for adding K’, so that regions of gel rich in detergent could be excised without risk of removing antibody. Further, the detergent could be redissolved and repositioned at will by simply reversing current to return Na’ to the buffer. Immunoassay of the desorbed primary antibody in step 4 proceeded as anticipated from studies by Bjerrum et al. (1973), except for need to maintain neutral pH with which the second antibody (swine anti-rabbit immunoglobulin) had zero mobility in the K’ buffer. This pH also proved suitable in studies using secondary antibody derived from rabbits, but not from goats. Detection of minor components of fibrinogen related antigens in specimens, as illustrated in Fig. ZB, depended on near complete removal of nonabsorbed portions of primary antibody. Portions left in the specimen contributed to the base of the precipitin line used for measurement of specifically retained antibody. When large portions were left a correspondingly large displacement of the baseline resulted, and irregularities in the distribution of antibody attained in loading would cause correspondingly irregular baselines. The distinct peaks observed in the illustrated results were presumed to be produced by antibody displaced from fibrinogen-related antigens in the specimens, because they could be seen by fluorescence to arise from appropriately shaped bands resembling immobilized protein in the specimen, were found in duplicate analyses, and were not observed in specimens of sera devoid of immunoreactive fibrinogen. Some minor components resolved in Fig. 2B corresponded to as little as 1% of the total fibrinogen, the equivalent of 20 ng or 6 X lo-i4 mole of fibrinogen in the specimen. DISCUSSION
The overall procedure differs from direct immunoelectrophoresis mainly in the use of zonal immobilization, made possible by the development of a medium that can be used interchangeably and sequentially either for separating or immobilizing proteins. We view the ability to array proteins on an immobilizing substrate in position indicative of physical properties of the protein as adding a new dimension to solid-phase technology that is anal-
239
ogous to the advance gained by applying electrophoresis to analysis of proteins in fluid phase. Although protein immobilization is widely used in immunoabsorption procedures (Robbins and Schneerson, 1974), reactivities of the immobilizing ligands have been such as to allow only random distribution of protein on the substrate. Thus, the substrates could be used only for immobilization of highly purified (monospecific) protein to obtain defined composition. Electrophoretic arraying of protein on glyoxyl agarose and subsequent immobilization with NaCNBH3 yield a dimensionally defined immunoabsorbent, step 1 of the procedure. As indicated before (Shainoff, 1980), the NaCNBH3 that is used to link the protein to the gel has no effect on either the protein or the gel separately, but acts in a highly specific manner (Borch et al., 1971) to drive reduction of the extremely weak and highly dissociable Schiff-base linkages that are formed between amines and simple alkanals. Thus, the protein and gel are left unaltered except at points of linkage catalyzed by the reducing agent. Removal of the NaCNBH3 returns the system to its non-reactive state, except for the immunoreactive or other specific epitopes imparted by the immobilized protein, the distribution of which is established by the pattern of antibody uptake (steps Z-4). As evidenced by the results obtained, negligible uptake of antibody by the gel occurs except through the imparted epitope even though the aldehyde groups of the gel are left unquenched. The ability to continue using the gel as a separation medium without quenching is an important advantage, because quenching reactions usually impart complications through added electrical charge. Since the immobilized state of the antigen preserves its distribution in the specimen, a battery of specific antibodies can be applied either to obtain consecutive details of composition, or to amplify sensitivity. Studies in progress are profiling not only the molecular weight distribution of fibrinogen-related antigens as illustrated here, but also the content of fibrinopeptides and neoantigenic determinants of the variant forms of the protein through sequential use of specific antibody (Plow et al. 1971; Nossel et al., 1976) to these epitopes. Sensitivity of the procedure is quite high. Based on the estimated retention of an average of two-thirds mole equivalent of primary antibody per antigenic determinant in fibrinogen examined in the present study, and the use of the retained IgG as a surrogate antigen having an effective valence of 8 for precipitation of the secondary antibody, we calculate that the method has about 5 times (2/3 X 8) greater sensitivity than direct immunoassay. If warranted to amplify sensitivity further the retained IgG could be immobilized with NaCNBH3 instead of measured directly, and then used as a second surrogate to cascade antibody uptake by another order of magnitude. It is important to emphasize that the procedure was not devised as a substitute for direct immunoelectrophoresis, but as an alternative when direct immunoprecipitation cannot be relied upon, a situation that arises when
240
studying either self-precipitating or non-immunoprecipitable antigens, and one that also arises in studies depending on use of monoclonal antibodies which usually do not yield precipitins. The 4 steps in the procedure entail twice the work of direct cross-immunoelectrophoresis. Additional effort may be required to ensure specificity of the antibody. The method depends critically on the use of purified antibody. Unlike direct immunoprecipitation methods with which unrelated antigens form separate precipitin lines, only one line is formed here - that produced by retained immunoglobulin. The need to purify antibody adds to cost of preparing it for use in the procedure, but is compensated manifold by the ability to recover all but specifically retained portion of the antibody applied in carrying out the procedure. By contrast, excess antibody spent in carrying out direct immunoprecipitation is usually lost, a consequence that adds considerably to the cost of the direct method. Several of the techniques developed for the procedure can be used to advantage in conjunction with other procedures. These include covalent immobilization to fix proteins for immunofluorescent staining or autoradiography, electrophoretic addition and removal of antibody to minimize losses in immunologic staining, and use of K’ instead of Lubrol@ (Converse and Papermaster, 1975) to limit migration of SDS. In addition, glyoxyl agarose provides an efficient protein immobilizing substrate for affinity purification of antibody, and for screening cell cultures for production of monoclonal antibodies. We view the techniques presented here as examples of ways in which zonal immobilization can provide a basis for the design of new immunologic procedures. ACKNOWLEDGEMENTS
Dolores Andrasic provided technical assistance. The work was supported in part by Grants HL-16361 and HL-19767 from the National Heart, Lung and Blood Institutes of the U.S. Public Health Service, and by an advanced research fellowship from the American Heart Association, North East Ohio Affiliate. REFERENCES Bjerrum, O.J., A. Ingold, H. Lowenstein and B. Weeke, 1973, Stand. J. Immunol. 2 (Suppl. l), 145. Borch, R.F., M.D. Bernstein and H.D. Durst, 1971, J. Am. Chem. Sot. 93, 2897. Converse, C. and D.S. Papermaster, 1975, Science 189, 469. Hoyer, L. and J.R. Shainoff, 1980, Blood 55, 1056. Laurell, C.B., 1965, Anal. Biochem. 10, 358. Levy, H.B. and H.A. Sober, 1960, Proc. Sot. Exp. Biol. Med. 103, 250. Nossel, H.L., V.P. Butler, Jr., G.D. Wilner, R.E. Canfield and E.J. Harfenist, 1976, Thromb. Haemostas. 35, 101.
241 Nussenzweig, V., M. Seligman, P. Grabar and M. Boucart, 1961, Ann. Inst. Pasteur 100, 490. Plow, E.F., C. Houghie and T.S. Edgington, 1971, J. Immunol. 107, 1496. Robbins, J.B. and R. Schneerson, 1974, in: Methods in Enzymology, Vol. 44, eds. W.B. Jackoby and M. Wilchak (Academic Press, NY) p. 703. Schafer Nielsen, C. and O.J. Bjerrum, 1975, Stand. J. Immunol. 4 (Suppl. 2), 73. Shainoff, J.R., 1980, Biochem. Biophys. Res. Commun. 95, 690. Shainoff, J.R. and W.E. Braun, 1973, Anal. Biochem. 55, 206. Weeke, B., 1973, Stand. J. Immunol. 2 (Suppl. l), 15.