INSTRUMENTATION
Advisory Panel Jonathan W. Amy Richard A. Durst G. Phillip Hicks
Donald R. Johnson Charles E. Klopfenstein Marvin Margoshes
Harry L. Pardue Howard J. Sloane Ralph E. Thiers
Two-Directional Immunoelectrophoresis LESLIE M. SHAW and DEAN A. ARVAN William Pepper Laboratory, Department of Pathology School of Medicine, University of Pennsylvania Philadelphia, Pa. 19104 The gap between the development and application of twodirectional immunoelectrophoresis and its potential use in the routine clinical laboratory as a diagnostic tool is closing. The technique is also becoming valuable in the basic research laboratory, where it provides the investigator with a powerful tool for the qualitative and quantitative analysis of proteins THLO CLASSICAL technique of im I xmunoelectrophoresis first introduced
by Grabar and Williams (1), agar gel zone electrophoresis is utilized initially to separate the proteins into various groups. This is followed by a double diffusion step in which the protein frac tions interact with antibody molecules diffusing in the opposite direction. The antibody molecules originate from a trough cut adjacent to the path of the electrophoresed proteins. As a result of this process, precipitin arcs form which correspond to individual protein com ponents. This technique has been extensively used in both clinical and research laboratories largely for the qualitative analysis of protein mixtures. In the clinical laboratory, for example, the confirmation of the presence of a para protein (myeloma protein or M-component) in a patient's serum and of the immunoglobulin class to which it be longs can be readily ascertained by Im munoelectrophoresis, although when the paraprotein is present in large excess, interpretation may be difficult owing to the formation of soluble antigen-anti body complexes. One of a number of useful applications of the technique has been in the research laboratory, where it has been used to determine the presence or absence of a protein or proteins in preparative frac tionation procedures. An obvious lim itation of Immunoelectrophoresis is that ordinarily it does not provide quantita tive information. Moreover, when
antiwhole human serum is used to analyze human serum proteins, it is frequently difficult, and at times impos sible, to see precipitin arcs correspond ing to some of the proteins because of extensive overlapping of some of the arcs. The first attempt to modify the classical technique was made by Ressler (β) to provide quantitative data and thus to improve its usefulness by achiev ing greater resolution of the overlapping precipitin arcs. In his original method, the initial electrophoresis separation in agar was followed by a second electro phoresis in a direction at right angles to the first. The separated proteins were thus forced into an agarose gel contain ing antibodies to these proteins. This method was further improved by Laurell (8) and again by Clarke and Freeman (4), who modified it into a truly quantitative technique. Laurell named this procedure "crossed electro phoresis," and some investigators refer to it as the "Laurell crossed electro phoresis" technique. At present a number of laboratories, including our own, are applying two-directional im munoelectrophoresis to clinical as well as basic research problems, utilizing es sentially the same conditions described by Clarke and Freeman. Principles of Method
The pattern illustrated in Figure 1 was obtained by use of normal human serum and antiwhole human serum. As shown, a number of rocket-like peaks
are obtained for the proteins in human serum, each peak corresponding to a different protein. Electrophoresis in the second direction causes proteins, which are negatively charged at the buffer pH (usually pH 8.6), to migrate through the agarose-antibody bed toward the anode. Since the antibody molecules (gamma globulins) migrate very little under the conditions of elec trophoresis, the negatively charged proteins (antigens) interact with their specific antibodies under conditions of relative stability in antibody concentra tion. As antigens interact with their respective antibodies, antigen-antibody complexes form, and when all of the migrating antigen molecules have complexed with antibodies, cone-shaped precipitin zones are formed and no further migration occurs. The following factors affect the size and shape of these peaks. Charge Density of Protein (Antigen). Proteins with high charge densities will move faster and form higher peaks than proteins with lower charge densities (e.g., prealbumin vs. transferrin in Figure 1). For the more highly charged antigens, a shorter time is necessary to form complete precipitin cones (5). Thus, the duration of the electrophoretic run in the second dimension is adjusted so as to obtain optimum precipitin cones with the less charged (slower) antigens. Quantity of Protein. Under a given set of electrophoresis conditions, both peak height and the area under a given protein peak are proportional to the concentration of that protein. Concentration of Antibody. Within certain limits of antigen-antibody ratios, both peak height and area are inversely proportional to the concentration of antibody to each protein. Electrophoretic Microheterogeneity. Proteins which are electrophoretically homogeneous (e.g., prealbumin, a\ acid glycoprotein, ceruloplasmin) have a symmetrically shaped peak, whereas those proteins which are electrophoreti cally heterogeneous (e.g., a 2 -macroglobulin, β-lipoprotein, /31C//31A globu lin in Figure 1) have asymmetrically shaped peaks.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
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Instrumentation
Figure 1. Human serum proteins resolved by two-directional Immunoelectrophoresis. I, II, and corresponding arrows refer to direction of first and second electrophoretic steps, respectively. Numbered peaks correspond to following proteins: 1, prealbu min; 2, αι-acid glycoprotein; 3, «i-lipoprotein; 4, albumin; 5, («-antitrypsin; 6, Gcglobulin; 7, haptoglobin; 8, ceruloplasmin; 9, a2-macroglobulin; 10, a2-HS glycoprotein; I I , hemopexin; 12, βΙΟ/βΙΑ globulin; 13, transferrin; 14, ^-lipoprotein; 15, IgG; 16, IgA; 17, IgM Experimental Procedure
The following conditions are those which we are currently using for this technique. For electrophoresis in the first direction, 10 ml of 1% agarose in 0.0125.V barbital buffer, pH 8.6, is pipetted onto an 8 X 10-cm glass plate in a Plexiglas holder. A well is punched at one corner of the gel with a punch having a diameter of 1 mm. The sample to be electrophoresed is inserted into the well with a Hamilton syringe (0.3-0.4 μΐ of undiluted human serum). The electrophoresis buffer is 0.0253Î barbital, pH 8.6. Electrophoresis is conducted at 250 V for i y 2 hr. The agarose plates are maintained at 15°C with a Plexiglas cooling plate during the run. After the first electrophoretic step is completed, the agarose plates are removed from the electrophoretic apparatus, and 20 ml of 1% agarose in 0.01253/ barbital, pH 8.6, containing the appropriate antiserum (2% v/v) is poured on top of the agarose plates. The second electrophoretic step is then carried out in the direction at right angles to the first at 100 V for 20 hr. Figure 2 shows some details of the apparatus designed and currently used in our laboratory. After this step the
agarose plate is soaked for 24 hr in 0.9% NaCl to remove excess protein. The agarose plate is then dried, stained with a protein stain (we use a mixture containing 0.05% azocarmine, 0.1% amido black, 0.1% light green in 2% acetic acid), and destained with methanolacetic acid-water (40-10-50). With a normal human serum as the antigen and antihuman serum as the antibody for the second direction, the pattern shown in Figure 1 is obtained. Depending upon the animal source and quality of the antiserum, up to 42 protein peaks may be detected (6) in normal human serum. A key question, of course, is how to identify the individual proteins in such a complex mixture. We and others (6) have found the following procedures useful in this regard: substituting a limited number (e.g., 3-5) of monospecific antisera of known composition and strength in place of antiwhole human serum and employing identical analytical conditions, so as to keep electrophoretic mobilities constant (Figure 3) ; adding monospecific antiserum to the sample and centrifuging the resulting antigenantibody precipitate prior to twodirectional electrophoresis. This leads
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to a selective reduction in the. height of the peak corresponding to the antigen in question; adding a small amount of the purified protein to the serum and observing the increase in peak height (e.g., transferrin, albumin) ; differential staining of a specific protein (e.g., ceruloplasmin, «i-lipoprotein, ^-lipoprotein) ; adding to the serum radioactively labeled compounds which bind to specific proteins (e.g., thyroxine or triiodothyronine binds primarily to thyroidbinding globulin and prealbumin). Following electrophoresis in the second direction, the plate is dried and the peaks containing the bound labeled molecules can be detected by radioautography; adding an agent which specifically binds to a protein resulting in a change in the electrophoretic mobility of that protein (e.g., hemoglobin added to serum binds to haptoglobin, resulting in a decreased electrophoretic mobility of the latter; this may serve as the basis for haptoglobin identification). Thus far, these procedures have been applied to human serum proteins, but it should be emphasized that they are applicable to any single antigen or antigen mixture for which appropriate antisera are available. Two procedures have been used for calculating the quantities of the individual proteins. The first takes advantage of the direct proportionality between protein concentration and the area under a given peak (4). The second utilizes the observed linear relationship between the protein concentration and peak height (7-9). One can measure the areas under the peaks manually or, more conveniently, with a semiautomatic planimeter and compare these areas directly to those obtained with an appropriate reference serum, such as the commercially available Standardized Stabilized Human Serum (Behring Diagnostics) or pooled normal serum. One can also measure the peak heights and compare these with the appropriate standard curves of the proteins in the reference serum. Both methods give reproducibility similar to that of other quantitative immunochemical methods, such as radial immunodiffusion and nephelometry. Applications of Technique
Thus far, two-directional Immunoelectrophoresis has been used primarily in studies of human serum proteins in health and disease. Clark and colleagues have quantitated twenty different proteins in sera from normal adults (4) as well as those from patients with a variety of disorders (10-19). Also, a number of reports have emphasized the usefulness and efficiency of this method for the measurement of sequential changes of serum proteins in a
Instrumentation
variety of diseases. This has been done, for example, in a study of inflammation a n d injury (13) a n d in a patient following a liver transplant (14)· An interesting application of this technique has been in t h e study of t h e genetic polymorphism of some proteins. Fagerhol and Laurell (15) found t h a t by substituting starch gel for agarose for t h e first electrophoretic step and using agarose and antibody as usual for t h e second step, t h e genetic variants of a serum protein known as cn-antitrypsin, recently found to be of importance in t h e development of familial pulmonary emphysema, could be resolved quite easily. A similar study has been carried out with the h u m a n serum protein (31C//31A globulin (the t h i r d component of complement) (16). As noted above, applications of this relatively new technique h a v e been devoted primarily to t h e analysis of h u m a n serum proteins, b u t this in no way precludes its potential use in a wide variety of other studies. A major prerequisite would be the availability of a sufficiently potent antiserum against the proteins or other antigens. An interesting study along these lines has recently appeared in t h e literature. I n t h e reinvestigation of t h e antigenic composition of the microorganism Candida albicans, Axelsen (17) applied two-
directional immunoelectrophoretic analysis. H e utilized t h e water-soluble fraction of t h e microorganism as a n antigen source and prepared a n a n t i serum to it. B y use of this technique, 68 antigen peaks were now demonstrable compared to t h e previously reported 1 5 16 antigens detectable with t h e classical immunoelectrophoretic technique. T h i s is b u t one demonstration of t h e high degree of resolution attainable with twodirectional Immunoelectrophoresis. Possible Future Developments
Since two-directional Immunoelectrophoresis is a relatively new technique, it is quite certain t h a t appropriate modifications will be made. W e are currently considering several future revisions t o improve t h e efficiency and reduce t h e cost of t h e measurements. Others h a v e reported modifications as well. For example, Stephan and F r a h m (9) have developed a technique utilizing 5 X 5-cm glass plates for the agarose support, t h u s requiring a smaller a m o u n t of agarose-antibody mixture for t h e second electrophoretic step. This procedure has t h e advantage of utilizing less antibody per plate, resulting in a lower cost per analysis. Although t h e authors made no specific mention of this in their paper, it is clear t h a t these plates could be photographed directly after electro-
Figure 2. " E x p l o d e d " view of apparatus speciaMy designed for two-directional Immunoelectrophoresis. 1, wick holder; 2, agarose gel plate holder; 3, Plexiglas cooling plate; 4, electrophoresis chamber. [Reproduced from Clin. Chem., 17, 745 (1971) by permission of the Editor.l
Figure 3. Two-directional Immunoelectrophoresis by use of a limited n u m b e r of monospecific antisera for the purpose of identifying various peaks. Three specific antisera were mixed with agarose in this experiment, giving peaks for 1, «i-antitrypsin; 2, a 2 -macroglobulin; 3, haptoglobin. This pattern was obtained by Vidmantas Raisys (present address: Department of Laboratory Medicine, University of Washington, Seattle, Wash.)
phoresis, utilizing, for example, a Cordis immunodiffusion camera. Although problems m a y be encountered in clearly visualizing faint peaks, considerable time could be saved by avoiding the rinsing, drying, and staining steps. One of t h e limitations of t h e technique as presently used in our laboratory is t h a t t h e immunoglobulins, particularly IgG, cannot be adequately evaluated (see Figure 1). T h i s results from t h e fact t h a t both antigen and antibody have similar electrophoretic mobilities. As noted above, during t h e second electrophoretic step those antigens with low charge densities move more slowly with respect t o t h e antibody t h a n antigens with high charge densities. One result of this slower migration is t h a t t h e antigens diffuse radially, t h u s resulting in a broader peak t h a n for t h e antigens with higher charge densities. An extreme example of this is seen with t h e IgG. T h i s protein not only has a low charge density b u t normally also consists of a group of molecules with variable electrophoretic mobilities. A proportion of normal IgG molecules has a sufficiently slow mobility so t h a t it is carried toward t h e cathode b y electroendosmotic effects and is responsible for producing an inverted peak (Figure 1). If one wishes to q u a n t i t a t e i m m u n o globulins by this technique, chemical modification of these proteins, which increases their net negative charge, can be employed. One such modification procedure (9) utilized /3-propiolactone, which increases t h e net negative charge of proteins by reacting with t h e imidazole group of t h e amino acid histidine and t h e £-amino group of lysine. By
ANALYTICAL CHEMISTRY, VOL. 4 4 , NO. 9, AUGUST 1972
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t h i s procedure, all of t h e serum proteins, including t h e immunoglobulins, can be q u a n t i t a t e d . Carbamylation of serum proteins with potassium c y a n a t e (18) is another m e t h o d which produces similar chemical modification. T h e efficiency with which these analyses are performed m a y be addi tionally improved b y t h e mechanization of some of t h e steps or b y t h e develop m e n t of an appropriate a p p a r a t u s t o handle a larger number of samples. T h u s , it is our conviction t h a t t h e gap between t h e development and applica tion of two-directional Immunoelectro phoresis and its potential use in t h e routine clinical chemistry laboratory as a diagnostic tool is closing. I n addition, t h e technique is now beginning t o find use in t h e basic research laboratory since it provides t h e investigator with a powerful tool for t h e qualitative a n d q u a n t i t a t i v e analysis of proteins. Acknowledgment
T h e a u t h o r s express their appreciation t o K a r l Linke and Chester M . Sinnett of t h e I n s t r u m e n t Division of t h e William Pepper L a b o r a t o r y for their invaluable assistance in t h e design and manufacture of t h e a p p a r a t u s used in this investiga tive work. References
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ANALYTICAL CHEMISTRY, VOL. 4 4 , NO. 9 , AUGUST 1972
(1) P. Grabar and C. A. Williams, Jr., Biochem. Biophys. Acta, 10, 193 (1953). (2) N . Ressler, Clin. Chim. Acta, 5, 795 (1960). (3) C. B. Laurell, Anal. Biochem., 10, 358 (1965). (4) H. G. M. Clarke and T. Freeman, Clin. Sci., 35, 403 (1968). (5) C. B. Laurell, Anal. Biochem., 15, 45 (1966). (6) B. Weeke, Scand. J. Clin. Lab. Invest., 25, 269 (1970). (7) V. A. Raisys and D. A. Arvan, Clin. Chem., 17, 745 (1971). (8) D . A. Arvan and L. M. Shaw, Separ. Sci., in press, 1972. (9) V. W. Stephan and TJ. Frahm, Z. Klin. Chem. Klin. Biochem., 9, 224 (1971). (10) H . G. M. Clarke, T. Freeman, and W. Ε. Μ. Pryse-Phillips, Clin. Chim. Acta, 30, 65 (1970). (11) A. H. Amin, H. G. Clarke, T. Free man, I. M. Lyon, P. M. Smith, and R. Williams, Clin. Sci., 38, 613 (1970). (12) H. G. M. Clarke, T. Freeman, R. Hickman, and W. Ε. Μ. Pryse-Phillips, Thorax, 25, 423 (1970). (13) H. G. M. Clarke, T. Freeman, and W. Ε. Μ. Pryse-Phillips, Clin. Sci., 40, 337 (1971). (14) I. M. Murrary-Lyon, R. Williams, T. Freeman, and H. G. M. Clarke, Nature, 231, 45 (1971). (15) M. K. Fagerhol and C. B. Laurell, Clin. Chim. Acta, 16, 199 (1967). (16) C. A. Alper and R. P. Propp, J. Clin. Invest., 47, 2181 (1968). (17) Ν . Η. Axelsen, Inf. Immun., 4, 525 (1971). (18) B. Weeke, Scand. J. Clin. Lab. Invest., 21, 351 (1968).
