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 t h e development and application of twodirectional immunoelectrophoresis and its potential use in t h e routine clinical laboratory as a diagnostic tool is closing. The technique is also becoming valuable in t h e basic research laboratory, where it provides t h e investigator with a powerful tool for t h e qualitative and quantitative analysis of proteins technique of imIbysmunoelectrophoresis first introduced Grabar and Williams ( I ) , agar gel THE
CLASSICAL
zone electrophoresis is utilized initially to separate the proteins int’o various groups. This is followed by a double diffusion step in which the protein fractions 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 individud protein components. This technique has been extensively used in both clinical and research laboratories largely for the qualitative analysis of protein mixtures. I n the clinical laboratory, for example, the confirmation of the presence of a paraprotein (myeloma protein or 11-componerit) in a patient’s serum and of the immunoglobulin class to which it belongs can be readily ascertained by immunoelectrophoresisJ although when the paraprotein is present in large excess, interpretation may be difficult owing t o the formation of soluble antigen-antibody complexes. One of a number of useful applications of t’hetechnique has been in the research laboratory, where it has been used to determine the presence or absence of a protein or proteins in preparative fractionation procedures. d n obvious limitation of immunoelectrophoresis is that ordinarily it does not provide quantitative information. Xoreover, when
antiwhole human serum is used to analyze human serum proteins, it is frequently difficult, and a t times impossible, to see precipitin arcs corresponding to some of the proteins because of extensive overlapping of some of t’he arcs. The first att’empt to modify the classical technique was made by Ressler (e) to provide quantitative data and thus to improve its usefulness by achieving greater resolution of the overlapping precipitin arcs. I n his original method, the initial electrophoresis separation in agar was followed by a second electrophoresis in a direction a t right angles t o bhe first. The separated proteins were thus forced into a n agarose gel containing antibodies to these proteins. This met,hod was further improved by Laurell (3) and again by Clarke and Freeman ( 4 ) , who modified it into a truly quantitative technique. baurell named t,his procedure “crossed electrophoresis,” and some investigators refer to it as the “Laurell crossed electrophoresis” technique. A t present a number of laboratories, including our own, are applying two-directional immunoelectrophoresis to clinical as well as basic research problems, utilizing essentially the same conditions described by Clarke and Freemm. 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 p H (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 electrophoresis, the negatively charged proteins (antigens) interact with their specific antibodies under conditions of relative stability in antibody concentration. As antigens interact with their respective antibodies, antigen-antibody complexes form, and when all of the migrating antigen molecules have cornplesed 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 ( A n t i g e n ) . Proteins with high charge densities ivill 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 t o 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. Q u a n t i f y 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 Il~icroheterogeneity. Proteins which are electrophoretically homogeneous (e.g., prealbumin, a1 acid glycoprotein, ceruloplasmin) have a symmetrically shaped peak, whereas those proteins which are electrophoretically heterogeneous (e.g., a2-niacroglobulin, P-lipoprotein, P1C p l d globulin in Figure 1) have asymmetrically shaped peaks.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
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Figure 1. Human serum proteins resolved by two-directional immunoelectrophoresis. I , 11, and corresponding arrows refer to direction of first a n d second electrophoretic steps, respectively. Numbered peaks correspond to following proteins: 1. prealbumin; 2, wacid glycoprotein: 3. a,-lipoprotein; 4, albumin; 5, al-antitrypsin: 6. Gcglobulin; 7. haptoglobin; 8. ceruloplasmin: 9, asmacroglobulin; 10, arHS glycoprotein: 11. hemopexin; 12. PlCiPlA 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 R x lO-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 t,he well with a Hamilton syringe (0.3-0.4 @I of undiluted human serum). The electrophoresis buffer is 0.025M barbital, pH 8.6. Electrophoresis is conducted at 250 V for 1*/2hr. 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 0.012551 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 augles to the first a t 100 V for 20 hr. Figure 2 shows some details of the apparatus designed and currently used in our laboratory. After this step the 58A
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 0.05Y0 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 he 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
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
to 8 selective reduction in the.height of the peak corresponding to the antigen in question; adding a small amount of t h e purified protein to the serum and observing the increase in peak height (e.& transferrin, albumin) ; differential staining of a specific protein (e.g., ceruloplasmin, a,-lipoprotein, 8-lipoprotein); adding to the serum radioactively labeled compounds which bind to specific proteins (e.g., thyroxine or triiodcthyronine binds primarily to thyroidbinding globulinand prealhumin). 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 hinds to haptoglobin, resulting in a decreased electrophoretic mobility of the latter; this may serve as the hasis for haptoglobin identification). Thus far, these procedures have been applied to human serum proteins, but i t should he 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 coucentration and peak height (7-57). 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 availahle 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, twodirectional 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 (10-28). Also, a number of reports have emphasized the usefulness and efficiencyof 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 intlammation and injury (IS) and in a patient following a liver transplant (14). An interesting application of this technique has been in the study of the genetic polymorphism of some proteins. Fagerhol and Laurel1 (16) found that by substituting starch gel for agarose for the first electrophoretic step and using agarose and antibody as usual for the second step, the genetic variants of a serum protein known as wantitrypsin, recently found to be of importance in the development of familial pulmonary emphysema, could be resolved quite easily. A similar study has been carried out with the human serum protein plC/,91A globulin (the third component of complement) (16). As noted above, applications of this relatively new technique have been devoted primarily to the analysis of human serum proteins, but 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 the literature. I n the reinvestigation of the antigenic composition of the microorganism Cam dida albieans, Axelsen (I7) applied two-
directional immunoelectrophoretic analysis. H e utilized the water-soluble fraction of the microorganism as an antigen source and prepared an antiserum to it. By use of this technique, 68 antigen peaks were now demonstrable compared to the previously reported 1516 antigens detectable with the classical immunoelectrophoretictechnique, This is but one demonstration of the high degree of resolution attainable with twodirectional immunoelectrophoresis. Possible Future Developments
Since twodirectional immunoelectrophoresis is a relatively new technique, it is quite certain that appropriate modifications will be made. We are currently considering several future revisions to improve the efficiency and reduce the cost of the measurements. Others have reported modifications as well. For example, Stephan and Frahm (9) have developed a technique utilizing 5 X 5-cm glass plates for the agarose support, thus requiring a smaller amount of agarose-antibody mixture for the second electrophoretic step. This procedure has the advantage of utilizing less antibody per plate, resulting in a lower cost per analysis. Although the authors made no specific mention of this in their paper, it is clear that these plates could be photographed directly after electro-
Figure 2. “Exploded” view of apparatus SpeciaHy designed for two-directional immunoelectrophoresis. 1, wick holder: 2, agarose gel plate holder: 3, Plexiglas cooling plate: 4, electrophoresischamber. [Reproduced from Clin. Chem., 17,745(1971) by permission of the Editor.]
Figure 3. Two-directional irnniunoelectroohoresis bv use of a l i inited number 01 monospecific antisera I()r the purpose 01 identifying varnous p eaks. Three specific antisera were mixeo with agarose in this experiment, giving peaks for 1, ol-antitrypsin: 2, ardnacroglobulin; 3, haptoglobin. This pattern wasobtained by Vidmantas Raisys (present address: Department of Laboratory Medicine, University of Washington, Seattle, Wash.) phoresis, utilizing, for example, a Cordis immunodiffusion camera. Although problems may he encountered in clearly visualizing faint peaks, considerable time could he saved by avoiding the rinsing, drying, and staining steps. One of the limitations of the technique as presently used in our laboratory is that the immunoglobulins, particularly IgG, cannot be adequately evaluated (see Figure 1). This results from the fact that both antigen and antibody have similar electrophoretic mobilities, As noted above, during the second electrophoretic step those antigens with low charge densities move more slowly with respect to the antibody than antigens with high charge densities. One result of this slower migration is that the antigens diffuse radially, thus resulting in a broader peak than for the antigens with higher charge densities. An extreme example of this is seen with the IgG. This protein not only has a low charge density but normally also consists of a group of molecules with variable electrophoretic mobilities. A proportion of normal IgG molecules has a sufficiently slow mobility so that it is carried toward the cathode by electroendosmotic effects and is responsible for producing an inverted peak (Figure 1). If one wishes to quantitate immunoglobulins by this technique, chemical modification of these proteins, which increases their net negative charge, can be employed. One such modification procedure (9) utilized ,9-propiolactone, which increases the net negative charge of proteins by reacting with the imidazole group of the amino acid histidine and the r-amino group of lysine. By
ANALYTICAL CHEMISTRY, VOL. 44, NO. 9, AUGUST 1972
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this procedure, all of the serum proteins, including the immunoglobulins, can be quantitated. Carbamylation of serum proteins with potassium cyanate (18) is another method which produces similar chemical modification. The efficiency with which these analyses are performed may be additionally improved by the mechanization of some of the steps or by the development of an appropriate apparatus t o handle a larger number of samples. Thus, it is our conviction that the gap between the development and application of two-directional immunoelectrophoresis and its potential use in the routine clinical chemistry laboratory as a diagnostic tool is closing. I n addition, the technique is now beginning t o find use in the basic research laboratory since it provides the investigator with a powerful tool for the qualitative and quantitative analysis of proteins. Acknowledgment
The authors express their appreciation to Karl Linke and Chester 11.Sinnett of the Instrument Division of the William Pepper Laboratory for their invaluable assistance in the design and manufacture of the apparatus used in this investigative work. References (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., 2 5 , 269 (1970). (7) V. A. Raisys and D. A. Arvan, Clin. Chem., 17, 745 (1971). (8) D. A. Arvan and L. kl. Shaw, Separ. Sci., in press, 1972. (9) V. W. Stephan and U. Frahm, 2. KEin. Chem. Klin. Bzochem., 9, 224 (1971). (10) H. G. hi. Clarke, T. Freeman, and W. E. >!I.Prvse-Phillim. Clin. Chim. Acta, 30, 65 (l"970). (11) A. H. Amin, H. G. Clarke, T. Freeman, 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. E. >I. Pryse-Phillips, Thorax. 25. 423 (1970). (13) H. G. i f . Clarke, T . Freeman, and W. E. M. Pryse-Phillips, Clin. Sci., 40, 337 (1971). (14) I. bl.blurrary-Lyon, R. Williams, T. Freeman, and H. G. 11.Clarke, ,\lature, 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. Inuest., 47, 2181 (1968). (17) N. H. Axelsen, Inj. Immun., 4, 525 (1971). (18) B. Weeke, Scand. J . Clin. Lab. Invest., 21, 351 (1968). _
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