PRECIPITATION OF MUSCLE EXTRACTS BY MYASTHENIA GRAVIS SERUM*

Raymond Lang, Sidney Shulman,? Ernst Beutner, Ernest Witebsky Department of Bacteriology and Immunology, School of Medicine, State

University of New York at Buffalo, Buffalo, N. Y.

A number of studies have revealed that serum from patients with my- asthenia gravis often contains antibodies to antigens in muscle tissue.'-' Definitive studies, using especially the technics of immunofluorescence, have shown that these antibodies are autoantibodies, since they are able to react with muscle tissue taken from the same patient.* In addition, the serum can react with muscle from other humans and also from other animals, especially the monkey. The use of muscle from rhesus monkey has provided a plentiful supply of material for exploring the antigen-antibody interaction by means of various immunological manifestations.* It had previously been difficult to prepare suitable muscle extracts and procedures for demonstrating the reaction by means of a precipitation phenomenon. We now wish to report on improved methods for preparation of muscle extracts for this purpose and to describe the methods that have been worked out for study of antigen-antibody precipitation. A preliminary report has appeared.?

Materials and Methods

Three methods for preparation of muscle extract have been developed, differing as to the extracting fluid utilized. In all cases, muscle was obtained from the hind legs of rhesus monkeys immediately after exsanguination. The tissue was suitably trimmed, minced and then mixed with an equal volume of the extracting fluid; i t was then homogenized in a Servall Omni- mixer. This type of product can be termed a 50 per cent suspension. The three methods of preparation involved different fluids. These were water, or saline (0.15 M NaCl), or hypertonic sucrose (1.5 M); in each type of extract, the protein concentration was found to be about 2.5 to 3.0 per cent, as judged by the biuret reaction.' One must distinguish carefully between water extract, saline extract and sucrose extract.

If extracts were maintained at room temperature, it was found that they became turbid and were likely to precipitate by the next day. This meant that control mixtures, that is, muscle extract mixed with saline or with normal human serum, also showed precipitation. Several procedures were tried for ageing and centrifuging the preparations. The routine practice is now to clarify the homogenate in the refrigerated centrifuge at 10,000 rpm for 30

'This investigation was supported in part by a grant from the Mae Stone Goode Estate.

+Recipient of a Research Career Award (K6-AI-1377) from the U. S. Public Health Service, Bethesda, Md.

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minutes, store i t in the cold and then to perform a second clarification just prior to using the material by again centrifuging a t 10,000 rpm for 30 minutes. This has proved to be a satisfactory procedure for all three types of extracts.

Similar extracts were occasionally made from human muscle, and saline extracts were made of a number of other rhesus monkey tissues. These included heart, lung, liver, spleen, pancreas, adrenal, uterus and thymus. The immunologic methods applied included precipitation in liquid, double diffusion plate tests and immunoelectrophoresis.

Results

Specific precipitation in the fluid phase will be considered first. The standard procedure was to mix one drop of serum with one drop of extract, preferably of the water or saline type, and to draw this into a thin capillary tube which was then sealed. When incubated a t room temperature, floccula- tion appeared within several hours, and in 12 hours a white fluffy precipitate

FIGURE 1. Capillary tube fluid precipitation test. In alternate tubes are seen two positive reactions (settled precipitates) and two negative reactions. The positive tubes contained a mixture of one drop of monkey muscle extract and one drop of myasthenic serum; the negative tubes contained a mixture of the same monkey muscle extract and one drop of normal human serum.

Lang et al.: Precipitation of Muscle Extracts

had formed in all tubes, regardless of whether the extract had been mixed with myasthenic serum or normal serum. Fortunately, however, it was found that nonspecific precipitations in control tubes could be considerably delayed by performing all experiments a t low temperature. In other words, control mixtures of muscle extract and normal serum or saline were always negative a t 24 hours of incubation a t 4OC. Therefore, the standard procedure now is to consider the results a t 24 hours a t 4OC. as the indicative results. When muscle extracts were tested against a number of normal sera, no visible pre- cipitate was formed, while a t the same time precipitation occurred with myasthenic serum mixed with the same extract. FIGURE 1 is a photograph of some typical tubes showing both positive and negative results. A total of 10 myasthenic sera have been examined in this way, and 3 of these were positive. This was done with water or saline extracts. Studies were also made on a large number of sera, about 100, from a great variety of pathological conditions. All of these were negative in these tests, except for two cases. In the past few weeks, additional clinical data have been received in one of these cases, and the evidence for myasthenia gravis has become much more suggestive, although the diagnosis is not really final. This is the same case that has been discussed a t some length in the preceding r e p ~ r t . ~

As another facet of specificity, a myasthenic serum was tested with all the monkey tissue extracts that were mentioned before; all but skeletal muscle were negative. In other words, smooth muscle and heart muscle were negative, along with various nonmuscle preparations. Eight muscle extracts of human origin were also shown to give precipitation.

The gel diffusion results were developed primarily in double diffusion plates. Preliminary experiments gave disappointing results. This seemed to be because of insufficient concentration of antigen. Various methods were used to increase the concentration of such extracts to approximately five per cent protein, and then it was found that a faint, but sharp line of precipitation was obtained. It has now been realized that the best precipitation patterns can be obtained by using agar a t 0.5 per cent concentration in 0.05 M NaCl, with antigen wells of 7 mm. diameter and serum wells of 3 mm. diameter, separated by 3 mm. of agar, and the whole system incubated a t 4°C. Under these conditions and using concentrated water or saline extracts of monkey muscle, satisfactory patterns could be obtained. Sometimes, various degrees of halo formation were seen around the wells, but the precipitation lines were sharp and readily discernible. In early studies, a single, somewhat broad, line curving toward the antigen well was seen. As the method for concentrat- ing the extract was improved and higher concentrations were reached, the broad line separated into two distinct lines as shown in FIGURE 2, between well 1, which contained a concentrated water extract, and well 3, which had the undiluted myasthenic serum. The use of the sucrose extract, however, was even more encouraging, for it was found quite unnecessary to concentrate this extract. Under the conditions that have just been stated, the ordinary

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FIGURE 2. Gel diffusion precipitation pattern of myasthenic serum and various muscle extracts. The wells were filled as follows: 1, water extract of monkey muscle; 2, sucrose extract of monkey muscle; and 3, myasthenic serum. The wells are sur- rounded to some extent by nonspecific halo effects.

FIGURE 3. Gel diffusion precipitation pattern of myasthenic serum and various muscle extracts. The wells were filled as follows: 1, sucrose extract of monkey muscle; 2, myasthenic serum; 3, water extract of monkey muscle; and 4, normal human serum.

FIGURE 4. Gel diffusion immunoelectrophoretic pattern of myasthenic serum and muscle extract. The well contained myasthenic serum, and electrophoretic migration was to the right. The upper trough contained a sucrose extract of monkey muscle. The lower trough contained rabbit antihuman serum.

Lang et al.: Precipitation of Muscle Extracts 605

sucrose extract gave a very heavy and clear line of precipitation, as shown in FIGURE 2 (well 2). Another set of precipitation lines is shown in FIG- URE 3. In this experiment, the negative reaction of normal serum (well 4) can be seen, in clear contrast to the precipitates formed by myasthenic serum (well 2) in the reactions with the muscle extracts. The line for well 1, a sucrose extract, was relatively narrow, and it appeared to be single. On the other hand, a double line was again observed with the concentrated water extract (well 3), and this appeared to coalesce with the other line.

Finally, a few comments may be given about immunoelectrophoresis. It has been found to be preferable to perform this procedure in the reverse sequence, presumably because of a diffusion problem with muscle extract. Myasthenic serum was separated by electrophoresis, and then muscle extract was allowed to diffuse from the troughs. Here, again, agar a t 0.5 per cent was used, along with a sucrose extract of muscle. FIGURE 4 shows an enlarge- ment of the gamma-beta globulin region of the protein distribution. As can be seen, there are, in fact, two arcs of precipitation (with muscle extract) in the gamma-globulin part of the electrophoretic field.

Additional investigations are in progress in order to develop enriched antigen preparations to improve the sensitivity of the tests, and to throw some light on the chemical nature of the antigen or antigens involved. Some further results will be presented elsewhere.

References

1. STRAUSS,A. J. L., B. C. SEEGAL, J. C. HSU, P. M. BURKHOLDER, W. L. NASTUK & K. E. OSSERMAN. 1960. Immunofluorescence demonstration of a muscle binding, complement-fixing serum globulin fraction in myasthenia gravis. Proc. Soc. Exp. Biol. Med. 106: 184.

2. BEUTNER, E. H., E. WITEBSKY, D. RICKEN & R. H. ADLER. 1962. Studies on autoantibodies in myasthenia gravis. J.A.M.A. 162: 47.

3. WHITE, R. G. & A. H. E. MARSHALL. 1962. The autoimmune response in myasthenia gravis. Lancet ii: 120.

4. NASTUK, W. L., 0. J. PLESCIA & K. E. OSSERMAN. 1960. Changes in serum complement activity in patients with myasthenia gravis. Proc. SOC. Exp. Biol. Med. 105 177.

5. SIMPSON, J. A. 1960. Myasthenia gravis: A new hypothesis. Scot. Med. J. 6: 419. 6. VAN DER GELD, H. & H. J. G. H. OOSTERHUIS. 1963. Muscle and thymus

antibodies in myasthenia gravis. Vox. Sang. 8 196. 7. LANG, R. & S. SHULMAN. 1964. Precipitation analysis of muscle antibodies in

human wrum. Fed. Proc. 23 342. 8. GORNALL A., C. BARDAWILL & M. DAVID. 1949. Determination of serum proteins

by means of biuret reaction. J. Biol. Chem. 177: 751. 9. BEUTNER, E. H., G. FAZEKAS, A. SCOTT & E. WITEBSKY. Direct fluores-

cent antibody studies of gamma globulin localization in muscle of patients with myasthenia gravis. Ann. N. Y. Acad. Sci. (This Annal.).