Patients with systemic lupus erythematosus (SLE) form a spectrum of antibodies which react in various test systems with native or altered autologous antigens (1-4). The stimuli eliciting antibody production and the pathogenetic role of these antibodies in SLE have been the subject of intensive investigation and debate. Earlier studies stressed the importance of such antibodies as direct cytotoxic agents (5-7). More recently, however, evidence has been accrued suggesting that circulating antigen-antibody complexes are formed during the course of SLE and are deposited in the kidneys of patients with the nephritis of SLE. Serum complement is depressed during active stages of nephritis (8, 9) and the clinical manifestations are similar to the chronic serum sickness syndrome elicited in animals by antigen-antibody complexes formed in vivo (10). Floccular and granular deposits of T-globulin and complement (11-13) have been localized in glomeruli utilizing the fluorescent antibody technique. These deposits resemble those found in experimental immune complex-induced nephritis of Dixon and associates (14). Antinudear antibodies have been eluted from the glomeruli of kidneys from patients with SLE nephritis (15-18). Recently, one potential antigen, deoxyribonucleic acid (DNA), has been observed in sera from certain patients with active nephritis (19). These data suggest that antigen-antibody complexes composed of nuclear antigen and antinuclear antibodies might well be involved in the pathogenesis of the nephritis.The present studies were undertaken to obtain further support for the antigen-antibody complex hypothesis. In particular, efforts were made not only to demonstrate antinuclear antibodies in glomeruli but to show by quantitative immunochemical techniques that they are concentrated at this site in association with nuclear antigens.
A number of different types of apparent receptor sites have been demonstrated on the surface membrane of lymphocytes. These include immunoglobulin receptors for a variety of antigens (1), a receptor for the C3 component of complement (2), and a possible receptor site for the Fc portion of the immunoglobulin molecule through which antibody alone or antigen-antibody complexes adhere to mouse lymphocytes (3,4). Attempts in the present study to demonstrate such a receptor for antigen-antibody complexes on human lymphocytes using fluorescein-labeled antigen or antibody presented a number of difficulties with different complexes. One particular antigen-antibody system consistently labeled a population of lymphocytes while several other systems were completely negative. The reason for this discrepancy was not apparent. As an alternative approach, aggregated T-globulin was employed for the study of this interaction. Such preparations of T-globulin are known to possess m a n y of the properties of antigen-antibody complexes and are considerably more stable. Materials and MethodsAggregated Human lmmunoglobulin.--Fraction II human y-globulin (Lederle Laboratories, Pearl River, N. Y.) was dissolved in phosphate-buffered saline (PBS), 1 pH 7.2, at a concentration of 50 mg/ml. This was heat inactivated at 56°C for 30 min, and fluorescein conjugated as previously described (5). Conjugated and unconjugated preparations were heat aggregated at 63°C for 15 rain, pelleted, and homogenized in PBS, pit 8.0. Just before use, aggregates were centrifuged at 1000 g for 30 min and adjusted to 1-3 mg/ml. Aggregates of isolated myeloma proteins were prepared in a similar manner except lower concentrations of protein were used to form the aggregates. Aggregates of specific size and 7S IgG were obtained by preparative ultracentrifugation.
The recent success in the separation of amino acids and peptides by filter paper chromatography has led numerous investigators to attempt to separate proteins in an analogous manner. These efforts have thus far not met with definite success. However the possibility of employing filter paper as a framework to support the liquid medium used for the separation of proteins through electrophoresis has proven useful. The observations of Durrum (1), Cremer and Tiselins (2), Wieland (3, 4), and Turba and Enenkel (5) have indicated that the serum proteins would separate on filter paper into components similar to those found in free electrophoresis. Furthermore by cutting the paper into segments and eluting the dye used to stain the separated serum proteins a curve could be obtained which resembled the usual electrophoretic pattern (2).Filter paper electrophoresis in addition to being extremely simple possesses advantages in some respects over the classical methods in free solution. First, absolute separation of components takes place rather than just separation into concentration gradients. Second, smaller amounts of material at lower concentrations can be investigated. Third, isolation of all the components which have been separated is possible. Fourth, the paper support makes it possible to develop techniques of two dimensional electrophoresis and devices for continuous flow preparative work (6, 7). Whether it is possible to achieve the accuracy obtained with the sensitive optical methods employed in free electrophoresis for localizing and quantitating components, is doubtful. In the present report a description is given of the separation and isolation of a large number of different proteins employing a method of paper electrophoresis between glass plates in which disturbing factors such as evaporation, heating, and buffer concentration gradients were reduced to a minimum. An attempt was made to employ the method for determining the mobilities of certain proteins. Material and MethodsIn a previous report from this laboratory (2) a method was described for filter paper electrophoresis in which the paper strip, immersed in buffer and containing the specimen to be analyzed, was clamped between glass plates and immersed under a solution of chlorobenzene and connected to electrode vessels. This apparatus was
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