IMMUNOCHEMICAL STUDIES OF HUMAN SERUM GAMMA GLOBULINS Solomon J. Zak, Robert A. Good J Clin Invest. 1959;38(3):579-586. https://doi.org/10.1172/JCI103835. Research Article Find the latest version: https://jci.me/103835/pdf IMMUNOCHEMICAL STUDIES OF HUMAN SERUM GAMMA GLOBULINS * By SOLOMON J. ZAK t AND ROBERT A. GOOD t (From the Minneapolis Veterans Administration Hospital, and the University of Minnesota Medical School, Minneapolis, Minn.) (Submitted for publication September 15, 1958; accepted October 10, 1958) The establishment of the quantitative precipi- the zone of antibody excess, has been well docu- tin reaction as an analytical-chemical procedure mented (7, 8). For the purposes of this study by Heidelberger and Kendall (1) was followed in it was felt that a physically or chemically homo- 1937 by Kendall's original use of the method in geneous gamma globulin preparation, possessing the estimation of human serum proteins in health immunologic homogeneity in the zone of anti- and disease (2, 3). A major drawback to the body excess, might be employed for preparation of use of this immunochemical method for obtaining antibody to be used in a quantitative precipitin meaningful human serum protein values has con- test for estimation of gamma-reactive globulin in cerned the purity or homogeneity of the specific human fluids. Because of the inadequacy of other protein used as the antigen (4). In particular, methods, it was considered likely that this tech- the heterogeneity of the human gamma globulin nique might be especially useful when the con- fraction, as defined electrophoretically, has been centration of the gamma globulin to be measured pointed out (5). Cohn, Deutsch and Wetter have is very low. demonstrated distinctive immunochemical be- The physical, chemical and immunological simi- havior in various subfractions of the human se- larities of the human gamma globulins and most rum gamma globulins (6). In these studies, specific antibodies are well documented (9, 10). however, the principal differences in immuno- On the biological level, the clinical entity agam- chemical behavior of the subfractions were noted maglobulinemia provides strong evidence in sup- in zones of antigen excess; this zone is not em- port of the concept that gamma globulin and ployed in the studies reported herein. most antibodies arise from a common source (11, Although numerous physical, chemical and im- 12). Accordingly, immunochemical estimations, munological criteria have been set forth for the patterned directly after the methods of Heidel- establishment of protein purity, the lack of in- berger and Kendall (1), have been applied to the formation regarding protein structure does not study of agammaglobulinemic and normal human permit a clear definition of a single human pro- sera, and reference comparisons to electrophoretic tein species. In general, the proteins comprising observations have been accomplished. The pur- the human serum gamma globulins separated by poses of the present study were several-fold and ethanol fractionation have an isoelectric distribu- included the following: 1. To establish the ef- tion of pH 5.7 to 7.5; their electrophoretic mo- ficacy of the quantitative precipitin reaction for bilities range from 0.97 to 2.64. The marked the accurate estimation of human serum gamma antigenic similarity, indeed almost antigenic iden- globulins. 2. To measure the serum concentra- tity, of the members of this family of proteins in tions of gamma-reactive globulin in normal hu- mans. 3. To describe the quantitative gamma *Aided by grants from the Minnesota Heart Associ- globulin deficit in patients with both congenital ation, the American Heart Association, the Helen Hay estab- Whitney Foundation, the Minnesota Division of the and acquired agammaglobulinemia. 4. To American Cancer Society, and the United States Public lish the biological survival time of parenterally Health Service (RG E-798 and H-2085). administered gamma globulin in humans by im- t Resident, Department of Internal Medicine, Minne- munochemical means. 5. To study the appearance apolis Veterans Administration Hospital. of gamma-reactive globulins in the newborn baby tAmerican Legion Memorial Heart Research Profes- sor of Pediatrics, University of Minnesota Medical of a mother with acquired agammaglobulinemia, School. and to relate the appearance of gamma globulin 579 580 SOLOMON J. ZAK Al.ND ROBERT A. GOOD to the time of onset of specific immune globulin performed as outlined by Kabat and Mayer (16). A synthesis and release; to study the disposition of highly purified gamma globulin preparation obtained by transmitted gamma globulin by the ethanol fractionation was kindly supplied by Dr. J. T. maternally Edsall, and served as antigen in these studies. The agammaglobulinemic offspring of an immunologi- method of Kendall (2) for alum precipitation of the cally normal female who had previously borne a antigen and that of Jager, Smith, Nickerson and Brown son afflicted with the congenital form of the di- (17) for the immunization schedule were adopted. Anti- sease. 6. To compare maternal and cord serum human gamma globulin was produced in albino mixed hy- concentrations of gamma-reactive globulin. brid rabbits weighing 4 to 5 Kg. The alum precipitated antigen was administered intravenously only and in a concentration of 1 mg. per ml. over a three week period. MATERIALS AND METHODS Each animal received a total of 12 mg. of the alum pre- cipitated material. On the eighth day following the Sixty immunologically normal children ranging in age last injection of antigen, each animal was exsanguinated from one day to 15 years: These children were patients by cardiac puncture under sterile conditions. The bloods at the University of Minnesota Hospitals, Minneapolis obtained were allowed to clot at room temperatures for General Hospital and the Ancker Hospital of St. Paul, one hour, placed at 2° C. overnight, the clots then who had been admitted for study or treatment of con- rimmed and the tubes centrifuged at 1,500 rpm for 20 genital anomalies or of trauma. None had been troubled minutes in an International PR-1 centrifuge; the sera with recurrent infections, and in no case was there rea- were decanted and pooled after preliminary testing for son to suspect an abnormality of protein synthesis or an antibody content. The pool was then recentrifuged at immunological deficit. Twenty-four immunologically 2,000 rpm for 20 minutes, decanted, and merthiolate was normal, healthy adults: These subjects were laboratory added to a final concentration of 1 part in 10,000. The and hospital personnel, ranging in age from 19 to 34 years. pool was then heated at 560 C. for 30 minutes, allowed Thirty-one maternal cord serum pairs: In all cases, the to come to room temperature, dispensed into sterile glass maternal blood was drawn within two hours of parturi- tubes, quick-frozen in an alcohol-acetone dry ice bath, and tion. Sera from 16 patients with agammaglobulinemia: stored at -26° C. until ready for study. Prior to use in Nine of these were patients with the congenital form of these and all subsequent determinations, sera and anti- the disease, and seven with the acquired form. The sub- sera were clarified by centrifugation for one hour in the jects chosen for study of gamma globulin half-life deter- cold at 3,000 rpm. minations included four congenitally agammaglobulinemic Absorption-precipitin study. A solution of the gamma patients and a normal newborn who was bled frequently globulin was prepared in nitrogen-free diluent, con- during the neonatal period. All subjects were free of sisting of 0.01 molar phosphate at pH 7.4, and brought clinical infections at the time these studies were per- to ionic strength 0.145 with sodium chloride. The con- formed. centration of gamma globulin in this solution was estab- After preinjection bleedings, each subject in the half- lished by repeated micro-Kjeldahl analyses; volumetric life study was given gamma globulin in various dos- dilutions of this standard were then prepared. Quanti- ages intramuscularly. Venous blood was drawn there- tative precipitin determinations were carried out by add- after at varying intervals. The bloods were allowed to ing 1 ml. of each antigen dilution to 1 ml. of antiserum clot at room temperatures for one hour, placed at 20 C. in standard heavy-walled precipitin tubes. After thor- overnight, the sera separated by centrifugation in the ough mixing by agitation, the tubes were capped with cold, quick-frozen at -70° C., and stored at -260 C. un- parafilm, and allowed to incubate for one hour at 370 C., til ready for use. then placed at 0 to 40 C. for eight days. The tubes The decline of maternally transmitted gamma globulin were agitated daily. They were then centrifuged for one was assessed by studying the male offspring of an ap- hour in the cold at 3,000 rpm, and the supernates poured parently normal mother who had previously borne a off and tested for the presence of antigen and antibody. congenitally agammaglobulinemic male. In this study, All procedures were carried out in the cold. The tubes blood samples were drawn from the jugular vein of the were allowed to drain over filter paper for 15 minutes, and baby in the neonatal period and at intervals through- the precipitates were washed by resuspension in 0.145 N out the first year. nitrogen-free NaCl. This washing procedure was re- of in the newborn and The appearance gamma globulin peated twice. The washed specific precipitates were its quantitative characteristics during the first year of ml. solution and for life were determined in the unique situation of a nor- digested with 0.5 digestion analyzed mal female offspring of a patient with acquired agam- nitrogen by the micro-Kjeldahl method. Repeated analy- maglobulinemia, reported upon in detail elsewhere (13, ses, using the same as well as different lots of antiserum, 14).