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OCR for page 179
IMMUNOLOGIC ASPECTS OF THE HUMAN HEMOGLOBINS AMOZ I. CHERNOF`F For many years the antigenicity of human hemoglobin was seriously ques- tioned. Most investigators now agree, however, that human hemoglobin, though weakly antigenic, can induce the formation of specific antibodies in a number of animal species. The immunologic specificity of normal adult and fetal hemoglobin precipitins was first described in 1940 by Darrow and associates) who noted striking qualitative differences between the two types of antibody. These studies were confirmed and extended by Vecchio and J 13arbagallo,2 by Campbell and Goodman and in our own laboratory as well as by several more recent studies carried out in this country and in Europe.5 A number of factors have contributed to the production of more specific as well as more potent antisera in recent years. Carefully crystallized hemo- globins, presumably free from contaminating proteins, have been prepared for immunization purposes by utilizing the techniques of DraDkin and others. Purified material separated by column chromatography and by starch electro- phoresis has also been used in the immunologic studies of the human hemo- globins. A wide range of animals has been tested. The rabbit, guinea pig, arid chicken have been found to be most sensitive as anti-hemoglobin anti- body producers. Furthermore, the recent interest in the use of adjuvants of various sorts has permitted the preparation of more potent sera than hereto- fore available. Finally, better techniques for quantitating antibody antigen reactions have permitted a more exact delineation of the activity of these sera. At present, only lIbs A, S and F have been studied exhaustively by im- munologic techniques although eve have in progress the preparation of anti- sera to Hbs C, D and E as well as to a new hemoglobin type tentatively re- ferred to as Durham No. 1. Only Hb F has been demonstrated to have im- rnlunologic specificity, while antisera to Hbs A and S. in the hands of most workers in this field, cross react completely. Furthermore, Hb C, D, E, H. I and Durham No. ~ have been tested against anti-lIb A and anti-Hb S sera and found to give a positive precipitin reaction. These results suggest there- fore that, with the exception of fIb F. all the other varieties of pigment have very similar or identical antigenic sites on the surface of the globin moeity. These observations do not, however, rule out the possibility that specific dif- fcrences in structure may be localized within individual portions of the pro- tein molecule, or that the differences are either too small to be detected, or are masked by a common antigenic structure. The lack of immunologic specificity between such compounds as Hb A and Hb S is somewhat surprising in view of the marked physical-chemical dis- ~ Some of the studies reported in this paper were supported by United States Public Health Service Grant No. A-1615. 179

OCR for page 179
180 PART III. ABNORMAL HEMOGLOBINS similarities between these two proteins. However, an explanation for this lack of specificity may be found in recent studies using chromatographic techniques which suggest that Hb S and Hb A differ primarily in the charge of one poly- peptide chain of the molecule.6 For some time we have been using Ouchterlony's technique of double dif- fusion in apart in an effort to obtain more specific information concerning the immunologic activity of the antihemoglobin sera. Similar studies have been reported recently by Ruggieri and Marchi.S The technique is as follows: Small agar plates are prepared which contain three wells as may be seen in figure 1. Solutions of the antigens and antibodies are introduced into the wells FIN. 1. Ouchterlony's double dif- fusion in agar technique. Upper wells ntain Hb A solution; lower well anti-Hb A serum. and permitted to diffuse through the agar. If a precipitin reaction occurs, it is manifest by a distinct line of precipitation within the substance of the agar. The number, position and type of lines which develop provide useful informa- tion for the evaluation of the precipitin reaction. Thus the number of lines indicates the minimum number of reactants in the system. The position of the zone of precipitation depends on the relative concentrations of the antibody and antigen. A line appearing about halfway between two wells would suggest that the reactants are present in equivalent amounts or that one is working in the zone of optimal proportions. If two different solutions are tested against the same antiserum, a continuous line arching as in figure 1 would suggest the same immunologic reactants in each of the upper two wells. Lines which in- tersect or are only partially shared indicate different antigens in the two test solutions. The results of our studies with the hemoglobins may be summarized as follows: Anti-Hb A sera react weakly with Hb F and strongly with all other hemoglobin types. In most instances two or three lines of precipitation become apparent after 10-14 days of reaction. At times the line of precipitation with Hb F seems to merge with one of the three lines seen with Hb A. Anti-Hb F sera react most strongly with specimens known to contain Hb F. but also

OCR for page 179
IMMUNOLOGIC ASPECTS CHERNOFF FLOODED WITH 6~ air ,,J H GB A ~, fit AGAR PLATE S fLOODED WITH HGB f 5/ ;_ HGB SOLUTION fROM NORMAL I I ADULT ERYTHROCYTES it_ HGB f SOLUT10N PREPARED FROM I CORD SLOOD WANDA VS ISEAR~o PFRODUCED 181 FIG. 2.Ouchterlony's double dif- fusion in agar Hb-anti Hb system. ( Hbg in the illustration is an alter- nate usage of the more familiar Hb for Hemoglobin.) weakly with many normal adult hemoglobin specimens. When the plates are flooded with an excess of Hb A or Hb F which places the reaction with that particular hemoglobin into the zone of antigen excess and thus inhibits pre- cipitation, the patterns diagrammed in figure 2 are seen. It was postulated that, since normal hemoglobin solutions contained some Hb F. the anti-A sera actually had some anti-Hb F antibodies. Hence the weak reaction between anti-A and Hb F. Similarly, anti-F would be expected to react with the small amount of Hb F present in the adult specimens. When the plates are flooded with Hb A the reaction anti-Hb A-Hb A is suppressed but not that of anti- IIb F-Hb 1~. Similarly, when Hb F is used to flood the plates, reactions be- tween anti-F and the corresponding hemoglobin are suppressed. It is interesting to speculate further as to the nature of the multiple lines seen in the reaction between anti-A sera and Hb A. At first these lines were considered as artifacts since they were slow in developing and quite faint, in spite of the fact that such multiple lines should indicate a minimum of at least the same number of reacting compounds. In the light of recent world faith starch electrophoresis and chromatography of adult hemoglobin solutions, it is apparent that as many as three components may be present normally. It will, therefore, be of interest to test aliquots of these fractions against the antisera, especially with the flooding technique, to determine which, if any, of these lines may be produced by the minor adult hemoglobin fractions. Simi- larly, it would seem important to test these antisera by the immunoelectrophor-