Primary interaction between antibody and components of Alternaria II. Antibodies in sera from normal, allergic, and immunoglobulin-deficient children Michael J. Schumacher, M.B., F.R.A.C.P., Richard S. Farr, M.D., J. Kenneth McClatchy, Ph.D., and Percy Minden, M.D. D~)ww’, ~'olo. illtermricr tenuis, a common soil fungus, is an important precipitant of bronchospasm in a significant number of patients with bronchial asthma.]-” Antibodies to components of &ternrk~r SIJ. have previously been observed in some sera from patients with asthma, in some sera from unselected hospital patients with nonallergic d&cases, and in small numbers of control sera.‘l-7 In normal scra, a high incidence of antibodies to certain fungi was suggested by previous studies from this laboratory. Antibodies to components of &~erqiZl~~~s furnigrrtus a.ntl ilsper~illus wid~clnw3were found in many sera from normal piTSOIlS, as well as in scra from patients infected with Aspet-gillus sp.“~ !’ Because spolw of /llfer)lnri,r ~1). are found in considerable numbers in the cnvironmrrlt,l’~-‘” the possibility that antibodies to antigenic components of this From the Depart,ment of Allergy and Clinical Immunology, National Jewish Hospital and Research Center. Supported by The Unitrtl States-Japan Co-operative Medical Science Program administered bv the Xntional Institute of Allergv and Infectious Diseases of the National Institutes of Iiealth, and by the United Rtatcs%blic Health Service Grants Nos. CA-15446, AI-08190, and AI-09481. Hweived for publication April 3, 1974. Reprint requests to: Dr. M. J. Schumacher, Children’s Medical Research Foundation, P.O. Box 61, Camperdown, N.S.W. 2050, Australia. Vol. 56, No. 1, pp. 54-63
Transcript
Primary interaction between antibody and components of Alternaria II. Antibodies in sera from normal, allergic, and
immunoglobulin-deficient children
Michael J. Schumacher, M.B., F.R.A.C.P., Richard S. Farr, M.D.,
J. Kenneth McClatchy, Ph.D., and Percy Minden, M.D. D~)ww’, ~'olo.
illtermricr tenuis, a common soil fungus, is an important precipitant of bronchospasm in a significant number of patients with bronchial asthma.]-” Antibodies to components of &ternrk~r SIJ. have previously been observed in some sera from patients with asthma, in some sera from unselected hospital patients with nonallergic d&cases, and in small numbers of control sera.‘l-7 In normal scra, a high incidence of antibodies to certain fungi was suggested by previous studies from this laboratory. Antibodies to components of &~erqiZl~~~s furnigrrtus a.ntl ilsper~illus wid~clnw3 were found in many sera from normal piTSOIlS, as well as in scra from patients infected with Aspet-gillus sp.“~ !’ Because spolw of /llfer)lnri,r ~1). are found in considerable numbers in the cnvironmrrlt,l’~-‘” the possibility that antibodies to antigenic components of this
From the Depart,ment of Allergy and Clinical Immunology, National Jewish Hospital and Research Center.
Supported by The Unitrtl States-Japan Co-operative Medical Science Program administered bv the Xntional Institute of Allergv and Infectious Diseases of the National Institutes of Iiealth, and by the United Rtatcs%blic Health Service Grants Nos. CA-15446, AI-08190, and AI-09481.
Hweived for publication April 3, 1974.
Reprint requests to: Dr. M. J. Schumacher, Children’s Medical Research Foundation, P.O. Box 61, Camperdown, N.S.W. 2050, Australia.
Vol. 56, No. 1, pp. 54-63
VOLUME 56 NUMBER 1
Interaction between antibody and Alternario. II 55
mold are similarly present in a large proportion of the normal population was considered to be of potential importance. This would hc particularly true if there were significant differences between antibodv levels in sera from normal persons and from those allergic to il. fewwis. Elcvatetl levels of antibotlics to thermophilic actinomycetes hare been postulated to play a tlircct role in the pathogenesis of some cases of hypersensitivity pneumonitis,” suggesting that hypersensitivity pneumonitis in malt workers with heavy exposure to sports of itsyergilhs sp.‘” may be explained by a similar mechanism. Although most reported cases of Arthus-type pulmonary reactivity to moltls art’ tluc to asptlr-
gill&‘* patients with hypersensitivity pneumonitis with tlcla-ctl ~cspo~lscs to
inhalational challenge with extracts of il. fe~~uis have also been itlcntifictl.” There are no reports of studies using trsts that measure the primary intcr-
action between antigens derived from A. te~uis and antibodies. These bincling procedures frequently demonstrate antibodics that are not dctectablc by tests such as hemagglutination and precipitin tests, which detect scconclar,v mani- festations of primary reactions between antigen and antihotl,v.17
In the present investigation, sera from normal and asthmatic chiltlren were tested employing a primary binding test and radiolabeled antigens as described in the accompanying paper.‘* Sera from normal infants and children at differentj age groups were tested to determine the incidence and possible pattern of devcl- opment of antibodies to components of ~4. feauis. Sera from hypogammagloh- ulinemic patients and from children with isolatecl IgA tleficiency wcrc similarl> studied.
MATERIALS AND METHODS Sera
Sera were obtained from 46 normal infants (2 months to 5 years of age), from 59
schoolchildren (6 to 16 years of age), and from cord blood at 5 normal deliveries. Donors were not screened for personal or family history of allergy and were not skin-tested.
Sera were also obtained from 142 children with bronchial asthma. Rlood samples were drawn prior to skin testing with an extract of A. tenwi.s (Greer Laboratories, Lenoir, North
Carolina). Prick tests were performed at an allergen concentration of 1:20 w/v, and
intradermal (ID) tests by injection of 0.02 ml of allergen at a concentration of 1,000
protein nitrogen units (PNU) per milliliter, employing standard techniques.19 Wheal diameters were recorded at 20 min, after subtraction of diameters of any wheal response to control
tests with buffered saline.
Hera were also obtained from 8 children with hypogamnmglobulinemia (IgG less than
200 mg/lOO ml, and IgA less than 15 mg/lOO ml) ; 4 children had infantile X-linked
agzmmaglobulinemia, 2 had immunodeficiency with hyper-TgM, and 2 had variable im-
munodeficiency (terms defined by WHO classification of immunodeficiency disease@). Sera
were also obtained from 10 asthmatic children with selective IgA deficiency (IgA less than
15 mg/lOO ml).
Antigens
Antigens derived from culture filtrates and homogenates of A. temis (CLF and HS,
respectively) were prepared and labeled with iodine-125 (1251) as described in the accompany-
ing paper.18 The radiolabeled components are referred to as 125I-CLF and 1251-HS. Other antigens were: Aspergillus fumi@u+- extract (Meridian Bio-Medical, Inc., Denver, Colorado) ;
*Per cent radiolabeled antigen (at 3 ng N/0.1 ml) bound by 0.1 ml of 1:5 dilution of sera.
tND: not done. $Significance of difference from normals: p < 0.005.
$Significance of difference from normals: p < 0.001; from negative skin test group: p < 0.01.
sp., and perennial rye grass (Greer Laboratories) ; and an extract of house dust (Endo Laboratories, Garden City, New York). An extract from Escherichin coli was prepared in
this laboratory as described previously.21
Measurement of antibodies
Binding between antibodies in 0.1 ml of 1:s dilutions of human sera in borate buffer, pH 8.3, and 0.1 ml of the l?sI-labeled antigens was studied employing goat antihuman IgG
(anti-HGG) to precipitate labeled antigen-antibody complexes. The quantitative binding data
reported are expressed as percentage of labeled antigen at 3 ng N/0.1 ml precipitated by anti-HFG. Details of the methods used for these studies as well as for the inhibition studies
that were employed have been described.% 22-24
Statistical analysis of data
Prior to the estimation of the significance of difference between sample means, the equality of the variances of the samples was tested by variance ratio (Snedecor’s F test).
VOLUME 56 NUMBER 1
Interaction between antibody and Alternaria. II 57
TABLE III. Effect of injections of Alternaria extract on capacity of sera to bind ‘251-CLF
Total dose of Alternaria Duration of
Binding to ‘V-CLF*
received treatment Before After Per cent Patients (PNUH (days) treatment treatment increase
*Per cent radiolabeled antigen (at 3 ng N per 0.1 ml) bound by 0.1 ml of 1:5 dilution of sera.
t PNU : protein nitrogen units. Details of treatment reported previously.nn IGiven injections of ragweed extract.
IGiven injections of dog epithelium extract.
Where the variances were not significantly different, Student’s t test was applied, using small
sample formulasa Where the variances were significantly different, appropriate formulas
for the calculation of an approximation to Student’s t were used.26
RESULTS Binding of Alternaria antigens by sera from normal children
Each test serum (diluted 1:5) was examined for binding to 12V-CLF and ‘251-IIS. One hundred and three of 105 sera from normal children up to 16 years of age bound more than 5% of each labeled antigen. The greatest amounts of l?“I-CLF and lz51-HS bound were 58% and SS%,, respectively.
The amount of each radiolabeled antigen bound by antibodies in cord sera was not significantly different from that observed by sera from children over the age of 8 years. Binding fell to relatively low levels during the first 8 months of life, and then progressively increased until the age of 12 years (Table I). The increase of binding with age between 30 months and 12 years, calculated by linear regression analysis, was significant (p < 0.05). When binding of lz51-CLF was compared to binding of lz51-HS, the differences were not statisti- cally significant (Table I).
Binding of Alternaria antigens by sera from asthmatic children
When binding of the two antigens was compared using sera from 23 asthmatics, binding to ‘251-CLF was slightly higher (mean, 38.4%) than binding to l”“I-HS (mean, 32.9%). The differences between these means were not st,atis- tically significant, and therefore it was convenient to carry out most experiments using the radiolabeled culture filtrate antigen (1251-CI~F) only.
AS with normal sera, virtually all sera from asthmatic children bound appreciable amounts of Y-CLF. Binding levels did not increase further beyond the age of 7 years (Table II).
The asthmatic patients were divided into 3 groups according to their im- mediate responses to skin testing with a commercial extract of A. tee&: (1) negative (smaller than 3 mm wheal diameter response to 1D test) ; (2) weakly
58 Schumacher et al. J. ALLERGY CLIN. IMMUNOL. JULY 1975
TABLE IV. Binding of ‘251-CLF by sera from immunoglobulin-deficient patients and controls
Number tested Mean f SD of binding
to ‘=4-CLF’
Asthmatics with selective IgA deficiency 10 44.5 * 15.3 Asthmatics, normal IgA 105 45.1 f 15.0 Hypogammaglobulinemiat 8 4.7 & 1.6
*Per cent radiolabeled antigen (at 3 ng N per 0.1 ml) bound by 0.1 ml of 1:5 dilution of sera.
Per cent ‘“‘I-HS at 3 ng N per 0.1 ml bound by 0.1 ml of sera without inhlbitor 35.7 39.5 30.1 44.0 57.1
“0.1 ml volumes added to 0.1 ml of sera.
t (Binding with inhibitor)-(binding without inhibitor)
Binding with inhibitor x 100. Binding calculated as above.
tPNU : protein nitrogen units.
$ND: not done.
positive (3 to 6 mm wheal response to ID test, or a 2 to 3 mm wheal after prick test) ; and (3) strongly positive (greater than 6 mm wheal after II) test or a wheal larger than 3 mm after prick test).
Sera from patients with negative skin test reactions did not show significant differences in binding from sera of normal subjects or from sera of patients with weakly positive skin test reactions. However, sera from patients with strongly positive skin tests bound significantly greater amounts of antigen than did sera from patients with negative reactions (p < 0.01) or sera from normal children (p < 0.001) (Table II).
Sera were obtained from 4 asthmatic children before and after a course of injection treatment with commercial extracts of A. tewwi9” and were studied for binding to ‘251-CI~F. These children were not treated with other antigens. Control sera from 3 children treated with extracts of dog epithelinm or ragweed were also studied. Increases in binding were seen in sera from patients treated with A. tenuis extracts, but not in sera from the 3 control children (Table III).
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Interaction between antibody and Alternaria. II 59
Binding of Alternaria antigens by sera from immunoglobulin-deficient patients
All sera from patients with hypogammaglobulinemia bound less than 8% of the lz51-CLF antigen. This was significantly less than the binding by sera of normal children at any age period (p < 0.001) (compare Tables IV and I). The mean binding of sera from asthmatic children with selective IgA deficiency was not significantly different from binding by sera, from the total unselected population of asthmatic patients in the same age group (4.5 to 15.0 yr) (see Table IV). Not shown in Table IV, sera from 2 patients with normal IgG, low IgA, and low IgM had reduced binding (18.1% and 11.2’$& of lz51-CLF).
Specificity of antibodies to Alternaria in human sera
The following experiment was carried out to determine whether antibodies to Y-HS in human sera would also bind to heterologous antigens. Sera from 5 normal children that had been shown to bind 35% to 55% of lz51-HS were selected and the capacity of 12 unlabeled substances to inhibit the binding of lz51-HS by 0.1 ml of I:5 dilutions of these scra was tested (Table V). As expected, unlabeled HS almost completely inhibited the binding. Stemphyliu~m
and Cwwdaria extracts also inhibited this binding to an appreciable degree in all 5 sera. All the other mold extra& tested partially inhibited these reactions, reflecting the known shared antigenicity among fungi.z7 Of special interest, house dust and perennial rye grass extracts also had significant inhibitory capacities, and the reactions were partially inhibited by an extract of E. coli in 4 sera. Methylcellulose did not inhibit any, and human group AB erythrocytes inhibited one reaction to a slight degree.
DISCUSSION
Results of the experiments described suggest that nearly all children have circulating antibodies to labeled antigens derived from A. tends, and that most, if not all, persons develop an immune response to this mold during early childhood. This is a reflection of the ubiquity of spores of Alternariu sp. in the atmosphere,l” soi1,28 and house dust,11-13 and, probably, the high frequency of isolation of Alternuriu sp. from normal foot skin in children.*” Also, stimulation of production of antibody to A. tenwis may have been due to shared antigens from other fungi and bacteria in the environment. In normal human sera, a high incidence of antibodies to components of mycobacteria,30 aspergilli,s, 31 and a variety of other microorganisms32-37 has similarly been reported. An increased incidence of antibodies against a variety of organic dust antigens has also been observed in sera from normal persons.F
The experiments in the accompanying paper’* indicated that the labeled antigens used showed only weak precipitation with antibodies. It is known that some populations of antibody may not precipitate with antigen, agglutinate antigen-coated erythrocytes, or fix complemcnt.17~ 38, 3g This, together with the sensitivity of the test procedure employed, probably accounts for the differences between our results and those of other investigators who used precipitin” and
60 Schumacher et al. J. ALLERGY CLIN. IMMUNOL. JULY 1975
hemagglutination tests and found fewer positive reactions in normal human sera.
The amount of antigen bound by antibodies in cord sera was comparable to t,he binding levels of antibodies from normal adult sera, as would be expected if t,he test employed detected mainly IgG antibody. After birth the binding levels fell to a minimum at around 9 months of age and then gradually rose toward adult levels. The age of occurrence of the least amount of binding was 3 to 6 months later than the usual age of hypogammaglobulinemia of infancy.4” Thus, the onset of production of antibody to d. te?&uis usually follows the onset of
physiological synthesis of IgG by several months, possibly reflecting the time of exposure of infants to these antigens.
Antibodies from sera of asthmatic patients with strongly positive skin tests to .4. te~is bound significantly more antigen than did antibodies from sera of skin test-negative pa,tients or from normal children. Previous cour.ses of im- munotherapy with extracts of A. tenu.G and other fungi, bacteria, and house dust may have stimulated production of antibodies that bound to ‘251-CLF and lz51-HS. As illustrated in this study, immunotherapy with an extract of A. tenuis elevated the binding capacity of sera from asthmatic patients (see Table III). Another explanation for higher binding by sera from some asthmatic patf&ts may be the increased ability of allergic patients t,o produce antibody in response to nasally administered a.ntigens,4* suggesting the possibility that patients sensitive to A. tenuis may have a decreased resistance to the penetration of the respiratory mucosa by antigens from mold spores. Production of antibody to 8. fewis, either due to natural exposure to spores, or from immunotherapy, might be further potentiated by the cross-reactivity, shown in this study, between antigenic components of A. texuis and extracts of other microorga- nisms and with house dust (see Table V) .
There was a markedly reduced capacity of sera from patients with hypo- gammaglobulinemia to bind Y-CLF. Such a finding might help alert one to the diagnosis of antibody deficiency syndromes such as agammaglobulinemia. Patients with IgA deficiency alone, who were not associated with a gross immune deficiency state, however, had sera that bound labeled antigens at levels com- parable to normal sera, suggesting that most of the binding observed in this study was probably by IgG antibody and not by IgA antibody.
The pathogenic activities of Altermria sp. seem to be primarily concerned with allergic disorders. Local or invasive forms of disease such as are caused by other fungi are not believed to occur frequently. However, an increase in the number of other fungal infections is being reported, especially those due to opportunistic fungi such as aspergilli and Caotdida albica~ns.42-44 It would not be surprising if disease states caused by Altermria sp. were to be observed in the future, especially in immunodefieient patients. Again, the observation that antigenic components were shared by A. tenuis and other fungi, including aspergilli and Candidu albicans (see Table V) , may turn out to be of significance, Some of the antibodies to such fungi are probably raised following exposure to antigens of A. tenuk
In many fungal diseases, the clinical and laboratory diagnosis is at times
VOLUME 56 NUMBER 1
Interaction between antibody and Alternaria. II 61
difficult. The mere isolation of fungi is not proof of infection and additional information is usually needed, such as that obtained h;- rocntgcnographic cxam- ination, skin tests, and antibody studies. Antibody tests to components of aspcr- gilli, for example, have been useful in the diagnosis of the myc+ctomal form of pulmonary aspergillosis.”
Hypersensitivity pneumonitis, a condition that has been linked with prc- cipitating antibodies in the TgC: class,45 may occur from sensitization to Alter- nn#ria sp.16 Bv contrast, antifungal antibodies in the IgQ class arc probably not pathogenic in patients with type I acute hypersensitivity diseases, and in some patients they may act as “blocking” antihodies.4”-4” E’unc%ional neutralization of allergen by “blocking” antibody in vivo is still controversia14” and rcquircs further study, which could employ techniques developed in this investigation. In addition, the biological significance of the near-universal presence in normal individuals of antibodies to A. te?ulis is not clear.
The availability of primary binding methods and the ease of preparation of the partially characterized and soluble antigens emplo,ved in this study shoultl assist in studying the relevance of anti-AZfer?z(1rin antibodies to disease states and should assist further study of humoral antibodies and other forms of the immune response such as cellular immune reactions in normal and allergic patients.
WC are indebted to Major L. Bernard Branch, Fitzsimons General Hospital, Denver, for providing sera from normal children, and to Drs. Elliot F. Ellis and David S. Pearlman
of this institution for supplying sern from children with asthms and immunoglobulin
deficiencies. We are grateful to Sally Winkler for assistance in collation of the skin test data
and to Janet Lightner for fine technical assistance.
REFERENCES
1 Hopkins, J. G., Benham, R. W., and Iiesten, B. M.: Asthma due to a fungus-Alternaria, J. A. M. A. 94: 6, 1930.
2 Feinberg, 8. M.: Mold allergy; its importance in asthma and hay fever, Wisconsin Med. J. 34: 254, 1935.
3 Rchonwald, P.: Allergenic molds in the Pacific Northwest, J. ALLERGY 9: 175, 1938. 4 Bonilla-Soto, O., Rose, N. R., and Arbesman, C. E.: Allergenic molds. Antigrnic and
allergenic properties of Alternarin tentis, J. ALLERGY 32: 246, 1961. 5 Reed, C. E., Busse, W., and Crandall, M.: Adverse rractions to immunotherapy with
Alternaria, J. ALLERGY CLIN. IXIMUNOI~. 51: 84, 1973. (Abst.)
6 Kawai, T., Salvaggio, J., Arquembourg, P., and Marsh, I).: Precipitating antibodies against organic dust antigens in human sera by counterimmunoclectrophoresis, Chest 64: 420, 1973.
7 Chmelik, F., Flaherty, D., and Reed, C. E.: Prevalence of precipitins to antigens associated with hypersensitivity pneumonitis, J. ALLERGY &IN. JMMVNOL. 53: 86, 1974. (Abst.)
8 Bxrdana, E. J., McClatchy, J. K., Farr, R. S., and Minden, P.: The primary interaction of anlibody to components of aspergilli. II. Antibodies in sern from normal persons and from patients with aspergillosis, J. ALLERGY CLIN. I~~MVNOI,. 50: 22’J 1972.
9 Bardan:~, E. J., Cianciulli, F. D., Noonan, M. J., and Sobti, K. L.: ?!he primary interaction of antibody to components of aspergilli in sera from patients with cystic fibrosis. Personal c0mmunicntion.
10 American Academy of Allergy: Statistical report of the pollen and mold committee, 1972. 11 Frey, D., Cross, D. O., and Durie, E. B.: lnvestigntion of a series of snmplos of 11ouse
dust for the presence of fungi: Correlation with previous investigations of air-borne, fungi and sensitivity tests on patients, Mycopathol. Mycol. Appl. 19: 83, 1963.
12 Sosm:~n, J. A., and Hirsch, S. R. : One-year survey of mold growth inside 12 homes, J. ALlXKGY CLIN. IMMUNOL. 53: 71, 1974. (Abst.)
62 Schumacher et al. I. ALLERGY CLIN. IMMUNOL. JULY 1975
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27 28
29
30
31
32
33
34
35
36
37
38
39
Solomon, W. R.: Assessing fungus prevalence in domestic interiors, J. ALLERGY CLIN. hlMVNOL. 53: 71, 1974. (Abst.)
Pepys, J.: Hypersensitivity diseases of the lungs due to fungi and organic dusts, in Monographs in allergy, Base& 1969, S. Knrger AG, vol. 4. Riddle, H. F. V., Channell, S., Blyth, W., VVeir, D. M., Lloyd, M., Amos, \V. M. G., and Grant, I. TV. B. : Allergic alveolitis in a maltworker, Thorax 23: 271, 1968. Schlueter, I). I’., Fink, J. N., and Henslry, G. T.: \Vood-pulp workers’ disease: A hypersensitivity pneumonitis caused by Blternarin, Ann. Intern. Med. 77: 907, 19i;Z. Minden, P., and Farr, It. S.: Antigen-antil,ody interactions and their measurement, in Samter, M., and Talmage, D. W., editors: Immunological cliseases; part I: Basic immunology, ed. 2, Boston, 1971, Little, Brown & Co., pp. 179201. Schumacher, M. J., McClatehy, J. K., Farr, R. S., and Mindeu, I’.: Primary interaction between antibody and components of Alternaria. I. lmn~unological and chemical char- acteristics of labeled antigens, J. ALLERGY &IN. IMM~~NOL. 56: 39, 1975. Vnnselo\v, N. A.: Skin testing, in Sheldon, J. M., Lovell, R. Cr., and Matthews, K. I’.: A manual of clinical allergy, ed. 2, Philadelphia, 1967, W. B. Saunders Co., pp. 55-66. Fudenberg, H., Good, R,. A., Goodman, H. C., et al.: Primary immunodeficiencies. Report of a World Health Organization committee, Pediatrics 4’7: 927, 1971. Mindeu, I’., McClatchy, J. K., Bardana, E. J., and Fnrr, R. 8.: Antigenic differences between ~ycobactertim bovis strain BCG and an isoniazid-resisbnt mutant, Infect. Immun. 3: 524, 1971.
Minden, P., and Farr, R,. S.: Binding between components of the tubercle bacillus and humoral antibodies, J. E-up. Med. 130: 931, 1969.
Minden, I’., Anthony, B. F., and Farr, R. S.: A comparison of seven procedures to detect the primary binding of antigen by antibody, J. Immunol. 102: 832, 1969. Minden, P., and Farr, R. S.: The ammonium sulphate method to measure antigen-binding capacity, irk Weir, D. M., editor: Handbook of experimental immunology, ed. 2., Philadelphia, 1973, F. A. Davis Co., chap. 15, pp. l-21. Snedecor, G. W.: Statistical methods applied to experiments in agriculture and biology, ed. 5, Ames, 1956, Iowa State College Press, chap. 4. Bailey, N. T. J.: Statistical methods in biology, London, 1959, English Universities Press, p. 51. Seeliger, II. P. R.: Advances in the serology of fungi, Trans. Br. Mycol. Sot. 43: 543, 1960. Gilman, J. C.: A manual of soil fungi, ed. 2, Ames, 1957, Iowa State University Press, p. 347.
Jones, H. E., Rinaldi, M. G., Chai, H., and Kahn, G.: Apparent, cross-reactivity of airborne molds and the dermatophytic fungi,J. ALLERGY CLIN. IYMUNOL.~~: 346,1973. Bardana, E. J., Jr., McClatchy, J. K., Farr, R. S., and Minden, P.: Universal occurrence of antibodies to tubercle bacilli in sera from non-tuberculous and tuberculous individuals, Clin. Exp. Immunol. 13: 65, 1973.
Bardana, E. J., Jr.: Measurement of humoral antibodies to aspergilli, Ann. N. P. Bead. Sci. 221: 64, 1974. Bradshaw, W., Schneerson, R., Parke, J. C., Jr., and Robbins, J. R.: Bacterial antigens cross-reactive with the capsular polysaccharide of IlarmophiZus infiw~zne type b, Lancet 1: 1095, 1971. Goldschneider, I., Gotschlich, E. C., and Artenstein, M. S.: Humau immunity to the meningococcus. I. The role of humoral antibodies, J. Exp. Med. 129: 1307, 1969. Goldschneider, I., Gotschlich, E. C., and Artenstein, M. R.: Human immunity to the meningococcus. II. Development of natural immunity, J. Exp. Med. 129: 1327, 1969. Neter, E., Drislane, A. M., Harris, A. II., and Gorzynski, E. A.: Study on antibodies against enteric pathogens in human gamma globulin, Am. J. Public Health 49: 1050, 1959. Rowley, M. J.: “Natural” antibody in man to flagellar antigens of $nZmonelln adehide, Aust. J. Exp. Biol. Med. Sci. 48: 249, 1970. Julian, A. J., Logan, L. C., Norins, L. C., and Cohen, I. R.: Imnmnoglobulin class of natural human antibodies reactive with Treponema pccllidum, Infect. Immun. 1: 555, 1970. Farr, R. 8.: A quantitative immunochemical measure of the primary interaction between I*BSA and antibody, J. Infect. Dis 103: 239, 1958. Ishizakn, T., Ishizaka, K., Salmon, S., and Fudenberg, H.: Biologic activities of aggregated y-globulin. VIII. Aggregated immunoglobulins of different classes, J. lmmunol. 99: 82, 1967.
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Interaction between antibody and Alternaria. II 63
40 Steihm, E. R., and Fudenberg, II. H.: Serum levels of immune globulins in health and disease: A survey, Pndiatrics 37: 715, 1966.
41 Halvaggio, J. E., Cavanaugh, J. J. A., Lowell, F. C., and Leskowitz, S.: A comparison of the immunologic responses of normal and atopic individuals to intmnasally administered antigen, J. ALLERGY 35: 62, 1964.
42 Orie, N. G. M., de Vries, G. A., and Kikstra, A.: Growth of Aspergillus in the human lung, Am. Rev. Respir. Dis. 82: 649, 1960.
43 Bodey, G. P. : Fungal infections complicating acute leukemia, J. Chron. Uis. 19: 667, lS66. 44 Young, R.. C., Bennett, J. E., Vogel, C. L., Carbone, P. I’., and BeVita, V. T.: Aspergillosis.
The spectrum of the disease in 98 patients, Medicine (Halt.) 49: 147, 1970. 45 Hayward, B. J., Augustin, R., and Longbottom, 5. I,. : Separation and c,ll:lrac~tc,riz:ltiorl
of antibodies to moulds in human sera. Acta Allergol. (Wm.) 15(Supp. 7): 87, IStiO. 46 Cooke, R. A., Barnard, J. II., Hebald, S., and Btull, A.: Herological evidence of immunity
with coexisting sensitization in a type of human allergy (hay fever), J. Exp. Med. 62: 733, 1935.
47 Loveless, M. II.: Tmmunologic studies of pollinosis. VI. Short,ening the treatment of hay fever, J. ALLERGY 15: 311, 1944.
48 Lichtenstein, 1,. M., and Osler, A. G.: Studies on the mechanisms of hypersensitivity phenomena. X11. An in vitro study of the reaction between ragwrcd pollen antigen, allergic human serum and ragweed-sensitive human leukocyt,es, J. Immunol. 96: 169, 1966.
49 May, C. I)., Lyman, M. S., Alberto, R., and Aduna, S.: On the measurement of blocking antibody, J. ALLERGY CLIN. IMMIJNOL. 49: 54, 1972.
56 Schumacher, M. J., and May, C. B.: Effects of injections of allergen extracts on antigenic release of histamine from leucocytes, skin tests and bronchial challenge in allergic children, Clin. Allergy 2: 345, 1972’.
