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IgE immune complexes
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 1, PART3
409
New Methodologies to Analyze IgG Subclass Distribution in Specific IgG Response and in lgG/lgE Immune Complexes. s Allauzen PhD. A Rosenber~ PhD. C Larue PhD. F
Abstracts
411
of Specific IgG Response in Response in Patients with Allergic
N e w M e t h o d o l o g y to M e a s u r e A v e r a g e A f f i n i t y
Disease. L Pierson MS. S Allauzen PhD. B Kravis BS. L Daniels MA. A Rosenbera PhD. Minneapolis, MN. The expression of average affinity of polyclonal antibody response in terms of free antigen concentration at 50% determinant saturation can be highly misleading if the affinity distribution is skewed. A more rewarding approach is to describe the affinities as the affinity probability distribution function. Binding isotherma contain information for numerical calculation of such functions (Hunston Anal Biochem 63, 99; 1975). For such calculations, however, we need binding data over at least four magnitudes ofligand concentration. We have developed a two-step multiple sandwich immunoassay in which the IgG from sera were captured by monoclonal antibodies directed against each IgG subclass. The binding of specific IgG to purified haptenated proteins in solution, such as Arab a I and Der p I, was detected using highly sensitive chemiluminescent technology. We compared the affinity distribution of Amb a I-specific IgG4 in allergen sensitive individuals who have been on and are undergoing immunotherapy. We found that while the antibody level rose 10-fold with immunotherapy, the affinity distribution function remained unchanged with an average affinity around 1.2 l0 g M". Thase data indicate that immunotherapy does not necessarily lead to the production of high-affinity antibodies but rather leads to an increase in the production of antibodies of selected specificity.
Total Serum IgD in Atopic Infants and Children. Y E1 Gamal MD, Z A w a d M D , M Zaghloul MD. A Fo3~act MS, Cairo, Egypt. A study of total serum IgD (by radial immunodiffusion), lgE (by ELISA) and absolute eosinophilic count (AEC) was conducted on 24 atopic infants and children: 15 with bronchial asthma and 9 with atopic dermatitis as well as 10 normal non atopic children as a control group. Measurement of peak expiratory flow rate (PEFR) was also done for asthmatics. A significantly higher serum IgD was found in asthmatic children both during free and wheezy states (mean 78.84 +83.9 and 52.17 +76.2 IU/ml respectively) and in infants and children with atopic dermatitis (mean 46.33 +78.9 IU/ml) as compared to the control group (mean 17.14 +15.8 IU/ml). Levels of serum lgD varied significantly with the degree of severity of asthma; values being highest in severe asthma (194.5 _+39.7 IU/ml) and lowest in mild asthma (17.6 _+13 IU/ml). No significant differences in serum IgD were observed between asthma and atopic dermatitis. Also, there were no significant differences between the levels in children with one versus those who co-expressedmore than one atopic condition. Serum IgD did not correlate with other laboratory parameters of atopy namely serum lgE and AEC. In conclusion, serum IgD is elevated in atopic infants and children and its levels can be a helpful marker of the severity of the condition, however, its exact role in the pathogenesis of atopy awaits further studies.
Bloeki PhD and MN Blumenthal MD. Minneapolis, MN. Two new immunoassays have been developed that detect either antiallergen specific lgG or IgG/IgE immune complexes. Both assays use paramagnetic particle solid phase coupled to monoclonal antibodies which are of selected specificity against each human lgG subclass. Capture of the individual IgG subclasses from the serum was used to try to increase the specificity of this test, as compared to currently used methodologies (ELISA, RAST, RIA) in which subclasses are detected after the binding of total lgs to the allergen. For determining allergen-specific IgG, patient samples, coupled paramagnetic particles, and haptenated allergens were incubated for 30 rain. The paramagnetic particles were then washed and an anti-hapten alkaline phosphatase (ALP) conjugate was added and incubated for 30 min. Bound allergen-specific lgG was measured utilizing a highly sensitive chemiluminescent detection. A second assay for the detection if lgG/IgE immune complexes used a modified protocol without allergen in which the anti-hapten ALP conjugate was replaced by an anti-IgE antibody ALP conjugate. IgG subclass distribution was analyzed in the sera from atopic patients (n=60), in patients receiving immunotherapy (n=10) and in healthy subjects (n=26). This study revealed that the lgG anti allergen activity was associated with lgG2 and IgG4 in atopic patients as well as in patients having received immunotherapy. On the other hand, circulating complexes of IgG antiIgE/IgE were restricted to IgG 1 and lgG4 isotypes and found only in the allergic population. These new assays should provide an excellent tool for unravelling relationships between immunoglobulin gene expression and specific globulin-mediated disorders such as allergy diseases.
410
285
412
THE EFFECTS OF INHALED PROSTAGLANDIN E 2 ON ALLERGEN-INDUCED AIRWAY RESPONSES GM Gauvreau. RM Watson. and PM O'Bvme. Asthma Research Group, McMaster University, Hamilton, Ontario, CANADA. Prostaglandin E2 (PGEz) inhaled immediately before allergen inhalation has been shown to be protective against allergen-induced asthmatic responses. We examined the effects of inhaled PGE2 on the early and late responses to allergen and methachoiine airway responsiveness in a double blind randomized crossover study. Eight non-smoking, atopic, mild asthmatics with documented dual asthmatic responses after inhaled allergen completed the study. Subject symptoms were controlled with [32agonist only, which was withheld for at least 8 hours before each visit. PGE; (100}ig) or placebo was inhaled for 8 minutes, followed by 2 doses of allergen within the next 20 minutes. FEVt was measured over the following 7 hours. Methacholine airway responsiveness, expressed as the provocation concentration of methacholine causing a 20% fall in FEV~ (PC20) was determined the day before and 24 hours after allergen. The area under the curve for the late response was significantly greater with PGF-,2inhalation than with placebo (p=0.03), as was the maximal drop in FEV~ (PGF.2 -28.1% (SEM 2.9%); placebo -20.7% (SEM 3.9%), p=0.0 l). Inhalation of PGE2 did not alter the maximal early response to allergen (PGE2 -26.8% (SEM 3.4%); placebo -26.9% (SEM 4.1%), p=0.98), nor the area under the curve of the early response (p=0.34). Also, there was no significant difference in the magnitude of allergen-induced airway responsiveness (p=0.22). These results indicate that inhalation of PGE2 does not have a protective effect against allergen-induced responses when allergen is inhaled within 20 minutes after PG~, and, in fact, worsens the late response. The therapeutic window for PGE2 on allergen-induced airway responses may be less than 20 minutes.
409
New Methodologies to Analyze IgG Subclass Distribution in Specific IgG Response and in lgG/lgE Immune Complexes. s Allauzen PhD. A Rosenber~ PhD. C Larue PhD. F
Abstracts
411
of Specific IgG Response in Response in Patients with Allergic
N e w M e t h o d o l o g y to M e a s u r e A v e r a g e A f f i n i t y
Disease. L Pierson MS. S Allauzen PhD. B Kravis BS. L Daniels MA. A Rosenbera PhD. Minneapolis, MN. The expression of average affinity of polyclonal antibody response in terms of free antigen concentration at 50% determinant saturation can be highly misleading if the affinity distribution is skewed. A more rewarding approach is to describe the affinities as the affinity probability distribution function. Binding isotherma contain information for numerical calculation of such functions (Hunston Anal Biochem 63, 99; 1975). For such calculations, however, we need binding data over at least four magnitudes ofligand concentration. We have developed a two-step multiple sandwich immunoassay in which the IgG from sera were captured by monoclonal antibodies directed against each IgG subclass. The binding of specific IgG to purified haptenated proteins in solution, such as Arab a I and Der p I, was detected using highly sensitive chemiluminescent technology. We compared the affinity distribution of Amb a I-specific IgG4 in allergen sensitive individuals who have been on and are undergoing immunotherapy. We found that while the antibody level rose 10-fold with immunotherapy, the affinity distribution function remained unchanged with an average affinity around 1.2 l0 g M". Thase data indicate that immunotherapy does not necessarily lead to the production of high-affinity antibodies but rather leads to an increase in the production of antibodies of selected specificity.
Total Serum IgD in Atopic Infants and Children. Y E1 Gamal MD, Z A w a d M D , M Zaghloul MD. A Fo3~act MS, Cairo, Egypt. A study of total serum IgD (by radial immunodiffusion), lgE (by ELISA) and absolute eosinophilic count (AEC) was conducted on 24 atopic infants and children: 15 with bronchial asthma and 9 with atopic dermatitis as well as 10 normal non atopic children as a control group. Measurement of peak expiratory flow rate (PEFR) was also done for asthmatics. A significantly higher serum IgD was found in asthmatic children both during free and wheezy states (mean 78.84 +83.9 and 52.17 +76.2 IU/ml respectively) and in infants and children with atopic dermatitis (mean 46.33 +78.9 IU/ml) as compared to the control group (mean 17.14 +15.8 IU/ml). Levels of serum lgD varied significantly with the degree of severity of asthma; values being highest in severe asthma (194.5 _+39.7 IU/ml) and lowest in mild asthma (17.6 _+13 IU/ml). No significant differences in serum IgD were observed between asthma and atopic dermatitis. Also, there were no significant differences between the levels in children with one versus those who co-expressedmore than one atopic condition. Serum IgD did not correlate with other laboratory parameters of atopy namely serum lgE and AEC. In conclusion, serum IgD is elevated in atopic infants and children and its levels can be a helpful marker of the severity of the condition, however, its exact role in the pathogenesis of atopy awaits further studies.
Bloeki PhD and MN Blumenthal MD. Minneapolis, MN. Two new immunoassays have been developed that detect either antiallergen specific lgG or IgG/IgE immune complexes. Both assays use paramagnetic particle solid phase coupled to monoclonal antibodies which are of selected specificity against each human lgG subclass. Capture of the individual IgG subclasses from the serum was used to try to increase the specificity of this test, as compared to currently used methodologies (ELISA, RAST, RIA) in which subclasses are detected after the binding of total lgs to the allergen. For determining allergen-specific IgG, patient samples, coupled paramagnetic particles, and haptenated allergens were incubated for 30 rain. The paramagnetic particles were then washed and an anti-hapten alkaline phosphatase (ALP) conjugate was added and incubated for 30 min. Bound allergen-specific lgG was measured utilizing a highly sensitive chemiluminescent detection. A second assay for the detection if lgG/IgE immune complexes used a modified protocol without allergen in which the anti-hapten ALP conjugate was replaced by an anti-IgE antibody ALP conjugate. IgG subclass distribution was analyzed in the sera from atopic patients (n=60), in patients receiving immunotherapy (n=10) and in healthy subjects (n=26). This study revealed that the lgG anti allergen activity was associated with lgG2 and IgG4 in atopic patients as well as in patients having received immunotherapy. On the other hand, circulating complexes of IgG antiIgE/IgE were restricted to IgG 1 and lgG4 isotypes and found only in the allergic population. These new assays should provide an excellent tool for unravelling relationships between immunoglobulin gene expression and specific globulin-mediated disorders such as allergy diseases.
410
285
412
THE EFFECTS OF INHALED PROSTAGLANDIN E 2 ON ALLERGEN-INDUCED AIRWAY RESPONSES GM Gauvreau. RM Watson. and PM O'Bvme. Asthma Research Group, McMaster University, Hamilton, Ontario, CANADA. Prostaglandin E2 (PGEz) inhaled immediately before allergen inhalation has been shown to be protective against allergen-induced asthmatic responses. We examined the effects of inhaled PGE2 on the early and late responses to allergen and methachoiine airway responsiveness in a double blind randomized crossover study. Eight non-smoking, atopic, mild asthmatics with documented dual asthmatic responses after inhaled allergen completed the study. Subject symptoms were controlled with [32agonist only, which was withheld for at least 8 hours before each visit. PGE; (100}ig) or placebo was inhaled for 8 minutes, followed by 2 doses of allergen within the next 20 minutes. FEVt was measured over the following 7 hours. Methacholine airway responsiveness, expressed as the provocation concentration of methacholine causing a 20% fall in FEV~ (PC20) was determined the day before and 24 hours after allergen. The area under the curve for the late response was significantly greater with PGF-,2inhalation than with placebo (p=0.03), as was the maximal drop in FEV~ (PGF.2 -28.1% (SEM 2.9%); placebo -20.7% (SEM 3.9%), p=0.0 l). Inhalation of PGE2 did not alter the maximal early response to allergen (PGE2 -26.8% (SEM 3.4%); placebo -26.9% (SEM 4.1%), p=0.98), nor the area under the curve of the early response (p=0.34). Also, there was no significant difference in the magnitude of allergen-induced airway responsiveness (p=0.22). These results indicate that inhalation of PGE2 does not have a protective effect against allergen-induced responses when allergen is inhaled within 20 minutes after PG~, and, in fact, worsens the late response. The therapeutic window for PGE2 on allergen-induced airway responses may be less than 20 minutes.