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784 Determination of total and specific IgE using the CAP-system (CAP)
VOLUME NUMBER
781
87 1. PART 2
Abstracts
IATION OF ELEVATED CORD BLOOD IGE WIT& RED ST-S. Robert F WainsMln. M.D., A.Lo&&&&DannisDwnbv.t&Q&jgdDavidR. pL9nnr.Ph0, sarhna&Mud[kboiLMicMpan.
SOX!SlWJk&MWBllWC-,
783 MEASUREMENT OF SPECIFIC IGE TO FIVE ALLERGENS FROM FINGER-STICK VOLUMES OF BLOOD. v Gwnby. MD, Detroit,
UOktlOWIltOEXWt
SUPPRESSIONOF I@ ANTIBODIES TO B-LACTOGLOBIJLIN BY THE ADMINISTRATION OF TOLRRDGENS R. Fritschk and Y. Bore&, Lausanne, Switzerland Conjugates made of isologous rat IgG coupled to fi-lactoglobulin (6LG) were constructed as previously described (.J.Immunol. methods, 126. 159-168, 1990). We determined whether we could suppress IgE to ,9LG (a major milk allergen) with these conjugates in the rat model. Sprague-Dawley rats were injected intravenously with 2 mg of either chromatographically purified tolerogen fractions or, as a control, with a mixture of unconjugated @G and rat IgG. Four days later, all rats were injected subcutaneously with 100 pg @LG in Al(OH),. Fourteen days later, rats were bled by cardiac puncture and the sera analyzed by ELISA for the levels of IgE and IgG anti-BLG antibodies. We found that the IgE response in the group of rats injected with tolerogen fraction 2 (insize) was depressed more than termediary 100 fold if compared to the other groups, injected with either tolerogen fraction 1 (large the control mixture (,$LG + IgG unconjusize), gated) or the group of rats that hasn't received any intravenous pretreatment. The data suggest that tolerogen administration can prevent or suppress appearance of IgE antibody to a potent cow's milk allergen.
Michigan
Researchon allergic diseasein infants often requires obtaining allergen-specificIgE data from micro-volumes of serum. We investigated two modifications of our ELISA to minim& the serumrequired for five allergen tests (cat, mite, ragweed,timothy grass,and dog). The first modification was reducing the serum volume from 50 ul to 25 ul/well. The second modification was to se uentially transfer serum from one allergen-coated we41 to another (eg from cat to mite to ragweed,etc). To evaluatethe first modification, 33 serawere tested in triplicate using 50 ul of serum/well or 25 ul of serum plus 75 ul of buffer/well for the five aller ens. A negativecontrol serumpool wastreated similarfy. When the resultsof the 33 serawere comparedby paired t-test, there was no significant difference (~~0.23) with cat, ragweedand timothy, 25 ul > 50 ul @=O.Ol) with dog and 50 ul > 25 ul (p=O.Ol) with mite. The mean coefficientsof variation (CVs) within replicateswere < 10% for all allergens. To evaluatethe secondmodification, the same33 sera were tested at the same two volumes with (w/) and without (w/o) transferring the sera from one set of allergen-coatedwells to another. The mean correlation between the results w/ and w/o transfer for four allergenswas 0.98. The meanCVs w/ and w/o transfer were all < 10%. We conclude that these two ELISA modifications combinedcan reduce the volume of serum re uired to measureallergen-specificIgE to five allergenssrom 750 ul to 75 ul (a 90% reduction) while maintaining accuracy.
profound affects on ths inwna system40cord blood samples omtalnlng lgEa.5 U/ml wara assayad for tsatcatarorm (T), wtradlol (E2), DHEAS (0). and 17XIH progastero~ (PI. Inaccordanwwlthpmviousstudles,msanTin2Omab cord blood samplas was higher than in 20 fwnala samples @a5); Pwas somewhat but not statistkalty, higher in mabs; and no sax difference was found in D or E2. T (p<.W), E2 (v.01). ratio of T/P (p401), and ratio of E2/P @402) wars slevatad in ths 10% of cord blood samples demon&a tlng bwedsof IGE exceeding 3.0 U/ml, compared to the rest In females, E2 (p<.oOl) and E2/P (p~.OOOl~wars incrsasad in thesa samplea, but not T or T/P. In males, T (~~02). T/P (p402). D (p<.O2), and DIP (p<.oo5) were lnerwwd In thsaa samplas. but not E2 or E2/P. Fbsuhs are eorsbtenl with preferantial enhancement of ths AMS-hydroxysteroid vs. A4Skatons pathway of steroidogsnasia in ths fetal adrenal. This finding: 1) suggests a relationship between atopic immune dsvelopmsnt and altered steroldogenasls and, 2) stranghthens the hypothesis of a common hormonal influancs on both nervous and lmmuns system development. resulting in neuroautonomic imbalance, shifted carebral domlnanoe, and IgE dysragulatbn of allergic disease.
782
335
784
DETERMINATION OF TOTAL AND SPECIFIC IGE USING THE CAP-SYSTEH (CAP). D.Abeck*, M.D. L
T-Schafer*, H.D., B.PrzVbilla*. M.D., J.Ring**, M.D., Ph.D., Hunich*,Hamburg**,Germany The determination allergen-specific
of ME
total IgE (tIgE1 and (sIgE1 is a well
established diagnostic method. In the automated CAP, allergens or anti-IgE, respectively, are
coupled to a novel solid phase (capsulated hydrophilic carrier polymer) which facilitates binding of antibodies. He assessed serum samples from 105 allergic individuals for sIgE (5 aeroallergens; 2 food allergens; hymenoptera venoms,HV) and from 268 individuals for tIgE using the CAP in comparison to the conventional radio-allergosorbent-test (RAST;for sIgE1 and an enzyme-iasuno-assay (EIA;for t IgE) . Determinations were done fivefold to calculated the intraand inter-assay variation coefficients (VCl . The results obtained for sIgE and tIgE correlated well (r=0.91/0.961, the CAP yielding mostly higher values. The mean intra-assay VC for the CAP was lower when compared to the RAST (7.57/12.38;p=O.O015) and higher compared to when the EIA (6.03/5.47;n.s.). Regarding sIgE there were 11 discordant positive results (CAP 9, RAST 21, among these some possibly falsely positive CAP results for EV. Thus, with regard to sensitivity, determination of IgE using the CAP mostly yields higher values, reproducibility is improved concerning sIgE. Additionally, there were technical advantages of this automated procedure.
781
87 1. PART 2
Abstracts
IATION OF ELEVATED CORD BLOOD IGE WIT& RED ST-S. Robert F WainsMln. M.D., A.Lo&&&&DannisDwnbv.t&Q&jgdDavidR. pL9nnr.Ph0, sarhna&Mud[kboiLMicMpan.
SOX!SlWJk&MWBllWC-,
783 MEASUREMENT OF SPECIFIC IGE TO FIVE ALLERGENS FROM FINGER-STICK VOLUMES OF BLOOD. v Gwnby. MD, Detroit,
UOktlOWIltOEXWt
SUPPRESSIONOF I@ ANTIBODIES TO B-LACTOGLOBIJLIN BY THE ADMINISTRATION OF TOLRRDGENS R. Fritschk and Y. Bore&, Lausanne, Switzerland Conjugates made of isologous rat IgG coupled to fi-lactoglobulin (6LG) were constructed as previously described (.J.Immunol. methods, 126. 159-168, 1990). We determined whether we could suppress IgE to ,9LG (a major milk allergen) with these conjugates in the rat model. Sprague-Dawley rats were injected intravenously with 2 mg of either chromatographically purified tolerogen fractions or, as a control, with a mixture of unconjugated @G and rat IgG. Four days later, all rats were injected subcutaneously with 100 pg @LG in Al(OH),. Fourteen days later, rats were bled by cardiac puncture and the sera analyzed by ELISA for the levels of IgE and IgG anti-BLG antibodies. We found that the IgE response in the group of rats injected with tolerogen fraction 2 (insize) was depressed more than termediary 100 fold if compared to the other groups, injected with either tolerogen fraction 1 (large the control mixture (,$LG + IgG unconjusize), gated) or the group of rats that hasn't received any intravenous pretreatment. The data suggest that tolerogen administration can prevent or suppress appearance of IgE antibody to a potent cow's milk allergen.
Michigan
Researchon allergic diseasein infants often requires obtaining allergen-specificIgE data from micro-volumes of serum. We investigated two modifications of our ELISA to minim& the serumrequired for five allergen tests (cat, mite, ragweed,timothy grass,and dog). The first modification was reducing the serum volume from 50 ul to 25 ul/well. The second modification was to se uentially transfer serum from one allergen-coated we41 to another (eg from cat to mite to ragweed,etc). To evaluatethe first modification, 33 serawere tested in triplicate using 50 ul of serum/well or 25 ul of serum plus 75 ul of buffer/well for the five aller ens. A negativecontrol serumpool wastreated similarfy. When the resultsof the 33 serawere comparedby paired t-test, there was no significant difference (~~0.23) with cat, ragweedand timothy, 25 ul > 50 ul @=O.Ol) with dog and 50 ul > 25 ul (p=O.Ol) with mite. The mean coefficientsof variation (CVs) within replicateswere < 10% for all allergens. To evaluatethe secondmodification, the same33 sera were tested at the same two volumes with (w/) and without (w/o) transferring the sera from one set of allergen-coatedwells to another. The mean correlation between the results w/ and w/o transfer for four allergenswas 0.98. The meanCVs w/ and w/o transfer were all < 10%. We conclude that these two ELISA modifications combinedcan reduce the volume of serum re uired to measureallergen-specificIgE to five allergenssrom 750 ul to 75 ul (a 90% reduction) while maintaining accuracy.
profound affects on ths inwna system40cord blood samples omtalnlng lgEa.5 U/ml wara assayad for tsatcatarorm (T), wtradlol (E2), DHEAS (0). and 17XIH progastero~ (PI. Inaccordanwwlthpmviousstudles,msanTin2Omab cord blood samplas was higher than in 20 fwnala samples @a5); Pwas somewhat but not statistkalty, higher in mabs; and no sax difference was found in D or E2. T (p<.W), E2 (v.01). ratio of T/P (p401), and ratio of E2/P @402) wars slevatad in ths 10% of cord blood samples demon&a tlng bwedsof IGE exceeding 3.0 U/ml, compared to the rest In females, E2 (p<.oOl) and E2/P (p~.OOOl~wars incrsasad in thesa samplea, but not T or T/P. In males, T (~~02). T/P (p402). D (p<.O2), and DIP (p<.oo5) were lnerwwd In thsaa samplas. but not E2 or E2/P. Fbsuhs are eorsbtenl with preferantial enhancement of ths AMS-hydroxysteroid vs. A4Skatons pathway of steroidogsnasia in ths fetal adrenal. This finding: 1) suggests a relationship between atopic immune dsvelopmsnt and altered steroldogenasls and, 2) stranghthens the hypothesis of a common hormonal influancs on both nervous and lmmuns system development. resulting in neuroautonomic imbalance, shifted carebral domlnanoe, and IgE dysragulatbn of allergic disease.
782
335
784
DETERMINATION OF TOTAL AND SPECIFIC IGE USING THE CAP-SYSTEH (CAP). D.Abeck*, M.D. L
T-Schafer*, H.D., B.PrzVbilla*. M.D., J.Ring**, M.D., Ph.D., Hunich*,Hamburg**,Germany The determination allergen-specific
of ME
total IgE (tIgE1 and (sIgE1 is a well
established diagnostic method. In the automated CAP, allergens or anti-IgE, respectively, are
coupled to a novel solid phase (capsulated hydrophilic carrier polymer) which facilitates binding of antibodies. He assessed serum samples from 105 allergic individuals for sIgE (5 aeroallergens; 2 food allergens; hymenoptera venoms,HV) and from 268 individuals for tIgE using the CAP in comparison to the conventional radio-allergosorbent-test (RAST;for sIgE1 and an enzyme-iasuno-assay (EIA;for t IgE) . Determinations were done fivefold to calculated the intraand inter-assay variation coefficients (VCl . The results obtained for sIgE and tIgE correlated well (r=0.91/0.961, the CAP yielding mostly higher values. The mean intra-assay VC for the CAP was lower when compared to the RAST (7.57/12.38;p=O.O015) and higher compared to when the EIA (6.03/5.47;n.s.). Regarding sIgE there were 11 discordant positive results (CAP 9, RAST 21, among these some possibly falsely positive CAP results for EV. Thus, with regard to sensitivity, determination of IgE using the CAP mostly yields higher values, reproducibility is improved concerning sIgE. Additionally, there were technical advantages of this automated procedure.