Validation of a Multiplex Assay for Measuring Specific IgG4.

PURPOSE: Specific IgG4 (sIgG4) increases with allergen specific immunotherapy and may reflect a state of immune tolerance in food allergy. While ImmunoCAP® has been widely used to measure sIgG4 to a single allergen, PROTIA™ Specific IgG4® has been designed as a multiplex assay for measuring sIgG4. This study sought to validate this assay in comparison to ImmunoCAP®. MATERIALS AND METHODS: Measurements of sIgG4 were compared between PROTIA™ Specific IgG4® and ImmunoCAP® using sera from 519 allergy patients (asthma: 114, allergic rhinitis: 318, food allergy: 146) with 731 paired tests. sIgG4 was measured against nine inhalant allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, cat dander, dog dander, birch pollen, oak pollen, ragweed pollen, mugwort pollen, and Alternaria alternata spores) and nine food allergens (egg white, casein, wheat, peanut, walnut, crab, shrimp, apple, and peach). RESULTS: PROTIA™ Specific IgG4® showed 95.6% agreement rate with ImmunoCAP® in the positivity comparison. For sIgG4 positivity to each individual allergen, an agreement rate of more than 84.8% was observed. In Cohen's kappa analysis, these assays displayed substantial correlations [Cohen's kappa coefficient (κ) ≥0.699], except for shrimp (κ=0.448). Furthermore, both assays displayed strong correlations in quantitative comparisons [correlation coefficients value (ρ) ≥0.8014], except for apple (ρ=0.6571, p=0.175). Serial dilution tests also showed consistency between the assays. CONCLUSION: PROTIA™ Specific IgG4® showed high consistency with ImmunoCAP® in measuring sIgG4. This assay is applicable to various clinical fields, including allergen immunotherapy and food allergy.

Immunological analysis of allergenic cross-reactivity between Cheyletus malaccensis and Dermatophagoides farinae, Dermatophagoides pteronyssinus and Blomia tropicalis / Análise imunológica da reatividade cruzada alergênica entre Cheyletus malaccensis e Dermatophagoides farinae, Dermatophagoides pteronyssinus e Blomia tropicalis

Objective: The mite Cheyletus malaccensis is cited in the literature as a predator of other mite species. Little is known about its protein composition, and few studies have evaluated its ability to trigger atopic respiratory allergic reactions. The present study aims to investigate the protein profile fingerprint present in Cheyletus malaccensis extract and to evaluate its immunologic reactivity in the presence of specific immunoglobulins (IgE) from the serum of individuals diagnosed with allergy to the mites Dermatophagoides farinae, Dermatophagoides pteronyssinus and Blomia tropicalis. These three species carry proteins responsible for the most cases of atopic respiratory allergies, hence the interest in comparing them to Cheyletus malaccensis. Methods: Samples of aspirated dust containing Cheyletus malaccensis were collected from households in the city of Rio de Janeiro, Brazil. From the collected mass of this mite, extracts were prepared for analysis. Proteins present in the extracts were identified by electrophoresis under denaturing conditions. Results: Proteins with a molecular mass of 24 kDa, 26 kDa, 12 kDa, 45 kDa and 70 kDa were visualized. The immunoblotting assay showed positive cross-reactivity for proteins of molecular mass ranging from 20 kDa to 45 kDa. These results indicate that specific links were established between IgE present in the serum of individuals allergic to the comparator mite and proteins from Cheyletus malaccensis. Conclusions: These findings are relevant for their potential clinical and immunotherapeutic applications, as well as information base for further studies.

Objetivo: O ácaro Cheyletus malaccensis é referido na literatura como um predador de outras espécies de ácaro. Pouco se sabe sobre sua composição proteica, e poucos estudos avaliaram sua habilidade de desencadear reações alérgicas respiratórias atópicas. O objetivo do presente estudo é investigar a impressão digital do perfil proteico presente em um extrato de Cheyletus malaccensis e avaliar sua reatividade imunológica na presença de imunoglobulinas (IgE) específicas do soro de indivíduos diagnosticados com alergia aos ácaros Dermatophagoides farinae, Dermatophagoides pteronyssinus e Blomia tropicalis. Essas três espécies carregam proteínas responsáveis pela maioria dos casos de alergias respiratórias atópicas, o que justifica o interesse em compará-las ao Cheyletus malaccensis. Métodos: Amostras de poeira aspirada contendo Cheyletus malaccensis foram coletadas de domicílios na cidade do Rio de Janeiro, no Brasil. A partir da massa coletada desse ácaro, extratos foram preparados para análise. As proteínas presentes nos extratos foram identificadas por eletroforese sob condições desnaturantes. Resultados: Proteínas com massa molecular de 24 kDa, 26 kDa, 12 kDa, 45 kDa e 70 kDa foram visualizadas. O ensaio imunoenzimático mostrou reatividade cruzada positiva para proteínas de massa molecular variando de 20 kDa a 45 kDa. Esses resultados indicam que ligações específicas foram estabelecidas entre a IgE presente no soro de indivíduos alérgicos ao ácaro usado como comparador e proteínas de Cheyletus malaccensis. Conclusões: Os achados são relevantes por seu potencial clínico e aplicações imunoterapêuticas, bem como sua base de informações para futuros estudos.

Nonirritant intradermal skin test concentrations of ciprofloxacin, clarithromycin, and rifampicin.

BACKGROUND: Intradermal skin testing of the clinically important antibiotics ciprofloxacin, clarithromycin, and rifampicin in the case of suspected allergies to antibiotics is poorly standardized. For clinical practice, standardized procedures and protocols are desired. METHODS: Fifteen healthy volunteers were tested with different concentrations of the antibiotics as well as with appropriate controls. Test readings included wheal area measured by digital image analysis and blood flow increase measured by laser Doppler flowmetry (LDF). To reduce interpersonal variability, test results were normalized with the individual controls using a novel protocol. RESULTS: Nonirritating concentrations of the three antibiotics (ciprofloxacin ~0.0067 mg/ml, clarithromycin ~0.05 mg/ml, rifampicin ~0.002 mg/ml) could be defined for healthy volunteers. Laser Doppler flowmetry generates comparable results to wheal area measurement. Normalization of the test results is necessary and can be applied in a practical algorithm. CONCLUSIONS: Standardized skin testing to detect sensitization to broadly used nonbetalactam antibiotics was presented and should be applied in truly sensitized patients. This approach should help to minimize the inter- and intraindividual differences in reactivity.

Comparison of two methods for the detection of oligoclonal bands in a large number of clinically isolated syndrome and multiple sclerosis patients.

OBJECTIVE: A novel oligoclonal band (OB) assay which consists of isoelectric focusing (IEF) and IgG immunodetection by alkaline phosphatase-labeled anti IgG antibody was reported to be very sensitive. It also accurately predicted conversion to MS in patients with CIS. The aim of our study was to compare sensitivity of a novel and the standard procedure with peroxidase immunodetection in a large number of CIS and MS patients. METHODS: OB were determined in serum and CSF samples in 161 patients (104 females), 47 with CIS and 114 with MS with median age 38 years (range 19-68) using both methods. RESULTS: Eighty-three percent of patients had CSF OB with the standard and 89% with the novel method. Median number of OB was 5 (range 0-17) with the peroxidase and 8 (range 0-18) with the alkaline phosphatase method; p = 0.001. Twenty-one percent of patients had ≥ 10 OB with the standard and 37% with the novel method of the detection; p = 0.021. Subjective impression of band clarity showed that 20% of patients had sharper and stronger bands when the peroxidase and 65% when the alkaline phosphatase method was used; p<0.0001. CONCLUSION: The alkaline phosphatase method is more sensitive than the peroxidase method and at the same time cheaper, easy to perform and less time consuming.

A comparison of techniques for evaluating IgE-mediated allergies.

Skin prick testing remains the most popular way to confirm an IgE-mediated allergic response. This study was performed to compare the accuracy and sensitivity of two skin testing modalities (serial endpoint titration and skin prick testing with 2 different in vitro assays). In 52 atopic patients serial endpoint titration showed a higher degree of sensitivity in evaluating skin response in less sensitive reactors. At higher degrees of reactivity in individual patients there was good correlation between the 3 modalities. Identifying patients who are less sensitive reactors is important, so that they can be started on immunotherapy. In fact, an informal survey of low-reacting patients treated with immunotherapy showed a high degree of success.

The patient with allergies.

Allergic disease affects a sizeable percentage of the general population, has a significant impact on patient quality of life, and exerts a significant financial burden on society. Atopic symptoms from inhalant allergens are among the most frequent complaints in outpatient medical visits. Key history and physical examination findings help to distinguish allergic rhinitis from other forms of chronic rhinosinusitis. Diagnostic testing may not be necessary unless immunotherapy is contemplated.

Childhood food allergy: a Singaporean perspective.

Food allergy is defined as reaction to a food which has an immunologic mechanism. Its prevalence is increasing in children globally and is therefore of increasing clinical importance. A useful clinical approach is to distinguish food allergic reactions by the timing of clinical reaction in relation to food exposure and classified as immediate (generally IgE-mediated) and delayed (generally non-IgE-mediated), with the exception of eczema and eosinophilic gastrointestinal disease, which, when associated with food allergy may be associated with either mechanism. This review is aimed at providing the clinician with a Singaporean perspective on the clinical approach and management of these disorders.

Oral desensitization to milk: how to choose the starting dose!

A renewed interest in oral desensitization as treatment for food allergy has been observed in the last few years. We studied a novel method based on the end point skin prick test procedure to establish the starting dose for oral desensitization in a group of 30 children highly allergic to milk. The results (in terms of reactions to the first dose administered) were compared with a group of 20 children allergic to milk as well. Such control group started to swallow the same dose of 0.015 mg/ml of milk. None reacted to the first dose when administered according to the end point skin prick test. On the other side, ten out of 20 children (50%) from the control group showed mild allergic reactions to the first dose of milk. In conclusion the end point skin prick test procedure results safe and easy to be performed in each single child in order to find out the starting dose for oral desensitization to milk, also by taking into account the individual variability.

Las pruebas cutáneas con bencilpenicilina siguen siendo importantes en el diagnóstico de las reacciones inmediatas de hipersensibilidad a las penicilinas / Benzylpenicillin skin testing is still important in diagnosing immediate hypersensitivity reactions to penicillins

Antecedentes. El hecho de que tanto Hollister-Stier como Allergopharma haya cesado la producción de penicilil-polilisina (PPL) y la mezcla de determinantes menores (MDM) en 2004, es un obstáculo importante en el diagnóstico de la hipersensibilidad a los ß-lactámicos y puede producir consecuencias negativas. Objetivo. Evaluar la contribución de las pruebas cutáneas con bencilpenicilina para el diagnóstico de la hipersensibilidad a penicilina mediada por IgE, con el fin de determinar cuánto podría compensar esta prueba cutánea la falta de disponibilidad de PPL y MDM. Métodos. Se seleccionaron pacientes con antecedentes de reacciones inmediatas a penicilina y test cutáneo para uno o más reactivos de penicilina (PPL, MDM o bencilpenicilina), una o más penicilinas semisintéticas (ampicilina, amoxicilina o piperacilina) o ambos. Resultados. Se seleccionaron en total 300 pacientes, 105 en el centro de Francia y 195 en los centros italianos. Las principales drogas responsables fueron la amoxicilina y ampicilina. La manifestación clínica más común fue la anafilaxia. Las pruebas cutáneas más frecuentemente positivas fueron con amoxicilina (188; 62,7%), ampicilina (151; 50,3%) y bencilpenicilina (111; 37,0%). Entre los 300 sujetos, 113 (37,7%) fueron positivos sólo para las penicilinas semisintéticas, 109 (36,3%) a ambas penicilinas semi-sintéticas y el clásico reactivo penicilina y 78 (26,0%) sólo a este último. En el último grupo, 64 (21,3% de los 300 sujetos) fueron positivos sólo a PPL y/o MDM y 14 (4,7%) a bencilpenicilina, de los cuales 8 (2,7%) fueron positivos sólo a este último Conclusiones. Las pruebas cutáneas con bencilpenicilina pueden compensar en parte la falta de PPL y de MDM. Por otra parte, puede aumentar ligeramente la sensibilidad del estudio diagnóstico alergológico y, por lo tanto, reducir el número de desafíos potencialmente peligrosos(AU)

Diagnosis of leptospirosis by recombinant antigen based single serum dilution ELISA.

BACKGROUND & OBJECTIVES: Leptospirosis, a zoonosis with a worldwide distribution is an acute febrile illness caused by spirochaetes of the pathogenic Leptospira interrogans. Microscopic agglutination test (MAT), the reference method for diagnosis was successively done to evaluate the modified ELISA which was developed with the recombinant LipL32 antigen for the detection of anti-leptospiral antibodies in human serum samples. METHODS: The recombinant LipL32 antigen was developed from the serovar Pomona strain Pomona of the pathogenic L. interrogans species. The predicted titre at a single working dilution was plotted against the observed antiserum titre. Subsequently, predicted antibody activity titres were determined directly from the standard curve by solving the regression line equation. The relative sensitivity, specificity and accuracy of the single dilution ELISA for the detection of anti-leptospiral antibodies were determined in comparison to the MAT. RESULTS: A linear relationship was found between the predicted antibody titres at a single working dilution of 1:250 and the corresponding observed serum titres by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. A high level of sensitivity of 96 per cent and specificity of 91 per cent between ELISA and MAT titres was found. The kappa value was almost 1.0 indicating perfect agreement. INTERPRETATION & CONCLUSIONS: The r LipL32 ELISA was proved to be sensitive, specific and accurate as compared to the standard MAT and the test could be efficiently utilized as a screening test for a large number of human serum samples for the detection of leptospiral antibodies.

Oral desensitization in children with immunoglobulin E-mediated cow's milk allergy--follow-up at 4 yr and 8 months.

Until now, the basic treatment for food allergy has been to avoid the offending item. This approach is difficult in the case of common foods and in the case where there is a risk of severe reaction after consuming the offending food, even inadvertently. This is the follow-up of a previous study aimed at desensitizing 21 children with immunoglobulin E (IgE)-mediated cow's milk (CM) allergy. This protocol was totally or partially successful in 85% of cases, but failed in the remaining 15%. Our aims were to study the long-term effectiveness and safety of oral CM desensitization, and the prognostic value of Skin Prick Test (SPT) and specific serum CM IgE. The 21 children were called back (one dropped out). The allergic history and other information on CM intake over the last 4-5 yr were recorded. Children underwent SPT, and end-point SPT, with casein and alpha-lactoalbumin. Specific CM IgE was also measured. At follow-up, 14/20 children totally (n = 13, 65%) or partially (n = 1, 5%) tolerated CM. None of the recalled children reported use of emergency care. SPT positivity to casein and/or alpha-lactoalbumin decreased significantly (p < 0.01), and all the negative SPT referred to the tolerant children. Cutaneous sensitivity to both casein and alpha-lactoalbumin (end-point SPT) significantly decreased after the 6-month desensitization period of the previous study (p < 0.001), but did not decrease significantly at follow-up. A significant reduction of serum-specific CM IgE was also observed (p < 0.05). Clinical tolerance induced by oral CM desensitization persists in time. Negativization of SPT and reduction of specific CM IgE could be considered prognostic indicators of CM tolerance. Oral CM desensitization seems to be a promising method to treat CM food allergy. This protocol is time-consuming but offers the advantage that it can be performed at home. This methodology must only be used by trained staff.

Comparison of intradermal dilutional testing, skin prick testing, and modified quantitative testing for common allergens.

OBJECTIVES: To compare and correlate wheal size using the Multi-Test II applicator with the endpoint obtained by intradermal dilutional testing (IDT) for 5 common allergens. To examine the safety of modified quantitative testing (MQT) for determining immunotherapy starting doses. STUDY DESIGN: Prospective comparative clinical study. SUBJECTS AND METHODS: A total of 134 subjects were simultaneously skin tested for immediate hypersensitivity using the Multi-Test II device and IDT. RESULTS: There was a 77% concordance between results from IDT and results from MQT. When there was a difference, MQT predicted a safer endpoint for starting immunotherapy in all but 2 cases. CONCLUSION: Wheal size by SPT is predictive of endpoint by IDT. MQT is nearly as effective as formal IDT in determining endpoint. SIGNIFICANCE: Modified quantitative testing appears to be a safe alternative to IDT for determining starting doses for immunotherapy.

Use of intradermal dilutional testing and skin prick testing: clinical relevance and cost efficiency.

OBJECTIVES/HYPOTHESIS: The objective was to determine the agreement of the positive results from a multiple skin prick test (SPT) device with the ability to determine a definable endpoint through intradermal dilutional testing (IDT) to compare semiquantitatively the degree of positivity of SPT results with quantitative results from IDT and to analyze the cost of immunotherapy based on SPT compared with IDT guided by SPT. STUDY DESIGN: Retrospective review of clinical data (random accrual). METHODS: One hundred thirty-four patients underwent allergy screening using a multiple SPT device. Antigens testing positive by skin prick device were tested using IDT on a separate day. Antigens testing negative by SPT were not evaluated by IDT. Regional allergy testing practice patterns were determined, and a cost analysis using Medicare rates was performed RESULTS: There was good agreement between an antigen testing positive by SPT and the determination of a definable endpoint (93.33%, n = 1,334 antigens). The degree of positivity from the SPT correlated poorly with the final endpoint concentration (r = 0.40, P < .0001). Blended testing techniques were similar in cost when compared with several commonly used allergy testing protocols. CONCLUSIONS: Antigens which show reactivity to a multiple SPT device usually have a treatable endpoint that is independent of the degree of positivity of the SPT result. IDT is an important step in the determination of the strongest starting dose of immunotherapy that may be safely administered. Initiating immunotherapy in this manner may potentially create significant health care savings by shortening the time required for a patient to reach their individual maximally tolerated dose. The use of a relatively large screening panel is cost effective and does not increase the average number of antigens treated by immunotherapy. Blended allergy testing techniques that include IDT in their protocol are comparable in cost with commonly used allergy testing protocols.

High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1.

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.

Comparison of intradermal dilutional testing with the Multi-Test II applicator in testing for mold allergy.

OBJECTIVES: To compare and correlate wheal size using the Multi-Test II applicator with the endpoint obtained by intradermal dilutional testing (IDT) for common mold allergens. To validate the safety and efficacy of modified quantitative testing (MQT) for determining immunotherapy starting doses. STUDY DESIGN AND SETTING: Prospective study of 86 subjects with Multi-Test II and IDT for 6 common mold antigens. RESULTS: There was 84% concordance between IDT results and the results expected from the MQT method. When IDT and MQT results differed, the MQT algorithm predicted a safer endpoint for starting immunotherapy in all but 2 cases. CONCLUSION: The correlation between Multi-Test II and IDT is not strong enough to infer IDT endpoint from Multi-Test II results for molds. MQT is nearly as effective as formal IDT in determining endpoint. SIGNIFICANCE: MQT appears to be a safe method for determining starting doses for immunotherapy with fungal allergens.

Prueba cutánea de la tuberculina PPD / PPD tuberculin skin test

La Tuberculosis sigue siendo enfermedad prevalente en el mundo y más aún en los países en desarrollo, ya que el 95% de los casos de tuberculosis están en éstos países. El PPD se ha convertido en una prueba mundialmente utilizada por los médicos para determinar la infección por tuberculosis. Presentamos una propuesta de estudio a una persona en contacto con paciente con Tuberculosis y la interpretación de los resultados de la prueba PPD.

Tuberculosis remains a prevalent disease in the world and even more so in developing countries, since 95% of tuberculosis cases are in these countries. PPD has become a worldwide test used by physicians to determine tuberculosis infection. We present a study proposal to a person in contact with a patient with Tuberculosis and the interpretation of the results of the PPD test.

Comparison of Multi-Test II skin prick testing to intradermal dilutional testing.

OBJECTIVES/HYPOTHESIS: Intradermal dilutional testing (IDT) has been considered more sensitive than prick testing for detecting low-level allergies. Multi-Test II is one of the most sensitive and reproducible prick testing methods available. This study compares Multi-Test II prick testing with IDT. DESIGN: Retrospective chart review and data analysis on patients who had allergy testing with both Multi-Test II and IDT. SETTING: Outpatient allergy clinic in the otolaryngology department of an academic tertiary care medical center. POPULATION: Forty adult patients had complete allergy testing with both Multi-Test II and IDT for a standard panel of twelve antigens between January 2002 and April 2002. INTERVENTION: Analysis and comparison of test results on Multi-Test II and IDT for each antigen separately and across all antigen tests together. RESULTS: Forty patients had complete testing for all antigens during the time period for the study. Six of these patients did not react to the positive control on Multi-Test II and were eliminated from the main analysis. Of the 34 patients included in the main analysis, all were positive for at least one antigen on IDT; one half (17/34) were negative for all antigens on Multi-Test II. A significantly greater number of patients were positive on IDT than Multi-Test II for all antigens except dog (P < 0.05). There was a statistically significant correlation between IDT endpoint and Multi-Test II score for several antigens (P < 0.05). Of 408 total antigen tests performed, 339 (83.1%) were negative on Multi-Test II. Of these 339 negative Multi-Test II tests, 148 (43.7%) were negative on IDT and 191 (56.3%) were positive on IDT; 174/339 (51.3%) had an IDT endpoint of 2 or 3 and 17/339 (5.0%) had an IDT endpoint > or = 4. A significantly greater number of antigen tests were positive on IDT than on Multi-Test II (P < 0.001). The overall Spearman correlation coefficient between IDT endpoint and Multi-Test II score was 0.370 (P < 0.001). CONCLUSION: Patients were more likely to have a positive test on IDT than on Multi-Test II. IDT therefore may be a more sensitive testing technique for inhalant allergies than Multi-Test II prick testing. In addition, Multi-Test II score may not be a good predictor of IDT endpoint. Although a statistically significant correlation is demonstrated between Multi-Test II score and IDT endpoint, the correlation coefficient is low enough that clinical application may not always be safe or appropriate. The clinical significance of positive IDT results in the presence of negative Multi-Test II results is not known.

Prevalence of allergy in patients with chronic rhinosinusitis.

OBJECTIVES: The purpose of this study was to provide further evidence that allergic rhinitis is an important factor in chronic and recurrent acute rhinosinusitis. Specifically, this study shows that perennial allergens play a more significant role than seasonal allergens. STUDY DESIGN AND SETTING: Census by chart review of patients with chronic and recurrent acute rhinosinusitis presenting to the Department of Otolaryngology at the MetroHealth Medical Center, Cleveland, OH. METHODS: All participants had allergy testing done either by RAST or intradermal skin endpoint titration utilizing a battery of seasonal and perennial antigens. RESULTS: Of the 48 voluntary participants analyzed in this study, 57.4% had a positive allergy test. Most patients in the study were sensitive to more than one allergen. Of the patients with a positive allergy test, 92% demonstrated sensitivity to one or more perennial allergens-most prominently, molds and dust mites. CONCLUSIONS: Perennial allergy has a statistically significant association with chronic and recurrent acute rhinosinusitis. SIGNIFICANCE: The diagnosis and management of perennial allergies may be beneficial when treating chronic sinus disease.