OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.
METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.
RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.
CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.
OBJECTIVE: The present study aimed to measure the agreement between BAT and immunoassay in diagnosis of penicillin allergy.
METHOD: BAT was performed using penicillin G (Pen G), penicillin V (Pen V), penicilloyl-polylysine (PPL), minor determinant mix (MDM), amoxicillin (Amx) and ampicillin (Amp) in 25 patients. Immunoassay of total IgE (tIgE) and specific IgE (sIgE) antibodies to Pen G, Pen V, Amx and Amp were quantified. Skin prick test (SPT) using PPL-MDM, Amx, Amp and Clavulanic acid were also performed.
RESULTS: Minimal agreement was observed between BAT and immunoassay (k=0.25). Of two BAT-positive patients, one patient is positive to Amx (59.27%, SI=59) and Amp (82.32%, SI=82) but sIgE-negative to all drug tested. This patient is also SPT-positive to both drugs. Another patient is BAT-positive to Pen G (10.18%, SI=40), Pen V (25.07%, SI=100) and Amp (19.52%, SI=79). In sIgE immunoassay, four patients were sIgE-positive to at least one of the drugs tested. The sIgE level of three patients was between low and moderate and they were BAT-negative. One BAT-positive patient had a high level of sIgE antibodies (3.50-17.5kU/L) along with relatively high specific to total IgE ratio ≥0.002 (0.004-0.007).
CONCLUSIONS: The agreement between BAT and immunoassay is minimal. Performing both tests provides little increase in the sensitivity of allergy diagnosis work-up for immediate reactions to penicillin.
METHODS: Malay, Chinese, and Indian AR patients (n = 138) with confirmed sensitivity to Dermatophagoides pteronyssinus, Dematophagoides farinae, and Blomia tropicalis were tested for mite-specific immunoglobulin E (sIgE) levels. A detailed questionnaire was used to collect data on nasal symptom score (NSS), ocular symptom score (OSS), sum of symptoms score (SSS), quality of life score (QLS), symptomatic control score (SCS), and total sum of scores (TSS) and correlate the derived data with patients' demography, mite-polysensitivity, and sIgE levels.
RESULTS: AR-related symptoms were most severe in Malays and least in Chinese (p < 0.01). Age (r = 0.516 to 0.673, p < 0.05) and duration of AR (r = 0.635 to 0.726, p < 0.01) correlated positively with severity domains (NSS, SSS, QLS, and TSS) in Chinese. Duration of concurrent allergies was highest in Malays (p < 0.05). Polysensitivity predicted increased sIgE levels in Malays (r = 0.464 to 0.551, p < 0.01) and Indians (r = 0.541 to 0.645, p < 0.05) but affected NSS, SSS, and TSS only in Indians (r = 0.216 to 0.376, p < 0.05). sIgE levels were lowest among Chinese but correlated strongly with NSS, OSS, SSS, and TSS (r = 0408 to 0.898, p < 0.05).
CONCLUSION: Clinical parameters in AR may be influenced by race. Symptoms were most severe among Malays but did not correlate with other variables examined. Although Indian ethnicity did not impact disease severity, duration of concurrent allergies and mite-polysensitivity was associated with more severe disease. Age, duration of disease, and sIgE levels may be useful indicators of disease severity in Chinese.
OBJECTIVES: This study aimed at describing the immunochemical properties of the BN allergens. Comparative studies between 3 commercially available sources (according to the country of origin) of BN were also made.
METHODS: Crude extracts of commercially available processed BN from Sarawak (Malaysia), Thailand, and Indonesia and fresh unprocessed BN from the caves of Sarawak were obtained by means of aqueous extraction. Specific IgE toward these sources were determined by using fluorescence allergosorbent tests (FASTs). Cross-reactivity studies between the 3 sources of commercially available processed BN were carried out by means of FAST inhibition. Immunochemical characterization by means of IgE immunoblot, periodate treatment, and heat stability studies were carried out on fresh unprocessed BN from Sarawak.
RESULTS: Serum from allergic patients showed differences in IgE binding to the 3 sources of commercially available BN, with the highest levels of specific IgE recorded with the Sarawak source (P
OBJECTIVE: We conducted a phase 1/2 clinical study to examine the safety and diagnostic accuracy (sensitivity and specificity) of nonammoniated latex, ammoniated latex, and rubber glove extracts as skin test extracts to identify the most efficacious source material for future skin test reagent development.
METHODS: Twenty-four adults not allergic to latex, 19 adults with hand dermatitis or pruritus, and 59 adults with a latex allergy were identified by clinical history. All provided blood and then received puncture skin tests and intradermal skin tests with nonammoniated latex, ammoniated latex, and rubber glove extracts from Malaysian H. brasiliensis latex by use of sequential titration. A glove provocation test and IgE anti-latex RAST were used to clarify positive history-negative skin test response and negative history-positive skin test response mismatches.
RESULTS: All three extracts were biologically safe and sterile. After normalization to 1 mg/ml of total protein, all three extracts produced equivalent diagnostic sensitivity and specificity in puncture skin tests and intradermal skin tests at various extract concentrations. Optimal diagnostic accuracy was safely achieved at 100 micrograms/ml for intradermal skin tests (e.g., nonammoniated latex: puncture skin test sensitivity 96%, specificity 100%; intradermal skin test sensitivity 93%, specificity 96%). The presence of IgE antibody in skin was highly correlated with IgE anti-latex in serum (nonammoniated latex: r = 0.98, p < 0.001; ammoniated latex: r = 0.94, p < 0.001; rubber glove extract: r = 0.96, p < 0.001). All five available subjects with a positive history, negative skin test response, and absence of IgE antibody in serum had a negative glove provocation test response, indicating no clinical evidence of latex allergy. No systemic or large local allergic reactions were observed with puncture skin tests or intradermal skin tests.
CONCLUSIONS: Equivalent diagnostic sensitivity and specificity were observed with the nonammoniated latex, ammoniated latex, and rubber glove extract skin test reagents after normalization for total protein; nonammoniated latex may be considered the reagent of choice on the basis of practical quality control and reproducibility considerations.
METHODS AND RESULTS: Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group.
CONCLUSIONS: Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect.
SIGNIFICANCE AND IMPACT OF THE STUDY: Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine.