Handgrip Strength and Timed Up-and-Go (TUG) Test are Predictors of Short-Term Mortality among Elderly in a Population-Based Cohort in Singapore.

OBJECTIVES: Asian studies on how physical tests predict short-term mortality in elderly are scarce. We assessed handgrip strength and timed-up-and-go (TUG) as such predictors among elderly Chinese in Singapore. DESIGN: Prospective cohort study. SETTING: Community-dwelling Chinese elderly in Singapore. PARTICIPANTS: We used data from 13,789 subjects in the prospective, population-based Singapore Chinese Health Study, who had a mean age of 74 (range 63 to 97) years at time of measurements. MEASUREMENTS: Subjects underwent assessment for handgrip strength and TUG. They were followed for mortality via linkage with nationwide death registry through 2018. RESULTS: In multivariable analyses, handgrip strength was inversely associated with risk of mortality in a dose-dependent manner: the hazard ratio (HR) [95% confidence interval (CI)] comparing extreme quartiles was 2.05 (1.44-2.90) (Ptrend<0.001). TUG was positively associated with mortality in a stepwise manner: the HR (95% CI) comparing extreme quartiles was 3.08 (2.17-4.38) (Ptrend<0.001). Compared to those with stronger handgrip and faster TUG, participants who either had weaker handgrip or slower TUG had a significant 1.59 to 2.11 fold increase in risk of mortality; while the HR (95% CI) for those who had both weaker handgrip and slower TUG was 3.93 (3.06-5.05). In time-dependent receiver operating characteristic curves, adding handgrip strength and TUG time to a Cox model containing sociodemographic and lifestyle factors, comorbidities, and body measurements significantly improved the area under the curve for the prediction of mortality from 0.5 to 2 years (P≤0.001). CONCLUSION: Among elderly in a Chinese population, handgrip strength and TUG test were strong and independent predictors of short-term mortality.

Long-term impacts of antibiotic allergy testing on patient perceptions and antibiotic utilization.

OBJECTIVES: To define the long-term impacts of antibiotic allergy testing (AAT) on patient allergy perception and antibiotic utilization. METHODS: Patients were identified from a prospective AAT database as having completed testing during a 15 month period beginning January 2017. Patients were contacted for a follow-up survey at least 12 months post-AAT. For those contacted, baseline demographics, antibiotic allergy label (AAL) history, age-adjusted Charlson comorbidity index, infection history, antibiotic de-labelling (≥1 AAL removed following AAT) and antibiotic usage for 12 months prior to testing (pre-AAT) and 12 months following testing (post-AAT) were recorded for each patient. RESULTS: From the follow-up survey of 112 patients post-AAT, 95.2% (59/62) of patients with complete AAL removal expressed willingness to use 'de-labelled' antibiotics and 91.9% (57/62) were adherent to allergy label modification. Comparing antibiotic utilization 12 months pre-AAT versus 12 months post-AAT, AAT was associated with a significant increase in preferred antibiotic therapy [adjusted odds ratio (aOR) 3.29, 95% CI 1.56-6.92] and reduction in restricted antibiotic utilization (aOR 0.42, 95% CI 0.19-0.93). CONCLUSIONS: An antimicrobial stewardship (AMS)-led AAT programme was safe and effective in the long term in the promotion of preferred and narrow-spectrum antibiotic usage, and favourable patient perception towards the AAT testing results was identified. This study further supports the routine incorporation of AAT into AMS programmes, confirming safety and durability of testing impacts on patients as well as increasing preferred antibiotic utilization.

The structure of the mite allergen Blo t 1 explains the limited antibody cross-reactivity to Der p 1.

The Blomia tropicalis (Blo t) mite species is considered a storage mite in temperate climate zones and an important source of indoor allergens causing allergic asthma and rhinitis in tropical and subtropical regions. Here, we report the crystal structure of one of the allergens from Blo t, recombinant proBlo t 1 (rproBlo t 1), determined at 2.1 Å resolution. Overall, the fold of rproBlo t 1 is characteristic for the pro-form of cysteine proteases from the C1A class. Structural comparison of experimentally mapped Der f 1/Der p1 IgG epitopes to the same surface patch on Blo t 1, as well as of sequence identity of surface-exposed residues, suggests limited cross-reactivity between these allergens and Blo t 1. This is in agreement with ELISA inhibition results showing that, although cross-reactive human IgE epitopes exist, there are unique IgE epitopes for both Blo t 1 and Der p 1.

Clinical utility of panfungal polymerase chain reaction for the diagnosis of invasive fungal disease: a single center experience.

The role of panfungal polymerase chain reaction (PCR) assays for diagnosis of invasive fungal disease (IFD) is inadequately defined. We describe the use of an internal transcribed spacer 1 (ITS-1) region-directed panfungal PCR in this context at a tertiary referral transplant center. A retrospective review of patients at Alfred Health, Melbourne, Australia (2009-2014) who had clinical samples referred for panfungal PCR testing was conducted. Baseline patient characteristics, antifungal drug history, fungal culture/histopathology, and radiology results were recorded. For bronchoalveolar lavage (BAL) fluid samples, identification of a fungus other than a Candida spp. was defined as a potential pathogen.Of 138 panfungal PCR tests (108 patients), 41 (30%) were positive for a fungal product. Ninety-seven percent (134/138) of specimens were from immunocompromised hosts. Thirteen percent (19/138) of panfungal PCR positive results were for potential pathogens and potential pathogens were detected more frequently in tissue as compared with BAL (12/13 vs. 6/26; P = .0001). No positive panfungal PCR results were obtained from CSF specimens. If histopathology examination was negative, panfungal PCR identified a potential pathogen in only 12% (11/94) of specimens. For the 20 culture negative/histopathology positive specimens, diagnosis of IFD to causative species level by panfungal PCR occurred in 35% (6/20).Sterile site specimens, in particular tissue, were more frequently panfungal PCR positive for potential pathogens than BAL. The utility of panfungal PCR appears greatest in tissue specimens, as an adjunct to histopathology to improve diagnostic sensitivity and specificity. Based on the results of this study we are now only testing tissue specimens by panfungal PCR.

Cotton fabric-based electrochemical device for lactate measurement in saliva.

Lactate measurement is vital in clinical diagnostics especially among trauma and sepsis patients. In recent years, it has been shown that saliva samples are an excellent applicable alternative for non-invasive measurement of lactate. In this study, we describe a method for the determination of lactate concentration in saliva samples by using a simple and low-cost cotton fabric-based electrochemical device (FED). The device was fabricated using template method for patterning the electrodes and wax-patterning technique for creating the sample placement/reaction zone. Lactate oxidase (LOx) enzyme was immobilised at the reaction zone using a simple entrapment method. The LOx enzymatic reaction product, hydrogen peroxide (H2O2) was measured using chronoamperometric measurements at the optimal detection potential (-0.2 V vs. Ag/AgCl), in which the device exhibited a linear working range between 0.1 to 5 mM, sensitivity (slope) of 0.3169 µA mM(-1) and detection limit of 0.3 mM. The low detection limit and wide linear range were suitable to measure salivary lactate (SL) concentration, thus saliva samples obtained under fasting conditions and after meals were evaluated using the FED. The measured SL varied among subjects and increased after meals randomly. The proposed device provides a suitable analytical alternative for rapid and non-invasive determination of lactate in saliva samples. The device can also be adapted to a variety of other assays that requires simplicity, low-cost, portability and flexibility.

Early onset wheeze associated with enhanced combined IL-1ß, IL-6, and IL-12/IL-23p40 in LPS-stimulated cord blood mononuclear cells.

BACKGROUND: Neonates with a family history of atopy are at higher risk for developing wheezing in early life. OBJECTIVE: From a birth cohort of at risk infants (first-degree family with atopic disease), we evaluated the influence of distinct intrinsic immunologic risk factors on wheezing disorders in the first 2 years of life. METHODS: Cord blood samples were collected from 195 eligible subjects of a birth cohort of 253 subjects. The subjects studied were those who developed wheezing (n=34) or eczema (n=29) in the first 2 years of life, and 65 healthy control infants. At the time of thawing the viability of the cells were median 70% (range 67.5%-72.5%). Cytokines from lipopolysaccharide (LPS)-stimulated mononuclear cells were analysed using fluorescent-activated cell sorting-array and their profiles were evaluated using factor analysis. RESULTS: Infants with wheeze were significantly associated with enhanced combined LPS stimulated IL-1ß, IL-6, and IL-12/IL-23p40 compared with healthy controls (P=0.003). This profile was also associated with the increased risk for wheeze at 2 years of age (OR=2.45; 95% CI=1.50-3.93, P=0.001). LPS-stimulated cytokine IL-8 was also significantly higher in the wheeze group compared with healthy controls and eczema (P=0.003). Intracellular staining showed that monocytes are main producers of IL-6 and IL-8 from cord blood mononuclear cells. Most of the subjects were non-atopic with 3/34 (9%) wheeze and 9/29 (31%) eczema subjects sensitized to the common dietary or inhalant allergens. CONCLUSION AND CLINICAL RELEVANCE: In infants at genetic risk of atopy, wheeze but not eczema in the first 2 years of life is associated with intrinsic hyperresponsive innate cytokine responses which might predispose infants to wheeze development. Distinct pre-symptomatic hyperresponsive innate immune responses risk factors were found to be associated with early onset wheeze disorders, but not eczema.

Dust mite infestation of flour samples.

BACKGROUND: Ingestion of flour contaminated with dust mite may trigger severe anaphylaxis in tropical and sub-tropical regions. AIMS: This study aimed to evaluate environmental factors that affect dust mite propagation in the tropics. MATERIALS & METHODS: Dust mites were introduced to a variety of flour samples and incubated at two different environmental conditions. RESULTS: It was found that dust mites populations flourished best in wheat flour compared to other varieties of flour, and at ambient temperatures with high humidity instead of the air conditioned environment. CONCLUSION: Dust mite infestation of flour is dependent on the presence of wheat and high ambient temperature in the tropics.

IgE cross-reactivity between Ascaris and domestic mite allergens: the role of tropomyosin and the nematode polyprotein ABA-1.

BACKGROUND: Analysis of cross-reactivity between the nematode Ascaris ssp. and dust mites, two important allergen sources in the tropics, will contribute in understanding their influence on asthma and atopy. The objective of this study was to investigate immunoglobulin E (IgE) cross-reactivity between Ascaris and two domestic mites in the tropics. METHODS: Sera from 24 asthmatic patients were used in ELISA and immunoblotting IgE-binding inhibition assays using Ascaris, Blomia tropicalis and Dermatophagoides pteronyssinus extracts and the recombinants Blo t 10, ABA-1 and Blo t 13 as competitors. Identification of Ascaris allergens was confirmed by mass spectrometry (LC-MS/MS). RESULTS: We detected at least 12 human IgE-binding components in Ascaris extract. Blomia tropicalis and D. pteronyssinus inhibited 83.3% and 79% of IgE-binding to Ascaris, while Ascaris inhibited 58.3% and 79.3% to B. tropicalis and D. pteronyssinus respectively. Mite tropomyosin inhibited 85% of IgE-binding to Ascaris. Affinity-purified human IgE to rBlo t 10 identified an allergen of 40 kDa in Ascaris extract, further confirmed as tropomyosin by LC-MS/MS. We found no evidence of IgE cross-reactivity between rABA-1 and any allergen component in mite extracts, including rBlo t 13. CONCLUSIONS: There is cross-reactivity between Ascaris and mites, determined by several allergens including tropomyosin and glutathione-S-transferase. In addition to its potential impact on asthma pathogenesis, Ascaris infection and mite allergy diagnosis relying on the determination of specific IgE could be affected by this cross-reactivity. ABA-1 has no cross-reactive counterpart in mite extracts, suggesting its usefulness as a more specific marker of Ascaris infection.

Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopes.

BACKGROUND: Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens. OBJECTIVE: To evaluate the differences in the IgE response against two Blo t 12 isoallergens. METHODS: The IgE-binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross-inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin-binding domain of the two isoallergens. RESULTS: The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE-binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin-binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes. CONCLUSION: In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE-reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy.

Cornulin, a marker of late epidermal differentiation, is down-regulated in eczema.

BACKGROUND: Eczema is a common chronic inflammatory skin disorder which shows strong genetic predisposition. To identify new potential molecular determinants of the disease pathogenesis, we performed a gene expression study in an eczema mouse model. This analysis identified a marked down regulation of the cornulin gene (CRNN), a member of the epidermal differentiation complex, in the eczema-like skin. We then investigated CRNN as an eczema candidate gene and studied its polymorphism and the expression in the skin of eczema patients. METHODS: An eczema-like phenotype was induced in mice by allergen (Der p2) patching. Gene expression analysis was performed with the subtractive suppression hybridization method and validated by real time PCR and the transmission disequilibrium test was used to test for genetic associations in 406 multiplex eczema families. RESULTS: Der p 2 patched mice developed a localized eczema and a Th 2 skewed systemic response. Real time PCR analysis confirmed a down regulation of CRNN mRNA in eczema-like skin in the mouse model and in human eczema. The CRNN polymorphism rs941934 was significantly associated with atopic eczema in the genetic analysis (P = 0.006), though only as part of an extended haplotype including a known associated variant (2282del4) in the filaggrin gene. CONCLUSIONS: CRNN mRNA expression is decreased in eczematous skin. Further studies are needed to verify whether the associated cornulin polymorphism contribute to the genetic susceptibility in eczema.

Anaphylaxis following the ingestion of flour contaminated by house dust mites--a report of two cases from Singapore.

This study presents two patients who developed anaphylaxis after eating mite-contaminated food, and also contains a survey of dust-mites contamination in flour samples from Singapore households. The clinical records of each patient was studied. Patient A developed anaphylaxis twenty minutes following the ingestion of home-made fried fish coated with Japanese flour, while Patient B developed similar life-threatening symptoms one hour after the ingestion of home baked scones. Both patients were NSAID-intolerant and had a history of allergic rhinitis. Skin prick tests showed a strong positive result for dust-mites and for extracts prepared from the ingested flour. Flour samples were also examined microscopically which revealed large numbers of live Dermatophagoides farinae dust-mites. A survey of 57 flour samples showed that 4 samples (7%) were contaminated with dust mites. The findings in the present study confirm that mite-contamination of flour exists in Singaporean households, and it may trigger anaphylaxis in susceptible individuals.

Distinctive immunoglobulin E anti-house dust allergen-binding specificities in a tropical Australian Aboriginal community.

BACKGROUND: There is evidence that the specificity of the IgE binding in allergy tests can vary for different populations. OBJECTIVE: We aimed to examine the allergenic specificity of IgE binding in sera from house dust mite (HDM)-atopic subjects in a tropical Australian Aboriginal community. METHODS: Sera shown to contain IgE antibodies to an HDM extract of Dermatophagoides pteronyssinus were examined for IgE binding to a panel of nine purified HDM allergens from this mite species by quantitative microtitre assays. IgG antibody binding (IgG1 and IgG4) was also measured. RESULTS: The IgE-binding activity in the sera from the Aboriginal community was not directed to the expected major groups 1 and 2 HDM allergens but instead to the group 4 amylase allergen. There was also little IgE binding to the potentially cross-reactive tropomyosin (Der p 10) or arginine kinase (Der p 20) allergens. The IgG4 antibody was rarely detected and limited to the Der p 4 allergen. IgG1 antibody binding was frequently measured to all the allergens regardless of an individual's atopic status, whereas in urban communities it is restricted to the major allergens and to atopic subjects. CONCLUSION: The high IgE anti-HDM response of Australian Aboriginals predominantly bound Der p 4 and not the Der p 1 and 2 allergens, showing a distinctive allergy that could affect the disease outcome and diagnosis.

Molecular characterization, expression and localization of tropomyosin and paramyosin immunodominant allergens from sheep scab mites (Psoroptes ovis).

cDNAs encoding the immunodominant allergens tropomyosin and paramyosin were amplified from RNA extracted from the sheep scab mite Psoroptes ovis. The tropomyosin cDNA contained an open reading frame (ORF) of 852 bp which encoded a predicted protein with 98% and 97% identity to the house dust mite allergens Der f 10 and Der p 10 respectively. The complete paramyosin ORF generated by RT-PCR was 2625 bp in length and encoded an 875aa predicted protein of 102.6 kDa with 97%, 95% and 89% identity to the paramyosins of Dermatophagoides pteronyssinus (Der p 11), Sarcoptes scabiei and Blomia tropicalis (Blo t 11) respectively. Full length tropomyosin and truncated and full-length paramyosin were expressed as recombinant proteins. IgG and IgE in sera from sheep with a 6-week duration primary infestation of P. ovis did not detect either full-length or truncated recombinant paramyosin. IgG in both infested and naïve sheep sera detected recombinant tropomyosin, suggesting cross-reactivity to tropomyosin and to other invertebrate species to which the sheep may have been exposed. Staining with antibodies directed against tropomyosin and paramyosin was observed throughout sections of P. ovis. Staining was especially prevalent in the anterior sections of the mites, possibly associated with locomotory muscles in this region.

Distinct pattern of commensal gut microbiota in toddlers with eczema.

BACKGROUND: Recent studies have demonstrated differences in the composition of gut microbiota in infants with and without allergic diseases, particularly eczema. METHODS: A case-control study involving 21 toddlers (age 3.0 +/- 0.5 years) with and 28 age-matched toddlers without eczema was conducted. Four groups of aerobic gut microbiota were identified and quantitated in stool samples grown on selective media. Three groups of anaerobes were enumerated by fluorescent in situ hybridization followed by quantitative flow cytometry. We also performed molecular typing of lactic-acid-producing bacteria (LAB) and enterococcal isolates to facilitate detailed analysis at species level by bacterial 16S rDNA sequencing. RESULTS: Toddlers with eczema harbored significantly lower counts of Bifidobacterium [(median 0.14 (25th and 75th percentile: 0.04 and 0.47) vs. 0.71% (0.16, 1.79) of cells acquired, p = 0.003)] and Clostridium [(0.28 (0.09, 0.78) vs. 0.83% (0.35, 1.82) of cells acquired, p = 0.012)] but significantly higher counts of total LAB [7.3 (6.1, 8.5) vs. 5.7 (4.4, 7.3) log CFU/g, p = 0.006] in particular enterococci [6.3 (4.8, 7.4) vs. 5.0 (3.4, 6.4) log CFU/g, p = 0.018]. There was no significant correlation between eczema severity score and bifidobacterial counts. CONCLUSION: The results further confirm previous reports that the gut microecosystem differs between children with and without eczema and extend them beyond infancy.

Characterization of glutathione S-transferase from dust mite, Der p 8 and its immunoglobulin E cross-reactivity with cockroach glutathione S-transferase.

BACKGROUND: Sensitization to mite and cockroach allergens is common, and diagnosis and therapy of allergy can be further complicated by the presence of allergen isoforms and panallergens. Purified recombinant and native allergens are useful for studies to resolve such problems. OBJECTIVE: To assess the allergenicity of native and recombinant mite glutathione S-transferase (GST) (Der p 8) and study the IgE cross-reactivity between Der p 8 and cockroach GST. METHODS: Der p 8 cDNA encoding a new isoform was isolated and expressed in yeast. Native Der p 8 was affinity purified from mite extract. IgE reactivity to native and recombinant Der p 8 was assessed by ELISA using sera from allergic subjects from Taiwan, Singapore and Malaysia. IgE cross-reactivity between Der p 8 and cockroach GST was examined by IgE inhibition assays. RESULTS: Our Der p 8 cDNA encoded a basic isoform (pI=8.5) containing six polymorphic residues located at positions 46, 106, 149, 160, 167 and 184. At least 8 isoforms of native Der p 8 were detected by two-dimensionalgel and immunoblot analyses. Sera from Taiwanese asthmatics showed 96% and 84% IgE reactivity to native Der p 8 and recombinant Der p 8, respectively. Native Der p 8 showed 75% and 65% IgE reactivity with sera from Malaysia and Singapore, respectively. CONCLUSIONS: A high frequency of sensitization to mite GST among allergic subjects was observed but the titres of IgE reactivity were low. The IgE cross-reactivity between mite and cockroach GST suggests that GST is a panallergen.

Quantification of Blo t 5 in mite and dust extracts by two-site ELISA.

BACKGROUND: Blomia tropicalis is an important mite species in the tropical and sub-tropical regions of the world. Blo t 5 is the major allergen with up to 70% sensitization rates in B. tropicalis allergic populations. METHODS: Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 5 gene with in vivo electroporation. Blo t 5 monoclonal antibodies were generated using methylcellulose-based hybridoma kit. Monoclonal antibody (mAb) 4A7 was characterized by two-dimensional electrophoresis immunoblotting. A specific quantitative two-site enzyme-linked immunosorbent assay (ELISA) was developed with mAb 4A7 and guinea pigs Blo t 5 polyclonal antibody as capture and detection antibodies, respectively. This system was tested with Blo t 5 in crude extracts and dust samples. RESULTS: A high-affinity mAb 4A7 recognizing several isoforms of Blo t 5 has been generated. Monoclonal antibody 4A7 is useful for immunoblotting and two-site ELISA. The two-site ELISA developed has a high sensitivity, with a detection limit of 10 pg/ml. The assay is species-specific and recognized the same epitopes on both native and recombinant Blo t 5. The assay developed is able to detect Blo t 5 in commercial diagnostic and therapeutic B. tropicalis extract. Blo t 5 quantification in dust samples showed that Blo t 5 is present in a high quantity in Singapore dust. CONCLUSIONS: A highly sensitive and specific two-site ELISA has been developed. The assay system developed is useful for the quantification of Blo t 5 in mite and environmental dust extracts.

Immunoglobulin E reactivity of native Blo t 5, a major allergen of Blomia tropicalis.

BACKGROUND: Blo t 5 is a major allergen of Blomia tropicalis and its complementary DNA (cDNA) has been expressed in both prokaryotic and eukaryotic expression systems. Although the recombinant Blo t 5 has been well characterized, relatively less is known about its native counterparts and the allergenicity comparison of the native and recombinant Blo t 5 allergens has not been reported. OBJECTIVE: The aims of this study are to characterize the native counterpart of Blo t 5, and compare the allergenicity of native and recombinant Blo t 5 by in vivo and in vitro assays. METHODS: Native Blo t 5 were purified by immuno-affinity chromatography and characterized by proteomic means. The allergenicity of the allergen was evaluated by skin prick tests, human IgE ELISA, ELISA inhibition and histamine release assays. RESULTS: Native Blo t 5 consists of at least five distinct isoforms, ranging from pI 3 to 5.5. Allergenicity assessment of recombinant and native Blo t 5 based on skin reaction, IgE-binding capacity and histamine release in allergic individuals indicated that there was a good correlation between both forms of Blo t 5 in general. However, data from IgE ELISA inhibition assay revealed the presence of additional unique IgE epitopes in native Blo t 5. CONCLUSIONS: At least five distinct isoforms of Blo t 5 have been identified. Comparative assessment of native and recombinant Blo t 5 revealed that the allergenicity of these two forms was similar but not completely identical suggesting that the various isoforms of native Blo t 5 may exhibit additional unique IgE epitopes.

Anaphylaxis after hamster bites--identification of a novel allergen.

BACKGROUND: Hamsters are popular household pets and anaphylaxis after their bites have described. However, the putative allergen has not been identified. OBJECTIVE: This study was conducted to identify the allergen causing dwarf hamster (Phodopus sungoris) bite-induced anaphylaxis. METHODS: Two children with hamster bite-induced anaphylaxis were enrolled. They both had negative results to skin testing and specific IgE to hamster epithelium. However, they were both allergic to Dermatophagoides pteronyssinus (Der p). Identification of the putative IgE-binding allergens from the hamster saliva was performed using immunoblot analysis. RESULTS: A specific IgE-binding component at 21 kD in the hamster saliva was identified. ELISA inhibition tests showed partial inhibition with Der p. CONCLUSIONS: The putative allergen from the hamster saliva causing dwarf hamster-induced anaphylaxis was identified. Possible cross-reactivity with Der p was demonstrated. Further studies will be needed to identify the exact nature and function of this allergen.

Production of monoclonal antibodies for immunoaffinity purification and quantitation of Blo t 1 allergen in mite and dust extracts.

BACKGROUND: Blo t 1 is a cysteine protease-like allergen from Blomia tropicalis. Recombinant Blo t 1 binds up to 90% of IgE from allergic patients and shows limited cross-reactivity to Der p 1. The generation of monoclonal antibodies (mAbs) against Blo t 1 is important for the detection, isolation and characterization of the native form of the allergen. METHODS: Mice were immunized intramuscularly with naked plasmid DNA encoding Blo t 1 gene with in vivo electroporation and boosted intraperitoneally with recombinant Blo t 1. mAbs against Blo t 1 were generated using a methylcellulose-based hybridoma cloning kit. The native Blo t 1 was isolated by mAb affinity purification and its allergenicity was determined by ELISA. A two-site ELISA for Blo t 1 was developed using the mAbs generated. RESULTS: A DNA-based immunization protocol induced high titre Blo t 1-specific antibodies in mice. Six stable hybridoma clones secreting mAbs recognizing the native and recombinant Blo t 1 were generated. The native Blo t 1 was affinity-purified from a B. tropicalis extract and its allergenicity was determined at 63% using a panel of Singaporean and Malaysian mite allergic patients' sera. A two-site ELISA was developed, which showed a detection limit of 10 ng/mL of Blot t 1. CONCLUSION: Six Blo t 1 mAbs were successfully generated by DNA immunization. These mAbs are useful for nBlo t 1 immunoaffinity isolation and quantitative immunoassays for Blo t 1 in mite and environmental dust extracts.

DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis.

BACKGROUND: Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA-based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production. OBJECTIVES: To use a DNA-based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs). METHODS: The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose-based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization - time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two-site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11. CONCLUSION: A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.