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.

Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin (Blo t 11).

BACKGROUND: The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms. METHODS: Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS. Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli. Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies. RESULTS: Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at approximately 66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart. CONCLUSIONS: This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.

Novel mutations in ndh in isoniazid-resistant Mycobacterium tuberculosis isolates.

Novel mutations in NADH dehydrogenase (ndh) were detected in 8 of 84 (9.5%) isoniazid (INH)-resistant isolates (T110A [n = 1], R268H [n = 7]), but not in 22 INH-susceptible isolates of Mycobacterium tuberculosis. Significantly, all eight isolates with mutations at ndh did not have mutations at katG, kasA, or the promoter regions of inhA or ahpC, except for one isolate. Mutations in ndh appear to be an additional molecular mechanism for isoniazid resistance in M. tuberculosis.

Use of ligase chain reaction and polymerase chain reaction on urine specimens to detect Chlamydia trachomatis infections in a sexually transmitted diseases clinic in Singapore.

This study was done to assess the specificity and sensitivity of the DNA amplification assays of ligase chain reaction (LCR) and polymerase chain reaction (PCR) on urine specimens to detect Chlamydia trachomatis infections in both male and female patients seen at a sexually transmitted diseases (STD) clinic in Singapore, compared with other diagnostic methods currently in use. A total of 100 patients were selected; 50 male patients diagnosed with non-gonococcal urethritis based on symptoms and a positive Gram-stained urethral smear and 50 female asymptomatic sex workers were assessed. Automated assays using LCR and PCR were used, and compared to enzyme immunoassays, chlamydial cell cultures and PCR of urethral and endocervical swab specimens. In male patients, LCR and PCR of urine specimens had sensitivities of 100%, compared to 87.0% for PCR of urethral swab specimen, 82.6% for enzyme immunoassay (EIA) and 91.3% for cell cultures. In female patients, LCR and PCR of urine samples achieved sensitivities of 77.8% and 88.9% respectively, compared with 55.6% for PCR of endocervical swab specimens, 22.2% for EIA and 66.7% for cell cultures. LCR and PCR of urine samples provided higher sensitivity compared to cell cultures, EIA and PCR of urethral and endocervical swab specimens. The use of LCR and PCR on urine as a non-invasive means of detecting chlamydial infections is viable, and may have a role to play in population-based screening programmes.