title: Quantification of Der f 1 in houses of patients allergic to house dust mite, Dermatophagoides farinae, using a locally produced detection reagents.
abstract: BACKGROUND
  House dust mite (HDM) allergen quantification in house dust samples before and after the allergen elimination is one means of convincing the target population about the health benefits of allergen removal from their environment.
  
  
  OBJECTIVE
  To produce local reagents for quantification of Der f 1 (major allergen of Dermatophagoides farinae) in dust samples from houses of HDM allergic Thai patients.
  
  
  METHODS
  Recombinant Der f 1 was used for immunization of a BALB/c mouse for hybridoma production. Polyclonal antibody (PAb) to whole body extract of D. farinae was prepared from an immunized rabbit. A sandwich ELISA (MAb-allergen-PAb) was used, in comparison with the commercialized reagents (Indoor Biotechnology, UK), to quantify Der f 1 in dust samples.
  
  
  RESULTS
  Two hybridoma clones, Dfl-1 and Dfl-2, were established. Their secreted MAbs (MAbDfl-1 and MAbDfl-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDfI-1 and MAbDfl-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf-1 had higher affinity to Der f 1 than the MAbDfl-2. A sandwich ELISA (MAbDfl-1-allergen-PAb) and commercialized reagents (MAbl-allergen-MAb2 sandwich ELISA) were used in comparison for quantification of Der f 1 in 42 dust samples collected from bedrooms and living rooms of 21 houses of the HDM allergic patients. All of the 42 dust samples measured by both ELISAs had the Der f 1 levels higher than 2 mg per gram of fine dust which is the HDM allergy sensitizing level. In addition, Der f 1 levels in 41 samples (except 1 sample from a living room) measured by the MAbDfI-1-PAb and MAbl-MAb2 sandwich ELISAs were higher than 10 mg per g of dust which is the morbidity level of HDM allergen. The local sandwich ELISA showed a high coefficient correlation (r = 0.91) in measuring known amounts of recombinant and native Der f 1. The results indicate that the reagents produced in the present study can be used for measuring the environmental levels of HDM Der f 1. The assay can also be used for standardization of the HDM extract for monitoring patient's allergenic status or for immunotherapeutic purpose.
authors.affiliation: Office for Research and Development, Siriraj Hospital, Mahidol University, Bangkok, 10400, Thailand. sinsru@mahidol.ac.th
authors.forename: Nitat
authors.initials: N
authors.lastname: Sookrung
authors.forename: Thanyarat
authors.initials: T
authors.lastname: Kamlanghan
authors.forename: Nitaya
authors.initials: N
authors.lastname: Indrawattana
authors.forename: Anchalee
authors.initials: A
authors.lastname: Tungtrongchitr
authors.forename: Pongsakorn
authors.initials: P
authors.lastname: Tantilipikorn
authors.forename: Chaweewan
authors.initials: C
authors.lastname: Bunnag
authors.forename: Kovit
authors.initials: K
authors.lastname: Pattanapanyasat
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides / Q000032:analysis* / Q000276:immunology; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases; D040001:Dermatophagoides farinae / Q000276:immunology; D004784:Environmental Monitoring / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D006967:Hypersensitivity; D007202:Indicators and Reagents; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D000939:Epitopes; D007202:Indicators and Reagents; D003546:Cysteine Endopeptidases; C451159:Dermatophagoides farinae antigen f 1