title: Intra-laboratory study to determine the reproducibility of LLNA:BrdU-ELISA for the prediction of the skin sensitizing potential of chemicals.
abstract: BACKGROUND
  The Local Lymph Node Assay (LLNA) has been designated as the first-choice in vivo assay for identification the skin sensitization potential of new chemicals. The LLNA:BrdU-ELISA is a validated non-radioactive modification to the LLNA. An intra-laboratory reproducibility study for the LLNA:BrdU-ELISA was conducted to demonstrate its adequate performance in our laboratory.
  
  
  METHODS
  Ten independent LLNA:BrdU-ELISAs with the preferred positive controls (PCs), i.e., 25% hexyl cinnamic aldehyde (HCA) and 25% eugenol, were conducted within a period of less than one year. In addition, different concentrations of 2,4-dinitrochlorobenzene (DNCB, an extreme sensitizer) (0.01, 0.1 and 0.3%), HCA (10, 25 and 50%) and eugenol (10, 25 and 50%), were tested to determine the EC1.6 values. Special Pathogen Free female CBA/J mice of 8-10weeks old were randomly allocated to the groups, each group having 4 mice. 25μl of AOO (vehicle, acetone: olive oil=4:1, v/v) or HCA, eugenol, DNCB at the needed concentration was applied to the dorsum of each ear of the mice, daily for 3 consecutive days. A single intraperitoneal injection of 0.5ml of BrdU solution (10mg/ml) was given on day 5. On day 6, a pair of auricular lymph nodes from each mouse was excised, and BrdU ELISA analysis was conducted.
  
  
  RESULTS
  The result for each group is expressed as the mean Stimulation Index (SI). The mean of the 10 mean SIs for 25% HCA (2.58±0.95) and 25% eugenol (3.51±1.25) was not significantly different to that from the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) (i.e., the data on the formal validation study for the LLNA:BrdU-ELISA by the ICCVAM) (3.03±2.00 for 25% HCA, 6.13±6.06 for 25% eugenol) (P>0.05), with even smaller Coefficient of Variations (CV) (36.8% for 25% HCA, 35.6% for 25% eugenol) than that from the ICCVAM (66.0% for 25% HCA, 98.8% for 25% eugenol). In addition, the EC1.6 values for HCA, eugenol and DNCB (15.2, 12.5 and 0.25%, respectively) were consistent with that from the ICCVAM (12.92, 8.85 and 0.34%, respectively).
  
  
  CONCLUSIONS
  The results indicate that the reliability for our laboratory to conduct the LLNA:BrdU-ELISA is successfully determined.
authors.affiliation: Department of Toxicology, National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Toxicology, National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China.
authors.forename: Caihong
authors.initials: C
authors.lastname: Xing
authors.affiliation: Department of Toxicology, National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China. Electronic address: houfenxia66@163.com.
authors.forename: Fenxia
authors.initials: F
authors.lastname: Hou
mesh_terms: D000171:Acrolein / Q000031:analogs & derivatives / Q000494:pharmacology; D000485:Allergens / Q000633:toxicity*; D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D017449:Dermatitis, Allergic Contact / Q000175:diagnosis; D004797:Enzyme-Linked Immunosorbent Assay; D005054:Eugenol / Q000494:pharmacology; D005260:Female; D059021:Laboratory Proficiency Testing / Q000379:methods*; D023441:Local Lymph Node Assay; D008198:Lymph Nodes / Q000166:cytology / Q000276:immunology; D008808:Mice, Inbred CBA; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000485:Allergens; C080043:hexyl cinnamic aldehyde; D005054:Eugenol; D000171:Acrolein; D001973:Bromodeoxyuridine
keywords: 2,4-Dinitrochlorobenzene (PubChem CID: 6); 5-Bromo-2-deoxyuridine (PubChem CID: 6035); Acetone (PubChem CID:180); Eugenol; Eugenol (PubChem CID: 3314); Hexyl cinnamic aldehyde; Hexyl cinnamic aldehyde (PubChem CID: 1,715,135); Local Lymph Node Assay:BrdU-ELISA; Methods; Reliability; Reproducibility; Skin sensitization