title: Hep-2 cell based indirect immunofluorescence assay for antinuclear antibodies as a potential diagnosis of drug-induced autoimmunity in nonclinical toxicity testing.
abstract: Antinuclear antibodies (ANAs) are important biomarkers in the diagnosis of autoimmune diseases in humans; however, the diagnostic performance of ANA in nonclinical safety studies are not well understood. Here, we studied the use of ANAs as potential nonclinical biomarkers for drug-induced autoimmunity (DIA) using a Hep-2 based indirect immunofluorescence assay (IFA). Initially, MRL-fas(lpr)/J mice and HgCl₂-treated rats were used as SLE-positive models. Serum samples obtained from 94 normal mice or 204 normal rats aged one to four months served as the negative control. The IFA effectively distinguished ANAs-positive samples in both species with a cut-off titer of 1:100. Brown Norway rats were treated with 450 mg/kg D-penicillamine for 30 consecutive days. ANAs were generated and corresponded with DIA development. Human Hep-2 cells, mice Neuro 2A cells, and Chinese Hamster Lung cells served as antigen from different species, which were found cross-reactive with ANA-positive serum samples from mice, rats, and humans without any differences in diagnosis. This methodology showed no species-specificity for ANA detection. Furthermore, we found approximately 20 percentage of the mice aged seven to eight months demonstrated age-related ANAs, which was consistent with humans. Overall, our findings demonstrated the use of ANA detection using IFA in the nonclinical diagnosis of murine drug-induced autoimmunity, and age-related ANAs should be considered when aged animals are used.
authors.affiliation: National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, China Food and Drug Administration, 100176 Beijing, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Hong
authors.affiliation: National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, China Food and Drug Administration, 100176 Beijing, People's Republic of China.
authors.forename: Ben
authors.initials: B
authors.lastname: Ma
authors.affiliation: National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, China Food and Drug Administration, 100176 Beijing, People's Republic of China.
authors.forename: Zhi
authors.initials: Z
authors.lastname: Lin
authors.affiliation: National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, China Food and Drug Administration, 100176 Beijing, People's Republic of China.
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Zhou
authors.affiliation: National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, China Food and Drug Administration, 100176 Beijing, People's Republic of China.
authors.forename: Xingchao
authors.initials: X
authors.lastname: Geng
authors.affiliation: National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, China Food and Drug Administration, 100176 Beijing, People's Republic of China.
authors.forename: Lianzhong
authors.initials: L
authors.lastname: Shen
authors.affiliation: National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, China Food and Drug Administration, 100176 Beijing, People's Republic of China. Electronic address: libo@nifdc.org.cn.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
mesh_terms: D000818:Animals; D000974:Antibodies, Antinuclear / Q000032:analysis*; D015551:Autoimmunity / Q000187:drug effects* / Q000276:immunology*; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D056945:Hep G2 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D011914:Rats, Inbred BN; D017208:Rats, Wistar; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D000974:Antibodies, Antinuclear
keywords: Age-related ANAs; Antinuclear antibodies (ANAs); Biomarker; Drug-induced autoimmunity (DIA)