title: Analysis and solution of false-positives when testing CVA16 sera using an antibody assay against the EV71 virus.
abstract: Hand, foot and mouth disease (HFMD) in humans is caused mainly by Enterovirus 71(EV71) and Coxsackievirus A16 (CVA16). EV71 is associated with severe HFMD cases but not CVA16. Use of IgM-capture enzyme-linked immunosorbent assay (ELISA) is important for the early diagnosis of EV71 infection, but cross-reactivity of the anti-CVA16 IgM antibody with EV71 produces false-positive results. In this report, we designed a new EV71 IgM-capture ELISA method using the EV71 VP1 peptide instead of the EV71 virion as the detectable antigen, and tested sera from patients infected with EV71 or CVA16. The results showed that acute sera from 76 EV71-infected patients had similar sensitivity for virus detection (98.68%) or VP1 detection (97.37%). When acute sera from patients infected with CVA16 were used, significant differences between the two methods were observed. The cross-reactivity rate of the virus detection method was 29.4% (5/17), but no cross-reactivity was observed using the VP1 detection method. Western immunoblotting demonstrated that EV71 VP3 cross-reacted with part of the CVA16 IgM antibody. The results demonstrate that EV71 VP3 is the cross-reactive antigen in the EV71 IgM-capture ELISA when testing CVA16 sera using the virus-antibody detection method. The problem of false-positive results was resolved by using the VP1 peptide as the detectable antigen.
authors.affiliation: Guangzhou Women and Children's Medical Center, Guangzhou Medical College, Guangzhou 510120, China. changbing2001@163.com
authors.forename: Changbing
authors.initials: C
authors.lastname: Wang
authors.forename: Aiping
authors.initials: A
authors.lastname: You
authors.forename: Xingui
authors.initials: X
authors.lastname: Tian
authors.forename: Mingqi
authors.initials: M
authors.lastname: Zhao
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Tao
authors.initials: T
authors.lastname: Lin
authors.forename: Jianbin
authors.initials: J
authors.lastname: Zheng
authors.forename: Misi
authors.initials: M
authors.lastname: Xiao
authors.forename: Yingying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Kuang
authors.forename: Zhenwen
authors.initials: Z
authors.lastname: Zhou
authors.forename: Bing
authors.initials: B
authors.lastname: Zhu
mesh_terms: D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral; D002648:Child; D002675:Child, Preschool; D003429:Cross Reactions; D003955:Diagnostic Tests, Routine / Q000379:methods*; D004770:Enterovirus / Q000276:immunology*; D029821:Enterovirus A, Human / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005189:False Positive Reactions; D006232:Hand, Foot and Mouth Disease / Q000175:diagnosis* / Q000821:virology*; D006801:Humans; D007075:Immunoglobulin M / Q000097:blood*; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods*
chemical_list: D000914:Antibodies, Viral; D000956:Antigens, Viral; D007075:Immunoglobulin M
keywords: 3,3′,5,5′-tetramethylbenzidine; 3,3′-diaminobenzidine; 50% tissue culture infective dose; CSF; CVA16; Coxsackievirus A16; Cross-reactivity; DAB; ELISA; EV71; Enterovirus 71; FBS; HFMD; HRP; OD; PBS; RT-PCR; SDS-PAGE; TCID(50); TMB; VP1; VP3; cerebrospinal fluid; enzyme-linked immunosorbent assay; hand, foot and mouth disease; horseradish peroxidase; optical density; phosphate-buffered saline; real-time polymerase chain reaction; rhabdomyosarcoma (RD) cells, fetal bovine serum; sodium dodecyl sulfate polyacrylamide gel electrophoresis