Enteroviruses (EVs) are common seasonal viruses that are associated with a variety of diseases. High-quality monoclonal antibodies (MAbs) are needed to improve the accuracy of EV diagnosis in clinical laboratories. In the present study, the full-length VP1 genes of poliovirus 1 (Polio 1) and coxsackievirus B3 (Cox B3) were cloned, and the encoded proteins were expressed and used as antigens in an attempt to raise broad-spectrum MAbs to EVs. Two pan-EV MAbs were isolated: one raised against Polio 1 VP1 and the other against Cox B3 VP1. The binding sites of both pan-EV MAbs were mapped to an amino acid sequence within a conserved region in the N terminus of Polio 1 VP1 by peptide and competition enzyme-linked immunosorbent assay. Two additional MAbs, an EV70-specific MAb and an EV71/Cox A16-bispecific MAb, developed against EV70 and 71 VP1 proteins, were pooled with the two pan-EV MAbs (pan-EV MAb mix) and tested for their sensitivity and specificity in the staining of various virus-infected cells. The pan-EV MAb mix detected all 40 prototype EVs tested and showed no cross-reactivity to 18 different non-EV human viruses. Compared with two commercially available EV tests, the pan-EV MAb mix exhibited higher specificity than one test and broader spectrum reactivity than the other. Thus, our study demonstrates that full-length Polio 1 VP1 and Cox B3 VP1 can serve as effective antigens for developing a pan-EV MAb and that the pan-EV MAb mix can be used for the laboratory diagnosis of a wide range of EV infections. Human enteroviruses (EVs) are classified into four species:Human enterovirus A (coxsackievirus A2 [Cox A2], A3, A5, A7, A8, A10, A12, A14, and A16 and EVs 71, 76, 89, 90, and 91), Human enterovirus B (Cox A9 and B1 to B6; echoviruses 1 to 7 [Echo 1 to 7], 9, 11 to 27, and 29 to 33; and EVs 69, 73 to 75, 77 to 88, 97, 100, and 101), Human enterovirus C (Cox A1, A11, A13, A17, A19 to A22, and A24; polioviruses 1 to 3 [Polio 1 to 3]; and EV96), and Human enterovirus D (EV68 and EV70) (14). Infection with these EVs causes a wide range of diseases in humans, from mild respiratory illness to severe aseptic meningitis. Diagnosis of EV infection depends mainly upon laboratory testing, since the clinical symptoms vary and overlap with other diseases. Laboratory diagnosis of EV infection is currently determined with either reverse transcription-PCR to detect EV RNA or by isolating the virus in cell culture followed by monoclonal antibody (MAb) staining (15,19). However, the two commercially available pan-EV MAbs used in the staining assay either fail to react with multiple EV serotypes (9, 19) or cross-react with other non-EVs (8, 22). The lack of availability of highly reactive and specific pan-EV MAbs for diagnosis of EV infection could be due to the difficulties in developing specific MAbs against the extensive antigenic diversity among EVs.EV capsid protein VP1 is one of four structural proteins of EV, and its antigenic homology among many different EV serotypes has been well documented (2,7,(16)(17)(18). The N te...