Initially, serum samples were classified as ASFV-seronegative and ASFV-seropositive based on their origin and their result on Ingezim PPA COMPAC and later all the samples were tested by the newly developed cELISA. (178 negative and 187 positive samples) were tested and a receiver-operating characteristic (ROC) analysis was applied to determine the cut-off value. Based on the ROC analysis, the area under the curve (AUC) was 0.982 (95% confidence interval: 96.9% to 99.4%), besides a sensitivity of 92.5% and a specificity of 98.9% was achieved when the percent inhibition of 20% was selected as a threshold. Moreover, the result showed an excellent agreement when compared to other commercially available blocking ELISA (kappa value = 0.912) and showed no reaction to other swine pathogens. Overall, the newly developed cELISA method offers a promising approach for a rapid and convenient ASFV serodiagnosis, which could be used as alternative to other serological assays for screening possible ASFV infection. Keywords: African swine fever virus, p54, monoclonal antibodies, competitive ELISA, diagnosis 1. Introduction Menaquinone-4 African swine fever (ASF) is a highly lethal hemorrhagic viral disease of swine that usually results to a mortality rate approaching 100% in domestic pigs and is classified as a notifiable disease by the World Organization for Animal Health (OIE). Currently, it is the major threat to global pig industry and is caused by African swine fever virus (ASFV), a large and complex double stranded DNA virus with icosahedral morphology [1,2]. Despite of the extensive ongoing research on ASFV, a safe Menaquinone-4 and effective vaccine is still lacking and its control and eradication solely depends on its rapid and accurate diagnosis. ASFV affects both domestic and wild pigs. However, in domestic pigs there is a variation in the clinical manifestations depending on the virus strains, which differs from an acute, highly lethal hemorrhagic disease to a mild inapparent infection [3,4]. On the contrary, it leads to a mild subclinical infection in the natural reservoirs hosts (wart hogs and bush pigs), which are the potential source of infection to domestic pigs. It is also important to note that, wild boars (the substantial reservoir of ASFV in Europe and IFI27 probably also in Asia) show a high susceptibility to ASFV with a disease development similar to domestic pigs [5]. Owing to the importance of the presence of seropositive animals to sub-acute or chronic form of ASFV, there is always a need of an accurate serological diagnosis. Additionally, in the recent years there have been a dramatic increase of ASF outbreak in some Asian and European countries which led to a renewed interest of combating the disease [6,7,8,9]. Therefore, to detect sub-acute or chronic nature of ASF either in domestic pigs or Menaquinone-4 the reservoir hosts, a range of sensitive serological investigations are needed and Menaquinone-4 antibody detection is a rational approach. Numerous ELISA-based serological assays integrating the major capsid protein p72/B646L, the structural and highly immunogenic protein p30/CP204L and polyprotein pp62/CP530R antigens are available for Menaquinone-4 ASFV antibody detection [10,11,12,13]. Alternatively, the structural and immunogenic p54/E183L protein is also a highly relevant antigen for serological diagnosis [11,14,15]. P54/E183L is a type II transmembrane protein and contains a potential membrane-spanning domain close to the N-terminus [16]. A two-dimensional gel electrophoresis study on ASFV particle, revealed that p54 has a molecular size of about 25 kDa with an isoelectric point of 6.5 [17]. Also, during cell culture adoption and propagation, ASFV generates virus sup-populations differing in p54 molecular size, which may have a role on the mechanism of genetic diversification of ASFV [16,17]. A number of studies have reported that p54 is one of the most important ASFV proteins and plays a key role in virus morphogenesis and viral infection. Anti-p54 sera were found to inhibit ASFV attachment.