Consistently, mAb 5-F9 demonstrated the highest binding activity in its interaction with the full-length PEDV-S1 protein (Fig.?4B). Open in a separate window Fig. and produce recombinant S1 protein. Through quantitative analysis, five monoclonal antibodies (mAbs) specifically targeting the recombinant S1 protein of PEDV were developed and subsequently evaluated using enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry assay (FCA). The results indicate that all five mAbs belong to the IgG1 isotype, and their half-maximal effective concentration (EC50) values measured at 84.77, 7.42, 0.89, 14.64, and 7.86 pM. All these five mAbs can be utilized in ELISA, FCA, and IFA for the detection of PEDV infection. MAb 5-F9 exhibits the highest sensitivity to detect as low as 0.3125 ng/mL of recombinant PEDV-S1 protein in ELISA, while only 0.096 ng/mL of mAb 5-F9 is required to detect PEDV in FCA. The results from antigen epitope analysis indicated that mAb 8-G2 is the sole antibody capable of recognizing linear epitopes. In conclusion, this study has yielded a highly immunogenic S1 protein and five high-affinity mAbs specifically targeting the S1 protein. These findings have significant implications for early detection of PEDV infection and provide a solid foundation for further investigation into studying virus-host interactions. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-024-04091-y. Keywords: Porcine epidemic diarrhea virus (PEDV), Recombinant PEDV S1 protein, Monoclonal antibody, High affinity Background Porcine epidemic diarrhea virus (PEDV) is an enteric RNA virus that belongs to the family [1, 2]. It is the causative agent of porcine epidemic diarrhea (PED), a diarrheal disease in swine. In the early 1970s, the swine industries of Europe and Asia experienced their first outbreak of PED, which subsequently spread to numerous other nations [3, 4]. China discovered new and highly virulent strains of PEDV in 2010 2010, leading to its widespread dissemination across multiple Cediranib (AZD2171) countries [2, 5]. The majority of pigs infected with PEDV exhibit symptoms such as vomiting, diarrhea, and dehydration [6]. This disease can affect pigs of all ages [7, 8], but poses Cediranib (AZD2171) a particularly high morbidity and mortality risk for suckling piglets. Globally, the infection of PEDV has resulted in substantial economic losses within the pig-breeding industry [9]. PEDV is a single-stranded RNA virus with a genome size of about 28?kb, and comprises four crucial structural proteins: spike (S), envelope, membrane, and nucleocapsid proteins [4, 10C12]. Among them, the S protein plays a critical role in the process of viral infection, which plays a crucial role in facilitating virus-cell recognition events and promoting viral entry into host cells [5, 13, 14]. As with other coronaviruses, the S protein of PEDV can be split into the S1 (1C735 aa) and S2 subunits (736C1,383 aa) [15, 16]. The neutralizing epitopes are primarily located in the S1 subunit [17C19], and the S1 subunit of S protein can Cediranib (AZD2171) induce protective immunity in pigs [20, 21]. Several S-based enzyme-linked immunosorbent assays (ELISAs) have been developed [22, 23] and demonstrated to be specific for PEDV without cross-reactivity with other swine coronaviruses. As a result, an S-based ELISA is more suitable for developing a specific detection method for PEDV. Although several monoclonal antibodies have been developed against the PEDV-S protein [17, 24C28], the researchers primarily focus on characterizing mAbs based on their specificity, neutralization capacity, or therapeutic potential, the quantitative analysis of the affinity activities of these monoclonal antibodies (mAbs) has been scarcely conducted. Current detection methods for PEDV suffer from issues such as low sensitivity, specificity, and difficulties in early detection. However, high-affinity antibodies can enhance the sensitivity of antibody-based viral detection by effectively binding to viral surface antigens, generating strong signals even at low viral concentrations. Thus, high-affinity antibodies are crucial for the sensitive detection of target proteins, which is particularly important in early PEDV infection diagnosis. In this study, we utilized a eukaryotic expression system to produce PEDV-S1 recombinant protein for immunization of mice in order to generate high-affinity antibodies against PEDV. Through quantitative analysis, five mAbs with high affinity were generated and their half-maximal effective concentration (EC50) values reached the ?10C13 M scale. These mAbs have the potential to be utilized for the early identification of PEDV infection, and our research provides a solid foundation for further investigation. Results Cediranib (AZD2171) The expression and purification of PEDV-S1 protein The gene encoding S1 subunit (234C736 amino acids) of PEDV S protein TSPAN7 was cloned into plasmid pcDNA3.4 with a six-histidine (6His)-tag at its C-terminus. The recombinant vector pcDNA3.4-S1 was transfected into.