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Luciferase Immunosorbent Assay Based on Multiple E Antigens for the Detection of Chikungunya Virus-Specific IgG Antibodies.

ABSTRACT: In recent years, the chikungunya virus (CHIKV) has continued to spread from local epidemics to nonnative habitats until eventually reaching pandemic status. Nonendemic areas such as China have also emerged as potential epidemic areas of CHIKV. Serological detection of CHIKV is the key to diagnosing and controlling the prevalence of this virus. In this study, we review the progress of the serological detection of the envelope (E) protein in CHIKV, and we provide a novel research assay and ideas for the serological detection of CHIKV. The luciferase immunosorbent assay (LISA) does not require species-specific labeled secondary antibodies for detection, making it universally suitable for tracking samples from various animals or carriers. At present, most research on CHIKV antigen detection technology tends to combine two or more proteins to avoid the decrease in detection ability caused by antigen mutation. Our results indicate that two or more kinds of CHIKV E antigens combined with LISA detection can improve the detection rate of anti-CHIKV immunoglobulin G (IgG) antibodies in CHIKV-infected patient sera and detect antibodies in the early stage of infection accurately and sensitively. After 235 days of infection, the anti-CHIKV IgG antibodies could still be detected in CHIKV-infected patients. All serum samples were tested with a detection rate of 100% after combining various recombinant CHIKV E antigens. Our proposed CHIKV-specific LISA could be a useful tool for serum diagnosis of CHIKV infection and serum epidemic research in areas where CHIKV is endemic, which would help to manage potential epidemics in the future. IMPORTANCE At present, chikungunya virus (CHIKV) is still circulating in some parts of the world, and mutated strains have emerged, making it easier for the virus to spread among humans. With the continuous variation of CHIKV, its antigen variation leads to the decline of detection ability. In addition, the risk of transmission of CHIKV in areas where CHIKV is not endemic, such as China, has increased dramatically, which compels us to enhance the detection capacity of CHIKV and continuously monitor CHIKV antibody levels in the population. Real-time quantitative PCR (RT-PCR) detection technology will not be reliable when the infection time is chronic or in subclinical infection due to decreases in virus concentration, and an antibody detection technology must be adopted. In this study, multiple CHIKV envelope (E) antigens were used to detect anti-CHIKV IgG antibodies in serum for the first time. The new assay is characterized by convenient operation, high detection rate, and high sensitivity and has significance for early warning and monitoring. Moreover, it contributes to the prevention and control of CHIKV.

SUBMITTER: Li X

PROVIDER: S-EPMC9045172 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Middle East respiratory syndrome (MERS) is a lethal zoonosis caused by MERS coronavirus (MERS-CoV) and poses a significant threat to public health worldwide. Therefore, a rapid, sensitive, and specific serologic test for detecting anti-MERS-CoV antibodies in both humans and animals is urgently needed for the successful management of this illness. Here, we evaluated various novel luciferase immunosorbent assays (LISA) based on nucleocapsid protein (NP) as well as fragments derived from spike protein (S) including subunit 1 (S1), N terminal domain (NTD), receptor-binding domain (RBD) and subunit 2 (S2) of S for the detection of MERS-CoV-specific IgG. Fusion proteins, including nanoluciferase (NLuc) and various fragments derived from the NP or S protein of MERS-CoV, were expressed in human embryonic kidney 293 T cells. LISAs that detected anti-MERS-CoV IgG were further developed using cell lysates expressing various fusion proteins. Panels of human or animal samples infected with MERS-CoV were used to analyze the sensitivity and specificity of various LISAs in reference to a MERS-CoV RT-PCR, commercial S1-based ELISA, and pseudovirus particle neutralization test (ppNT). Our results showed that the S1-, RBD-, and NP-LISAs were more sensitive than the NTD- and S2-LISAs for the detection of anti-MERS-CoV IgG. Furthermore, the S1-, RBD-, and NP-LISAs were more sensitive (by at least 16-fold) than the commercially available S1-ELISA. Moreover, the S1-, RBD-, and NP-LISA specifically recognized anti-MERS-CoV IgG and did not cross-react with samples derived from other human CoV (OC43, 229E, HKU1, NL63)-infected patients. More importantly, these LISAs proved their applicability and reliability for detecting anti-MERS-CoV IgG in samples from camels, monkeys, and mice, among which the RBD-LISA exhibited excellent performance. The results of this study suggest that the novel MERS-CoV RBD- and S1- LISAs are highly effective platforms for the rapid and sensitive detection of anti-MERS-CoV IgG in human and animal samples. These assays have the potential to be used as serologic tests for the management and control of MERS-CoV infection.

Project description:BACKGROUND:Porcine epidemic diarrhea virus (PEDV) infection is a highly contagious infectious disease causing watery diarrhea, vomiting, dehydration and high mortality rate in newborn piglets. PEDV infection can cause high economic losses in pig industry. In Japan, a PEDV outbreak occurred with high mortality from 2013 to 2015. Even though until now, PEDV infection occurs sporadically. For the control and monitoring of PEDV infection, not only symptomatic pigs, but also asymptomatic pigs should be identified. The objective of this study is to develop and optimize novel indirect ELISA as a simple, rapid, sensitive and specific method for the detection of anti-PEDV antibodies and evaluate the efficacy of the assay as a diagnostic method for PED. RESULTS:One hundred sixty-two serum samples, consisting of 81 neutralization test (NT) positive and 81 NT negative sera, were applied to the assay. Indirect ELISA test based on whole virus antigen (NK94P6 strain) derived from Vero cell culture was evaluated by receiver operating characteristic (ROC) analysis with neutralization test (NT) as a reference method, and cut-off value was determined as 0.320 with sensitivity and specificity of 92.6 and 90.1%, respectively. The area under curve (AUC) was 0.949, indicating excellent accuracy of indirect ELISA test. There was significant positive correlation between indirect ELISA and neutralization test (R = 0.815, P < 0.05). Furthermore, the kappa statics showed the excellent agreement between these two tests (kappa value = 0.815). In addition, the sensitivity and specificity of preserved plates with different periods (1 day, 2 weeks, 1, 2, 3, 4, 5 and 6 months) after drying antigen coated plates were 100% and 80-100%, respectively. CONCLUSIONS:The developed indirect ELISA test in our study would be useful as a reliable test for serological survey and disease control of PEDV infection, and our pre-antigen coated ELISA plates can be preserved at 4 °C until at least 6 months.

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Luciferase Immunosorbent Assay Based on Multiple E Antigens for the Detection of Chikungunya Virus-Specific IgG Antibodies.

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