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.

Keywords: CHIKV antibodies; chikungunya virus (CHIKV); envelope (E) antigens; envelope protein; luciferase immunosorbent assay (LISA).

FIG 1

(A) A brief discourse on CHIKV structure. (B) Cloning strategy of recombinant CHIKV E glycoprotein in pNLF1-N. Schematic diagram showing the chimeras used in sero-neutralization. Cloning strategy of recombinant CHIKV E2 glycoprotein in pNLF1-N. Schematic representation of the E2 and E1 glycoprotein, domain A, domain B, domain C, β-ribbon, transmembrane region (TM), and cytoplasmic tail (C tail). (C) A vector map of pNLF1-N with EcoRI/EcoRV and Notl/ XbaI restriction sites. (D) Mouse anti-His (α-His, at 1:7,000 dilution) are used to detect recombinant protein. (E) Schematic of the LISA and its validation based on different recombinant proteins.

FIG 2

Serum samples from healthy subjects (“healthy control”) and patients with CHIKV (“panel A”) were assessed for anti-CHIKV IgG antibodies by LISA. (A) LISA can detect samples with E1-full (numbers 16 and 17), E1-C1 (numbers 7, 16, and 17), and E1-C2 (numbers 7, 16, and 17). (B) LISA can detect samples with E2 full-length (numbers 1, 3, 5, 6, 10, and 19), E2-C1 (numbers 3, 10, 12, 13, 16, 17, 18, 19, and 20), E2-C2 (numbers 2, 3, 8, 10, 11, 12, 14, 16, 17, and 19), E2-C3 (numbers 1, 4, 5, 6, 7, 11, 15, 16, and 17), and E2-C4 (numbers 3, 6, 9, 11, 12, 14, 16, 17, 18, 19, and 20).

FIG 3

(A) We defined the anti-CHIKV IgG antibody-positive RFI cutoff value as 5 × 10⁴ and consider it a positive result when CHIKV-infected patients were tested with the LISA assay. The eight assays could detect anti-CHIKV IgG antibodies in patient sera by LISA; serum data were collected. (B) Comparison of CHIKV E1 antigen and CHIKV E2 antigen detection sample volume. The C2 antigen group with the largest sample size detected by CHIKV E1, the E2-full antigen group with the lowest sample size detected by CHIKV-E2, and the C2-C4 antigen group with the largest sample size detected by CHIKV E2 were statistically analyzed. Data that are statistically significant are marked with an asterisk; *, P < 0.05.

FIG 4

(A) Serum samples from a patient with CHIKV infection, collected on day 3 (number 16) and day 235 (number 17) after admission in the hospital, were serially diluted, and the relative fluorescence intensity (RFI) was determined using the method as described above. The cutoff value was decided based on values from 21 healthy donors (numbers 66 to 87). (B) Sensitivity comparison between LISA and ELISA. (C) Coefficient of variation for LISA. (D) Antigen specificity and cross-reactivity (P > 0.1).

FIG 5

(A) ELISA detection of anti-CHIKV IgM and IgG antibodies in CHIKV-infected sera. (B) ELISA and LISA E2 detection assays to detect the number of CHIKV IgG antibody samples. CHIKV IgG antibody was detected by LISA E2 and LISA E2. Data that are statistically significant are marked with an asterisk; *, P < 0.05.