Zika Virus Non-Structural Protein 1 Antigen-Capture Immunoassay

Abstract

Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.

Identification of Zika Virus NS1-Derived Peptides with Potential Applications in Serological Tests

“…The assays based on rabbit anti-ZIKV NS1 pAb and mAb 2F2 showed similar detection limits to DENV-NS1 capture ELISAs, such as SD Bioline’s detection limit of between 16 and 63 ng/mL NS1 [ 48 ]. Moreover, our results are in line with an antigen-capture ELISA configured with the affinity-purified anti-ZIKV NS1 pAb, for which the reported limit of detection was between 1.95 and 7.8 ng/mL [ 42 ].…”

“…The mentioned similarities guided us to test a small ZIKV sera panel obtained from infected patients and validated by qRT-PCR. The results showed an accuracy of 77.2%, which was superior to the one previously obtained by Beddingfield et al (2021) [ 42 ], for which the detection limit was similar to the one obtained by us (55.5% vs. 55.7%). Additionally, other studies have been published with similar approaches based on capturing ZIKV-NS1 [ 45 ].…”

“…It has a reported sensitivity and specificity of 86.8% and 97.8%, respectively [ 40 ]; however, one potential issue may be the lack of sensitivity in secondary dengue cases, as reported in one study indicated 100% sensitivity in primary infections and 10% sensitivity in secondary infections [ 41 ]. Even so, the successful detection of DENV through NS1-based assays offers an attractive option for the development and optimization of immunoassays against other flaviviruses [ 42 ].…”

“…Previous studies have identified similar linear and discontinuous flavivirus epitopes from naturally infected humans [ 49 ]. The epitope REGYRT mapped on immunodominant region three of the NS1 loop has been previously described [ 42 ]. Through alignments of the NS1 full epitope sequence between flavivirus, there is a predominance of residues that are fully conserved, in addition to others, with a conservation between groups of strongly and weakly similar properties [ 50 , 51 ].…”

Multiplexed rapid antigen tests developed using multicolored nanoparticles and cross-reactive antibody pairs: Implications for pandemic preparedness

“…The data in Table S4 demonstrate that the lateral flow tests detect Dengue NS1 proteins in the range of 30-100 ng/ml, while the concentration of circulating Dengue NS1 protein has been reported to reach 15-50 micrograms/ml [5]. The limit of detection for Zika virus NS1 protein is approximately 220 ng/ml (Table S4), while circulating Zika virus NS1 levels has been reported to reach about 2 micrograms/ml [16].…”