Immunoassay platform for the serological discrimination of closely related viruses
Inventors
Zhou, Yan • Zhou, Zhiguo • Li, Qian
Assignees
Publication Number
US-12196755-B2
Publication Date
2025-01-14
Expiration Date
2041-12-02
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Abstract
An immunoassay platform and methodology are described for discriminatively detecting target microorganism antibodies to a target antigen in a biological sample derived from a host animal, from among antibodies of cross-reactively-related microorganism(s) potentially present in the biological sample, by use of contemporaneous assays conducted with and without blocking antibodies exogenous to the host animal. Attenuation of detection reagent signal between the contemporaneous assays may be used to determine a target antigen previously infected or non-infected status of the host animal. An assay system is described, including: a single-use cartridge containing test strips constituted to perform the immunoassay; an integrated sample collection and processing device engageable with the single-use cartridge for delivery of sample thereto, and for operatively initiating immunoassay performance in the single-use cartridge; and a portable immunoassay reader for reading immunoassay output signals from the test strips of the single-use cartridge.
Core Innovation
The invention provides an immunoassay platform and methodology for discriminatively detecting target microorganism antibodies in a biological sample from a host animal, particularly to distinguish specific virus infections among closely related viruses that exhibit high levels of cross-reactivity. The method involves contemporaneous assays conducted on pairs of immunoassay substrates with target microorganism antigens immobilized, where one assay includes exogenous blocking antibodies and the other does not. Signal attenuation between the two assays is used to determine the infection status of the host animal with respect to the target antigen.
The problem being solved addresses the difficulty in serological discrimination of viruses that are closely related and have high cross-reactivity between antibodies, such as the Flavivirus family including Dengue virus, Zika virus, and West Nile virus. Conventional immunoassays using traditional viral antigens are unable to effectively differentiate infections due to shared conserved epitopes causing cross-reactivity. Existing efforts to develop mutated recombinant antigens specific to target viruses are complex, time-consuming, and costly. This invention aims to provide a rapid, low-cost, simple and effective immunoassay platform that overcomes these challenges to specifically detect target virus antibodies in the presence of cross-reactive antibodies.
Claims Coverage
The patent includes two independent claims addressing immunoassays for discriminating antibodies specific for a target virus from cross-reactive antibodies in human and non-human samples, respectively, by using blocking antibodies in paired assays.
Immunoassay method for discriminating human antibodies specific to a target virus from cross-reacting human antibodies
The immunoassay comprises immobilizing target virus antigens on two substrates; introducing the same biological sample to each; applying non-human antibodies against the target virus antigens to one substrate while applying blank solution to the other; allowing competition of non-human blocking antibodies with human antibodies on the first substrate; introducing a human antibody detection reagent to both; and generating signals reflective of human antibodies bound to each substrate. The difference in signal levels between substrates indicates presence or absence of target virus antibodies.
Immunoassay method for discriminating non-human antibodies specific to a target virus from cross-reacting non-human antibodies
The method similarly immobilizes target virus antigens on two immunoassay substrates; introduces a non-human sample to both; applies blocking antibodies from other species to the first substrate but not the second; the blocking antibodies compete with non-human antibodies in the sample to bind antigen on the first substrate; non-human species antibody detection reagent is used on both substrates; and signals generated reflect antibody binding levels. A predetermined difference in signals between substrates indicates presence or absence of target virus antibodies in the non-human sample.
The independent claims cover inventive immunoassays that use paired assays with and without non-host species blocking antibodies competing with sample antibodies on immobilized target viral antigens, detection of host or non-host species antibodies, and analysis of differential output signals to discriminate target virus specific antibodies from cross-reactive related virus antibodies in biological samples.
Stated Advantages
The immunoassay platform is rapid, simple, low cost, and can be conducted in under 20 minutes, enabling point-of-care use.
It provides high specificity discrimination between target virus antibodies and cross-reactive antibodies from closely related viruses using traditional viral antigens without requiring development of mutant antigens or specially modified reagents.
The method is universally applicable across different viruses and host species, and can differentiate closely related viruses exhibiting high levels of serological cross-reactivity.
The platform can assess prior infection status, monitor vaccine immunogenicity, and track epidemiology and progression of viral infections.
Documented Applications
Discrimination of Dengue virus infection from other closely related flaviviruses such as Zika virus and West Nile virus in human biological samples.
Measurement of IgG antibodies against Dengue virus Non-Structural Protein 1 (NS1) to inform vaccine administration decisions.
General serological discrimination of any target virus from closely related cross-reactive viruses in biological samples of humans or non-human species, including viruses in families such as Flaviviridae, Coronaviridae, Herpesviridae, Orthomyxoviridae, Hepadnaviridae, and others.
Application on lateral flow immunoassay platforms, ELISA platforms, and microparticle enzyme immunoassay platforms using a single-use cartridge system, integrated sample collection and processing devices, and portable readers.
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