Universal capture array for multiplexed subtype-specific quantification and stability determination of influenza proteins
Inventors
Rowlen, Kathy • KUCK, Laura R.
Assignees
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Publication Number
US-10732180-B2
Publication Date
2020-08-04
Expiration Date
Abstract
A universal array for multiplexed quantification of variable hemagglutinin such as subcomponents of multivalent annual influenza vaccines that is robust to variations in proteins such as mutations and is capable of quantifying degradation of proteins. Universal capture array (100) comprises one or more substrates (102) and a low-density microarray (104) of sub-arrays (108) comprising spots (106a-c). The microarray (104) is contacted with one or more targets (202) at one or more unknown concentrations, and bound complexes (203) are formed and subsequently quantified with a suitable method. Quantified signals are compared to calibration curves to obtain one or more unknown concentrations and/or quantify degradation of the one or more targets (202). Other embodiments are described and shown.
Core Innovation
The described invention provides a method for determining one or more unknown concentrations of hemagglutinin proteins in a vaccine sample using one or more substrates comprising at least one microarray. Each microarray includes at least one positive control spot and at least one negative control spot, and includes more than one subarray with a plurality of capture agent spots, where each capture agent spot includes a murine antibody configured to bind to a linear or conformational epitope of an influenza hemagglutinin protein.
In the method, the microarray is contacted with the sample to form one or more bound complexes, and one or more signals corresponding to an amount of the bound complexes are generated at each spot. The signals are quantified to generate one or more quantified signals, and one or more calibration curves establish a one-to-one relationship between a concentration of a reference hemagglutinin protein and the quantified signals. The quantified signals are compared to the calibration curves to determine the one or more unknown concentrations of the hemagglutinin proteins in the sample.
A plurality of capture agents that are different but related targets different subsets of a class of antigen, and the sub-arrays include anti-A/H1 monoclonal antibodies for first and second conformational epitopes, anti-A/H3 monoclonal antibodies for third and fourth conformational epitopes, anti-B/Yamagata monoclonal antibodies for fifth and sixth conformational epitopes, and anti-B/Victoria monoclonal antibodies for seventh and eighth conformational epitopes. The sub-arrays collectively enable detection of hemagglutinin proteins associated with influenza of at least one of A/H1, A/H3, B/Yamagata, or B/Victoria over a period selected from the range of one to six years despite antigenic drift.
The method is additionally described as simultaneously determining concentration and stability of hemagglutinin proteins. A single label agent capable of binding to hemagglutinin associated with each predetermined influenza subtype and/or lineage is used, and the step of comparing simultaneously determines the hemagglutinin concentration and a stability of the hemagglutinin protein, or the concentration of hemagglutinin proteins from each predetermined influenza subtype and/or lineage.
Claims Coverage
The partial content contains three independent claims (clm-00001, clm-00018, clm-00019). Together they cover three inventive features: microarray-based quantification of unknown hemagglutinin concentrations, simultaneous determination of hemagglutinin concentration and stability using a single label agent, and simultaneous determination of hemagglutinin concentration for each predetermined subtype/lineage using a single label agent.
Universal microarray quantification of unknown hemagglutinin concentrations with control spots and calibration curves
Providing substrates comprising at least one microarray, each microarray having positive control spots, negative control spots, and more than one subarray with capture agent spots including murine antibodies configured to bind linear or conformational epitopes of influenza hemagglutinin, generating signals corresponding to bound complexes, quantifying the signals, providing calibration curves establishing a one-to-one relationship between reference hemagglutinin concentration and quantified signals, and comparing to determine unknown hemagglutinin protein concentrations.
Related-but-different capture agents across multiple influenza subtypes/lineages for detection despite antigenic drift
Configuring the microarray with a plurality of different but related capture agents that target different subsets of a class of antigen, with sub-arrays including anti-A/H1 monoclonal antibodies for first and second conformational epitopes, anti-A/H3 monoclonal antibodies for third and fourth conformational epitopes, anti-B/Yamagata monoclonal antibodies for fifth and sixth conformational epitopes, and anti-B/Victoria monoclonal antibodies for seventh and eighth conformational epitopes, such that the sub-arrays collectively detect hemagglutinin proteins associated with influenza of at least one of A/H1, A/H3, B/Yamagata HA or B/Victoria antigen over a period selected from one to six years despite antigenic drift.
Single label agent contacting and simultaneous concentration-and-stability determination
During generating by contacting bound complexes with a single label agent capable of binding to hemagglutinin associated with each predetermined influenza subtype and/or lineage, and in a step of comparing, simultaneously determining a concentration and a stability of the hemagglutinin protein.
Single label agent contacting and simultaneous subtype/lineage concentration determination
During generating by contacting bound complexes with a single label agent capable of binding to hemagglutinin associated with each predetermined influenza subtype and/or lineage, and in a step of comparing, simultaneously determining a concentration of the hemagglutinin proteins from each of the predetermined influenza subtypes and/or lineages.
The claim set centers on microarray-based quantification of unknown hemagglutinin concentrations using murine antibody capture agents with positive/negative controls and calibration curves, and extends this with a universal multi-subtype/lineage capture-agent layout designed to operate over one to six years despite antigenic drift. Additional independent coverage specifies the use of a single label agent to enable simultaneous determination of concentration and stability, or concentration for each predetermined subtype/lineage.
Stated Advantages
Eliminates a need to change the plurality of capture agents to adapt to seasonal influenza mutations while enabling detection over a period selected from one to six years despite antigenic drift.
Simultaneously determines a concentration and a stability of the hemagglutinin protein.
Simultaneously determines the concentration of hemagglutinin proteins from each predetermined influenza subtype and/or lineage.
Documented Applications
Determining unknown concentrations of hemagglutinin proteins in a vaccine sample using hemagglutinin-binding microarrays.
Simultaneous determination of hemagglutinin concentration and stability.
Multiplex detection of hemagglutinin proteins associated with influenza of at least one of A/H1, A/H3, B/Yamagata HA, or B/Victoria antigen over a period selected from one to six years despite antigenic drift.
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