# Patent

## Title
Arrayed imaging reflectometry (air) sensor chip comprising influenza hemagglutinin (HA) polypeptides suitable for the detection of antiviral immune responses

## Abstract

A sensor chip for detecting an immune response against an influenza virus, the sensor chip including a substrate having a surface and a plurality of hemagglutinin polypeptides bound to discrete locations on the surface of the substrate, each hemagglutinin polypeptide having a hemagglutinin epitope. Detection devices containing the sensor chip and methods of detecting influenza immune responses are also described herein.

## Background
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/050,039, filed May 2, 2008, which is hereby incorporated by reference in its entirety.


This invention was made with government support under grant number R24-AL054953 awarded by the National Institutes of Health. The government has certain rights in this invention.


FIELD OF THE INVENTION
This invention relates to sensor chips designed for the detection of an immune response to a particular antigen, which can be used either to detect an individual's prior exposure to a pathogen that possesses the antigen, for epidemiological purposes to survey the extent of infection among a population of individuals, or to measure the efficacy of a vaccine directed against the pathogen.
BACKGROUND OF THE INVENTION
The influenza virus is a membrane-enclosed RNA virus, whose genome consists of discretized segments of negative-sense RNA, and is primarily an infectious agent of avians and humans (Lamb et al., “The Gene Structure and Replication of Influenza Virus,” Annu Rev Biochem 52:467-506 (1983)). The influenza proteins responsible for docking, hemagglutinin (HA), and budding, neuraminidase (NA), are anchored in the viral lipid membrane and are the major antigenic focus of the prevention and treatment of infection. There are three distinct genera of the influenza virus—denoted A, B, and C, and typified by differences in structural proteins—that cause immunological effects of variable severity (Cox et al., “The Molecular Epidemiology of Influenza Viruses,” Semin Virol 6:359-370 (1995)), such as the typical symptoms associated with the flu (Monto et al., “Clinical Signs and Symptoms Predicting Influenza Infection,” Arch Intern Med 160:3243-3247 (2000)). Influenzas A and B are the most immunologically relevant due to their co-circulating seasonal infectivity in human populations (Lin et al., “Recent Changes Among Human Influenza Viruses,” Virus Res 103:47-52 (2004)), and influenza A has been responsible for all recorded pandemics. Influenza A infects many different animal species besides humans, including ducks, chickens, pigs, whales, horses, and seals. Influenza B viruses generally only infect humans.
All three types of influenza virus have genomes composed of eight different RNA helices, which encodes a single gene and are bound by a nucleoprotein that determines the viral type: A, B, or C. In effect, the influenza genome is made up of eight separate pieces of nucleic acid that can come together to form viruses with new combinations of viral genes when cells become co-infected by more than one viral type. Two of these RNA helices encode the important viral surface proteins hemagglutinin and neuraminidase, which are embedded in the lipid bilayer of a mature virus particle.
Variations in the viral hemagglutinin and neuraminidase determine the viral subtype. Hemagglutinin is responsible for entry of the virus into the host cell, while neuraminidase is important in the release of newly formed viruses from the infected cells. Antibodies to hemagglutinin can neutralize the virus and are the major determinant for immunity. Antibodies to neuraminidase do not neutralize the virus but may limit viral replication and the course of infection. Host antibodies to specific types of hemagglutinin and neuraminidase prevent and generally ameliorate future infection by the same viral strain. However, since the genetic makeup of viral strains is dynamic and ever-changing, immunity gained through successful resistance to one strain gained during an infection one year may be useless in combating a new, recombined, variant strain the next year.
Epidemics of influenza are thought to result when viral strains change over time by the process of antigenic drift. Antigenic drift (caused by mutations in the principal viral antigen genes, especially in the hemagglutinin or neuraminidase genes) results in small changes in surface antigens, and occurs essentially continuously over time. When these changes occur in the right places in the genes, they render the new antigens unrecognizable by the antibodies raised against other influenza virus strains during previous infections.
Influenza pandemics (or worldwide epidemics) occur as a result of “antigenic shift.” Antigenic shift is an abrupt, major change in an influenza A virus that results from a new hemagglutinin and/or new hemagglutinin and neuraminidase protein appearing in an influenza A strain. Such shifts are generally thought to occur when a new combination of viral genomic RNAs is created, possibly in a non-human species, and that new combination is passed to humans. When such an antigenic shift occurs, most humans have little or no protection against the virus, and an infection can prove lethal.
Periodically, unique strains of the influenza virus may emerge, and the antigenic novelty of the pathogen results in enhanced rates of infectivity, transmission, and morbidity. History has shown that these influenza pandemics may be extremely deadly. For example, the “Spanish Flu” (Johnson et al., “Updating the Accounts: Global mortality of the 1918-1920 “Spanish” Influenza Pandemic,” Bull Hist Med 76:105-115 (2002)) (H1N1; 1918-1920 upwards of 50 million deaths), the “Asian Flu” (Rajagopal et al., “Pandemic (avian) Influenza,” Semin Respir Crit. Care 28:159-170 (2007)) (H2N2; 1957-1958, 1 million deaths), and the “Hong Kong Flu” (Shalala et al., “Collaboration in the Fight Against Infectious Diseases,” Emerg Infect Dis 4:354-357 (1998)) (H3N2; 1968-1969, 700,000 deaths) demonstrated the efficiency of the virus and its ability to quickly spread worldwide; additionally, the rapidity in which infectious strains may emerge, and our innate resistance to them, was clearly highlighted. To combat these pandemics, the first mono- and bivalent influenza vaccines were developed in the mid-1940s, which consisted of deactivated influenza viruses (Henle et al., “Demonstration of the Efficacy of Vaccination Against Influenza Type A by Experimental Infection of Human Beings,” J Immunol 46:163-175 (1943), Francis et al., “Protective Effect of Vaccination Against Induced Influenza A,” J Clin Invest 24:536-546 (1945), Salk et al., “Protective Effect of Vaccination Against Induced Influenza B,” J Clin Invest 24:547-553 (1945)). Trivalent influenza vaccines have since been become standard and consist of three deactivated strains (Halperin et al., “Safety and Immunogenicity of a Trivalent, Inactivated, Mammalian Cell Culture-derived Influenza Vaccine in Healthy Adults, Seniors, and Children,” Vaccine 20:1240-1247 (2002)), or three live, attenuated strains (Belshe et al., “The Efficacy of Live Attenuated, Cold-adapted, Trivalent, Intranasal Influenzavirus Vaccine in Children,” New Engl J Med 338:1405-1412 (1998))—two of influenza A and one of influenza B—in order to provide broader preventative measures against co-circulating seasonal strains. Since these initial efforts, much research has been performed towards developing improved, more enveloping vaccines based on recombinant technology to not only immunize against current viral strains (Kilbourne et al., “Future Influenza Vaccines and the Use of Genetic Recombinants,” Bull World Health Org 41:643-645 (1969), Webby et al., “Are We Ready for Pandemic Influenza?,” Science 302:1519-1522 (2003), Treanor et al., “Safety and Immunogenicity of a Baculovirus-Expressed Hemagglutinin Influenza Vaccine: A Randomized Controlled Trial,” J Am Med Assoc 297:1577-1582 (2007)), but also provide protection against past epidemic strains as well.
Avian influenza, of the H5N1 designation, is currently the subject of major international research efforts. Past influenza pandemics have proven that in the absence of proper safeguards, new and highly pathogenic strains of influenza can be extremely deadly. With the rise in the global population and the advent of international travel and commerce, the repercussions of a pandemic would be devastating. Since it was initially isolated in 1997 (de Jong et al., “A Pandemic Warning?” Nature 389:554 (1997)), there have been a reported 380 cases of H5N1 that have resulted in 240 deaths (World Health Organization, “Epidemic and Pandemic Alert and Response: Avian Influenza,” accessed online from the WHO on Apr. 16, 2008). The majority of these reported cases are transmitted from avians to humans, but isolated cases of human-to-human transmission have been reported (Ungchusak et al, “Probable Person-to-Person Transmission of Avian Influenza A (H5N1),” N Engl J Med 352:333-340 (2005)).
Very recently, there have been reports of an H1N1, type A, strain of swine influenza that has unique genetic properties and is capable of human-to-human transmission. The initial outbreak appeared in Mexico, but cases have now been reported in a number of urban centers across the United States and elsewhere in the world. As of Apr. 30, 2009, the World Health Organization has raised the Alert Level to Phase 5.
Vaccines are essential as preventative measures against disease, but traditional drug-based therapeutics are also required in the event that the vaccine supply is limited or not yet available, scenarios that are especially worrisome in highly virulent pandemic viral strains such as with avian influenza H5N1 (Kilpatrick et al., “Predicting the Global Spread of H5N1 Avian Influenza,” Proc Natl Acad Sci USA 103:19368-19373 (2006)). For example, neuraminidase, the influenza enzyme that controls the release of the newly packaged virus from the host cell membrane (Wagner et al., “Interdependence of Hemagglutinin Glycosylation Neuraminidase as Regulators of Influenza Virus Growth: A Study by Reverse Genetics,” J Virol 74:6316-6323 (2000)), is an attractive drug target in the influenza lifecycle. Oseltamivir (TAMIFLUTM) (Kim et al., “Influenza Neuraminidase Inhibitors Possessing a Novel Hydrophobic Interaction in the Enzyme Active Site Design, Synthesis, and Structural Analysis of Carboxylic Acid Sialic Acid Analogues with Potent Anti-Influenza Activity,” J Am Chem Soc 119:681-690 (1997)) is an orally active antiviral that acts as a transition state mimic of the active site of neuraminidase. It is currently suggested that world centers begin stockpiling supplies of TAMIFLUTM (and other antivirals, such as Zanamivir (Itzstein et al., “Rational Design of Potent Sialidase-Based Inhibitors of Influenza Virus Replication,” Nature 363:418-423 (1993)) in the event of a sudden pandemic (Moscona et al., “Neuraminidase Inhibitors for Influenza,” New Engl J Med 353:1363-1373 (2005)). While antivirals are capable therapies, preventative rather than reactive measures will ultimately ensure long-term success against deadly influenza virus pandemics since drug-resistant forms of influenza are readily appearing (de Jong et al., “Oseltamivir Resistance During Treatment of Influenza A (H5N1) Infection,” New Engl J Med 353:2667-2672 (2005)).
An ancillary development stemming from researchers' ability to produce and amplify recombinant proteins, and the genes from which they are encoded, is the high-throughput microarray. While initial applications of high-throughput screening focused on genomic arrays (Schena et al., “Quantitative Monitoring of Gene Expression Patterns With a Complementary DNA Microarray,” Science 270:467-470 (1995), Lipshutz et al., “High density Synthetic Oligonucleotide Arrays,” Nat Genet. 21:20-24 (1999)), the protein microarray has found a variety of significant uses as well. For example, proteome profiling via protein microarrays has unveiled a myriad of novel interactions (MacBeath et al., “Printing Proteins as Microarrays for High-Throughput Function Determination,” Science 289:1760-1763 (2000), Michaud et al., “Analyzing Antibody Specificity With Whole Proteome Microarrays,” Nat Biotech 21:1509-1512 (2003), Chan et al., “Protein Microarrays for Multiplexed Analysis of Signal Transduction Pathways,” Nat Med 10: 1390-1396 (2004)). Protein microarrays have been used to discover antigenic proteins and monitor human immunological responses to them (Davies et al., “Profiling the Humoral Immune Response to Infection by Using Proteome Microarrays: High-Throughput Vaccine and Diagnostic Antigen Discovery,” Proc Natl Acad Sci USA 102:547-552 (2005), Li et al., “Protein Microarray for Profiling Antibody Responses to Yersinia pestis Live Vaccine,” Infect Immun 73:3734-3739 (2005), Qiu et al., “Antibody Responses to Individual Proteins of SARS Coronavirus and Their Neutralization Activities,” Microbes Infect 7:882-889 (2005)). This tactic has not been used previously for immobilization of multiple isoforms of the influenza antigen hemagglutinin. Moreover, in each of these reports, detection was achieved using labeled reagents.
It would be desirable to provide an array of immobilized antigen isoforms that can be used to screen for antibodies against infectious agents and vaccines involving multiple similar specificities, e.g., distinguishing between different strains of an infectious agent such as influenza based on the immune response generated by these infectious agents, or vaccines against them, using unlabeled reagents. In view of the possibility of influenza pandemic, it would also be desirable to develop a system capable of screening putative vaccine therapies for efficacy and/or cross-protection against various strains of influenza. Furthermore, a system able to rapidly screen for the presence of avian influenza or other strains in wildlife and livestock would be of considerable utility in monitoring the status and spread of the disease.
The present invention is directed to overcoming these and other deficiencies in the art.
SUMMARY OF THE INVENTION
A first aspect of the present invention relates to a sensor chip for detecting an immune response against an influenza virus. The sensor chip includes a substrate having a surface and a plurality of hemagglutinin polypeptides bound to discrete locations on the surface of the substrate, each hemagglutinin polypeptide having a hemagglutinin epitope, preferably the immunodominant epitope thereof. The sensor chip may optionally include a plurality of neuraminidase polypeptides bound to discrete locations on the surface of the substrate, each neuraminidase polypeptide having a neuraminidase epitope.
In preferred embodiments, the sensor chip is suitable for use in an Arrayed Imaging Reflectometry (“AIR”) detection system, a surface plasmon resonance (“SPR”) detection system, a Brewster Angle Straddle Interferometry (“BASI”) detection system, and ellipsometry detection systems.
A second aspect of the present invention relates to a detection system that includes a sensor chip according to the first aspect of the present invention. The detection system preferably includes a light source that is positioned to illuminate the chip and a detector that is positioned to detect light reflected from the surface of the chip, and thereby determine whether an antibody binds to the hemagglutinin polypeptide.
A third aspect of the present invention relates to a flow cell useful for detecting anti-influenza antibodies in a sample. The flow cell includes a base having an inlet and an outlet; a light transmissive cover mounted to the base in a substantially fluid-tight manner, and forming with the base a compartment through which fluid may pass from the inlet to the outlet; and a sensor chip according to the first aspect of the present invention, which is positioned in the compartment and exposed through the light transmissive cover to incident light, whereby incident light used to illuminate the chip surface at an appropriate angle of incidence achieves a condition of near perfect destructive interference in the absence of antibody binding to a hemagglutinin polypeptide and a substantial change in light reflectivity in the presence of antibody binding to a hemagglutinin polypeptide.
A fourth aspect of the present invention relates to a detection system that includes: a flow cell according to the third aspect of the present invention; a fluid sample source in fluid communication with the inlet of the flow cell; a light source that is positioned to illuminate the chip; and a detector that is positioned to detect light reflected from the surface of the chip, wherein the angle of incidence of the illuminating light is suitable to produce a condition of near perfect destructive interference in the absence of antibody binding to a hemagglutinin polypeptide and a substantial change in light reflectivity in the presence of antibody binding to a hemagglutinin polypeptide.
A fifth aspect of the present invention relates to a method for sensing an anti-influenza antibody in a sample. This method includes providing a detection system according to the second or fourth aspects of the present invention; directing light at a surface of the sensor chip; contacting the sensor chip with a sample under conditions effective to allow an anti-influenza antibody in the sample to bind specifically to a hemagglutinin polypeptide recognized by the antibody; and detecting light reflected from the chip under conditions effective to identify hemagglutinin polypeptides bound by an antibody of the sample.
In preferred embodiments, detection is carried out using an AIR detection system, an SPR detection system, a BASI detection system, or ellipsometry detection system.
A sixth aspect of the present invention relates to a method for sensing an anti-influenza antibody using the detection system according to the fourth aspect of the present invention, which method includes the steps of directing light at the sensor chip in a manner effective to result in a condition of near perfect destructive interference; introducing a fluid sample into the flow cell; measuring light reflected from the chip; and providing an output identifying the hemagglutinin polypeptides bound by an antibody of the fluid sample based on the measured reflected light.
A seventh aspect of the present invention relates to a method for screening efficacy of an influenza vaccine. This method includes the steps of administering an influenza vaccine to one or more individuals; obtaining a serum sample for the one or more individuals; and performing the method according to the fifth or sixth aspects of the present invention to measure the anti-influenza immune response generated by the influenza vaccine.
The sensor chips of the present invention, containing arrays of influenza hemagglutinin polypeptides, have been demonstrated to produce detection results using a label-free, “reagentless” technique in less than 30 minutes, and the results are shown to be consistent with those derived via a comparative ELISA assay, which takes longer to perform and requires additional labeled reagents to be introduced following reaction of the sample to the sensor chip. Consequently, the present invention offers a faster, reliable detection system for measuring vaccine efficacy and cross-reactivity for influenza virus strains. The examples herein demonstrate that with implementation via an AIR detection system, antibody titers over a wider dynamic range can be screened in a single experiment as compared to ELISA.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of an AIR detection system.
FIG. 2 is a cross-sectional view a flow cell of the invention which includes a sensor chip suitable for use in an AIR detection system for aqueous environments.
FIG. 3 is a schematic illustration of an ellipsometry detection system.
FIG. 4A is a schematic illustration of an SPR detection system. FIG. 4B illustrates the output of SPR.
FIGS. 5A-B are an alignment of hemagglutinin sequences from Genbank Accession ABW80979 (strain A/New Calcdonia/20/1999, H1N1) (SEQ ID NO: 1), AY531033 (strain A/Wyoming/3/2003, H3N2) (SEQ ID NO: 2), EF541403 (strain A/Vietnam/1203/2004, H5N1) (SEQ ID NO: 3), AF250479 (strain A/Teal/Hong Kong/W312/199, H6N1) (SEQ ID NO: 4), and NC—004908 (strain A/Hong Kong/1073/1999, H9N2) (SEQ ID NO: 5), each of which is hereby incorporated by reference in its entirety). The alignment was prepared using Multalin version 5.4.1 (Corpet, “Multiple Sequence Alignment with Hierarchical Clustering,” Nucl. Acids Res., 16(22):10881-10890 (1988), which is hereby incorporated by reference in its entirety. In the consensus sequence (SEQ ID NO: 6), the symbol “!” represents I or V, the symbol “$” represents L or M, the symbol “%” represents F or Y, and the symbol “#” represents one of N, D, Q, E, B, or Z. X can be any amino acid. Capital letters in the consensus represent absolutely conserved amino acid residues (100% identity), whereas lower case letters represent predominant residues (50-99% identity).
FIG. 6 shows a three-dimensional structure of hemagglutinin, differentially colored to represent sequence homology across five HA isoforms: H1N1, H3N2, H5N1, H6N1, and H9N2. ‘Black’ represents complete identity, ‘dark gray’ denotes sequence similarity, and ‘light gray’ symbolizes divergent sequences. The left structure is a side-on view of the HA extraviral domain spike and the right structure is a top-down view of the HA receptor binding site.
FIG. 7 is a schematic illustrating the key for a manually prepared hemagglutinin array of the present invention.
FIGS. 8A-B are images of an arrayed influenza test chip pre- and post-treatment, respectively, with undiluted serum from a vaccine trial subject. Results based on the difference in reflected intensity for selected antigen spots are illustrated graphically in FIG. 9.
FIG. 9 is a graph illustrating the quantification of reflectance changes for all HA isoforms upon exposure to serum from six clinical subjects. Refer to the array key in FIG. 7 for the full antigen description; the α-fluorescein control is depicted as “anti-F” in the graph.
FIG. 10 is graph illustrating the comparative analysis of AIR data for subject samples whose ELISA and western blot results corroborated. Subject 1 is subject 056; Subject 2 is subject 080; the placebo control is subject 079. The H5 hemagglutinin is H5,3 (Vietnam/1203/2004).
FIG. 11 is a graph illustrating H5N9 avian antiserum titration results. All reflectance changes are normalized to changes in the α-fluorescein negative control spot. Recombinant GFP served as a secondary negative control and a supplementary positive spike control An IgY antibody acted as the experimental positive control. A second set of chips exposed to buffer alone was the 0.0% control assay.
FIG. 12 is an AIR image of an HA microarray for H7N3 antiserum experiments. The chip was exposed to a 10% solution of H7N3 antiserum. Each probe type is printed eight times in a column. From left to right, probe molecules are: H1, H3, H5, H6, blank, α-IgY, α-IgG, and α-fluorescein. Note that α-fluorescein negative control is not easily visible, and that α-IgY is barely active at this concentration of antiserum.
FIG. 13 is a graph illustrating H7N3 avian antiserum titration results performed as sequential log3 dilutions. All changes were normalized to the α-fluorescein control spot. Each concentration was repeated three times.
FIG. 14 is a graph showing the results for subject antisera screened at a 5% dilution. The “buffer” column corresponds to the average and standard deviation of the MPBS-ET control chips from six experiments. The HA array responses are labeled according to subject number and dose of H5 received (in micrograms).
FIG. 15 is a graph showing a comparison of pre- and post-inoculation reflectance changes for subject 06FRO033 for log5 antiserum dilutions from 5% to 0.04%.
FIG. 16 is a graph illustrating the results of subject 06FRO033 antiserum titrations from 5% to 0.04%.
FIGS. 17A-B show robotically printed HA microarray for human antiserum experiments. A background image (FIG. 17A) and an image of an experimental array exposed to 5% antiserum (FIG. 17B; subject 071) are depicted. From left to right, spot identities are α-IgG, α-IgM, α-fluorescein, HSA, blank, H1, H3, H5, H6, and H9.
FIG. 18 shows an antiserum titration for placebo subject 064.



DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention concerns a sensor chip that is useful for detecting an immune response against an influenza virus. The sensor chip includes a substrate having a surface and a plurality of hemagglutinin polypeptides bound to discrete locations on the surface of the substrate, each hemagglutinin polypeptide having a hemagglutinin epitope, preferably the immunodominant epitope thereof.
The overall design and construction of the sensor chip can be varied according to the particular detection system in which it is to be employed. These include, for example and without limitation, sensors designed for use with AIR detection systems, SPR detection systems, BASI detection systems, and ellipsometry detection systems, as well as any other label-free or fluorescence labeled array technique.
An AIR detection system is described in U.S. Pat. No. 7,292,349 to Miller et al., which is hereby incorporated by reference in its entirety. This setup is illustrated in FIG. 1. The system 10 includes a light source 12, a polarizer 14, a receptor 16 (i.e., the functionalized sensor chip of the present invention), and a detector 18. The light source 12 generates and transmits a light (L) at a set wavelength towards a surface of the receptor. One or more lenses and filters can be employed to optimize the system. AIR exploits interference between reflections from the medium/coating and coating/substrate interfaces on the receptor, exhibiting changes in reflectivity upon binding of biomolecules to the coating. In practice, using a silicon wafer having an oxide coating, judicious choice of incident angle and wavelength can be used with s-polarized light to obtain near complete destructive interference (i.e., reflectivity that is preferably less than about 10−5 or even 10−6 under some circumstances) in the absence of a target, in this case the anti-hemagglutinin antibodies. The condition of near complete (or near perfect) destructive interference is removed upon target binding. Thus, highly sensitive detection of even small quantities of anti-hemagglutinin antibodies is possible.
While AIR using s-polarized light has proven to be a highly sensitive, simple analytical method for the quantitative detection of a variety of biomolecular analytes, the system described in the above-referenced U.S. Pat. No. 7,292,349 to Miller et al. is much more easily carried out in a dry state, that is, with an air/oxide interface rather than with an aqueous/oxide interface. An improved system for performing AIR in an aqueous environment is described in co-pending U.S. patent application Ser. No. 12/261,818 to Mace et al., and PCT International Patent Application No. PCT/2008/081804 to Mace et al., which are hereby incorporated by reference in their entirety. Basically, the flow cell as described therein allows for coupling of the s-polarized light into the aqueous environment for detection of target binding. Use of this same flow cell, containing a sensor chip functionalized with the plurality of hemagglutinin polypeptides, in contemplated herein.
The flow cell is illustrated in FIG. 2. The flow cell includes a base 112, a light transmissive cover 114 in the form of a 900 prism, a gasket 116 positioned between the base and cover, and one or more mounting braces 118 that are utilized to secure the base and cover in a substantially fluid-tight manner. The base 112 includes a well 120 formed in one face thereof, as well as inlet 122 and outlet 124 that communicate with the well via passages 126 and 128, respectively. Inlet 122 and outlet 124 are formed on opposite ends of the base such that the passages 126 and 128 that communicate with the well 120 ensure fluid flow over chip 130 when it is placed in the well. To assist with fluid flow in this respect, a notch 131 is formed in the sidewall of well 120 at each end of the well such that fluid can easily flow into the well from passage 126 and from the well via passage 128. The passages 126 and 128 are preferably provided with fittings 132 that allow conduits or other forms of tubing to be coupled to the flow cell. For example, the fluid sample source can be coupled to the inlet 122 and the outlet 124 can be coupled to additional fluid analyzers or simply to a waste reservoir. The chip 130 is preferably supported in the well 120 by an angled chip support 140.
In both the wet and dry AIR systems, the sensor chip has the same fundamental construction, with a substrate, one or more coating layers on the substrate, and then the probe molecules—in this case the hemagglutinin polypeptides—bound to the coating surface. As described in the above-referenced U.S. Pat. No. 7,292,349 to Miller et al., U.S. patent application Ser. No. 12/261,818 to Mace et al., and PCT International Patent Application No. PCT/2008/081804 to Mace et al., a number of different materials can be selected for the substrate and coating(s). Any suitable combination of substrates and coatings is contemplated for the sensor chip to be used in an AIR detection system.
The BASI detection system is described in U.S. Patent Publication No. 20070076214 to Rothberg, which is hereby incorporated by reference in its entirety. The BASI system, like the AIR system, exploits interference between reflections from the medium/coating and coating/substrate interfaces, and exhibits changes in reflectivity upon binding of biomolecules to the coating. The basic design of the system is similar to that illustrated in FIG. 1 (for AIR), but the structure of the sensor chip differs. The BASI system is functional with any substrate/coating combinations where the coating is very thin (e.g., a native oxide film on silicon) and when the incidence angle on one of two interfaces (substrate/coating or coating/medium) is greater than its Brewster angle and the incidence angle on the other of the two interfaces is less than its Brewster angle. Unlike AIR systems being commercially developed for use with incident s-polarized light, the BASI system relies on the detection of p-polarized light. As a result of using Brewster angle straddle and p-polarized light, where the coating thickness is <<λ, a phase flip of the reflected polarization allows nearly complete destructive interference (where reflectivity is preferably less than about 10−4 or even 10−5 in the absence of target binding). As with the AIR detection system, sensitive detection of even small quantities of anti-hemagglutinin antibodies is possible.
Ellipsometric detection systems measure the polarization component of reflected light as a measure of changes in coating thickness on the surface of the sensor chip. Ellipsometry sensitively measures the change of the state of polarization when electromagnetic radiation is reflected or transmitted by a sample. A classical embodiment of such an ellipsometric detection system, illustrated in FIG. 3, includes a light source that emits a collimated light beam passing a variable polarization controller given by the combination of a linear polarizer (P) and a compensator in the form of a quarter-wave plate (C). The polarized light beam is incident on the sensor surface (S) under a known oblique angle, reflected from the sample surface and analyzed by a second linear polarizer coupled to a suitable photodetector (A, collectively). In this ellipsometer setup, the measurement may be done by changing the azimuths of the components P and A, while the optical axis of C is kept at a constant azimuth, e.g., at 45° with respect to the plane of incidence, until the photodetector receives a minimum of intensity. The azimuthal angles of the components P, C and A for this “nulling” condition may be used to calculate the ellipsometric angles Delta and Psi, which are specific for the optical parameters of the sample at a given angle of incidence and wavelength of light. Using a suitable optical model and numerical regression, the quantities Delta and Psi may be recalculated in terms of the thickness of the optical layer, or changes thereof during a growth process. The application of ellipsometry for monitoring of binding reactions of biological molecules dates back to 1942 (Rothen et al., “Serological Reactions of Protein Films and Denatured Proteins,” J. Exp. Med. 76:437 (1942), which is herby incorporated by reference in its entirety), where the amount of adsorbed biological material at a surface during a binding reaction may be recalculated from the quantities Delta and Psi.
Imaging ellipsometry, as described for example in U.S. Pat. No. 5,076,696 to Cohn et al., which is hereby incorporated by reference in its entirety, uses spatially resolving detector and imaging optics to allow for a massively parallel measurement of ellipsometric data, e.g., in the form of Delta and/or Psi maps. Such maps may in turn be converted into surface maps of layer thickness, optical index of refraction, chemical composition or the amount of adsorbed material for each spot on an array. Imaging ellipsometry with its intrinsic parallel detection scheme may be used advantageously as a detection technique for these so-called biochips, microarrays or microplates (Eing et al., Imaging Ellipsometry in Biotechnology, ISBN 3-9807279-6-3 (2002), which is hereby incorporated by reference in its entirety).
Imaging ellipsometry has been demonstrated with light employed for the measurement impinging on the surface to be measured coming from the ambient medium. Other measurement setups are based on total internal reflection as described for example in U.S. Pat. No. 6,594,011 to Kempen, which is hereby incorporated by reference in its entirety. Here, the light from a light source is directed through an internal reflection element to reflect off the specimen to be detected.
Enhancement of the detection signal can be achieved using SPR ellipsometry, illustrated in FIG. 4A. The substrate 232 employed during SPR ellipsometry uses a thin metal layer 234 to allow the excitation and propagation of surface plasmons. While one side of the metal layer 234 is in contact with a transparent support structure 236, usually attached to a prism 238 allowing light to couple-in under an oblique angle, the other side of the layer is exposed to the ambient medium 240. Changes in the optical index of refraction in the ambient by the formation of an adsorbent layer (e.g., antibodies 244 binding to surface-bound hemagglutinin polypeptide 242) are monitored as a shift in the angle of incidence (Δθ) that generates surface plasmon resonance, causing a change of reflected light intensity (see FIG. 4B). For SPR based sensors it is known that an intermediate dielectric layer between the metal film and the probed surface may act as a means to further increase the sensitivity.
One exemplary SPR substrate is described in U.S. Pat. No. 7,332,329 to Wark et al., which is hereby incorporated by reference in its entirety. This SPR substrate is particularly suited for biomolecular arrays of hemagglutinin polypeptides, where the substrate includes a plurality of a metallic islands surrounded by a hydrophobic layer or a dielectric material, and the hemagglutinin polypeptides are bound to the metallic islands.
Regardless of the sensor chip substrate or the detection system in which the substrate is intended to be used, the sensor chip includes a plurality of hemagluttinin polypeptides bound to the surface of the sensor chip. Each hemagluttinin polypeptide bound to the surface of the sensor chip can be a full length protein or a polypeptide fragment thereof.
A trait inherent to all biosensors, regardless of labeling status or means of signal transduction, is probe immobilization. The role of the terminal hydroxyl of a silicon dioxide surface is highly flexible as it may act as a nucleophile (Bikiaris et al., “Compatibilisation effect of PP-g-MA copolymer on iPP/SiO2 nanocomposites prepared by melt mixing,” Eur Polym J 41:1965-1978 (2005); Tripp et al., “Chemical Attachment of Chlorosilanes to Silica: A Two-step Amine-promoted Reaction,” J Phys Chem 97:5693-5698 (1993), each of which is hereby incorporated by reference in its entirety) or support adsorption. For this reason, silicon dioxide is readily derivatized through a variety of chemical methods. These chemical reactions result in the effective transformation of the hydroxyl group to any of a number of chemical functionalities including, but not certainly not limited to, amines (Huang et al., “Directed Assembly of One-dimensional Nanostructures Into Functional Networks,” Science 291: 630-633 (2001), which is hereby incorporated by reference in its entirety) or halides (Hergenrother et al., “Small-molecule Microarrays: Covalent Attachment and Screening of Alcohol-containing Small Molecules on Glass Slides,” J Am Chem Soc 122: 7849-7850 (2001), which is hereby incorporated by reference in its entirety). From each initial reaction, a secondary chemical can be added to further alter the surface reactivity or probes may be directly coupled. Moreover, a multitude of functionalized silanes, molecules that couple to and self-assemble on silicon dioxide (Onclin et al., “Engineering Silicon Oxide Surfaces Using Self-assembled Monolayers,” Angew Chemie Int Ed 44:2-24 (2005), each of which is hereby incorporated by reference in its entirety), are commercially available, and may confer a diverse chemical landscape to the surface of the substrate (amines, epoxides, alkenes, etc.). A number of these approaches are generally described in U.S. Pat. No. 7,226,733 to Chan et al. and U.S. Pat. No. 7,292,349 to Miller et al., each of which is hereby incorporated by reference in its entirety.
U.S. Provisional Patent Application Ser. No. 61/101,831 to Mace et al., which is hereby incorporated by reference in its entirety, teaches the use of a non-nucleophilic additive in a formulation containing a probe molecule to be bound to an array surface. The non-nucleophilic additive is used in an amount effective to avoid or reduce the severity of surface morphological anomalies caused by non-homogeneous distribution of the reactant across a spot on the array where the reactant is bound. These surface morphological anomalies include bright center spots and “coffee stain” rings (or halos) that can interfere with accurate detection of target molecule binding at a particular spot. In other words, the use of effective amounts of the non-nucleophilic additive promotes substantially homogeneous distribution of the reactant across each of the spots on the array where the probe is located. By homogeneous distribution, it is intended that the variance of reactant concentration across the surface of a spot is minimized (relative to spots prepared in the absence of the non-nucleophilic additives). Stated another way, there is preferably less than about 10 percent pixel variation across the array spot, more preferably less than 5 percent variation, most preferably less than about 3 percent variation, 2 percent variation, or even less than about 1 percent variation.
Any effective amount of non-nucleophilic additive can be used. Typically, such an effective amount is between about 0.001 to about 3 percent v/v, more preferably between about 0.01 to about 1 percent v/v.
One embodiment of the non-nucleophilic additive includes compounds having a structure of formula (I) as follows:

R1—O—[(CH2)mO]n—R2  (1)

where, n is an integer from 0 to about 250; m is an integer from 1 to 3, preferably 1 or 2; and R1 and R2 are independently selected from the group of a C1 to C3 alkyl, or R1 and R2 together form a C1 to C3 alkyl, in which case the compound of formula (I) has a cyclic structure. R1 and R2 are preferably methyl or ethyl, or together form an ethyl group. These additives preferably have a molecular weight that is about 5000 Da or less, more preferably about 4000 Da or less, or about 3000 Da or less, most preferably about 2000 Da or less, or even about 1000 Da or less. Exemplary non-nucleophilic additives of formula (I) include, without limitation, crown ethers (18-Crown-6,15-Crown-5, 12 Crown-4, etc.), bis(2-methoxyethyl)ether, dialkyl ethers, and polyethylene glycol.

According to another embodiment, the non-nucleophilic additive is dimethylsulfoxide (DMSO).
The benefit of employing the non-nucleophilic additives, which do not participate in the chemical coupling of a reactant (or probe precursor) to the functionalized chip substrate, is that the additives promote better dispersion of the probe molecules across their respective discrete locations on the array. This improved dispersion consequently minimizes or entirely avoids the presence of surface morphological anomalies that can decrease the sensitivity of the detection system. As a result, improved sensitivity for the detection of target molecules can be achieved.
Hemagglutinin, a 222 kDa transmembrane homotrimer, is the influenza antigen responsible for mediating host cell recognition via sialic acid receptors (Copeland et al., “Assembly of Influenza Hemagglutinin Trimers and Its Role in Intracellular Transport,” J Cell Biol 103:1179-1191 (1986), which is hereby incorporated by reference in its entirety). Currently, there are sixteen major HA isoforms that have been identified (Fouchier et al., “Characterization of a Novel Influenza A Virus Hemagglutinin Subtype (H16) Obtained from Black-headed Gulls,” J Virol 79:2814-2822 (2005), which is hereby incorporated by reference in its entirety), and isolated viral serotypes are, along with neuraminidase, categorized based on this determinant. When host contact has been made, the cell attempts to endocytose the virus. The lower pH environment of the endosome causes a drastic structural rearrangement in HA (Skehel et al., “Changes in the confirmation of Influenza Virus Hemagglutinin at the pH Optimum of Virus-mediated Membrane Fusion,” Proc Natl Acad Soc USA 79:968-972 (1982), which is hereby incorporated by reference in its entirety) that results in membrane fusion and, ultimately, delivery of the viral payload (Skehel et al., “Receptor Binding and Membrane Fusion in Virus Entry: the Influenza Hemagglutinin,” Annu Rev Biochem 69:531-569 (2000), which is hereby incorporated by reference in its entirety).
The region of the viral RNA genome that encodes for HA is highly susceptible to mutation (Plotkin et al., “Codon Bias and Frequency-dependent Selection on the Hemagglutinin Epitopes of Influenza A Virus,” Proc Natl Acad Sci USA 100:7152-7157 (2003), which is hereby incorporated by reference in its entirety), and is the primary cause for the influenza virus' ability to evade host defenses. Evolution of this type is categorized as either ‘antigenic drift’ or ‘antigenic shift’: ‘Antigenic drift’ is the natural accumulation of mutations in genes that encode antigenic proteins, whereby an alteration in the immunological properties of the antigen results (Jin et al., “Two Residues in the Hemagglutinin of A/Fujian/411/02-like Influenza Viruses are Responsible for Antigenic Drift from A/Panama/2007/99,” Virology 336:113-119 (2005), which is hereby incorporated by reference in its entirety). ‘Antigenic shift’, on the other hand, is a drastic genetic recombination that occurs between two viral strains simultaneously infecting a host cell, thus generating an immunologically distinct antigen (Laver et al., “Studies on the Origin of Pandemic Influenza III. Evidence Implicating Duck and Equine Influenza Viruses as Possible Progenitors of the Hong Strain of Human Influenza,” Virology 51:383-391 (1973), which is hereby incorporated by reference in its entirety). By understanding and anticipating both evolutionary pathways, it is then possible to phylogenetically trace the progression of one influenza serotype to another through sequencing antigenic Determinants (Lindstrom et al., “Genetic Analysis of Human H2N2 and Early H3N2 Influenza Viruses, 1957-1972: Evidence for Genetic Divergence and Multiple Reassortment Events,” Virology 328:101-119 (2004), which is hereby incorporated by reference in its entirety). For example, the divergence of the human pandemic H2N2 virus to pandemic H3N2 virus was mapped in this manner (Scholtissek, et al., “On the Origin of the Human Influenza Virus Subtypes H2N2 and H3N2,” Virology 87:13-20 (1978), which is hereby incorporated by reference in its entirety).
The sensor chip arrays of the present invention are intended to include any two or more hemagglutinin polypeptides, but preferably any one or more H1 polypeptides (such as those from H1N1-H1N9), any one or more H2 polypeptides (such as those from H2N1-H2N9), any one or more H3 polypeptides (such as those from H3N1-H3N9), any one or more H4 polypeptides (such as those from H4N1-H4N9), any one or more H5 polypeptides (such as those from H5N1-H5N9), any one or more H6 polypeptides (such as those from H6N1-H6N9), any one or more H7 polypeptides (such as those from H7N1-H7N9), any one or more H8 polypeptides (such as those from H8N1-H8N9), any one or more H9 polypeptides (such as those from H9N1-H9N9), any one or more H10 polypeptides (such as those from H10N1-H10N9), any one or more H11 polypeptides (such as those from H11N1-H11N9), any one or more H12 polypeptides (such as those from H12N1-H12N9), any one or more H13 polypeptides (such as those from H13N1-H13N9), any one or more H14 polypeptides (such as those from H14N1-H14N9), any one or more H15 polypeptides (such as those from H15N1-H15N9), any one or more H16 polypeptides (such as those from H16N1-H16N9), and all possible combinations thereof. Any newly discovered hemagglutinin variants can also be incorporated onto the sensor chip of the present invention.
In addition to hemagglutinin polypeptides, the sensor chip arrays of the present invention can also include any two or more neuraminidase polypeptides. Preferably, the sensor chip arrays include any one or more N1 polypeptides (such as those from H1N1-H16N1), any one or more N2 polypeptides (such as those from H1N2-H16N2), any one or more N3 polypeptides (such as those from H1N3-H16N3), any one or more N4 polypeptides (such as those from H1N4-H16N4), any one or more N5 polypeptides (such as those from H1N5-H16N5), any one or more N6 polypeptides (such as those from H1N6-H16N6), any one or more N7 polypeptides (such as those from H1N7-H16N7), any one or more N8 polypeptides (such as those from H1N8-H16N8), any one or more N9 polypeptides (such as those from H1N9-H16N9), and all possible combinations thereof. Any newly discovered neuraminidase variants can also be incorporated onto the sensor chip array of the present invention.
As is appreciated by persons of skill in the art, antigens subject to post-translational modifications such a glycosylation may also be included on the array. For example, recombinant expression of hemagglutinin or neuraminidase polypeptides (to be bound to the array surface) in mammalian cells or Baculovirus cells should result in their glycosylation.
As will be appreciated by those of skill in the art, the amount of hemagglutinin or neuraminidase bound to each discreet location on the chip can be optimized based on the surface area of the location where detection is to occur. By way of example, it is believed that optimal results can be achieved with a concentration of hemagglutinin or neuraminidase polypeptide per location of about 100 ng/mm2 to about 100 ng/mm2, preferably about 1 pg/mm2 to about 10 ng/mm2.
The experimental HA arrays (described below) that are utilized in the present invention are of distinct isoforms and isolates. This does not, however, prohibit the cross-reactivity of specifically raised antisera to surface-immobilized recombinant hemagglutinins. Therefore, to obtain a molecular understanding of the subtle differences between the hemagglutinins in the antigen array, a multiple amino acid sequence alignment of hemagglutinins from the H1N1, H3N2, H5N1, H6N1, and H9N2 strains (obtained from Genbank—see Materials and Methods, infra) was performed. As shown in FIG. 5, these five protein sequences were found to have complete identity of 24.5% of amino acids and an additional 42.4% sequence similarity. This is seemingly a high percentage of sequence conservation, but it does not guarantee that common epitopes will be available on the protein's surface to be recognized by neutralizing antibodies.
Therefore, structural information was obtained to supplement sequential analysis. To this end, the known structure of the H1N1 hemagglutinin from the 1918 influenza pandemic (Gamblin et al., “The Structure and Receptor Binding Properties of the 1918 Influenza Hemagglutinin,” Science 303:1838-1842 (2004), which is hereby incorporated by reference in its entirety) was used as an HA scaffold to three-dimensionally represent sequence homology (FIG. 6). The HA structure was colored to visually represent identity (black), similarity (dark gray), and divergence (light gray). The results of the alignment studies (FIG. 5) illustrate that a considerable extent of sequence similarity lies on the surface of hemagglutinin as accessible, potential epitopes. Thus, a certain degree of inter-isoform cross-reactivity can be expected for antisera, especially when subjects (human or otherwise) are vaccinated with recombinant, extraviral domains and not native, virus-anchored hemagglutinin. Additionally, the top-down view (FIG. 6) of the hemagglutinin active site reveals a highly dissimilar region of the protein between isoforms. Consequently, antibodies or small molecule inhibitors of the receptor binding site of one isoform are not likely to be broadly active against other hemagglutinins.
Hemagglutinin and neuraminidase polypeptides can be coupled to the array surface using any suitable chemistry for coupling polypeptides. Several different binding chemistries are described in the above-referenced U.S. Pat. No. 7,292,349 to Miller et al., which is hereby incorporated by reference in its entirety. A preferred approach, particularly for oxide coatings, involves the use of an aminoalkyl trialkoxysilane, followed by glutaraldehyde, which affords an amino-reactive surface capable of binding the hemagglutinin or neuraminidase polypeptides.
Binding of the hemagglutinin or neuraminidase polypeptides to each discrete location can be carried out manually or using an automated system. For manual arraying, polypeptide solutions can be arrayed in a volume of  ̃1 μL at a final concentration of 1-100 μg/mL, preferably 10-60 μg/mL, after a 1:1 dilution from a 2× stock (in modified phosphate-buffered saline (“MPBS”)) into a solution containing 10% glycerol and 0.01% Tween-20. After incubating for 10 minutes in an ambient environment, the chips can be immersed in a solution of blocking buffer (1 mg/mL bovine serum albumin (“BSA”) in Hepes buffered saline (“HBS”)) for 45 minutes and then rinsed with MPBS buffer containing an additional 3 mM EDTA and 0.005% Tween-20 (“MPBS-ET”). For automated arraying, the polypeptide solutions can be arrayed by robotically printing using a Virtek ChipWriter Pro or comparable device at a final concentration of 1-100 μg/mL, preferably 40-60 μg/mL, after a 1:1 dilution from a 2× stock (in MPBS) into a solution containing 0.01-1% (v/v) 12-crown-4 ether in MPBS. After incubating for 60 minutes at 70° F. and 70% relative humidity in the microarray chamber, the chips can be immersed in a solution of blocking buffer (300 μg/mL BSA in HBS) for 60 minutes and then rinsed with MPBS-ET.
Once the array is prepared, the sensor chip can be exposed to serum samples obtained from individuals (or diluted serum samples), and then the presence (or absence) of one or more antibodies for a particular influenza strain can be determined based on the detection of a change (or lack of change) in the detector output following exposure of the sensor chip to the serum sample. As is well known in the art, the absence of a detectable signal does not necessarily mean that the antibodies are not present but rather that they are below detectable limits and, therefore, are not likely to be present. The image capture can be achieved by any of the detection systems described above, but preferably via an image array detector that captures an image of at least a substantial portion of the surface of the chip. For arrays of hundreds to hundreds of thousands of probes, an automated chip reader can be programmed to assess the change in reflectivity for each spot on an array based on the captured image.
As used herein, the individual from which serum samples are obtained can be any animal that is susceptible to infection by influenza, including humans and non-human primates, livestock, domesticated animals, and wild animals (particularly birds). Screening of livestock is particularly desired, because it is useful for monitoring the spread of influenza by wildlife. The serum sample can be obtained from both living individuals and a corpse post-mortem.
The arrays of the present invention are particularly useful for screening the efficacy of an influenza vaccine. Basically, the array is preferably used to screen pre- and post-immunization serum obtained from individuals to whom a vaccine has been administered. Following adequate time to allow for an immune response, post-immunization samples will be obtained and then screened against the array of the present invention. Dilution of the serum sample, typically from about 1:20 to about 1:2500, can be optimized based on the amount of hemagglutinin or neuraminidase loaded onto each discrete location of the array. Nevertheless, following exposure of the sample to the array, detection of antibody-hemagglutinin or antibody-neuraminidase reactivity can be assessed using the detection system of the system employed to read the sensor chip surface by AIR, SPR, BASI, ellipsometry, etc. A quantitative measurement of the immunoreactivity can be assessed.
If desired or required, sensitivity can be further enhanced by introduction of a secondary antibody, for example an antibody specific for IgG.
Further analysis can include, without limitation, ELISA, PCR, realtime-PCR, mass spectrometry, and liquid chromatography-NMR spectroscopy. Moreover, after detecting the presence of an antibody during use of the chip, the antibody itself can be dissociated from the hemagglutinin or neuraminidase polypeptide to which it was bound during use of the device. Dissociation can be achieved by any of a variety of ways including, without limitation, a glycine solution at low pH, a low pH water solution, a high pH water solution, a detergent solution (with low, moderate, or high concentrations), a low concentration denaturant solution (e.g., urea). After dissociation, the antibody (now free from the chip surface) can be recovered and then analyzed, if desired. Depending on the approach of subsequent down-stream analyses, it is possible to use the eluted samples directly or following one or more steps for concentration of the antibodies of interest.
Once the sensor chip is cleared of previously bound antibodies, the sensor chip can be re-used to screen other serum samples for the presence of anti-influenza antibodies.
EXAMPLES
The present invention may be further illustrated by reference to the following examples.
Materials and Methods for Example 1-3
Hemagglutinins
For the HA arrays, the following isoforms were used for manual and robotic arrays (isolated from humans unless specified otherwise): A/New Calcdonia/20/1999 (H1N1), A/Wyoming/3/2003 (H3N2), A/Hong Kong/56/1997 (H5N1), A/Hong Kong/213/2003 (H5N1), A/Vietnam/1203/2003 (H5N1), A/Teal/Hong Kong/W312/1997 (H6N1), A/Hong Kong/1073/1999 (H9N2). All HA's were purchased from Protein Sciences, Corp. (Meriden, Conn.) and/or provided by the research group of David Topham (University of Rochester, Rochester, N.Y.).
Human, Avian, and Mouse Samples
Human antisera to H5 vaccination were acquired from the University of Rochester Retrovirology Lab and Vaccine Evaluation Unit. The nine subjects studied were marked: 031, 033 (pre and post), 036, 037, 038, 064, 067, 069, and 071. The potential pathogenic states of the sera are unknown (i.e., information on tests for HIV 1/2, etc. are not known), and, as such, extreme caution was taken when handling the samples in a BSL-2 laboratory. Chicken antisera to avian influenza strains H5N9 and H7N3 were courteously donated by the Whittaker lab of the College of Veterinary Medicine at Cornell University. Negative control mouse plasma was obtained from the Pearce Laboratory at the University of Rochester Medical Center. Whole blood samples from five four-month old, female, 129Sv/J mice (original source Taconic, now bred in-house) were collected following an IP injection with pentobarbital and a heart right ventricle puncture. The blood was pooled over heparin and centrifuged to remove red blood cells.
Hemagglutinin Sequence Alignment
The complete amino acid sequences for all studied hemagglutinins were located through NCBI (Genbank) searches and conversion from the genomic nucleotide sequence (except H1N1, which was deposited as a peptide sequence). The Genbank accession numbers for each sequence, each of which is hereby incorporated by reference in its entirety, are as follows: ABW80979 (A/New Calcdonia/20/1999, H1N1) (Bragstad et al., “The Evolution of Human Influenza A Viruses from 1999 to 2006—A Complete Genome Study,” Virol J 5:40 (2008), which is hereby incorporated by reference in its entirety), AY531033 (A/Wyoming/3/2003, H3N2) (Bragstad et al., “New Avian Influenza A Virus Subtype Combination H5N7 Identified in Danish Mallard Ducks,” Virus Res. 109:181-190 (2005), which is herby incorporated by reference in its entirety), EF541403 (A/Vietnam/1203/2004, H5N1) (World Health Organization Global Influenza Program Surveillance Network, “Evolution of H5N1 Avian Influenza Viruses in Asia,” Emerg. Infect. Dis. 11:1515-1521 (2005), which is hereby incorporated by reference in its entirety), AF250479 (A/Teal/Hong Kong/W312/1997, H6N1) (Hoffmann et al., “Characterization of the Influenza A Virus Gene Pool in Avian Species in Southern China: Was H6N1 a Derivative or a Precursor of H5N1?,” J. Virol. 74:6309-6315 (2000), which is hereby incorporated by reference in its entirety), and NC—004908 (A/Hong Kong/1073/1999, H9N2) (Lin et al., Avian-to-Human Transmission of H9N2 Subtype Influenza A Viruses: Relationship Between H9N2 and H5N1 Human Isolates,” Proc. Natl. Acad. Sci. USA 97:9654-9658 (2000), which is hereby incorporated by reference in its entirety). The primary structure alignment software Multalin (v5.4.1) (Corpet, F., “Multiple Sequence Alignment with Hierarchial Clustering,” Nucl Acids Res 16:10881-10890 (1988); Combet et al., “NPS@: Network Protein Sequence Analysis,” Trends Biochem Sci. 25:147-150 (2000), which are hereby incorporated by reference in their entirety) was used to align each HA sequence of approximately 560-570 amino acids. Multalin parameters were set to use the identity symbol comparison table and the identity scoring method. The upper and lower limits of sequence conservation were set to 100% and 50%, respectively. The alignment output is shown in FIG. 5.
For graphical representation, the sequence of the solved hemagglutinin structure H1N1 (Combet et al., “NPS@: Network Protein Sequence Analysis,” Trends Biochem Sci. 25:147-150 (2000), which is hereby incorporated by reference in its entirety) (PDB ID: 1RUZ) was used as a scaffold and rendered using MacPYMOL (DeLano, W. L., The PyMOL Molecular Graphics System, DeLano Scientific, Palo Alto, Calif., USA (2002), which is hereby incorporated by reference in its entirety). Its sequence was added to the Multalin alignment algorithm in order to verify the position of each conserved/similar amino acid residue; in MacPYMOL, each residue that was 100% conserved was colored ‘black’, 50%-99% conserved was colored ‘dark gray’, and <50% conserved was colored ‘light gray’. The hemagglutinin homology structure was then ray traced in order to display the ‘spike’ (side on) and ‘active site’ (top down) orientations (see FIG. 6).
Manual Hemagglutinin Array Experiments
AIR substrates were prepared to a starting SiO2 thickness of 1381 Å and functionalized using PITC general amine attachment chemistry. The HAs used for manual array experiments were H1N1, H3N2, all three H5N1s, and H6N1. HAs were manually arrayed in a volume of 1 μL at a final concentration of 20 μg/mL after a 1:1 dilution from a 2× stock (in MPBS) into a solution containing 10% glycerol and 0.01% Tween-20. Human IgG (positive control) and fluorescein antibodies were arrayed at a final concentration of 50 μg/mL in the same volume and buffer dilution. Probe solutions were allowed to incubate for 10 minutes in an ambient environment, after which the chips were immediately immersed in a solution of blocking buffer (1 mg/mL BSA in HBS) for 45 minutes. The chips were then rinsed with MPBS-ET, and 150 μL of 100% human serum samples were pipetted onto the surface (some variable sample dilution will have occurred). After a 45 minute incubation period, the chips were rinsed with MPBS-ET and added to a shaking bath of MPBS-ET for 5 minutes. The chips were then rinsed with ddH2O, dried under nitrogen, and imaged on a G3 reflectometer. Reflectance values for each spot were compared to the reciprocal spot on a negative control chip (MPBS-ET only) and normalized to the α-fluorescein negative control.
Microarrayed Hemagglutinin Experiments
AIR substrates were prepared to a starting SiO2 thickness of 1393 Å and functionalized using glutaraldehyde general amine attachment chemistry. The difference between the oxide thicknesses required for macro- and microarrayed HA assays are presumably caused by the slight broadening of the AIR reflectance minimum due to the inefficient coupling of reflected light to the G4 reflectometer detector. All probe spots were printed in eight replicates. The HAs used for manual array experiments were H1N1, H3N2, H5N1 (Vietnam/1203/2003), H6N1, and H9N2 (human antiserum experiments only). HAs were robotically printed using a Virtek ChipWriter Pro at a final concentration of 40 μg/mL (50 μg/mL for H9N2) after a 1:1 dilution from a 2× stock (in MPBS) into a solution containing 0.1% 12-crown-4 in MPBS. Human IgG and human IgM antibodies (positive controls) were arrayed at 100 μg/mL. Human serum albumin (HSA) and α-fluorescein (negative controls) were arrayed at final concentrations of 200 μg/mL and 10 μg/mL, respectively, in the same buffer dilution. Probe solutions were allowed to incubate for 60 minutes at 70° F. and 70% relative humidity in the microarray chamber. Afterwards, the chips were immediately immersed in a solution of blocking buffer (300 μg/mL BSA in HBS) for 60 minutes. The chips were then rinsed with MPBS-ET, and 50 μL of human serum samples were pipetted onto the surface (some variable sample dilution will have occurred). After a 60 minute incubation period, the chips were rinsed with MPBS-ET and added to a shaking bath of MPBS-ET for 5 minutes. The chips were then rinsed with ddH2O, dried under nitrogen, and imaged on a G4 reflectometer. Reflectance values for each spot were compared to the reciprocal spot on a negative control chip (MPBS-ET only) and normalized to HSA negative control.
For chicken H5N9 and H7N3 antiserum experiments, all methods were kept the same except for the exclusion of H9N2 as a probe (lack of availability), removal of HSA as a negative control, removal of α-IgM as a positive control, and the exchange of α-IgY for α-IgG as a positive control. Also, recombinant green fluorescent protein (rGFP) was arrayed as a secondary control at a concentration of 50 μg/mL. In the presence of chicken antiserum, rGFP served as an additional negative control; at low antiserum dilutions, α-rGFP was supplemented at a concentration of 5 μg/mL as a positive spike control.
Example 1
Manually Prepared Hemagglutinin Arrays
Initial experiments utilizing manually arrayed hemagglutinins at high concentrations were performed to study both the arrayability of these proteins and the proper starting chip thickness. At 100 μg/mL of A/Vietnam/1203/2004 (H5N1), an oxide of 1360 Å was determined to be the ideal film thickness to give suitable unbound probe intensities. While these spots were resolvable, an early experimental concern lay in the general amine attachment chemistry: HAs are homotrimeric and contain roughly 30 solvent accessible amines per monomer. The orientation of the immobilized molecule, therefore, will be completely random. The oligomeric state may ensure that there is a solvent accessible face available to bind to specific antibodies, but orientations that result in an effectively inactive sensor are also possible.
Attempts to observe signal changes upon the addition of an antibody specific to the A/Vietnam/1203/2004 (H5N1) hemagglutinin (enzyme product number IA-005-01000) failed at these high HA concentrations presumably due to steric crowding at the surface that impeded epitope recognition by large antibodies. Ideally, since each HA isoform is identical in size—differing only slightly in sequence—the solution concentrations used to array each should also be identical. The A/New Calcdonia/20/1999 (H1N1) isoform was supplied at the lowest stock concentration of 66 μg/mL, and, thus, was the limiting probe for this study; all probe molecules were diluted such that they could be supplemented with a sufficient amount of stabilization solution consisting of glycerol and Tween-20. Due to this, subsequent experiments were performed using 20 μg/mL as a final concentration for all HAs. This low concentration, and a subtle alteration to the washing protocol during the surface derivitization, caused the new optimum chip starting thickness to increase to 1381 Å. A test array was created and consisted of the aforementioned H1N1 and H5N1 experimental hemagglutinin probe spots, as well as positive and negative control spots of α-IgG and α-fluorescein, respectively. These arrays were screened against a specific A/New Calcdonia/20/1999 (H1N1) antibody (Fitzgerald product number M32210) as well as the H5N1 antibody. An interesting result of these experiments was that the H1N1 antibody seemed to preferentially recognize the H5N1 isoform, while the H5N1 antibody only moderately recognized both HAs. This was not completely unforeseen, though, since antiserum cross-reactivity was predicted by sequence homology.
A full HA array, containing six isoforms and a positive and negative control, was manually generated in preparation for a small-scale clinical trial (FIG. 7). Antisera were obtained from six different subjects: five were inoculated with various amounts of A/Hong Kong/156/1997 (H5N1) on two separate visits, and one subject was given a placebo injection only (see Table 1). All tests were performed blind.







TABLE 1







Tabulated Results from Traditional Vaccination Efficacy Tests


for Each Subject Inoculated with Varying Amounts of Antigen













Subject
Amount
ELISA
Western
Neut.
HAI, 1
HAI, 3


No.
(μg)
OD
Blot
Titer
Titer
Titer
















056
90/90
1.748
positive
453
2,560 
20


068
90/10
0.652
negative
14
320
10


076
25/25
1.677
negative
20
n/p
n/p


079
placebo
0.235
negative
10
n/p
n/p


080
25/25
1.856
positive
80
n/p
n/p


081
90/60
1.160
negative
14
160
20





n/p = experiment not performed






Aliquots of undiluted serum were utilized on a full HA array by AIR using a detection system of the type described in U.S. patent application Ser. No. 10/282,274 to Miller et al., filed Oct. 28, 2002, now U.S. Pat. No. 7,292,349, issued Nov. 6, 2007, which is hereby incorporated by reference in its entirety. Representative pre-exposure and post-exposure images are shown in FIG. 8A-B, while the full quantification of reflectance changes from all chips is shown in FIG. 9.

It is important to make known that these studies were performed blind, with no prior knowledge of the amount of antigen each subject was inoculated with, the results of antiserum response as monitored by traditional means, or the identity of the placebo sample. Traditional assays ELISA, western blot against antiserum, viral neutralization, and hemagglutinin inhibition (HAI)—were performed on sera collected from the subjects seven days after their second immunization. These results are shown in Table 1.
A few traits are immediately apparent from the clinical antiserum data that corroborate the studies with purified antibodies. Firstly, there is a high degree of correlation between the responses to the New Calcdonia/20/1999 and Vietnam/1203/2004 hemagglutinins. Secondly, there is limited response from the Hong Kong/156/1997 spot regardless of subject. Thirdly, specific antisera appear to have elicited a higher response to general HA epitopes than others. It is peculiar, however, that very little response is seen at the Hong Kong/156/1997 spot, because this was the recombinant isoform that was used to inoculate the patient population. However, while this isotype has confirmed activity in T-cell assays, it is unlikely to exist in a native, folded conformation. In addition, the reflectance changes that occur are specific to the HA probes since the α-fluorescein control spot intensity changes were negligible for all assays (average value of 5.4 units). This is an exceptional result considering the experiments utilized undiluted antiserum.
Comparing the AIR results with the conventional analyses shown in Table 1, a high degree of similarity is observed. Samples from subjects 056 and 080 show a high AIR reflectance (FIGS. 9 and 10), ELISA OD, a good neutralization titer, and a positive western blot. Subject 056 shows a high New Calcdonia/20/1999 cross-reactivity (FIG. 9). Subject 068 had high HA cross-reactivity as seen by AIR and HA1 titers (FIG. 9). An important facet of the blind study was to determine whether the placebo subject could be resolved by AIR. Based on the AIR results, the three candidates for the placebo sample were Subjects 068, 076, and 079. Subject 068 was identified due to low responses, aside from New Calcdonia/20/1999 and Vietnam/1203/2004 HAs. Subject 076 was identified because this sample generated one of the lowest two overall reactivities. Subject 079 was also identified due to low activity across all H5 hemagglutinin spots. Indeed, the identity of the placebo sample was later disseminated to be from subject 079.
The low reactivity for subject 068 may be explained by the smaller second inoculation dosage that could have led to a decrease in the secondary immune response and, therefore, a lesser amount of antibody generation. The results from subject 076 are more difficult to explain. It is possible that the ELISA OD is misleading and this subject had a very weak immune response to vaccination, or the immunogenic HA epitope for this subject is not available when the Hong Kong/156/1997 hemagglutinin is surface-immobilized. One dissimilarity, however, was subject 081. By traditional methods, subject 081 appeared to be a poor candidate for successful vaccination because their antiserum neutralization titer was low and the western blot was negative, but by AIR subject 081 garnered some of the highest responses across the array. There is the potential, however, that by tethering these HAs to the substrate surface that certain epitopes are preferentially exposed or obscured. The reflectance values observed, therefore, would be distorted with respect to solution-based, or matrix-based, assays.
Importantly, these results—obtained using a label-free, “reagentless” technique in less than 30 minutes—were entirely consistent with those derived via a comparative ELISA assay.
Example 2
Viral Surveillance in Avian Flocks
Avian-to-human contact is the main route of H5N1 influenza virus transmission (Sandrock et al., “Clinical Review: Update of Avian Influenza A Infections in Humans,” Crit. Care 11:209 (2007), which is hereby incorporated by reference in its entirety). Therefore, the ability to monitor global poultry populations is critical for protecting human health. Moreover, surveillance is of agricultural and humane concerns as well: as of the end of 2006, over 240 million poultry have been preventatively destroyed to stem the spread of the avian influenza virus (World Organization for Animal Health, Avian influenza: Fact Sheet, accessed Apr. 16, 2008, which is hereby incorporated by reference in its entirety).
Samples of chicken H5N9 and H7N3 antisera were obtained from the Whittaker lab at the College of Veterinary Medicine, Cornell University. The H5N9 antiserum would serve as a substitution for avian H5N1, while the H7N3 virus has also shown the potential to be infectious to humans (Tweed et al., “Human Illness from Avian Influenza H7N3, British Columbia,” Emerg. Infect. Dis. 10:2196-2199 (2004), which is hereby incorporated by reference in its entirety). In addition, avian antiserum experiments would serve as an excellent model system to study concurrently with a modification of AIR methodology. These experiments were performed with robotically printed arrays as described above.
For the avian antiserum experiments, arrays containing New Calcdonia/20/1999 (H1N1), Wyoming/3/2003 (H3N2), Vietnam/1203/2004 (H5N1), and Teal/Hong Kong/W312/1997 (H6N1) were created. To these arrays, an α-IgY positive control, and α-fluorescein and recombinant green fluorescent protein (rGFP) negative controls were added. The rGFP spot would simultaneously serve as a positive spike control for dilute solutions. Unfortunately, the H5N9 antiserum was supplied in a limited volume and only a preliminary titer could be acquired, shown in FIG. 11. A fairly large reflectance increase was observed for H3, H5, and H6 at higher concentrations of antiserum. H3 appeared to be less specific since it did not titrate well as dilutions were introduced, while H5 and H6 changes corresponded well to concentration changes. The H1 hemagglutinin showed negligible reflectance changes for all screened concentrations. The IgY antibody appeared to be less active than anticipated, given the large serum immunoglobulin concentration; for example, when the array was supplemented with an additional column of α-human IgG, the human immunoglobulin-specific antibody displayed a greater sensitivity than the antibody for chicken immunoglobulin (in the context of H7N3 antiserum experiments). Recombinant GFP showed little non-specific adsorption towards the chicken antiserum, but was ‘active’ in that it was readily recognized by an addition of α-GFP to a dilute antiserum solution. A 50% H5N9 antiserum dilution was also performed on a set of chips, but nonspecific binding to the surface precluded repeatable quantification of binding.
While the H5N9 antiserum was quickly depleted, at the very least it afforded a titration window in which to effectively study H7N3 antiserum activity. For these experiments, the dilution window was focused to range from 10% to 0.37% (three log3 dilutions). Handling more dilute antiserum concentrations allowed for conservation and for a larger amount of replicate experiments to be performed. FIG. 12 shows an image of a hemagglutinin microarray imaged on the G4 AIR reflectometer after exposure to 10% H7N3 antiserum. The spot morphologies are typical of each probe type. The large flare in the bottom right corner of the image is scatter caused by particulates on the reflectometer's imaging system. This variety of reflectivity abnormality is easily circumvented during image processing. The results of the H7N3 antiserum titration was quite different than that of the H5N9 antiserum, as a large H3 cross-reactivity was observed (FIG. 13). Unfortunately, a recombinant H7N3 hemagglutinin is not commercially available and therefore unable to be implemented into this array. Modest reflectance increases were observed for all arrayed hemagglutinins, and all HA signals titrate well with reducing antiserum concentration. The sensitivity of AIR is clearly demonstrated by the statistically distinct reflectance changes for substrates exposed to 10% antiserum versus 3.33% antiserum. The standard deviations in the average observed signal change are large for the more dilute solutions, but this can be accounted for by the systematic errors stemming from additional sample dilution caused by introducing target solutions to wet chips.
As expected, stark differences were observed between H5N9 and H7N3 avian antisera. Comparison of a single high antiserum dilution (as performed for the human antisera described below) establishes trends in cross-reactivities between arrayed HAs: H5N9 antiserum is specific for H5 and, to a lesser extent, H6, while displaying no reactivity towards either H1 or H3 hemagglutinins; H7N3 antiserum, on the other hand, exhibits reactivity over the entire hemagglutinin panel, with a slight specificity for H1 and H3. These results serve to prepare for more extensive avian surveillance studies to be performed in the future with the inclusion of a larger HA array and more diverse poultry population.
Example 3
Larger Hemagglutinin Arrays
Arrays were prepared with the automated microarray printer using five hemagglutinin isoforms as follows: H1=A/New Calcdonia/20/1999 (H1N1); H3=A/Wyoming/3/2003 (H3N2); H5=A/Vietnam/1203/2004 (H5N1); H6=A/Teal/Hong Kong/W312/1997 (H6N1); and H9=A/Hong Kong/1073/1999 (H9N2). Several controls were also included on the array, including human serum albumin (HSA) as the “normalizing” negative control, and anti-fluorescein as a secondary negative control element. Antibodies to human IgG and human IgM serve as positive controls.
While the preliminary human antiserum results were successful in proving that AIR data could augment the results of traditional vaccination assays, there were two major limitations that could not be overcome by conventional manual arraying techniques: expanding the array to include an increased probe redundancy and removing the “human variable” by robotically controlling the volume and morphology of printed arrays. As was demonstrated with the avian antiserum experiments of the preceding section, utilizing a robotic microarrayer solves both technique inadequacies in a controlled and repeatable manner. Additionally, the A/Hong Kong/1073/1999 (H9N2) hemagglutinin was supplemented into the full array, along with two more control probes: an antibody to human IgM would serve as a second positive control (Lacroix-Desmazes et al., “Analysis of Antibody Reactivities Toward Self Antigens of IgM of Patients with Waldenstrom's Macroglobulinemia,” Int Immunol 9:1175-1183 (1997), which is hereby incorporated by reference in its entirety), and human serum albumin, through solution-phase competition effects, would be an effective additional negative control (FIG. 14). FIG. 17B shows a representative image of a hemagglutinin microarray imaged on the G4 AIR reflectometer after exposure to 5% antiserum of subject 071.
From assays performed on serum from a randomly selected subject from the clinical population, the effective experimental titration range for the human antiserum experiments was determined to be between 5% (1:20 dilution) and 0.04% (1:2500 dilution). 5% antiserum was dilute enough to negate signals from non-specific binding while retaining a large, non-saturating reflectance increase, and 0.04% (a 1:2,500 dilution) was dilute enough to, in effect, titrate out reflectance changes to zero for most samples. This dilution series was performed in log5 steps for all titrated samples. The majority of the antisera, however, were studied at a 5% dilution alone to quantify trends in cross-reactivity and to identify potential placebo subjects. Based on low H5 reflectance changes at the upper 5% titer, samples from 06FRO037 and 06FRO069 are likely recipients of placebo (FIG. 14). Also, the buffer controls arrays for antiserum experiments (N=6) were analyzed to quantify array-to-array reproducibility. The largest reflectance variations between control chips were observed for α-IgG spots (3.05+/−8.1), while the most reproducible spots were α-fluorescein (2.66+/−1.8). The “noise” of the assay is then approximately 3 units, and, therefore, reflectance changes in this range are considered negligible.
One subject also had pre-inoculation sera aliquoted to quantify innate resistance and determine the actual protective effect of the H5 inoculation. Reflectance changes derived from subject 06FRO033 indicated modest responses to H3 hemagglutinin, potentially due to memory immunity derived from a prior influenza infection, and slight cross-reactivities to H1 and H9 hemagglutinins (FIG. 15). However, only basal recognition of H5 and H6 hemagglutinins were observed in the pre-inoculation antiserum aliquot, and, since these interactions were not titratable over the concentration range of the experiment, they were presumably not specific to those isoforms. Nevertheless, upon inoculation with H5, subject 06FRO033 gained considerable specific protective effects against H5 and all HAs in the array (FIG. 16). Moreover, since these interactions titrated well over the screened concentrations-four log5 serial dilutions (5%, 1%, 0.2%, and 0.04%), they are assuredly a consequent of specific immunological responses. As expected, there was a dose-dependent change in signal over the course of dilutions. The sensitivity of AIR is clearly demonstrated by the statistically distinct reflectance changes for substrates exposed to as little as 0.2% antisera.
The placebo sample was identified as being derived from subject 064, which is clearly observed in the reflectance data displayed in FIG. 14. A full antiserum titration was then performed in order to monitor the rate of signal depletion against the HAs. Similar to what was observed with the pre-inoculation sample of subject 033, there is very little basal recognition of H5 and H6 in the non-vaccinated population; however, antibodies to H1, H3, and H9 hemagglutinins are present in normal human serum. This is shown in FIG. 18.
Through the National Institutes of Health, John Treanor (University of Rochester, Rochester, N.Y.) provided H5 hemagglutinin inhibition data for all subjects screened by AIR (Table 2). With the exception of subjects 036 and 067, there appears to be little correlation between the amount of H5 used to inoculate a subject and their ability to generate inhibitory antisera as observed through the H5 HA1 titer. Interestingly, subjects 031 and 036 had higher HAI titers six months subsequent to the conclusion of the study than those detected during the course of vaccination (post-vaccination visits 1 and 2 in Table 2). AIR results, however, were less discriminating. There are definite discrepancies between dosage, HAI titer (post-vaccination visit 2), and AIR reflectance change as typified by the results from samples derived from subjects 036, 067, and 071. This is expected, though, since all antibodies generated for hemagglutinin-specific epitopes have the potential to cause reflectance increases, and not just those that inhibit activity. However, it is not possible to conclude that the HA arrays, as monitored by AIR, are clearly capable of discerning pre- and post-inoculation antisera and samples derived from placebo and experimental subjects. Moreover, these results are consistent with what would be expected from similarly performed ELISA and western blot experiments, with respect to agreement with HAI titers and inoculation dosage, while vastly increasing the amount of data that can be acquired from a single subject-derived sample.







TABLE 2







Comparison of H5 Hemagglutinin Inhibition Titers Performed


at Various Stages Along the Clinical Study














HAI
HAI
HAI



Subject
Dose (μg)
post-vac 1
post-vac 2
6 months
AIR Result















064
0
5.0
5.0
5.0
7.11


037
7.5
5.0
5.0
5.0
42.78


031
7.5
5.0
5.0
10.0
56.94


033
7.5
5.0
7.1
5.0
61.57


071
7.5
5.0
5.0
5.0
77.87


069
15
5.0
5.0
5.0
38.81


038
45
5.0
5.0
5.0
65.83


036
90
28.3
28.3
56.6
59.28


067
90
5.0
160.0
5.0
103.75





AIR data acquired at a 5% antiserum dilution; samples screened by AIR are equivalent to the samples studied for HAI during post-vaccination visit 2 (both results are bolded).






Although the preceding experiments were conducted using AIR as a label-free sensor platform, it should be appreciated by those of skill in the art that AIR is merely a preferred sensing platform because of its simplicity, sensitivity, and adaptability to large arrays of probe molecules (in this example, hemagglutinin isoforms). Indeed, use of an arrays sensor suitable for use in surface plasmon resonance (“SPR”) detection systems, Brewster Angle Straddle Interferometry (“BASI”), ellipsometry, and any other label-free or fluorescence labeled array techniques is contemplated.
The rapid differentiation between clinically relevant naïve and generated immune states is a significant capability for any diagnostic assay, and more so if the platform is highly parallelized and quantitative. Towards this end, the utility of antigenic arrays for the determination of vaccine efficacy and viral surveillance of high-risk populations has been demonstrated by AIR. Like most biomolecular detection techniques (ELISA and SPR included), this simple direct assay lacks the ability to differentiate between inhibitory and broad, yet specific, antibody-ligand interactions; however, the tremendous sensitivity and selectivity of AIR can be exploited to probe the immunological contents of complex, subject-derived samples and augment the data acquired from traditional immune assays.
The incorporation of a robotic microarray instrument allowed arrays to be printed reproducibly, and in a high density, to allow more for more complex and informative assays to be performed. Moreover, the development of the G4 reflectometer permitted the microarrays to be clearly resolved and imaged. This proved to be a major technological advancement over the G3 reflectometer and a significant improvement toward optimization of these antigenic arrays.
Having thus described the basic concept of the invention, it will be rather apparent to those skilled in the art that the foregoing detailed disclosure is intended to be presented by way of example only, and is not limiting. Various alterations, improvements, and modifications will occur and are intended to those skilled in the art, though not expressly stated herein. Additionally, the recited order of processing elements or sequences, or the use of numbers, letters, or other designations therefore, is not intended to limit the claimed processes to any order except as may be specified in the claims. These alterations, improvements, and modifications are intended to be suggested hereby, and are within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims and equivalents thereto.

## Claims

1. An arrayed imaging reflectometry (AIR) sensor chip suitable for the detection of influenza immune responses comprising:
a multilayer substrate comprising a surface and hemagglutinin polypeptides from different influenza strains or isolates, wherein said hemagglutinin polypeptides from each individual influenza strain or isolate comprise a hemagglutinin epitope and are covalently attached to the chip surface at different locations;
wherein said hemagglutinin polypeptides retain conformational epitopes that are capable of binding to anti-influenza antibodies;
wherein said surface with hemagglutinin polypeptides covalently attached at the different locations forms a coating on the chip surface that results in destructive interference of polarized light illuminating said surface at an appropriate incident angle and wavelength in the absence of antibody binding; and
wherein exposure of the surface to a sample comprising anti-influenza antibodies produces a detectable change in reflectance at a location upon antigen-antibody binding.

2. The sensor chip according to claim 1, wherein the hemagglutinin polypeptides are full length hemagglutinin proteins.

3. The sensor chip according to claim 1, wherein the hemagglutinin polypeptides are fragments of full length hemagglutinin proteins.

4. The sensor chip according to claim 3, wherein the hemagglutinin polypeptide fragments each comprise an immunodominant hemagglutinin epitope of their respective native influenza strain.

5. The sensor chip according to claim 1, wherein the hemagglutinin polypeptides are covalently attached to the substrate via a glutaraldehyde linker.

6. The sensor chip according to claim 1, wherein each of said locations comprises a concentration of hemagglutinin polypeptide of about 100 fg/mm2 to about 100 ng/mm2.

7. The sensor chip according to claim 1 further comprising:
neuraminidase polypeptides from different influenza strains or isolates, wherein said neuraminidase polypeptides from each individual strain or isolate comprise a neuraminidase epitope and are covalently attached to the chip surface at different locations;
wherein said neuraminidase polypeptides retain conformational epitopes that are capable of binding to anti-influenza antibodies; and
wherein said surface with neuraminidase polypeptides covalently attached at the different locations forms a coating on the chip surface that results in destructive interference of polarized light illuminating said surface at an appropriate incident angle and wavelength in the absence of antibody binding.

8. The sensor chip according to claim 7, wherein the neuraminidase polypeptides are full length neuraminidase proteins.

9. The sensor chip according to claim 7, wherein the neuraminidase polypeptides are fragments of full length neuraminidase proteins.

10. The sensor chip according to claim 9, wherein the neuraminidase polypeptide fragments each comprise an immunodominant neuraminidase epitope of their respective native influenza strain.

11. A method for detecting anti-influenza antibodies using arrayed imaging reflectometry (AIR), said method comprising:
providing a sensor chip according to claim 1;
contacting the sensor chip with a sample under conditions that permit specific anti-influenza antibody binding to the hemagglutinin polypeptides present on the chip surface;
rinsing the sensor chip to remove any unbound antibody; and
detecting light reflected off the surface of the chip under conditions effective to identify anti-influenza antibody-antigen binding.

12. The method according to claim 11, wherein the sample is obtained from a human, a non-human primate, a domesticated animal, or a wild animal.

13. The method according to claim 11, wherein said detecting comprises:
measuring light reflected from the chip and
providing an output identifying the hemagglutinin polypeptides bound by an antibody of the sample based on the measured reflected light.

14. The method according to claim 13, wherein the measuring the reflected light further comprises capturing an image of at least a substantial portion of the surface of the chip.