Method for determining DNA concentration by biolayer interferometry

Abstract

The present invention provides methods of determining the concentration of a nucleic acid of interest in a liquid sample. The method uses a capture oligonucleotide and one or more signal oligonucleotides, which hybridize with different regions of the nucleic acid of interest in a liquid phase. The hybridized complex is then captured by a solid phase through the capture oligonucleotide, and detected through the one or more signal oligonucleotides. In one embodiment, bio-layer interferometry (BLI) technique is used for detecting DNA concentrations. BLI is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip.

Description

[0001] METHOD FOR DETERMINING DNA CONCENTRATION BY BIOLAYER INTERFEROMETRY

[0002] FIELD OF THE INVENTION

[0003] The present invention related to a DNA hybridization assay based on interferometry . The present invention provides biolayer interferometry (BLI) methods to determine DNA concentration in a sample by hybridization in a liquid phase. The present invention further provides a method of determining the percentage of full virus capsids in a sample containing a DNA virus.

[0004] BACKGROUND OF THE INVENTION

[0005] Adenoviruses are medium-sized, nonenveloped viruses with an icosahedral nucleocapsid containing a double-stranded DNA genome. The adenovirus particle consists of an icosahedral protein shell (capsids) surrounding a protein core that contains the linear, double-stranded DNA genome. The shell, which is 70 to 100 nm in diameter, is made up of 252 structural capsomeres.

[0006] Herpes simplex virus type 1 (HSV-1) has a large linear double-stranded DNA genome in an icosahedral capsid shell, a cell-derived lipid envelope and a proteinaceous tegument layer. There are over fifty viral proteins and many host proteins identified in HSV-1 virions.

[0007] Adeno-associated viruses (AAVs) have emerged as vectors of choice for gene therapy clinical trials because of their long-term expression and lack of pathogenicity in humans.1. The wild-type adeno-associated virus (AAV) consists of a single-stranded DNA genome up to 4.8 kb in size that is flanked by inverted terminal repeats (ITRs), and this genome is encapsidated within a protein shell that is assembled from 60 proteins at a molar VP1:VP2:VP3 ratio of approximately 1: 1: 10.

[0008] AAV vectors that contain a DNA transgene packaged into a protein capsid have shown tremendous therapeutic potential in recent years. An inherent characteristic of the AAV manufacturing process is production of capsids that are not packaged with the therapeutic transgene and are therefore referred to as empty capsids. In general, up to 95% of the capsids produced upstream in the cell culture may be empty capsids, and this percentage has been shown to vary significantly between independent vector preparations. In its natural life cycle, wild-type AAVs have also been shown to produce a large proportion of empty capsids. The clinical effect of these empty capsids is not well understood, but it has been suggested that there could be elevated immune responses to high concentrations of viral particles and potential impairment of potency through receptor competition. Empty capsids are unable to provide the intended therapeutic benefit and are therefore considered to be a product-related impurity. In addition to empty capsids, a heterogeneous population of partially filled (or intermediate) capsids may also be produced during the manufacturing process, containing packaged process-related impurities or truncated genomes. Therefore, it is essential to have analytical techniques that are capable of providing information regarding the content distribution of AAV capsids, referred to capsid content.

[0009] Effectively quantifying AAV titers and determining the DNA transgene content by empty / full (E / F) capsid ratios is a major challenge for developing and manufacturing gene therapy vectors. A list of currently available tools includes AUC, TEM, ELISA. PCR-based methods and more. TEM (transmission electron microscopy) and AUC (analytical ultracentrifugation) methods are based the capsid size difference under the assumption there is a correlation to the DNA transgene content. qPCR is an established technique but can take hours and require development and optimization for each AAV vector. Digital droplet PCR (ddPCR) is more precise than qPCR but has a smaller dynamic range and requires exact sample dilutions.

[0010] Current methods require that the AAV sample be purified prior to analysis. AAV purification methods are multi-step, labor intensive, and time consuming. A typical AAV purification process includes multiple steps involving filtration, column chromatography, and centrifugation. For example, a commercial scale AAV production includes the steps: thaw HEK cells, expand HEK cells and transfect, mechanical cell lysis (release vector from cells), freeze, depth filtration (clarification), cross flow filtration (concentration), lodixanol centrifugation (full capsid separation), affinity chromatography, and fill and finish. The complexity of the purification process is a major limitation for the accuracy, reproducibility, and adoption of E / F capsid measurements. Also, purified samples are not necessarily very' pure, possibly containing trace contaminants capable of interfering with the analytical method.

[0011] There is a need to have analytical techniques that provides information regarding the content distribution of virus capsids, which is referred to as capsid contents.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 A depicts a biosensor interferometer that includes a light source, a detector, a waveguide, and an optical assembly (also referred to as a “probe”). FIG. IB depicts an example of a probe. FIG. 1C depicts another example of a biosensor interferometer probe.

[0014] FIG. 2A shows a spectral interference pattern generated by two light signals reflected from boundaries between first and second refracting surfaces. FIG. 2B shows the spectral interference pattern shifting from TO to Tl, when analyte molecules bind to the analytebinding molecules on the distal surface of the interference layer.

[0015] FIG. 3 illustrates a method for determining DNA concentration by oligonucleotide hybridization with one biotin on the signal oligonucleotide. The capture oligonucleotide comprises fluorescein (F) to be captured by a probe immobilized with an anti-F antibody. The signal oligonucleotide comprises one biotin to react with anti-biotin-horse radish peroxidase (HRP) to amplify the signals for BLI detection.

[0016] FIG. 4 illustrates a method for determining DNA concentration by oligonucleotide hybridization with two biotins on the signal oligonucleotide.

[0017] FIG. 5 illustrates hybridization pairs with six biotins on several signal oligonucleotides.

[0018] FIG. 6 illustrates a method for determining DNA concentration by oligonucleotide hybridization with six biotins on several signal oligonucleotides.

[0019] FIG. 7 illustrates a method using a first anti- AAV probe to capture AAVs, and then determining the DNA concentration in the lysed AAVs by oligonucleotide hybridization with six biotins on several signal oligonucleotides captured on a second probe.

[0020] FIG. 8 shows BLI signals plotted against GFP concentrations.

[0021] FIG. 9 shows BLI signals with different numbers of biotins per hybridized complex.

[0022] FIG. 10 plots a standard curve of BLI signals against AAV concentrations.

[0023] FIG. 11 shows BLI signals using crosslinked HRP conjugate vs. commercial HRP conjugate.

[0024] FIG. 12 plots BLI signals vs. empty / full ratios of AAV samples.

[0025] FIG. 13 shows BLI signal curves of different full ratios of AAVs captured on the AAVX probe at different time.

[0026] FIG. 14 shows the hybridization signals of the captured AAV samples at different full ratios.

[0027] FIG. 15 plots the normalized shift values against the full ratios to show the standard curve.

[0028] FIG. 16 shows the AAVX quantitation results.

[0029] FIG. 17 shows the GFP hybridization results.

[0030] FIG. 18 illustrates a method using an anti-AAV magnetic particles to capture AAVs, and then determining the DNA concentration in the lysed AAVs by oligonucleotide hybridization with six biotins on several signal oligonucleotides captured on a probe.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] Definitions

[0033] Terms used in the claims and specification are to be construed in accordance with their usual meaning as understood by one skilled in the art except and as defined as set forth below.

[0034] ‘‘About,” as used herein, refers to within ± 10% of the recited value.

[0035] An "analyte-binding" molecule, as used herein, refers to any molecule capable of participating in a specific binding reaction with an analyte molecule. Examples include but are not limited to, (i) antigen molecules, for use in detecting the presence of antibodies specific against that antigen; (ii) antibody molecules, for use in detecting the presence of antigens; (iii) protein molecules, for use in detecting the presence of a binding partner for that protein; (iv) ligands, for use in detecting the presence of a binding partner; or (v) single stranded nucleic acid molecules, for detecting the presence of nucleic acid binding molecules.

[0036] An “aspect ratio” of a shape refers to the ratio of its longer dimension to its shorter dimension.

[0037] A “binding molecule,” refers to a molecule that is capable to bind another molecule of interest.

[0038] “A binding pair,” as used herein, refers to two molecules that are attracted to each other and specifically bind to each other. Examples of binding pairs include, but not limited to, an antigen and an antibody against the antigen, a ligand and its receptor, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectin and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and antifluorescein, digioxigenin / anti-digi oxigenin.

[0039] A “full AAV capsid” contains complete genomic materials, while an “empty AAV capsid” contains no genomic material.

[0040] “Immobilized,” as used herein, refers to reagents being fixed to a solid surface. When a reagent is immobilized to a solid surface, it is either be non-covalently bound or covalently bound to the surface.

[0041] “A monolithic substrate,” as used herein, refers to a single piece of a solid material such as glass, quartz, or plastic that has one refractive index. An “oligonucleotide” or an “oligo”, as used herein, refers to single-stranded nucleotides of 4-1000 nucleotides, preferably 10-500, or 10-100, or 10-60 nucleotides. An “oligonucleotide” or an “oligo” is used interchangeably in this application.

[0042] A “capture oligonucleotide” as used herein, refers to an oligonucleotide that causes the DNA comprising the oligonucleotide to hybridize to its complimentary oligonucleotide on a solid phase and be captured by the solid phase. A “capture oligonucleotide” in general comprises 10-2000 nucleotides or 30-1000 nucleotides, preferably 10-500, 20-500, 10-200, 20-200, 10-100, 20-100, or 30-100 nucleotides.

[0043] A “signal oligonucleotide” as used herein, refers to an oligonucleotide that attaches to a signal molecule that is not an oligonucleotide, and the signal molecule is able to generate a signal for detection, directly or non-indirectly. A “signal oligonucleotide” in general comprises 10-1000 nucleotides or 30-1000 nucleotides, preferably 10-500, 20-500, 10-200, 20-200, 10-100, 20-100, 30-100, or 30-50 nucleotides.

[0044] A BLI “probe,” as used herein, refers to a monolithic substrate having as aspect ratio (length-to-width) of at least 2 to 1 with a thin-film layer coated on the sensing side. A probe is typically made of a material suitable for BLI detection such as glass. A probe has a distal end and a proximal end. The proximal end (also refers to probe tip in the application) has a sensing surface coated with a thin layer of analyte-binding molecules.

[0045] A “waveguide” refers to a device (e g., a duct, coaxial cable, or optic fiber) designed to confine and direct the propagation of electromagnetic waves (as light).

[0046] The present invention provides methods of determining the concentration of a nucleic acid of interest in a liquid sample. The method uses a capture oligonucleotide and one or more signal oligonucleotides, which hybridize with different regions of the nucleic acid of interest in a liquid phase. The hybridized complex is then captured by a solid phase through the capture oligonucleotide, and detected through the one or more signal oligonucleotides. In one embodiment, the liquid sample contains viruses. In one embodiment, bio-layer interferometry (BLI) technique is used for detecting DNA concentrations. BLI is an optical technique for measuring macromolecular interactions by analyzing interference paterns of white light reflected from the surface of a biosensor tip.

[0047] The invention further provides methods to determine DNA concentrations in viruses and determine empty vs. full (E / F) virus capsids ratio. The initial step is to capture specific virus by capturing a defined amount of capsid particles on the surface of a first solid phase. The first solid phase is designed for specific virus capture with negligible non-specific binding from interfering substances in samples. The second step is to lyse the capsid to release the virus DNA from the first solid phase into a lysis solution. After separating the lysis solution from the first solid phase, the third step is to hybridize the DNAs in the lysis solution with a capture oligonucleotide and one or more signal oligonucleotides, and then contacting the hybridized DNAs with a second solid surface. The second solid phase captures and measures the total amount of DNA derived from the captured capsid. Since the amount of capsid particles is known in the initial capture step and / or is consistent from sample to sample, the total DNA assay signal is proportional to the E / F ratio. By comparing the DNA signal to a calibration curve of of known mixtures of E / F ratios, one can derive the actual E / F ratio in the sample.

[0048] Biosensor Interferometer Systems

[0049] FIG. 1A depicts a biosensor interferometer 100 (or simply “interferometer”) that includes a light source 102, a detector 104, a waveguide 106, and an optical assembly 108 (also referred to as a “probe”). The probe 108 may be connected to the waveguide 106 via a coupling medium.

[0050] The light source 102 may emit white light that is guided toward the probe 108 by the w aveguide 106. For example, the light source 102 may be a light-emitting diode (LED) that is configured to produce light over a range of at least 50 nanometers (nm), 100 nm, or 150 nm within a given spectrum (e.g., 400 nm or less to 700 nm or greater). Alternatively, the interferometer 100 may employ a plurality of light sources having different characteristic wavelengths, such as LEDs designed to emit light at different wavelengths in the visible range. The same function could be achieved by a single light source with suitable filters for directing light with different wavelengths onto the probe 108.

[0051] The detector 104 is preferably a spectrometer, such as an Ocean Optics USB4000, that is capable of recording the spectrum of interfering light received from the probe 108. Alternatively, if the light source 102 operates to direct different wavelengths onto the probe 108, then the detector 104 can be a simple photodetector capable of recording intensity at each wavelength. In another embodiment, the detector 104 can include multiple filters that permit detection of intensity at each of multiple wavelengths.

[0052] The waveguide 106 can be configured to transport light emitted by the light source 102 to the probe 108. and then transport light reflected by surfaces within the probe 108 to the detector 104. In some embodiments the waveguide 106 is a bundle of optical fibers (e.g., single-mode fiber optic cables), while in other embodiments the waveguide 106 is a multimode fiber optic cable.

[0053] As shown in FIG. IB, the probe 108 includes a monolithic substrate 114, a thin-film layer (also referred to as an “interference layer”), and a biomolecular layer (also referred to as a “biolayer”) comprised of analyte molecules 122 that have bound to analyte-binding molecules 120. The monolithic substrate 114 is comprised of a transparent material through which light can travel. The interference layer is also comprised of a transparent material. When light is shone on the probe 108, the proximal surface of the interference layer may act as a first reflecting surface and the biolayer may act as a second reflecting surface. As further described below, light reflected by the first and second reflecting surfaces may form an interference pattern that can be monitored by the interferometer 100.

[0054] The interference layer normally includes multiple layers that are combined in such a manner to improve the detectability of the interference pattern. For example, the interference layer is comprised of a tantalum pentoxide (TazOs) layer 116 and a silicon dioxide (SiO2) layer 118. The tantalum pentoxide layer 116 may be thin (e.g., on the order of 10-40 nm) since its main purpose is to improve reflectivity at the proximal surface of the interference layer. Meanwhile, the silicon dioxide layer 118 may be comparatively thick (e.g., on the order of 650-900 nm) since its main purpose is to increase the distance between the first and second reflecting surfaces.

[0055] To illustrate a simple interferometry test, the probe 108 can be suspended in a well 110 that includes a sample 112. Analyte molecules 122 will bind to the analyte-binding molecules 120 along the distal end of the probe 108 over the course of the diagnostic test, and these binding events will result in an interference pattern that can be observed by the detector 104. The interferometer 100 can monitor the thickness of the biolayer formed along the distal end of the probe 108 by detecting shifts in a phase characteristic of the interference pattern.

[0056] FIG. 1C illustrates another biosensor interferometer probe. The probe includes a monolithic substrate that has a first and a second surfaces arranged substantially parallel to one another at opposite ends of the monolithic substrate, an interference layer coated on the second surface of the monolithic substrate, and a layer of analyte-binding molecules coated on the interference layer. The interference layer will generally be comprised of magnesium fluoride (MgFz). A first interface between the monolithic substrate and the interference layer acts as a first reflecting surface when light is shone on the interferometric sensor, while a second interface between a biolayer formed by analyte molecules in a sample binding to the analyte-binding molecules and a solution containing the sample acts as a second reflecting surface when the light is shone on the probe. As described above, the thickness of the biolayer can be estimated based on the interference pattern of light reflected by the first and second reflecting surfaces.

[0057] The probe 200 includes an interference layer 204 that is secured along the distal end of a monolithic substrate 202. Analyte-binding molecules 206 can be deposited along the distal surface of the interference layer 204. Over the course of a biochemical test, a biolayer will form as analyte molecules 208 in a sample bind to the analyte-binding molecules 206.

[0058] As shown in FIG. 1C, the monolithic substrate 202 has a proximal surface (also referred to as a '‘coupling side”) that can be coupled to, for example, a waveguide of an interferometer and a distal surface (also referred to as a “sensing side”) on which additional layers are deposited. Generally, the monolithic substrate 202 has a length of at least 3 millimeters (mm), 5 mm. 10 mm, or 15 mm. In a preferred embodiment, the aspect ratio (length-to-width) of the monolithic substrate 202 is at least 5 to 1. In such embodiments, the monolithic substrate 202 may be said to have a columnar form. The cross section of the monolithic substrate 202 may a circle, oval, square, rectangle, triangle, pentagon, etc. The monolithic substrate 202 preferably has a refractive index that is substantially higher than the refractive index of the interference layer 204, such that the proximal surface of the interference layer 204 effectively reflects light directed onto the probe 200. The preferred refractive index of the monolithic substrate may be higher than 1.5, 1.8, or 2.0. Accordingly, the monolithic substrate 202 may be comprised of a high-refractive-index material such as glass (refractive index of 2.0) rather than a low-refractive-index material such as quartz (refractive index of 1.46) or plastic (refractive index of 1.32-1.49).

[0059] The interference layer 204 is comprised of at least one transparent material that is coated on the distal surface of the monolithic substrate 202. These transparent material(s) are deposited on the distal surface of the monolithic substrate 202 in the form of thin films ranging in thickness from fractions of a nanometer (e.g., a monolayer) to several micrometers. The interference layer 204 may have a thickness of at least 500 nm, 700 nm, or 900 nm. An exemplary thickness is between 500-5,000 nm (and preferably 800-1,200 nm). Here, for example, the interference layer 204 has a thickness of approximately 900-1,000 nm, or 940 nm.

[0060] In contrast to conventional probes, the interference layer 204 has a substantially similar refractive index as the biolayer. This ensures that the reflection from the distal end of the probe 200 is predominantly due to the analyte molecules 208 rather than the interface between the interference layer 204 and the analyte-binding molecules 206. In some embodiments, the interference layer 204 is comprised of magnesium fluoride (MgF2), while in other embodiments the interference layer 204 is comprised of potassium fluoride (KF), lithium fluoride (LiF), sodium fluoride (NaF), lithium calcium aluminum fluoride (LiCaAlFe), sodium aluminum fluoride (Na3AlFe), strontium fluoride (SrF2), aluminum fluoride (AIF3), sulphur hexafluoride (SFe), etc. Magnesium fluoride has a refractive index of 1.38, which is substantially identical to the refractive index of the biolayer formed along the distal end of the probe 200. For comparison, the interference layer of conventional probes is normally comprised of silicon dioxide, and the refractive index of silicon dioxide is approximately 1.4-1.5 in the visible range. Because the interference layer 204 and biolayer have similar refractive indexes, light will experience minimal scattering as it travels from the interference layer 204 into the biolayer and then returns from the biolayer into the interference layer 204.

[0061] In one embodiment, the probe 200 includes an adhesion layer that is deposited along the distal surface of the interference layer 204 affixed to the monolithic substrate 202. The adhesion layer may be comprised of a material that promotes adhesion of the analyte-binding molecules 206. One example of such a material is silicon dioxide. The adhesion layer is generally very7thin in comparison to the interference layer 204, so its impact on light traveling toward, or returning from, the biolayer will be minimal. For example, the adhesion layer 310 may have a thickness of approximately 3-10 nm, while the interference layer 304 may have a thickness of approximately 800-1,000 nm. The biolayer formed by the analytebinding molecules 306 and analyte molecules 308 will normally have a thickness of several nm.

[0062] When light is shone on the probe 200, the proximal surface of the interference layer 204 may act as a first reflecting surface and the distal surface of the biolayer may act as a second reflecting surface. The presence, concentration, or binding rate of analyte molecules 208 to the probe 200 can be estimated based on the interference of beams of light reflected by these two reflecting surfaces. As analyte molecules 208 attach to (or detach from) the analyte-binding molecules 206, the distance between the first and second reflecting surfaces will change. Because the dimensions of all other components in the probe 200 remain the same, the interference pattern formed by the light reflected by the first and second reflecting surfaces is phase shifted in accordance with changes in biolayer thickness due to binding events.

[0063] In operation, an incident light signal 210 emitted by a light source is transported through the monolithic substrate 202 toward the biolayer. Within the probe 200, light will be reflected at the first reflecting surface resulting in a first reflected light signal 212. Light will also be reflected at the second reflecting surface resulting in a second reflected light signal 214. The second reflecting surface initially corresponds to the interface between the analytebinding molecules 206 and the sample in which the probe 200 is immersed. As binding occurs during the biochemical test, the second reflecting surface becomes the interface between the analyte molecules 208 and the sample.

[0064] The first and second reflected light signals form a spectral interference pattern, as show n in FIGs. 2A and 2B. When analyte molecules bind to the analyte-binding molecules on the distal surface of the interference layer, the optical path of the second reflected light signal will lengthen. As a result, the spectral interference pattern shifts from TO to T1 as shown in FIG. 2B. By measuring the phase shift after a period of time, the association of an analyte molecule to an analyte-binding molecule immobilized on the distal surface of the interference layer 204 can be used to calculate analyte concentration in the sample. By measuring the phase shift continuously in real time, a kinetic binding curve can be plotted as the amount of shift versus the time. The association rate of an analyte molecule to an analyte-binding molecule immobilized on the distal surface of the interference layer can be used to calculate analyte concentration in the sample. Hence, the measure of the phase shift is the detection principle of a thin-film interferometer.

[0065] Methods for Determining Concentration of Nucleic Acid of Interest by Solution Phase Hybridization and BLI Detection

[0066] The present invention is directed to methods of determining the concentration of a nucleic acid of interest in a liquid sample and using BLI technique. The nucleic acid can be DNA or RNA.

[0067] One Signal Oligo

[0068] In this aspect, the method uses one capture oligonucleotide to hybridize to one region of the nucleic acid (e g., DNA) of interest and to immobilize the hybridized complex to a BLI probe, and one signal oligonucleotide to hybridize to another region of the nucleic acid of interest and to generate the signals for BLI detection.

[0069] The method comprises the steps of: (a) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the nucleic acid of interest; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5 ’-end or 3 ’-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’ - and / or 3 ’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens; (b) adding the capture oligonucleotide and the signal oligonucleotide to the liquid sample comprising the nucleic acid of interest and heating the mixture to denature nucleic acids in the sample to single-stranded nucleic acids; (c) cooling the heated sample to hybridize the capture oligonucleotide and the signal oligonucleotide to the single-stranded nucleic acid of interest, (d) dipping a probe comprising a second member of the first binding pair on the probe tip to the hybridized sample, wherein the probe captures hybridized nucleic acid of interest through the first binding pair; (e) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair; (f) dipping the probe in a HRP substrate solution to chemically react the substrate with the HRP in the conjugate for a period of time; and (g) determining the concentration of the nucleic acid of interest by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

[0070] In one embodiment, the first and the second binding pairs are selected from the group consisting of: biotin and streptavidin, biotin and avidin, biotin and anti-biotin, fluorescein and anti-fluorescein, digioxigenin / anti-digioxigenin.

[0071] One specific embodiment of the method is illustrated in FIG. 3. Another specific embodiment of the method is illustrated in FIG. 4.

[0072] In one embodiment, the liquid sample comprises nucleic acids such as DNAs or RNAs.

[0073] In another embodiment, the liquid sample comprises a virus sample that comprise nucleic acids. The virus may be a DNA virus such as adeno-associated viruses (AAV), adenovirus, or herpes simplex virus type (HSV-1), or a RNA virus such as Lentivirus.

[0074] In step (a), a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the nucleic acid of interest are obtained. The capture oligonucleotide further comprises a first member of a first binding pair at the 5’-end or 3’-end, to immobilize the hybridized complex to a BLI probe. The signal oligonucleotide further comprises a first member of a second binding pair at the 5 ’-end and / or 3 ’-end.

[0075] FIG. 3 illustrates the capture oligonucleotide comprises fluorescein (F) to be captured by a probe immobilized with an anti-F antibody. FIG. 3 also illustrates the signal oligonucleotide comprises one biotin to react with anti-biotin-horse radish peroxidase (HRP) or streptavidin-HRP to amplify the signals for BLI detection.

[0076] FIG. 4 illustrates the signal oligonucleotide comprises two biotins at both 5 ’-end and 3 ’-end to react with anti-biotin-HRP or streptavidin-HRP to further amplify the signals for BLI detection.

[0077] The tip of probe for detecting ssDNA may have a smaller surface area with a diameter < 5 mm, preferably < 2 mm or < 1 mm. The small surface of the probe tip provides several advantages. In solid phase immunoassays, a probe having a small surface area has less nonspecific binding and thus produces a lower background signal. Further, the reagent or sample carried over on the probe tip is extremely small due to the small surface area of the tip. This feature makes the probe tip easy to wash and results in negligible contamination in the wash solution since the wash solution has a larger volume. Negligible contamination of the wash solution and small consumption of the reagents enable the reagents and the wash solution to be re-used many times, if desired, for example, 3-10 times, 3-15 times, or 3-20 times.

[0078] Methods to immobilize a protein such as an antibody to a solid phase (the sensing surface of the probe tip) are common in immunochemistry . A protein can bind directly to the solid phase through adsorption or it can bind indirectly to the solid phase through a binding pair.

[0079] In step (b), the capture oligonucleotide and the signal oligonucleotide are added to the liquid sample comprising the nucleic acid of interest and the mixture is heated to denature nucleic acids in the sample to single-stranded nucleic acids without secondary structures. The techniques of denaturing DNA are known in the art. The heating step is typically at a temperature 50 -90°C, or 70 -90°C, or 80-95°C for ssDNA (e.g., AAV), and 80-95°C or 90- 100°C for dsDNA. A lysis solution containing a detergent having a concentration at least 1% or 2%, for example, 2-4% of Tween 20, can be added to reduce the temperature of time for heating. The heating time is typically 2-20 minutes or 2-10 minutes. If the liquid sample contains visuses such as AAVs, the liquid sample is preferably heated in a lysis solution to lyse the capsids of the viruses to release the nucleic acids into the solution. In step (c), the heated sample is cooled down in order to hybridize the capture oligonucleotide and the signal oligonucleotide to the single-stranded nucleic acid of interest. In general, the hybridization temperature should be at least 6-8 °C lower (e.g., 10-15 °C low er) than the melting temperature of the melting temperature of the complementary nucleic acids. For example, in step (c), the hybridization can be performed at room temperature for about 15-25 minutes, or at 10-20 °C for 5-10 minutes, or at 2-8 °C for 1-5 minutes.

[0080] In step (d), a BLI probe comprising a second member of the first binding pair on the probe tip is dipped into the hybridized sample to capture the hybridized nucleic acid of interest through the first binding pair. The reaction time is about 2-30 minutes, preferably 2- 20 minutes. 5-20 minutes, or 5-15 minutes.

[0081] The probe is optionally washed in a wash solution 1-3 times for a period of time (e.g., 5 seconds to 5 minutes, 10 seconds to 2 minutes, or 15 seconds to 1 minute). The wash solution comprises an aqueous solution preferably having pH of 6.0-8.5. The aqueous solution can be water or a buffer having pH between 6.0 to 8.5. Preferably, the aqueous solution contains 1-10 mM or 1-100 mM of phosphate buffer, tris buffer, citrate buffer or other buffer suitable for pH between 6.0-8.5.

[0082] In step (e), the probe is dipped in a conjugate solution comprising a second member of the second binding pair (such as streptavidin or anti-biotin) and horse radish peroxidase (HRP) for a period of time (e.g., 10 seconds to 5 minutes, 15 seconds to 1 minute, or 20 seconds to 2 minutes), to bind the conjugate to the probe.

[0083] In one embodiment, the conjugate is a high molecular conjugate comprising multiple second members of the second binding pair, which increases the signal and sensitivity of the assay. In general, the high molecular weight conjugate has a molecular weight of at least 200,000, or at least 400,000, or at least 500,000, or at least 1 million Daltons, or at least 2 million Daltons. For example, the conjugate has a molecular weight of 200,000 to 10 million Daltons, 200,000 to 10 million Daltons, 500,000 to 10 million Daltons, 1 to 10 million Daltons, or 2-10 million Daltons.

[0084] In one embodiment, the high molecular weight conjugate comprises multiple second members of the second binding pair (for example, streptavidins, anti-biotins) and multiple crosslinked HRP, wherein the conjugate comprises at least 2, or at least 4, or at least 5, or at least 10 HRPs. For example, the conjugate comprises 2-10 HRPs.

[0085] In one embodiment, the high molecular weight conjugate comprises anti- biotin / streptavidin and HRP, where streptavidin and HRP are crosslinked together. The conjugate comprises at least 2, or at least 4, at least 5, or at least 10 HRPs, and the molar ratio of streptavidin to HRP is between about 1 :2 to about 2: 1, or about 1: 1.5 to about 1.5 to 1, e.g., about 1: 1.

[0086] In a preferred embodiment, the high molecular weight conjugate comprises a second member of the second binding pair, HRP and a polymer. In the conjugate, the second member of the second binding pair and HRP may bind to the polymer. The polymer in general has a molecular weight of 200,000 to 10 million Daltons, or 500,000 to 10 million Daltons, or 1 million to 10 million Daltons, or 2 million to 10 million Daltons. The polymer can be a polysaccharide (e.g., dextran, amylose, polysucrose), a dendrimer, or a polyethylene glycol. In one preferred embodiment, the polymer is FICOLL® (copolymers of sucrose and epichlorohydrin). The conjugate comprises at least 2, or at least 4, at least 5, or at least 10 HRPs. For example, the conjugate comprises 2-10 HRPs. The molar ratio of the second member of the second binding pair such as streptavidin to HRP is between about 1:2 to about 1 : 10.

[0087] After binding, the probe is then optionally washed in a wash solution as for 1-3 times. The wash solution is described in step (d) above.

[0088] In step (f), the probe is dipped in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time (e.g., 30 seconds to 10 minutes).

[0089] When the product of the HRP / substrate reaction binds to the surface of the optical layer, the optical layer increases its thickness and produces a nanometer wavelength shift due to light interference, and the wavelength shift is proportional to the amount of HRP on the probe. Any HRP substrate whose product binds to solid phase surfaces is suitable for the present invention. A precipitating substrate is preferred for BLI assay. Suitable substrates include 3,3'-diaminobenzidine tetrahydrochloride (DAB), 3,3',5,5'-tetramethylbenzidine (TMB). benzamidine, 4-chloro-l-naphthol, nitro-blue tetrazolium chloride and 5-bromo-4- chloro-3'-indolyphosphate p-toluidine salt. Preferred substrates are chloronapthol, benzamidine, and tetramethylbenzidine, and chloronapthol is more preferred.

[0090] In step (g), the single-stranded nucleic acid concentration in the sample is determined by measuring the wavelength shift due to light interference, and the wavelength shift is quantitated against a calibration curve to determine the ssDNA concentration. The phase shift can be monitored either kinetically or determined by the difference between starting time point (TO) and end time point (Tl) (see FIG. 2B).

[0091] In the above binding and washing steps, the reaction is optionally accelerated by agitating or mixing the solution in the vessel. For example, a flow such as a lateral flow or an orbital flow of the solution across the probe tip can be induced in one or more reaction vessels, including sample vessel, reagent vessel, wash vessels, and conjugate vessel, to accelerates the binding reactions, disassociation. For example, the reaction vessels can be mounted on an orbital shaker and the orbital shaker is rotated at a speed at least 50 rpm, preferably at least 200 rpm or at least 500 rpm, such as 50-200 or 500-1,500 rpm. Additionally, the probe tip can be moved up and down and perpendicular to the plane of the orbital flow, at a speed of 0.01 to 10 mm / second, in order to induce additional mixing of the solution above and below the probe tip.

[0092] Multiple Signal Oligos

[0093] In a second aspect for determining the concentration of nucleic acid of interest in a liquid sample, the method uses one capture oligonucleotide to hybridize to one region of the nucleic acid of interest and to immobilize the hybridized complex to a BLI probe, and a first signal oligonucleotide to hybridize to another region of the nucleic acid of interest, and one or more additional signal oligonucleotides to hybridize to the first signal oligonucleotide, wherein the multiple signal oligonucleotides comprising signal generating molecules to increase the signals for BLI detection.

[0094] The method comprises the steps of: (a) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary to a first region of the nucleic acid of interest and a first member of a first binding pair at the 5 ’-end or the 3 ’-end; (b) obtaining a first signal oligonucleotide comprising (i) a second oligonucleotide sequence complementary to a second region of the nucleic acid of interest, wherein the first and the second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourth oligonucleotide each between the second oligonucleotide and the first member of the second binding pair each at one end of the first signal oligonucleotide, wherein the first and the second binding pairs are different, and the first members of the first and the second binding pairs are haptens; (c) obtaining a second signal oligonucleotide which sequence is complementary' to the third oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends; (d) obtaining a third signal oligonucleotide which sequence is complementary' to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends; (e) adding the capture oligonucleotide, the first signal oligonucleotide, the second signal oligonucleotide, and the third signal oligonucleotide to the liquid sample comprising the nucleic acid of interest and heating the mixture to denature DNAs in the sample to single-stranded DNAs; (f) cooling the heated sample to hybridize (i) the first oligonucleotide and the second oligonucleotide to the single-stranded nucleic acid of interest, (ii) the second signal oligonucleotide to the third oligonucleotide, and (iii) the third signal oligonucleotide to the fourth oligonucleotide; (g) dipping a probe comprising a second member of the first binding pair on the probe tip to the hybridized sample, wherein the probe captures hybridized nucleic acid of interest through the first binding pair; (h) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair; (i) dipping the probe in a HRP substrate solution to chemically react the substrate with the HRP in the conjugate for a period of time; an (j ) determining the concentration of the nucleic acid of interest by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

[0095] In the method, the first and the second binding pairs are selected from the group consisting of: biotin and streptavidin, biotin and avidin, biotin and anti-biotin, fluorescein and anti-fluorescein, digioxigenin / anti-digioxigenin.

[0096] The hybridization pairs of Steps (a) to (d) are illustrated in FIG. 5.

[0097] The method of determining DNA concentration in a sample is illustrated in FIG. 6.

[0098] In this method using multiple signal oligos, details of the steps that are similar or the same to those of the method using a single signal oligo have been described above, and thus are not repeated here.

[0099] The third oligonucleotide and the fourth oligonucleotide can be the same or different. Preferably, the third oligonucleotide and the fourth oligonucleotide are the same.

[0100] In one embodiment, the third oligonucleotide and / or the fourth oligonucleotide is poly A, poly T, or Poly(ATGG).

[0101] Methods for Determining Nucleic Acid Concentration in Viruses by BLI Detection

[0102] The present invention is further directed to a method of determining nucleic acid concentration in a virus. In one embodiment, the method determines DNA concentration in DNA viruses such as adeno-associated viruses (AAVs), adenovirus, and Herpes simplex virus t pe 1 (HSV-1), by solution phase hybridization and bio-layer interferometry detection.

[0103] The method first uses a solid phase to capture the vims, and then lyses and denatures the nucleic acids from virus. The denatured nucleic acids separate from the solid phase and diffuse into the hybridization reagent where hybridization with the capture oligo and signal oligo occurs. One capture oligonucleotide is used to hybridize to one region of the virus nucleic acid (e.g., DNA) and to immobilize the hybridized complex to a BLI probe, and one or more signal oligonucleotides is / are used to hybridize to another region of the nucleic acid of interest and to generate the signals for BLI detection.

[0104] The methods include the same steps for determining the concentration of DNA in a liquid sample as described above.

[0105] One Signal Oligo

[0106] In this aspect, the method uses one capture oligonucleotide to hybridize to one region of the virus nucleic acid and to immobilize the hybridized complex to a BLI probe, and one signal oligonucleotide to hybridize to another region of the virus nucleic acid and to generate signals for BLI detection.

[0107] In this aspect for determining nucleic acid concentration in a virus, the method comprises the steps of: (a) obtaining a first probe having a fixed amount of an anti-virus antibody immobilized on the tip of the probe, wherein the anti-virus antibody is against a virus in the sample; (b) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the virus nucleic acid; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5 ’-end or 3 ’-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’- and / or 3’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens; (c) dipping the first probe tip in a sample solution comprising a virus sample to capture the virus on the probe; (d) dipping the first probe in a wash solution to wash the first probe tip; (e) dipping the first probe in a lysis solution containing the capture oligonucleotide and the signal oligonucleotide and heating the lysis solution to lyse the virus from the first probe tip and to denature nucleic acids in the virus to single-stranded nucleic acids; (f) removing the first probe from the heated lysis solution; (g) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotide to its complementary region of the singlestranded virus nucleic acid; (h) dipping a second probe comprising a second member of the first binding pair on the probe tip to the solution of (g), wherein the second probe captures hybridized nucleic acids through the first binding pair; (i) dipping the second probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP). to bind the conjugate to the probe through the second binding pair; (j ) dipping the second probe in a HRP substrate solution to bind the substrate to the HRP in the conjugate for a period of time; and (k) determining the concentration of the virus nucleic acid by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

[0108] The first probe to capture the virus can be replaced by other solid phases including magnetic particles such as magnetic beads; tubes; microwells such as microplates containing 96 well or 384 wells in ELISA format; polystyrene beads or latex beads in micron and nanometer sizes; nanospheres; microspheres; Sepharose, agarose, dextran and other carbohydrate base resins; glass / silica, borate silicate / quartz materials in slide, pin, fiber, microsphere, or bead format; magnetic particles with latex, dextran, polymer matrices in nano, microsphere, or rod format; quantum dots nanocrystals, Europium Chelates conjugates with or without polymer coating on Europium surfaces. In one preferred embodiment, the solid phase is magnetic particle such as magnetic bead.

[0109] In step (c) of the method, the first probe tip is dipped in a sample solution comprising a virus sample for a first period of time to capture a defined amount of viruses on the probe in a defined binding condition. Alternatively, a sample solution comprising a virus sample is incubated with a solid phase, such as magnetic particles immobilized with anti-virus antibody, for a first period of time to capture a defined amount of viruses on the probe in a defined binding condition. The binding time is from 10 minutes, 20 minutes, or 30 minutes to 1 or 2 hours. For example, the binding time is from 10 minutes to 1 hour. In a preferred embodiment, the amount of the anti-virus on the probe is fixed and the binding condition is fixed, so about the same amount of viruses is capture in different samples. In general, viruses in the sample exceed the maximum binding capacity of anti-virus on the solid phase. In one embodiment, the virus binding is allowed to approach the maximum binding with the antivirus on the solid phase.

[0110] This step provides an important aspect of the invention that the capture of virus is standardized to the same number of viral particles from sample to sample.

[0111] In step (e) of the method, the first probe tip is dipped into a lysis solution, and the lysis solution is heated to remove nucleic acids from the capsids, release the nucleic acids into the lysis reagent, and denature the nucleic acids into single-strand. There are different methods to lyse viruses and to release nucleic acid that can be used in this invention; heating plus detergent lysis is preferred due to its speed. The lysis solution in general contains a detergent having a concentration at least 1% or 2%. For example, the lysis solution contains 2-4% of a non-ionic polysorbate detergent such as Tween 20. The heating step is typically at a temperature 50 -90°C, or 70 -90°C, or 80-95°C for ssDNA (e.g., AAV), and 80-95°C or 90- 100°C for dsDNA. The heating time is typically 2-5 minutes or 2-10 minutes. After the DNA is lysed from the first probe, the first probe is removed from the lysis solution.

[0112] In step (I), the first probe or the solid phase is removed from the cooled solution.

[0113] In order to accelerate the hybridization time, the lysis solution of step (e) which is also the hybridization solution in step (g) optionally contains a high salt of concentration higher than IM, e.g., NaCl concentration of > IM, such as 1.2-2M. High salt promotes hybridization. However, a capture probe does not bind to its counterpart of capture oligo at such high salt concentration. When high salt is used for hybridization, before adding the capture probe (the second probe), an aliquot of water is mixed with the sample to lower the salt concentration (e.g.. < 0.5 M) to facilitate the capture step of (h). which the second probe captures hybridized DNAs through the first binding pair.

[0114] In steps (c) to (f), when other solid phases (e.g., magnetic beads) instead of a probe are used, the reactions are carried out by reacting the solutions with the solid phase, and then separating the solutions from the solid phase.

[0115] The remaining steps are similar to those described above in Methods for Determining Concentration of nucleic acid of Interest by Solution Phase Hybridization and BLI Detection.

[0116] Multiple Signal Oligos

[0117] In this aspect, the method uses one capture oligonucleotide to hybridize to one region of the virus nucleic acids and to immobilize the hybridized complex to a BLI probe, and multiple signal oligonucleotides to hybridize to another region of the virus DNA and to generate signals for BLI detection. FIG. 7 is an illustration of one embodiment.

[0118] In this method for determining nucleic acid concentration in a sample containing a virus, the method the steps of: (a) obtaining a first probe having a fixed amount of an antivirus antibody immobilized on the tip of the probe, wherein the anti-virus antibody is against a virus in the sample; (b) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary to a first region of the virus nucleotide and a first member of a first binding pair at the 5 '-end or the 3 '-end; (c) obtaining a first signal oligonucleotide comprising (i) a second oligonucleotide sequence complementary’ to a second region of the virus DNA, wherein the first and the second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourth oligonucleotide each between the second oligonucleotide and the first member of the second binding pair at one end of the first signal oligonucleotide; wherein the first and the second binding pairs are different, and the first members of the first and the second binding pairs are haptens; (d) obtaining a second signal oligonucleotide which sequence is complementary to the third oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends; (e) obtaining a third signal oligonucleotide which sequence is complementary to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5'- and 3'- ends; (f) dipping the first probe tip in the sample comprising the virus to capture the virus on the probe; (g) dipping the first probe in a wash solution to wash the first probe tip; (h) dipping the first probe in a lysis solution containing the capture oligonucleotide and the first, the second, and the third signal oligonucleotides and heating the lysis solution to lyse the virus from the first probe tip and to denature DNAs in the virus to single-stranded DNAs; (i) removing the first probe from the heated lysis solution; (j) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotides to its complementary region of the single-stranded virus DNA; (k) dipping a second probe comprising a second member of the first binding pair on the probe tip to the solution of (j), wherein the second probe captures the hybridized nucleotides through the first binding pair; (1) dipping the second probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and HRP, to bind the conjugate to the second probe through the second binding pair; (m) dipping the second probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time; and (n) determining the concentration of the virus DNA by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

[0119] The details of each step are similar to the details of similar steps described above. The first probe to capture the viruses may be replaced by other solid phases, for example, magnetic beads, as described above.

[0120] Method for Determining Empty vs. Full Virus Capsids by BLI Detection

[0121] The present invention is directed to a method for measuring the percentage of full virus capsid in a vims (e.g., AAV) sample; also called measuring empty (empty vims capsid) vs. full (full virus capsid), or measuring a full ratio.

[0122] One Signal Oligo and Multiple Signal Oligos

[0123] The BLI signal of capturing DNA viruses (e.g., AAVs) or RNA virus is directly proportional to the number of capsids captured in real time. In theory, if the standards and samples have the same virus (e.g., AAV) concentration or the same number of capsids captured, the BLI signals of virus binding are the same, and the ssDNA signals are proportional to the empty / full capsid ratio and the empty / full capsid ratio of a virus (e.g., AAV) sample can be determined from the standard curve, in which calibrators comprises mixtures of empty and full capsids of different ratios. The calibrators are run at the same virus concentration.

[0124] In a method of determine the percentage of full virus capsids in a sample, the method comprises first determining the single-stranded (ss) nucleic acid concentration in a virus sample according to the method described above, and then converting the ss nucleic acid concentration to % of full virus capsid using a calibration curve having DNA concentration plotted against % of full virus capsid standards. The calibration curve can be established with results of calibrators that run together with unknown samples, or with results of calibrators from a separate run.

[0125] Method for Determining Empty vs. Full Virus Capsids by BLI Detection with Normalization

[0126] If the standards and samples have different virus concentrations or different numbers of capsids captured, the BLI signals of virus (e.g., AAV) binding may be different; which may then bias the data analysis in the DNA concentration assay to derive empty / full ratio in a virus sample. The present invention provides a method to normalize the BLI signals of virus binding and align the sample concentration difference in order to determine the empty capsid / full capsid ratio.

[0127] In using normalization for the empty / full capsid ratio determination, the virus binding signal in each sample is normalized by comparison with the virus binding signal of one or more calibrators to give a normalization factor. The ssDNA signal (wavelength) of a sample is multiplied by the normalization factor to yield the normalized wavelength shift value of a sample. The calibrators are run at the same virus concentration and are used to generate a standard curve of BLI signal vs. empty / full virus capsid ratio. DNA virus samples are run together with the calibrators and the wavelength shift values of a sample is normalized by the normalization factor, then the empty / full ratio of the sample is determined from the standard curve. As the normalized wavelength shift values take into account the capture difference due to different virus concentration in samples, the normalization may be more accurate to calculate the empty / full ratio in some situation.

[0128] One Signal Oligo / Normalization

[0129] In a first aspect, the method uses one capture oligonucleotide to hybridize to one region of the virus nucleic acid and to immobilize the hybridized complex to a BLI probe, and one signal oligonucleotide to hybridize to another region of the virus nucleic acid and to generate the signals for BLI detection.

[0130] The method comprises the steps of: (a) obtaining a first probe having a fixed amount of anti-virus antibody immobilized on the tip of the probe, wherein the anti-virus antibody is against the virus in the sample;(b) dipping the first probe tip in the sample to capture the virus on the probe in a defined binding condition for a first period of time to measure a first wavelength shift due to light interference as a result of the binding virus on the probe; (c) determining a normalization factor based on the first wavelength shift of the sample in comparison with (i) the first wavelength shift of a calibrator, or (ii) the average of the first wavelength shifts of all calibrators; (d) dipping the first probe in a wash solution to wash the first probe tip; (e) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the virus nucleic acid; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5’-end or 3’-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’- and / or 3’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens; (f) dipping the first probe in a lysis solution containing the capture oligonucleotide and the signal oligonucleotide and heating the lysis solution to lyse the virus from the first probe tip and to denature nucleic acids in the virus to single-stranded nucleic acids; (g) removing the first probe from the heated lysis solution; (h) cooling the heated lysis solution to hybridize the capture oligonucleotide and the signal oligonucleotide to the complementary regions of the singlestranded virus nucleic acid; (i) dipping a second probe comprising a second member of the first binding pair on the probe tip to the heated lysis solution of (g), wherein the second probe captures the hybridized nucleic acids through the first binding pair; (j) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP). to bind the conjugate to the probe through the second binding pair; (k) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time, and measuring the second wavelength shift due to light interference; (1) applying the normalization factor to the second wavelength shift to produce a normalized wavelength shift; and (m) quantitating the normalized wavelength shift against a calibration curve having normalized wavelength shift plotted against % of full virus capsids to determine the percentage of full virus capsids in the sample.

[0131] In step (c), the normalization factor is determining by comparing the first wavelength shift of virus binding to the probe of the sample with that of one or more calibrators. A calibrator is a virus (e.g., AAV) sample with known empty / full capsid ratio and captured at the same virus concentration as the other calibrators. The calibrators can be run together with the samples under the same assay conditions. Alternatively, the calibrators can be run separately from the samples under the same assay conditions. In general, 1-6, 2-6, 3-6, 2-5, or 3-5 calibrators are used to establish a normalization factor. The normalization factor is determined by the ratio of the first wavelength shift of the sample to (i) the first wavelength shift of a calibrator, or (ii) the average of the first wavelength shifts of all calibrators. In one preferred embodiment, the normalization factor is determined by the ratio of the first wavelength shift of the sample to the average of the first w avelength shifts of all calibrators.

[0132] In step (1), a normalized wavelength shift of the second wavelength shift is calculated by applying the normalized factor to the second wavelength shift.

[0133] In step (m), the percentage of full virus capsids in the sample is determined by quantitating the normalized w avelength shift against a calibration curve. The calibration cun e is established by measuring the normalized shifts of calibrator samples with known percentages of full virus capsids and plotting normalized wavelength shifts against percentages of full virus capsids.

[0134] Typically, the samples and calibrators are run in the same run, but they can be run in different runs on the same day. Running calibrators on a different day is possible providing the samples and the instrument are stable.

[0135] In this method, the details of other steps (a), (b), and (d)-(k) are similar to the corresponding steps described above in a method of determining DNA concentration in a virus. Multiple Signal Oligos / Normalization

[0136] In a second aspect, the method uses one capture oligonucleotide to hybridize to one region of the virus DNA and to immobilize the hybridized complex to a BLI probe, and multiple signal oligonucleotides to hybridize to another region of the virus DNA and to generate the signals for BLI detection.

[0137] The method comprises the steps of: (a) obtaining a first probe having a fixed amount of anti-virus antibody immobilized on the tip of the probe; (b) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary to a first region of the virus nucleic acid and a first member of a first binding pair at the 5 '-end or the 3 '-end; (c) obtaining a first signal oligonucleotide comprising: (i) a second oligonucleotide sequence complementary to a second region of the virus nucleic acid, wherein the first and the second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourth oligonucleotide each between the second oligonucleotide and the first member of the second binding pair at one end of the first signal oligonucleotide; wherein the first and the second binding pairs are different, and the first members of the first and the second binding pairs are haptens; (d) obtaining a second signal oligonucleotide which sequence is complementary to the third oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends; (e) obtaining a third signal oligonucleotide which sequence is complementary’ to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends; (f) dipping the first probe tip in a sample solution comprising a virus sample to capture the virus on the probe in a defined binding condition for a first period of time to measure a first w avelength shift due to light interference as a result of the binding virus on the probe; (g) determining a normalization factor based on the first wavelength shift of the sample in comparison with (i) the first wavelength shift of a calibrator, or (ii) the average of the first wavelength shifts of all calibrators; (h) dipping the first probe in a wash solution to wash the first probe tip; (i) dipping the first probe in a lysis solution containing the capture oligonucleotide and the first, the second, and the third signal oligonucleotides and heating the lysis solution to lyse the virus from the first probe tip and to denature nucleic acids in the virus to single-stranded nucleic acids; (j) removing the first probe from the heated lysis solution; (k) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotides to its complementary region of the single- stranded virus nucleic acid; (1) dipping a second probe comprising a second member of the first binding pair on the probe tip to the solution of (k), wherein the second probe captures the hybridized nucleic acids through the first binding pair; (m) dipping the second probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair; (n) dipping the second probe in a HRP substrate solution to react the substrate with the HRP in the conjugate for a period of time, and measuring the second wavelength shift due to light interference; (o) applying the normalization factor to the second wavelength shift to produce a nonnalized wavelength shift; and (p) quantitating the normalized wavelength shift against a calibration curve having normalized wavelength shift plotted against % of full AAV capsids to determine the percentage of full AAV capsids in the sample.

[0138] The invention is illustrated further by the following examples that are not to be construed as limiting the invention in scope to the specific procedures described in them.

[0139] EXAMPLES

[0140] Oligonucleotides

[0141] All oligonucleotides were purchased from Integrated DNA Technologies and HPLC purified. Oligonucleotide modification by fluoresceination or biotinylation were requested during purchase.

[0142] Oligo 1 (synthetic GFP positive stranded oligonucleotide. Gene of Interest, See FIG. 3) 5 -CAA CGT CTA TAT CAT GGC CGA CAA GCA GAA GAA CGG CAT CAA GGT GAA CTT CAA GAT CCG CCA CAA CAT CGA GGA CGG CAG CGT GCA GCTCGC CGA CCA CTA CCA GCA GAA CAC CCC CAT CGG CGA CGG CCC CGT GCT GCT GCC CGA CAA CCA CTA CCT GAG CAC CCA GTC CGC CCT GAG CAA AGA CCC CAA CGA G-3’ (SEQ ID NO: 1)

[0143] The second underlined sequence CC CGA CAA CCA CTA CCT GAG CAC CCA GTC CGC CCT GAG CA (SEQ ID NO: 2) is hybridization Region B for Oligo 2.

[0144] The first underlined sequence C CGA CAA GCA GAA GAA CGG CAT CAA GGT GAA CTT CAA G (SEQ ID NO: 3) is hybridization Region A for Oligos 3, 4, and First Signal Oligo. Oligo 2 (5 ’-fluorescein GFP hybridizing oligonucleotide, for capturing, See FIG. 3) hybridizes to Region B of Oligo 1.

[0145] 5’F*-TGC TCA GGG CGG ACT GGG TGC TCA GGT AGT GGT TGT CGG G-3’ (SEQ ID NO: 4)

[0146] Oligo 3 (5 ’-biotinylated GFP hybridizing oligonucleotide, for signaling, see FIG. 3) hybridizes to Region A of Oligo 1.

[0147] 5’B*-CTT GAA GTT CAC CTT GAT GCC GTT CTT CTG CTT GTC GG-3’ (SEQ ID NO: 5)

[0148] Oligo 4 (5’ and 3’ end dual biotinylated GFP hybridizing oligonucleotide, for signaling), which hybridizes to Region A of Oligo 1 (see FIG. 4).

[0149] 1stSignal Oligo (see FIG. 5): 5’ and 3’ end dual biotinylated GFP hybridizing oligonucleotide with poly T (3rd and 4th Oligo in FIG. 5) between biotin and the hybridizing sequence in both ends). The 1stSignal Oligo hybridizes to Region A of Oligo 1 (see FIG. 5).

[0150] 5’B*-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-CTT GAA GTT CAC CTT GAT GCC

[0151] GTT CTT CTG CTT GTC GG-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-B*3’ (SEQID

[0152] NO: 7)

[0153] 2ndand 3rdSignal Oligo (see FIG. 5): 5’ and 3’ end dual biotinylated poly A oligonucleotide.

[0154] 5B*-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-B*3’ (SEQ ID NO: 8)

[0155] Probes and Other Reagents

[0156] Anti-Fluorescein (anti-F) probes were made by dipping Gator H probes in PBS for 30 sec, lOOug / ml of Reagent 1 (Gator Bio, Cat# 5001) for 300 sec, coating buffer (0.5% BSA. 0.05% Tween-20, 0.05% proclin in PBS) for 60 sec and 15% sucrose solution in PBS for 30 sec. The probes were ready for use after being dried at 37°C for 30 min.

[0157] The lysis hybridization buffer (LH buffer) was 5.5M NaCl, 5% Tween, lOrnM EDTA in PBS at pH 7.2. Anti-biotin antibody HRP conjugate was from Thermo Fisher Scientific (Cat# PAI-30595), which was used in Examples 1, 2, 3A and 3B, and 5-8.

[0158] Chloronaphthol solution was from Thermo Fisher Scientific (Cat# 34012).

[0159] PCR tubes were from Eppendorf (Cat# 951010022). Q buffer (2 mg / ml BSA, PBS, 0.05% Tween 20) and MAX plate were from Gator Bio. 384-well plates were from Greiner (Cat# 781900).

[0160] AAV8 full capsids were from Virovek (Cat# AAV8-CMV-GFP). Pluronic F-68 from Sigma (Cat# Pl 300)

[0161] Example 1: Single Biotinylated Oligonucleotide

[0162] First, we validated the hybridization assay by testing the short synthetic green fluorescent protein (GFP) oligonucleotide (Oligo 1 of FIG. 3). In the assay, fluorescein labelled-GFP hybridizing oligonucleotide (Oligo 2 of FIG. 3) and biotinylated-GFP hybridizing oligonucleotide (Oligo 3 of FIG. 3) should hybridize to the synthetic GFP positive stranded oligonucleotide (Oligo 1) to form a stable hybridized complex as shown in FIG. 3. In each assay, 0.5 pL of 800 nM Oligo 2, 0.5 pL of 800nM Oligo 3, and 8 pL of lysis hybridization buffer were added into the PCR tubes. Water was added to adjust the volume to 10 pL. Lastly, 10 pL of Oligo 1 at different concentrations was added into the PCR tubes. The final volume is 20 pL. Negative control has no Oligo 1 . The samples were heated in a heating block at 95°C for 5 min. The samples were then cooled at 4°C for 2 min to allow the hybridized complex formation. Finally, 80 pL water was added into the PCR tubes to adjust the sample volume to approximately 100 pL for BLI assay.

[0163] In order to accelerate the hybridization time, the lysis hybridization buffer contains 5.5M NaCl (about 2M NaCl total in the hybridized complex mix). High salt promotes hybridization. However, the anti-F probe does not bind to the fluorescein labelled oligonucleotide in the hybridized complex at such high salt concentration. Before adding the anti-F probe, an aliquot of water is mixed with the sample, lowering the salt concentration to 0.4M.

[0164] The hybridized complex is quantified by using BLI assay. To set up the 384-well plate, 100 pL of each sample was transferred into the appropriate wells of the first column. BLI probe containing anti-F was added in each well of the first column and move from column to column.

[0165] 100 pL of 10 pg / ml of anti-biotin antibody HRP conjugate was diluted by Q buffer in column 2. 100 pL of chloronaphthol solution was added in column 3. To set up the MAX plate, each had 250 pL of Q buffer in columns 1-5 and 250 pL of PBS in columns 6-7. Anti-F probes was pre-incubated with Q buffer in column 1 for at least 5 min. To program the Gator instrument, the anti-F probe binding step was set up for 10 min followed by three Q buffer washes, anti-biotin HRP conjugate binding step was for 10 min, followed by one Q buffer wash and two PBS washes, and finally the chloronaphthol substrate BLI reading step was for 5 min. Each wash is set to 10 sec. The Gator instrument protocol is shown in Table 1 . Table 1.

[0166] Result of Using Synthetic GFP Oligonucleotide

[0167] BLI signals at different concentrations of the synthetic GFP oligonucleotide (Oligo 1), at 300 seconds are shown in Table 2.

[0168] Table 2.

[0169] The BLI signals are plotted against the synthetic GFP concentration in FIG. 8 to show the standard curve, which shows that BLI signals are almost directly proportional to the concentration of the positive stranded GFP in the assay.

[0170] Example 2: Using an Oligo with Dual Biotins

[0171] This example used an oligonucleotide probe with two biotins each on the 5’- or 3’- end. We replaced the 5 ’-end single biotinylated GFP hybridizing oligonucleotide (Oligo 3) in Example 1 with 5 ’-end and 3 ’-end dual biotinylated GFP hybridizing oligonucleotide (Oligo 4, see FIG. 4). This oligonucleotide had the same hybridizing sequence as Oligo 3 but it had 2 biotin molecules.

[0172] To compare the BLI signal of using Oligos 3 (one biotin) and 4 (two biotins), we used the same assay procedure and condition as described in Example 1. In brief, 0.5 pL of 800 nM fluorescein or biotinylated oligo was used. 10 pL of 100 pM of the synthetic positive stranded GFP oligonucleotide was used except for the negative controls.

[0173] The data show that the hybridization assay using dual biotinylated Oligo 4 had almost 2 times higher BLI signal than that using single biotinylated Oligo 3 at 300 seconds. This result shows that the BLI signal increases with the number of biotins in the hybridized complex.

[0174] Example 3: Using an Oligo Complex with 6 Biotins (Suplex)-Poly T

[0175] Since the BLI signal in the hybridization assay increases with the number of biotins in the hybridized complex, we constituted up to 6 biotin molecules per hybridized complex (See FIG. 5). To achieve this, we used Poly T as the third and the fourth oligos and Poly A as its complementary counterpart in the second and the third signal oligos.

[0176] To test the BLI signal of the hybridized complex with 6 biotins, we used the same assay procedure and condition as described in Example 1 except that first, secnd, and third signal oligos were added together (see FIG. 5). The concentrations of the fluorescein and biotinylated oligos used were 800 nM. In the assay, 0.5 pL of capture oligo and 1 pL of signal oligos were mixed with 8 pL of hybridization buffer. 10 pL of 100 pM of the synthetic positive stranded GFP oligonucleotide was used except for the negative controls. For comparison, we also tested the hybridized complex with single biotin (Example 1) or two biotins (Example 2). Using this protocol, BLI signals with different numbers of biotins per hybridized complex are shown in FIG. 9.

[0177] The results show that BLI signal was the highest when the hybridized complex had 6 biotins. This results show that the BLI signal significantly increased with the number of biotin molecules per hybridized complex. We used the 6-biotin assay format for the following experiments. Example 4. Using an Oligo Complex with 6 Biotins (Suplex)-Poly T and Poly(ATGG)

[0178] Materials

[0179] Oligonucleotides (See FIG. 5)

[0180] First Signal Oligo (5’ and 3’ end dual biotinylated GFP oligonucleotide with one poly T region and one poly ATGG region between biotin and the hybridizing sequence) hybridizes to Region A of Oligo 1 (GFP transgene).

[0181] 5’B*-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-CTT GAA GTT CAC CTT GAT GCC

[0182] 2ndSignal Oligo (5’ and 3’ end dual biotinylated poly A oligonucleotide):

[0183] 5B*-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-B*3’ (SEQ ID NO: 8)

[0184] 3rdSignal Oligo (5’ and 3’ end dual biotinylated poly CCAT oligonucleotide): 5B*-CCAT CCAT CCAT CCAT CCAT CCAT CCAT CCAT-B*3’ (SEQ ID NO: 10)

[0185] Example 4 is similar to Example 3 except both poly T and poly(ATCG) were used in the first signal oligo. Second signal oligo hybridized to poly A, and third signal oligo hybridized to Poly(ATCG) in the first signal oligo.

[0186] Example 4 used similar protocols as described in Example 3. The concentrations of the fluorescein and biotinylated oligos used were 800 nM. In the assay, 0.5 pL of 0.5 pL of capture oligo, and 0.5 pL of each first, second, and third signal oligos were mixed w ith 8 pL of hybridization buffer. 10 pL of 100 pM of the synthetic positive stranded GFP oligonucleotide was used as gene of interest (GOI). lOul of the AAV sample at 5E11 vp / ml or PBS blank was used. The samples were heated and lysed at 95°C for 5 min and then cooled down at 4°C for 2 min. 80 pL of water was added into the samples. 100 pL of the samples was used for the analysis by the hybridization assay. The BLI signals at 300 seconds of Examples 3 and 4 are summarized in Table 3.

[0187] Table 3.

[0188] Examples 3 and 4 had similar BLI results. The results show that the hybridization pairs are not only limited to poly A / poly T interaction and they can be extended to other reasonable complementary sequences.

[0189] Example 5: Capturing AAV Capsids

[0190] Preparation of HRP / anti-biotin / CxFicoll conjugate Anti-biotin-HRP-CxF Conjugate

[0191] Anti-biotin antibody (Biotin Polyclonal Antibody, Invitrogen, goat IgG, polyclonal, lyophilized, Cat# Thermo Scientific 31852) was dissolved into PBS (IX) to obtain (Img / mL) solution. Anti-biotin solution was passed through Zeba™ Spin Desalting Columns (5mL. 7k MWCO, Thermo Scientific). The purified anti-biotin PBS solution was reacted with SMCC (molar coupling ratio (MCR)=7.5, 20mg / mL in DMF, 0.84 pL, Thermo Scientific) for 1 hour at room temperature RT) and then purified with Zeba™ Spin Desalting Columns (5mL, 7K MWCO, Thermo Scientific).

[0192] Horseradish peroxidase (HRP, Thermo Scientific, MCR=12.5 to anti-biotin) (3.3 mg in 200 pL PBS) was reacted with SMCC (MCR=7.5, 20mg / mL in water, 10.54 pL, Thermo Scientific) for 1 hour at R.T. and then purified with Zeba™ Spin Desalting Columns (2mL, 7k MWCO. Thermo Scientific). SPDP-Labeled Crosslinked FICOLL® 400-SPDP was prepared according to Example 2 of WO2017 / 035343. DTT (dithiothreitol) (MCR=550, 38mg / mL in PBS, 7.34 pL) was reacted with SPDP-Labeled Crosslinked FICOLL® 400- SPDP (1.33mg in 200 pL PBS, MCR=0.5 to anti-biotin) for 1 hour at RT and then purified with Zeba™ Spin Desalting Columns (2mL, 7k MWCO, Thermo Scientific). The resulting Crosslinked FICOLL® (CxF) was mixed with the anti-biotin-SMCC and HRP-SMCC solutions. The mixture was vortexed for 30 seconds, reacted for 3 hours, and then quenched with 12.5 pL of N-ethyl maleimide in water (20 mg / mL) for 0.5 hour. After passing the conjugate with CL-4B Columns in a AKTA purification system with PBS as the eluent, anti- biotin-HRP-Crosslinked FICOLL® in PBS was obtained. The concentrations of anti-biotin and HRP were calculated based on their extinction coefficients and UV-vis absorbances as 280nm and 405nm. In this example, a high molecular weight of HRP / anti-biotin / crosslinked FICOLL® conjugate was used. The molar ratio of HRP / anti-biotin / Crosslinked FICOLL® was 20 / 2 / 1.

[0193] AAV Sample Assay

[0194] Next we tested the hybridization assay by using AAV samples. AAV8 CMV-GFP full capsids from Virovek were used, which was lysed and heated to released ssDNA for hybridization.

[0195] To prepare the samples, AAV8 at 2E13 vg / ml was diluted by PBS with 0.005% Pluronic F-68 to different concentrations (vg / ml). In the assay, 10 pL of the AAV samples at different concentrations was mixed with 0.5 pL of Oligo 2, 0.5 pL of 1st Signal Oligo, 0.5 pL of 2nd Signal Oligo, and 0.5 pL of 3rd Signal Oligo (see FIG. 5 amd Example 4), and 8 pL of hybridization buffer in a final volume of 20 pL The samples were heated and lysed at 95°C for 5 min and then cooled down at 4°C for 2 min. 80 pL of water was added into the samples. 100 pL of the samples was used for the analysis by the hybridization assay. The workflow is shown in FIG. 6. Using this protocol, the BLI signals at different concentrations of AAV capsids are shown in below.

[0196] The data at 300 seconds are summarized in Table 4.

[0197] Table 4.

[0198] BLI signals at 300 seconds are plotted against the AAV concentration to show the standard curve in FIG. 10. The standard curve shows that BLI signals are almost directly proportional to AAV concentrations (vg / ml). This results indicate when an unknown AAV sample is tested along with the standards, the BLI signal of the unknown can be plotted against a standard curve to determine the AAV concentration of the unknown sample. EXAMPLE 6. Comparing the Present High Molecular Weight vs. Commercial HRP / Anti-biotin Conjugates

[0199] The commercial anti -biotin HRP conjugate from ThermoFisher usually has a limited activity because the conjugate is made by conjugating HRP to the antibody directly and therefore the antibody has a small number of HRP molecules attached.

[0200] By crosslinking the anti-biotin antibody and HRP to a high molecular weight and highly branched FICOLL® polymer, we prepared a conjugate of HRP / anti-biotin / Crosslinked FICOLL® with molar ratio of 20 / 2 / 1 (see Example 5).

[0201] This example compares the performance of the high molecular weight (Example 5) vs ThermoFisher’s HRP / Anti-biotin Conjugates.

[0202] Protocols and Result

[0203] In this assay, 10 pL of the AAV samples at 1E10 vg / ml was mixed with 0.5 pL of Oligo 2, 0.5 pL of First Signal Oligo, 1 pL of second Signal Oligo (see FIG. 5 and Example 3), and 8 pL of hybridization buffer in a final volume of 20 pL.

[0204] The samples were heated and lysed at 95°C for 5 min and then cooled down at 4°C for 2 min. 80 pL of w ater was added into the samples. 100 pL of each sample was used for assay. In the HRP conjugate binding step, 10 pg / ml of the crosslinked or commercial conjugate was used. The BLI signals are shown in FIG. 11. The data indicates that the crosslinked HRP conjugate shows 3-4 times higher signal than the commercial conjugate.

[0205] Example 7: Use of the Hybridization Assay to Determine the Empty / Full Ratio of a Purified AAV Sample with the Same Concentration

[0206] Next we applied the hybridization assay to determine empty / full ratio of a purified AAV sample. In theory, assuming AAV samples at the same concentration, the AAV sample with a higher full ratio should generate a higher hybridization signal. Consequently, the hybridization signal can be used to determine the empty / full ratio of an unknown. To test the concept, we prepared 1E11 vp / ml of AAV8 samples at different full ratios such as 100%, 80%, 60%, 40%, 20% and 0% full. The protocol is referred to Example 4. 10 pL of AAV samples with different full ratios w ere mixed with 8 pL of a hybridization buffer, 0.5 pL of Oligo 2, 0.5 pL of 1st Signal Oligo, 0.5 pL of 2nd Signal Oligo, and 0.5 pL of 3rd Signal Oligo (see Example 4). The mixtures w ere heated at 95°C for 5 min and cooled down at 4°C for 2 min. 80 pL of water was added and then 100 pL of the sample was used for analysis. The BLI signals at 300 seconds are summarized in Table 5.

[0207] Table 5.

[0208] The BLI signals are plotted against the full ratios in FIG. 12, which shows that BLI signals were proportional to the full ratios of the AAV samples. This result indicates when an AAV sample with an unknown full ratio is tested along with standards, the hybridization signal of the unknown can be used to determine the full ratio based on the standard curve.

[0209] Example 7: Use of the AAVX Probes Followed by Hybridization Assay to Determine the Full Ratio

[0210] The workflow of this experiment is shown in FIG. 7.

[0211] In this experiment, 100 pL of AAV8 samples at 1E11 vg / ml with the full ratio of 0%, 5%, 10%, 20%, 40%, 60%, 80% and 100% was pipetted into each of the appropriate wells of the first column in a 384- well plate. Then AAVX probes were programmed by using an Gator instrument to capture the AAV capsids in these wells for approximately 15 min using the following protocol.

[0212] FIG. 13 show s BLI signal curves of different full ratios of AAVs captured on the AAVX probe at different time.

[0213] After AAV capture, the AAVX probes were dipped into PCR tubes containing 20 pL of the hybridization oligonucleotide mixture, which consisted of 8 pL of hybridization buffer, 0.5 pL of the 1st Signal Oligo, 0.5uL of 2nd Oligo, 0.5uL of 3rd Signal, and 0.5uL of Capture Oligo (all at 800nM), and 10 pL of w ater (see Example 4). The 2ndOligo and 3rdOligo are the same which is PolyA oligonucleotide. The samples in the PCR tubes were heated at 95°C for 5 min and cooled down at 4°C for 2 min. 80 pL of water is added into the PCR tubes. 100 pL of sample was used for the hybridization assay. The hybridization assay protocol is shown in Table 6.

[0214] Table 6. *Q buffer: 2 mg / ml BSA, PBS, 0.05% Tween 20

[0215] The hybridization signals of the captured AAV samples at different full ratios are shown in FIG. 14.

[0216] The AAV Capture and BLI signals in the whole normalization workflow process are summarized in Table 7.

[0217] Table 7.

[0218] The AAV capture signals were normalized to generate the normalization ratios. Then the normalization ratios were multiplied by the detected BLI signals in the hybridization assay to generate the normalized shift values. The normalized shift values were plotted against the full ratio to show the AAV standard curve (FIG. 15).

[0219] The above AAV standard curve shows that after AAV capture, the hybridization signal was linearly proportional to the full ratio of the AAV sample. Since the AAV capture step can purify AAV capsids from the crude or unpurified medium for the assay of empty / full ratio, the results indicate that AAV capture followed by the hybridization assay is able to determine the empty / full ratio of crude or unpurified AAV samples.

[0220] Example 8: Using AAVX Probe Followed by Hybridization Assay to Determine the Empty / Full Ratio in a Crude AAV Sample In this example, we tested the robustness of the AAV capture followed by the hybridization assay (see Example 7 protocols) to determine the full ratio of a mocked crude sample. This was done by spiking AAV samples into PBS or a crude lysate of HEK 293 cells and then comparing the hybridization signal and determined empty / full ratio in these two conditions. In the experiment, AAV8 samples with approximately 25%, 50% and 75% adjusted full ratios were spiked into PBS or 1 / 10 diluted crude medium. The crude medium was produced by culturing HEK293 cell in DMEM medium (Thermo Fisher Scientific). After cell culture, the growing medium was spun at 3000 rpm and the supernatant was collected as the mocked crude medium. The AAV8 samples in duplicates were captured by AAVX probes and then assayed by the hybridization assay using the same protocol as previously described. The data of the AAV capture and hybridization assay are summarized in the Table 8.

[0221] Table 8.

[0222] In Table 8, the AAV capture signals are normalized to generate the normalization ratios which are then multiplied by detected BLI signals to yield the normalized shift signals. Finally, the normalized shift signals are averaged and compared in PBS vs. 1 / 10 diluted mocked crude lysate.

[0223] The data show that the average normalized shift values of the 25%, 50% and 75% spiked AAV samples in PBS vs. 1 / 10 diluted crude lysate display <10% variation, indicating that the hybridization signals are not significantly influenced by the crude condition. We converted the normalized shift values into the calculated full ratios by using the AAV standard curve generated from Table 7. Since the calculated full ratios in PBS and 1 / 10 crude condition were very close within 10% variation and the recoveries were very good ranging from 89-107%, these results suggest that the AAV capture followed by hybridization assay is reliable to determine the empty / full ratio of the unpurified or crude AAV samples.

[0224] Example 9: Anti-AAV Magnetic Particles Preparation for Capturing AAVs

[0225] Materials:

[0226] HiSur Carboxyl Beads, 1 pm, Ocean NanoTech, Part No:HC1000

[0227] EDC. Thermo Scientific. Part No: 22980

[0228] N-Hydroxysuccinimide (NHS), ChemCruz, Part No: sc-219162

[0229] MES buffer, 50 mM pF! 6

[0230] SA XT, Gator Bio, Part No: 110051

[0231] Tris-HCl buffer, ph8

[0232] Biotin-anti-AAVX, CaptureSelect, Part No: 7193522500

[0233] Centrifuge tubes

[0234] Orbital mixer

[0235] Magnet tube rack

[0236] Protocols:

[0237] 1. Allowed reagents to come to room temperature

[0238] 2. Diluted FliSur Carboxyl bead slurry to 1 mg / mL with MES buffer, vortexed 30 seconds

[0239] 3. Used magnet to separate magnetic beads for 2 minutes, then removed supernatant with ImL pipette

[0240] 4. Washed magnetic beads with MES buffer at 1 mg / mL, vortexed 30 seconds, repeated (step 3) 5. Prepared 1 mg / mL slurry of magnetic beads with 4mM EDC and lOmM NHS in MES buffer, vortexed 30 seconds

[0241] 6. Incubated mixture at room temperature for 30 minutes with orbital mixing at 20 rpm

[0242] 7. Used magnet to separate magnetic beads for 2 minutes, then removed supernatant with ImL pipette

[0243] 8. Washed magnetic beads with PBS buffer at 1 mg / mL, vortexed 30 seconds, repeated (step

[0244] 7)

[0245] 9. Added 50 pg / mL crosslinked streptavidin (SA XT) to 1 mg / mL magnetic beads slurry in PBS buffer, vortexed for 30 seconds

[0246] 10. Incubated mixture for 2 hours with orbital mixing at 20 rpm

[0247] 11. Used magnet to separate magnetic beads for 2 minutes, then removed supernatant with ImL pipette

[0248] 12. Washed magnetic beads with PBS buffer at 1 mg / mL, vortexed 30 seconds, repeated (step H)

[0249] 13. Resuspended magnetics beads with Tris-HCl buffer to make 1 mg / mL slurry, vortexed 30 seconds

[0250] 14. Incubated slurry' for 30 minutes with orbital mixing at 20 rpm

[0251] 15. Used magnet to separate magnetic beads for 2 minutes, then removed supernatant with ImL pipette

[0252] 16. Washed magnetic beads with PBS buffer at 1 mg / mL, vortexed 30 seconds, repeated (step

[0253] 15)

[0254] 17. Resuspended magnetic beads in 1 mg / mL slurry with 833 nM Biotin-anti- AAVX

[0255] 18. Incubated mixture at room temperature for 30 minutes with orbital mixing at 20 rpm

[0256] 19. Used magnet to separate magnetic beads for 2 minutes, then removed supernatant with ImL pipette

[0257] 20. Resuspended with PBS buffer and vortexed

[0258] 21. Repeated (step 19-20) 3 more times to wash unbound ligand

[0259] 22. Stored at 2-8 °C

[0260] Example 10: Magnetic Particle Capture of AAV Capsids with GFP Hybridization

[0261] FIG. 16 depicts the assay format where magnetic particles coated with anti-AAV capture AAV capsid, followed by immersion in a lysis / hybridization reagent with heating to 95°C then rapid cooling, after which an anti-F coated BLI probe is inserted into the sample for BLI detection of the hybridized complex (see Example 3).

[0262] MATERIALS

[0263] Biotin-anti AAVX from Thermo Fisher Scientific

[0264] Gator AAVX Probes, Gator Bio, Part No: 160017

[0265] 2mL centrifuge tube

[0266] PCR tubes (lysis tubes)

[0267] Phosphate buffer

[0268] 384-well plate, Greiner, Part No: 781209

[0269] Max Plate, Gator Bio, Part No: 130062

[0270] AAV full capsid, Virovek, Part No: AAV8-CMV-GFP

[0271] Anti-AAVX magnetic beads (see Example 3)

[0272] Magnet tube rack

[0273] LH Buffer: 5.5M NaCl, 50 mM phosphate, 5 mM EDTA, 7.3% Tween20, pH 7.4

[0274] INSTRUMENTS

[0275] Gator Plus

[0276] Heating block

[0277] Orbital mixer

[0278] PROTOCOLS

[0279] Rapid Titer Measurement of AAV Vectors by AAVX Probes, GatorBio Application Note ,

[0280] APP-1 Version 1

[0281] Hybridization protocol, see Example 3

[0282] PROCEDURE:

[0283] AAV Capture

[0284] 1. AAV stock dilutions were prepared by adding phosphate buffer to make 4.00E+09, 2.600+09, 8.00E+08 and 0 vp / mL concentrations.

[0285] 2. Magnetic capture sample: 50, 25 and 12.5 pL of Img / mL anti-AAVX magnetic bead preparation was added to 300pL aliquots of the make 4.00E+09, 2.600+09, 8.00E+08 vp / mL AAV stock dilutions respectively, then incubated at RT for 10 minutes with orbital shaking. 2a. Tubes were placed into the magnet tube rack for 2 minutes to separate the beads from solution.

[0286] 3. Samples without magnetic capture (‘'starting” sample): 50, 25 and 12.5 pL of phosphate buffer was added to 300pL aliquots of the 4.00E+09, 2.600+09, 8.00E+08 vp / mL AAV stock dilutions in centrifuge tubes, then incubated at RT for 10 minutes with orbital shaking.

[0287] 4. Follow ing the “Sample Plate Setup” section of the AAVX Titer Protocol (GatorBio Doc APP-1,V1, Rapid Titer Measurement of AAV Vectors), lOOpL of AAV stock dilutions (step 1) were pipetted into the 384 black plate

[0288] 5. Following the “Sample Plate Setup"’ section of the AAVX Titer Protocol, lOOpL of the supernatant of the magnetic capture samples (step 2a) and

[0289] 6. Following the '‘Sample Plate Setup” section of the AAVX Titer Protocol, lOOpL of the samples without magnetic capture (step 3) were pipetted into the 384 black plate

[0290] 7. AAVX Probes w ere prepared in Max Plate following the “Max Plate Setup” section of the AAVX Titer Protocol [Example A]

[0291] 8. The '‘Assay Setup” section of the AAVX Titer Protocol [Example 1] was follow ed to run the AAV quantitation assay in the GatorOne software a. The AAV stock dilutions (step 4) were designated as “standards” b. The samples (step 5) and (step 6) were designated as “unknown”

[0292] 9. After completion of the assay, quantitative results were obtained per the “Data Analysis” section of the AAVX Titer Protocol (GatorBio Doc APP-1,V1, Rapid Titer Measurement of AAV Vectors). Result is output as “Calculated concentration”

[0293] GatorBio Doc APP-1,V1, Rapid Titer Measurement of AAV Vectors

[0294] GFP Transgene Quantitation

[0295] 10. The remaining supernatant was removed from the magnetically pelleted samples (Step 2a).

[0296] 11. Magnetic particles and captured AAV capsids w ere resuspended in 20pL (LH buffer, F and B oligos as in Example 3)

[0297] Note: Remaining steps followed the lysis / hybridization and BLI detection steps in Example

[0298] 3. RESULTS

[0299] The AAV capture results are summarized in Tables 9 and 10.

[0300] Table 9.

[0301] Table 10.

[0302] The AAVX quantitation results are shown in FIG. 17. The GFP hybridization results are shown in FIG. 18.

[0303] The invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

Claims

WHAT IS CLAIMED IS:

1. A method of determining the concentration of a nucleic acid of interest in a liquid sample, comprising the steps of:(a) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the nucleic acid of interest; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5'-end or 3'-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’- and / or 3’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens;(b) adding the capture oligonucleotide and the signal oligonucleotide to the liquid sample comprising the nucleic acid of interest and heating the mixture to denature nucleic acids in the sample to single-stranded nucleic acids;(c) cooling the heated sample to hybridize the capture oligonucleotide and the signal oligonucleotide to the single-stranded nucleic acid of interest,(d) dipping a probe comprising a second member of the first binding pair on the probe tip to the hybridized sample, wherein the probe captures hybridized nucleic acid of interest through the first binding pair;(e) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair;(f) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time; and(g) determining the concentration of the nucleic acid of interest by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

2. The method of Claim 1, wherein the liquid sample comprises viruses and the mixture of (b) is heated in a lysis solution containing a detergent to lyse the viruses and to denature the virus nucleic acids.

3. A method for determining nucleic acid concentration in a sample containing a virus,comprising the steps of:(a) obtaining a first probe having a fixed amount of an anti-vims antibody immobilized on the tip of the probe, wherein the anti-vims antibody is against a virus in the sample;(b) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the virus nucleic acid; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5‘- end or 3 ’-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’- and / or 3’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens;(c) dipping the first probe tip in the sample comprising the virus to capture the virus on the probe;(d) dipping the first probe in a wash solution to wash the first probe tip;(e) dipping the first probe in a lysis solution containing the capture oligonucleotide and the signal oligonucleotide and heating the lysis solution to lyse the virus from the first probe tip and to denature nucleic acids in the virus to single-stranded nucleic acids;(f) removing the first probe from the heated lysis solution;(g) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotide to its complementary region of the single-stranded virus nucleic acid;(h) dipping a second probe comprising a second member of the first binding pair on the probe tip to the solution of (g), wherein the second probe captures the hybridized nucleic acids through the first binding pair;(i) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair;(j) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time; and(k) determining the concentration of the virus nucleic acid by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

4. A method of determine the percentage of full virus capsids in a sample containing a virus, comprising the steps of: determining the concentration of the virus nucleic acid according to Claim 6, and converting the nucleic acid concentration to % of full virus capsid using a calibration curve having nucleic acid concentration plotted against % of full virus capsid standards.

5. A method of determine the percentage of full virus capsids in a sample containing a virus, comprising the steps of:(a) obtaining a first probe having a fixed amount of anti-virus antibody immobilized on the tip of the probe, wherein the anti-virus antibody is against the virus in the sample;(b) dipping the first probe tip in the sample to capture the virus on the probe in a defined binding condition for a first period of time to measure a first wavelength shift due to light interference as a result of the binding virus on the probe;(c) determining a normalization factor based on the first wavelength shift of the sample in comparison with (i) the first wavelength shift of a calibrator, or (ii) the average of the first wavelength shifts of all calibrators;(d) dipping the first probe in a wash solution to wash the first probe tip;(e) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the virus nucleic acid; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5’- end or 3 ’-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’- and / or 3’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens;(f) dipping the first probe in a lysis solution containing the capture oligonucleotide and the signal oligonucleotide and heating the lysis solution to lyse the virus from the first probe tip and to denature nucleic acids in the virus to single-stranded nucleic acids;(g) removing the first probe from the heated lysis solution;(h) cooling the heated lysis solution to hybridize the capture oligonucleotide and the signal oligonucleotide to the complementary regions of the single-stranded virus nucleic acid;(i) dipping a second probe comprising a second member of the first binding pair onthe probe tip to the solution of (g), wherein the second probe captures the hybridized nucleic acids through the first binding pair;(j) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair;(k) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time, and measuring the second wavelength shift due to light interference;(l) applying the normalization factor to the second wavelength shift to produce a normalized wavelength shift; and(m) quantitating the normalized wavelength shift against a calibration curve having normalized wavelength shift plotted against % of full virus capsids to determine the percentage of full virus capsids in the sample.

6. The method of claim 5, wherein the normalization factor in step (c) is determined by comparing the first wavelength shift of the sample in comparison with the average of the first wavelength shifts of all calibrators.

7. The method of claim 5 or 6, wherein the calibration curve is established by measuring the normalized shifts of calibrator samples with known percentages of full virus capsids and plotting normalized wavelength shifts against percentages of full virus capsids.

8. A method of determining the concentration of nucleic acid of interest in a liquid sample, comprising the steps of:(a) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary to a first region of the nucleic acid of interest and a first member of a first binding pair at the 5 ’-end or the 3 ’-end;(b) obtaining a first signal oligonucleotide comprising (i) a second oligonucleotide sequence complementary to a second region of the nucleic acid of interest, wherein the first and the second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourtholigonucleotide each between the second oligonucleotide and the first member of the second binding pair at one end of the first signal oligonucleotide, wherein the first and the second binding pairs are different, and the first members of the first and the second binding pairs are haptens;(c) obtaining a second signal oligonucleotide which sequence is complementary to the third oligonucleotide sequence and having the first member of the second binding pair at both 5‘- and 3‘- ends;(d) obtaining a third signal oligonucleotide which sequence is complementary to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(e) adding the capture oligonucleotide, the first signal oligonucleotide, the second signal oligonucleotide, and the third signal oligonucleotide to the liquid sample comprising the nucleic acid of interest and heating the mixture to denature nucleic acids in the sample to single-stranded nucleic acids;(f) cooling the heated sample to hybridize (i) the first oligonucleotide and the second oligonucleotide to the single-stranded nucleic acid of interest, (ii) the second signal oligonucleotide to the third oligonucleotide, and (iii) the third signal oligonucleotide to the fourth oligonucleotide;(g) dipping a probe comprising a second member of the first binding pair on the probe tip to the hybridized sample, wherein the probe captures hybridized nucleic acid of interest through the first binding pair;(h) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair;(i) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time; and(j) determining the concentration of the nucleic acid of interest by measuring the wavelength shift due to tight interference, and quantitating the wavelength shift against a calibration curve.

9. The method of Claim 8, wherein the liquid sample comprises viruses and the mixture of (e) is heated in a lysis solution containing a detergent to lyse the viruses and to denature the virus nucleic acids.

10. The method of claim 1 or 8, wherein the diameter of the tip surface of the probe is < 5 mm.

11. The method of claim 1 or 8, wherein the first and the second binding pairs are selected from the group consisting of: biotin and streptavidin, biotin and avidin, biotin and anti-biotin, fluorescein and anti-fluorescein, digioxigenin / anti-digioxigenin.

12. The method of claim 1 or 8, wherein the conjugate further comprises a polymer having a molecular weight over 1 million.

13. The method of claim 12, wherein the polymer is crosslinked copolymers of sucrose and epichlorohydrin.

14. A method for determining nucleic acid concentration in a sample containing a virus, comprising the steps of:(a) obtaining a first probe having a fixed amount of an anti-virus antibody immobilized on the tip of the probe, wherein the anti-virus antibody is against a virus in the sample;(b) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary to a first region of the virus nucleic acid and a first member of a first binding pair at the 5’-end or the 3’-end;(c) obtaining a first signal oligonucleotide comprising: (i) a second oligonucleotide sequence complementary to a second region of the virus nucleic acid, wherein the first and the second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourth oligonucleotide each between the second oligonucleotide and the first member of the second binding pair at one end of the first signal oligonucleotide; wherein the first and the secondbinding pairs are different, and the first members of the first and the second binding pairs are haptens;(d) obtaining a second signal oligonucleotide which sequence is complementary to the third oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(e) obtaining a third signal oligonucleotide which sequence is complementary' to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(f) dipping the first probe tip in the sample comprising the virus to capture the virus on the probe;(g) dipping the first probe in a wash solution to wash the first probe tip;(h) dipping the first probe in a lysis solution containing the capture oligonucleotide and the first, the second, and the third signal oligonucleotides and heating the lysis solution to lyse the virus from the first probe tip and to denature nucleic acids in the virus to singlestranded nucleic acids;(i) removing the first probe from the heated lysis solution;(j) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotides to its complementary' region of the single-stranded virus nucleic acids;(k) dipping a second probe comprising a second member of the first binding pair on the probe tip to the solution of (j), wherein the second probe captures the hybridized nucleic acids through the first binding pair;(l) dipping the second probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and HRP, to bind the conjugate to the second probe through the second binding pair;(m) dipping the second probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time; and(n) determining the concentration of the virus nucleic acid by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a15. A method of determine the percentage of full virus capsids in a sample containing a virus, comprising the steps of: determining the concentration of the virus nucleic acid according to Claim 14, and converting the nucleic acid concentration to % of full virus capsid using a calibration curve having nucleic acid concentration plotted against % of full virus capsid standards.

16. A method for determining the percentage of full virus capsids in a sample containing a virus, comprising the steps of:(a) obtaining a first probe having a fixed amount of anti-virus antibody immobilized on the tip of the probe;(b) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary' to a first region of the virus nucleic acid and a first member of a first binding pair at the ’-end or the 3 ’-end;(c) obtaining a first signal oligonucleotide comprising: (i) a second oligonucleotide sequence complementary' to a second region of the virus nucleic acid, wherein the first and the second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourth oligonucleotide each between the second oligonucleotide and the first member of the second binding pair at one end of the first signal oligonucleotide; wherein the first and the second binding pairs are different, and the first members of the first and the second binding pairs are haptens;(d) obtaining a second signal oligonucleotide which sequence is complementary to the third oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(e) obtaining a third signal oligonucleotide which sequence is complementary to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(!) dipping the first probe tip in a sample solution comprising a virus sample to capture the virus on the probe in a defined binding condition for a first period of time to measure a first wavelength shift due to light interference as a result of the binding virus on the probe;(g) determining a normalization factor based on the first wavelength shift of the sample in comparison with (i) the first wavelength shift of a calibrator, or (ii) the average of the first wavelength shifts of all calibrators;(h) dipping the first probe in a wash solution to wash the first probe tip;(i) dipping the first probe in a lysis solution containing the capture oligonucleotide and the first, the second, and the third signal oligonucleotides and heating the lysis solution to lyse the virus from the first probe tip and to denature nucleic acids in the virus to singlestranded nucleic acids;(j ) removing the first probe from the heated lysis solution;(k) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotides to its complementary region of the single-stranded virus nucleic acid;(l) dipping a second probe comprising a second member of the first binding pair on the probe tip to the solution of (k), wherein the second probe captures the hybridized nucleic acids through the first binding pair;(m) dipping the second probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair;(n) dipping the second probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time, and measuring the second wavelength shift due to light interference;(o) applying the normalization factor to the second wavelength shift to produce a normalized wavelength shift; and(p) quantitating the normalized wavelength shift against a calibration curve having normalized wavelength shift plotted against % of full AAV capsids to determine the percentage of full AAV capsids in the sample.

17. The method of claim 16, wherein the normalization factor in step (c) is determined by comparing the first wavelength shift of the sample in comparison with the average of the first wavelength shifts of all calibrators.

18. The method of claim 16 or 17, wherein the calibration curve is established by measuring the normalized shifts of calibrator samples with known percentages of full virus capsids and plotting normalized wavelength shifts against percentages of full virus capsids.

19. A method for determining nucleic acid concentration in a sample containing a virus, comprising the steps of:(a) obtaining a solid phase having a fixed amount of an anti-virus antibody immobilized on the tip of the probe, wherein the anti-virus antibody is against a virus in the sample;(b) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the virus nucleic acid; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5’- end or 3 ’-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’- and / or 3’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens;(c) contacting the solid phase with the sample comprising the virus to capture the virus on the probe;(d) washing the solid phase;(e) contacting the solid phase with a lysis solution containing the capture oligonucleotide and the signal oligonucleotide and heating the lysis solution to lyse the virus from the solid phase and to denature nucleic acids in the virus to single-stranded nucleic acids;(f) removing the solid phase from the heated lysis solution;(g) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotide to its complementary region of the single-stranded virus nucleic acid;(h) dipping a probe comprising a second member of the first binding pair on the probe tip to the solution of (g), wherein the second probe captures the hybridized nucleic acids through the first binding pair;(i) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind theconjugate to the probe through the second binding pair;(j ) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time; and(k) determining the concentration of the virus nucleic acid by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

20. A method of determine the percentage of full virus capsids in a sample containing a virus, comprising the steps of: determining the concentration of the virus nucleic acid according to Claim 21, and converting the nucleic acid concentration to % of full virus capsid using a calibration curve having nucleic acid concentration plotted against % of full virus capsid standards.

21. A method of determine the percentage of full virus capsids in a sample containing a virus, comprising the steps of:(a) obtaining a solid phase having a fixed amount of anti-virus antibody immobilized on the tip of the probe, wherein the anti-virus antibody is against the virus in the sample;(b) contacting the solid phase with the sample to capture the virus on the probe in a defined binding condition for a first period of time to measure a first wavelength shift due to light interference as a result of the binding virus on the probe;(c) determining a normalization factor based on the first wavelength shift of the sample in comparison with (i) the first wavelength shift of a calibrator, or (ii) the average of the first wavelength shifts of all calibrators;(d) contacting the solid phase in a wash solution to wash the solid phase;(e) obtaining a capture oligonucleotide and a signal oligonucleotide each comprising a nucleotide sequence complementary to a different region of the virus nucleic acid; wherein the capture oligonucleotide further comprises a first member of a first binding pair at the 5’- end or 3 ’-end, and the signal oligonucleotide further comprises a first member of a second binding pair at the 5’- and / or 3’end, wherein the first and the second binding pair are different, and the first members of the binding pairs are haptens;(f) contacting the solid phase in a lysis solution containing the capture oligonucleotideand the signal oligonucleotide and heating the lysis solution to lyse the virus from the solid phase and to denature nucleic acids in the virus to single-stranded nucleic acids;(g) removing the solid phase from the heated lysis solution;(h) cooling the heated lysis solution to hybridize the capture oligonucleotide and the signal oligonucleotide to the complementary regions of the single-stranded virus nucleic acid;(i) dipping a probe comprising a second member of the first binding pair on the probe tip to the solution of (g), wherein the probe captures the hybridized nucleic acids through the first binding pair;(j) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair;(k) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time, and measuring the second wavelength shift due to light interference;(l) applying the normalization factor to the second wavelength shift to produce a normalized wavelength shift; and(m) quantitating the normalized wavelength shift against a calibration curve having normalized wavelength shift plotted against % of full virus capsids to determine the percentage of full virus capsids in the sample.

22. A method for determining nucleic acid concentration in a sample containing a virus, comprising the steps of:(a) obtaining a solid phase having a fixed amount of an anti-virus antibody immobilized on the solid phase, wherein the anti-virus antibody is against the virus in the sample;(b) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary to a first region of the virus nucleic acid and a first member of a first binding pair at the 5’-end or the 3’-end;(c) obtaining a first signal oligonucleotide comprising: (i) a second oligonucleotide sequence complementary to a second region of the virus nucleic acid, wherein the first andthe second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourth oligonucleotide each between the second oligonucleotide and the first member of the second binding pair at one end of the first signal oligonucleotide; wherein the first and the second binding pairs are different, and the first members of the first and the second binding pairs are haptens;(d) obtaining a second signal oligonucleotide which sequence is complementary to the third oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(e) obtaining a third signal oligonucleotide which sequence is complementary' to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(f) contacting the solid phase with the sample comprising the virus to capture the virus on the probe;(g) dipping the solid phase in a wash solution to wash the solid phase;(h) dipping the solid phase in a lysis solution containing the capture oligonucleotide and the first, the second, and the third signal oligonucleotides and heating the lysis solution to lyse the virus from the solid phase and to denature nucleic acids in the virus to singlestranded nucleic acids;(i) removing the solid phase from the heated lysis solution;(j) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotides to its complementary' region of the single-stranded virus nucleic acids;(k) dipping a probe comprising a second member of the first binding pair on the probe tip to the solution of (j), wherein the second probe captures the hybridized nucleic acids through the first binding pair;(l) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and HRP, to bind the conjugate to the second probe through the second binding pair;(m) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate in the conjugate for a period of time; and(n) determining the concentration of the virus nucleic acid by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve.

23. A method of determine the percentage of full virus capsids in a sample containing a virus, comprising the steps of: determining the concentration of the virus nucleic acid according to Claim 22, and converting the nucleic acid concentration to % of full virus capsid using a calibration curve having nucleic acid concentration plotted against % of full virus capsid standards.

24. A method for determining the percentage of full virus capsids in a sample containing a virus, comprising the steps of:(a) obtaining a solid phase having a fixed amount of anti-virus antibody immobilized on the solid phase; (b) obtaining a capture oligonucleotide comprising a first oligonucleotide sequence complementary to a first region of the virus nucleic acid and a first member of a first binding pair at the 5’-end or the 3’-end;(c) obtaining a first signal oligonucleotide comprising: (i) a second oligonucleotide sequence complementary to a second region of the virus nucleic acid, wherein the first and the second regions are different, (ii) two first members of a second binding pair each at one end of the first signal oligonucleotide, (iii) a third oligonucleotide and a fourth oligonucleotide each between the second oligonucleotide and the first member of the second binding pair at one end of the first signal oligonucleotide; wherein the first and the second binding pairs are different, and the first members of the first and the second binding pairs are haptens;(d) obtaining a second signal oligonucleotide which sequence is complementary to the third oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(e) obtaining a third signal oligonucleotide which sequence is complementary' to the fourth oligonucleotide sequence and having the first member of the second binding pair at both 5’- and 3’- ends;(f) dipping the solid phase in the sample to capture the virus on the probe in a defined binding condition for a first period of time to measure a first wavelength shift due to light interference as a result of the binding virus on the probe;(g) determining a normalization factor based on the first wavelength shift of the sample in comparison with (i) the first wavelength shift of a calibrator, or (ii) the average of the first wavelength shifts of all calibrators;(h) dipping the solid phase in a wash solution to wash the solid phase;(i) dipping the solid phase in a lysis solution containing the capture oligonucleotide and the first, the second, and the third signal oligonucleotides and heating the lysis solution to lyse the virus from the solid phase and to denature nucleic acids in the virus to singlestranded nucleic acids;(j) removing the solid phase from the heated lysis solution;(k) cooling the heated lysis solution to hybridize each of the capture oligonucleotide and the signal oligonucleotides to its complementary region of the single-stranded virus nucleic acid;(l) dipping a probe comprising a second member of the first binding pair on the probe tip to the solution of (k), wherein the second probe captures the hybridized nucleic acids through the first binding pair;(m) dipping the probe in a conjugate solution comprising a conjugate comprising a second member of the second binding pair and horse radish peroxidase (HRP), to bind the conjugate to the probe through the second binding pair;(n) dipping the probe in a HRP substrate solution to chemically react the HRP in the conjugate with the substrate for a period of time, and measuring the second wavelength shift due to light interference;(o) applying the normalization factor to the second wavelength shift to produce a normalized wavelength shift; and(p) quantitating the normalized wavelength shift against a calibration curve having normalized wavelength shift plotted against % of full AAV capsids to determine the percentage of full AAV capsids in the sample.

25. The method of any one of claims 19-24, wherein the solid phase is magnetic particles.

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