Method for determining dna concentration in dna virus

A method of determining DNA concentration in DNA viruses, 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 virus is AAV, adenovirus, or HSV-1;
• (b) dipping the first probe tip in a sample solution comprising a virus sample to capture the virus on the probe;
• (c) dipping the first probe in a wash solution to wash the first probe tip;
• (d) dipping the first probe in a lysis solution and lysing the virus from the first probe tip to obtain single-stranded DNA (ssDNA), and then removing the first probe from the lysis solution;
• (e) dipping a second probe comprising a first protein on the probe tip in the lysis solution, wherein the first protein binds to ssDNA;
• (f) dipping the second probe in a reagent solution comprising biotin-labelled second protein, wherein the second protein binds to ssDNA;
• (g) dipping the second probe in a conjugate solution comprising a conjugate comprising streptavidin and horse radish peroxidase (HRP), to bind the conjugate to the second probe,
• (h) 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
• (i) determining the ssDNA concentration in the sample by measuring the wavelength shift due to light interference, and quantitating the wavelength shift against a calibration curve to determine the ssDNA concentration.

A method of measuring the percentage of full virus capsids in a sample, comprising the steps of:

• (a) obtaining a first probe having a fixed amount of anti-DNA virus antibody immobilized on the tip of the probe, wherein the virus is adeno-associated virus (AAV), adenovirus, or herpes simplex virus type 1 (HSV-1);
• (b) dipping the first probe tip in a sample solution comprising a DNA virus sample to capture the virus capsid on the probe in a defined binding condition for a 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) dipping the first probe in a lysis solution and lysing the AAV from the first probe tip to obtain single-stranded DNA (ssDNA), and then removing the first probe from the lysis solution;
• (f) dipping a second probe comprising a first protein on the probe tip in the lysis solution, wherein the first protein binds to ssDNA;
• (g) dipping the second probe in a reagent solution comprising HRP-labelled second protein, wherein the second protein binds to ssDNA;
• (h) dipping the second probe in a HRP substrate solution to bind the substrate to the HRP bound on the second probe for a period of time and measuring the second wavelength shift due to light interference;
• (i) applying the normalization factor to the second wavelength shift to produce a normalized second wavelength shift, and
• (j) quantitating the normalized second 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.

A method of determining DNA concentration in DNA viruses, comprising the steps of:

• (a) obtaining a first solid surface immobilized with a fixed amount of anti-virus antibody, wherein the virus is adeno-associated virus (AAV), adenovirus, or herpes simplex virus type 1 (HSV-1);
• (b) contacting the first solid surface with a sample solution comprising a virus sample to capture the virus on the first solid surface in a defined binding condition;
• (c) washing the first solid surface with a wash solution to remove the non-bound materials from the solid surface;
• (d) contacting the first solid surface with a lysis solution and lysing the virus from the first solid surface to obtain single-stranded DNA (ssDNA) in the lysis solution, and then collecting the lysis solution;
• (e) contacting the ssDNA-containing lysis solution with a second solid surface immobilized with a first protein, wherein the first protein binds to ssDNA;
• (f) washing the second solid surface;
• (g) contacting the second solid surface with a second protein conjugated with a fluorescent label;
• and
• (h) detecting the fluorescent label and determining the DNA concentration in the virus;
• wherein the first protein and the second protein are independently SSB or anti-DNA antibody.