NanoLogix Inc (NNLX)

[soupoftheday]

SUMMARY OF THE INVENTION

The present invention fulfills this need by providing a diagnostic method for rapid, highly specific and sensitive, detection and quantification of a virus, such as SARS-CoV-2, which causes COVID-19 disease. The method comprises the steps of coating a plurality of microtiter wells in a microtiter plate with a host receptor protein contained in a coating buffer; incubating the plurality of microtiter wells overnight; washing the microtiter wells; adding a blocking solution to the plurality of microtiter wells; washing the plurality of microtiter wells three times; adding a viral substrate to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; washing the plurality of microtiter wells three times; adding an antibody directed against the viral substrate to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; adding a horseradish peroxidase (HRP)-conjugated antibody directed against the anti-viral substrate antibody to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; washing the plurality of microtiter wells three times; adding a TMB solution to the plurality of microtiter wells; adding a stop solution to the plurality of microtiter wells; and detecting the viral substrate in the microtiter wells by observing those microtiter wells that undergo a color change, or quantifying the concentration of the viral substrate by reading optical density at 450 nm, wherein color change is observed in about five minutes and the method is completed by a user in about one hour.

In another aspect of the present invention, there is provided a diagnostic, point-of-care method for rapid, highly specific and sensitive, detection and quantification of a virus from an individual suspected of being infected with a virus. The method comprises the steps of coating a plurality of microtiter wells with a host receptor protein contained in a coating buffer; incubating the plurality of microtiter wells overnight; washing the microtiter wells; adding a blocking solution to the plurality of microtiter wells; washing the plurality of microtiter wells three times; adding a viral substrate, the viral substrate obtained via a specimen collected from an individual suspected of being infected by a virus or possibly exposed to someone infected with a virus, to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; washing the plurality of microtiter wells three times; adding an antibody (i.e., primary antibody) directed against the viral substrate to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; adding a horseradish peroxidase (HRP)-conjugated antibody (i.e., secondary antibody) directed against the primary antibody to the plurality of microtiter wells; incubating the plurality of microtiter wells for 20 minutes; washing the plurality of microtiter wells three times; adding a TMB solution to the plurality of microtiter wells; adding a Stop solution to the plurality of microtiter wells; and detecting the viral substrate in the microtiter wells by observing those microtiter wells that undergo a color change, or quantifying the concentration of the viral substrate by reading optical density at 450 nm, wherein the color change is observed about five minutes and the method is completed by a user in about one hour.

In both the above-described methods, after adding a blocking solution to the microtiter wells, the microtiter plate may be stored, after which it can be shipped for use to another site, as the assay start time begins only when the viral substrate is added to the microtiter wells.

The host receptor protein may be, without limitation, ACE2; the viral substrate may be, without limitation, a SARS-CoV-2 Spike protein or a recombinant Spike protein; and the suspected virus may be, without limitation, SARS-CoV-2.

The primary antibody may be, without limitation, a rabbit polyclonal antibody directed against the SARS-CoV-2 Spike protein or the recombinant Spike protein; and the HRP-conjugated antibody directed against the primary antibody may be, without limitation, an HRP-conjugated anti-rabbit polyclonal goat antibody. Tags other than HRP directed against the primary antibody may be used in the invention, including, without limitation, alkaline phosphatase, His, FLAG, or a fluorescent tag. The invention contemplates that any antibodies used, whether they are primary or secondary antibodies, can be either polyclonal or monoclonal, IgG, or IgM, and may be derived from any suitable antibody-producing animal.

The specimen obtained from the individual suspected of being infected by a virus may include, without limitation, a nasopharyngeal swab, cerebrospinal fluid, amniotic fluid, serum, plasma, whole blood, bronchopulmonary lavage, nares, vaginal sampling, semen, or rectal/stool sampling.

In a further aspect of the invention, there is provided a test kit for rapid, highly specific and sensitive, point-of-care detection of a virus from an individual suspected of being infected with the virus. The test kit comprises a plurality of microtiter wells in a microtiter plate, the microtiter wells coated with a host receptor protein specific for the virus deposited on surfaces of the plurality of microtiter wells; a primary antibody directed against the viral substrate; a wash liquid for washing the plurality of microtiter wells and for preparing a mixture consisting of the wash liquid, an HRP-conjugated secondary antibody directed against the primary antibody and a specimen obtained from an individual suspected of being infected with the virus, said mixture made into one or more serial dilutions which are deposited atop the coating in the plurality of microtiter wells; a TMB solution; and a STOP solution, wherein the detection of the virus in the specimen is achieved by observing those microtiter wells that undergo a color change, wherein the color change is observed in about five minutes and the test is completed by a user in about thirty minutes.