DIAGNOSTICS

AIM #2

Aim #2: Establish quantitative sandwich ELISA and Lateral Flow Assay for QSOX-L from urine using antibodies from Aim #1.

Immunoblotting and immunoprecipitation: QSOX1-L will be detected in urine using standard immunodetection techniques such as immunoblotting or immunoprecipitation. In immunoblotting, protein preparations from urine will be electrophoresed through polyacrylamide gels and transferred onto nitrocellulose or PVDF membrane. The QSOX1-L will be detected with polyclonal antibodies from Aim #1. In immunoprecipitation, a urine sample will be incubated with antibody against QSOX1-L covalently coupled with agarose beads. Following washing the bound QSOX1-L will be detected by immunoblotting or ELISA.

Quantitative sandwich ELISA assay: Sandwich ELISA enables quantifying levels of proteins that will allow us to set threshold for basal levels of QSOX1-L in urine. Stored de-identified urine from 100 BC patient samples will be utilized in this study. Dr. Ho will also identify and provide urine from 100 patients with non-malignant conditions.  Urine will be serially diluted with a blocking buffer in triplicate followed by incubation in ELISA plates coated with anti-QSOX-L capture Ab from Aim #1.  After 1-hour incubation at 37C, plates will be washed followed by addition of biotinylated anti-QSOX-L detection antibody. Streptavidin-HRP will be used to generate dose dependent signal. A standard curve will be obtained for each plate using recombinant QSOX1-L protein spiked into urine that has been depleted of QSOX1-L using affinity chromatography column conjugated with anti-sera against 100aa peptide in Aim #1. Concentrations of QSOX1-L will be calculated based on the standard curve for each plate. To establish a reference range for QSOX1-L levels in urine from patients and individuals without malignant disease, we will calculate the mean concentrations, ±2 SD. 

Quantitative Lateral Flow Assay (LFA): Lateral flow assays are essentially sandwich ELISA run on a nitrocellulose membrane. One antibody specific for QSOX1-L will be conjugated onto detector beads. Another antibody, specific for another epitope of QSOX1-L will either be immobilized on a test line or biotinylated. If QSOX1-L is present in a sample, a sandwich complex will form, resulting in a signal on the test line when the detector beads accumulate at the test line as a result of direct sandwich formation or sandwich capture by the streptavidin zone.

Strip design: The proposed LFA will be manufactured (American Bionostica, LLC) by affixing four overlapping pads to a 300 mm wide self-adhesive backing card. The card will be cut into 3 mm wide strips that are each inserted into a plastic cassette. The four different components of the assembly will be: (1) a filter to remove particulates from urine; (2) the conjugate pad made of glass fiber, onto which assay reagents will be deposited and dried; (3) the nitrocellulose membrane that will contain two lines, a test line composed of either an anti-QSOX1-L antibody or a poly-streptavidin to capture biotinylated antibody, and a control  

line made of deposited anti-rabbit antibody to capture escaped beads functionalized with rabbit Ab. The control line ensures that the fluid flows properly through the membrane and the beads are released from conjugate pad; (4) absorbent pad that wicks away the moisture and promotes capillary flow on the nitrocellulose membrane.

LFA configuration: We will explore two configurations: (1) standard LFA configuration where one antibody is conjugated to detector beads and the other is dispensed as a test line; (2) alternative configuration where one antibody is conjugated to a detector bead and another is biotinylated while Polystreptavidin dispensed as a test line serves as a capture reagent for the sandwich formed by the two antibodies.

Reagent preparation: Each antibody from Aim #1 will be conjugated to blue latex beads following a standard EDC/NHS conjugation chemistry and blocked with a proprietary blocking solution. Same antibodies will either be dispensed as a test line or biotinylated with Thermo EZ-link sulfo-NHS-LC2-Biotin (PN21343) per manufacturer’s instructions. All possible working combinations of antibody pairs will be tested to ensure optimal performance of the assay. The metrics of optimal performance will be highest dynamic range, lowest LOD and absence of non-specific binding.

LFA configuration: We will explore two configurations: (1) standard LFA configuration where one antibody is conjugated to detector beads and the other is dispensed as a test line; (2) alternative configuration where one antibody is conjugated to a detector bead and another is biotinylated while Polystreptavidin dispensed as a test line serves as a capture reagent for the sandwich formed by the two antibodies.

Reagent preparation: Each antibody from Aim #1 will be conjugated to blue latex beads following a standard EDC/NHS conjugation chemistry and blocked with a proprietary blocking solution. Same antibodies will either be dispensed as a test line or biotinylated with Thermo EZ-link sulfo-NHS-LC2-Biotin (PN21343) per manufacturer’s instructions. All possible working combinations of antibody pairs will be tested to ensure optimal performance of the assay. The metrics of optimal performance will be highest dynamic range, lowest LOD and absence of non-specific binding.

Data acquisition: During optimization the tests will be done in wet assay mode, where beads are dispensed in solution instead of being dried on a conjugate pad. The drying of the test and assembly into cassettes will be relegated to Phase II. The aim is to generate standard curves with recombinant QSOX1-L in depleted urine and to determine limits of detection and dynamic range of each assay configuration. A universal reader RDS-1500 (Detekt Biomedical, Austin, TX) will be used to quantify signals on the strips. The data will be directly correlated to ELISA to ensure the two methods yield comparable results.

Milestones: (1) A correlation between sandwich ELISA and LF assays is established with at least one antibody pair; (2) Reproducibility, accuracy, limits of detection, and linear dynamic range of quantitative sandwich ELISA and LFA tests are determined. The tests are available for screening samples in Aim #3.