Rap1-GTP, Rac1-GTP and FMS-like Tyrosine Kinase 3 ligand (FLT3-L) As Biomarkers For Early Detection of Sepsis

A lateral flow assay comprising:

• i) at least one sample and buffer zone where a sample is to be introduced onto the assay;
• ii) a detector reagent or primary capture zone where Flt3-L and Rap1-GTP analytes become bound to detector reagents comprising reporter antibodies or reporter proteins which are analyte specific and wherein each analyte specific antibody or protein is conjugated to a reporter to provide a Flt3-L antibody or protein reporter conjugate or a Rap1-GTP antibody or protein reporter conjugate after binding, each of which analyte bound antibody or protein report conjugate is capable of flowing through said assay;
• iii) an analyte capture zone comprising analyte capture lines for Flt3-L and Rap1-GTP each of which capture lines comprises a support which is conjugated to a capture reagent comprising a capture antibody or protein which is specific for binding said Flt3-L antibody or protein reporter conjugate or said Rap1-GTP antibody or protein reporter conjugate and wherein said Flt3-L antibody or protein reporter conjugate or said Rap1-GTP antibody or protein reporter conjugate becomes concentrated after binding to said capture reagent at said capture lines;
• and
• iv) a terminal wick zone which allows wicking of solution which has flowed through the assay to its terminal zone.

A multiplex lateral flow assay comprising:

• i) a lateral flow cassette with two or three lanes for determining the concentration of analytes in a biological sample obtained from a patient wherein said analytes comprising Flt-3L and Rap1-GTP, and optionally Rac1-GTP;
• ii) a sample and buffer zone in each lane where a sample is to be introduced onto the assay;
• iii) a detector or primary capture zone in each lane where FLT3L or a Rap1-GTP analytes become bound to detector reagents wherein said detector reagents comprise antibodies or proteins which are analyte specific and wherein each detector reagent is conjugated to a reporter and each of said detector regents is capable of flowing through said lane of said assay after binding said analyte;
• an analyte capture zone comprising analyte capture lines for FLT3L and Rap1-GTP each of which capture lines comprises a support which is conjugated to a capture reagent comprising a capture antibody or protein which is specific for binding said FTL3L detector reagent said Rap1-GTP detector reagent and wherein said FLT3L detector reagent or said Rap1-GTP detector reagent becomes concentrated at said capture line after binding to said capture reagent;
• and
• vi) a terminal wick zone which allows wicking of solution which has flowed through the assay to its terminal zone.

A Modular Multiplex G-Trap assay kit for measuring Rap1, Rac1 and optionally, other GTPase targets in a sample comprising reagents for up to 96 assays/target and up to 384 assays for 4 targets in multiplex format comprising:

• 1) 1-4 sets of 96 assays with red color-coded beads, each functionalized with effector proteins for a desired GTPase target;
• 2) RIPA buffer, buffer bottle;
• PMSF;
• and protease inhibitors;
• 3) Rinse and flow buffer, buffer bottle;
• 4) Optional primary antibodies (these may be user provided);
• 5) Optional secondary antibody conjugated (may be user provided);
• 6) GST-GFP for troubleshooting, 1 mL vial;
• and
• 7) Manual, user guide with detailed protocols and troubleshooting suggestions.

A lateral flow assay kit comprising:

• 1) A lateral flow cassette with single or multiple lanes for determining user specified analytes Flt3-L, Rap1-GTP and optionally, Rac1-GTP;
• 2) Bioconjugate markers comprising reporters (e.g., color beads, quantum dots, colloidal gel) functionalized with effector proteins or antibodies, for each of the analytes to be measured;
• 3) Lateral Flow cassettes available for qualitative analysis (color readout) or quantitative analysis (fluorescence readout);
• 4) Working Buffer reagents;
• 5) Instructions for using the assay;
• and
• 6) A graph setting forth standards for correlating an observed pattern of analyte captured at analyte capture lines in the serial barrier zones of said lateral flow cassettes, to analyte concentration in the test sample.