Methods for analysing a urine sample

Abstract

The present invention provides methods for analysing a urine sample from a subject comprising exposing the urine sample to a lysis buffer which is capable of releasing at least one biomarker from cells in the urine sample. The present invention further provides kits, devices, and apparatuses that can be used in these methods. Finally, the present invention provides methods for detecting the presence of a urological cancer in a subject comprising performing an assay on a sample from a subject to determine the concentration of an Mcm protein.

1 - 113 . (canceled) 
     
     
         114 . A kit comprising a lysis buffer which is capable of releasing a minichromosome maintenance (Mcm) protein from cells in a urine sample, a capture antibody and a detection antibody wherein the capture antibody and the detection antibody bind to Mcm5. 
     
     
         115 . The kit of  claim 114  wherein:
 (a) the lysis buffer is capable of releasing Mcm5 from cells in the urine sample and does not substantially denature the Mcm5 protein; or 
 (b) the lysis buffer does not denature an antibody. 
 
     
     
         116 . The kit of  claim 114  wherein:
 (a) the lysis buffer comprises a detergent; 
 (b) the lysis buffer comprises a detergent and the detergent comprises or consists of polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether; 
 (c) the lysis buffer comprises a detergent and the detergent comprises or consists of polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether at a concentration between 0.01% and 25%, between 0.01% and 10%, between 0.05% and 5%, or around 1%; 
 (d) the lysis buffer comprises a detergent and the detergent comprises sodium dodecyl sulphate; 
 (e) the lysis buffer comprises a detergent and the detergent comprises sodium dodecylsulphate at a concentration between 0.1% and 20%; optionally wherein the detergent comprises sodium dodecylsulphate at a concentration of 0.1%; 
 (f) the lysis buffer comprises a detergent and the detergent comprises sodium deoxycholate; and/or 
 (g) the lysis buffer comprises a detergent and the detergent comprises sodium deoxycholate at a concentration between 0.1% and 20%, between 0.5% and 10%, between 0.5% and 5%, between 0.75% and 1.25%, or around 1%. 
 
     
     
         117 . The kit of  claim 114  wherein:
 (a) the lysis buffer comprises a buffer component; 
 (b) the lysis buffer comprises a buffer component and the buffer component is, comprises or consists of tris(hydroxymethyl)methylamine or tris(hydroxymethyl)methylamine hydrochloride; 
 (c) the lysis buffer comprises a buffer component and the buffer component comprises or consists of tris(hydroxymethyl)methylamine at a concentration greater than 5 mM, between 5 mM and 350 mM, between 10 mM and 250 mM, between 10 mM and 50 mM, or around 25 mM; and/or 
 (d) the lysis buffer comprises a buffer component and the buffer component maintains the pH of the buffer between pH 4 and pH 9, between pH 5 and pH 8, between pH 6 and pH 8, or around pH 7.6. 
 
     
     
         118 . The kit of  claim 114  wherein:
 (a) the lysis buffer comprises a salt selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, sodium sulphate, potassium sulphate, magnesium sulphate, sodium acetate, potassium acetate, magnesium acetate, sodium phosphate, potassium phosphate and magnesium phosphate; 
 (b) the lysis buffer comprises a salt and the salt is at a concentration between 20 mM and 300 mM, between 150 mM and 300 mM, between 100 mM and 200 mM or around 200 mM; and/or 
 (c) the lysis buffer comprises a salt and the lysis buffer or salt comprises or consists of sodium chloride at a concentration between 20 mM and 300 mM, between 150 mM and 300 mM, between 100 mM and 200 mM, or around 150 mM. 
 
     
     
         119 . The kit of  claim 114  wherein the lysis buffer has an ionic strength of between 1 mM and 500 mM, between 50 mM and 450 mM, between 100 mM and 250 mM, or between 100 mM and 175 mM. 
     
     
         120 . The kit of  claim 114  wherein the lysis buffer comprises or consists of:
 (a) (i) between 10 mM and 50 mM tris(hydroxymethyl)methylamine;
 (ii) between 100 mM and 200 mM sodium chloride; 
 (iii) between 0.75% and 1.25% sodium deoxycholate; 
 (iv) between 0.1% and 20% sodium dodecyl sulphate; and/or 
 (v) between 0.05% and 5% polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether; 
 
 (b) (i) between 10 mM and 25 mM tris(hydroxymethyl)methylamine;
 (ii) between 100 mM and 200 mM sodium chloride; 
 (iii) around 1% sodium deoxycholate; 
 (iv) between 0.1% and 20% sodium dodecyl sulphate; and/or 
 (v) between 0.05% and 1% glycol p-( 1 , 1 , 3 , 3 ,-tetramethylbutyl)-phenyl ether; 
 
 (c) (i) 25 mM tris(hydroxymethyl)methylamine;
 (ii) between 100 mM and 200 mM sodium chloride; 
 (iii) around 1% sodium deoxycholate; 
 (iv) 0.1% sodium dodecyl sulphate; and/or 
 (v) 1% glycol p-(1,1,3,3,-tetramethylbutyl)-phenyl ether; or 
 
 (d) (i) 25 mM tris(hydroxymethyl)methylamine;
 (ii) 150 mM sodium chloride; 
 (iii) around 1% sodium deoxycholate; 
 (iv) 0.1% sodium dodecyl sulphate; and/or 
 (v) 1% polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether. 
 
 
     
     
         121 . The kit of  claim 114 , wherein:
 (a) the lysis buffer comprises radioimmunoprecipitation assay (RIPA) buffer; and/or   (b) the lysis buffer is Cytobuster Protein Extraction Reagent.   
     
     
         122 . The kit of  claim 114  further comprising a calibrator; optionally wherein the calibrator is a solution of Mcm5 of a known concentration. 
     
     
         123 . The kit of  claim 114  further comprising at least one substrate reagent which can be used to detect the concentration of the detection antibody; optionally wherein:
 (a) the detection antibody is conjugated to horse radish peroxidase the substrate reagent comprises TMB (3, 3′, 5, 5′-tetramethylbenzidine), DAB (3, 3′-diaminobenzidine) or ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); or 
 (b) at least one substrate reagent is peroxide and TMB. 
 
     
     
         124 . The kit of  claim 114  further comprising a wash solution and/or a stop solution. 
     
     
         125 . The kit of  claim 114  further comprising an ELISA plate. 
     
     
         126 . The kit of  claim 114  wherein the capture antibody:
 (a) is an antibody bound to the surface of a solid support; 
 (b) is immobilised on an ELISA plate. 
 
     
     
         127 . The kit of  claim 114  wherein:
 (a) the kit is suitable for performing an immunoassay to detect an Mcm protein or determine the concentration of an Mcm protein; and/or 
 (b) the kit is suitable for performing an immunoassay to detect an Mcm protein or determine the concentration of an Mcm protein and the immunoassay is a sandwich immunoassay, an enzyme-linked immunosorbent assay (ELISA), a sandwich ELISA, a radioimmunoassay (RAI), an enzyme immunoassay (EIA), a lateral flow/immunochromatographic strip test, or a particle-based immunoassay such as a particle-based immunoassay comprising gold, silver, or latex particles and magnetic particles or Q-dots. 
 
     
     
         128 . The kit of  claim 127  wherein the kit is suitable for performing a sandwich ELISA. 
     
     
         129 . The kit of  claim 114  wherein:
 (a) the detection antibody is conjugated to a label; optionally wherein the label is directly attached or attached via a linker, such as Adipic Acid Dihyrazide or ADH; 
 (b) the detection antibody is conjugated to Horse Radish Peroxidase; or 
 (c) the detection antibody is conjugated to Europium. 
 
     
     
         130 . The kit of  claim 114 , comprising:
 (a) an anti-Mcm5 antibody coated microplate comprising twelve 8-well strips coated with mouse monoclonal anti-Mcm5 antibody;   (b) a wash solution at 20× concentrate;   (c) an anti-MCM5 antibody-HRP conjugate;   (d) a TMB substrate reagent;   (e) a stop solution;   (f) a lysis buffer; optionally a negative control; and   (e) a positive control.   
     
     
         131 . The kit of  claim 114  wherein the urine sample:
 (a) comprises urinary sediment; and/or 
 (b) comprises urinary sediment obtained from first catch urine collected after prostatic massage. 
 
     
     
         132 . A method for analysing a urine sample from a subject comprising:
 (a) exposing the urine sample to a lysis buffer wherein the lysis buffer is capable of releasing at least one biomarker from cells in the urine sample; and   (b) performing an assay to determine the concentration of the at least one biomarker in the urine sample.