Methods and kits

Abstract

The present invention relates to methods for detecting the presence of Mcm5 in a subject, the method comprising preparing a sample from said subject by exposing the sample to a lysis buffer and performing an assay to determine the concentration of Mcm5 by exposing the sample to a first monoclonal antibody and/or a second monoclonal antibody and measuring the amount of Mcm5 that binds to the first monoclonal antibody and/or the second monoclonal antibody. The invention further relates to kits that can be used in the methods.

1 - 97 . (canceled) 
     
     
         98 . A method for detecting the presence of minichromosome maintenance complex component 5 (Mcm5) in a subject, the method comprising:
 (a) preparing a sample from said subject wherein preparing the sample comprises exposing the sample to a lysis buffer, wherein the lysis buffer is capable of releasing Mcm5 from cells in the sample;   (b) performing an assay to determine the concentration of Mcm5 by exposing the sample to a first monoclonal antibody and/or a second monoclonal antibody and measuring the amount of Mcm5 that binds to the first monoclonal antibody and/or the second monoclonal antibody; and   (c) comparing the concentration of Mcm5 determined in step (b) to reference values;   wherein the first monoclonal antibody and the second monoclonal antibody bind to Mcm5,   wherein the first monoclonal antibody binds to a polypeptide comprising an amino acid sequence of SEQ ID NO: 1, and the second monoclonal antibody binds to a polypeptide comprising an amino acid sequence of SEQ ID NO: 2.   
     
     
         99 . The method of  claim 98  wherein the first monoclonal antibody is an antibody which:
 (i) comprises at least one Complementary Determining Region (CDR) selected from the group consisting of: 
 (a) 12A7 CDRH1 which has a sequence consisting of SEQ ID NO: 9 or a sequence that differs from SEQ ID NO:9 by a single amino acid substitution;
 (b) 12A7 CDRH2 which has a sequence consisting of SEQ ID NO: 11 or a sequence that differs from SEQ ID NO:11 by a single amino acid substitution; 
 
 (c) 12A7 CDRH3 which has a sequence consisting of SEQ ID NO: 13 or a sequence that differs from SEQ ID NO:13 by a single amino acid substitution; 
 (d) 12A7 CDRL1 which has a sequence consisting of SEQ ID NO: 3 or a sequence that differs from SEQ ID NO:3 by a single amino acid substitution; 
 (e) 12A7 CDRL2 which has a sequence consisting of SEQ ID NO: 5 or a sequence that differs from SEQ ID NO:5 by a single amino acid substitution; and 
 (f) 12A7 CDRL3 which has a sequence consisting of SEQ ID NO: 7 or a sequence that differs from SEQ ID NO:7 by a single amino acid substitution; 
 (ii) comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 29; 
 (iii) comprises a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 27; or 
 (iv) competes with the antibody of (i), (ii), or (iii); and/or the second monoclonal antibody is an antibody which: 
 (i) comprises at least one Complementary Determining Region (CDR) selected from the group consisting of: 
 (a) 4B4 CDRH1 which has a sequence consisting of SEQ ID NO: 21 or a sequence that differs from SEQ ID NO:21 by a single amino acid substitution; 
 (b) 4B4 CDRH2 which has a sequence consisting of SEQ ID NO: 23 or a sequence that differs from SEQ ID NO:23 by a single amino acid substitution; 
 (c) 4B4 CDRH3 which has a sequence consisting of SEQ ID NO: 25 or a sequence that differs from SEQ ID NO:25 by a single amino acid substitution; 
 (d) 4B4 CDRL1 which has a sequence consisting of SEQ ID NO: 15 or a sequence that differs from SEQ ID NO:15 by a single amino acid substitution; 
 (e) 4B4 CDRL2 which has a sequence consisting of SEQ ID NO: 17 or a sequence that differs from SEQ ID NO:17 by a single amino acid substitution; and 
 (f) 4B4 CDRL3 which has a sequence consisting of SEQ ID NO: 19 or a sequence that differs from SEQ ID NO:19 by a single amino acid substitution; 
 (ii) comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 33; 
 (iii) comprises a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 31; or 
 (iv) competes with the antibody of (i), (ii), or (iii). 
 
     
     
         100 . The method of  claim 98  wherein the first monoclonal antibody and/or the second monoclonal antibody is Fab′ 2 , F′(ab) 2 , Fv, a single chain antibody or a diabody. 
     
     
         101 . The method of  claim 98  wherein
 (a) the first monoclonal antibody comprises a 12A7 CDRH1 which has a sequence consisting of SEQ ID NO: 9, a 12A7 CDRH2 which has a sequence consisting of SEQ ID NO: 11, and a 12A7 CDRH3 which has a sequence consisting of SEQ ID NO: 13; and/or 
 (b) the first monoclonal antibody comprises a 12A7 CDRL1 which has a sequence consisting of SEQ ID NO: 3, a 12A7 CDRL2 which has a sequence consisting of SEQ ID NO: 5 and a 12A7 CDRL3 which has a sequence consisting of SEQ ID NO: 7. 
 
     
     
         102 . The method of  claim 98  wherein
 (a) the second monoclonal antibody comprises a 4B4 CDRH1 which has a sequence consisting of SEQ ID NO: 21, a 4B4 CDRH2 which has a sequence consisting of SEQ ID NO: 23, and a 4B4 CDRH3 which has a sequence consisting of SEQ ID NO: 25; and/or 
 (b) the second monoclonal antibody has a 4B4 CDRL1 which has a sequence consisting of SEQ ID NO: 15, a 4B4 CDRL2 which has a sequence consisting of SEQ ID NO: 17 and a 4B4 CDRL3 which has a sequence consisting of SEQ ID NO: 19. 
 
     
     
         103 . The method of  claim 98  wherein
 (a) the first monoclonal antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 29; 
 (b) the first monoclonal antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 29; 
 (c) the first monoclonal antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27; 
 (d) the first monoclonal antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 27; 
 (e) the second monoclonal antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 33; 
 (f) the second monoclonal antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 33; 
 (g) the second monoclonal antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 31; and/or 
 (h) the second monoclonal antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 31. 
 
     
     
         104 . The method of  claim 98  wherein
 (a) the assay is not an immunofluorometric assay; 
 (b) an abnormal value for the concentration of Mcm5 indicates an increased likelihood of a urological cancer in said subject; 
 (c) the assay to determine the concentration of Mcm5 is an enzyme-linked immunosorbent assay (ELISA assay), optionally wherein the ELISA assay is a sandwich ELISA assay; 
 (d) the assay to determine the concentration of Mcm5 comprises capturing the Mcm5 in the sample using a capture antibody and detecting the concentration of the Mcm5 using a detection antibody, wherein the capture antibody is the first monoclonal antibody and the detection antibody is the second monoclonal antibody or the capture antibody is the second monoclonal antibody and the detection antibody is the first monoclonal antibody, optionally wherein the capture antibody is immobilised on an ELISA plate, optionally wherein the detection antibody is conjugated to Horse Radish Peroxidase; 
 (e) the method further comprises diagnosing a patient as having the urological cancer where the concentration of the Mcm5 determined in step (b) is higher than the mean value from healthy patients plus a multiple of the standard deviation shown by the values derived from healthy subjects; 
 (f) the sample comprises urine; 
 (g) the sample or the urine sample comprises urinary sediment; 
 (h) the sample or urine sample comprises urinary sediment obtained from first catch urine collected after prostatic massage; 
 (i) the method is an in vitro method; 
 (j) an abnormal value for the concentration of Mcm5 indicates an increased likelihood of a urological cancer in said subject and said urological cancer comprises transitional cell carcinoma, optionally wherein said transitional cell carcinoma comprises:
 (i) bladder cancer; 
 (ii) prostrate cancer; or 
 (iii) cancer of the pelvis or the kidney; 
 
 (k) the lysis buffer is capable of releasing Mcm5 from cells in the urine sample; 
 (l) preparing the sample comprises a step of exposing the sample to a lysis buffer and:
 (i) the lysis buffer is not PBS containing 0.4% sodium deoxycholate and 0.02% sodium azide; 
 (ii) the method does not comprise incubation of the sample at a temperature greater than 95° C. for around 45 minutes; 
 (iii) the method does not comprise shearing the nucleic acids by passing the sample through a 21 gauge needle; 
 (iv) the method does not comprise digesting the nucleic acids by exposing the sample to Dnase I or Rnase A; and/or 
 (v) the method does not comprise centrifuging the sample at 15,000 g for 10 minutes; 
 
 (m) preparing the sample comprises a step of concentrating cells in the urine sample; and/or 
 (n) the sample comprises cells and preparing the sample consists of the following steps:
 (i) centrifugation of the sample; and 
 (ii) resuspension of the pelleted cells from the sample in the lysis buffer. 
 
 
     
     
         105 . The method of  claim 98  wherein
 (a) the first monoclonal antibody is a capture antibody and the second antibody is a detection antibody; or 
 (b) the second monoclonal antibody is a capture antibody and the first antibody is a detection antibody. 
 
     
     
         106 . The method of  claim 105  wherein
 (a) the capture antibody is bound to an ELISA plate; and/or 
 (b) the detection antibody is conjugated to horseradish peroxidase. 
 
     
     
         107 . The method of  claim 98  wherein:
 (a) the lysis buffer is capable of releasing Mcm5 from cells in the sample and does not substantially denature the Mcm5 protein; 
 (b) the lysis buffer does not denature an antibody; 
 (c) the lysis buffer comprises a detergent; 
 (d) the lysis buffer comprises a detergent and the detergent comprises or consists of polyethylene glycol p-(1,1,3,3,-tetramethylbutyl)-phenyl ether; 
 (e) 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%, between 0.05% and 1% , between 0.05% and 0.5% or around 0.1%; 
 (f) the lysis buffer comprises a detergent and the detergent comprises sodium deoxycholate or sodium dodecylsulphate; 
 (g) the lysis buffer comprises a detergent and the detergent comprises sodium deoxycholate or sodium dodecylsulphate at a concentration between 0.1% and 20%, between 0.5% and 10%, between 0.5% and 5% or around 1%; 
 (h) the lysis buffer comprises a buffer component; 
 (i) the lysis buffer comprises a buffer component and the buffer component is, comprises or consists of Trizma or Tris buffer; 
 (j) the lysis buffer comprises a buffer component and the buffer component comprises or consists of Tris buffer at a concentration greater than 5 mM, between 5 mM and 350 mM, between 200 mM and 300 mM, between 10 mM and 25mM, around 10 mM or around 250 mM; 
 (k) 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; 
 (l) the lysis buffer comprises 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 200 mM; 
 (m) 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; 
 (n) the lysis buffer comprises or consists of:
 (i) between 1 mM and 100 mM Tris; 
 (ii) between 100 mM and 300 mM sodium chloride; 
 (iii) between 1% and 5% BSA; 
 (iv) between 0.01% and 1% glycol p-(1,1,3,3,-tetramethylbutyl)-phenyl ether; and/or 
 (v) between 0.01% and 0.1% sodium azide; 
 optionally wherein the lysis buffer comprises or consists of: 
 (i) around 10 mM Tris; 
 (ii) around 200 mM sodium chloride; 
 (iii) around 2.5% BSA; 
 (iv) around 0.1% glycol p-(1,1,3,3,-tetramethylbutyl)-phenyl ether; and/or 
 (v) around 0.09% sodium azide; 
 
 (o) the lysis buffer comprises or consists of:
 (i) between 10 mM and 25 mM Tris; 
 (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; 
 
 (p) the lysis buffer comprises or consists of:
 (i) 25 mM Tris; 
 (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; and/or 
 
 (q) the lysis buffer comprises or consists of:
 (i) 25 mM Tris; 
 (ii) 150 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. 
 
 
     
     
         108 . The method of  claim 98  wherein
 (a) the method does not comprise incubation of the sample at a high temperature, optionally wherein the high temperature is a temperature greater than 50° C., greater than 60° C., greater than 70° C., greater than 80° C., greater than 90° C., between 50° C. and 120° C., between 60° C. and 110° C., between 70° C. and 100° C. or between 80° C. and 100° C.; 
 (b) the method does not comprise incubation of the sample at a high temperature for more than 30 minutes, more than 35 minutes, more than 40 minutes, more than 45 minutes, between 30 minutes and 2 hours, between 35 minutes and 2 hours, or between 40 minutes and 2 hours; 
 (c) the method does not comprise shearing nucleic acids in the sample by mechanical shearing; 
 (d) the method does not comprise exposing the sample to enzymes which digest nucleic acids; and/or 
 (e) the method does not comprise centrifuging the sample at 15,000 g for 10 minutes. 
 
     
     
         109 . A kit comprising a lysis buffer which is capable of releasing Mcm5 from cells in a sample, a first monoclonal antibody and a second monoclonal antibody wherein the first monoclonal antibody is an antibody which:
 (i) binds to a polypeptide having an amino acid sequence of SEQ ID NO: 1;   (ii) comprises at least one Complementary Determining Region (CDR) selected from the group consisting of:
 (a) 12A7 CDRH1 which has a sequence consisting of SEQ ID NO: 9 or a sequence that differs from SEQ ID NO:9 by a single amino acid substitution; 
 (b) 12A7 CDRH2 which has a sequence consisting of SEQ ID NO: 11 or a sequence that differs from SEQ ID NO:11 by a single amino acid substitution; 
 (c) 12A7 CDRH3 which has a sequence consisting of SEQ ID NO: 13 or a sequence that differs from SEQ ID NO:13 by a single amino acid substitution; 
 (d) 12A7 CDRL1 which has a sequence consisting of SEQ ID NO: 3 or a sequence that differs from SEQ ID NO:3 by a single amino acid substitution; 
 (e) 12A7 CDRL2 which has a sequence consisting of SEQ ID NO: 5 or a sequence that differs from SEQ ID NO:5 by a single amino acid substitution; and 
 (f) 12A7 CDRL3 which has a sequence consisting of SEQ ID NO: 7 or a sequence that differs from SEQ ID NO:7 by a single amino acid substitution; 
   (iii) comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 29;   (iv) comprises a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 27; or   (v) competes with the antibody of (i), (ii), (iii) or (iv);   and the second monoclonal antibody is an antibody which:   (i) binds to a polypeptide having an amino acid sequence of SEQ ID NO: 2;   (ii) comprises at least one Complementary Determining Region (CDR) selected from the group consisting of:
 (a) 4B4 CDRH1 which has a sequence consisting of SEQ ID NO: 21 or a sequence that differs from SEQ ID NO:21 by a single amino acid substitution; 
 (b) 4B4 CDRH2 which has a sequence consisting of SEQ ID NO: 23 or a sequence that differs from SEQ ID NO:23 by a single amino acid substitution; 
 (c) 4B4 CDRH3 which has a sequence consisting of SEQ ID NO: 25 or a sequence that differs from SEQ ID NO:25 by a single amino acid substitution; 
 (d) 4B4 CDRL1 which has a sequence consisting of SEQ ID NO: 15 or a sequence that differs from SEQ ID NO:15 by a single amino acid substitution; 
 (e) 4B4 CDRL2 which has a sequence consisting of SEQ ID NO: 17 or a sequence that differs from SEQ ID NO:17 by a single amino acid substitution; and 
 (f) 4B4 CDRL3 which has a sequence consisting of SEQ ID NO: 19 or a sequence that differs from SEQ ID NO:19 by a single amino acid substitution; 
   (iii) comprises a heavy chain variable region having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 33;   (iv) comprises a light chain variable region sequence having a sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 31; or   (v) competes with the antibody of (i), (ii), (iii) or (iv).   
     
     
         110 . The kit of  claim 109  wherein the first monoclonal antibody and/or the second monoclonal antibody is Fab'2, F′(ab)2, Fv, a single chain antibody or a diabody. 
     
     
         111 . The kit of  claim 109  wherein
 (a) the first monoclonal antibody comprises a 12A7 CDRH1 which has a sequence consisting of SEQ ID NO: 9, a 12A7 CDRH2 which has a sequence consisting of SEQ ID NO: 11, and a 12A7 CDRH3 which has a sequence consisting of SEQ ID NO: 13; and/or 
 (b) the first monoclonal antibody comprises a 12A7 CDRL1 which has a sequence consisting of SEQ ID NO: 3, a 12A7 CDRL2 which has a sequence consisting of SEQ ID NO: 5 and a 12A7 CDRL3 which has a sequence consisting of SEQ ID NO: 7. 
 
     
     
         112 . The kit of  claim 109  wherein
 (a) the second monoclonal antibody comprises a 4B4 CDRH1 which has a sequence consisting of SEQ ID NO: 21, a 4B4 CDRH2 which has a sequence consisting of SEQ ID NO: 23, and a 4B4 CDRH3 which has a sequence consisting of SEQ ID NO: 25; and/or 
 (b) the second monoclonal antibody has a 4B4 CDRL1 which has a sequence consisting of SEQ ID NO: 15, a 4B4 CDRL2 which has a sequence consisting of SEQ ID NO: 17 and a 4B4 CDRL3 which has a sequence consisting of SEQ ID NO: 19. 
 
     
     
         113 . The kit of  claim 109  wherein
 (a) the first monoclonal antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 29; 
 (b) the first monoclonal antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 29; 
 (c) the first monoclonal antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 27; 
 (d) the first monoclonal antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 27; 
 (e) the second monoclonal antibody comprises a heavy chain variable region having a sequence at least 95% identical to SEQ ID NO: 33; 
 (f) the second monoclonal antibody comprises a heavy chain variable region having a sequence at least 98% identical to SEQ ID NO: 33; 
 (g) the second monoclonal antibody comprises a light chain variable region having a sequence at least 95% identical to SEQ ID NO: 31; and/or 
 (h) the second monoclonal antibody comprises a light chain variable region having a sequence at least 98% identical to SEQ ID NO: 31. 
 
     
     
         114 . The kit of  claim 109  wherein
 (a) the first monoclonal antibody is a capture antibody and the second antibody is a detection antibody; or 
 (b) the second monoclonal antibody is a capture antibody and the first antibody is a detection antibody. 
 
     
     
         115 . The kit of  claim 114  wherein
 (i) the capture antibody is bound to an ELISA plate; and/or 
 (ii) the detection antibody is conjugated to horseradish peroxidase. 
 
     
     
         116 . The kit of  claim 109  wherein
 (a) the lysis buffer is capable of releasing Mcm5 from cells in the sample and does not substantially denature the Mcm5 protein; 
 (b) the lysis buffer does not denature an antibody; 
 (c) the lysis buffer comprises a detergent; 
 (d) the lysis buffer comprises a detergent and the detergent comprises or consists of polyethylene glycol p-(1,1,3,3,-tetramethylbutyl)-phenyl ether; 
 (e) 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%, between 0.05% and 1% , between 0.05% and 0.5% or around 0.1%; 
 (f) the lysis buffer comprises a detergent and the detergent comprises sodium deoxycholate or sodium dodecylsulphate; 
 (g) the lysis buffer comprises a detergent and the detergent comprises sodium deoxycholate or sodium dodecylsulphate at a concentration between 0.1% and 20%, between 0.5% and 10%, between 0.5% and 5% or around 1%; 
 (h) the lysis buffer comprises a buffer component; 
 (i) the lysis buffer comprises a buffer component and the buffer component is, comprises or consists of Trizma or Tris buffer; 
 (j) the lysis buffer comprises a buffer component and the buffer component comprises or consists of Tris buffer at a concentration greater than 5 mM, between 5 mM and 350 mM, between 200 mM and 300 mM, between 10 mM and 25mM, around 10 mM or around 250 mM; 
 (k) 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; 
 (l) the lysis buffer comprises 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 200 mM; 
 (m) 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; 
 (n) the lysis buffer comprises or consists of:
 (i) between 1 mM and 100 mM Tris; 
 (ii) between 100 mM and 300 mM sodium chloride; 
 (iii) between 1% and 5% BSA; 
 (iv) between 0.01% and 1% glycol p-(1,1,3,3,-tetramethylbutyl)-phenyl ether; and/or 
 (v) between 0.01% and 0.1% sodium azide; 
 optionally wherein the lysis buffer comprises or consists of: 
 (i) around 10 mM Tris; 
 (ii) around 200 mM sodium chloride; 
 (iii) around 2.5% BSA; 
 (iv) around 0.1% glycol p-(1,1,3,3,-tetramethylbutyl)-phenyl ether; and/or 
 (v) around 0.09% sodium azide; 
 
 (o) the lysis buffer comprises or consists of:
 (i) between 10 mM and 25 mM Tris; 
 (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; 
 
 (p) the lysis buffer comprises or consists of:
 (i) 25 mM Tris; 
 (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; and/or 
 
 (q) the lysis buffer comprises or consists of:
 (i) 25 mM Tris; 
 (ii) 150 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. 
 
 
     
     
         117 . The kit of  claim 109  further comprising
 (a) a calibrator, optionally wherein the calibrator is a solution of Mcm5 of a known concentration; 
 (b) a substrate reagent which can be used to detect the concentration of the detection antibody, optionally wherein the substrate reagent is peroxide and TMB; and/or 
 (c) a wash solution and/or a stop solution.