METHODS FOR DIAGNOSIS, DIFFERENTIATION AND MONITORING USING URINE PROTEINS AS MARKERS IN IGA NEPHROPATHY

Abstract

The present invention relates to use of markers selected from a group consisting of alpha-IB-glycoprotein (A1BG), alpha-1-acid glycoprotein 1 (ORM1), Ig lambda-2 chain C regions (IGLC2) and serotransferrin (TF) in methods for diagnosis, monitoring and differentiation of IgA nephropathy. In addition corresponding diagnostic kits are provided.

Claims

1. A method of diagnosis of IgA nephropathy in a subject, comprising (a) a step of identification of the of at least two, three or four of the markers selected from a group consisting of AIBG, ORM1, IGLC2 and TF, wherein said markers also comprise the non-full-length fragments thereof, in a urine sample from said subject and (b) a step of quantitative or semi-quantitative comparison of the markers identified in step (a) with the markers identified in a healthy individual.

2. The method of claim 1, wherein a GP6 marker is further identified and compared.

3. The method of claim 1, wherein the markers are identified using techniques comprising mass spectrometry, or an antibody-based test, or a combination of said techniques.

4. The method of claim 3, wherein the antibody-based test is Western blot.

5. A method of monitoring a response to an IgA nephropathy treatment, wherein (a) in a first point in time the quantitative or semi-quantitative analysis of at least two, three or four of the markers selected from a group consisting of AIBG, ORM1, IGLC2 and TF, wherein said markers also comprise the non-full-length fragments thereof, in a urine sample of a subject is performed; (b) subsequently the same analysis is carried out at a later point of time, and (c) the response to the IgA nephropathy treatment is assessed based on comparison of the results obtained in step (a) and (b), wherein the lower marker expression is indicative of a response to the treatment.

6. The method of claim 5, wherein in steps (a)-(c) GP6 is used as a further marker.

7. The method of claim 5, wherein (a), (b) and (c) are repeated.

8. A kit for diagnosis, differentiation and monitoring of IgA nephropathy in a subject, which comprises at least two antibodies that specifically bind to at least two markers selected from the group consisting of AIBG, ORM1, IGLC2 and TF, wherein said markers also comprise the non-full-length fragments thereof, and wherein said kit is equipped with means of identification of markers that bind the antibodies in said kit.

9. The kit of claim 8, wherein it comprises a further antibody for detection of a GP6 marker.

10. The kit of claim 8, wherein the antibody or antibodies are conjugated to a label.

11. The kit of claim 8, wherein it comprises a microfluidic chip.

12-13. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0031] The invention was described in relation to the following figures of drawings in which

[0032] FIG. 1 present sample results of Western blot analysis of A1BG, ORM1, IGLC2 and TF content in urine samples derived from patients with renal diseases versus healthy controls;

[0033] FIG. 2A shows evaluation by Western blotting of the A1BG protein in selected urine samples of study participants with renal diseases (left-hand side of the blots) as compared to healthy subjects (right hand side of the blots). The presence of several subforms of the protein within various molecular weight can be noticed. Densitometry measurements were done for the upper, middle and bottom ranges separately. As shown in FIG. 2B, each of the molecular ranges correlated differently with the clinical diagnosis and also as compared to the cumulative assessment of all ranges (FIG. 2C);

[0034] FIG. 3 shows a summary of densitometry readings from Western blotting of the ORM1 protein in selected urine samples of study participants with renal diseases as compared to healthy subjects.

[0035] FIG. 4 shows a summary of densitometry readings from Western blotting of the IGLC2 protein (30 kDa) in selected urine samples of study participants with renal diseases as compared to healthy subjects.

[0036] FIG. 5 shows a summary of densitometry readings from Western blotting of the TF protein in selected urine samples of study participants with renal diseases as compared to healthy subjects.

[0037] FIG. 6 (A) present sample results of Western blot analysis of GP6 content in urine samples derived from patients with renal diseases versus healthy controls, and (B) shows a summary of densitometry readings from Western blotting of the GP6 protein in selected urine samples of study participants with renal diseases as compared to healthy subjects.

[0038] FIG. 7 presents a list of possible combinations of the markers for use according to the invention.

EXAMPLES

Example 1

Discovery Phase

[0039] The methodological approach of the discovery phase has been described in Mucha et al. Below, the most pertinent information is listed.

Patients Characteristics

Groups of Patients

[0040] The study included 30 patients with IgAN and 30 healthy age- and sex-matched volunteers serving as controls. Demographic and clinical data of both groups are presented in Mucha, et al. (Supplementary material online, Table S1). Briefly, patients with biopsy-proven IgAN at different stages of chronic kidney disease (CKD) and older than 18 years were included. The inclusion criteria for the control group were as follows: age older than 18 years and absence of any kidney disease or other chronic diseases requiring treatment. The exclusion criteria for both groups included: active infection, history of malignancy, previous organ transplantation, or current pregnancy. To estimate GFR, we used the Chronic Kidney Disease Epidemiology Collaboration equations, which are the most accurate, have been evaluated in large diverse populations, and are applicable for clinical use. The study protocol was approved by the local ethics committee and informed consent was obtained from all participants. The study was performed in accordance with the Declaration of Helsinki.

Urine Collection

[0041] Samples were collected from all individuals according to a uniform study protocol, following the recommendations on urine proteomic sample collection. The second- or third-morning midstream urine was collected to sterile urine containers 1 to 3 h after previous urination. The pH of each sample was stabilized at 7.2 by addition of 1/10th vol. of 1M HEPES pH 7.2 immediately after collection. Then, samples were vortexed for 2 min, centrifuged at 3000g at room temperature for 10 min to clear the debris, filtered (0.4-m filter, Rotilabo-Spritzenfilter, Roth, Karlsruhe, Germany), and portioned into 1-ml aliquots that were stored at 80 C. before further use.

Sample Filtration

[0042] Membrane filters of the 10 kDa cut-off (Amicon Ultra-0.5, UFC501096, Millipore, Billerica, United States) were washed twice with MilliQ (MQ) water prior to use. Urine was centrifuged through the membrane at 14,000g for 15 min. Next, 500 l MQ was added to the retentate and centrifugation step was repeated. To recover the concentrated and desalted sample, the filter was placed upside down and centrifuged in a clean microcentrifuge tube for 2 min at 1000g. The protein concentration was measured by the Bradford method. Aliquots of samples were stored at 80 C.

Sample Preparation

[0043] 30 IgAN samples were divided into 3 disease pooled samples (DPSs I, II, and III), and similarly, 30 control samples were divided into 3 control pooled samples (CPSs I, II, and III). Age and sex matching was preserved within the 3 pairs of pooled sample groups. All DPSs and CPSs were obtained in 2 technical replicates (marked A and B) each, making a set of 12 pooled samples to be compared after isobaric tags for relative and absolute quantitation (iTRAQ) labeling. As 4-plex iTRAQ was used, 2 technical replications of DPSs and CPSs were compared in 1 isoelectric focusing/liquid chromatography-mass spectrometry/mass spectrometry (IEF/LC-MS/MS) experiment. To analyze 12 samples, we conducted a set of 3 independent IEF/LC-MS/MS experiments. Aliquots with extracted peptides were stored at 80 C. for the IEF/LC-MS/MS analysis.

Mass Spectrometry

[0044] Qualitative MS/MS data processing The MS/MS data were pre-processed with Mascot Distiller (v. 2.3.2.0, Matrix Science, London, United Kingdom). Data search using the MASCOT search engine was conducted on the Swiss-Prot database with the taxonomy restricted to Homo sapiens (20,236 sequences) in a 3-step procedure described elsewhere to calculate MS and MS/MS measurement errors and to recalibrate the data for the repeated MASCOT to remove systematic bias. Protein ratios were calculated as the median ratio of their peptide's ratios. The statistical significance of a single protein ratio was assessed by an in-house program, Diffprot. Calculated P values were adjusted for multiple testing using a false discovery rate-con-trolling procedure, yielding protein ratio q values.

Results of the Discovery Phase

[0045] As a result of qualitative analysis (peptide and protein identification) in each of the 3 IEF/LC-MS-MS/MS experiments, 761, 951, and 956 proteins were identified, respectively, each represented by 2 or more peptides. The results of this observations were partially presented in Mucha et al. The discovery of alpha-1B-glycoprotein (A1BG), alpha-1-acid glycoprotein 1 (AGP1, ORM1), Ig lambda-2 chain C regions (IGLC2) and serotransferrin (TF) as markers for IgAN is being reported in the current invention (Table 2).

TABLE-US-00002 TABLE2 Peptideread-outsofurineproteomicsspecificforalpha-1B-glycoprotein(A1BG), alpha-1-acidglycoprotein1(AGP1,ORM1),Iglambda-2chainCregions(IGLC2) andserotransferrin(TF)obtainedinadiscoveryphaseoftheinvention. Numberof ID1 Proteinname Gene peptides P04217 Alpha-1B-glycoprotein A1BG 51 Peptides q-value SEQIDNO:1 SLPAPW 4.26E03 SEQIDNO:2 ITPGLK 4.66E03 SEQIDNO:3 GVTFLLR 2.84E03 SEQIDNO:4 SWVPHTF 5.71E03 SEQIDNO:5 LLELTGPK 0.00E+00 SEQIDNO:6 SWITPGLK 4.57E04 SEQIDNO:7 ATWSGAVLAGR 0.00E+00 SEQIDNO:8 LELHVDGPPPR 7.51E04 SEQIDNO:9 SLPAPWLSMAPV 0.00E+00 SEQIDNO:10 VAPLSGVDFQLR 0.00E+00 SEQIDNO:11 IFVGPQHAGNYR 6.31E05 SEQIDNO:12 HQFLLTGDTQGR 0.00E+00 SEQIDNO:13 LETPDFQLFK 0.00E+00 SEQIDNO:14 SGLSTGWTQLSK 0.00E+00 SEQIDNO:15 SMAPVSWITPGLK 1.67E03 SEQIDNO:16 HGESSQVLHPGNK 0.00E+00 SEQIDNO:17 SLPAPWLSMAPVSW 6.55E03 SEQIDNO:18 LELHVDGPPPRPQL 3.58E04 SEQIDNO:19 HHGESSQVLHPGNK 7.56E05 SEQIDNO:20 HQFLLTGDTQGRYR 7.25E03 SEQIDNO:21 GVAQEPVHLDSPAIK 0.00E+00 SEQIDNO:22 LELIFVGPQHAGNYR 0.00E+00 SEQIDNO:23 LELHVDGPPPRPQLR 2.78E04 SEQIDNO:24 IFFHLNAVALGDGGHY 0.00E+00 SEQIDNO:25 NLELIFVGPQHAGNYR 0.00E+00 SEQIDNO:26 TFESELSDPVELLVAES 7.70E04 SEQIDNO:27 GAAANLELIFVGPQHAGNYR 0.00E+00 SEQIDNO:28 SLPAPWLSMAPVSWITPGLK 0.00E+00 SEQIDNO:29 TPGAAANLELIFVGPQHAGNYR 0.00E+00 SEQIDNO:30 SWVPHTFESELSDPVELLVAES 0.00E+00 SEQIDNO:31 TVRTPGAAANLELIFVGPQHAGNYR 0.00E+00 SEQIDNO:32 LHDNQNGWSGDSAPVELILSDETLPAPEFSPEPESGR 1.74E03 SEQIDNO:33 TDGEGALSEPSATVTIEELAAPPPPVLMHHGESSQVLHPGNK 0.00E+00 SEQIDNO:34 SWVPHTFE 6.44E03 SEQIDNO:35 VGPQHAGNYR 6.31E05 SEQIDNO:36 STGWTQLSK 9.51E03 SEQIDNO:37 HQFLLTGDTQ 0.00E+00 SEQIDNO:38 PVSWITPGLK 0.00E+00 SEQIDNO:39 HVDGPPPRPQLR 1.02E03 SEQIDNO:40 LSMAPVSWITPGLK 6.31E05 SEQIDNO:41 MHHGESSQVLHPGNK 3.73E04 SEQIDNO:42 SGLSTGWTQLSKLLELTGPK 3.87E05 SEQIDNO:43 GPPPRPQLR 7.28E03 SEQIDNO:44 SLPAPWLSMA 5.85E04 SEQIDNO:45 LELHVDGPPPRPQ 4.34E04 SEQIDNO:46 IFFHLNAVALGDGGH 0.00E+00 SEQIDNO:47 NGVAQEPVHLDSPAIK 3.87E05 SEQIDNO:48 TPGAAANLELIFVGPQHAGN 0.00E+00 SEQIDNO:49 LPAPWLSMAPVSWITPGLK 3.87E05 Numberof ID2 Proteinname Gene peptides P02763 Alpha-1-acidglycoprotein1 ORM1 29 Peptides q-value SEQIDNO:50 TYMLAF 1.964E03 SEQIDNO:51 AHLLILR 0.000E+00 SEQIDNO:52 NWGLSVY 6.310E05 SEQIDNO:53 TYLNVQR 5.215E03 SEQIDNO:54 YVGGQEHF 6.264E03 SEQIDNO:55 FAHLLILR 1.789E04 SEQIDNO:56 TTYLNVQR 4.565E04 SEQIDNO:57 YVGGQEHFA 5.451E04 SEQIDNO:58 YVGGQEHFAH 0.000E+00 SEQIDNO:59 EHFAHLLILR 6.704E03 SEQIDNO:60 SDVVYTDWK 0.000E+00 SEQIDNO:61 YVGGQEHFAHL 0.000E+00 SEQIDNO:62 MLAFDVNDEK 0.000E+00 SEQIDNO:63 YVGGQEHFAHLL 3.866E05 SEQIDNO:64 GQEHFAHLLILR 0.000E+00 SEQIDNO:65 SVYADKPETTK 0.000E+00 SEQIDNO:66 TYMLAFDVNDEK 0.000E+00 SEQIDNO:67 GLSVYADKPETTK 3.866E05 SEQIDNO:68 EQLGEFYEALDCLR 0.000E+00 SEQIDNO:69 YVGGQEHFAHLLILR 0.000E+00 SEQIDNO:70 WGLSVYADKPETTK 3.866E05 SEQIDNO:71 NWGLSVYADKPETTK 0.000E+00 SEQIDNO:72 DVNDEKNWGLSVYADKPETTK 0.000E+00 SEQIDNO:73 TYMLAFDVNDEKNWGLSVYADKPETTK 0.000E+00 SEQIDNO:74 VVYTDWK 7.322E03 SEQIDNO:75 VYADKPETTK 1.877E03 SEQIDNO:76 VGGQEHFAHLLILR 2.390E04 SEQIDNO:77 IPKSDVVYTDWK 6.846E03 SEQIDNO:78 GGQEHFAHLLILR 3.901E03 Numberof ID3 Proteinname Gene peptides P0CG05 Iglambda-2chainCregions IGLC2 4 Peptides q-value SEQIDNO:79 ADSSPVK 5.85E04 SEQIDNO:80 GVETTTPSK 1.61E04 SEQIDNO:81 AGVETTTPSK 0.00E+00 SEQIDNO:82 KAGVETTTPSK 8.03E04 Numberof ID4 Proteinname Gene peptides P02787 Serotransferrin TF 76 Peptides q-value SEQIDNO:83 VYIAGK 4.49E03 SEQIDNO:84 DSGFQMN 1.79E04 SEQIDNO:85 HSTIFEN 1.05E03 SEQIDNO:86 GLLYNK 3.85E03 SEQIDNO:87 SAGWNIPI 9.31E03 SEQIDNO:88 PDPWAK 5.33E03 SEQIDNO:89 MYLGYEY 4.17E04 SEQIDNO:90 NPDPWAK 1.79E04 SEQIDNO:91 DSAHGFLK 0.00E+00 SEQIDNO:92 FGYSGAFK 2.78E04 SEQIDNO:93 VAEFYGSK 0.00E+00 SEQIDNO:94 KDSGFQMN 9.23E04 SEQIDNO:95 EFQLFSSPH 2.43E04 SEQIDNO:96 KPVEEYAN 6.13E04 SEQIDNO:97 DGAGDVAFVK 0.00E+00 SEQIDNO:98 SAGWNIPIGLL 0.00E+00 SEQIDNO:99 EDLIWELLN 3.73E04 SEQIDNO:100 YLGEEYVK 3.87E05 SEQIDNO:101 HSTIFENLAN 0.00E+00 SEQIDNO:102 GYYGYTGAFR 0.00E+00 SEQIDNO:103 KPVDEYK 4.57E04 SEQIDNO:104 IPMGLLYNK 3.87E05 SEQIDNO:105 DSGFQMNQLR 0.00E+00 SEQIDNO:106 PVVAEFYGSK 0.00E+00 SEQIDNO:107 LAQVPSHTVVAR 0.00E+00 SEQIDNO:108 KPVDEYKD 8.27E04 SEQIDNO:109 EGYYGYTGAFR 3.87E05 SEQIDNO:110 SAGWNIPIGLLY 6.31E05 SEQIDNO:111 NIPMGLLYNK 2.78E04 SEQIDNO:112 HQTVPQNTGGK 0.00E+00 SEQIDNO:113 QYFGYSGAFK 6.31E05 SEQIDNO:114 TAGWNIPMGLLY 6.31E05 SEQIDNO:115 SLDGGFVYIAGK 0.00E+00 SEQIDNO:116 SASDLTWDNLK 0.00E+00 SEQIDNO:117 HSTIFENLANK 0.00E+00 SEQIDNO:118 EFQLFSSPHGK 0.00E+00 SEQIDNO:119 TAGWNIPMGLLYN 3.87E05 SEQIDNO:120 SKEFQLFSSPH 2.29E03 SEQIDNO:121 KDSGFQMNQLR 0.00E+00 SEQIDNO:122 EDLIWELLNQAQ 1.61E04 SEQIDNO:123 MYLGYEYVTAIR 0.00E+00 SEQIDNO:124 KEGYYGYTGAFR 0.00E+00 SEQIDNO:125 LKPVVAEFYGSK 0.00E+00 SEQIDNO:126 KSASDLTWDNLK 0.00E+00 SEQIDNO:127 TAGWNIPMGLLYNK 0.00E+00 SEQIDNO:128 STLNQYFGYSGAFK 6.31E05 SEQIDNO:129 NLKPVVAEFYGSK 0.00E+00 SEQIDNO:130 EDPQTFYYAVAVVK 0.00E+00 SEQIDNO:131 SKEFQLFSSPHGK 0.00E+00 SEQIDNO:132 AIAANEADAVTLDAGLVYDAY 0.00E+00 SEQIDNO:133 LAPNNLKPVVAEFYGSK 0.00E+00 SEQIDNO:134 EDLIWELLNQAQEHFGK 0.00E+00 SEQIDNO:135 IMNGEADAMSLDGGFVYIAGK 3.87E05 SEQIDNO:136 AIAANEADAVTLDAGLVYDAYLAPN 0.00E+00 SEQIDNO:137 GKEDLIWELLNQAQEHFGK 0.00E+00 SEQIDNO:138 GGKEDLIWELLNQAQEHFGK 0.00E+00 SEQIDNO:139 EDLIWELLNQAQEHFGKDK 0.00E+00 SEQIDNO:140 SMGGKEDLIWELLNQAQEHFGK 0.00E+00 SEQIDNO:141 AIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSK 0.00E+00 SEQIDNO:142 APNHAVVT 8.70E03 SEQIDNO:143 APNHAVVTR 6.31E05 SEQIDNO:144 SAGWNIPIGL 8.24E03 SEQIDNO:145 QVPSHTVVAR 1.06E03 SEQIDNO:146 STIFENLANK 0.00E+00 SEQIDNO:147 HLAQVPSHTVVAR 0.00E+00 SEQIDNO:148 GWNIPMGLLYNK 6.31E05 SEQIDNO:149 MYLGYEYVTAIRNLR 3.86E03 SEQIDNO:150 PNNLKPVVAEFYGSK 5.33E03 SEQIDNO:151 HSTIFENLA 5.85E04 SEQIDNO:152 ADRDQYELL 1.81E03 SEQIDNO:153 QLFSSPHGK 1.75E03 SEQIDNO:154 LGYEYVTAIR 2.69E03 SEQIDNO:155 HSTIFENLANKADR 5.85E04 SEQIDNO:156 HQTVPQNTGGKNPDPWAK 3.87E05 SEQIDNO:157 KEDLIWELLNQAQEHFGK 1.52E03 SEQIDNO:158 GLVYDAYLAPNNLKPVVAEFYGSK 3.87E05

Example 2

Validation Phase

[0046] The primary difference between the discovery and validation phases is the transition from the assessment of the pooled urine samples (i.e. the discovery phase) to the individual evaluation of each protein in a given patient or a healthy person and a direct correlation of these results with the known clinical parameters in each case (i.e. the validation phase).

Patient Characteristics

[0047] The study included 133 renal disease patients and 19 healthy controls. Renal disease included IgAN (77 cases), ADPKD (29) and LN (27).

Sample Collection

[0048] Urinary samples were collected according to the protocol standardized in the Transplantation Institute, Medical University of Warsaw.

SDS-PAGE

[0049] Samples were defrozen to room temperature (23 C.), then suspended by intensive pipetting or mixing using a vortex. Leammli buffer was added to urine samples to achieve final concentrations as follows: 2% SDS; 10% glycerol; 5% -mercaptoethanol; 0.002% bromophenol blue; 0.125 M Tris-HCl; pH 6.8. Samples were boiled at 95 C. for 2 min. 10 l of each sample was loaded on the Mini-PROTEAN TGX 4-15% gradient gel.

Western Blotting Analysis

[0050] The method developed by the Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences (patent application no P.415033) allows to assay all the proteins of interest in the urine samples. It permits the analysis of all the selected protein biomarkers with the accuracy not reachable by classical methods. To date, the proteome analysis of urine in medicine starts from centrifugation of the sample (in line with the European Confederacy of Laboratory Medicine guidelines), which result losing of protein which are insoluble, and exist as aggregates or degradants. This solid fraction is crucial because proteins progress into insoluble forms randomly, depending of protein, state of patient, his diet and properties of urine. For that reason, in the current study we used the whole urine in form of suspension, which next were analyzed by Western blotting technique. That gives an opportunity to examine all of proteins present in a given in urine sample. Advantages of this method are important for medicinal diagnostics. Method is noninvasive for patients, it allows collecting the samples from the patients even few times per day, and it is relatively little time consuming. For Western blotting analysis, the urine protein samples were separated on SDS-PAGE gels, as described above, and transferred to a nitrocellulose membrane. Membranes were incubated in appropriate blocking buffer (either 5% low-fat dry milk or bovine serum albumin in TBS-Tween 20 (TBST)). After an incubation in the primary antibody (A1BG (F-9); catalog number sc-374415; Santa Cruz) the cells were washed in TBST, and incubated with a horseradish peroxidase-conjugated secondary antibody. The chemiluminescence reaction for HRP was developed using SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and visualized with Stella 8300 bioimager Densitometry read-outs were carried out for each of the bands in the blots. Eight randomly chosen patients samples were pooled together and used on each Western blot as a benchmark. Densitometry read-outs from other band on a given Western blot membrane were divided by the read-out of the respective benchmark. The results were referred to as a relative band density.

[0051] List of antibodies used for Western blotting: A1BG (cat # sc-374415, Santa Cruz), ORM1 (sc-69753, Santa Cruz), IGLC2 (sc-33134, Santa Cruz), TF (sc-21011, Santa Crus), GP6 (sc-20149, Santa Cruz).

Results

[0052] The results of representative Western blotting analysis for A1BG, ORM1, IGLC2 and TF presence in urine samples are presented in FIG. 1.

[0053] A1BG Based on the Western blotting analysis, it becomes evident that study participants with kidney diseases exhibit the presence of different forms of the protein (for the purpose of this invention segregated into within the high [80 kDa], medium [45 kDa] and low [15 kDa] molecular weight range), occurring in different proportions. A direct comparison of selected samples from kidney disease versus heathy patients is presented in FIG. 2A. Mutual relations between the visible forms may be important in the pathophysiology of the given disease. Indeed, as presented in FIG. 2B, various subforms of A1BG correlate differently with the type of kidney disease and also differently as compared to the cumulative assessment of the protein (FIG. 2C). Notably, the bottom-range bands tend to be most prominently elevated in IgAN patients.

[0054] Validation phase as described above was also performed for the other markers, in particular ORM1, IGLC2 and TF and the results are presented in FIGS. 3-5. The results are also presented for GP6 (FIG. 6). Although GP6 is on average expressed at higher levels in LN than IgA, on the basis of our results and data, the compilation of 4 proteins reported in the GP6 compilation may increase the sensitivity and specificity of the test.