Method of early diagnosis of immune-mediated inflammatory disease
11047856 · 2021-06-29
Assignee
Inventors
Cpc classification
G01N2800/105
PHYSICS
G01N33/564
PHYSICS
International classification
G01N33/564
PHYSICS
Abstract
An in-vitro method for early diagnosing or prediction of immune-mediated inflammatory diseases, comprising: —obtaining a sample from a subject; —quantifying simultaneously by one LC/MS-MS analysis of said sample, a presence of V65 vitronectin fragment or fragment, variant or degradation product thereof; and a presence of a complement C3f or fragment or variant or degradation products thereof.
Claims
1. A method, comprising obtaining a biological sample from a subject that has or is suspected of having an immune-mediated inflammatory disease; and quantifying simultaneously (a) a V65 vitronectin or fragment, variant or degradation product thereof and (b) a complement C3f or fragment, variant or degradation product thereof by LC/MS-MS analysis of said biological sample, wherein quantifying comprises: (i) mixing the biological sample with an acidic aqueous solution comprising trifluoro acetic acid (TFA); (ii) loading the resulting mixture on a trapping LC column; (iii) eluting the column with an alcoholic mixture and obtaining an eluate; (iv) drying the eluate; (v) dispersing the dried eluate in an acetonitrile aqueous solution; (vi) loading the resulting dispersion on a reversed phase LC column and separating two mobile phases; and (vii) injecting by continuous flux in MS-MS, both mobile phases and simultaneously quantifying the V65 vitronectin or fragment, variant or degradation product thereof and the complement C3f or fragment, variant or degradation product thereof.
2. The method according to claim 1, wherein the subject is a human.
3. The method according to claim 1, wherein the biological sample is blood, serum, plasma, urine, or synovial fluid.
4. The method according to claim 1, wherein the V65 vitronectin fragment comprises the following amino sequence: SQRGHSRGRNQNSRRPS (SEQ ID NO:1).
5. The method according to claim 1, wherein the complement C3f comprises the following amino sequence: SSKITHRIHWESASLLR (SEQ ID NO:2).
6. The method according to claim 1, wherein the immune-mediated inflammatory diseases is osteoarthritis.
7. The method according to claim 1, wherein the immune-mediated inflammatory diseases is lupus.
8. The method according to claim 1, wherein the immune-mediated inflammatory diseases is sclerodermia.
9. The method according to claim 1 wherein the LC/MS-MS analysis comprises microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry analysis.
10. The method according to claim 1, wherein the acidic aqueous solution comprises 1% v trifluoro acetic acid (TFA).
11. The method according to claim 1, wherein the alcoholic mixture is methanol/water/TFA.
12. The method according to claim 11, wherein the mixture methanol/water/TFA is in a ratio from 90:7:3 to 90:9:1 v/v/v.
13. The method according to claim 1, wherein the acetonitrile aqueous solution comprises TFA.
14. The method according to claim 13, wherein the acetonitrile aqueous solution comprising TFA is in a ratio acetonitrile/water/TFA from 3:97:0.1 to 1:99:0.1 v/v/v.
15. The method according to claim 1, wherein the complement C3f or fragment, variant or degradation product thereof are quantified at m/z 459.3, 530.7 and 646.3 and wherein the V65 vitronectin or fragment, variant or degradation product thereof are quantified at m/z 434.9, 445.2, 460.5, 466.2, 469.7, 480.3, 485.3.
16. The method according to claim 11, wherein the mixture methanol/water/TFA is in a ratio of 90:9:1 v/v/v.
17. The method according to claim 13, wherein the acetonitrile aqueous solution comprising TFA is in a ratio acetonitrile/water/TFA of 1:99:0.1 v/v/v.
18. The method according to claim 1, wherein the trapping LC column is washed prior to eluting the column.
Description
BRIEF DESCRIPTION OF FIGURES
(1)
(2)
(3)
(4)
(5) Samples were classified into 7 groups: HV (n=12), K&O (n=18), K&L1 (n=17), K&L2 (n=25), K&L3 (n=48), K&L4 (n=8) and RA (n=19). Peak intensities of C3f and vitronectin fragments obtained by SELDI-TOF-MS are illustrated in
(6) Concentrations of C3f and vitronectin fragments obtained by LC-MS/MS according to the invention are illustrated in
(7) Both methods, SELDI-TOF-MS and LC-MS/MS (also called LC-chip-MS/MS), are then compared in
(8)
DETAILED DESCRIPTION
(9) Definitions of Abbreviations Used in the Present Description
(10) OA: osteoarthritis HV: healthy volunteers RA: rheumatoid arthritis SLE: Systemic lupus erythematosus UC: Ulcerative colitis SELDI-TOF-MS: surface enhanced laser desorption/ionization-time of flight mass spectrometry JSN: joint space narrowing LC: liquid chromatography MeOH: methanol ACN: acetonitrile FA: formic acid TFA: trifluoroacetic acid NH.sub.4OAc: ammonium acetate NH.sub.4OH: ammonium hydroxide ESI: electrospray ionization source m/z: mass-to-charge ratio EIC: extracted ion current NSAID: non steroidal anti-inflammatory drug LLOQ: lower limit of quantitation HLB: hydrophilic-lipophilic balance MCX: mixed-mode cationic exchange PE: process efficiency ME: matrix effect ER: extraction recovery EY: extraction yield
Classification According to Grade of Severity
(11) The Kellgren and Lawrence (K&L) score (0-4) as reported in Annals of the rheumatic disease 1957; 16: 494-502 is used to classify biomarker expression levels detected in samples of patients with knee OA according to their grade of severity. The K&L classification is running from 1 to 4 after X-ray examination. K&L0, 1 and 2 scores represent the first stages of OA development, for which X-ray cannot detect joint narrowing space and bone sclerosis. K&L3 and 4 are further associated to the late stages of OA with marked joint space narrowing, bone sclerosis and presence of multiple osteophytes.
(12) Early stage of diagnosis as defined in the present invention corresponds to K&L0 for OA. No corresponding K&L classification is reported for the other IMIDs.
(13) LC/MS-MS
(14) The method according to the invention starts with a weak cation exchange such as WCX SPE 96 well plates approach that was selected for the purification and enrichment process of both fragments. A Liquid Chromatography (LC) is then carried out for a separation of peptides fragments or fragment, variant or degradation product thereof and is continuously followed by a double MS detection of respectively the peptides and their fragments or variant or degradation products thereof. Several parameters for LC separation and for MS/MS detection were optimized to get two independent time windows for C3f and vitronectin fragments quantitation. The developed method for C3f and vitronectin fragment quantitation in sample was fully validated. After having selected the most appropriate regression model on the basis of the accuracy profiles, method selectivity, trueness, precision, accuracy and linearity were demonstrated according to a well known method with e-noval software as disclosed for example in U.S. Pat. No. 8,494,818B2.
(15) This new absolute quantitative method according to the invention, was applied to the analysis of different IMID samples previously analyzed by SELDI-TOF-MS. IMIDs were classified according to severity. It has been surprisingly found that the new absolute method according to the invention allows to detect vitronectin fragment and complement C3f fragment in the early stage of IMID samples.
Example 1: Application of the Method According to the Invention to Osteoarthritis
(16) For example this new absolute quantitative method according to the invention, was applied to the analysis of different samples such as serum samples (n=147) previously analyzed by SELDI-TOF-MS on OA serum samples. OA samples were classified according to OA severity, which is characterized by the K&L classification from 1 to 4 after X-ray examination. OA serum samples were also compared to RA serum samples to assess the specificity of C3f and vitronectin fragments. Healthy Volunteers (HV) serum samples were also included to determine the starting concentration of C3f and vitronectin fragments in normal serum.
(17) Similarly to the SELDI-TOF-MS analysis, we observed by LC-MS/MS that C3f and vitronectin fragments levels increased with OA severity. Compared to HV, C3f fragment showed statistically increased expression in stages of OA where Joint Space Narrowing (JSN) is definite (K&L3-4 scores) compared to HV. Compared to RA, it was already statistically increased in the earlier stage with definite osteophyte and possible JSN (K&L2 score) of OA. Further, within OA patients, C3f fragment showed statistically increased expression in the K&L3-4 scores compared to K&L1-2 scores. C3f biomarker could therefore approach the definition of a “burden of disease” biomarker assessing disease severity in individuals with OA. Compared to HV, vitronectin fragment showed statistically increased expression in most K&L scores. Vitronectin fragment biomarker could therefore approach the definition of a “diagnostic” biomarker distinguishing between individuals with and without OA.
(18) In this comparison study, we observe that both C3f and vitronectin fragments increase with OA severity. We also observe that C3f fragment is more related to the severity of OA, whereas vitronectin fragment is more related to early OA detection.
(19) Materials and Methods
(20) Methods Summary:
(21) Microfluidic liquid chromatography coupled to a nanoelectrospray source ion trap mass spectrometry was used for the absolute and simultaneous quantitation of C3f and vitronectin fragments in serum. The method was first carefully optimized and then validated in serum biological matrix according to FDA guidelines. Stable isotopes for the two biomarkers of interest were used as internal standards. Microelution solid phase extraction in 96-well plate format was used to purify and concentrate C3f and vitronectin fragments. Parameters for liquid chromatophraphy and for mass spectrometry were simultaneously optimized for C3f and vitronectin fragments quantitation. Serum samples (n=147) classified in 7 groups [(healthy volunteers, OA with 5 grades of severity and rheumatoid arthritis (RA) patients] were analyzed with our new quantitative method.
(22) Chemicals
(23) Water, methanol (MeOH), acetonitrile (ACN) and formic acid (FA) 99% were all at LC/MS grade and were purchased from Biosolve. Trifluoroacetic acid (TFA) was obtained from Fluka. Ammonium hydroxide (NH.sub.4OH) was purchased from Merck and ammonium acetate (NH.sub.4OAc) 98.9% from VWR. Helium and nitrogen (Alphagaz 2) were obtained from Air Liquide. Human synthetic complement C3f fragment (.sub.1304SSKITHRIHWESASLLR.sub.1320), vitronectin fragment (.sub.381SQRGHSRGRNQNSRRPS.sub.397) and the internal standard [.sup.13C.sub.6, .sup.15N.sub.2]Lys.sup.3, [.sup.13C.sub.6, .sup.15N.sub.4]Arg.sup.17-Complement C3f, [.sup.13C.sub.6, .sup.15N.sub.4]Arg.sup.3,15-vitronectin fragment were purchased from Eurogentec.
(24) Instruments
(25) Solid-phase extraction procedure was carried out by Oasis μElution weak cation exchange (WCX) 96 well plates with a vacuum manifold (Waters Inc.) for peptides extraction and enrichment. Sample evaporation was performed on a vacuum concentrator (Labconco). A 1200 series LC-system including nanoflow pump, a capillary pump, a well plate sampler and a LC/MS interface was used for chromatographic separation. ChemStation (Agilent Technologies) is a software package to control Agilent liquid chromatography system. Protonated peptides detection was performed by Ion Trap mass spectrometry combined with a nanoelectrospray ionisation source operating in positive mode (Agilent Technologies). TrapControl (Bruker Daltonik GmbH) determined the mass spectrometry detection parameters. Raw data obtained by mass spectrometry were processed using DataAnalysis and QuantAnalysis softwares (Bruker Daltonik GmbH).
(26) Internal Standards and Calibration Standards
(27) All peptides (labeled and unlabeled) were dissolved in a H.sub.2O/ACN/FA (80:20:0.1; v/v/v) solution to reach a concentration of 1 mg/mL, then were aliquoted and stored at −80° C. Isotopically labeled peptides ([.sup.13C.sub.6, .sup.15N.sub.2]Lys.sup.3, [.sup.13C.sub.6, .sup.15N.sub.4]Arg.sup.17-complement C3f and [.sup.13C.sub.6, .sup.15N.sub.4]Arg.sup.3, 15-vitronectin fragment) were used as internal standard for the two biomarkers of interest (complement C3f and vitronectin fragments) since they share the same physicochemical properties. Internal standard at a final concentration of 25 ng/mL and 10 ng/mL for complement C3f and vitronectin fragments, respectively, were prepared in H.sub.2O/TFA (99:1; v/v) solution. Calibration standards of complement C3f and vitronectin fragments were then diluted in H.sub.2O/TFA (99:1; v/v) solution in the range of 2.5 to 200 ng/mL at seven concentration levels (2.5, 5, 10, 20, 50, 100 and 200 ng/mL) for C3f and in the range of 2.5 to 100 ng/mL at six concentration levels (2.5, 5, 10, 20, 50 and 100 ng/mL) for vitronectin fragment.
(28) Solid Phase Extraction Procedure
(29) Serum samples were thawed at room temperature, vortexed and centrifuged during 5 min at 13,400 rpm. A Waters Oasis μElution WCX 96 well plate was used to purify and concentrate peptides of interest before chromatographic separation and mass spectrometry analysis. The sorbent conditioning for the OASIS-WCX was first 300 μL of MeOH followed by 300 μL of water. 100 μl of diluted human serum samples were prepared as follows: 10 μL of human serum+50 μL of H.sub.2O/TFA (99:1; v/v) solution+40 μL of internal standards (of C3f and vitronectin fragments). 100 μL of calibration standards were prepared as follows: 10 μL of calibration standards at expected concentration (of C3f and vitronectin fragments)+10 μL of serum bovine (Sigma, B9433)+40 μL of in H.sub.2O/TFA (99:1; v/v) solution+40 μL of internal standards (of C3f and vitronectin fragments). The 100 μL of diluted serum or calibration standards were then transferred in the well and drawn through the sorbent with a vacuum manifold. The plate was then washed with 200 μL NH.sub.4OAc 25 mM, pH6.8, 200 μL of MeOH/H.sub.2O/NH.sub.4OH (50:47.5:2.5, v/v/v) followed by 200 μL of water. The extracts were eluted with 2×50 μL of MeOH/H.sub.2O/TFA (90:9:1, v/v/v). The eluates were then evaporated in a vacuum evaporator at 30° C. for 65 min, reconstituted in 100 μL ACN/H.sub.2O/TFA (1:99:0.1, v/v/v) and vortexed 15 min at room temperature.
(30) LC-MS/MS Analysis
(31) LC-Conditions
(32) ProtID with a 40 nL trapping column and a 43 mm×75 μm analytical column, both packed with a Zorbax 300SB 5 μm C18 phase (Agilent Technologies) were used for chromatographic separation. The mobile phase A [H.sub.2O/TFA (100:0.1, v/v] and the mobile phase B [ACN/H.sub.2O/TFA (90:10:0.1, v/v/v] for the capillary pump and the mobile phase A [H.sub.2O/FA (100:0.1, v/v] and the mobile phase B [ACN/H.sub.2O/FA (90:10:0.1, v/v/v] for the nanopump, were degassed by ultrasonication for 15 min before use. During the analytical process, the sample was first loaded on the trapping column during an isocratic enrichment of the column by capillary pump with 1% of the mobile phase B at a flow rate of 4 μL/min. A flush volume of 1 μL of 1% mobile phase B was used to remove unretained components. Then, a gradient of elution starting at 1% of the mobile phase B and linearly ramping up to 90% of mobile phase B in 5 min was performed in backflush mode using the nanopump. Ninety % of mobile phase B was maintained for 2 min before coming back to 1%. Ten column volumes were finally used to re-equilibrate the column before the next injection. The time for one run was 14 min. Five μL of sample was injected. The injection needle was also thoroughly rinsed twice from inside and outside with a mix solution of ACN/H.sub.2O/TFA (60:40:0.1, v/v/v).
(33) MS Detection
(34) Optimization of MS detection parameters was performed by infusion experiments at a flow rate of 36 μL/h with 1 μg/mL of C3f and vitronectin fragment and their internal standards solutions. NanoESI source was operating in a positive mode. Ions optics were optimized using the Smart Ramp tool included in Trap-Control software in order to maximize precursor ion intensity for C3f, vitronectin fragment and their internal standards. Capillary voltage was set at 1800V and the endplate offset at 500V. Nitrogen was used as drying gas at a flow rate of 4 L/min and the source temperature was set at 325° C. MS.sup.2 spectra were acquired in the mass-to-charge (m/z) ratio from 375 to 700 for C3f and in the m/z range of 350 to 600 for the vitronectin fragment.
(35) The MS and MS/MS experimental parameters were optimized to be as sensitive and selective as possible. Reference peptides were first infused one by one in order to optimize source parameters and to select the most intense precursor ions ([M+4H].sup.4+ for both). Then, after fragmentation energy optimization, the most abundant fragment ions were selected for quantitation: m/z=459.3, 530.7 and 646.3 for C3f and m/z=434.9, 445.2, 460.5, 466.2, 469.7, 480.3, 485.3 for the V65 vitronectin fragment.
(36) Total ion current chromatogram was handled to yield an extracted ion current (EIC) chromatogram that summed the intensities of the selected MS/MS ions for C3f and vitronectin fragment. EIC were smoothed using a 1×2 Gauss algorithm and subsequently integrated. Area ratios (peptide vs. labeled peptide) were considered for quantitation.
(37) Serum Cohort—Epidemiological Data
(38) The 116 OA patients from University of Bristol cohort were included to validate the quantitation of C3f and the V65 vitronectin fragment by LC-MS/MS analysis. The demographic, clinical and radiographic data of these patients were previously published in Annals of the rheumatic diseases 2011. None of the OA patients was on intra-articular steroids or any potential disease-modifying drugs, the majority was on analgesics or non-steroidal anti-inflammatory drugs (NSAIDs) that are unlikely to affect serum or synovial fluid levels of investigated markers. Nineteen RA patients fulfilling the 1987 American College of Rheumatology criteria (as described in Journal of Chromatography A 2013; 1314:199-207) and 12 healthy individuals referred to as healthy volunteers (HV) were included in the study as control subjects.
(39) Statistics
(40) The e.noval software (Arlenda, Liège, Belgium) was used to compute trueness, precision, accuracy for all concentrations of calibration standards, as well as lower limit of quantitation (LLOQ). It was also used to choose the most appropriate regression model.
(41) Peak intensities (by SELDI-TOF-MS) and concentrations (by LC-MS/MS) of C3f and vitronectin fragments were compared in all groups of OA to HV and RA groups by the non-parametric Kruskal-Wallis test with a post-hoc test of Dunn's. K&L1-2 values were compared to K&L3-4 values by the non-parametric Mann-Whitney U test. P-values were statistically significant at P<0.05.
(42) Correlation coefficients were calculated by the non-parametric Spearman analysis to correlate SELDI-TOF-MS data to LC-MS/MS data. Deming regression was used for regression analysis.
(43) Results
(44) Results Summary:
(45) A weighted 1/x.sup.2 quadratic regression for C3f and a weighted 1/x quadratic regression for vitronectin peptide were selected for calibration curves. Trueness (with a relative bias <10%), precision (repeatability and intermediate precision <15%) and accuracy (risk <15%) of the method were successfully demonstrated. The linearity of results was validated in the dosing range of 2.5-200 ng/mL for C3f and 2.5-100 ng/mL for vitronectin fragment.
(46) Optimization of MS/MS Detection
(47) Synthetic peptides for the C3f (.sub.1304SSKITHRIHWESASLLR.sub.1320) and vitronectin (.sub.381SQRGHSRGRNQNSRRPS.sub.397) fragments were first infused at a concentration of 1 μg/mL in 0.1% of TFA and analyzed in full scan mode into the ion trap in the range of m/z 200-1500. Voltages were optimized to detect the highest relative intensity using the Smart Ram tool. [M+4H].sup.4+ at m/z 506.3 and at m/z 496.0 were the most abundant ions observed for C3f and vitronectin synthetic peptides (
(48) Labeled peptides were synthesized accordingly: [.sup.13C.sub.6, .sup.15N.sub.2]Lys.sup.3, [.sup.13C.sub.6, .sup.15N.sub.4]Arg.sup.17-Complement C3f and[.sup.13C.sub.6, .sup.15N.sub.4]Arg.sup.3,15-vitronectin fragment. Both unlabeled and labeled peptides were simultaneously infused for the C3f and for the vitronectin fragment. Two precursor ions, [M+4H].sup.4+ at m/z 506.3 and [M+4H].sup.4+ at m/z 510.8, were distinctly observed for the unlabeled and labeled C3f peptides (
(49) LC Method Development
(50) In this work, the method was developed for the simultaneous detection of C3f and vitronectin fragments in one single analysis of serum by LC-MS/MS method. All experimental conditions (mobile phases and dissolution medium composition) were optimized to reach the highest peak intensity in MS for both peptides. Synthetic peptides were diluted in a solution containing 0.1% of TFA (any retention being observed with 0.1% of formic acid for the vitronectin fragment). The dissolution solution also contained 1% ACN as the peak intensity rapidly decreased at higher percentage of ACN for the vitronectin fragment (
(51) Solid Phase Extraction
(52) Several Oasis μElution well plates such as WCX, HLB and MCX were tested for the purification and enrichment of C3f and vitronectin peptides before chromatographic separation and mass spectrometry analysis. Oasis WCX was the most efficient in term of recovery for both peptides. Extraction conditions were optimized and summarized in the Materials and Methods section.
(53) Validation of the Method in Biological Fluid
(54) Validation of this new method was then implemented on serum, the selected biological fluid.
(55) Bovine serum was first tested to confirm that any detection of C3f and vitronectin fragment was observed by our new method. Calibration curves were then set up by preparing calibration standards of C3f and vitronectin fragments in bovine serum as described in the solid phase extraction procedure of the Materials and Methods section. Each solution was submitted to the entire process including the extraction phase in Oasis μElution WCX well plates, nano-LC retention process and injection in the nano-LC-MS/MS for analysis. For each calibration curve, all calibration standards were processed in duplicates during three days. A weighted 1/x.sup.2 quadratic regression for the C3f and a weighted 1/x quadratic regression for the vitronectin peptide were selected showing the highest accuracy and trueness index and the widest dosing range (Table 1). As summarized in Table 2, trueness (with a relative bias <10%), precision (repeatability and intermediate precision <15%) and accuracy [(risk <15%, except for the 2.5 ng/mL calibration standard of vitronectin (17,11%)] of the method were demonstrated for all concentrations of calibration standards, as well as at the lower limit of quantitation (LLOQ=2.5 ng/mL). The linearity of the results for C3f and vitronectin fragment quantitation was validated in the dosing range of 2.5-200 ng/mL for C3f and 2.5-100 ng/mL for vitronectin. The reproducibility for the retention time of the LC-chip system was also very good: 3.5 min (0.6% RSD) for the V65 vitronectin fragment and 6.0 min (0.1% RSD) for the C3f.
(56) The assessment of the biological matrix effect in our quantitative bio-analytical assay was also performed according to the following references J. of chromatography A 2010; 1217:3929-37 or Analytical chemistry 2003; 75: 3019-30 and Clinical biochemistry 2005; 38:328-34. Process efficiency (PE), matrix effect (ME), extraction recovery (ER) and extraction yield (EY) were summarized in
(57) Quantitation in Biological Sample
(58) For quantitation, 3 fragment ions (m/z=459.3, 530.7 and 646.3) for C3f and 7 fragment ions (m/z=434.9, 445.2, 460.5, 466.2, 469.7, 480.3 and 485.3) for the V65 vitronectin fragment were selected. Both unlabeled and labeled peptides were simultaneously injected for the C3f and for the V65 vitronectin fragment. Chromatogram area ratios (peptide vs. labeled peptide) were considered for all biological samples as well as for the calibration curve. C3f and vitronectin quantitation was performed by LC-MS/MS on serum samples (n=147) provided from the previous cohort used for the SELDI-TOF-MS validation study. These samples were classified into 7 groups: HV (n=12), K&O (n=18), K&L1 (n=17), K&L2 (n=25), K&L3 (n=48), K&L4 (n=8) and RA (n=19). Peak intensities of C3f and vitronectin fragments obtained by SELDI-TOF-MS, were illustrated for all serum samples (n=147) in
(59) Both methods, SELDI-TOF-MS and LC-MS/MS, were then compared in
(60) Discussion for Osteoarthritis
(61) We have developed a new method by LC-MS/MS that combines a simultaneous quantitation of C3f (.sub.1304SSKITHRIHWESASLLR.sub.1320) and vitronectin (.sub.381SQRGHSRGRNQNSRRPS.sub.397) fragments in serum sample. Combination of both quantifications is of high interest as it considerably decreases analysis time. Further, it allows to cover two different processes in OA: innate immunity/inflammation for C3f and extracellular matrix degradation for vitronectin fragment. WCX SPE 96 well plates approach was selected for the purification and enrichment process of both fragments. Several parameters for LC separation and for MS/MS-MS detection were optimized to get two independent time windows for C3f and vitronectin fragments quantitation. The developed method for C3f and vitronectin fragment quantitation in serum was fully validated. After having selected the most appropriate regression model on the basis of the accuracy profiles, method selectivity, trueness, precision, accuracy and linearity were demonstrated according to FDA guidelines.
(62) This new absolute quantitative method was applied to the analysis of serum samples (n=147) previously analyzed by SELDI-TOF-MS. OA serum samples were classified according to OA severity, which is characterized by the K&L classification from 1 to 4 after X-ray examination. K&L0, 1 and 2 scores represent the first stages of OA development, for which X-ray cannot detect joint narrowing space and bone sclerosis. K&L3 and 4 are further associated to the late stages of OA with marked joint space narrowing, bone sclerosis and presence of multiple osteophytes. OA serum samples were also compared to RA serum samples to assess the specificity of C3f and vitronectin fragments. HV serum samples were also included to determine the starting concentration of C3f and vitronectin fragments in normal serum.
(63) We observed by LC-MS/MS that C3f and vitronectin fragments levels increased with OA severity. Compared to HV, C3f fragment showed statistically increased expression in stages of OA where JSN is definite (K&L3-4 scores) compared to HV. Compared to RA, it was already statistically increased in the earlier stage with definite osteophyte and possible JSN (K&L2 score) of OA. Further, within OA patients, C3f fragment showed statistically increased expression in the K&L3-4 scores compared to K&L1-2 scores. C3f biomarker could therefore approach the definition of a “burden of disease” biomarker assessing disease severity in individuals with OA. Compared to HV, vitronectin fragment showed statistically increased expression in all K&L scores, except for K&L0, but was only statistically increased in K&L3 score compared to RA. Vitronectin fragment biomarker could therefore approach the definition of a “diagnostic” biomarker distinguishing between individuals with and without OA.
(64) With this new absolute quantitative method, we observe that both C3f and vitronectin fragments increase with OA severity. We also observe that C3f fragment is more related to the severity of OA, whereas vitronectin fragment is more related to early OA detection.
Example 2: Application of the Method According to the Invention to Systemic Lupus Erythematosus and Sclerodermia
(65) The same experimental conditions were applied to the analysis of samples such as serum samples provided from rheumatoid arthritis (RA, n=46), spondylarthropathies (n=27), systemic lupus erythematosus (SLE, n=23), sclerodermia (n=20), ulcerative colitis (UC, n=27) and Crohn's patients (n=24). Healthy volunteers (n=41) samples were also included to determine the starting concentration of C3f and vitronectin fragments in serum samples.
(66) We observe that median range of concentration for C3f and V65 vitronectin fragment is increased compared to healthy controls but also compared to other pathologies such as RA, spondylarthropathies, UC and Crohn's disease. C3f and V65 vitronectin fragment are present in all IMIDs tested. (
Conclusions: Experimental Conditions to Carry Out the Method According to the Invention
(67) In the Solid-Phase Extraction Procedure (SPE)
(68) The method according to the invention starts with a weak cation exchange such as WCX SPE 96 well plates approach that was selected for the purification and enrichment process of both fragments.
(69) TABLE-US-00002 Experimental Conditions SPE on WCX Sequence Conditioning 300 μL MeOH Equilibration 300 μL H.sub.2O Loading 100 μL 10 μL of human serum + 50 μL of H.sub.2O/TFA (99:1; v/v) solution + 40 μL of internal standards. Wash 1 200 μL NH4OAc 25 mM pH 6.8 Wash 2 200 μL MeOH/H.sub.2O/NH.sub.4OH (50:47.5:2.5) (v/v/v) Wash 3 200 μL H.sub.2O 100% Elution 2 × 50 μL MeOH/H.sub.2O/TFA (90:9:1) (v/v) Vacuum evaporator 65 min à 30° C. Resuspention 100 μl ACN/H.sub.2O/TFA (1:99:0.1, v/v/v) Vortexed 15 min at room temperature
In the Liquid Chromatography Separation
(70) The method according to the invention include a liquid chromatography separation step with a hydrophobic column such as C18 approach that was selected for the separation of both fragments.
(71) For the capillary pump: the mobile phase A [H2O/TFA (100:0.1, v/v] and the mobile phase B [ACN/H2O/TFA (90:10:0.1, v/v/v].
(72) For the nanopump: the mobile phase A [H2O/FA (100:0.1, v/v] and the mobile phase B [ACN/H2O/FA (90:10:0.1, v/v/v].
ACKNOWLEDGEMENTS
(73) This research was funded by the “Fond National de la Recherche Scientifique” (Projet de Recherche #23592988) for the Laboratory of Rheumatology and the Laboratory for the Analysis of Medicines, ULg, Liege.
(74) TABLE-US-00003 TABLE 1 Regression models: related accuracy and trueness indexes and dosing ranges Accuracy Trueness Dosing range Index Index (ng/mL) Model for C3f Weighted 1/x.sup.2 quadratic regression 0.8392 0.9782 2.50-200.0 Weighted 1/x quadratic regression 0.7923 0.8976 27.53-200.0 Weighted 1/x.sup.2 linear regression 0.6966 0.7621 21.43-140.1 Weighted 1/x linear regression 0.6285 0.4528 30.06-200.0 Model for vitronectin fragment Weighted 1/x quadratic regression 0.8649 0.9880 2.50-100.0 Weighted 1/x.sup.2 quadratic regression 0.8609 0.9872 2.57-100.0 Weighted 1/x.sup.2 linear regression 0.8106 0.9927 2.82-100.0 Weighted 1/x linear regression 0.7990 0.9821 3.90-100.0
(75) TABLE-US-00004 TABLE 2 A) C3f and B) vitronectin fragments quantitation - method validation: trueness, precision, accuracy and linearity properties A. C3f fragment quantitation - Method Validation Weighted quadratic regression model calibration Response function range (m = 7): 2.5-200 ng/mL (k = 3) Series 1 Series 2 Series 3 Quadratic term 0.0001165 0.00009036 0.00008730 Slope 0.03261 0.02053 0.03655 Intercept −0.3955 −0.02173 −0.04982 Weight 1/x.sup.2 1/x.sup.2 1/x.sup.2 r.sup.2 0.9909 0.9924 0.9885 Trueness (k = 3, n = 2) (ng/mL) Relative bias (%) 2.5 7.949 5.0 2.060 10 −5.575 20 2.715 50 4.251 100 3.443 200 −1.307 Precision Repeatability Intermediate precision (k = 3, n = 2) (ng/mL) (RSD %) (RSD %) 2.5 7.01 7.39 5.0 12.09 14.80 10 8.54 8.74 20 6.16 13.02 50 5.59 5.59 100 1.61 5.76 200 4.41 8.82 Accuracy ß-expectation (k = 3, n = 2) (ng/mL) tolerance limits (%) Risk (%) 2.5 [−4.08, 19.97] 2.56 5.0 [−23.22, 27.34] 14.50 10 [−19.68, 8.53] 3.36 20 [−22.89, 28.32] 15.61 50 [−4.72, 13.22] 0.57 100 [−8.62, 15.51] 4.06 200 [−8.38, 5.77] 0.18 Linearity (k = 3, n = 2) (ng/mL) Range 2.5-200 Slope 0.9922 Intercept 0.8931 r.sup.2 0.9962 LOD 0.76 LLOQ 2.5 B. Vitronectin fragment quantitation - Method validation Weighted quadratic regression model calibration range (m = 6): 2.5-100 ng/mL Response function (k = 3) Series 1 Series 2 Series 3 Quadratic term 0.0001232 −0.00001847 0.00002566 Slope 0.05878 0.07712 0.06873 Intercept 0.06105 0.01201 0.02518 Weight 1/x 1/x 1/x r.sup.2 0.9957 0.9673 0.9905 Trueness (k = 3, n = 2) (ng/mL) Relative bias (%) 2.5 2.425 5.0 5.496 10 1.250 20 −1.875 50 3.130 100 −3.690 Precision Intermediate precision (k = 3, n = 2) (ng/mL) Repeatability (RSD %) (RSD %) 2.5 8.53 14.48 5.0 6.74 6.74 10 10.52 10.52 20 7.39 7.91 50 5.85 5.99 100 3.56 3.94 ß-expectation tolerance Accuracy (k = 3, n = 2) (ng/mL) limits (%) Risk (%) 2.5 [−24.77, 29.62] 17.11 5.0 [−5.31, 16.30] 1.32 10 [−15.63, 18.13] 4.83 20 [−14.81, 11.06] 2.15 50 [−6.54, 12.80] 0.74 100 [−10.20, 2.82] 0.19 Linearity (k = 3, n = 2) (ng/mL) Range 2.5-100 Slope 0.9677 Intercept 0.6698 r.sup.2 0.9954 LOD 0.76 LLOQ 2.5 k: Number of days of experiments (series); m: number of concentration levels; n: number of replicates per concentration level and per series.