Monoclonal antibody specific for gamma-glutamyl-l-epsilon-lysine for the monitoring of apoptosis

Abstract

The present invention concerns an ex vivo method for the monitoring of apoptosis which is based on the detection of free gamma-glutamyl-L-epsilon-Lysine (GGEL) in a biological sample of a subject with a monoclonal antibody specific to GGEL. The invention also relates to the monoclonal antibody specific to GGEL, as well as to diagnostic kits containing such a ligand.

Claims

1. An isolated monoclonal antibody specific for gamma-glutamyl-L-epsilon-Lysine (GGEL) which comprises CDR-H1 of sequence SEQ ID NO:3, CDR-H2 of sequence SEQ ID NO:4, CDR-H3 of sequence SEQ ID NO:5, CDR-L1 of sequence SEQ ID NO:6, CDR-L2 of sequence SEQ ID NO:7, and CDR-L3 of sequence SEQ ID NO:8.

2. The isolated monoclonal antibody according to claim 1 which comprises a variable domain of heavy chain of sequence SEQ ID NO:1, or a sequence at least 85% identical to SEQ ID NO: 1.

3. The isolated monoclonal antibody according to claim 1 which comprises a variable domain of light chain of sequence SEQ ID NO:2, or a sequence at least 85% identical to SEQ ID NO:2.

4. A method for measuring the level of gamma-glutamyl-L-epsilon-Lysine (GGEL) in a sample, which comprises: a) contacting a sample with the monoclonal antibody specific for GGEL according to claim 1; and b) measuring the level of complexes formed with the monoclonal antibody specific for GGEL; wherein the level of GGEL in the sample is deduced from the level of complexes formed with the monoclonal antibody specific for GGEL.

5. An ex vivo method for the monitoring of apoptosis in a subject, which comprises: a) measuring the level of free gamma-glutamyl-L-epsilon-Lysine (GGEL) isopeptide in a plasma sample of the subject with an immunoassay using the monoclonal antibody according to claim 1; b) comparing said measured level of free GGEL with a control; and c) monitoring apoptosis in said subject based on the comparison with the control.

6. The method according to claim 5, wherein monitoring apoptosis in said patient based on the comparison with the control is performed by: (i) if the control is derived from a healthy subject or population of healthy subjects, determining that apoptosis is upregulated in the subject if the level of free GGEL in the plasma sample of the subject is greater than the level of free GGEL in the control, or determining that apoptosis is downregulated in the subject if the level of free GGEL in the plasma sample of the subject is lower than the level of free GGEL in the control; or (ii) if the control is derived from a subject or population of subjects suffering from disease associated with upregulated apoptosis, determining that apoptosis is upregulated in the subject if the level of free GGEL in the plasma sample of the subject is equal or greater than the level of free GGEL in the control; or (iii) if the control is derived from a subject or population of subjects suffering from disease associated with downregulated apoptosis, determining that apoptosis is downregulated in the subject if the level of free GGEL in the plasma sample of the subject is equal or lower than the level of free GGEL in the control.

7. The method according to claim 5, wherein monitoring of apoptosis enables for diagnosing a disease associated with dysregulated apoptosis, upregulated or downregulated apoptosis.

8. The method according to claim 7, wherein monitoring of apoptosis enables for diagnosing sepsis.

9. The method according to claim 4, wherein the level of free GGEL is measured by an ELISA, indirect, competitive or sandwich.

10. A method for monitoring effectiveness of an apoptosis inducing treatment in a subject, which comprises: a) measuring the level of free gamma-glutamyl-L-epsilon-Lysine (GGEL) isopeptide in a plasma sample of a subject undergoing an apoptosis inducing treatment, with a method according to claim 4; b) repeating the measurement of step a) in time; and c) deducing that the apoptosis inducing treatment is effective if the level of free GGEL increases over time, or that the apoptosis inducing treatment is ineffective if the level of free GGEL is unchanged or decreases over time.

11. A method for monitoring effectiveness of an apoptosis inhibiting treatment in a subject is provided, which comprises: a) measuring the level of free gamma-glutamyl-L-epsilon-Lysine (GGEL) isopeptide in a plasma sample of a subject undergoing an apoptosis inhibiting treatment, with a method according to claim 4; b) repeating the measurement of step a) in time; and c) deducing that the apoptosis inhibiting treatment is effective if the level of free GGEL decreases over time, or that the apoptosis inhibiting treatment is ineffective if the level of free GGEL is unchanged or increases over time.

12. A method of treating a disease associated with dysregulated apoptosis in a subject in need thereof, which comprises: a) administering an apoptosis modulating treatment to a subject treating suffering from a disease associated with dysregulated apoptosis b) monitoring if said treatment modulates apoptosis in the subject by implementing the method of monitoring of apoptosis according to claim 5; and c) continuing or modifying the apoptosis modulating treatment based on the result of monitoring of step b).

13. A kit for the monitoring of apoptosis which comprises: a) a monoclonal antibody specific for gamma-glutamyl-L-epsilon-Lysine (GGEL) as defined in claim 1; and b) a control.

14. A method of treating sepsis in a subject in need thereof, which comprises: a) diagnosing sepsis in a subject by an ex vivo method of diagnostic of sepsis according to claim 8; and b) administering a therapeutic treatment against sepsis to the subject diagnosed as suffering from sepsis.

15. A lateral flow immunoassay device which comprises a monoclonal antibody specific for gamma-glutamyl-L-epsilon-Lysine (GGEL) as defined in claim 1.

Description

FIGURES

(1) FIG. 1. Formation of N-ε-(γ-glutamyl)-L-lysine isopeptide bond by transglutaminase reaction.

(2) FIG. 2. Quantification of GGEL concentration using standard curve. Standardized BZGO with known amount of GGEL were used as standard for the quantification of GGEL. Standard curve was calculated using 4-Parameter line using SoftMax Pro 6.5.

(3) FIG. 3. GGEL quantification after induction of apoptosis by staurosporin 1 μmol/L (STS) in HL60 cells. GGEL concentration in apoptosis-induced cell group was significantly different from GGEL concentration in cell group non-induced into apoptosis. (P value<0.05).

(4) FIG. 4. Results of GGEL quantification in sepsis plasma (PBS-T20: Phosphate Buffer Saline with Tween 20=negative control, Blank plasma=control plasma, Plasma sepsis D1=plasma from sepsis patient at day+1, Plasma sepsis D3=plasma from sepsis patient at day+3).

(5) FIG. 5. Blank plasma were used in order to determinate the threshold of the test (V=μ×3 SD). Specificity and sensitivity data were obtained resulting in a specificity of 91% for D1 group, 100% for D+3 group and 95.45% for the merged group (D1+D+3). ROC curve of the test was obtained. All plasma have been diluted at 1/20.

(6) FIG. 6. Exemplary lateral flow immunoassay device.

(7) FIG. 7. GGEL quantification between different pathologies.

(8) FIG. 8. GGEL plasma concentration between time points T1 and T2 in different pathologies.

EXAMPLE

Example 1: Development of a Monoclonal Antibody Specific to Gamma-Glutamyl-L-Epsilon-Lysine (GGEL)

(9) Materials & Methods

(10) Development of mAb, Preparation of Immunogen and Immunization Regime.

(11) Mice were immunized with gamma-glutamyl-L-epsilon-Lysine (GGEL) coupled on Keyhole limpet hemocyanin (KLH) via glutaraldehyde and then dialysed against Phosphate Buffer Saline 1×(PBS).

(12) 25 μg of antigenic solution was dissolve in 100 μL of PBS. Six-weeks-old BALB/c and SJL female white mice were given four intranodal injection of immunogen at 1 week intervals and a single intraperitoneal injection three days before fusion.

(13) Production and Screening of Hybridomas.

(14) Hybridoma cells were produced by the method described elsewhere Galfre and Milstein (1981). The supernatant were screened by enzyme-linked immunosorbent assay (ELISA) against GGEL coupled to bovine Serum Albumine (BSA) immobilized to the wells of Maxisorp microtiter plates. Wells containing immobilized antigens were incubated with hybridomas supernatant for 1 h, followed with goat anti-mouse (H+L) peroxidase conjugate diluted 1 in 2000 in PBS containing Tween 20 0.05% (PBST) for one hour. Bound antibody was visualized by incubating wells with tetramethyl benzidine substrate (TMB) for five minutes and reaction were stopped with H.sub.2SO.sub.4, 2N. Absorbance values were determined at 450 nm with Spectramax i3® automated microplate reader (Molecular Devices, Sunnydale, USA). Wells were given four rinses with PBST between each incubations. Working volumes were 100 μL per well, and control wells were incubated with culture medium. All incubation were performed at 37° C. Threshold for detection of antibody in ELISA were determined from negative control means.

(15) Determination of Ig Subclass and Cloning Procedure.

(16) The Ig class of mAbs was determined with a commercial mouse mAb isotyping kit (ISO-1) according to the manufacturer's instructions (Sigma). All antibody that was developed for this project have been IgM. Hybridoma cells lines were cloned by limiting dilution, and cell lines Were grown in bulk in a non-selective medium, preserved by slowly freezing in fetal bovine serum/dimethyl sulfoxide (92:8 [vol/vol]), and stored in liquid nitrogen.

(17) Antibody Precipitation.

(18) Selected hybridomas were cultivated in RPMI-1640 supplemented of Fecal Calf Serum (FCS) 10%. After 2-weeks cultivation in incubator 37° C., 5% CO2 and 95% of humidity, supernatant were dialysis against water in order to precipitate IgM three times. After centrifugation of 2000 g 30 minutes at +4° C., pellet were resuspended in phosphate buffer 20 mM pH8.00 supplemented with 1 M NaCl. Then dialysis against PBS three times. Concentrations of precipitated antibody were calculated using absorbance at 280 nm with Spectradrop® automated microplate reader (Molecular Devices, Sunnydale, USA).

(19) Antibody Specificity Determination. Synthesis of Antigenic Protein.

(20) For specificity validation of each selected hybridoma, antigenic proteins that mimic the isopeptide (GGEL) were synthetized: GGEL were coupled on bovine serum albumin (BSA) using glutaraldehyde as crosslinking agent (GGEL-BSA). To that end, a solution of GGEL/NaOH 1 M 0.36 mg/ml, 151 nM of BSA and 2.5% of glutaraldehyde (10 mg/ml) were mixed and incubated overnight. Dialysis was performed against PBS three times. N-Alpha-Cbz-L-Lysine Methyl Ester (Z-GluOme) were coupled to BSA (BZGO) using (EDC) and N-hydroxysuccinimide (NHS) as crosslinking agent (LysOMe-BSA). N-alpha-Cbz-Glutamic acid Methyl Ester coupled to BSA (BZGO); GGEL isopeptide bounds were created between Z-GluOme and BSA lysines' using N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and NHS. EDC 490 μmol/L/NHS 524 μmol/L and Z-GluOme 295 mmol/L were diluted in dimethylformamide (DMF) and incubated 15 min at room temperature. BSA 2 mg/ml diluted in a 0.1 M phosphate buffer pH 8.00 was added and incubated overnight. Dialysis was performed against PBS, three times. N-alpha-Cbz-Glutamic acid-Tert-Butyl Ester coupled to BSA (GluOter-BSA); GGEL isopeptide bounds were created between Z-GluOter and BSA lysines' using N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and NHS. EDC 490 μmol/L/NHS 524 μmol/L and Z-GluOter 295 mmol/L were diluted in dimethylformamide (DMF) and incubated 15 min at room temperature. BSA 2 mg/ml diluted in a 0.1 M phosphate buffer pH 8.00 was added and incubated overnight. Dialysis was performed against PBS, three times.

(21) Antigenic proteins that mimic unwanted isopeptide (alpha-Glycine-L-espilon-Lysine, ubiquitin isopeptide), acetylation of Lysine or polyamine crosslink were synthetized: N-Alpha-Boc-Glycine were coupled to BSA using EDC and NHS as coupling agent (Boc-Gly-BSA), Spermidine was coupled to poly-L-Glutamic using EDC/NHS (Spd-pGlu), BSA was treated with anhydride acetic to for acetylation on primary amine of the protein (Lysine) (BSA-Ac).

(22) In order to evaluate the quantity of GGEL isopeptide created on BSA, a quantification of primary amine with 2,4,6-trinitrobenzene sulfonic acid (TNBS) test was performed. Proteins were diluted at 100 μg/mL in a 0.1 mol/L bicarbonate solution supplemented with 0.01% TNBS. After 2 h of incubation at 37° C., reaction was stopped with hydrogen chloride (HCl) 1N. Optical densities were read at 335 nm with Spectramax i3® automated microplate reader (Molecular Devices, Sunnydale, USA).

(23) TABLE-US-00003 TABLE 2 Antigenic proteins used for specificity determination that mimic GGEL isopeptide. GGEL on protein BSA GGEL coupled with glutaraldehyde (GGEL-BSA) GGEL formed by coupling Cbz-Lys-Ome to BSA (ZLysOme-BSA) GGEL formed by coupling Cbz-Glu-Oter to BSA (ZGluOter-BSA)

(24) TABLE-US-00004 TABLE 3 Antigenic proteins used for specificity determination as negative control. Spd coupled to polyGlutamic acid bis- (poly(glutamic acid))- spermidine mimic polyamine crosslink bis-Glu-Spd Boc-Glycine coupled to BSA (Boc-Gly-BSA) mimic Ubiquitinylation/Sumolytation crosslink Acetylated BSA (BSA-Ac) mimic acetylation of lysine

(25) Antibody Specificity Determination. Competition ELISA.

(26) Selected clones were tested using a competitive ELISA test in order to determine the specificity of the clones. Therefore BSA-GGEL was adsorbed on microtiter plate at a concentration of 10 μg/mL in a bicarbonate buffer pH 9.50. A concentration of purified antibody (1G1h1 at 2 μg/mL or AB424 at 0.5 μg/mL) was incubated with each competitor antigenic protein of Tables 2 and 3, diluted in cascade two by two with a starting concentration of 5 μg/mL. After 1 hour of incubation at 37° C., the microtiter plate was washed and a goat anti-mouse (H+L) peroxidase conjugate was added for 30 minutes at 37° C. Revelations were performed using TMB for 5 minutes and reaction were stopped using H.sub.2SO.sub.4, 2N. Absorbance values were determined at 450 nm with Spectramax i3® automated microplate reader (Molecular Devices, Sunnydale, USA). Wells were given four rinses with PBST between each incubation. Working volumes were 100 μL per well, and control wells were incubated with culture medium. The comparison of each antigenic protein were performed by calculated the inhibitory concentration of 50% of the initial signal (without any competitor).

(27) Results

(28) A so-called 1G1h1 anti-GGEL monoclonal antibody comprising a variable domain of heavy chain of sequence

(29) TABLE-US-00005 (SEQ ID NO: 1) QVQLQQPGTELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGN INPSNGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSG LLLWSPWFAYWGQGTLVTVS,
and a variable domain of light chain of sequence

(30) TABLE-US-00006 (SEQ ID NO: 2) DIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVY NAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPFT FGSGTKLEIKR
was isolated. CDRs, as identified according to the IMGT definition, are represented in bold and underlined characters.

(31) Specificity of the isolated 1G1h1 antibody was determined and compared to the previously characterized commercial AB424 antibody (Thomas et al. 2004, J. Immunol. Methods 292, 83-95).

(32) It was determined that both 1G1h1 and AB424 antibodies detect D-dimer, with a non-significant difference in their titers (2.5 μg/mL for 1 G1 h1 and 0.6 μg/mL for AB424).

(33) Specificity of 1G1h1 and AB424 antibodies was further determined using antigenic proteins described in Tables 2 and 3 in a competitive ELISA against GGEL. The results are shown in Table 4.

(34) An IC.sub.50 is considered as significantly different from another IC.sub.50 when one log of difference is observed between the two IC.sub.50.

(35) TABLE-US-00007 TABLE 4 Specificity of anti-GGEL antibodies 1G1h1 and AB424 IC.sub.50 in nM GGEL like proteins Negative controls GGEL- ZGluOter- ZLysOme- Spd- BSA- Boc-Gly- mAb BSA BSA BSA pGlu Ac BSA 1G1h1 1.09 3.89 3.47 >25000 >45.3 >45.3 AB424 8.68 >400 4.89 2700 >45.3 >45.3

(36) IC.sub.50 of each antigenic protein was evaluated, and for 1G1h1 one log of decrease was observed between GGEL-like proteins (Table 2) and negative control proteins (Table 3).

(37) It was concluded that the 1G1h1 antibody is specific for GGEL-BSA, ZGluOter-BSA and ZLysOme-BSA antigenic proteins, therefore that the antibody is specific for the GGEL isopeptide versus other crosslinked or modified lysines.

(38) Conversely, the AB424 antibody cross-reacts with the isopeptide N.sup.1,N.sup.8bis(gamma-glutamyl) spermidine but does not bind the GGEL competitor antigen ZGluOter-BSA.

(39) Accordingly, that 1G1h1 antibody has enhanced specificity for GGEL compared to the commercial AB424 antibody.

Example 2: Quantification of Gamma-Glutamyl-L-Epsilon-Lysine (GGEL) with the 1G1h1 Monoclonal Antibody

(40) Materials & Methods

(41) GGEL Quantification by Competitive ELISA. Competitive ELISA.

(42) BSA-GGEL was adsorbed on microtiter plate at a concentration of 10 μg/mL in a 50 mM bicarbonate solution pH 9.50. Plates were incubated overnight at laboratory temperature. Saturation was performed with a phosphate buffer 0.1 M supplemented of BSA 0.5% and sucrose 5% (FIG. 3, step 1). Samples diluted were added in presence of antibody solution for 1 hour at 37° C., BZGO with a precise number of GGEL “coated” on were diluted two by two and used as standard (FIG. 3, step 2 and 3). After three washes with PBST, secondary antibody diluted 1 in 2000 PBST was incubated 30 minutes at 37° C. Revelation were performed using TMB for 5 minutes and reaction were stopped using H.sub.2SO.sub.4, 2N (FIG. 3, step 4). Absorbance values were determined at 450 nm with Spectramax i3® automated microplate reader (Molecular Devices, Sunnydale, USA). GGEL quantification was performed using standard plotted on a 4 parameters line using GrapPad Prism version 5.0 (GraphPad software, San Diego, USA). The threshold for positivity of results was determined considering the mean of the blank added to 3.33 standard deviation.

(43) Results

(44) The 1G1h1 antibody was used for GGEL quantification by a competitive ELISA Standardized N-alpha-Cbz-Glutamic acid Methyl Ester (Z-GluOme) coupled to BSA (BZGO) with known amounts of GGEL were used as standard for the quantification of GGEL. FIG. 2 displays the standard curve for quantification of GGEL concentration, which was calculated using 4-Parameter line using SoftMax Pro 6.5.

(45) Curve fit using 4-Parameter line was as follows:

(46) $y=D+\frac{A-D}{1+{\left(\frac{x}{C}\right)}^B}$

(47) TABLE-US-00008 TABLE 5 GGEL standard curve parameters Estimated Std. Parameter value error Confidence interval Standard curve A 0.840 0.047 [0.691; 0.989] R.sup.2 = 0.998 B 1.193 0.180 [0.621; 1.766] EC.sub.50 = 3.274 C 3.274 0.356 [2.142; 4.406] D 0.079 0.028 [−0.011; 0.170] 

(48) This anti-GGEL 1G1h1 monoclonal antibody was further used in the following experiments.

Example 3: In Vitro Detection of Free GGEL Released by Apoptotic Cells

(49) Materials & Methods

(50) Cell Lines.

(51) Cell line used for apoptosis induction was Human promyelocytic leukemia cells, HL-60. HL-60 were cultured in RPMI media supplemented with 10% Fetal Calf Serum (FCS), 1% Streptomycin/Penicillin and 200 mM L-Glutamine.

(52) Induction of apoptosis.

(53) Cells were plated at 1×10E6 cells/mL in cell culture medium described before. Induction of apoptosis were done using staurosporine at a concentration of 1 μmol/L during 8 hours. Staurosporine is an alkaloid isolated from the culture broth of Streptomyces staurosporesa. It is a potent, cell permeable protein kinase C inhibitor and other kinases such as PKA, PKG, CAMKII and Myosin light chain kinase (MLCK). At 0.2-1 μM, staurosporine induces cell apoptosis. After treatment, cells were then centrifuged at 800 g, 10 min and supernatant were used for GGEL quantification.

(54) GGEL Quantification

(55) GGEL quantification by competitive ELISA. Competitive ELISA. BSA-GGEL were adsorbed on microtiter plate at a concentration of 10 μg/mL in a 50 mM bicarbonate solution pH9.50. Plates were incubated overnight at laboratory temperature. Saturation was perform with a phosphate buffer 0.1 M supplemented of BSA 0.5% and sucrose 5% (FIG. 3, step 1). Cells supernatant were added in presence of antibody solution for 1 hour at 37° C., BZGO with a precise number of GGEL “coated” on were diluted two by two and used as standard (FIG. 3, step 2 and 3). After three washes with PBST, secondary antibody diluted 1 in 2000 PBST was incubated 30 minutes at 37° C. Revelation were performed using TMB for 5 minutes and reaction were stopped using H2SO4, 2N (FIG. 3, step 4). Absorbance values were determined at 450 nm with Spectramax i3® automated microplate reader (Molecular Devices, Sunnydale, USA). GGEL quantification was performed using standard plotted on a 4 parameters line using GrapPad Prism version 5.0 (GraphPad software, San Diego, USA).

(56) Statistical Analysis.

(57) Values are expressed as mean±SD or frequencies and proportions. Differences between groups were determined by unpaired t test, Chi-square, Fisher's exact test or ANOVA, where appropriate. P<0.05 was considered statistically significant. Analysis was performed using GraphPad prism version 5.0 (GraphPad software, San Diego Calif. USA).

(58) Results

(59) FIG. 3 shows the results of GGEL quantification with the 1G1h1 antibody in HL-60 cells after induction of apoptosis by staurosporin, or without staurosporin treatment. GGEL concentration in HL-60 cells induced into apoptosis was significantly different from HL-60 cells non-induced into apoptosis.

Example 4: Identification of Gamma-Glutamyl-L-Epsilon-Lysine (GGEL) as a Biomarker of Early Sepsis

(60) Materials & Methods

(61) The inventors have sought to determine whether GGEL in plasma can be measured in an Enzyme Immuno Assay (EIA). The protocol was based on a GGEL quantification competitive EIA (see example 2 & 3) with plasma samples diluted at 1/20. Control plasma samples from 10 healthy individuals were used to determine the background level of GGEL in normal plasma. The tests were performed on 22 sepsis samples, obtained at day 1 and day 3 from 11 patients. Day 1 is the day on which the patient was hospitalized and was detected with fever and a symptomatic feature of SIRS (see table 1). In order to distinguish positive from negative tests, a cut-off value was calculated from the 10 control plasma samples.

(62) Comparison of each group was evaluated using ANOVA 1-way.

(63) ROC curve Analysis was performed using GraphPad prism version 5.0 (GraphPad software, San Diego Calif. USA). Blank plasma was designated as controls and sepsis plasmas as patients.

(64) Results

(65) Using this test, it was demonstrated that sepsis plasma can be discriminated from blank plasma as a significant enhancement of GGEL concentration was observed for group D+1 and D+3 in comparison to blank samples (FIG. 4). Accordingly, GGEL presence is an effective biomarker of early sepsis.

(66) However, day 1 plasma may not be discriminated from day 3 samples. Yet by analyzing the results for each patient, an increase of GGEL biomarker from day 1 to day 3 has been detected and observed for 8 patients out of 11.

(67) The marker diagnostic performance could be characterised by sensitivity, which represents its ability to detect the sepsis population, and specificity, which represents its ability to detect the control population.

(68) Blank plasma were used in order to determinate the threshold of the test (V=μ×3 SD). Specificity and sensitivity were obtained resulting in a specificity of 91% for D+1 group, 100% for D+3 group and 95.45% for the merged group (D+1+D+3) (Table 6). A ROC curve of the test was further obtained (FIG. 5 and Table 7).

(69) TABLE-US-00009 TABLE 6 Specificity of GGEL quantitation for sepsis diagnosis True False True False positive positive negative negative Group (TP) (FP) (TN) (FN) specificity D1 10 0 0 1  91% D3 11 0 0 0 100% D1 + D3 21 0 0 1 95.45%.sup. 

(70) TABLE-US-00010 TABLE 7 Diagnosis potential (mROC approach) of GGEL for sepsis diagnosis Area under the ROC curve 0.9773 Std. Error 0.02730 99% confidence interval 0.9069 to 1.048 P value 0.002851

Example 5: Dosage of Gamma-Glutamyl-L-Epsilon-Lysine (GGEL) in Different Pathologies

(71) Materials and Methods

(72) Samples

(73) Plasma were obtained from the Centre de Ressources Biologiques from Hôpital Lariboisière, Paris, France. 364 plasmas sample were obtained representing 4 different clinical pathology: heart/respiratory failure (HRF) (n=106), Trauma (n=90), Septic shock (n=130) or Severe Sepsis (n=38), at two different time points, T1 which were collected at the entry in hospital, and T2 for the coming out of the hospital.

(74) GGEL Quantification by Competitive ELISA. Competitive ELISA

(75) Antigen BSA-GGEL was adsorbed on a microtiter plate with a 50 mM bicarbonate solution pH 9.50. Plates were incubated overnight at room temperature. Saturation was performed with a phosphate buffer 0.1M supplemented with BSA 0.5% and sucrose 5%. Diluted samples were added in presence of the anti-GGEL antibody solution for 1 hour at 37° C., BZGO with a precise number of GGEL “coated” on were diluted two by two and used as standard. After three washes with PBST, revelation was performed using TMB for 10 minutes and reaction was stopped using H.sub.2SO.sub.4, 2N. Absorbance values were determined at 450 nm with Spectramax i3® automated microplate reader (Molecular Devices, Sunnydale, Calif., USA). GGEL quantification was performed using standard plotted on a semi-log line using SoftMaxPro 6.5.1 (Molecular Devices, Sunnydale, USA).

(76) Statistical Analysis

(77) Variables were analyzed by descriptive statistics to evaluate the clinical characteristics of the cases. All are reported as mean±S.D, and were analyzed using one-way analysis of variance to estimate the presence of any statistical difference between the studied samples. Two-tailed probability values are listed in the table and the level of statistical significance was set up at p<0.05. All statistical analyses were performed with GraphPad Prism version 5.0 (GraphPad software, San Diego, Calif., USA) or JMP software version 12 (SAS Institute Inc. Cary, N.C., USA).

(78) Results

(79) Comparison of GGEL Concentration Between Different Pathologies

(80) The level of GGEL concentration in plasma was significantly higher in Trauma group than in the others (p<0.01). A higher level of GGEL concentration than in Normal samples (22 μmol.Math.L.sup.−1) was observed in Septic Shock (39.29 μmol.Math.L.sup.−1) and Severe Sepsis samples (28.47 μmol.Math.L.sup.−1) but results were not significant (FIG. 7).

(81) Comparison of GGEL Concentration Between the Different Time Point by Pathologies

(82) The level of GGEL significantly increase from T1 to T2 in Trauma from a T1-mean of 45.61±19.72 to 111.14±24 μmol.Math.L.sup.−1 (FIG. 8). For the other pathologies, a non-significant increase of GGEL concentration in plasma was observed.

(83) Comparison a GGEL Concentration Per Patient by Pathologies

(84) For each patient plasma, the modification of GGEL concentration was calculated as follow ΔGGEL=[GGEL].sub.T2−[GGEL].sub.T1. The mean of ΔGGEL per pathologies were analyzed with result for Trauma and Septic shock respectively 38.75±22.78 and 21.58±10.2 μmol.Math.L.sup.−1, higher than HRF and Severe sepsis (Table 8). However non-significant results were noticed between the different pathologies.

(85) TABLE-US-00011 TABLE 8 ΔGGEL concentration per patient by pathologies Pathologies Mean (μmol .Math. L.sup.−1) Standard Deviation (μmol .Math. L.sup.−1) HRF 3.5325 6.451 Septic Shock 21.5827 10.021 Severe Sepsis 9.7431 7.899 Trauma 38.7504 22.782