IMMUNOASSAY FOR THE DETECTION OF PROCALCITONIN

Abstract

The present invention relates to an in vitro method for the detection of Procalcitonin or a fragment thereof of at least 20 amino acid residues in length in a biological sample derived from a bodily fluid obtained from a subject, comprising the steps of: (i) contacting said sample with at least two antibodies or functional fragments thereof directed against different epitopes within Procalcitonin, and (ii) qualitatively or quantitatively detecting binding of said at least two antibodies to Procalcitonin or said fragment thereof, wherein binding indicates the presence or concentration of Procalcitonin or said fragment in said sample, wherein at least one antibody or functional fragment thereof is directed against an epitope comprised in the sequence spanning amino acid residues 2 to 52 of Procalcitonin. The invention also pertains to antibodies directed against an N-terminal epitope of Procalcitonin and kits comprising antibodies directed against PCT.

Claims

1. In vitro method for the detection of Procalcitonin or a fragment thereof of at least 20 amino acid residues in length in a biological sample derived from a bodily fluid obtained from a subject, comprising the steps of: a. contacting said sample with at least two antibodies or functional fragments thereof directed against different epitopes within Procalcitonin, and b. qualitatively or quantitatively detecting binding of said at least two antibodies to Procalcitonin or said fragment thereof, wherein binding indicates the presence or concentration of Procalcitonin or said fragment in said sample, wherein at least one antibody or functional fragment thereof is directed against an epitope comprised in the sequence spanning amino acid residues 2 to 52 of Procalcitonin.

2. Method according to claim 1, wherein the antibody or functional fragment thereof, which is directed against an epitope comprised in the sequence spanning amino acid residues 2 to 52 of Procalcitonin, is a monoclonal antibody.

3. The method according to claim 1, wherein one other antibody or functional fragment thereof is directed against an epitope comprised in the sequence spanning amino acid residues 53 to 116 of Procalcitonin.

4. The method according claim 1, wherein the epitope comprised in the sequence spanning amino acid residues 2 to 52 of Procalcitonin is an epitope comprised in the sequence spanning amino acid residues 16 to 40 of Procalcitonin.

5. The method according to claim 4, wherein the epitope comprised in the sequence spanning amino acid residues 16 to 40 of Procalcitonin is selected from a group consisting of an epitope comprised in the sequence spanning amino acid residues 21 to 40 of Procalcitonin, an epitope comprised in the sequence spanning amino acid residues 16 to 35 of Procalcitonin and an epitope comprised in the sequence spanning amino acid residues 25 to 37 of Procalcitonin.

6. The method according to claim 3, wherein the epitope comprised in the sequence spanning amino acid residues 53 to 116 of Procalcitonin is an epitope comprised in the sequence spanning amino acid residues 96 to 116 of Procalcitonin or an epitope comprised in the sequence spanning amino acid residues 60 to 91 of Procalcitonin.

7. The method according to claim 1, wherein the antibody or functional fragment thereof, which is directed against an epitope comprised in the sequence spanning amino acid residues 53 to 116 of Procalcitonin, is a monoclonal antibody.

8. Antibody or a functional fragment thereof directed against an epitope comprised in the sequence spanning amino acid residues 16 to 40 of Procalcitonin.

9. Antibody or a functional fragment thereof according to claim 8, wherein the antibody or functional fragment thereof is directed against an epitope is selected from a group consisting of an epitope comprised in the sequence spanning amino acid residues 21 to 40 of Procalcitonin, an epitope comprised in the sequence spanning amino acid residues 16 to 35 of Procalcitonin and an epitope comprised in the sequence spanning amino acid residues 25 to 37 of Procalcitonin.

10. Antibody according to claim 8, wherein the antibody is monoclonal.

11. Antibody according to claim 10, wherein the antibody is produced by a hybridoma cell line that is deposited at the DSMZ under accession number DSM ACC2993, DSM ACC2996 or DSM ACC2997.

12. Kit comprising at least a. a first antibody or a functional fragment thereof directed against an epitope comprised in the sequence spanning amino acid residues 2 to 52 of Procalcitonin, and b. a second antibody or a functional fragment thereof directed against an epitope comprised in the sequence spanning amino acid residues 53 to 116 of Procalcitonin.

13. Kit according to claim 12, wherein the first antibody is directed against an epitope comprised in the sequence spanning amino acid residues 16 to 40 of Procalcitonin, preferably against an epitope that is selected from a group consisting of an epitope comprised in the sequence spanning amino acid residues 21 to 40 of Procalcitonin, an epitope comprised in the sequence spanning amino acid residues 16 to 35 of Procalcitonin and an epitope comprised in the sequence spanning amino acid residues 25 to 37 of Procalcitonin.

14. Kit according to claim 12, wherein the second antibody is directed against an epitope comprised in the sequence spanning amino acid residues 60 to 91 of Procalcitonin or directed against an epitope comprised in the sequence spanning amino acid residues 96 to 116 of Procalcitonin.

15. A method of performing a sandwich immunoassay for the detection and or quantification of Procalcitonin in a biological sample from a bodily fluid comprising employing a kit of claim 12.

16. The method according to claim 1 for the determination of the presence or absence of Procalcitonin or a fragment thereof or for the quantification of Procalcitonin or a fragment thereof in a biological sample from a bodily fluid.

17. The method according to claim 16 for the diagnosis, prognosis, risk stratification, therapy monitoring, therapy guidance, or stratification for application of therapeutic measures of a disease or condition associated with elevated Procalcitonin levels.

18. The method according to claim 17, wherein the disease or condition is selected from the group of local bacterial infections, sepsis, severe sepsis, septic shock, non-infectious disease including cardiovascular diseases (acute coronary syndrome, heart failure, coronary artery disease, atherosclerosis, stroke), cancer, diabetes, chronic gastrointestinal diseases, chronic renal diseases, hypertension, orthopaedic diseases including osteoporosis, and neurodegenerative diseases including Alzheimer's disease.

19. The hybridoma cell line deposited at the DSMZ under accession number DSM ACC2993, DSM ACC2996 or DSM ACC2997.

Description

DESCRIPTION OF DRAWINGS

[0050] FIG. 1: Schematic representation of assays (C, D and E) used in comparison to existing assays (A and B: B RAHMS PCT LIA and B RAHMS PCT sensitive LIA, respectively). PCT with its calcitonin and katacalcin moieties is depicted, and antibodies with their epitopes are shown. A and B: One antibody is directed against the calcitonin moiety and the other antibody is directed against the katacalcin moiety of PCT; C: Assay, wherein one antibody is directed against an epitope in the sequence spanning amino acid residues 21-40 of PCT and the other antibody is directed against the katacalcin moiety of PCT. D, E: Assay, wherein one antibody is directed against an epitope in the sequence spanning amino acid residues 21-40 of PCT and the other antibody is directed against the calcitonin moiety of PCT.

[0051] FIG. 2: PCT immunoreactivity profiles of size-fractionated PCT containing sera. Fractions were measured in Assays A (designation as in FIG. 1), and measured values were related to the maximal measured value for each assay within each fractionation run. Shown are the means+standard error (SEM).

[0052] FIG. 3: PCT immunoreactivity profiles of size-fractionated PCT containing sera. Fractions were measured in Assays C and D (Panels A and B, respectively; designations as in FIG. 1), and measured values were related to the maximal measured value for each assay within each fractionation run. Shown are the means+standard error (SEM).

[0053] FIG. 4: Dose response curves for three PCT sandwich immunoassays. The assays were incubated for 30 minutes (panel A) or 2 hours (panel B). PCT LIA and PCT LIA sens. correspond to BRAHMS PCT LIA and BRAHMS PCT sensitive LIA, respectively (designated A and B in FIG. 1). FX1G5/anti-Calc. represents assay Assay E.

[0054] FIG. 5: Amino acid sequence of Procalcitonin (PCT) (SEQ ID NO:1)

EXAMPLES

Example 1

[0055] Material and Methods

[0056] A. Development of Monoclonal Antibodies

[0057] Monoclonal antibodies against PCT were generated by genetic immunization following principally a described procedure (Costagliola et al., J Immunol 1998; 160:1458-65). In brief, the PCT coding sequence was cloned by standard procedures in vector pcDNAIII (Invitrogen, Karlsruhe, Germany). BALB/c mice were injected in the anterior tibialis muscle on day 0 with 100 mg of pcDNAIII-PCT in 25% sucrose. Injections were repeated 3 and 6 wk thereafter. Blood samples were obtained from retro-ocular capillaries 8 and 11 wk after the initial immunization and at sacrifice, which was after 18 wk, when the spleens and thyroids were also removed. Spleen cells were fused with SP2/0 myeloma cells to generate hybridoma cell lines. Cell lines were screened for their ability to secrete antibodies that would bind to immobilized recombinant human PCT (InVivo GmbH, Hennigsdorf, Germany). With this approach, cell lines secreting monoclonal antibodies FX7A7 (produced by the hybridoma cell line deposited on Jun. 4, 2009 at the DSMZ under accession number DSM ACC2997), FW5H6 (produced by the hybridoma cell line deposited on Jun. 4, 2009 at the DSMZ under accession number DSM ACC2996) and FX1G5 (produced by the hybridoma cell line deposited on Apr. 29, 2009 at the DSMZ under accession number DSM ACC2993) were generated.

[0058] B. Epitope Mapping

[0059] The mapping of epitopes within PCT of the three monoclonal antibodies FX7A7, FW5H6 and FX1G5 was done on peptide microarrays by standard procedures (JPT GmbH, Berlin, Germany). The peptide microarray was composed of 74 peptides displayed as overlapping peptide scans (format 13/11: 53 peptides; format 20/15: 21 peptides) and thus covering the entire PCT sequence on a glass surface. The microarrays were pre-treated with blocking buffer (Pierce, Superblock; 2 h at room temperature) followed by washings with TBS buffer pH 8 and water (3 times each). Each pre-treated microarray was scanned using Axon Genepix 4000B Scanner for background control (no signals could be detected). Individual microarrays were incubated with antibodies in assay buffer (final concentration 60 g/mL in Pierce Superblock buffer; total assay volume 350 L, incubation time 3 h). Microarrays were washed with TBS buffer pH 8 followed by an incubation with fluorescence labelled secondary antibody (anti-mouse-Dylight-647; Pierce 31015, 1 g/mL, incubation time 45 min). Control incubation with fluorescence labelled secondary antibody (anti-mouse-Dylight-647; Pierce 31015, 1 g/mL, incubation time 45 min) were performed in parallel to the described experiment. Microarrays were scanned using Axon Genepix 4000B Scanner with appropriate wavelength settings. SPOT recognition software package ArrayPro was used for data analysis. Mean of signal intensities (corrected for local background) from 3 identical subarrays on each microarray image were used for data evaluation.

[0060] C. Immunoassays

[0061] Sandwich immunoassays in the chemiluminesce-/coated tube format were set up as follows: Assay A: A commercially available sandwich assay for PCT was used (BRAHMS PCT LIA sensitive), which uses one antibody directed against the katacalcin moiety of PCT as solid phase, and one antibody directed against the calcitonin moiety of PCT as labeled antibody (BRAHMS AG, Hennigsdorf, Germany). Recombinant PCT in various concentrations is used as standards. For the comparison with Assay E (see below), incubation conditions were adapted to those described for Assay E; i.e. 50 l sample and 200 l labeled antibody solution were used and incubated in a one step reaction in test tubes for 30 minutes or 2 hours.

[0062] Assay B:

[0063] A commercially available sandwich assay for PCT was used (BRAHMS PCT LIA), which uses one antibody directed against the katacalcin moiety of PCT as solid phase, and one monoclonal antibody directed against the calcitonin moiety of PCT as labeled antibody (BRAHMS AG, Hennigsdorf, Germany). Recombinant PCT in various concentrations is used as standards. For the comparison with Assay E (see below), incubation conditions were adapted to those described for Assay E; i.e. 50 l sample and 200 l labeled antibody solution were used and incubated in a one step reaction in test tubes for 30 minutes or 2 hours.

[0064] For the other assays, assay components were generated as follows:

[0065] Labeling of Antibodies

[0066] Labeling of antibody FX1G5 was done by standard procedures (EP 1488209, EP 1738178): The concentration of the purified antibody was adjusted to 1 g/L, and the antibody was labeled by incubation with the chemiluminescent label MACN-Acridinium-NHS-Ester (1 g/L; InVent GmbH, Hennigsdorf, Germany) in a 1:5 molar ratio for 20 min at room temperature. The reaction was stopped by addition of 1/10 volume of 50 mmol/L glycine for 10 min at room temperature. Labeled antibody was separated from free label by size-exclusion chromatography on a NAP-5 column (GE Healthcare, Freiburg, Germany) and a Bio-Sil SEC-400-5 HPLC column (BIO-RAD).

[0067] Coating of Antibodies

[0068] Coating of a monoclonal antibody directed against the calcitonin moiety of PCT (BRAHMS AG, Hennigsdorf, Germany) was done by standard procedures (EP 1488209, EP 1738178): Polystyrene startubes (Greiner) were coated with purified antibody (per tube, 2 g of antibody in 300 L of 10 mmol/L Tris, 100 mmol/L NaCl, pH 7.8) overnight at 22 C. Tubes were then blocked with 10 mmol/L sodium phosphate (pH 6.5) containing 30 g/L Karion FP (Merck), 5 g/L bovine serum albumin protease free (Sigma) and lyophilized

[0069] With these components the following assays were set up:

[0070] Assay C:

[0071] Tubes coated with an anti-katacalcin antibody and standards (recombinant PCT) were taken from the assay B.R.A.H.M.S PCT LIA sensitive (B.R.A.H.M.S AG, Hennigsdorf, Germany). MACN labeled antibody FX1G5 was used as labeled antibody. The assay buffer was 300 mmol/L potassium phosphate, pH 7.0, 100 mmol/L NaCl, 10 mmol/L EDTA, 0.9 g/L sodium azide, 5 g/L bovine serum albumin protease free (Sigma), 1 g/L nonspecfic bovine IgG, 1 g/L nonspecific sheep IgG, 1 g/L nonspecific mouse IgG and contained 210.sup.6 relative light units (RLU) of MACN-labeled antibody per 200 l. 100 l standards or samples and 200 l assay buffer containing the MACN-labeled antibody were pipetted in the coated tubes. Tubes were incubated 2 hours at 22 C. under agitation. Then, the tubes were washed 5 times with 1 mL of B.R.A.H.M.S washing solution (B.R.A.H.M.S AG, Hennigsdorf, Germany) and bound chemiluminescence was measured for 1 s per tube with a LB952T luminometer (Berthold). Concentrations of samples were calculated using the Software MultiCalc (Spline Fit).

[0072] Assay D:

[0073] Tubes coated with an anti-calcitonin antibody were used. Standards (recombinant PCT) were taken from the assay BRAHMS PCT LIA sensitive (BRAHMS AG, Hennigsdorf, Germany) MACN labeled antibody FX1G5 was used as labeled antibody. The assay buffer was 300 mmol/L potassium phosphate, pH 7.0, 100 mmol/L NaCl, 10 mmol/L EDTA, 0.9 g/L sodium azide, 5 g/L bovine serum albumin protease free (Sigma), 1 g/L nonspecfic bovine IgG, 1 g/L nonspecific sheep IgG, 1 g/L nonspecific mouse IgG and contained 210.sup.6 relative light units (RLU) of MACN-labeled antibody per 200 l. 100 l standards or samples and 200 l assay buffer containing the MACN-labeled antibody were pipetted in the coated tubes. Tubes were incubated 2 hours at 22 C. under agitation. Then, the tubes were washed 5 times with 1 mL of B.R.A.H.M.S washing solution (B.R.A.H.M.S AG, Hennigsdorf, Germany) and bound chemiluminescence was measured for 1 s per tube with a LB952T luminometer (Berthold). Concentrations of samples were calculated using the Software MultiCalc (Spline Fit).

[0074] Assay E:

[0075] Tubes coated with FX1G5 antibody were used. Standards (recombinant PCT) and labeled polyclonal anti-Calcitonin antibody were taken from the assay BRAHMS PCT LIA sensitive (BRAHMS AG, Hennigsdorf, Germany) 50 l standards or samples and 200 l assay buffer containing the MACN-labeled antibody were pipetted in the coated tubes. Tubes were incubated for either 30 minutes or 2 hours at 22 C. under agitation. Then, the tubes were washed 5 times with 1 mL of B.R.A.H.M.S washing solution (B.R.A.H.M.S AG, Hennigsdorf, Germany) and bound chemiluminescence was measured for 1 s per tube with a LB952T luminometer (Berthold).

[0076] D. Size Exclusion Chromatography

[0077] Plasma samples from nine patients with elevated PCT concentrations (including patients with sepsis) were fractionated using a Bio-Sil SEC-125-5 HPLC column (BIO-RAD) HPLC column. The sample volume was 100 The running buffer was PBS pH 7.4. The flow rate was 0.8 mL/min 0.4 mL fractions were collected measured in assays A, C, D. The following peptides were used as calibrators: recombinant PCT (MW=ca. 13 kDa; InVivo GmbH, Hennigsdorf, Germany), preproADM 45-92 (Sequence ELRMSS SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RV; MW=5.1 kDa; JPT GmbH, Berlin, Germany), Vitamin B12 (MW 1.3 kDa). Recombinant PCT and preproADM 45-92 were resolved in standard matrix obtained from the assays BRAHMS PCT LIA sensitive and BRAHMS MR-proADM LIA (BRAHMS AG, Hennigsdorf, Germany), and their elution profile of the size fractionation HPLC was determined using these assays. Vitamin B12 was diluted in running buffer and subjected to chromatography; absorption at 280 nm was recorded.

[0078] E. Measurement of Samples

[0079] Thirty serum samples of patients with local bacterial infections, sepsis, septic shock were measured in assays A, C, D.

[0080] Results

[0081] Monoclonal Antibodies

[0082] Three mouse monoclonal antibodies were generated by genetic immunization employing the entire PCT coding sequence. The epitope mapping revealed similar, albeit not identical results for all three antibodies (Table 2). Antibodies FW5H6 and FX7A7 showed maximal binding to peptide EARLLLAALVQDYVQMKASE (pos. 21-40 within PCT), and for antibody FX1G5 maximum binding was observed on a peptide derived from the previous one, i.e. LLAALVQDYVQMK (pos. 25-37). Outside these regions, no other significant binding sites within the PCT sequence were identified for the three antibodies. The immunization method used here is only one example. Other methods are well known, which could be applied alternatively to generate antibodies against an epitope in the described regions, and more generally upstream from position 53, for instance chemically synthesized peptides conjugated to a carrier protein could be used as antigen.

[0083] Size Exclusion Chromatography

[0084] The apparent molecular weight of native PCT and the detectability with various sandwich immunoassays was assessed by fractionation of serum samples from patients with elevated native PCT concentrations (including sepsis patients) using size exclusion HPLC. Essentially the same immunoreactivity profile was observed, whether fractions were measured with assay A, C or D (FIG. 1): The elution time of native PCT was indistinguishable from that of recombinant PCT (13 kDa) (FIGS. 2 and 3). Virtually no PCT immunoreactivity corresponding to a molecular weight smaller than 13 kDa was detected by any of the three assays. Most notably, no PCT immunoreactivity corresponding to a molecular weight of ca. 6 kDa was detected by Assay A; this would have been expected, if the assumptions in the state of art were correct, that PCT can be split just upstream from the calcitonin moiety of PCT. These results demonstrate that, opposed to speculations in the state of the art, in patients with elevated PCT concentrations (excluding medullary thyroid carcinoma) PCT is not detectably cleaved in the middle of the molecule, and that sandwich immunoassays of the A, C or D-type detect the same antigen.

[0085] Measurement of Samples

[0086] Thirty serum samples of patients with local bacterial infections, sepsis, septic shock were measured in assays A, C, D. The Spearman correlation coefficients came out as follows: Assay A vs. C: r=0.9893; Assay A vs. D: r=0.9844. These ideal correlation coefficients derived from the measurement of a significant number of samples from patients having infections at various degrees of severity clearly confirm the results obtained by size exclusion chromatography so that one has to conclude generally that PCT, when elevated over normal (excluding medullary thyroid carcinoma), is not cleaved in the middle of the molecule.

[0087] Assay Characteristics

[0088] The use of one of the antibodies described in the present invention, FX1G5 having an epitope corresponding to positions 25-37 of PCT, in a sandwich assay employing an anti-Calcitonin antibody as second antibody (Assay E), was analyzed in comparison to state-of-art PCT assays, which utilize the same detection technology (coated tube/chemiluminescence label); i.e. BRAHMS PCT LIA sensitive (Assay A) and BRAHMS PCT LIA (Assay B). Surprisingly, Assay E exhibited considerably more dynamic dose-response-curves than both established assays, independent from the incubation time (FIG. 4).

TABLE-US-00002 TABLE1 Describedanti-PCTantibodiesandtheiruseinimmunoassays Immunogen Epitope (numbersrefer (numbersrefer testedin tested toamino toamino sandwich with acidpositions acidpositions immuno- native Name Source inPCT1-116) inPCT1-116) assay PCT Reference anti- Sheep Calcitonin GTYTQDFNKFH; yes yes (Morgenthaler, Calcitonin 69-79 etal.Clin Chem 2002;48:788- 90) anti- mouse Katacalcin ERDHRPHVSM; yes yes (Morgenthaler, katacalcin 102-111 etal.Clin (QN05) Chem 2002;48:788- 90) PROC1 rat FRSALESSPADPATL n.d. yes no (Kramer,etal. 3G3 SEDE;3-20 AnalBioanal Chem 2008;392:727- 36) PROC4 rat SDLERDHRPHV;99- n.d. yes no (Kramer,etal. 6C6etc 109 AnalBioanal Chem 2008;392:727- 36) R2B7 rabbit Amino-ProCT; n.d. no yes (Whang,etal.J antiserum 1-57 Clin Endocrinol Metab 1998;83:3296- 301) 295/3H12 mouse APFRLSALESC; n.d.otherthanN- yes yes DE102007 etc. 1-9 terminalAlanin 009751 beingrequired 98-47/44 mouse DSPRSKRCGNLS; n.d. yes yes US6451311 53-64 98-31/04 mouse VGAPGKKRDMSS; n.d. yes yes US6451311 88-99 CT08 mouse Calcitonin TYTQDFN;70- yes yes (Assicot,etal. 76 Lancet 1993;341:515- 8;Ghillaniet al,CancerRes 1989;49:6845- 51) KC01 mouse Katacalcin DMSSDLERDHR; yes yes (Assicot,etal. 96-106 Lancet 1993;341:515- 8;Ghillani,et al.CancerRes 1989;49:6845- 51)

TABLE-US-00003 TABLE2 Epitopemappingresults:Observedbindingsignals forthethreeantibodiestotheshownpeptides representingsubsequencesoftheentirePCT sequencewererelatedtothemaximumbinding obtainedperantibody(B/Bmax). pep- tide # sequence FX1G5 FW5H6 FX7A7 1 APFRSALESSPAD 0.0% 0.0% 0.0% 2 FRSALESSPADPA 0.0% 0.0% 0.0% 3 SALESSPADPATL 0.0% 0.0% 0.0% 4 LESSPADPATLSE 0.0% 0.0% 0.0% 5 SSPADPATLSEDE 0.0% 0.0% 0.0% 6 PADPATLSEDEAR 0.0% 0.0% 0.0% 7 DPATLSEDEARLL 0.0% 0.0% 0.0% 8 ATLSEDEARLLLA 0.0% 0.1% 0.0% 9 LSEDEARLLLAAL 3.0% 0.0% 0.0% 10 EDEARLLLAALVQ 0.3% 0.0% 0.0% 11 EARLLLAALVQDY 1.7% 0.0% 0.0% 12 RLLLAALVQDYVQ 25.0% 57.3% 0.2% 13 LLAALVQDYVQMK 100.0% 59.5% 62.7% 14 AALVQDYVQMKAS 11.9% 14.7% 0.0% 15 LVQDYVQMKASEL 0.0% 0.0% 0.0% 16 QDYVQMKASELEQ 0.0% 0.0% 0.0% 17 YVQMKASELEQEQ 0.0% 0.0% 0.0% 18 QMKASELEQEQER 0.0% 0.0% 0.0% 19 KASELEQEQEREG 0.0% 0.0% 0.0% 20 SELEQEQEREGSS 0.0% 0.0% 0.0% 21 LEQEQEREGSSLD 0.0% 0.1% 0.0% 22 QEQEREGSSLDSP 0.0% 0.0% 0.0% 23 QEREGSSLDSPRS 0.0% 0.0% 0.0% 24 REGSSLDSPRSKR 0.0% 0.1% 0.0% 25 GSSLDSPRSKRCG 0.0% 0.3% 0.1% 26 SLDSPRSKRCGNL 0.0% 0.2% 0.3% 27 DSPRSKRCGNLST 0.0% 0.0% 0.2% 28 PRSKRCGNLSTCM 0.0% 0.0% 0.2% 29 SKRCGNLSTCMLG 0.0% 0.0% 0.0% 30 RCGNLSTCMLGTY 0.0% 0.0% 0.2% 31 GNLSTCMLGTYTQ 0.1% 0.0% 0.0% 32 LSTCMLGTYTQDF 0.0% 0.0% 0.0% 33 TCMLGTYTQDFNK 0.0% 0.0% 0.0% 34 MLGTYTQDFNKFH 0.0% 3.4% 0.0% 35 GTYTQDFNKFHTF 0.0% 1.9% 0.0% 36 YTQDFNKFHTFPQ 0.0% 0.1% 0.0% 37 QDFNKFHTFPQTA 0.4% 0.0% 0.0% 38 FNKFHTFPQTAIG 0.0% 0.1% 0.0% 39 KFHTFPQTAIGVG 0.2% 0.0% 0.0% 40 HTFPQTAIGVGAP 0.0% 0.0% 0.0% 41 FPQTAIGVGAPGK 0.1% 0.0% 0.0% 42 QTAIGVGAPGKKR 1.0% 0.1% 0.1% 43 AIGVGAPGKKRDM 0.0% 0.0% 0.0% 44 GVGAPGKKRDMSS 0.0% 0.0% 0.0% 45 GAPGKKRDMSSDL 0.0% 0.6% 0.0% 46 PGKKRDMSSDLER 0.0% 0.3% 0.1% 47 KKRDMSSDLERDH 0.0% 0.0% 0.0% 48 RDMSSDLERDHRP 0.0% 1.5% 0.0% 49 MSSDLERDHRPHV 1.8% 1.5% 1.9% 50 SDLERDHRPHVSM 0.4% 1.5% 0.9% 51 LERDHRPHVSMPQ 1.3% 1.5% 2.8% 52 RDHRPHVSMPQNA 0.0% 0.1% 0.2% 53 DHRPHVSMPQNAN 0.0% 0.0% 0.0% 54 APFRSALESSPADPATLSED 0.2% 0.0% 0.0% 55 ALESSPADPATLSEDEARLL 0.3% 0.1% 0.0% 56 PADPATLSEDEARLLLAALV 0.0% 0.0% 0.0% 57 TLSEDEARLLLAALVQDYVQ 64.4% 64.7% 49.9% 58 EARLLLAALVQDYVQMKASE 74.6% 100.0% 100.0% 59 LAALVQDYVQMKASELEQEQ 2.8% 2.7% 0.1% 60 QDYVQMKASELEQEQEREGS 0.7% 0.0% 0.1% 61 MKASELEQEQEREGSSLDSP 0.6% 0.0% 0.1% 62 LEQEQEREGSSLDSPRSKRC 0.0% 0.4% 0.1% 63 EREGSSLDSPRSKRCGNLST 0.0% 0.2% 0.0% 64 SLDSPRSKRCGNLSTCMLGT 0.5% 0.0% 0.0% 65 RSKRCGNLSTCMLGTYTQDF 0.9% 0.2% 0.0% 66 GNLSTCMLGTYTQDFNKFHT 0.0% 0.5% 0.0% 67 CMLGTYTQDFNKFHTFPQTA 0.0% 0.1% 0.0% 68 YTQDFNKFHTFPQTAIGVGA 0.0% 0.0% 0.0% 69 NKFHTFPQTAIGVGAPGKKR 4.8% 0.3% 0.9% 70 FPQTAIGVGAPGKKRDMSSD 0.0% 0.0% 0.0% 71 IGVGAPGKKRDMSSDLERDH 0.0% 0.1% 0.0% 72 PGKKRDMSSDLERDHRPHVS 0.7% 0.1% 2.3% 73 DMSSDLERDHRPHVSMPQNA 0.2% 0.1% 0.3% 74 MSSDLERDHRPHVSMPQNAN 0.3% 0.4% 0.2%