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. An in vitro method for the detection of SEQ ID NO:1 (Procalcitonin), or a fragment of SEQ ID NO:1 having a length of at least 20 amino acid residues, in a biological sample derived from a bodily fluid obtained from a subject, said fragment of SEQ ID NO:1 containing an epitope in the sequence spanning amino acid residues 25 to 37 of SEQ ID NO:1 and: an epitope in the sequence spanning amino acid residues 96 to 116 of SEQ ID NO:1, and/or an epitope in the sequence spanning amino acid residues 60 to 91 of SEQ ID NO:1; said method comprising: a. contacting said sample with at least two antibodies or antigen binding fragments thereof directed against different epitopes within SEQ ID NO:1 (Procalcitonin), and b. qualitatively or quantitatively detecting binding of said at least two antibodies to SEQ ID NO:1 (Procalcitonin), or said fragment thereof, wherein binding indicates the presence or concentration of Procalcitonin or said fragment in said sample, wherein one of said at least two antibodies or antigen binding fragment thereof is directed against an epitope in the sequence spanning amino acid residues 25 to 37 of SEQ ID NO:1, wherein said one antibody is produced by a hybridoma cell line that is deposited at the DSMZ under accession number DSM ACC2993, and wherein another of said at least two antibodies or antigen binding fragment thereof is directed against an epitope in the sequence spanning amino acid residues 96 to 116 of SEQ ID NO:1, or is directed against an epitope in the sequence spanning amino acid residues 60 to 91 of SEQ ID NO:1, and wherein said another of said at least two antibodies is a monoclonal antibody.

2. The method of claim 1, wherein said another of said at least two antibodies or antigen binding fragment thereof is directed against an epitope in the sequence spanning amino acid residues 96 to 116 of SEQ ID NO:1.

3. The method according to claim 1, wherein said another of said at least two antibodies or antigen binding fragment thereof is directed against an epitope in the sequence spanning amino acid residues 60 to 91 of SEQ ID NO:1.

4. An antibody or an antigen binding fragment thereof directed against an epitope in the sequence spanning amino acid residues 25 to 37 of SEQ ID NO:1 (Procalcitonin), wherein the antibody is produced by a hybridoma cell line that is deposited at the DSMZ under accession number DSM ACC2993.

5. A kit comprising: a first antibody or an antigen binding fragment thereof directed against an epitope in the sequence spanning amino acid residues 25 to 37 of SEQ ID NO:1, wherein said first antibody is produced by a hybridoma cell line that is deposited at the DSMZ under accession number DSM ACC2993, and a second antibody or an antigen binding fragment thereof directed against an epitope in the sequence spanning amino acid residues 96 to 116 of SEQ ID NO:1 or 60 to 91 of SEQ ID NO:1, and wherein said second antibody is a monoclonal antibody.

6. The kit of claim 5, wherein the second antibody is directed against an epitope in the sequence spanning amino acid residues 60 to 91 of SEQ ID NO:1.

7. A method of performing a sandwich immunoassay for the detection and/or quantification of Procalcitonin in a biological sample from a bodily fluid, said method comprising detecting or determining the amount of Procalcitonin in said biological sample using the kit of claim 5.

8. The hybridoma cell line deposited at the DSMZ under accession number DSM ACC2993.

9. The kit of claim 5, wherein the second antibody is directed against an epitope in the sequence spanning amino acid residues 96 to 116 of SEQ ID NO:1.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1: Schematic representation of assays (C, D and E) used in comparison to existing assays (A and B: BRAHMS PCT LIA and BRAHMS 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.

(2) 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).

(3) 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).

(4) 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.

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

EXAMPLES

Example 1

(6) Material and Methods

(7) A. Development of Monoclonal Antibodies

(8) 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.

(9) B. Epitope Mapping

(10) 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.

(11) C. Immunoassays

(12) Sandwich Immunoassays in the Chemiluminesce-/Coated Tube Format were Set Up as Follows:

(13) Assay A:

(14) 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.

(15) Assay B:

(16) 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.

(17) For the other assays, assay components were generated as follows:

(18) Labeling of Antibodies

(19) 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).

(20) Coating of Antibodies

(21) 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.

(22) With these components the following assays were set up:

(23) Assay C:

(24) 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 nonspecific 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).

(25) Assay D:

(26) 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 nonspecific 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).

(27) Assay E:

(28) 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).

(29) D. Size Exclusion Chromatography

(30) 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 l. 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 (SEQ ID NO: 2); 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.

(31) E. Measurement of Samples

(32) Thirty serum samples of patients with local bacterial infections, sepsis, septic shock were measured in assays A, C, D.

(33) Results

(34) Monoclonal Antibodies

(35) 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 (SEQ ID NO: al (pos. 21-40 within PCT), and for antibody FX1G5 maximum binding was observed on a peptide derived from the previous one, i.e. LLAALVQDYVQMK (SEQ ID NO: 4) (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.

(36) Size Exclusion Chromatography

(37) 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.

(38) Measurement of Samples

(39) 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.

(40) Assay Characteristics

(41) 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).

(42) TABLE-US-00002 TABLE 1 Described anti-PCT antibodies and their use in immunoassays Epitope Immunogen (numbers refer tested in tested (numbers refer to to amino acid sandwich with amino acid positions positions in PCT immuno- native Name Source in PCT 1-116) 1-116) assay PCT Reference anti- Sheep Calcitonin GTYTQDFNKFH; yes yes (Morgenthaler, Calcitonin 69-79 et al. Clin (SEQ ID NO: 5) Chem 2002; 48: 788-90) anti- mouse Katacalcin ERDHRPHVSM; yes yes (Morgenthaler, katacalcin 102-111 et al. Clin (QN05) (SEQ ID NO: 6) Chem 2002; 48: 788-90) PROC1 rat FRSALESSPADPATLSEDE; n.d. yes no (Kramer, et al. 3G3 3-20 Anal Bioanal (SEQ ID NO: 7) Chem 2008; 392: 727-36) PROC4 rat SDLERDHRPHV; 99-109 n.d. yes no (Kramer, et al. 6C6 etc (SEQ ID NO: 8) Anal Bioanal Chem 2008; 392: 727-36) R2B7 rabbit Amino-ProCT; 1-57 n.d. no yes (Whang, et al. J antiserum Clin Endocrinol Metab 1998; 83: 3296-301) 295/3H12 mouse APFRLSALESC; 1-9 n.d. other than N- yes yes DE 10 2007 etc. (SEQ ID NO: 9) terminal Alanin 009 751 being required 98-47/44 mouse DSPRSKRCGNLS; n.d. yes yes U.S. Pat. No. 6,451,311 53-64 (SEQ ID NO: 10) 98-31/04 mouse VGAPGKKRDMSS; n.d. yes yes U.S. Pat. No. 6,451,311 88-99 (SEQ ID NO: 11) CT08 mouse Calcitonin TYTQDFN; 70-76 yes yes (Assicot, et al. (SEQ ID NO: 12) Lancet 1993; 341: 515-8; Ghillani et al, Cancer Res 1989; 49: 6845-51) KC01 mouse Katacalcin DMSSDLERDHR; yes yes (Assicot, et al. 96-106 Lancet (SEQ ID NO: 13) 1993; 341: 515-8; Ghillani, et al. Cancer Res 1989; 49: 6845-51)

(43) TABLE-US-00003 TABLE2 Epitopemappingresults:Observedbindingsignalsforthethree antibodiestotheshownpeptidesrepresentingsubsequencesof theentirePCTsequencewererelatedtothe maximumbindingobtainedperantibody(B/Bmax). SEQID peptide# sequence NO: FX1G5 FW5H6 FX7A7 1 APFRSALESSPAD 14 0.0% 0.0% 0.0% 2 FRSALESSPADPA 15 0.0% 0.0% 0.0% 3 SALESSPADPATL 16 0.0% 0.0% 0.0% 4 LESSPADPATLSE 17 0.0% 0.0% 0.0% 5 SSPADPATLSEDE 18 0.0% 0.0% 0.0% 6 PADPATLSEDEAR 19 0.0% 0.0% 0.0% 7 DPATLSEDEARLL 20 0.0% 0.0% 0.0% 8 ATLSEDEARLLLA 21 0.0% 0.1% 0.0% 9 LSEDEARLLLAAL 22 3.0% 0.0% 0.0% 10 EDEARLLLAALVQ 23 0.3% 0.0% 0.0% 11 EARLLLAALVQDY 24 1.7% 0.0% 0.0% 12 RLLLAALVQDYVQ 25 25.0% 57.3% 0.2% 13 LLAALVQDYVQMK 26 100.0% 59.5% 62.7% 14 AALVQDYVQMKAS 27 11.9% 14.7% 0.0% 15 LVQDYVQMKASEL 28 0.0% 0.0% 0.0% 16 QDYVQMKASELEQ 29 0.0% 0.0% 0.0% 17 YVQMKASELEQEQ 30 0.0% 0.0% 0.0% 18 QMKASELEQEQER 31 0.0% 0.0% 0.0% 19 KASELEQEQEREG 32 0.0% 0.0% 0.0% 20 SELEQEQEREGSS 33 0.0% 0.0% 0.0% 21 LEQEQEREGSSLD 34 0.0% 0.1% 0.0% 22 QEQEREGSSLDSP 35 0.0% 0.0% 0.0% 23 QEREGSSLDSPRS 36 0.0% 0.0% 0.0% 24 REGSSLDSPRSKR 37 0.0% 0.1% 0.0% 25 GSSLDSPRSKRCG 38 0.0% 0.3% 0.1% 26 SLDSPRSKRCGNL 39 0.0% 0.2% 0.3% 27 DSPRSKRCGNLST 40 0.0% 0.0% 0.2% 28 PRSKRCGNLSTCM 41 0.0% 0.0% 0.2% 29 SKRCGNLSTCMLG 42 0.0% 0.0% 0.0% 30 RCGNLSTCMLGTY 43 0.0% 0.0% 0.2% 31 GNLSTCMLGTYTQ 44 0.1% 0.0% 0.0% 32 LSTCMLGTYTQDF 45 0.0% 0.0% 0.0% 33 TCMLGTYTQDFNK 46 0.0% 0.0% 0.0% 34 MLGTYTQDFNKFH 47 0.0% 3.4% 0.0% 35 GTYTQDFNKFHTF 48 0.0% 1.9% 0.0% 36 YTQDFNKFHTFPQ 49 0.0% 0.1% 0.0% 37 QDFNKFHTFPQTA 50 0.4% 0.0% 0.0% 38 FNKFHTFPQTAIG 51 0.0% 0.1% 0.0% 39 KFHTFPQTAIGVG 52 0.2% 0.0% 0.0% 40 HTFPQTAIGVGAP 53 0.0% 0.0% 0.0% 41 FPQTAIGVGAPGK 54 0.1% 0.0% 0.0% 42 QTAIGVGAPGKKR 55 1.0% 0.1% 0.1% 43 AIGVGAPGKKRDM 56 0.0% 0.0% 0.0% 44 GVGAPGKKRDMSS 57 0.0% 0.0% 0.0% 45 GAPGKKRDMSSDL 58 0.0% 0.6% 0.0% 46 PGKKRDMSSDLER 59 0.0% 0.3% 0.1% 47 KKRDMSSDLERDH 60 0.0% 0.0% 0.0% 48 RDMSSDLERDHRP 61 0.0% 1.5% 0.0% 49 MSSDLERDHRPHV 62 1.8% 1.5% 1.9% 50 SDLERDHRPHVSM 63 0.4% 1.5% 0.9% 51 LERDHRPHVSMPQ 64 1.3% 1.5% 2.8% 52 RDHRPHVSMPQNA 65 0.0% 0.1% 0.2% 53 DHRPHVSMPQNAN 66 0.0% 0.0% 0.0% 54 APFRSALESSPADPATLSED 67 0.2% 0.0% 0.0% 55 ALESSPADPATLSEDEARLL 68 0.3% 0.1% 0.0% 56 PADPATLSEDEARLLLAALV 69 0.0% 0.0% 0.0% 57 TLSEDEARLLLAALVQDYVQ 70 64.4% 64.7% 49.9% 58 EARLLLAALVQDYVQMKASE 71 74.6% 100.0% 100.0% 59 LAALVQDYVQMKASELEQEQ 72 2.8% 2.7% 0.1% 60 QDYVQMKASELEQEQEREGS 73 0.7% 0.0% 0.1% 61 MKASELEQEQEREGSSLDSP 74 0.6% 0.0% 0.1% 62 LEQEQEREGSSLDSPRSKRC 75 0.0% 0.4% 0.1% 63 EREGSSLDSPRSKRCGNLST 76 0.0% 0.2% 0.0% 64 SLDSPRSKRCGNLSTCMLGT 77 0.5% 0.0% 0.0% 65 RSKRCGNLSTCMLGTYTQDF 78 0.9% 0.2% 0.0% 66 GNLSTCMLGTYTQDFNKFHT 79 0.0% 0.5% 0.0% 67 CMLGTYTQDFNKFHTFPQTA 80 0.0% 0.1% 0.0% 68 YTQDFNKFHTFPQTAIGVGA 81 0.0% 0.0% 0.0% 69 NKFHTFPQTAIGVGAPGKKR 82 4.8% 0.3% 0.9% 70 FPQTAIGVGAPGKKRDMSSD 83 0.0% 0.0% 0.0% 71 IGVGAPGKKRDMSSDLERDH 84 0.0% 0.1% 0.0% 72 PGKKRDMSSDLERDHRPHVS 85 0.7% 0.1% 2.3% 73 DMSSDLERDHRPHVSMPQNA 86 0.2% 0.1% 0.3% 74 MSSDLERDHRPHVSMPQNAN 87 0.3% 0.4% 0.2%