# Patent

## Title
Vibrio cholerae lipoprotein 15 (Lp15) variants as anti-interference additive in TpN17-based immunoassays for detection of anti-Treponema antibodies

## Abstract

The invention relates to a method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample wherein a peptide sequence of Vibrio cholerae lipoprotein 15 (VcLp15) or a partial sequence thereof is used as a reagent for reduction of interference, i.e. for minimizing false positive results. In addition the invention relates to fusion polypeptides comprising a VcLp15 peptide sequence and a chaperone, to their use as an additive in an immunoassay for said reduction of interferences and for minimizing false positive results and to a reagent kit for detecting antibodies against Treponema pallidum antigens in an isolated sample comprising a TpN17 antigen and said VcLp15-chaperone fusion polypeptide.

## Background
RELATED APPLICATIONS
This application claims the benefit of European Patent Application No. 13003633.8 filed Jul. 18, 2013, the disclosure of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to a method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample wherein a peptide sequence of Vibrio cholerae lipoprotein 15 (VcLp15) is used as a reagent for reduction of interference and for minimizing false positive results. In addition the invention relates to fusion polypeptides comprising a VcLp15 peptide sequence and a chaperone, to their use as an additive in an immunoassay for reduction of interferences and for minimizing false positive results and to a reagent kit for detecting antibodies against Treponema pallidum antigens in an isolated sample comprising a TpN17 antigen and said VcLp15-chaperone fusion polypeptide.
BACKGROUND OF THE INVENTION
Syphilis, also called Lues, is a severe infectious disease which is caused by Treponema pallidum, belonging to the bacterial family of spirochetes. It is mainly transmitted by sexual contact but can also be passed from an expectant mother to the unborn during pregnancy. The disease is characterized by distinct clinical stages and long periods of latent, asymptomatic infection. Many infected individuals do not notice symptoms and thus are unaware of their syphilis infection for years. The primary infection is confined and usually causes a small painless ulcer (primary stage, “Lues I”). If left untreated by penicillin, the disease proceeds to the secondary stage Lues II (about eight weeks after infection), which entails flu-like symptoms, non-itchy skin rash and swollen lymph nodes. After some years, at stage Lues III, syphilitic nodes appear throughout the body. The final stage (Lues IV) is characterized by destruction of the central nervous system eventually leading to neurological and cardiological disorders, general paralysis, ataxia, dementia and blindness.
Although effective therapies have been available since the introduction of penicillin in the mid-20th century, syphilis still remains an important global health problem with estimated 12 million new infections worldwide each year. It is necessary to reliably identify patients with Treponema infection in order to initiate antibiotic therapy and thus to prevent the further spread of syphilis. As a consequence, it is necessary to provide reliable diagnostic tools such as immunoassays for the detection of antibodies against Treponema pallidum. Yet, in order to be used as specific compounds in serological applications, recombinant-derived proteins have to meet several requirements such as solubility, stability and antigenicity.
TpN17 (Treponema pallidum strain Nichols, 17 kDa), a small protein that consists of 134 amino acid residues in its mature form, is the immunodominant antigen of Treponema pallidum, the causative agent of Syphilis (J. Clin. Lab. Immunol. (1998), 50, 27-44; Folia Microbial. (2003) 48 (4), 549-553). Antibodies towards TpN17 are frequent and abundant in Treponema-infected individuals, and thus it is advantageous to use TpN17 in certain embodiments of an immunoassay that aims at the sensitive and reliable detection of Treponema infections.
However, we observed that an immunoassay using TpN17 as an antigen tends to show false positive results, i.e. it provides a seemingly positive signal although in fact no antibodies against Treponema are present in that sample. These interferences are a rare but significant phenomenon. They compromise the specificity of the immunoassay and they are clearly due to the use of the Treponema pallidum antigen TpN17, which is virtually indispensable in a Syphilis immunoassay.
The problem underlying the current invention can therefore be seen in providing means and methods for avoiding false positive results and increasing the specificity of TpN17-based immunoassays for the detection of anti-Treponema antibodies.
SUMMARY OF THE INVENTION
The problem is solved by the current invention as characterized by the claims. In particular, the invention concerns a method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample wherein a peptide sequence of Vibrio cholerae lipoprotein 15 (VcLp15) or a partial sequence thereof is used as a reagent for reduction of interference, i.e. for minimizing false positive results. Said partial sequence of the VcLp15 polypeptide sequence can comprise amino acids 26-163 of SEQ ID NO. 1. In a further embodiment said VcLp15 peptide sequence or partial sequence thereof is fused to a chaperone.
The invention also relates to a fusion polypeptide comprising a VcLp15 peptide sequence according to SEQ ID NO. 1 or a partial sequence thereof and a chaperone. In certain embodiments the chaperone fused to the VcLp15 peptide sequence is selected from the group consisting of SlyD, SlpA, FkpA, and Skp.
In another embodiment the fusion polypeptide comprises SEQ ID NO. 3 which is a fusion polypeptide of E. coli SlyD and VcLp15 (EcSlyD-VcLp15).
Also encompassed by the current invention is the use of a fusion polypeptide comprising a VcLp15 peptide and optionally a chaperone as an additive in an immunoassay for reduction of interferences and for minimizing false positive results.
In a further embodiment the invention concerns a reagent kit for the detection of antibodies against Treponema pallidum antigens in an isolated sample by an immunoassay, comprising a TpN17 antigen and a fusion polypeptide comprising a VcLp15 peptide and optionally a chaperone.
The current invention also relates to a method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample, said method comprising
a) forming an immunoreaction admixture by admixing a body fluid sample with a specific binding partner that can be specifically bound by said antibodies present in said sample
b) adding a fusion polypeptide comprising a VcLp15 peptide and optionally a chaperone to said immunoreaction admixture either before, at the same time or after said specific binding partner is added to said sample
c) maintaining said immunoreaction admixture for a time period sufficient for allowing the antibodies present in said body fluid sample to immunoreact with said specific binding partner to form an immunoreaction product; and
d) detecting the presence and/or the concentration of any of said immunoreaction product.



BRIEF DESCRIPTION OF THE FIGURES, TABLES AND SEQ ID NOS.
FIG. 1 shows near UV CD spectra (ultra-violet circular dichroism spectra) of the fusion polypeptide EcSlyD-VcLp15 according to the invention; for more details refer to example 4 describing the thermally induced unfolding of EcSlyD-VcLp15 as monitored by CD spectroscopy.
FIG. 2 shows melting curves of the fusion polypeptide EcSlyD-VcLp15 according to the invention, as monitored by CD-spectroscopy in the near UV region. For details refer to example 4.



Table 1 shows protein parameters of the fusion polypeptide variants used in this study (example 2)
Tables 2 to 5 show the results of the experiments performed according to example 5 on the anti-interference activity of Vibrio cholerae Lp15 in a syphilis immunoassay.
Table 2 shows the results for oligomeric TpN17 as Treponema-specific antigen wherein monomeric VcLp15 (fused with SlyD as a chaperone) is added for interference reduction.
Table 3 shows the results for monomeric TpN17 as Treponema-specific antigen wherein monomeric VcLp15 (fused with SlyD as a chaperone) is added for interference reduction.
Table 4 shows the results for oligomeric TpN17 as Treponema-specific antigen wherein oligomeric VcLp15 (fused with Skp as a chaperone) is added for interference reduction.
Table 5 shows the results for monomeric TpN17 as Treponema-specific antigen wherein oligomeric VcLp15 (fused with Skp as a chaperone) is added for interference reduction.
SEQ ID NO. 1 shows the complete amino acid sequence (163 residues) of Vibrio cholerae lipoprotein 15 (VcLp15) as retrievable from the public database UniProt, accession no. Q9KQN6. Amino acid residues 1-25 constitute the signal sequence; the mature VcLp15 comprises amino acid residues 26-163 (underlined).







MMKKSIFALS ALTLILVGCD NQQDAKVEVE KVVDVAAAPA


 


EQSAAQPSTA SVDAAHNAQN SLDWAGIYQG TLPCADCGGI


 


ETELTLNADG TYALTEKYLD KEGEPFASQG TFVWNEAGNI


 


VTLQTGDQTG RQFMVGENTL SHLDMEGKVI EGELAEFYVL SKQ






SEQ ID NO. 2 shows the VcLp15 sequence (26-163) as used in fusion with different chaperone modules. The mature VcLp15 sequence (amino acid residues 26-163) is lacking the N-terminal signal sequence (amino acid residues 1-25) and is devoid of cysteine residues. The two genuine cysteine residues of VcLp15 at the positions 74 and 77 [numbering of precursor protein] were replaced by alanine residues—underlined—in order to facilitate the refolding process and to suppress disulfide adduct formation. VcLp15 bears a hexa-histidine tag at the C-terminal end (underlined) in order to facilitate purification and to enable matrix-coupled refolding via a metal column (Ni, Zn, Cu).









KVEVEKVVDV AAAPAEQSAA QPSTASVDAA HNAQNSLDWA



 



GIYQGTLPAA DAGGIETELT LNADGTYALT EKYLDKEGEP



 



FASQGTFVWN EAGNIVTLQT GDQTGRQFMV GENTLSHLDM



 



EGKVIEGELA EFYVLSKQLE HHHHHH






SEQ ID NO. 3 shows EcSlyD-VcLp15 which is a fusion polypeptide according to the invention, comprising one molecule of E. coli SlyD as a chaperone and the mature VcLp15 sequence (26-163, underlined).









MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS 



 



LISGLETALE GHEVGDKFDV AVGANDAYGQ YDENLVQRVP



 



KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVD



 



GNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH



 



DHDHDGGGSG GGSGGGSGGG SGGGSGGGKV EVEKVVDVAA



 



APAEQSAAQP STASVDAAHN AQNSLDWAGI YQGTLPAADA



 



GGIETELTLN ADGTYALTEK YLDKEGEPFA SQGTFVWNEA



 



GNIVTLQTGD QTGRQFMVGE NTLSHLDMEG KVIEGELAEF



 



YVLSKQLEHH HHHH






SEQ ID NO. 4 shows EcSkp-VcLp15 which is a fusion polypeptide according to the invention, comprising one molecule of E. coli Skp as a chaperone and the mature VcLp15 sequence (26-163, underlined).









MADKIAIVNM GSLFQQVAQK TGVSNTLENE FRGRASELQR



 



METDLQAKMK KLQSMKAGSD RTKLEKDVMA QRQTFAQKAQ



 



AFEQDRARRS NEERGKLVTR IQTAVKSVAN SQDIDLVVDA



 



NAVAYNSSDV KDITADVLKQ VKGGGSGGGS GGGSGGGSGG



 



GSGGGKVEVE KVVDVAAAPA EQSAAQPSTA SVDAAHNAQN



 



SLDWAGIYQG TLPAADAGGI ETELTLNADG TYALTEKYLD



 



KEGEPFASQG TFVWNEAGNI VTLQTGDQTG RQFMVGENTL



 



SHLDMEGKVI EGELAEFYVL SKQLEHHHHH H






SEQ ID NO. 5 shows the complete TpN17 sequence (amino acid residues 1-156) of Treponema pallidum as retrieved from the public database UniProt, accessible under UniProt ID P29722.









MKGSVRALCA FLGVGALGSA LCVSCTTVCP HAGKAKAEKV



 



ECALKGGIFR GTLPAADCPG IDTTVTFNAD GTAQKVELAL



 



EKKSAPSPLT YRGTWMVRED GIVELSLVSS EQSKAPHEKE



 



LYELIDSNSV RYMGAPGAGK PSKEMAPFYV LKKTKK






SEQ ID NO. 6 shows the TpN17 sequence as used in example 5. For this immunoassay, the mature TpN17 protein (amino acid residues 23-156) lacking the signal sequence (amino acid residues 1-22) was used. We found the four genuine cysteine residues of TpN17 to be dispensable for the antigenicity of the protein (data not shown). Thus, the cysteine residues at the positions 25, 29, 42 and 58 (numbering of the precursor protein) were replaced by alanine residues (underlined) in order to facilitate the refolding process and to suppress detrimental side reactions such as disulfide adduct formation.









VSATTVAPHA GKAKAEKVEA ALKGGIFRGT LPAADAPGID



 



TTVTFNADGT AQKVELALEK KSAPSPLTYR GTWMVREDGI



 



VELSLVSSEQ SKAPHEKELY ELIDSNSVRY MGAPGAGKPS



 



KEMAPFYVLK KTKK






SEQ ID NO. 7 shows the TpN17 sequence amino acid residues 23-156 (see also SEQ ID NO. 6) as used in fusion with different chaperone modules. TpN17 bears a hexa-histidine tag at the C-terminal end (underlined) in order to facilitate purification and to enable matrix-coupled refolding via a metal column (Ni, Zn, Cu). The N-terminal signal sequence of TpN17 (amino acid residues 1-22) was omitted in order to obtain the mature (processed) form of the protein in its native-like conformation.









VSATTVAPHA GKAKAEKVEA ALKGGIFRGT LPAADAPGID



 



TTVTFNADGT AQKVELALEK KSAPSPLTYR GTWMVREDGI



 



VELSLVSSEQ SKAPHEKELY ELIDSNSVRY MGAPGAGKPS



 



KEMAPFYVLK KTKKLEHHHH HH






SEQ ID NO. 8 shows the TpN17 sequence (underlined) to which two molecules of E. coli SlyD (“tandem SlyD”) have been fused N-terminally; this molecule is also named EcSlyD-EcSlyD-TpN17 or EcSS-TpN17.









MKVAKDLVVS LAYQVRTEDG VLVDESPVSA PLDYLHGHGS



 



LISGLETALE GHEVGDKFDV AVGANDAYGQ YDENLVQRVP



 



KDVFMGVDEL QVGMRFLAET DQGPVPVEIT AVEDDHVVVD



 



GNHMLAGQNL KFNVEVVAIR EATEEELAHG HVHGAHDHHH



 



DHDHDGGGSG GGSGGGSGGG SGGGSGGGKV AKDLVVSLAY



 



QVRTEDGVLV DESPVSAPLD YLHGHGSLIS GLETALEGHE



 



VGDKFDVAVG ANDAYGQYDE NLVQRVPKDV FMGVDELQVG



 



MRFLAETDQG PVPVEITAVE DDHVVVDGNH MLAGQNLKFN



 



VEVVAIREAT EEELAHGHVH GAHDHHHDHD HDGGGSGGGS



 



GGGSGGGSGG GSGGGVSATT VAPHAGKAKA EKVEAALKGG



 



IFRGTLPAAD APGIDTTVTF NADGTAQKVE LALEKKSAPS



 



PLTYRGTWMV REDGIVELSL VSSEQSKAPH EKELYELIDS



 



NSVRYMGAPG AGKPSKEMAP FYVLKKTKKL EHHHHHH






SEQ ID NO. 9 shows the TpN17 sequence (underlined) to which two molecules of Pasteurella multocida SlyD (“tandem SlyD”) have been fused N-terminally; this molecule is also named PmSlyD-PmSlyD-TpN17 or PmSS-TpN17.









MKIAKNVVVS IAYQVRTEDG VLVDEAPVNQ PLEYLQGHNN



 



LVIGLENALE GKAVGDKFEV RVKPEEAYGE YNENMVQRVP



 



KDVFQGVDEL VVGMRFIADT DIGPLPVVIT EVAENDVVVD



 



GNHMLAGQEL LFSVEVVATR EATLEEIAHG HIHQEGGGGS



 



GGGSGGGGGS GGGSGGGKIA KNVVVSIAYQ VRTEDGVLVD



 



EAPVNQPLEY LQGHNNLVIG LENALEGKAV GDKFEVRVKP



 



EEAYGEYNEN MVQRVPKDVF QGVDELVVGM RFIADTDIGP



 



LPVVITEVAE NDVVVDGNHM LAGQELLFSV EVVATREATL



 



EEIAHGHIHQ EGGGGSGGGS GGGSGGGSGG GSGGGVSATT



 



VAPHAGKAKA EKVEAALKGG IFRGTLPAAD APGIDTTVTF



 



NADGTAQKVE LALEKKSAPS PLTYRGTWMV REDGIVELSL



 



VSSEQSKAPH EKELYELIDS NSVRYMGAPG AGKPSKEMAP



 



FYVLKKTKKL EHHHHHH






SEQ ID NO. 10 shows the TpN17 sequence (underlined) to which one molecule of E. coli FkpA has been fused N-terminally; this molecule is also named EcFkpA-TpN17.







MAEAAKPATT ADSKAAFKND DQKSAYALGA SLGRYMENSL


 


KEQEKLGIKL DKDQLIAGVQ DAFADKSKLS DQEIEQTLQA


 


FEARVKSSAQ AKMEKDAADN EAKGKEYREK FAKEKGVKTS


 


STGLVYQVVE AGKGEAPKDS DTVVVNYKGT LIDGKEFDNS


 


YTRGEPLSFR LDGVIPGWTE GLKNIKKGGK IKLVIPPELA


 


YGKAGVPGIP PNSTLVFDVE LLDVKPAPKA DAKPEADAKA


 


ADSAKKGGGS GGGSGGGSGG GSGGGSGGGV SATTVAPHAG


 


KAKAEKVEAA LKGGIFRGTL PAADAPGIDT TVTFNADGTA


 


QKVELALEKK SAPSPLTYRG TWMVREDGIV ELSLVSSEQS


 


KAPHEKELYE LIDSNSVRYM GAPGAGKPSK EMAPFYVLKK


 


TKKLEHHHHH H






SEQ ID NO. 11 shows the TpN17 sequence (underlined) to which one molecule of E. coli Skp has been fused N-terminally; this molecule is also named EcSkp-TpN17.









MADKIAIVNM GSLFQQVAQK TGVSNTLENE FRGRASELQR



 



METDLQAKMK KLQSMKAGSD RTKLEKDVMA QRQTFAQKAQ



 



AFEQDRARRS NEERGKLVTR IQTAVKSVAN SQDIDLVVDA



 



NAVAYNSSDV KDITADVLKQ VKGGGSGGGS GGGSGGGSGG



 



GSGGGVSATT VAPHAGKAKA EKVEAALKGG IFRGTLPAAD



 



APGIDTTVTF NADGTAQKVE LALEKKSAPS PLTYRGTWMV



 



REDGIVELSL VSSEQSKAPH EKELYELIDS NSVRYMGAPG



 



AGKPSKEMAP FYVLKKTKKL EHHHHHH






SEQ ID NO. 12 shows the amino acid sequence of the glycine-rich spacer (comprising triple glycine units separated by a serine) that can be used as a flexible, soluble and protease-resistant spacer or linker between several chaperone moieties.









GGGSGGGSGG GSGGGSGGGS GGG






DETAILED DESCRIPTION OF THE INVENTION
Immunoassays for the detection of antibodies against Treponema pallidum tend to show false positive results as could be demonstrated by the inventors. In particular when the Treponema antigen TpN17 is used, the number of false positive signals is significantly elevated. This phenomenon has been observed with human sera that had definitely been characterized as anti-Treponema negative: when using the TpN17 antigen, a significant number of false positives were found (see example 5). Yet, TpN17 is a crucial immunogen in Treponema infections and a paramount antigen in syphilis serology. As a consequence, it is not a viable option to circumvent this interference problem by simply omitting the TpN17 antigen.
We therefore started out with the design of a recombinant TpN17 variant which enables the reliable and sensitive detection of anti-TpN17 antibodies. More precisely, we fused TpN17 to a solubility-conferring chaperone (tandem SlyD, namely EcSlyD-EcSlyD and PmSlyD-PmSlyD) via a flexible linker rich in glycine and serine residues. Due to the beneficial effects of the fused folding helpers, the resulting fusion polypeptide meets all the physicochemical and immunological requirements of a good antigen for serological purposes (i.e. for use in an immunoassay).
The chaperone-TpN17 fusion proteins that we designed for an automated syphilis immunoassay are highly soluble and reactive and are advantageously used in a double antigen sandwich format. As mentioned above, during the feasibility studies for a syphilis immunoassay it turned out that TpN17 is indeed an immunodominant Treponema antigen with outstanding diagnostic significance. In other words, it is imperative to use a TpN17 variant in a Syphilis immunoassay in order to warrant the desired sensitivity. Yet, when chaperone polypeptide fusion constructs of TpN17 were used in the double antigen sandwich (DAGS) format, the problem became evident: even though the TpN17 fusion constructs had been designed in an asymmetric fashion (i.e., the symmetry of the DAGS format was deliberately abolished by the use of different fusion partners on the biotin side and the ruthenium side), and despite the use of chaperone polymers as anti-interference additives, quite a number of positive results occurred in a panel of well-characterized anti-Treponema negative human sera, leading to a substantial worsening of the assay specificity. Obviously, some of the anti-Treponema negative human sera contained at least one unknown factor which was able to interact specifically with the TpN17 antigen.
To our surprise, it turned out that addition of recombinant-derived protein Lp15 from the human pathogen Vibrio cholerae (VcLp15)—which is, despite certain sequence homologies, an organism quite unrelated to Treponema pallidum—to the immunoassay mixture reduced the elevated signals of the false positives to the signal level of negative sera as can be seen in example 5 and tables 2-5. We conclude that VcLp15 (when added in an unlabeled form) is able to recognize, bind and quench the unknown interference factor(s) which are directed towards TpN17. It has indeed turned out that VcLp15 is an invaluable tool for reducing false positive results and for improving the specificity of syphilis immunoassays based on the Treponema antigen TpN17.
In detail, the current invention relates to a method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample wherein a peptide sequence of Vibrio cholerae lipoprotein 15 (VcLp15) or a partial sequence thereof is used as a reagent for reduction of interference and for minimizing false positive results.
Any TpN17 antigen or variant thereof can be used provided that the antigen's conformation is native-like enough to be recognized by the antibodies present in the sample. In its natural host, T. pallidum, the N-terminal signal sequence of TpN17 (residues 1-22) is cleaved off the precursor protein to allow folding of the mature TpN17 part into its native conformation. In other words, the signal sequence is dispensable when TpN17 is produced recombinantly in a prokaryotic host such as E. coli. It rather impedes proper folding of the target molecule and is thus omitted. In some embodiments, a peptide sequence according to UniProt ID P29722 (SEQ ID NO. 5) or SEQ ID NO. 6 or a partial sequence of SEQ ID NOs. 5 or 6 is used. The partial sequence comprises at least about 100 amino acids of SEQ ID NOs 5 or 6. Another useful sequence is an amino acid sequence comprising amino acid residues 23-156 of SEQ ID NO. 5 or amino acid residues 1-134 of SEQ ID NO. 6.
A useful TpN17 antigen is a polypeptide according to SEQ ID NOs. 7 to 11 wherein TpN17 has been fused to various chaperone peptide sequences. To facilitate the refolding process after purification and to suppress disulfide adduct formation the cysteine residues in all envisaged TpN17 antibodies may be replaced by other amino acid residues such as alanine or serine. These residues replace the oxidation-sensitive thiol moiety of the cysteine side chain but almost equal the cysteine residue in size. Therefore, they usually fit into the overall three-dimensional protein structure and do not severely compromise folding and stability of the cysteine-free protein variant.
According to the method of the current invention a peptide sequence of Vibrio cholerae lipoprotein 15 (VcLp15) or a partial sequence thereof is used as a reagent for reduction of interference, i.e. for minimizing false positive results. In some embodiments said partial sequence of VcLp15 comprises amino acids comprises amino acid residues 26-163 of SEQ ID NOs. 1 or 2. The N-terminal signal sequence comprising residues 1-25 of SEQ ID NO. 1 is dispensable. In a further mode of the invention said VcLp15 partial sequence of amino acid residues 26-163 of SEQ ID NOs. 1 or 2 can be truncated by 1 to 5 amino acids at its N-terminal or C-terminal end or at both ends. In another embodiment said VcLp15 partial sequence of amino acid residues 26-163 of SEQ ID NOs. 1 or 2 can be modified in such a way that conservative amino acid substitutions can be introduced like e.g. substitution of an alanine residue by a serine residue or cysteine. Any of these three amine acids can be replaced by the other two amino acids. Other examples of conservative amino acid substitutions known by a person skilled in the art are Serin/Cystein/Alanin, Isoleucin/Valin or Phenylalanin/Tyrosin. For any of these modifications it is important that the three-dimensional structure of the Vibrio cholerae lipoprotein 15 (VcLp15) remains unchanged.
In some embodiments, the VcLp15 peptide sequence or partial sequence thereof used in the above-described method is fused to a chaperone to provide high expression yields and to facilitate the refolding process after purification.
A further aspect of the invention is a fusion polypeptide comprising a VcLp15 peptide sequence according to SEQ ID NO. 1 or 2 or a partial sequence of SEQ ID NOs. 1 or 2 and a chaperone.
The use of polypeptide fusion proteins wherein chaperones are fused to difficult target antigen sequences to solubilize them and make them more benign is well-known in the art and has been described in great detail before such as in the international patent application WO 2003/000878. Known and well-documented examples of useful fusion chaperones are SlyD, FkpA, Skp and SlpA, see also European Patent Application EP2127678A1.
A further aspect of the invention therefore is a fusion polypeptide comprising a VcLp15 peptide sequence and a chaperone. In certain embodiments the chaperone is selected from the group consisting of SlyD, SlpA, FkpA and Skp. These chaperones may originate from various organisms, and in certain embodiments the chaperone sequences are derived from E. coli. 
In another embodiment of the invention the fusion polypeptide comprising a VcLp15 peptide sequence comprises SEQ ID NO. 3 (EcSlyD-VcLp15).
The use of a fusion polypeptide comprising a VcLp15 peptide sequence and a chaperone as an additive in an immunoassay for reduction of interferences and for minimizing false positive results is also an aspect of the current invention.
Another aspect of the invention is a reagent kit for the detection of antibodies against Treponema pallidum antigens in an isolated sample by an immunoassay, comprising a TpN17 antigen and a fusion polypeptide comprising a VcLp15 peptide sequence and a chaperone as described in detail further above.
Moreover, the invention covers a method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample, said method comprising the steps of
a) forming an immunoreaction admixture by admixing a body fluid sample with a specific binding partner that can be specifically bound by said antibodies present in said sample
b) adding a fusion polypeptide comprising a VcLp15 peptide sequence as defined above to said immunoreaction admixture either before, at the same time or after said specific binding partner is added to said sample
c) maintaining said immunoreaction admixture for a time period sufficient for allowing the antibodies present in said body fluid sample to immunoreact with said specific binding partner to form an immunoreaction product; and
d) detecting the presence and/or the concentration of any of said immunoreaction product.
The fusion polypeptide of the invention can be added to the immunoassay admixture (comprising sample and a binding partner specifically binding to the analyte antibodies in the sample) either before, at the same time or after said specific binding partner is added to the sample. In some embodiments, the fusion polypeptide is added to the test reagents before the body fluid sample containing the analyte antibodies, is brought into contact with the specific binding partners.
In one embodiment of the invention the immunoassay for detecting anti-Treponema antibodies in an isolated sample is performed according to the so-called double antigen sandwich concept (DAGS). Sometimes this assay concept is also termed double antigen bridge concept, because the two antigens are bridged by an antibody analyte. In such an assay the ability of an antibody to bind at least two different molecules of a given antigen with its two (IgG, IgE), four (IgA) or ten/twelve (IgM) paratopes is required and used.
In more detail, an immunoassay for the determination of anti-Treponema antibodies according to the double antigen bridge format is carried out by incubating a sample containing the anti-Treponema antibodies with two different TpN17 antigens, i.e. a first (“solid phase”) TpN17 antigen and a second (“detection”) TpN17 antigen, wherein each of the said antigens binds specifically to said anti-Treponema antibodies. The first antigen is or can be bound directly or indirectly to a solid phase and usually carries an effector group which is part of a bioaffine binding pair like biotin/avidin. For example, if the first antigen is conjugated to biotin the solid phase is coated with either avidin or streptavidin. The second antigen carries a detectable label. Then an immunoreaction admixture is formed comprising the first antigen, the sample antibody and the second antigen. A solid phase to which the first antigen can be bound is added either before the addition of the sample to said antigens or after the immunoreaction admixture is formed. This immunoreaction admixture is maintained for a time period sufficient for allowing anti-Treponema antibodies against said TpN17 antigens in the body fluid sample to immunoreact with said TpN17 antigens to form an immunoreaction product. Next step is a separation step wherein the liquid phase is separated from the solid phase. Finally, the presence of any of said immunoreaction product is detected in the solid or liquid phase or both.
In said DAGS immunoassay the basic structures of the “solid phase antigen” and the “detection antigen” are the same. It is also possible to use similar but different TpN17 antigens, which are immunologically cross-reactive in a double antigen bridge assay. The essential requirement for performing such assays is that the relevant epitope or the relevant epitopes are present on both antigens. According to the invention it is desirable to use different fusion moieties for each TpN17 antigen (e.g. EcFkpA is fused to TpN17 on the solid phase side and EcSkp is fused to TpN17 on the detection side) as such variations break the symmetry of the DAGS format and thus reduce the problem of antibody-mediated bridging of the fusion chaperones which would lead to a false positive outcome of the immunoassay. In brief, the use of structurally distant fusion partners on both sides of a DAGS format reduces unwanted immunological cross-reactions and thus improves specificity.
The current invention therefore also relates to a method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample wherein a peptide sequence of VcLp15 is used as a reagent for reduction of interference and for minimizing false positive results. Said method is further characterized in that the assay is carried out in the double-antigen sandwich format (DAGS). Moreover, said assay uses two TpN17 antigen fusion polypeptides—a first and a second TpN17 antigen—wherein both TpN17 antigens are identical or at least immunologically cross-reactive against the same antibodies so that a bridging between both antigens by the antibodies present in the sample is possible. In addition, the first and the second antigens are fused to different chaperones as described in the preceding paragraph.
Moreover, the use of specific chaperone fusion partners like Skp and FkpA may facilitate a considerably improved IgM recognition and detection. Due to their avidity mode of binding, IgM molecules can only react with polymeric antigens possessing a medium to high epitope density. Both Skp and FkpA are oligomeric chaperones serving a role as folding helpers in the periplasm of Gram negative bacteria. To our surprise, we find that the quartery structure of Skp and FkpA is maintained when large target molecules are fused to the C-termini of the chaperones. As a consequence, the FkpA-TpN17 and Skp-TpN17 fusion proteins reproducibly form natural oligomers with defined epitope densities that are sufficient to detect IgM molecules. Sensitive and specific IgM detection is a very important feature warranting reliable detection of early and primary syphilis infections. Since we aim at developing an immunoassay for total immunoglobulin detection (i.e. the detection of both IgG and IgM), the oligomeric antigen modules FkpA-TpN17 and Skp-TpN17 may be used advantageously as specifiers on both sides of a DAGS format (e.g. FkpA-TpN17-biotin and Skp-TpN17-ruthenium). Since FkpA and Skp are very distinct from each other in terms of structure, the risk of unwanted immunological cross-reaction and bridging via the fusion partners is very low. It is further reduced by adding chemically polymerized FkpA and Skp anti-interference additives to the assay.
Various additional formats and principles of immunoassays for detecting analytes and different modes of detection have been widely described and are familiar to a person skilled in the art.
According to the invention any biological isolated sample in which Treponema antibodies might be detectable can be used. In particular human blood, serum, plasma or saliva are suitable as sample material.
The invention is further illustrated in the examples section.
Example 1
Cloning and Purification of TpN17 and VcLp15 Chaperone Fusion Polypeptides
Cloning of Expression Cassettes
On the basis of the pET24a expression plasmid of Novagen (Madison, Wis., USA), expression cassettes encoding TpN17 and VcLp15 fusion proteins were obtained essentially as described (Scholz, C. et al., J. Mol. Biol. (2005) 345, 1229-1241). The sequences of the TpN17 and VcLp15 antigens were retrieved from the SwissProt database (SwissProt ID P29722 and Q9KQN6, respectively). A synthetic gene encoding mature TpN17 aa 23-156 (the signal peptide spanning amino acid residues 1-22 was omitted) with a glycine-rich linker region fused in frame to the N-terminus was purchased from Medigenomix (Martinsried, Germany). The cysteine residues of TpN17 at positions 25, 29, 42 and 58 were changed to alanine residues in order to prevent unwanted side-effects such as oxidation or intermolecular disulfide bridging. BamHI and XhoI restriction sites were at the 5′ and the 3′ ends of the TpN17-coding region, respectively. A further synthetic gene encoding two EcSlyD units (residues 1-165 according to SEQ ID NO. 1, SwissProt accession no. P0A9K9) connected via a glycine-rich linker region and encompassing part of a further linker region at the C-terminus were likewise purchased from Medigenomix. NdeI and BamHI restriction sites were at the 5′ and 3′ ends of this cassette, respectively. The genes and the restriction sites were designed to enable the in frame fusion of the chaperone part EcSlyD-EcSlyD and the TpN17 antigen part by simple ligation. In order to avoid inadvertent recombination processes and to increase the genetic stability of the expression cassette in the E. coli host, the nucleotide sequences encoding the EcSlyD units were degenerated as were the nucleotide sequences encoding the extended linker regions. i.e., different codon combinations were used to encode identical amino acid sequences.
The pET24a vector was digested with NdeI and XhoI and the cassette comprising tandem-SlyD fused in frame to Treponema TpN17 23-156 was inserted. Expression cassettes comprising Pasteurella multocida SlyD (1-156, SwissProt ID Q9CKP2) or E. coli Skp (21-161, SwissProt ID P0AEU7) or FkpA (26-270, SwissProt ID P45523) were constructed accordingly, as well as expression cassettes comprising target polypeptides different from TpN17, notably the Vibrio cholerae lipoprotein Lp15 (26-163, SwissProt ID Q9KQN6). As with TpN17, the genuine cysteine residues of VcLp15 at positions 74 and 77 (precursor Lp15 numbering) were changed to alanine residues in order to prevent unwanted side-effects such as oxidation or intermolecular disulfide bridging. All recombinant fusion polypeptide variants contained a C-terminal hexahistidine tag to facilitate Ni-NTA-assisted purification and refolding. QuikChange (Stratagene, La Jolla, Calif., USA) and standard PCR techniques were used to generate point mutations, deletion, insertion and extension variants or restriction sites in the respective expression cassettes.
The drawing below shows a scheme of the Treponema TpN17 full length antigen 23-156 bearing two SlyD chaperone units fused in frame to its N-terminal end. To denote the E. coli origin of the SlyD fusion partner, the depicted fusion polypeptide has been named EcSlyD-EcSlyD-TpN17 (23-156); see also SEQ ID NO. 8.




The insert of the resulting plasmid was sequenced and found to encode the desired fusion protein. The complete amino acid sequence of the TpN17 and VcLp15 fusion polypeptides is shown in SEQ ID NOs. 2 to 4 (VcLp15) and 7 to 11 (TpN17). The amino acid sequence of the linker L is shown is SEQ ID NO. 12.
Purification of Fusion Proteins Comprising TpN17 or VcLp15
All TpN17 and VcLp15 fusion protein variants were purified by using virtually identical protocols. E. coli BL21 (DE3) cells harboring the particular pET24a expression plasmid were grown at 37° C. in LB medium plus kanamycin (30 μg/ml) to an OD600 of 1.5, and cytosolic overexpression was induced by adding 1 mM isopropyl-β-D-thiogalactoside. Three hours after induction, cells were harvested by centrifugation (20 min at 5000 g), frozen and stored at −20° C. For cell lysis, the frozen pellet was resuspended in chilled 50 mM sodium phosphate pH 8.0, 7.0 M GdmCl, 5 mM imidazole and the suspension was stirred for 2 h on ice to complete cell lysis. After centrifugation and filtration (0.45 μm/0.2 μm), the crude lysate was applied onto a Ni-NTA column equilibrated with the lysis buffer including 5.0 mM TCEP. The subsequent washing step was tailored for the respective target protein and ranged from 5 to 15 mM imidazole (in 50 mM sodium phosphate pH 8.0, 7.0 M GdmCl, 5.0 mM TCEP). At least 10-15 volumes of the washing buffer were applied. Then, the GdmCl solution was replaced by 50 mM potassium phosphate pH 8.0, 100 mM KCl, 10 mM imidazole, 5.0 mM TCEP to induce conformational refolding of the matrix-bound protein. In order to avoid reactivation of copurifying proteases, a protease inhibitor cocktail (Complete® EDTA-free, Roche) was included in the refolding buffer. A total of 15-20 column volumes of refolding buffer were applied in an overnight reaction. Then, both TCEP and the Complete® EDTA-free inhibitor cocktail were removed by washing with 3-5 column volumes 50 mM potassium phosphate pH 8.0, 100 mM KCl, 10 mM imidazole. Subsequently, the imidazole concentration—still in 50 mM potassium phosphate pH 8.0, 100 mM KCl—was raised to 25-50 mM (depending on the respective target protein) in order to remove unspecifically bound protein contaminants. The native protein was then eluted by 500 mM imidazole in the same buffer. Protein-containing fractions were assessed for purity by Tricine-SDS-PAGE and pooled. Finally, the proteins were subjected to size-exclusion-chromatography (Superdex HiLoad, Amersham Pharmacia) and the protein-containing fractions were pooled and concentrated to 10-20 mg/ml in an Amicon cell (YM10).
After the coupled purification and refolding protocol, protein yields of roughly 10-30 mg could be obtained from 1 g of E. coli wet cells, depending on the respective target protein.
Example 2
Spectroscopic Measurements
Protein concentration measurements were performed with an Uvikon XL double-beam spectrophotometer. The molar extinction coefficients (ε280) were determined by using the procedure described by Pace (1995), Protein Sci. 4, 2411-2423. The molar extinction coefficients (εM280) used for the distinct fusion polypeptides are specified in table 1.







TABLE 1







Protein parameters of the fusion polypeptide variants


used in this study. All parameters are referring to the


respective protein monomers.













length of
molecular






target
weight of






protein
fusion


Abs0.1%



(aa
polypeptide

εM280
(=1 mg/


fusion protein
residues)
(Da)
pI
M−1cm−1
ml)










TpN17 variants












EcSlyD-EcSlyD-
23-156
54048
5.0
23380
0.433


TpN17







PmSlyD-PmSlyD-
23-156
52171
4.9
23380
0.448


TpN17







EcFkpA-TpN17
23-156
42995
8.3
27390
0.637


EcSkp-TpN17
23-156
32461
9.3
12950
0.399







VcLp15 variants












EcSlyD-VcLp15
26-163
35156
4.6
22920
0.652


EcSkp-VcLp15
26-163
33010
5.3
18450
0.559









The amino acid sequences of the fusion polypeptide variants are shown in SEQ ID NOs. 3, 4, 8, 9, 10 and 11, respectively.
Example 3
Coupling of Biotin and Ruthenium Moieties to the TpN17 Fusion Proteins
The lysine ε-amino groups of the TpN17 fusion polypeptides were modified at protein concentrations of 10-30 mg/ml with N-hydroxy-succinimide activated biotin and ruthenium label molecules, respectively. The label/protein ratio varied from 2:1 to 5:1 (mol:mol), depending on the respective fusion protein. The reaction buffer was 150 mM potassium phosphate pH 8.0, 100 mM KCl, 0.5 mM EDTA. The reaction was carried out at room temperature for 15 mM and stopped by adding buffered L-lysine to a final concentration of 10 mM. To avoid hydrolytic inactivation of the labels, the respective stock solutions were prepared in dried DMSO (seccosoiv quality, Merck, Germany). DMSO concentrations up to 25% in the reaction buffer were well tolerated by all fusion proteins studied. After the coupling reaction, unreacted free label was removed by passing the crude protein conjugate over a gel filtration column (Superdex 200 HiLoad).
Example 4
CD-Detected Thermally Induced Unfolding of EcSlyD-VcLp15
Near-UV CD spectra were recorded with a Jasco-720 spectropolarimeter with a thermostatted cell holder and were converted to mean residue ellipticity. The buffer was 50 mM potassium phosphate pH 7.0, 250 mM KCl, 0.5 mM EDTA. The pathlength was 0.2 cm, the protein concentration was  ̃74 μM (2.6 mg/ml). The measuring range was 250-330 nm, the band width was 1.0 nm, the scanning speed was 20 nm/min at a resolution of 0.5 nm, and the response was 1 s. In order to improve the signal-to-noise ratio, spectra were measured nine times and averaged.
Circular dichroism spectroscopy (CD) is the method of choice to assess both the secondary and the tertiary structure of proteins. Ellipticity in the aromatic region (260-320 nm) reports on tertiary contacts within a protein (i.e., the globular structure of a regularly folded protein) and is considered as the fingerprint region of a native-like fold (conformation).
Near UV CD spectra of EcSlyD-VcLp15 were monitored to address the question whether the fusion protein adopts an ordered conformation after the matrix-coupled refolding procedure which is the crucial step in the purification process. The answer is quite clear-cut: the near UV CD signal of EcSlyD-VcLp15 unequivocally reports an orderly tertiary structure of the fusion polypeptide. The aromatic residues of EcSlyD-VcLp15 are obviously embedded in the lipophilic protein core and thus experience asymmetric surroundings which strongly points to a native-like conformation of both EcSlyD and VcLp15 within the fusion construct (FIG. 1).
In order to address the question whether the thermally induced unfolding of EcSlyD-VcLp15 is reversible, melting curves were monitored in the near UV region at a detection wavelength of 281 nm. The temperature range was 20-80° C., the band width was 1.0 nm, the temperature ramp was 1° C./min and the response was 4 s (see FIG. 2).
The thermally-induced unfolding was monitored at 281 nm (which is the wavelength of the maximal signal amplitude for EcSlyD-VcLp15). Upon heating, the non-covalent contacts which stabilize the native conformation of the EcSlyD-VcLp15 molecule become loose and finally break down. This thermally induced unfolding is reflected in an increase in the CD signal as shown in FIG. 2. At 80° C., EcSlyD-VcLp15 is fully unfolded. Strikingly, the native-like CD signal is restored again when the protein solution is chilled down to 20° C. Despite a slight hysteresis, the unfolding curve and the refolding curve virtually superimpose, strongly indicative of a reversible refolding behavior of EcSlyD-VcLp15. It must be admitted that the cooperativity of unfolding is rather low and that the typical sigmoidal shape of a protein melting curve is not observed in the case of EcSlyD-VcLp15. Yet, we unambiguously find that EcSlyD-VcLp15 is able to readopt its native-like conformation when the protein solution is chilled from 80° C. to 20° C. Indeed, the near UV CD spectra monitored prior to and after the thermally induced unfolding, virtually superimpose (see FIG. 1). In conclusion, EcSlyD-VcLp15 possesses robust folding properties which are outstanding for a fusion polypeptide and which are highly desired for a molecule that serves as an anti-interference additive in an immunoassay. These benign physicochemical properties, in combination with an outstanding solubility (>130 mg/ml in phosphate-buffered saline) and the anti-interference potential in an anti-Treponema immunoassay makes EcSlyD-VcLp15 a very attractive molecule warranting a high specificity in syphilis serology.
Example 5
Anti-Interference Activity of Vibrio cholerae Lp15 in a Syphilis Immunoassay
The anti-interference activity of the polypeptide fusion variants of Vibrio cholerae Lp15 was assessed in an automated Elecsys® 2010 analyzer (Roche Diagnostics GmbH). Elecsys® is a registered trademark of the Roche group. Measurements were carried out in the double antigen sandwich format.
Signal detection in Elecsys® 2010 is based on electrochemiluminescence. The biotin-conjugate (i.e. the capture-antigen) is immobilized on the surface of a streptavidin coated magnetic bead whereas the detection-antigen bears a complexed Ruthenium cation (switching between the redox states 2+ and 3+) as the signaling moiety. In the presence of a specific immunoglobulin analyte, the chromogenic ruthenium complex is bridged to the solid phase and emits light at 620 nm after excitation at a platinum electrode. The signal output is in arbitrary light units.
The recombinant anti-interference Lp15 polypeptides were assessed in a double antigen sandwich (DAGS) immunoassay format. To this end, recombinant Treponema antigen TpN17 was used as a biotin and a ruthenium conjugate, respectively, to detect anti-TpN17 antibodies in human sera. TpN17 is one of the immunodominant antigens of Treponema pallidum, and soluble variants of TpN17—as disclosed in this patent application—are invaluable tools for the detection of syphilis infections. In order to detect anti-TpN17 IgG molecules, EcSlyD-EcSlyD-TpN17-biotin and PmSlyD-PmSlyD-TpN17-ruthenium were used in R1 (reagent buffer 1) and R2 (reagent buffer 2), respectively. In order to detect both anti-TpN17 IgM and IgG molecules, EcFkpA-TpN17-biotin and EcSkp-TpN17-ruthenium were used in R1 (reagent buffer 1) and R2 (reagent buffer 2), respectively. The concentrations of the antigen conjugates in R1 and R2, respectively, were 200 ng/ml each.
In a first experiment, human sera negative for Treponema were assessed with the aforementioned DAGS immunoassay setup. In order to get a hint to the incidence rate of false positives which are solely targeted to the TpN17 moiety, the screening was performed in the presence of SS-Helix (GDA,P), a GDA-crosslinked soluble heterogeneous SlyD polymer which is customarily used as an anti-interference substance. In addition, EcSkp-EcSlyD-EcSlyD, another anti-interference module, was included in the assay buffer in order to rule out interference phenomena due to moieties different from the very TpN17 part. SS-Helix (GDA,P) and EcSkp-EcSlyD-EcSlyD were added to R1 (reagent 1 buffer containing the biotin conjugate) in large excess amounts (10 μg/ml). 75 μl R1 (reagent 1 buffer, biotin conjugate and anti-interference polymers), 75 μl R2 (reagent 2 buffer, ruthenium conjugate), 10 μl sample (human serum) and 40 μl bead suspension are then mixed and incubated to yield a reaction volume of roughly 200 μl.
By means of this approach, approximately 8 human sera could be found (out of  ̃2500 negative human sera) which clearly were negative for anti-Treponema pallidum antibodies but exhibited elevated signals in the DAGS format when using TpN17 as an antigen. Elecsys® signals were, however, not increased when other Treponema antigens such as TpN15 or TpN47 were used instead of TpN17 (data not shown). This finding points to a specific anti-TpN17 interference factor.
Table 2 illustrates the anti-interference effect of unlabeled monomeric EcSlyD-VcLp15 in an oligomeric TpN17 assay setup. EcFkpA-TpN17-Bi(DDS) and EcSkp-TpN17-BPRu(SK(2)DSS) were used as oligomeric detection antigens, and unlabeled EcSlyD-VcLp15 was added to R1 in increasing concentrations. It is obvious that the signals of the true positive sera are significantly quenched upon addition of Lp15. Yet, the positive signals clearly remain positive, even when VcLp15 is added in high concentrations such as 1 μg/ml to R1. As expected, well-characterized human sera (Trina Bioreactives AG, Nänikon, Switzerland) for which Treponema infections have clearly been ruled out, exhibit very low signals close to the system-inherent background signal ( ̃450 counts), irrespective of the addition of VcLp15. Interference sera such as C131839, C132663, C132723, R183554 and C132976 exhibit significantly elevated signals when no VcLp15 anti-interference module is added. Since the averaged signal of the negative sera amounts to  ̃770 counts, it is evident that signal levels of 15,146 counts (for human serum C132663) or 8,503 counts (for human serum C132723) would point to a true positive result raising the suspicion of a syphilis infection. It is remarkable that addition of VcLp15 at a concentration of 1.0 μg/ml in R1 reduces the elevated signals to the normal background signal level, revealing the putatively positive results as false positive. The same holds true when unlabeled monomeric EcSlyD-VcLp15 is added in a monomeric TpN17 DAGS setup (Tab. 3). Again, significantly elevated signal levels as with human serum C132663 are reduced from 11,128 counts to 651 counts when EcSlyD-VcLp15 is added to R1 at a concentration of 1.0 μg/ml. Conversely, oligomeric VcLp15 (EcSkp-VcLp15) efficiently quenches elevated and false positive signals, respectively, to the negative signal background both in the oligomeric (Tab. 4) and in the monomeric (Tab. 5) TpN17 DAGS setup.
TpN17 from Treponema pallidum and lipoprotein 15 from Vibrio cholerae share 34% sequence identity and 55% homology over a stretch of 97 amino acid residues. It is probably via this shared sequence (and probably structural) motif that the immunological cross-reaction takes place, evoking high signals and thus pretending a positive outcome in Treponema assays that are based on TpN17 antigen. Addition of the VcLp15 anti-interference protein to the assay mixture reduces the elevated signals to normal negatives, as shown in Tab. 2-5. The disclosed data provide compelling evidence that even very strong interferences, i.e. high false positives, may be efficiently eliminated by addition of Vibrio cholerae lipoprotein 15, in a monomeric or oligomeric form. The bottom line of the results shown in tables 2-5 is the following: interferences due to the use of recombinant TpN17 antigen in a DAGS immunoassay are rather frequent (>1 in 500 negative sera) and they can efficiently be mitigated by the addition of lipoprotein 15 from Vibrio cholerae, which is a bacterial genus phylogenetically rather distant from Treponema pallidum. Put simply, this application discloses that the addition of an easily available recombinant protein significantly improves the specificity of a TpN17-based Syphilis immunoassay for the detection of anti-Treponema antibodies.







TABLE 2







Oligomeric TpN17; addition of monomeric VcLp15









EcSlyD-VcLp15














R1

0
0.1
0.2
0.3
0.5
1.0


c (μg/ml)

counts
counts
counts
counts
counts
counts

















positive sera









PLTP_124
undil.
1,274,726
1,171,468
1,166,110
1,092,657
973,974
793,911


PLTP_124
1:50
154,146
156,165
136,898
131,248
101,395
62,840


PLTP_121
1:50
223,230
211,697
205,298
190,606
151,374
93,737


PLTP_121
1:100
112,031
105,466
103,909
91,727
72,926
47,972


BM 146027
SC_056
1,408,728
1,357,227
1,258,085
1,217,480
1,149,624
1,133,749


BM 146623
SC_058
1,187,013
1,158,037
1,083,204
1,083,952
1,078,868
1,050,374


negative sera









Trina #0642
neg.
778
716
722
698
698
661


Trina #0645
neg.
725
703
722
682
715
706


Trina #0646
neg.
797
795
817
802
784
784


Trina #0647
neg.
774
786
764
773
762
742


interference sera









C131839
false positive
5,795
5,090
4,464
3,861
2,851
1,555


C132663
false positive
15,146
2,116
1,110
878
765
724


C132723
false positive
8,503
7,725
6,811
5,357
2,298
941


R183554
false positive
11,072
10,030
9,142
7,413
4,488
2,534


C132976
false positive
5,687
5,174
4,446
3,690
2,509
1,419





R1 EcFkpA-TpN17-Bi(DDS)


R2 EcSkp-TpN17-BPRu(SK(2)DSS)













TABLE 3







Monomeric TpN17; addition of monomeric VcLp15









EcSlyD-VcLp15














R1

0
0.1
0.2
0.3
0.5
1.0


c (μg/ml)

counts
counts
counts
counts
counts
counts

















positive sera









PLTP_124
undil.
1,467,523
1,374,990
1,272,604
1,272,720
1,208,354
1,164,404


PLTP_124
1:50
167,294
155,940
153,077
136,428
124,244
89,204


BM 146624
undil.
1,451,271
1,423,730
1,376,393
1,359,799
1,370,884
1,369,198


BM 146624
1:10
436,534
403,003
384,434
365,742
329,370
259,984


BM 145855
SC_055
901,047
816,640
766,258
707,030
616,574
511,011


BM 146027
SC_056
1,721,399
1,708,261
1,678,979
1,651,647
1,641,513
1,641,787


negative sera









Trina #0642
neg.
657
613
638
626
623
610


Trina #0643
neg.
698
703
699
704
687
665


Trina #0644
neg.
660
653
638
636
620
616


Trina #0646
neg.
634
617
634
628
621
613


Trina #0647
neg.
623
614
606
620
618
623


interference sera









C131839
false positive
4,206
3,707
3,364
2,996
2,309
1,383


C132663
false positive
11,128
1,812
1,119
895
729
651


R179865
elev. signal
1,630
1,338
1,219
1,114
1,000
873


R183554
elev. signal
1,015
894
883
819
790
740


C132976
false positive
4,866
4,041
3,454
2,911
2,057
1,230





R1 EcSS-TpN17-Bi(DDS)


R2 PmSS-TpN17-BPRu(SK(2)DSS)













TABLE 4







Oligomeric TpN17; addition of oligomeric VcLp15









EcSkp-VcLp15











R1

0
0.1
1.0


c (μg/ml)

counts
counts
counts














positive sera






PLTP_121
1:50
230,387
225,976
104,039


PLTP_121
1:100
116,383
112,747
55,150


BM 140149
SC_052
1,136,096
1,077,292
508,996


BM 200680
SC_054
826,355
737,483
354,883


BM 145855
SC_055
745,762
704,713
322,241


BM 146027
SC_056
1,446,006
1,380,464
1,192,282


negative sera






Trina #0642
neg.
750
757
744


Trina #0646
neg.
840
847
815


Trina #0872
neg.
846
805
750


Trina #0873
neg.
764
763
754


interference sera






R179865
false positive
3,951
1,580
782


C131839
false positive
6,142
4,805
1,587


C132663
false positive
16,565
2,510
755


C132723
false positive
7,868
4,406
875


R183554
false positive
10,511
8,747
1,306


C132976
false positive
6,274
3,062
1,088





R1 EcFkpA-TpN17-Bi(DDS)


R2 EcSkp-TpN17-BPRu(SK(2)DSS)













TABLE 5







Monomeric TpN17; addition of oligomeric VcLp15









EcSkp-VcLp15











R1

0
0.1
1.0


c (μg/ml)

counts
counts
counts














positive sera






PLTP_121
1:50
235,946
221,210
112,238


PLTP_121
1:100
118,333
113,770
60,259


BM 140149
SC_052
1,142,874
1,048,211
637,784


BM 200680
SC_054
757,518
672,745
382,894


BM 145855
SC_055
861,637
823,346
475,266


BM 146027
SC_056
1,550,267
1,553,275
1,516,740


negative sera






Trina #0642
neg.
673
673
632


Trina #0646
neg.
651
659
646


Trina #0872
neg.
680
683
639


Trina #0873
neg.
690
661
651


interference sera






C132221
elev. signal
1,301
1,204
950


C131839
false positive
3,928
3,108
1,144


C132663
false positive
11,457
2,270
671


C132927
elev. signal
1,551
1,284
877


R183554
elev. signal
1,003
858
672


C132976
false positive
4,476
2,373
829





R1 EcSS-TpN17-Bi(DDS)


R2 PmSS-TpN17-BPRu(SK(2)DSS)

## Claims

1. A method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample comprising contacting the sample with a peptide sequence of Vibrio cholerae lipoprotein 15 (VcLp 15) comprising SEQ ID NO:1.

2. A method according to claim 1 wherein a peptide sequence according to UniProt ID Q9KQN6 (SEQ ID NO. 1) or SEQ ID NO. 2 or a partial sequence of SEQ ID NOs. 1 or 2 is used the peptide sequence of Vibrio Cholera lipoprotein 15 (VcLpl5) is SEQ ID NO: 1 or SEQ ID NO: 2.

3. A method according to claim 2 wherein said partial sequence of VcLp15 comprises amino acids 26-163 of SEQ ID NOs. 1 or 2.

4. A method according to claim 1 wherein said VcLp15 peptide sequence or partial sequence thereof is fused to a chaperone.

5. A method for detecting antibodies against the TpN17 antigen of Treponema pallidum in an isolated sample, said method comprising
a) forming an immunoreaction admixture by admixing a body fluid sample with a specific binding partner that can be specifically bound by said antibodies present in said sample;
b) adding SEQ ID NO. 1 or 2 and a chaperone to said immunoreaction admixture either before, at the same time or after said specific binding partner is added to said sample;
c) maintaining said immunoreaction admixture for a time period sufficient for allowing the antibodies present in said body fluid sample to immunoreact with said specific binding partner to form an immunoreaction product; and
d) detecting the presence and/or the concentration of any of said immunoreaction product.

6. A method according to claim 5 wherein two TpN17 antigens are used as specific binding partners for the antibodies to be detected in the isolated sample,
a first TpN17 antigen that comprises a TpN17 sequence and a first chaperone wherein said first TpN17 antigen can be bound to a solid phase,
a second TpN17 antigen that comprises a TpN17 sequence and a second chaperone wherein said second TpN17 antigen carries a detectable label,
wherein both TpN17 antigen are identical or immunologically cross-reactive so that they can be bound specifically by the antibodies present in the sample, and
wherein the first and the second chaperones are different.

7. A method according to claim 6 wherein the first TpN17 antigen comprises an E. coli FkpA as a chaperone and the second TpN17 antigen comprises E. coli Skp as a chaperone.