NZ577689A - Method of diagnosing Treponema palladium infection with lipid linked antigen - Google Patents

Abstract

Disclosed is a method for determining the likelihood of infection of a subject with Treponema palladium comprising the steps of: Contacting a sample of the plasma or serum of the subject with a suspension of cells modified to incorporate a water soluble peptide-lipid construct of the structure F-S-L or L-S-F to provide a mixture; Incubating the mixture for a time and at a temperature sufficient to allow agglutination; and Determining the degree of agglutination of the cells in the mixture; where: F is a peptide comprising the sequence: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu (SEQID NO: 3); S is a spacer covalently linking F to L; L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerophospholipids; and the degree of agglutination indicates the likelihood of infection of the subject.

Description

Patents Form No. 5 IPPC ref. K01.102NZ *10059249585* INTELLtCTUAL PROPERTY! OFFICE OF N.Z. I t 0 SEP 2010 I RECEI VEn) Patents Act 1953 Complete Specification We, KODE Biotech Limited of 19 Mount Street, Scott Laboratory Building, Auckland Univerity of Technology, Auckland , a New Zealand Company, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in the following statement: 1 Received at IPONZ on 26 November 2011 KOI.102NZ methods for the diagnosis of infection with TREPONEMA PALLADIUM field of invention The invention relates to methods for the diagnosis of infection with Treponema palladium. In particular, the invention relates to methods for the diagnosis of infection with Treponema palladium in donors of blood intended for transfusion. background art The risk of transfusion transmitted infections (TTIs) requires the testing of donor units of blood for the presence of pathogens. The testing methods employed need to be sensitive, specific and cost effective. The requirement for cost effectiveness is of particular importance given that many TTIs 15 are prevalent in economically disadvantaged regions of the world.

A pathogen of particular concern in transfusion medicine is Treponema palladium. This organism is the causal agent of syphilis. Research has been performed to identify those 20 antigens of Treponema palladium that may be responsible for eliciting an immunogenic response. The presence of antibody in the sera of donors is indicative of infection.

Known assays for the serological diagnosis of syphilis include the rapid plasma reagin (RPR) card test (Becton Dickinson 25 Microbiology Systems, Cockeysville, MD) and the solid phase erythrocyte adherence (SPEA) assay CAPTURE-S™ (Immucor, Inc., Norcross, GA) (Stone et al (1997)).

These two assays compare favourably in terms of sensitivity and specificity. The solid phase erythrocyte adherence assay 30 provides additional benefits of ease of use, accommodation of high-volume testing, and the potential for automation, e.g. the GALILEO ECHO™ platform (Immucor, Inc., Norcross, GA). 2 Received at IPONZ on 26 November 2011 KOI.102NZ The specification accompanying international application no, PCT/NZ2008/000239 (publ. no. WO 2009/035347) describes a diagnostic method based on the use of peptide-lipid constructs that is adaptable for use on existing tube serology platforms. 5 Additional peptide-lipid constructs suitable for use in the diagnostic method disclosed are described in the specification accompanying international application no. PCT/NZ2008/000266 (publ. no. WO 2009/048343).

It is an object of the invention to provide an improved assay for serological diagnosis of infection with Treponema palladium.

It is an object of the invention to provide an improved assay for the detection of antibodies in donated blood that are indicative of the donor being infected with Treponema palladium.

These objects are to be read disjunctively with the object to at least provide a useful choice. statements of invention In a first aspect the invention provides a method for determining the likelihood of infection of a subject with Treponema palladium comprising the steps of: • Contacting a sample of the plasma or serum of the subject with a suspension of cells modified to incorporate a water soluble peptide-lipid construct of the structure F-S-L or L-S-F to provide a mixture; • Incubating the mixture for a time and at a temperature sufficient to allow agglutination; and • Determining the degree of agglutination of the cells in the mixture; where: F is a peptide comprising the sequence: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu (SEQID NO: 3); 3 Received at IPONZ on 26 November 2011 KOI.102NZ S is a spacer covalently linking F to L; L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerophospholipids; and the degree of agglutination indicates the likelihood of 5 infection of the subject.

Preferably, the method includes prior to determining the degree of agglutination of the cells of the mixture the intermediate step of: • Adding an anti-subject globulin antibody to the mixture. 10 Preferably, the subject is a human.

Preferably, the cells are RBCs.

Preferably, the plasma or serum of a subject is obtained from a donated sample of blood.

Preferably, the anti-subject globulin antibody is anti-human 15 globulin (AHG) antibody.

S is selected to provide a construct that is water soluble. [followed by page 5] 4 Received at IPONZ on 26 November 2011 KOI.102NZ Preferably, the peptide-lipid construct is of the structure; AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeuCys ,=CT . ' ,Vi o.

•(CH 0 N- °\ V-NH 0 NH*' o. r(fH. i OH 0 NH-^ >=° ^ 0 < / NH vn^° /—NH /—N ,0 nh-^t ^ OH 0 /— 0 OH _/-nh V M—' ^-N v\J ■N 0 NHJ °.\ r\ 0 \ 0 /-NH 0 OH ^"(CH2)n ^NH ,0 0 o=< 0=< R- R, where: Rx and R2 are independently selected from the group 5 consisting of: alkyl or alkenyl substituents of the fatty acids trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-ll-octadecenoic acid, cis-11-10 octadecenoic acid, cis-ll-eicosenoic acid or cis-13- docsenoic acid; m is the integer 3, 4 or 5; n is the integer 1, 2 or 3; and M is a monovalent cation such as H+, Na+, K+ or NH4+. 15 Preferably, m is the integer 5 and n is the integer 2, Received at IPONZ on 26 November 2011 KOI.102NZ In an embodiment of the first aspect the invention provides a method for determining the likelihood of infection of a human subject with Treponema palladium comprising the steps of: • Contacting a sample of the plasma or serum of the subject 5 with a suspension of RBCs modified to incorporate the water soluble peptide-lipid construct designated FSL-SYPH3; • Incubating the contacted sample of the plasma or serum and the suspension of RBCs for a time and at a temperature sufficient to allow binding of antibodies present in the sample to the RBCs; • Preparing a suspension of washed RBCs; • Adding an amount of anti-human globulin (AHG) antibody to the suspension to provide a mixture; • Incubating the mixture for a time and at a temperature sufficient to allow agglutination; and • Determining the degree of agglutination of the cells in the mixture to indicate the likelihood of infection.

In a second aspect the invention provides a method of testing donated blood for the presence of antibodies indicative of the donor being infected with Treponema palladium comprising the steps of: • Contacting a sample of plasma or serum of the donated blood with an immobilized layer of modified RBCs; • Incubating the contacted sample of plasma or serum and the immobilized layer of modified RBCs for a time and at a temperature sufficient to allow binding of the antibodies; • Washing the immobilized layer of modified RBCs to remove the sample of plasma or serum; • Contacting the immobilized layer of modified RBCs with a suspension of anti-immunoglobulin coated indicator cells; • Incubating the immobilized layer of modified RBCs and the suspension of anti-immunoglobulin coated indicator cells for a time and at a temperature sufficient to allow 6 Received at IPONZ on 26 November 2011 KOI.102NZ binding of the anti-immunoglobulin coated indicator cells to the immobilized layer of modified RBCs; and • Determining the degree of adherence of the antiimmunoglobulin coated indicator cells to the immobilized layer of modified RBCs, where the modified RBCs have been modified to incorporate a water soluble peptide-lipid construct of the structure F-S-L where: F is a peptide comprising the sequence: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu (SEQID NO: 3); S is a spacer covalently linking F to L; L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerophospholipids; and the degree of adherence is indicative of the likelihood of the antibodies being present.

Preferably, the determining the degree of adherence of the antiglobulin coated indicator cells to the immobilized layer of modified RBCs is by centrifugation to pellet unbound antiglobulin coated indicator cells.

S is selected to provide a construct that is water soluble, [followed by page 8] 7 Received at IPONZ on 26 November 2011 KOI.102NZ Preferably, the peptide-lipid construct is of the structure; AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeuCys ,=CT . ' ,Vi o.

•(CH 0 N- °\ V-NH 0 NH*' o. r(fH. i OH 0 NH-^ >=° ^ 0 < / NH vn^° /—NH /—N ,0 nh-^t ^ OH 0 /— 0 OH _/-nh V M—' ^-N v\J ■N 0 NHJ °.\ r\ 0 \ 0 /-NH 0 OH ^"(CH2)n ^NH ,0 0 o=< 0=< R- R, where: Rx and R2 are independently selected from the group 5 consisting of: alkyl or alkenyl substituents of the fatty acids trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-ll-octadecenoic acid, cis-11-10 octadecenoic acid, cis-ll-eicosenoic acid or cis-13- docsenoic acid; m is the integer 3, 4 or 5; n is the integer 1, 2 or 3; and M is a monovalent cation such as H+, Na+, K+ or NH4+. 15 Preferably, m is the integer 5 and n is the integer 2, 8 Received at IPONZ on 26 November 2011 KOI.102NZ In a third aspect the invention provides a water soluble peptide-lipid construct of the structure F-S-L where: F is a peptide comprising the sequence: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu 5 (SEQID NO:3); S is a spacer covalently linking F to L; L is a lipid selected from the group consisting of diacyl-and dialkyl-glycerophospholipids.

Preferably, the water soluble peptide-lipid construct is of the 10 structure: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeuCy^ °<r N-<\ /-(CH2)n° OH 0. NH"^ to ^ o < ! NH /—NH VNIi /—N ,0 nh^t ^ OH 0 / 0 OH fj \ ' NH 0 N' °v\ ^ V-NH 0 NH-' °x\ ^ v-N o NH ) ^=\ o r~~^\ \ 0 y-NH 0 OH ^<CH2' ^,NH o'V u K -P °=< °=< R-_ R2 where: Ri and R2 are independently selected from the group consisting of: alkyl or alkenyl substituents of the fatty 15 acids trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-ll-octadecenoic acid, cis-11- 9 Received at IPONZ on 26 November 2011 KOI.102NZ octadecenoic acid, cis-ll-eicosenoic acid or cis-13-docsenoic acid; m is the integer 3, 4 or 5; n is the integer 1, 2 or 3; and M is a monovalent cation such as H+, Na+, K+ or NH4+.

Preferably, m is the integer 5 and n is the integer 2.

"Antibody reactive to an antigen" means an immunoglobulin, the presence of which in the serum of a subject is indicative of a phenotype or pathological condition of the subject.

"PCV" or "pcv" denotes packed cell volume.

"Plasma" means the colourless fluid part of blood or lymph, in which corpuscles or fat globules are suspended.

"RBC" or "rbc" means red blood cell.

"Saline" means a solution of one or more salts.

"Serum" means the amber-coloured, protein-rich liquid which separates out when blood coagulates.

"Solid phase immunoassay" means an assay in which one component of the immunological reaction, either antigen or antibody, is immobilized onto the surface of a solid phase support and in this context the term "antigen" includes mammalian cells such as erythrocytes (RBCs), leukocytes, lymphocytes, platelets and components of such cells including components prepared by lysing the cells .

"Solid phase support" means an article of manufacture composed 25 of an organic polymer such as polystyrene, polypropylene, polyvinylchloride or nylon, or other materials that are capable of binding a monolayer of mammalian cells, or capable of being adapted to bind a monolayer of mammalian cells, such as glass.

"Synthetic" means made by chemical synthesis.

Received at IPONZ on 26 November 2011 KOI.102NZ "Water soluble" means a stable, single phase system is formed when the construct is contacted with water or saline (such as PBS) at a concentration of at least 100 jig/ml and in the absence of organic solvents or detergents. The terms "soluble" and 5 "dispersible" are used synonymously.

The amino acid residues of peptides are identified according to Table 3 of Appendix 2 of Annex C of the Administrative Instructions under the Patent Cooperation Treaty dated 7 February 2007 and in accordance with the convention: H2N-XaaXaaXaa XaaXaaXaa-COOH Exemplifying embodiments of the invention will now be described in detail with reference to the Figures of the accompanying drawings pages. brief description of drawings Figure 1. The structure of a peptide-lipid construct (F-S-L) of the invention comprising the peptide sequence: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu(SEQID NO: 3) with conjugation via the sulfhydryl residue of a Cys residue located at the carboxy terminus of the sequence designated FSL-SYPH3.

Figure 2. Degrees of adherence of anti-immunoglobulin coated indicator cells to an immobilized layer of modified RBCs in Microtitre plate wells following centrifugation. detailed description Many peptide sequences are known to be immunogenic, i.e. capable 25 of eliciting the production of antibody. However, immunogenic peptide sequences are not equally capable of interacting with elicited antibody and therefore providing the basis for a diagnostic assay.

The following peptide has been shown to be particularly capable 30 of interacting with antibodies, the presence of which in the serum of a subject is considered to be indicative of infection by Treponema palladium: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu (SEQID NO:3) 11 Received at IPONZ on 26 November 2011 KOI.102NZ This selected peptide sequence (designated SYPH3) has been demonstrated to be superior to the following peptide sequences when used in the diagnostic method described in the specification accompanying international application no. 5 PCT/NZ2008/000239 (publ. no. WO 2 00 9/035347): ArgValMetTyrAlaSerSerGly (SEQID NO:l) designated SYPH1 and: ProGluLysAlaPheArgGluLeu (SEQID NO:2) designated SYPH2.

The peptides were prepared in the form of peptide-lipid constructs (F-S-L) as described in the specification accompanying international application no. PCT/NZ2008/000266 (publ. no. WO 2009/048343). Each peptide was conjugated via the sulfhydryl residue of a Cys residue located at the carboxy 15 terminus of the sequence (cf. Figure 1). example 1 In a first study, a comparison of RBCs modified to incorporate peptide-lipid constructs (F-S-L) comprising one of the peptide (F) sequences designated SYPH1, SYPH2 and SYPH3 was performed.

RBCs were modified by contacting a suspension of the cells (1 part pcv) with a dispersion of construct at a concentration of 100 jig/mL or 50 jig/mL (2 parts pcv) at 37 °C for 1 hour.

Modified cells (kodecytes) were washed three times in phosphate buffered saline (PBS) and resuspended.

The reactivity of clinical samples (n=95) indicated to be positive for Syphilis by EIA (BioKit or ICE) was assessed employing the diagnostic method described in the specification accompanying international application no. PCT/NZ2008/000239 (publ. no. WO 2009/035347).

Each of the 95 samples was assessed against RBCs modified to incorporate the FSL-SYPH3 construct at concentrations of either 12 Received at IPONZ on 26 November 2011 KOI.102NZ 100 jig/mL (SYPH3-100 kodecytes) or 50 pg/mL (SYPH3-50 kodecytes).

In addition 10 of the samples were assessed against RBCs modified to incorporate the FSL-SYPH1 construct at a 5 concentration of 100 jig/mL (SYPH1-100 kodecytes) and 41 of the samples were assessed against RBCs modified to incorporate the FSL-SYPH2 construct at a concentration of 100 pg/mL (SYPH2-100 kodecytes).

Briefly, a suspension of 3% (pcv/v) kodecytes is prepared in PBS 10 and 30 jil of the suspension mixed with 30 jil of sample plasma or serum. The mixtures are then incubated for 45 min at 37 °C. Following incubation the kodecytes are centrifuged for 10 s in an Immufuge™ (setting: "high") and observed for agglutination before being washed 3 times with PBS.

After washing one drop of Epiclone™ anti-human globulin (AHG) is added and the tubes then centrifuged for 10 s in an Immufuge™ (setting: "high"). Tubes are then read and serology scores recorded.

The reactivity of the samples is recorded in Table 1.

Method n Reactive % Reactive EIA (BioKit or ICE) 95 95 100 TPHA 86 79 92 TPPA 22 19 86 RPR 94 53 56 SYPH1-100 kodecytes 0 0 SYPH2-100 kodecytes 41 0 0 SYPH3-100 kodecytes 42 37 CO CO SYPH3-50 kodecytes 95 56 59 Table 1. Reactivity of EIA Syphilis positive clinical samples confirmed positive by reactivity with either TPHA or TPP. 13 Received at IPONZ on 26 November 2011 KOI.102NZ Although none of the tested samples were reactive when assessed using the SYPH1-100 kodecytes and SYPH2-100 kodecytes, rates of reactivity when assessed using the SYPH3-100 kodecytes were comparable with those obtained for the confirmatory assays RPR, 5 TPHA and TPPA.

Notably when the reactivity of samples determined to be false positives (negative confirmatory assay) was assessed against SYPH3-100 kodecytes all samples were determined to be negative. example 2 In a second study, the reactivity of syphilis Qualification Panel samples (n=5) (SeraCare Life Sciences) confirmed to be positive using the Olympus PK TP System, and syphilis validation samples (n=20) (Plasma Services Group, Inc) confirmed to be positive using the Arlington Scientifice RPR and EuroimmunTP IgG 15 and IgM ELISA assays, was compared in two solid phase immunoassays ("Test Method 1" and "Test Method 2").

Both Test Method 1 and Test Method 2 were performed on a GALILEO ECHO™ automated blood bank instrument (Immucor Gamma). In Test Method 1 the reactivity of the samples (n=25) was evaluated 20 using CAPTURE-S™ plates (Immucor Gamma). In Test Method 2 the reactivity of the samples (n=25) was evaluated using SYPH3-10 kodecytes immobilized onto the surface of plates (CAPTURE-FSL-SYPH3™ plates).

SYPH3-10 kodecytes were created by suspending a packed cell 25 volume (pcv) of group O red blood red cells in an equal volume of a solution containing the FSL-SYPH3 construct at a concentration of 10 pg/ml and incubating for 120 mins at 37°C, followed by washing.

The CAPTURE-FSL-SYPH3™ plates were then prepared mutatis 30 mutandis according to the methods described in Sinor et al (1989), Sinor et al (1990) and Sinor and Eatz (1991).

Briefly, a MICROTITRE™ plate having a plurality of recessed wells is first treated with a sufficient quantity of Alcian 14 Received at IPONZ on 26 November 2011 KOI.102NZ yellow at a concentration of 0,1 mg/ml so that all of the wells are covered by the Alcian yellow solution. The Alcian yellow solution is allowed to remain in contact with the MICROTITRE™ plate for about 30 minutes and excess dye solution is removed.

A monolayer of the SYPH3-10 kodecytes is formed on the stained plate by addition of 100 jiL of a 0.2% (v/v) suspension of the SYPH3-10 kodecytes in saline or reagent RBC diluent per well. The SYPH3-10 kodecytes are allowed to settle by gravity for one hour at room temperature. The plate is then washed at least two 10 times with isotonic saline to remove unbound SYPH3-10 kodecytes by an automatic washing apparatus or by manual methods.

One hundred microlitres (100 jiL) of a drying solution containing 1.0 M dextrose and 154 mM sodium chloride is added to each well. Immediately following addition of the drying solution, each well 15 is aspirated to remove any excess drying solution.

The MICROTITRE™ plate is then placed in an inverted position into a foil packet or pouch having at least two, 2 g capacity molecular sieve dessicant packets therein. The pouch is sealed with heat and dried for seven days at 2°to 8°C.

The CAPTURE-S plates and the CAPTURE-FSL-SYPH3 plates are each used according to the standard test method. Briefly, the foil package containing each plate is opened and one drop of a biological fluid such as serum or plasma or a control is added to each. Two drops of a 19 g/L solution of glycine with a 25 preservative and a dye for colour is also added to each well.

The solutions are allowed to incubate in the wells at 37°C for 15 minutes. After the incubation period, the wells of the MICROTITRE™ plate are washed three times with saline, preferably by an automatic washing apparatus such as the Bio-Tek model EL-30 402.

One drop of anti-IgG coated indicator RBCs is then added to each well. The MICROTITRE™ plate is centrifuged at 450 x G for one minute. The plate is then examined for adherence or lack of Received at IPONZ on 26 November 2011 KOI.102NZ adherence of the indicator RBCs to the erythrocyte cell monolayer (Figure 2).

A positive reaction is seen by adherence of the indicator red cells over the reaction surface. A negative reaction forms a 5 discreet button of indicator red cells at the bottom of the wells showing no adherence (Figure 2).

Thus, if the biological fluid being tested has antibodies directed towards the erythrocyte cell monolayer, it binds to the erythrocyte monolayers. The anti—IgG coated indicator RBCs 10 correspondingly bind to the antibody thus bound when they are added to the wells. This gives the positive reaction.

If no antibodies directed toward the erythrocytes are present in the biological fluid being tested, the anti-IgG coated indicator RBCs will have no complimentary immunologically component to 15 bind to and will collect at the bottom of the well as a discreet button.

The results obtained from Test Method 1 (CAPTURE-S) and Test Method 2 (CAPTURE-FSL-SYPH3) are presented in Table 2.

Sample +ve (n=2 5) -ve (n=32) Test method 1 (CAPTURE-S) +ve assay 0 -ve assay 32 Test method 2 (CAPTURE-FSL-SYPH3) +ve assay 24 0 -ve assay 1 32 Table 2. Comparison of sensitivity and specificity of two solid phase immunoassays ("Test Method 1" and "Test Method 2").

Test Method 2 (CAPTURE-FSL-SYPH3) demonstrated improved sensitivity over Test Method 1 (CAPTURE-S). Test Method 2 (CAPTURE-FSL-SYPH3) 25 provides the additional advantage that the introduced antigen (F) of the FSL construct is expressed against the background antigens of the naturally occurring RBCs that are modified to provide the kodecytes. 16 Received at IPONZ on 26 November 2011 KOI.102NZ Although the invention has been described by way of examples it should be appreciated that variations and modifications may be made to the claimed methods without departing from the scope of the invention. It will be understood that for a non-specific 5 interaction, such as the interaction between the diacyl- or dialkyl- glycerolipid portion of the functional-1ipid constructs and a membrane, structural and stereo-isomers of naturally occurring lipids can be functionally equivalent.

Where known equivalents exist to specific features, such 10 equivalents are incorporated as if specifically referred to in this specification. For example, it is contemplated that diacylglycerol 2-phosphate could be substituted for phosphatidate (diacylglycerol 3-phosphate) and that the absolute configuration of phosphatidate could be either R or S. 17 Received at IPONZ on 26 November 2011 Enlarged representation of the structural formula presented in the foregoing Statements of Invention and following Claimsi AlaSerClyAlaLysCluCluAluCluLysLysAlaAluCluClriArgAluLeuLeuCy^ on n r('di2) -NH V -NH /—N .C nh^t ^ _ OH o /— o OH Nh. o \ r\ NH > q, 0=\ 0 y-NH O on hah).

J.TH ,0 .0 0=< 0=< Ri R, 18 Received at IPONZ on 26 November 2011 KOI.102NZ references Antoni et al(1996) Detection of antigenic determinants in the Treponema pallidum membrane protein TmpA using overlapping synthetic peptides J Immunol Methods, 189(1), 137-140.

Baughn et al(1996) Epitope mapping of B-cell determinants on the 15-kilodalton lipoprotein of Treponema pallidum (Tppl5) with synthetic peptides Infect Immun, 64(7), 2457-2466.

Coombs et al (1956) Specific mixed agglutination: Mixed erythrocyte-platelet anti-globulin reaction for the detection of platelet 10 antibodies Brit. J. Haemat., 2,84-94.

IEDB (2007) Immune epitope database and analysis report.

Manavi et al (2006) The sensitivity of syphilis assays in detecting different stages of early syphilis Int J STD AIDS, 17(11), 768-771.

McKevitt et al (2005) Genome scale identification of Treponema 15 pallidum antigens Infect Immun, 73(7), 4445-4450.

Morgan et al (2002) Segregation of B and T cell epitopes of Treponema pallidum repeat protein K to variable and conserved regions during experimental syphilis infection J Immunol, 169(2), 952-957.

Purcell et al (1990) Lipid modification of the 15 kiloDalton major 20 membrane immunogen of Treponema pallidum Mol Microbiol, 4(8), 1371-1379 .

Ratnam (2005) The laboratory diagnosis of syphilis Can J Infect Dis Med Microbiol, 16(1), 45-51.

Sinor and Eatz (1989) Solid phase indicator red blood cells and method 25 United States Patent No. 4,816,413.

Sinor et al (1990) Article for preforming [sic] immunological assays utilizing organic dyes and method for producing and utilizing same United States Patent No. 4,963,478.

Sinor et al (1991) Method for drying mammalian cells for use in solid 30 phase immunoassays and articles incorporating same United States Patent No. 5,030,560.

Stone et al (1997) Capture-S, a nontreponemal solid-phase erythrocyte adherence assay for serological detection of syphilis J Clinical Microbiology, 35(1), 217-222.

WHO (2008) 10 Facts on blood transfusion from http://www.who.int/bloodsafety/FactFile2 00 8.pdf 19

Claims (16)

Received at IPONZ on 26 November 2011 KOI.102NZ what we claim is:

1) A method for determining the likelihood of infection of a subject with Treponema palladium comprising the steps of: 5 • Contacting a sample of the plasma or serum of the subject with a suspension of cells modified to incorporate a water soluble peptide-lipid construct of the structure F-S-L or L-S-F to provide a mixture; 10 • Incubating the mixture for a time and at a temperature sufficient to allow agglutination; and • Determining the degree of agglutination of the cells in the mixture; where: 15 F is a peptide comprising the sequence: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu (SEQID NO: 3); S is a spacer covalently linking F to L; L is a lipid selected from the group consisting of 20 diacyl- and dialkyl-glycerophospholipids; and the degree of agglutination indicates the likelihood of infection of the subject.

2) The method of claim 1 where the method includes prior 25 to determining the degree of agglutination of the cells of the mixture the intermediate step of:

• Adding an anti-subject globulin antibody to the mixture. 30 3) The method of claim 1 where the subject is a human,

4) The method of claim 1 where the cells are RBCs.

5) The method of claim 1 where the plasma or serum of a 35 subject is obtained from a donated sample of blood. 20 Received at IPONZ on 26 November 2011 KOI.102NZ

6) The method of claim 2 where the anti-subject globulin antibody is anti-human globulin (AHG) antibody.

7) The method of claim 1 where the peptide-lipid construct is of the structure: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeuCys °<r ,n-\ r{ ch2 ) n° OR 0. NH-^ >=° ^ 0 < i nh i—nh vk /—N ,0 nh' oh 0. jn v\ y w-7 "o oh / -NH o' N— ' •nh 0 nh'' ~0 nh-' ) 0- °=v 0 ^-NH 0 /"(CH2 ) n ^NH p 0 °=< 0^ r- r, where: Ri and R2 are independently selected from the group consisting of: alkyl or alkenyl substituents of the 10 fatty acids trans-3-hexadecenoic acid, cis-5- hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-15 11-eicosenoic acid or cis-13-docsenoic acid; m is the integer 3, 4 or 5; n is the integer 1, 2 or 3; and M is a monovalent cation such as H+, Na+, K+ or NH4+. 20

8) The method of claim 7 where m is the integer 5 and n is the integer 2. 21 Received at IPONZ on 26 November 2011 KOI.102NZ

9) A method for determining the likelihood of infection of a human subject with Treponema palladium comprising the steps of: • Contacting a sample of the plasma or serum of the 5 subject with a suspension of RBCs modified to incorporate the water soluble peptide-lipid construct designated FSL-SYPH3; • Incubating the contacted sample of the plasma or serum and the suspension of RBCs for a time and at 10 a temperature sufficient to allow binding of antibodies present in the sample to the RBCs; • Preparing a suspension of washed RBCs; • Adding an amount of anti-human globulin (AHG) antibody to the suspension to provide a mixture; 15 • Incubating the mixture for a time and at a temperature sufficient to allow agglutination; and • Determining the degree of agglutination of the cells in the mixture to indicate the likelihood of infection. 20

10) A method of testing donated blood for the presence of antibodies indicative of the donor being infected with Treponema palladium comprising the steps of: • Contacting a sample of plasma or serum of the 25 donated blood with an immobilized layer of modified RBCs; • Incubating the contacted sample of plasma or serum and the immobilized layer of modified RBCs for a time and at a temperature sufficient to allow 30 binding of the antibodies; • Washing the immobilized layer of modified RBCs to remove the sample of plasma or serum; • Contacting the immobilized layer of modified RBCs with a suspension of anti-immunoglobulin coated 35 indicator cells; 22 Received at IPONZ on 26 November 2011 KOI.102NZ • Incubating the immobilized layer of modified RBCs and the suspension of anti-immunoglobulin coated indicator cells for a time and at a temperature sufficient to allow binding of the anti- 5 immunoglobulin coated indicator cells to the immobilized layer of modified RBCs; and • Determining the degree of adherence of the antiimmunoglobulin coated indicator cells to the immobilized layer of modified RBCs, 10 where the modified RBCs have been modified to incorporate a water soluble peptide-lipid construct of the structure F-S-L where: F is a peptide comprising the sequence: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu 15 (SEQID NO: 3); S is a spacer covalently linking F to L; L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerophospholipids; and the degree of adherence is indicative of the likelihood 20 of the antibodies being present.

11) The method of claim 10 where the determining the degree of adherence of the anti-globulin coated indicator cells to the immobilized layer of modified RBCs is by 25 centrifugation to pellet unbound anti-globulin coated indicator cells. 23 Received at IPONZ on 26 November 2011 KOI.102NZ

12) The method of claim 10 where the peptide-lipid construct is of the structure: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeuCys °<r r{ch2)° oh 0, nh^v, )=0 ^ ° ( j nh /—NH VNIi /—N ,0 NH^ oh 0 / 0 oh fr\ v-nh 0^O °\\_ r~^ ° o. r~NH o ^<ch2)n ^NH nhy °a r~{ f~\ 0 •NH J 0 N- Q> r~^ -P P °=< °=< R: r2 where: 5 Ri and R2 are independently selected from the group consisting of: alkyl or alkenyl substituents of the fatty acids trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-10 octadecenoic acid, trans-9-octadecenoic acid, trans- 11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid; m is the integer 3, 4 or 5; n is the integer 1, 2 or 3; and 15 M is a monovalent cation such as H+, Na+, K+ or NH4+.

13) The method of claim 12 where m is the integer 5 and n is the integer 2. 20

14) A water soluble peptide-lipid construct of the structure F-S-L where: F is a peptide comprising the sequence: 2 4 Received at IPONZ on 26 November 2011 KOI.102NZ AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeu (SEQID NO:3); S is a spacer covalently linking F to L; L is a lipid selected from the group consisting of 5 diacyl- and dialkyl-glycerophospholipids.

15) The construct of claim 14 where the water soluble peptide-lipid construct is of the structure: AlaSerGlyAlaLysGluGluAlaGluLysLysAlaAlaGluGlnArgAlaLeuLeuCy^ °<r r{ch2)° oh 0, nh^v, )=o f-' o < t nh /—NH VH /—N ,0 nh-^ oh 0 / 0 oh nh' °a r~^ J~\ 0 nh j r\ 0=\ jri X\ -y w—' J-WA '0 / -nh 6' n- ' 0- \ 0 ^-nh 0 oh ^"(ch2)n ^NH P 0 °=< o=< r- r, 10 where: Ri and R2 are independently selected from the group consisting of: alkyl or alkenyl substituents of the fatty acids trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-15 hexadecenoic acid, cis-6-octadecenoic acid, cis-9- octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid; m is the integer 3, 4 or 5; 20 n is the integer 1, 2 or 3; and M is a monovalent cation such as H+, Na+, K+ or NH4+. 25 Received at IPONZ on 26 November 2011 KOI.102NZ

16) The construct of claim 15 where m is the integer 5 and n is the integer 2 . By our attorney Stephen R. Parker, PhD 26