WO2009074808A1 - Syphilis detection reagent - Google Patents

Abstract

An isolated Treponema pallidum protein for use in an assay to detect the presence of Treponema pallidum in a sample, characterised in that the protein is reduced and/or denatured. Also disclosed is a reduced and/or denatured T. pallidum protein conjugate, methods of making a reduced and/or denatured T. pallidum protein and T. pallidum protein conjugates, use of same, a kit comprising same for the detection of T. pallidum in a sample, and a method of detecting T. pallidum in a sample.

Description

Syphilis detection reagent

The present invention relates to the fields of microbiology and immunology and more specifically relates to the detection of Treponema pallidum, and a reagent which can be used for detecting, diagnosing and monitoring the treatment of syphilis. In particular, the invention pertains to a modified T. pallidum antigen and its use in immunoassays.

Syphilis is a bacterial infection that is usually sexually transmitted, but may also be passed from an infected mother to her unborn child. Syphilis is one of a group of diseases caused by spirochaete organisms of the genus Treponema. Spirochaetes is a phylum of distinctive Gram-negative bacteria, which have long, helically coiled cells.

Sexually acquired syphilis occurs worldwide and is caused by Treponema pallidum subspecies pallidum. Related treponemes cause the non-venereal treponematoses bejel, or endemic syphilis (T. pallidum endemicum), yaws (T. pallidum pertenue), and^r pinta (T. carateum).

The natural history of syphilis is very variable. The course of the infection spans many years and may lead to various clinical presentations, which are classified into early (infectious) and late (non-infectious) stages (Egglestone S.I. and Turner AJ. L., Communicable Disease and Public Health 2000; 3: 158-162).

The first symptoms of syphilis may not be seen immediately. The primary stage begins within 10 days to three months of being infected and is characterised by a lesion, ulcer or sore - called a chancre - which may be painless and which disappears within a few weeks. Secondary syphilis begins three to six weeks after the chancre appears. It is marked by a skin rash and other symptoms such as fever and sore throat. If untreated, syphilis may continue into a latent stage, during which there are no symptoms and where the patient is no longer contagious. However, about one-third of all cases will progress into the complications of late, or tertiary, syphilis. In these cases, the bacteria can damage the heart, eyes, brain, nervous system, bones, joints, or almost any other part of the body. This stage can last for years, with the final stage leading to mental illness, blindness, other neurological problems, heart disease, and death.

The effects of syphilis in pregnant women are serious because of the consequential effects on the unborn child. It is likely that an untreated pregnant woman with active syphilis will pass the infection to her unborn child, and about one quarter of these pregnancies result in stillbirth or neonatal death. As infected infants become older they may develop the symptoms of late- stage syphilis including bone, tooth, eye, ear, and brain damage. An estimated 12.22 million cases of syphilis occurred worldwide in 1999 ("Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections Overview and Estimates", World Health Organization, 2001 ). T. pallidum is sensitive to penicillin, which continues to be the drug of choice for syphilotherapy (Deka,RK.,Machius, M., Norgard, MV., and Tomchick, DR., J. Biol. Chem., Vol. 277, Issue 44, 41857-41864, November 1 , 2002). Despite the availability of effective antibiotics, syphilis continues to result in worldwide morbidity and mortality because the disease can go undiagnosed, and can continue to be transmitted sexually. Moreover, by going undetected or undiagnosed, the disease can exert extensive damage, which may not be resolved by therapy. This can be a particular problem in the case of cardiovascular syphilis, neurosyphilis, and the congenital form of the disease. Though treatment for treponemal infection is available, control of treponemal diseases is managed by eliminating person to person spread. Accordingly, early detection of treponemal infection is vital for reducing widespread dissemination of related diseases.

As detection of T. pallidum by dark-field microscopy or direct immunofluorescence is possible only in the early stages of the disease, serology has become the method of choice for syphilis diagnosis. Serological tests for syphilis are widely used, for example in the screening of pregnant women; screening genitourinary medicine clinic attendees at recent risk of acquiring a sexually transmitted infection; screening blood and organ/tissue donors; detecting or excluding current or past syphilis in patients with HIV infection; testing patients whose history or clinical signs are consistent with syphilis, confirmatory testing of specimens reactive in screening tests for syphilis; and assessment of the stage of infection and monitoring the therapeutic response.

Serology cannot distinguish between the different treponematoses (syphilis, yaws, pinta, and bejel).

The immune response to syphilis involves production of antibodies to a broad range of antigens, including non-specific antibodies (cardiolipin or lipoidal antibody) and specific treponemal antibodies (Egglestone S.I. & Turner AJ. L., Communicable Disease and Public Health 2000; 3: 158-162). Serological tests for syphilis include non-treponemal tests and treponemal tests, which detect non-specific treponemal antibody and specific treponemal antibody, respectively (Young H., Int. J STD AIDS 1992; 3: 391-413).

The most common screening tests are the rapid plasma reagin (RPR) and Venereal Disease Research Laboratory (VDRL) tests, both of which test for the presence of antilipoidal antibodies. The antilipoidal antibodies recognize lipid material released from damaged host cells, and from T. pallidum. Because neither of these tests utilise syphilis-specific antibodies, there are problems associated with both their specificity and their sensitivity. Irrespective of the screening method employed, confirmation of a reactive screening test is essential, especially as false-positive results are associated with conditions such as pregnancy, autoimmune diseases, borreliosis, human immunodeficiency virus infection, malignancy, and chronic liver disease. The confirmatory test should ideally have equivalent sensitivity and greater specificity than the screening test and be independent methodologically, so as to reduce the chance of coincident false positive reactions (Egglestone S.I. & Turner A.J. L., Communicable Disease and Public Health 2000; 3: 158- 162).

Confirmatory tests include FTA- Abs (fluorescent treponemal antibody absorption test), MHA- TP (micro-heamagglutination assay for T. pallidum), and TPHA (T. pallidum heamagglutination assay), which use crude T. pallidum antigens; tests using whole T. pallidum antigen extracts; and a variety of T. pallidum recombinant protein tests, for example enzyme immunoassay (EIA) tests.

The combination of VDRL and TPHA tests provides sensitive and specific screening for all stages of syphilis other than very early primary infection but it is more labour intensive than a single screening test, requires subjective interpretation, and cannot readily be automated (Young H., Dermatol Clin 1998; 16: 691-8). With these practical disadvantages, and with the recent commercial availability of EIAs, the VDRL and TPHA combination for screening is being replaced increasingly in UK diagnostic microbiology laboratories by the use of EIA tests that detect treponemal IgG or IgG and IgM. EIAs have several practical advantages as a screening test and laboratories with large workloads, (usually above 20 000 tests a year) typically use this approach.

T. pallidum cannot be readily cultured in vitro and EIA tests typically use recombinant and/or synthetic polypeptide antigens derived from T. pallidum proteins. The outer membrane of T. pallidum has very few surface proteins - there are only 22 lipoproteins - and few antibodies can be generated against this organism. This paucity of membrane associated proteins allows T. pallidum to evade host defence mechanisms, makes serological diagnostic tests ambiguous and is the reason why there is no vaccine for syphilis. T. pallidum antigens identified to date have a molecular mass of 47kDa, 17kDa and 15kDa, respectively. Other membrane proteins include the 42kDa membrane protein TmpA (treponemal membrane protein A) and the 34 kDa membrane protein (TmpB ).

The 47kDa protein (or antigen), hereinafter referred to as Tp47, is a cytoplasm membrane lipoprotein, which was first noted in early molecular studies of T. pallidum, due largely to its abundance and profound immunogenicity. The protein was thus targeted for study as a potential syphilis serodiagnostic reagent, and further to the cloning and sequencing of the Tp47 DNA sequence, many newer generation serological tests for syphilis now include Tp47 as a principal, if not sole, antigenic component. The 47kDa protein is not localized to just one pathogenic subspecies, in that T. pallidum subspecies pertenue, endemicum, and Treponema carateum all possess cognate 47-kDa antigens.

Published data show that screening with a treponemal IgG EIA gives comparable results to the VDRL and TPHA combination (Egglestone S.I. & Turner A.J. L., Communicable Disease and Public Health 2000; 3: 158-162; Young H, Moyes A, McMillan A, Robertson DHH, Genitourin Med 1989; 65: 72-8; and Young H, Moyes A, McMillan A, Patterson J, J Clin Pathol 1992; 45: 37-41 ). Several treponemal EIAs with acceptable performance characteristics are known, however the assays vary in their performance, and as is generally the case with other EIAs, these tests can produce false positives, or they lack the sensitivity to detect T. pallidum in the sample being tested, or if they do so, it may be deemed an equivocal result which requires a follow-up test. In light of these disadvantages, there is a need to establish an improved and optimally performing single assay for the sensitive and specific unequivocal detection of T. pallidum infection in a sample. Also needed is a simple, inexpensive assay that can be used to monitor the success of syphilis treatment.

The present inventors have sought to address the above stated problems by developing a reagent which, when incorporated into a treponemal EIA, provides improved performance and increased sensitivity over known and existing EIAs.

According to a first aspect of the present invention, there is provided an isolated Treponema pallidum protein for use in an assay to detect the presence of T. pallidum in a sample, characterised in that the protein is reduced and/or denatured.

The present inventors have surprisingly discovered that a reduced/denatured T. pallidum protein provides greater sensitivity in T. pallidum detection assays, perhaps due to the improved availability of epitopes within the reduced/denatured - and therefore less structured - protein for binding to anti-7. pallidum antibodies within a sample.

By protein it is meant the full length protein, or one or more protein fragments or amino acid sequences which retain the ability to bind to anti- T. pallidum antibodies in a sample, and which provide greater sensitivity in T. pallidum detection assays than the equivalent non- reduced/non-denatured fragments or sequences.

The T. pallidum protein may be selected from the group consisting of: Tp47, Tp17, Tp15, TmpA, or TmpB.

The protein may be partially, or fully, chemically reduced, chemically denatured, heat denatured, or combinations thereof. By reduced it is meant that one or more disulphide bonds (or cystine residues) are reduced, i.e. one or more of the single covalent bonds derived from the coupling of the thiol groups within the cysteine residues is reduced.

By partially reduced it is meant that at least one disulphide bond remains in the protein. By fully reduced it is meant that all of the disulphide bonds in the protein are reduced.

By denatured, it is meant that the secondary and tertiary structures are altered but the peptide bonds between the amino acids are left intact. Denaturation may be induced using heat, acid, or alkali. The protein may be partially, or fully denatured.

The reduction and denaturation may be reversible or irreversible.

The T. pallidum protein may be Tp47.

The T. pallidum protein may be chemically reduced.

The protein may be T. pallidum Tp47 protein which is chemically reduced. The Tp47 protein may be fully or partially chemically reduced. Preferably, the Tp47 protein is fully chemically reduced.

The protein may be suitable for use in an assay to screen for, monitor, or confirm a T. pallidum infection. The protein may be suitable for use in an assay to screen for, monitor, or confirm a syphilis infection. The protein may be suitable for use in a screening and/or confirmatory test for the detection of syphilis, and/or the monitoring of a syphilis infection, which may be being treated.

The infection may be being treated.

The assay may be an EIA.

The reduced and/or denatured T. pallidum protein may exhibit greater quantitatively detectable binding to anti- 7^". pallidum antibodies in a sample, compared to the same non- reduced/denatured or partially reduced/denatured T. pallidum protein.

The reduced Tp47 protein may exhibit greater quantitatively detectable binding to anti- T. pallidum antibodies in a sample, compared to non-reduced/denatured Tp47 protein. The reduced Tp47 protein may exhibit greater quantitatively detectable binding to anti- T. pallidum antibodies in a sample, compared to partially-reduced/denatured Tp47 protein. The protein may be a recombinant protein, or a protein isolated and purified from T. pallidum extracts. The protein or epitopes within the protein or one or more amino acid sequences may be chemically synthesised.

The protein may be in a powder or freeze-dried form. The protein may be pure, or substantially pure.

The protein may be conjugated to at least one detection moiety selected from the group consisting of: a radioisotope, a fluorescent label, an enzyme, a lectin, avidin, biotin, a chemiluminescent tag, or flavine adenine dinucleotide, or combinations thereof.

The protein may be conjugated to an enzyme selected from the group consisting of: horseradish peroxidase, alkaline phosphatase, urease, and luciferase. Preferably, the enzyme is horseradish peroxidase.

According to a second aspect of the present invention, there is provided a composition comprising a T. pallidum protein of the first aspect of the present invention. The protein may be Tp47. The protein may be comprised within a diluent or buffer, which may for example be PBS, or which may comprise bicarbonate, carbonate, and/or EDTA. The diluent or buffer may comprise detergent or surfactant.

The protein may be soluble or partially soluble in the diluent or buffer.

The protein may be comprised within a solution which may be concentrated or dilute, or a suspension. The protein may be fully or partially soluble in aqueous and/or organic solvents.

The composition may further comprise at least one other T. pallidum protein, which may be a membrane protein, a cytoplasmic protein, or a lipoprotein. The protein is preferably immunogenic, and may be selected from the group consisting of: Tp47, Tp17, Tp15, TmpA, or TmpB. The protein may be fully or partially reduced, or in its native or folded state. Each protein may be a recombinant protein, a synthetic protein, a protein purified from T. pallidum extracts, or one or more fragments thereof.

The composition may further comprise a sample to be tested for the presence of T. pallidum. The sample is preferably a sample to be tested for the presence of a syphilis infection. The sample may be an ex vivo biological sample obtained from a patient.

According to a third aspect of the present invention, there is provided a method of preparing a T. pallidum protein according to the first aspect of the present invention, comprising contacting the protein, or a composition or solution comprising the protein with a reducing agent.

There is provided a method of preparing a reduced Tp47 protein according comprising contacting a Tp47 protein, composition or solution comprising a Tp47 protein with a reducing agent.

The method may further comprise heating the protein.

The reducing agent may be selected from the group consisting of: Tris(2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), β-mercaptoethanol (2-ME), 2-mercaptoethylamine (2- MEA, or Cysteamine), and dithioerythritol (DTE).

DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. Due to air oxidation, DTT is a relatively unstable compound whose useful life can be extended by refrigeration and handling in an inert atmosphere. Since protonated sulphurs have lowered nucleophilicities, DTT becomes less potent as the pH lowers. DTE is an epimer of DTT and is slightly less effective at reducing than DTT.

β-mercaptoethanol (also 2-Mercaptoethanol) is also used to reduce disulfide bonds. It is widely used as a reducing agent because the hydroxyl group confers solubility in water and lowers the volatility. Due to its diminished vapour pressure, its odour, while unpleasant, is less objectionable than related thiols.

Preferably the reducing agent comprises TCEP or TCEP hydrochloride.

TCEP, or TCEP hydrochloride is a preferred alternative which is more stable and works even at low pH. TCEP selectively and completely reduces even the most stable water-soluble alkyl disulfides over a wide pH range. Reductions frequently require less than 5 minutes at room temperature (RT). Compared to DTT and β-mercaptoethanol, TCEP has the advantage of being odourless, a more powerful reductant, an irreversible reductant, more hydrophilic and more resistant to oxidation in air. TCEP is also advantageous because it does not reduce metals used for metal chelation purification.

The use of other chemical reducing agents is envisaged, and these will be well known to a person skilled in the art.

The denaturing agent may be an acid or alkali. The TCEP may be used at a final concentration of between about 0.1mM and about 100OmM. The TCEP may be used at a final concentration of between 1 mM and 50OmM₁ or between 5mM and 25OmM₁ or between 1OmM and 25OmM, or between 2OmM and 20OmM.

The TCEP may be comprised within a PBS buffer, which may further comprise EDTA.

The chemical reduction may also be performed under denaturing conditions. Reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g. at high temperatures, or in the presence of a strong denaturant such as 6M guanidinium hydrochloride, guanidinium isothiocyanate, 8 M urea, or 1% Sodium dodecylsulfate). The reducing agent may therefore be a denaturing agent.

The denaturing agent may be guanidinium hydrochloride, guanidinium isothiocyanate, urea, or sodium dodecylsulfate (SDS), or combinations thereof. The working concentrations of each denaturing agent are well established, and will be well known to a person skilled in the art. For example, guanidinium hydrochloride and guanidinium isothiocyanate may be used at 6M, urea may be used at 8M, and SDS may be used at 1 %. The use of other chemical denaturing agents is envisaged, and these will be well known to a person skilled in the art.

The disulphide bonds in the T. pallidum protein may be reduced using a combination of one or more reducing agents, which may be in combination with one or more denaturing agents. The chemical reduction and/or the chemical denaturation may be performed at temperatures which also result in heat denaturation of the T. pallidum protein, for example above 37⁰C. Denaturation of the protein may be effected or promoted by heating the protein, for example by incubating the protein at a temperature above 37⁰C, e.g. up to 45⁰C, up to 55⁰C, or up to, or over 65⁰C, or at any temperature which results in an at least partial heat denaturation of the protein.

The method may comprise incubating the T. pallidum protein, composition or solution comprising a T. pallidum protein at a temperature over 37°C.

Where the T. pallidum protein is Tp47, the method may comprise incubating the Tp47 protein, or a composition or solution comprising the Tp47 protein at a temperature over 37⁰C.

The incubation may provide an at least partial heat denaturation of the protein.

The protein, composition or solution comprising the protein may be contacted with the reducing agent and/or the denaturing agent for between about 5 seconds and about 24 hours, or between about 10 seconds and about 12 hours, or between about 30 seconds and about 6 hours, or between 1 minute and 4 hours, or between 5 minutes and 2 hours. The protein, composition or solution comprising the protein may be contacted with the reducing agent and/or the denaturing agent at a temperature of between about 15⁰C and about 78⁰C, or between about 2O⁰C and about 65⁰C, or between about 25⁰C and about 55⁰C, or between about 3O⁰C and about 42⁰C. The temperature may be about 37⁰C.

Where a higher temperature is used the incubation period for full or partial reduction/denaturation may be less than that required when a lower incubation temperature is used.

The protein, composition or solution comprising the protein may be contacted with the reducing agent at a pH of between 1 and 14. TCEP will work as a reducing agent within this pH range. Preferably, the pH is physiological, for example between about 6 and about 8, and more preferably between 7.0 and 8.0, for example, pH 7.0, pH 7.1 , or 7.2, or 7.3, or 7.4, or 7.5, or 7.6, or 7.7, or 7.8, or 7.9, or 8.0.

The protein, composition or solution comprising the protein may be contacted with a reducing agent comprising TCEP having a pH of about 7.2, at a final concentration of about 20OmM for about 2 hours at a temperature of about 37⁰C.

The method may further comprise solubilising the protein in a diluent or buffer prior to contacting the reducing agent. The diluent or buffer may be formulated to stabilise the protein before, during or after the reduction/denaturation, or it may promote or facilitate the reduction /denaturation by the reducing agent/denaturing agent being used.

The method may further comprise purifying the protein before and/or after contacting the protein with the reducing agent and/or the denaturing agent. The purification may be performed using established protein purification techniques well known to a person skilled in the art. Chromatography may be used, for example using a NAP-5 column.

The method may further comprise concentrating the protein before and/or after contacting the protein with the reducing agent and/or the denaturing agent.

The purification and concentration steps may be performed together, for example by using chromatography may be used, e.g. a NAP-5 column, and eluting the protein from the purification column in a volume of buffer or diluent to give a solution with a higher concentration than the starting material. The method may further comprise quantitating the amount of protein before and/or after contacting the protein with the reducing agent and/or the denaturing agent.

The protein may be conjugated to at least one detection moiety selected from the group consisting of: radioisotope, a fluorescent label, an enzyme, a lectin, avidin, biotin, a chemiluminescent tag, or flavine adenine dinucleotide, or combinations thereof.

The protein may be conjugated to an enzyme selected from the group consisting of: horseradish peroxidase (HRP), alkaline phosphatase, urease, and luciferase. In a preferred embodiment, the enzyme is HRP.

When the protein is conjugated to HRP, the protein conjugate may be in an HRP stabilisation buffer, for example in an HRP stabilisation buffer manufactured by Dako.

According to a fourth aspect of the present invention there is provided a method of preparing a reduced and/or denatured T. pallidum conjugate, comprising taking at least one detection moiety selected from the group consisting of: a radioisotope, a fluorescent label, an enzyme, a lectin, avidin, biotin, a chemiluminescent tag, or flavine adenine dinucleotide, or combinations thereof, and conjugating the at least one detection moiety to the reduced and/or denatured T. pallidum protein according to the first aspect of the present invention.

The T. pallidum protein may be Tp47.

It is envisaged that many detection moieties such as fluorescent labels, enzymes etc. would be suitable for conjugation to the T. pallidum protein. Such moieties are well known in the art, and the substitution of one moiety for another would be routine and obvious to a person skilled in the art. Similarly, each detection moiety may have various ways in which it may be detected, and the scope of the invention encompasses all of these. For example, where the detection moiety is an enzyme, the use of several substrates may be possible, or where the detection moiety is a radiolabel, the use of different radioisotopes may be possible.

The detection moiety may be HRP. HRP may be conjugated to Tp47 by first activating the HRP, for example with sodium periodate, and then mixing the HRP and the Tp47 together, for example in a ratio of 1 :2 (protein:HRP).

The HRP and protein may be incubated for between about 1 and about 4 hours, preferably about 2 hours.

According to a fifth aspect of the present invention there is provided a use of a T. pallidum protein according to the first aspect of the present invention, or the composition according to the second aspect of the present invention in the manufacture of a medicament for the detection of T. pallidum in a sample.

The sample may be an ex vivo biological sample obtained from a patient.

There is provided a use of a T. pallidum protein according to the first aspect of the present invention, or the composition according to the second aspect of the present invention in the manufacture of a medicament for the detection of syphilis, and/or the monitoring of a syphilis infection, which may be being treated.

According to a sixth aspect of the present invention there is provided a method of detecting T. pallidum in an ex vivo biological sample obtained from a patient, the method comprising contacting the reduced and/or denatured T. pallidum protein according to the first aspect of the present invention or a composition according to the second aspect of the present invention, with the sample, and detecting immune-complexes formed between anti- T. pallidum antibodies within the sample and the T. pallidum protein.

The protein may be Tp47, which is preferably reduced, and more preferably is chemically reduced.

There is provided a method of detecting T. pallidum in an ex vivo biological sample obtained from a patient, the method comprising contacting a reduced Tp47 protein or a composition comprising same, with the sample, and detecting immune-complexes formed between antibodies within the sample and the protein.

The sample may be diluted before contacting the T. pallidum protein.

In a preferred method, the antibodies within the sample may be immobilised to a surface prior to contacting the T. pallidum protein. The sample may be diluted before immobilisation to the surface.

The antibodies within the sample may be directly or indirectly immobilised to the surface. For indirect immobilisation, the antibodies within the sample may be immobilised to the surface by reaction with anti-human antibodies, the anti-human antibodies being immobilised to the surface. The anti-human antibodies may be rabbit antibodies.

The surface may be a surface of a reaction vessel, a well, a glass slide, latex particles, or filter paper. The surface may be the wells of a microtiter or ELISA plate. The surface may comprise plastic, or polystyrene, or polystyrene coated plastic. The surface may have an affinity for proteins. In alternative embodiments, the surface may exhibit no affinity for proteins.

The method may be an EIA, where anti-T. pallidum antibodies within the patient sample are detectable because they form detectable immune-complexes with the reduced and/or denatured T. pallidum protein.

The method may be a screening test or a confirmatory test.

The method may comprise one or more wash steps to remove incompletely adsorbed material, for example antibodies used for immobilisation of the sample antibodies, the antibodies within the sample, the T. pallidum protein, or the reagent used to detect immune- complexes.

The method may comprise binding or coating a non-specific protein such as bovine serum albumin (BSA) or casein onto the surface that is known to be antigenically neutral. This allows for blocking of non-specific adsorption sites on the immobilizing surface and thus reduces the background caused by non-specific binding of the T. pallidum protein onto the surface.

After indirect or direct binding of the sample antibodies to the surface, the method may comprise coating the surface with a non-reactive material to reduce background, and washing to remove unbound material.

The surface may be contacted with the T. pallidum protein in a manner conducive to immune complex (antigen/antibody) formation. Such conditions preferably include diluting the T. pallidum protein with diluents such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.

Immune-complex formation may be allowed to occur by incubation for from about 2 to about 4 hours, at temperatures preferably in the order of between about 20⁰C to about 40⁰C, and preferably between about 25°C to about 37°C. Following incubation, the T. pallidum protein- contacted surface may be washed so as to remove non-immune-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer.

To provide a detecting means, the T. pallidum protein is preferably conjugated to a detection moiety, which is preferably an associated enzyme that will generate a colour development upon incubating with an appropriate chromogenic substrate. In a preferred embodiment, the reduced T. pallidum protein may be conjugated to HRP. Following formation of specific immune-complexes between the bound antibodies within the sample and the T. pallidum protein, and subsequent washing, the amount of bound T. pallidum protein may be quantified. Where the T. pallidum protein is conjugated to HRP, for example Tp47-HRP, quantitation may be performed by incubation with a chromogenic substrate which may be urea peroxide and tetramethyl benzidine (TMB)₁ or urea and bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS) and H₂O₂,

Quantification may then be achieved by measuring the degree of colour generation, e.g. using a visible spectra spectrophotometer. TMB is oxidized during the enzymatic degradation of

H₂O₂ by HRP. The oxidized product of TMB has a deep blue colour. A clear yellow colour is formed after addition of an acidic stop solution. For detection of oxidized TMB, the optical density (OD) of the yellow colour in a standard ELISA plate reader may be determined at

450nm when the reaction has been stopped with acid. If the reaction is not stopped with acid, the blue colour can be measured at 655 nm.

In an alternative embodiment of the method, the T. pallidum protein may be immobilised onto a selected surface, and antibodies within the sample bound to the immobilised protein. With this method, the T. pallidum protein may be conjugated to a detection moiety, or, a secondary antibody - used to bind to antibodies within the sample - may itself be conjugated to a detection moiety. As with the other embodiments of this invention, the detection moiety may be a radioisotope, a fluorescent label, an enzyme, a lectin, avidin, biotin, a chemiluminescent tag, or flavine adenine dinucleotide, or combinations thereof.

After binding of the T. pallidum protein to the surface, the method may comprise coating and washing steps as described previously. Following formation of specific immune-complexes between the antibodies in the sample and the bound T. pallidum protein, and subsequent washing, the occurrence of immune-complex formation may be determined by subjecting same to a second antibody having specificity for the first. Since the test sample will typically be of human origin, the second antibody will preferably be an antibody having specificity in general for human antibodies, i.e. an anti-human Ig antibody. To provide a detecting means, the second antibody may have an associated detection moiety, preferably an enzyme such as HRP, which will preferably generate a colour development upon incubating with an appropriate chromogenic substrate. Alternatively, where the T. pallidum protein is itself conjugated to a detection moiety, the amount of label can be determined as described previously, or using detection methods known in the art.

The antibodies within the sample may be anti-Tp47 antibodies, anti-7^". pallidum and/or anti- antibodies produced in response to a syphilis infection. The antibodies within the sample and/or the anti-human antibodies may be IgG or IgM antibodies, or both IgG and IgM antibodies.

The method may comprise contacting the sample with a plurality of T. pallidum proteins, one or more of which may be reduced and/or denatured. Various permutations and combinations of T. pallidum proteins are envisaged, the proteins being selected from the group consisting of: Tp47, Tp17, Tp15, TmpA, or TmpB. The method may comprise contacting the sample with Tp47 and Tp17, or Tp47 and Tp15, or Tp47, Tp17 and Tp15. The Tp47 is preferably reduced and/or denatured. In a preferred embodiment, the Tp47 is chemically reduced.

According to a seventh aspect of the present invention there is provided a kit for the detection of T. pallidum in an ex vivo sample obtained from a patient, the kit comprising the T. pallidum protein according to the first aspect of the present invention, or a composition according to the second aspect of the present invention, and at least one component selected from the group consisting of: detection means to detect the formation in the sample of immune-complexes between the protein and anti-7. pallidum antibodies, sample dilution buffer, conjugate dilution buffer, wash buffer, a positive control, a negative control, at least one reaction vessel, and instructions for use of the kit to determine whether the sample comprises anti-7. pallidum antibodies.

The sample may be a biological sample selected from the group consisting of: whole blood, lesion exudate, cerebrospinal fluid, serum, plasma, urine, amniotic fluid, synovial fluid, or tissue homogenate.

Where the sample is whole blood, serum or plasma, the sample may comprise anticoagulants, for example EDTA, sodium citrate or heparin.

The reaction vessel may comprise a surface suitable for immobilisation of antibodies and/or proteins. The surface may comprise plastic, or polystyrene, or polystyrene coated plastic. The surface may have an affinity for proteins. In alternative embodiments, the surface may exhibit no affinity for proteins. The surface may be coated with anti-human antibodies, which may be IgM antibodies. The reaction vessel may be a tube, or well, the well optionally being comprised within an 8- well strip, a 12-well strip, a 96-well plate, or a 384-well plate. The plate may be an ELISA or microtiter plate.

The sample dilution buffer may comprise protein and surfactant. The sample dilution buffer may comprise PBS, BSA, and Tween 20. The sample buffer may comprise PBS containing 1% BSA, and 0.5% Tween 20.

The T. pallidum protein may be in a concentrated form and may require to be diluted prior to contact with the sample.

The T. pallidum protein may be conjugated to a detection moiety, which may be HRP.

The conjugate dilution buffer may comprise surfactant and at least one stabiliser. The conjugate dilution buffer may be Dako HRP stabilisation buffer. The conjugate dilution buffer may comprise protein, which may be casein.

The wash buffer may comprise saline and surfactant. The wash buffer may comprise PBS, and Tween 20. The wash buffer may comprise PBS containing 0.05% Tween 20.

The positive and/or negative control may comprise human serum. The positive control preferably comprises human antibodies which form immune-complexes with the T. pallidum protein. The negative control preferably does not comprise human antibodies which form immune-complexes with the T. pallidum protein.

The detection means may comprise a substrate, which may be chromogenic, and which may comprise urea peroxide and tetramethyl benzidine (TMB).

The T. pallidum protein is preferably Tp47.

The kit may further comprise an at least one additional different T. pallidum protein selected from the group consisting of: Tp47, Tp17, Tp15, TmpA, or TmpB.

The kit may further comprise Tp17 and/or Tp15.

The one or more additional T. pallidum proteins may be in their folded or native forms, or may be partially or fully reduced, or partially or fully denatured. The additional T. pallidum proteins may be recombinant proteins.

The one or more additional T. pallidum proteins may be conjugated to a detection moiety. The kit may be a kit for the detection of T. pallidum in a sample, the kit comprising a reduced Tp47 protein, or a composition comprising same, and a substrate comprising urea peroxide and tetramethyl benzidine to detect the formation in the sample of immune-complexes between the HRP conjugated protein and anti-T. pallidum antibodies, sample dilution buffer comprising protein and surfactant, conjugate dilution buffer comprising surfactant and stabilisers, wash buffer comprising saline and surfactant, a positive control comprising human serum, a negative control comprising human serum, at least one reaction vessel comprising a plurality of reaction wells, the each well having a polystyrene surface being coated with anti-human IgM antibodies, and optionally instructions for use of the kit to determine whether the sample comprises T. pallidum.

The Treponema pallidum may be selected from the group consisting of Treponema pallidum pallidum, Treponema pallidum pertenue, Treponema pallidum endemicum.

The presence of Treponema pallidum in the sample may cause a disease comprising syphilis, yaws, or bejel in the patient from which the sample was obtained.

The patient may be a human. The patient may be a neonate or a pregnant woman, or an attendee at a GUM clinic. The patient may be being treated for a T. pallidum infection.

The contents of each of the references discussed herein, including the references cited therein, are herein incorporated by reference in their entirety.

The present invention will be further apparent from the following description, which shows, by way of example only, specific embodiments of the protein, composition, methods, use, and kit, and experimentation therewith.

Experimental

The following experimental methodology details, by way of example only, a process for preparing a reduced T. pallidum protein, and its conjugation to a detection moiety. Further experiments detail the use of the protein conjugate in an EIA to detect T. pallidum in a sample.

Manufacture of a reduced Tp47, and conjugation to a detection moiety

1. Introduction The procedure below describes the manufacture of a reduced Tp47 protein conjugate for use in a Syphilis IgM EIA. The method can be completed in a single full day and is based on 0.5 mg of Tp47 which will produce sufficient conjugate for 300 plates (96-well).

2. Materials & Equipment

Recombinant Tp47 protein was obtained from a commercial source. TCEP was obtained from Sigma-Aldrich, Inc. Horseradish peroxidase (HRP) was obtained from Kem-en-Tec Diagnostics A/S.

3. Methodology

The experimental procedure involves (i) an initial Tp47 protein buffer exchange, (ii) Tp47 protein chemical reduction, (iii) periodate activation/purification of HRP, and (iv) coupling of Tp47 protein to HRP.

3.1 Tp47 protein buffer exchange

Remove the Tp47 protein from storage (-7O⁰C) and thaw. The protocol is based for the conjugate of 0.5 mg.

Equilibrate a NAP-5 column with PBS/EDTA buffer (0.1 M PBS/10mM EDTA) (minimum 10 ml). Add 0.5 mg Tp47 protein (volume up to 0.5 ml; if volume is less allow entering gel bed and then adding buffer so that the combined volume is 0.5 ml, allowing buffer to completely enter gel bed). Elute the purified sample with 1.0 ml buffer, collect 1 ml fraction in 1.5ml microtube.

Concentrate the purified Tp47 protein using a Vivaspin micro concentrator, 10,000 Molecular Weight Cut Off (Sartorius Ltd.) to ~200μl - 400μl and store in a 1.5ml microtube. Add 0.5 ml and spin at 10,000 rpm for 3 minutes, add a further 0.25 ml, spin for 3 minutes and then add final 0.25 ml and spin until volume is approximately 0.3 ml. Remove from micro concentrator and store in a 1.5 ml microtube, record volume.

3.2 Tp47 protein reduction

Weigh 1.72 g of TCEP into a labelled 30ml universal, add 5 ml of PBS/EDTA buffer to dissolve, and then adjust pH to 7.2. This will require a significant amount of 3M sodium hydroxide to be added.

Add the required volume of TCEP solution (120OmM) to the concentrated Tp47 protein (3.1 above) in order to obtain a final TCEP concentration of 20OmM.

Incubate the Tp47 protein/TCEP solution mixture (Tp47/TCEP) at about 37⁰C for 2 hours. Near to the end of the Tp47/TCEP incubation, equilibrate a NAP-5 column with bicarbonate/carbonate/EDTA buffer (minimum 10 ml). Add reduced Tp47 protein (volume up to 0.5 ml, if volume is less allow entering gel bed and then adding buffer so that the combined volume is 0.5 ml; allowing buffer to completely enter bed). Elute the purified sample with 1.0 ml buffer, collect whole 1 ml fraction in 1.5 ml microtube.

Concentrate the purified/reduced Tp47 protein using a Vivaspin micro concentrator (10,000 MWCO) to ~200μl - 400μl. Add 0.5 ml of reduced Tp47 and spin at 10,000 rpm for 3 minutes, add a further 0.25 ml, spin for 3 minutes and then add final 0.25 ml and spin until volume is approximately 0.3 ml. Remove from concentrator and store in a 1.5 ml microtube.

3.3 Periodate activation and purification of HRP

Remove HRP from storage (-7O⁰C) and allow to reach RT (25⁰C +/-5⁰C), then weigh 5 mg into a 1.5ml microtube, add 0.5 ml of deionised water and dissolve.

Weigh 95 mg of sodium periodate into a 30 ml universal, add 5 ml of deionised water and dissolve. Cover with foil to protect from light inactivation.

Add 50μl of the periodate solution to the microtube containing the HRP solution, cover with foil, and incubate at RT (25⁰C +/-5⁰C) for 20 minutes.

Whilst the periodate and HRP are reacting, equilibrate a NAP-5 column with bicarbonate/carbonate/EDTA buffer (minimum 10 ml), and add 0.5 ml of the activated HRP to the NAP-5 column. Elute with 1.0 ml of bicarbonate/ carbonate/EDTA buffer, collect the whole of the 1.0 ml fraction in a 1.5 ml microtube.

The theoretical concentration of the concentrated/purified/reduced Tp47 protein can be estimated by calculating:

Vol. Tp47 Ag. Initial ml/ Vol. Tp47 Ag: Final ml x Tp47 Ag. Initial mg/ml = Tp47 Ag. Final mg/ml.

3.4 Coupling of Tp47 protein to HRP

The reduced Tp47 protein and the activated HRP are mixed in a ratio of 1 :2 to form the conjugate. The amount (in mg) of Tp47 Ag can be calculated by determining the Vol. Tp47 protein Final ml x Tp47 protein Final mg/ml. Twice as much activated HRP (in mg) is conjugated to Tp47 protein (in mg). The conjugate is incubated at RT for 2 hours. During last 30 minutes of conjugate incubation prepare 5M sodium cyanoborohydride solution. Weigh out 314 mg and dissolve in 1.0 ml deionised water. At the end of the 2 hour conjugate incubation add 1 μl of sodium cyanoborohydride per 100 μl of Tp47 protein /HRP conjugate mix. Incubate for 30 minutes at RT. During the incubation with sodium cyanoborohydride prepare 1M ethanolamine solution, by adding 300 μl ethanolamine to 5 ml deionised water, and adjust pH to 9.6 by the addition of concentrated HCI. Add 5 μl of 1M ethanolamine per 100 μl of Tp47 protein /HRP conjugate and mix. Incubate for 30 minutes at RT.

Dilute the Tp47 protein-HRP conjugate to a final Tp47 protein concentration of 0.1 mg/ml in Dako HRP stabilisation buffer in an 8 ml Nalge container.

Use of a reduced Tp47 conjugate in a Syphilis IgM antibody enzyme immunoassay

Rationale

In infections with the spirochaete Treponema pallidum the primary response to the organism is the production of IgM class antibodies. This is followed by the appearance of IgG class antibodies which are detectable throughout all stages of the disease. Specific IgM antibodies tend to decline and eventually disappear - their presence is therefore a useful indicator of whether an infection is recent.

In conjunction with screening tests such as the Newmarket Laboratories Syphilis Total Antibody EIA the Syphilis IgM EIA can be used to aid in the diagnosis and investigation of cases of syphilis. The contents of the kit are listed in Table 1.

Maternal syphilis can be transmitted to the unborn foetus. IgM class antibodies cannot pass through the placenta - therefore detection of T.

IgM antibodies in neonatal blood samples can aid in the diagnosis of congenital syphilis.

Principle

Microtitration plate wells are supplied coated with anti-human IgM antibodies. IgM specific antibodies to Treponema pallidum present in a sample of plasma or serum will bind when the sample is incubated in the coated well. After unbound material is washed away, peroxidase- conjugated treponemal proteins are added, and in turn attach to any specific human antibodies captured.

After another washing step, a substrate/chromogen mixture is added to the wells, and the presence of the enzyme label is revealed by a colour change in the chromogen.

The optical absorbance of each well is read at appropriate wavelengths, and compared to controls to determine the presence or absence of anti- Treponema pallidum IgM antibodies. Table 1 - Syphilis IgM Antibody Enzyme Immunoassay kit contents

Storage and Stability

All reagents as supplied may be used up to their expiry date if stored at 2-8oC in their original containers (provided no contamination of one with another has occurred). Diluted wash buffer is stable for 4 weeks at 2-8oC. Make up sufficient diluted conjugate only for the number of strips to be run; the diluted conjugate is stable for 12 hours. Coated strips removed from the original packaging and stored in the bag provided at 2-8oC may be used for up to 4 weeks after opening. Equipment Required

Properly calibrated and maintained pipetting devices capable of delivering volumes of 10, 50 and 100 microlitres (specimens and reagents) and approx 300 microlitres (wash fluids).

Plate or strip reader to read at 450 nm, 550 nm and 620 nm filters.

Incubator at 37⁰C.

Specimens Serum or plasma (collected into EDTA₁ sodium citrate or heparin) samples may be used. They may be stored at 2-8⁰C for 1 week before testing. If longer storage is required, the samples should be frozen at -20⁰C or lower and be well mixed after thawing.

Samples that are haemolysed, icteric or lipaemic may be run in the assay but the sample indicator system may not be relied upon. Samples that are contaminated with bacteria or contain visible particulate matter should NOT be tested. Samples that have been heated to 56⁰C for viral inactivation may be used in the assay.

Assay Protocol (Manual) Allow all reagents and samples to warm to 18 - 25⁰C prior to use. Immediately after use return to recommended storage conditions.

Dilute wash buffer 1 in 20 with distilled or deionised water prior to use.

Take as many strips of wells (R1 ) as are needed for the number of samples to be tested, plus 5 wells for the controls (Negative control in triplicate and Positive control in duplicate). Store unused strips in the re-sealable plastic bag provided, and return to storage at 2 - 8°C.

Assay Controls The Negative controls must be tested three times with each lot of tests, and the Positive control twice.

Sample Addition

Pipette 100 μl sample dilution buffer R2 into a well followed by 10μl of sample or control into each well as appropriate. Ensure the contents of the wells are mixed after sample addition.

The sample dilution buffer will visually change colour from red/pink to clear/yellow within the well upon addition of sample. This colour change may also be monitored by reading the plate at 550 nm after addition of sample dilution buffer and after addition of sample at 450 nm. An increase in optical density when compared to the blank reading of the sample addition buffer will occur for wells that have had sample added.

The colour change is not quantitative for the amount of sample added. Quality control sera which have been diluted during their manufacture may not give this colour change.

Incubate at 37⁰C for 30 minutes, covered to reduce evaporation.

Wash

Dilute a sufficient quantity of wash buffer R5 1 in 20 with distilled or deionised water, plus the volume necessary for filling tubing, priming pumps, etc.

Aspirate the well contents and wash x 5 with working strength wash buffer ensuring complete filling and emptying at each cycle. If required, finally tap the inverted plate sharply on a layer of absorbent paper to remove any residual fluid.

Conjugate Preparation

The conjugate is prepared by adding one part peroxidase conjugated Treponema pallidum antigens R3 to 10 parts conjugate dilution buffer R4. Only prepare sufficient conjugate for the number of samples and controls being run; 50 μl of diluted conjugate is added per well. Discard any excess diluted conjugate after 12 hours.

For example, for 2 strips add 100 μl peroxidase conjugated Treponema pallidum proteins R3 to 1 ml conjugate dilution buffer R4. Only dilute the quantity necessary for the assays.

Conjugate Addition To each well pipette 50 μl of diluted conjugate. Addition of conjugate is verified by reading at 550 nm, a well with conjugate added must have an absorbance greater than 0.080.

Incubate at 37⁰C for 60 minutes, covered to reduce evaporation.

Wash

Aspirate the well contents and wash x 5 with working strength wash buffer ensuring complete filling and emptying at each cycle. If desired, finally tap the inverted plate sharply on a layer of absorbent paper to remove any residual fluid.

Substrate Addition Add 50 μl_ substrate/chromogen mixture R8 to each well. Addition of substrate is verified by reading at 550 nm, a well with substrate added must have an absorbance greater than 0.080.

Incubate at 18 - 25⁰C for 30 minutes, protected from light.

Stop Colour Development

Pipette 50 μl_ stop solution R9 to each well, and mix (blue colour changes to yellow).

Within 10 minutes, read and record the absorbance of each well at 450 nm, preferably with a reference reading at 600 - 630 nm to eliminate the effects of any optical imperfection in the wells.

Interpretation of Results Validity of Controls

Mean Absorbance of the Negative controls must be <0.100 and give an antibody index O.7.

Mean Absorbance of the Positive controls must be >1.000 and give an antibody index >7.0.

If any of the controls give results lying outside of these limits, the test should be repeated.

Calculation of Results

The Cut-Off Point (COP) is calculated as the mean of the Negative controls (NC) + 0.100 absorbance units.

i.e. (NC1 + NC2 + NC3) + 0.100 3

The antibody index (Al) is determined by dividing the absorbance of the control or test sample by the cut-off point (COP).

Al = absorbance of control or test sample Cut-off point

Samples giving Al values less than 0.9 are NEGATIVE for Syphilis IgM antibody.

Samples giving Al values greater than 1.1 are POSITIVE for Syphilis IgM antibody. Samples giving Al values between 0.9 and 1.1 are EQUIVOCAL. If an equivocal result is obtained, the samples must be retested in duplicate. If upon retest equivocal results are obtained a fresh specimen should be requested.

The diagnosis of an active syphilis infection should not be made on the sole basis of a positive syphilis IgM result but in conjunction with other tests and clinical evidence.

Performance Characteristics

Precision Intra and inter-assay precision was assessed at 3 levels using a negative sample and 2 positive samples representing an intermediate and strong IgM response. Intra-assay precision was calculated using 48 replicate measurements at the levels indicated. Inter-assay precision was calculated by running the samples in quadruplicate over 5 different assays.

Specificity

Clinical specificity was assessed by testing 87 plasma and serum samples from blood donors. The samples were tested in the Newmarket Laboratories syphilis IgM assay and a competitor assay. The Newmarket Laboratories syphilis IgM assay scored all samples as negative thus giving a specificity of 100%.

Sensitivity

Several studies were conducted comparing the performance of this kit with other commercially available (CE) marked kits on samples submitted to reference laboratories for specialist syphilis serology.

The combined results were as follows:

Additional sensitivity studies were performed using a syphilis seroconversion panel (Serologicals Inc., USA); 100% agreement between the Newmarket Laboratories syphilis IgM kit and two other commercially available syphilis IgM kits was obtained.

Experiment 1 - Comparison of reduced and non-reduced conjugated Tp47 protein with a syphilis positive control

The effect of using a reduced and non-reduced conjugated Tp47 protein with a primary syphilis positive control sample was tested. In summary, the protocol used was as follows:

I . Allow plates and reagents to reach RT 2. Add 10Oul diluent to each well

3. Add 2ul sample (1/50 dilution)

4. Mix for 60 seconds

5. Incubate for 30 mins at 37°C

6. Wash 5x Buffer Il 7. Add 100ul conjugate (diluted in Dako HRP stabilisation buffer)

8. Incubate for 30 mins at 37°C

9. Wash 5x Buffer Il

10. Add 5OuI TMB

I 1. Incubate for 30 mins at RT in dark 12. Add 5OuI stop solution

13. Read plate 450/620nm

The sample used was Sc3 (Sero conversion 3), a Syphilis IgM positive control, indicative of primary syphilis. The cut-off value was 0.174 and a positive result was a value greater than the cut-off value, and a negative result was a value less than the cut-off value.

Results

Table 2 lists the OD values obtained comparing the reduced and non-reduced Tp47 protein with the Sc3 sample. Figure 1 shows these results graphically.

Table 2 - Comparison of using a reduced and non-reduced conjugated Tp47 protein with a Syphilis positive control

As can be seen from the results in table 2, the Syphilis positive sample produced a negative reading using the non-reduced Tp47 conjugate. In contrast, a positive reading was obtained using the reduced Tp47 conjugate. Experiment 2 - Comparison of reduced and non-reduced conjugated Tp47 in combination with Tp17 protein in syphilis IgM test

The effect of using a reduced and non-reduced Tp47 protein in combination with Tp17 was investigated using the Syphilis IgM test. Reduced Tp47 was produced as described previously. In summary, the protocol used was as follows:

1. Allow plates and reagents to reach RT

2. Add 100ul diluent to each well

3. Add 2ul sample (1/50 dilution)

4. Mix for 60 seconds

5. Incubate for 30 mins at 37°C

6. Wash 5x Buffer Il

7. Add 100ul conjugate (diluted in Dako HRP stabilisation buffer)

8. Incubate for 30 mins at 37°C

9. Wash 5x Buffer Il

10. Add 5OuI TMB

11. Incubate for 30 mins at RT in dark

12. Add 5OuI stop solution

13. Read plate 450/620nm

The two samples used were positive and negative, respectively, for syphilis. The cut-off value was 0.174 and a positive result was a value greater than the cut-off value, and a negative result was a value less than the cut-off value.

Results

Table 3 lists the OD values obtained comparing the reduced and non-reduced Tp47 protein with the Sc3 sample. Figure 2 shows these results graphically.

Table 3 - Comparison of using a reduced and non-reduced conjugated Tp47 in combination with Tp17 protein in Syphilis IgM test

As can be seen from the results in table 3 and Figure 2, the Syphilis negative sample produced a low OD value - a negative result - with both the non-reduced Tp47 conjugate and the reduced Tp47 conjugate. The non-reduced Tp47 conjugate also produced a negative result (0.085) with the positive sample, which was an incorrect result. In contrast, the reduced Tp47 conjugate produced a good positive result (an OD value of 0.356) with the positive sample. The reduction of the protein therefore made the difference between providing a correct, positive result and an incorrect, negative result with the syphilis infected sample.

The combination of Tp47 and Tp17 was also tested. The non-reduced Tp47 conjugate in combination with Tp17 produced a negative reading with the negative control, but was strongly positive with the positive control sample. This was in contrast to the results obtained using only the non-reduced Tp47 conjugate (i.e. with no Tp17), which provided a false negative result with the positive control sample. These results were mirrored with the reduced Tp47 conjugate in combination with Tp17, which produced a negative reading with the negative control, but was strongly positive with the positive control sample. In each case the OD value associated with the reduced Tp47 conjugate was higher than that obtained with the non-reduced Tp47 conjugate. These results clearly show that Tp17 increases the sensitivity of the assay, and that the reduced Tp47 conjugate has a greater sensitivity than the non-reduced Tp47 conjugate.

Experiment 3 - Comparison of using a reduced and non-reduced conjugated Tp47 protein with syphilis samples in Syphilis IgM test

Reduced and non-reduced Tp47 protein in combination with Tp17 was tested on samples using the Syphilis IgM test. Reduced Tp47 was produced as described previously. The reduced and non-reduced Tp47 proteins were tested in the presence of Tp17 and were conjugated to HRP, as detailed above. In summary, the protocol used was as follows:

1. Allow plate and reagents to reach RT

2. In an off line dilution add 6ul sample to 30OuI diluent 3. Add 100ul of the diluted (1/50) sample

4. Incubate for 30 mins at 37⁰C

5. Wash plate x 5 EIA wash Il

6. Add 50μl conjugate (diluted in Dako HRP stabilisation buffer)

The samples were Biomedical Resources Samples (7th March 06), using a negative control, and Sc3 (Sero conversion 3), a Syphilis IgM positive control, indicative of primary syphilis.

The cut-off values were 0.149 for non-reduced and 0.124 for reduced. A positive reading was obtained when the OD value was greater than the cut-off value, and a negative reading was obtained when the OD value was less than the cut-off value.

Results

Table 4 lists the OD values obtained with the 4 samples. Figure 3 shows these results graphically.

Table 4 - Comparison of reduced and non-reduced Tp47 protein in a Syphilis IgM test

As can be seen from the results in table 4 and Figure 3, the syphilis negative sample produced low OD values with both the reduced and non-reduced Tp47 conjugates. The Sc3 (positive control) sample produced a low, barely positive reading using non-reduced Tp47, however, use of the reduced Tp47 protein was associated with a considerably higher OD value (a positive result), indicating that the sensitivity of the reduced Tp47 protein is significantly greater than that of the non-reduced protein in this assay. This increased sensitivity was also seen using the two test samples, BM144717 and BM142357. BM142357 was clearly positive using both the reduced and non-reduced Tp47 conjugates; however, the OD value using the reduced Tp47 was nearly three times greater than the OD value using non-reduced Tp47. For BM144717, the OD value using non-reduced Tp47 was low and barely over the cut-off value. In contrast, the OD value using reduced Tp47, was higher and indicated a clear and definite positive result.

Conclusions

The following conclusions can be drawn from the above described experiments:

• The use of non-reduced Tp47 in an EIA can produce incorrect, negative results with syphilis infected (positive control) samples.

• Reduced Tp47 can produce correct, positive results with a syphilis infected sample which tests negative with a non-reduced Tp47 protein.

• Compared to non-reduced Tp47, the use of reduced Tp47 can cross the threshold between providing an incorrect, negative result and a correct, positive result on a syphilis infected (positive control) sample.

• The sensitivity of an EIA using reduced Tp47 is greater than an EIA using non- reduced Tp47.

• This sensitivity is enhanced when the conjugate is used in combination with Tp17.

• This improved sensitivity means that syphilis infected samples which might previously have tested negative or equivocal results will now correctly test as positive. • Reduced Tp47 provides a considerably improved reagent for the detection of syphilis and kits/assays incorporating the reduced reagent represent an improvement over known prior art testing kits.

• A clearer, stronger result is advantageous because it reduces the need for repeat and multiple testing, thereby saving time and resources. References

Egglestone S.I. & Turner AJ. L. Serological Diagnosis of syphilis, Communicable Disease and Public Health 2000; 3: 158-162.

Deka,RK.,Machius, M., Norgard, MV., and Tomchick, DR., Crystal Structure of the 47-kDa Lipoprotein of Treponema pallidum Reveals a Novel Penicillin-binding Protein, J. Biol. Chem., Vol. 277, Issue 44, 41857-41864, November 1 , 2002

Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections Overview and Estimates", World Health Organization, 2001

Young H. Syphilis: new diagnostic directions, lnt J STD AIDS 1992; 3: 391 -413.

Young H. Syphilis serology. Dermatol Clin 1998; 16: 691-8.

Young H, Moyes A, McMillan A, Robertson DHH. Screening for treponemal infection by a new enzyme immunoassay. Genitourin Med 1989; 65: 72-8.

Young H, Moyes A, McMillan A, Patterson J. Enzyme immunoassay for anti-treponemal IgG: screening or confirmatory test? J Clin Pathol 1992; 45: 37-41.

Claims

Claims

1. An isolated Treponema pallidum protein for use in an assay to detect the presence of Treponema pallidum in a sample, characterised in that the protein is reduced and/or denatured.

2. A T. pallidum protein as claimed in claim 1 wherein the T. pallidum protein is selected from the group consisting of: Tp47, Tp17, Tp15, TmpA, or TmpB.

3. A T. pallidum protein as claimed in claim 1 or claim 2 characterised in that the protein is partially or fully chemically reduced, chemically denatured, heat denatured, or combinations thereof.

4. A T. pallidum protein as claimed in any one of claims 1-3 wherein the T. pallidum protein is Tp47.

5. A T. pallidum protein as claimed in any one of claims 1-4, characterised in that the protein is chemically reduced.

6. A T. pallidum protein as claimed in any one of claims 1-5, characterised in that the protein is suitable for use in an assay to screen for, monitor, or confirm a syphilis infection.

7. A T. pallidum protein as claimed in any previous claim, wherein the protein is a recombinant protein.

8. A T. pallidum protein as claimed in any previous claim, characterised in that the protein exhibits greater quantitatively detectable binding to anti-I. pallidum antibodies in a sample, compared to the same non-reduced/denatured or partially reduced/denatured T. pallidum protein.

9. A T. pallidum protein as claimed in claim 1 , wherein the protein is purified, recombinant chemically reduced Tp47.

10. A T. pallidum protein as claimed in any previous claim, characterised in that the protein is conjugated to at least one detection moiety selected from the group consisting of: a radioisotope, a fluorescent label, an enzyme, a lectin, avidin, biotin, a chemiluminescent tag, or flavine adenine dinucleotide, or combinations thereof.

11. A T. pallidum protein as claimed in claim 10, characterised in that the protein is conjugated to an enzyme selected from the group consisting of: horseradish peroxidase, alkaline phosphatase, urease, and luciferase.

12. A T. pallidum protein as claimed in claim 11 , characterised in that the enzyme is horseradish peroxidase.

13. A composition comprising a T. pallidum protein as claimed in any one of claims 1-12.

14. A composition as claimed in claim 13, wherein the protein is comprised within a diluent or buffer.

15. A composition as claimed in claim 13 or claim 14, the composition further comprising at least one other T. pallidum protein selected from the group consisting of: Tp47, Tp17, Tp15, TmpA, or TmpB.

16. A composition as claimed in any one of claims 13-15, further comprising a sample to be tested for the presence of T. pallidum.

17. A method of preparing a T. pallidum protein as claimed in any one of claims 1-12, comprising contacting the protein, or a composition or solution comprising the protein with a reducing agent and/or a denaturing agent, and optionally heating the protein.

18. A method as claimed in claim 17 wherein the reducing agent is selected from the group consisting of: Tris(2-carboxyethyl) phosphine (TCEP), Dithiothreitol (DTT), β-

^* mercaptoethanol (2-ME), 2-mercaptoethylamine (2-MEA, or Cysteamine), and dithioerythritol (DTE).

19. A method as claimed in claim 18 wherein the reducing agent comprises TCEP or TCEP hydrochloride.

20. A method as claimed in claim 19 wherein the TCEP is used at a final concentration of between about 0.1 mM and about 100OmM.

21. A method as claimed in any one of claims 17-20, characterised in that the protein, composition or solution is contacted with the reducing agent and/or the denaturing agent for between about 5 seconds and about 24 hours.

22. A method as claimed in any one of claims 17-21 , characterised in that the protein, composition or solution is contacted with the reducing and/or the denaturing agent at a temperature of between about 15⁰C and about 78⁰C.

23. A method as claimed in any one of claims 17-22, characterised in that the protein, composition or solution is contacted with the reducing agent and/or the denaturing agent at a pH of between about 6 and about 8.

24. A method as claimed in claim 17, wherein the protein, composition or solution is contacted with a reducing agent comprising TCEP having a pH of about 7.2, at a final concentration of about 20OmM for about 2 hours at a temperature of about 37⁰C.

25. A method as claimed in any one of claims 17-24, further comprising solubilising the protein in a diluent or buffer prior to contacting the reducing agent and/or the denaturing agent.

26. A method as claimed in any one of claims 17-25, further comprising purifying the protein before and/or after contacting the protein with the reducing agent and/or the denaturing agent.

27. A method as claimed in any one of claims 17-26, further comprising concentrating the protein before and/or after contacting the protein with the reducing agent and/or the denaturing agent.

28. A method of preparing a reduced and/or denatured T. pallidum protein conjugate, comprising taking at least one detection moiety selected from the group consisting of: a radioisotope, a fluorescent label, an enzyme, a lectin, avidin, biotin, a chemiluminescent tag, or flavine adenine dinucleotide, or combinations thereof, and conjugating the at least one detection moiety to a reduced and/or denatured T. pallidum protein as claimed in any one of claims 1-9.

29. The method as claimed in claim 28, characterised in that the T. pallidum protein is Tp47.

30. The method as claimed in claim 28 or claim 29, characterised in that the detection moiety is horseradish peroxidase.

31. Use of a T. pallidum protein as claimed in any one of claims 1-12, or the composition as claimed in any one of claims 13-15, in the manufacture of a medicament for the detection of T. pallidum in a sample.

32. Use of a 7^". pallidum protein as claimed in claim 31 , for the detection of syphilis, and/or the monitoring of a syphilis infection.

33. A method of detecting T. pallidum in an ex vivo biological sample obtained from a patient, the method comprising contacting the reduced and/or denatured T. pallidum protein of any one of claims 1-12 or the composition as claimed in any one of claims 13-15, with the sample, and detecting immune-complexes formed between anti- T. pallidum antibodies within the sample and the T. pallidum protein.

34. The method as claimed in claim 33 characterised in that the T. pallidum protein is Tp47.

35. A method as claimed in claim 33 or claim 34, characterised in that the sample is diluted before contacting the T. pallidum protein.

36. A method as claimed in any one of claims 33-35, characterised in that antibodies within the sample are immobilised to a surface prior to contacting the T. pallidum protein.

37. A method as claimed in claim 36, characterised in that the antibodies within the sample are immobilised to the surface by reaction with anti-human antibodies, the anti-human antibodies being immobilised to the surface.

38. A method as claimed in claim 36 or claim 37, wherein the surface is a surface of a reaction vessel, a well, a glass slide, latex particles, or filter paper.

39. A method as claimed in claim 38, wherein the surface comprises plastic, or polystyrene, or polystyrene coated plastic.

40. The method of any one of claims 33-39 characterised in that the sample is a biological sample selected from the group consisting of: whole blood, lesion exudate, cerebrospinal fluid, serum, plasma, urine, amniotic fluid, synovial fluid, or tissue homogenate.

41. A method as claimed in any one of claims 33-40, wherein the antibodies within the sample are anti-Tp47 antibodies, anti- 7^". pallidum and/or antibodies produced in response to a syphilis infection.

42. A method as claimed in any one of claims 33-41 , wherein the antibodies within the sample and/or the anti-human antibodies are IgG or IgM, or IgG and IgM antibodies.

43. A kit for the detection of T. pallidum in an ex vivo sample obtained from a patient, the kit comprising the T. pallidum protein as claimed in any one of claims 1-12, or the composition of any one of claims 13-15, and at least one component selected from the group consisting of: detection means to detect the formation in the sample of immune-complexes between the protein and anti-V. pallidum antibodies, sample dilution buffer, conjugate dilution buffer, wash buffer, a positive control, a negative control, at least one reaction vessel, and instructions for use of the kit to determine whether the sample comprises anti-7^". pallidum antibodies.

44. A kit as claimed in claim 43, the reaction vessel having a surface suitable for immobilisation of antibodies and/or proteins, the surface comprising polystyrene, plastic, polystyrene coated plastic.

45. A kit as claimed in claim 44, characterised in that the surface is coated with anti- human antibodies.

46. A kit as claimed in claim 45, the anti-human antibodies being of the class IgM.

47. A kit as claimed in any one of claims 43-46, wherein the reaction vessel is a tube, or well, the well being comprised within an 8-well strip, a 12-well strip, a 96-well plate, or a 384- well plate.

48. A kit as claimed in any one of claims 43-47, wherein the T. pallidum protein is in concentrated form.

49. A kit as claimed in any one of claims 43-48 wherein the T. pallidum protein is conjugated to horseradish peroxidase.

50. A kit as claimed in any one of claims 43-49, wherein the detection means comprises a chromogenic substrate.

51. A kit as claimed in claim 50, wherein the substrate comprises urea peroxide and tetramethyl benzidine.

52. A kit as claimed in any one of claims 43-51 , characterised in that the T. pallidum protein is Tp47.

53. A kit as claimed in any one of claims 43-52, the kit further comprising an at least one different T. pallidum protein selected from the group consisting of: Tp47, Tp17, Tp15, TmpA, or TmpB.

54. A kit as claimed in claim 52, further comprising Tp17.

55. The protein, composition, method, use, or kit of any previous claims wherein the Treponema pallidum is selected from the group consisting of Treponema pallidum pallidum, Treponema pallidum pertenue, Treponema pallidum endemicum.

56. The method of any one of claims 33-42, or the kit of any one of claims 43-55 wherein the presence of Treponema pallidum in the sample causes a disease comprising syphilis, yaws, or bejel in the patient from which the sample was obtained.