WO1995002186A1 - Nouveau dosage diagnostique destine a la detection de la syphilis - Google Patents

Nouveau dosage diagnostique destine a la detection de la syphilis Download PDF

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Publication number
WO1995002186A1
WO1995002186A1 PCT/GB1994/001486 GB9401486W WO9502186A1 WO 1995002186 A1 WO1995002186 A1 WO 1995002186A1 GB 9401486 W GB9401486 W GB 9401486W WO 9502186 A1 WO9502186 A1 WO 9502186A1
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WO
WIPO (PCT)
Prior art keywords
antibodies
treponema
binding agent
reaction vessel
plasma
Prior art date
Application number
PCT/GB1994/001486
Other languages
English (en)
Inventor
Ian Kenneth Grant
Jane Elizabeth Mijovic
Geoffrey Sheppard
Original Assignee
Shield Diagnostics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shield Diagnostics Limited filed Critical Shield Diagnostics Limited
Priority to AU71285/94A priority Critical patent/AU7128594A/en
Publication of WO1995002186A1 publication Critical patent/WO1995002186A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/571—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses for venereal disease, e.g. syphilis, gonorrhoea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G01N33/555—Red blood cell

Definitions

  • This invention relates to a novel assay for the diagnosis of syphilis using human serum or plasma.
  • Syphilis is a sexually transmitted disease caused by the spirochaete micro-organism Trepojiej ⁇ a pallidum.
  • Antibodies are produced in infected patients. Use of the invention results in a new and efficient means for detecting these antibodies, and hence infection.
  • Syphilis has been recognised as a specific disease for over 500 years, it was not until the beginning of this century that the causative organism, Treponema pallidum, was first identified.
  • the primary means for detection of the disease was limited to visual identification of the organism in human syphilitic material.
  • Syphilis is transmitted chiefly by direct contact, the organism entering the body through minute pores in the skin or mucous membranes. In practice the most common route of infection is as a sexually transmitted disease. Of particular concern is that the disease may also be transmitted congenitally from mother to foetus.
  • ELISA tests have been developed which use specific antigens bound to a labelling enzyme. These offer good specificity but can be expensive and time-consuming.
  • FTA-ABS fluorescent Treponema antibody absorption test
  • FTA-ABS fluorescent Treponema antibody absorption test
  • Haemagglutination is one of the most commonly used screening tests.
  • the test uses Treponema antigens bound to red blood cells.
  • the presence of anti-T. pallidum antibodies in the test serum leads to extensive cross-linking which results in agglutination of the red blood cells to form a visible mat.
  • These are known generally as either Treponema pallidum Haemagglutination Assays (TPHA) or Micro- Haemagglutination Assay - Treponema pallidum (MHA-TP) .
  • TPHA Treponema pallidum Haemagglutination Assays
  • MHA-TP Micro- Haemagglutination Assay - Treponema pallidum
  • test cells avian or ovine erythrocytes coated with T. pallidum antigens
  • the agglutination of the erythrocytes can be seen either with the naked eye or by fully automated optical methods.
  • TPHAs One of the inherent problems of prior art TPHAs is the requirement for the dilution step.
  • the need for a dilution step makes the assay harder to automate, increases the time to complete the assay, increases the cost of the procedure and limits the overall sensitivity of the assay.
  • Another disadvantage is the potential loss of integrity of the patient's sample in a two-stage process.
  • the need for a dilution step arises as a result of a number of non-specific binding reactions which can occur between the test sample and the antigens bound to the erythrocytes.
  • the main sources of these non-specific binding reactions are as follows;
  • Treponema pallidum organisms are grown in rabbit testes. Subsequent purification of the antigen is never completely effective with the result that some rabbit antigens co-purify with the T. pallidum . The presence in the test serum of any anti-rabbit antibodies could lead to the development of false positives.
  • the erythrocytes on which the T. pallidum antigens are bound have antigenic sites themselves. Any antibodies to these antigens present in the test serum could also lead to the development of false positives.
  • T. pallidum antigens are not completely specific to anti-T pallidum antibodies; other commonly found antibodies, particularly those related to commonly found non-pathogenic related species can react, so giving false positives.
  • T pallidum antibodies A reliable assay for detection of T pallidum antibodies must therefore ensure that these three means of interference are overcome, otherwise a significant number of false positives will result.
  • this is generally achieved by the addition of neutralising substances to a diluent.
  • the test sample is then mixed with the diluent so neutralising the various non-specific binding sites.
  • the rabbit antigens are neutralised by the addition of rabbit serum and the erythrocyte antigens are neutralised using a complex mixture of fragmented red blood cells such as commercially available 'ox stroma'.
  • the reaction of non-T. pallidum antibodies with the T. pallidum antigens is reduced by adding an autoclaved mixture of related species of Treponema .
  • Prior art TPHAs have generally added these neutralising reagents to a diluent. This results in two liquid handling operations; addition of test sample to diluent and the addition of a proportion of the diluent to the activated red blood cells. This dilution step is also thought to assist in reduction of non-specific binding by diluting out contaminating antibodies.
  • TPHA/MHA-TP which does not require a dilution step, but nevertheless offers a highly specific diagnostic assay.
  • substantially undiluted sample means a sample which is a test serum or plasma in essentially the form in which it is taken from a patient, in contrast to a sample diluted according to the prior art TPHA/MHA-TP practices.
  • TPHAs when used as a preliminary screen are carried out in small reaction vessels such as microtitre plates.
  • small reaction vessels such as microtitre plates.
  • automated liquid handling systems can be used with microtitre plates, the requirement for a dilution step adds significantly to the complexity of the assay, with concomitant repercussions on time, cost and sample integrity.
  • the present invention thus enables a single stage TPHA assay to be used to routinely screen large numbers of samples which can be readily automated.
  • the removal of the dilution step has the added advantage that the assay can also be made more sensitive, so enabling earlier detection of syphilis.
  • a polystyrene microtitre plate is used as the reaction vessel coated with hydrolysed gelatin as the binding agent.
  • Other binding agents which have been found to be effective include foetal calf serum, lactose, bovine serum albumin, rabbit serum and casein digest. It has also been found beneficial to mix certain of these binding agents together, for example a mixture of hydrolysed gelatin and lactose has been found to be a particularly effective binding agent.
  • the reaction vessel in another embodiment of the invention in order to prevent the collapse of high titre positive samples, is coated with T. pallidum.
  • the invention can be used in a number of different types of reaction vessel made of differing materials. Thus microtitre plates, strip-well plates, cell culture wells, cuvettes, test-tubes and the like can all be coated with binding agent and used to carry out a single stage TPHA assay.
  • These reaction vessels can be made of a number of materials including polystyrene, polypropylene, polyvinyl chloride, polycarbonate, polyethylene, terephthalate G copolymer or glass.
  • TPHAs have generally been carried out using serum samples it has been found that a slight modification allows efficient use of plasma samples.
  • the single stage TPHA can be improved by the presence of certain anti-coagulants, particularly heparin; such anti-coagulant will usually be included in the test cell mixture or formulation (the mixture of all other components - coated erythrocytes, neutralising agents etc - to which the test sample is added) ; its concentration will generally be appreciably greater than that convention for anti-coagulation purposes - eg at least 340 units/ml of test cell formulation.
  • Example 1 shows the effects of using an uncoated polystyrene microtitre plate in a single stage assay of 20 serum samples found to be negative for T pallidum antibodies using a standard two stage TPHA. Of the 20 true negatives only 2 yielded a 'compact button' indicative of a true negative with 1 indeterminate result. This type of result would be clinically unacceptable for unambiguous diagnosis of syphilis.
  • Example 2 shows the effect of coating the microtitre plates with a number of different binding agents. Although 2 show improvement over the uncoated plates, hydrolysed gelatin gives the best improvement, increasing the number of 'compact buttons' from 2 to 18, and thus improving the quality of the negative patterns.
  • Example 3 shows that hydrolysed gelatin, Bovine Serum Albumin (BSA) and Foetal Calf Serum (FCS) all give promising results when mixed with Tween and lactose. All coated plates show an improvement over the uncoated plates.
  • BSA Bovine Serum Albumin
  • FCS Foetal Calf Serum
  • Example 4 shows that the addition of sugars other than lactose to the protein mixture gives good results, though lactose is marginally better than the other sugars tested.
  • Example 5 plasma samples are used in place of the serum samples tested previously. Surprisingly, the results are not as good. This deficiency is readily overcome by the addition of heparin at a concentration of 340 - 1700 units/ml.
  • Example 6 shows that with a specimen containing a high concentration of anti-T. pallidum antibody, collapse of the agglutination pattern is seen unless the plate is coated with T. pallidum.
  • a variety of substances coated onto the reaction vessel prevent the formation of false positives and the collapse of high titre positives, and so allow a single-stage TPHA to be used as a screening method for both serum and plasma samples.
  • a polystyrene microtitre plate is coated with T. pallidum, 2% (w/w) hydrolysed gelatin and 2% (w/w) lactose solution.
  • Test Cells A formulation of Test Cells is made by mixing together the following reagents:
  • Agglutination of the cells is interpreted as a positive result. Strong positives may show some folding at the edge at the cell mat. Setting of the cells into a compact button or a button with a pinprick hole in the middle is interpreted as a negative result. Cells showing partial agglutination resulting in a ring with a large hole in the middle are interpreted as a +/- indeterminate reaction.
  • Each of the binding agents is added to distilled water and sodium azide (0.1%w/v) is fully dissolved and mixed. 200 ⁇ l of this solution is added to each well of a polystyrene microtitre plate. The plate is then incubated at 37"C, aspirated and dried. The Test Cell Formulation, Specimens and Procedure are as described in Example 1. The results shown are compared to the uncoated plate in Example 1.
  • Hydrolysed gelatin is one example of a complex protein mixture.
  • the data below show the effect of different protein mixtures when combined with a sugar such as lactose (2% (w/v)) and a surfactant such as Tween 20 (0.01% v/v).
  • a sugar such as lactose (2% (w/v))
  • a surfactant such as Tween 20 (0.01% v/v).
  • the Test Cell Formation, Procedure and Specimens are as described in Example 1.
  • the procedure for coating the plates is as described in Example 2.
  • T. pallidum initial concentration 6.0 x 10 8 organisms/ml
  • 100-200 ⁇ l is added to each well of a polystyrene microtitre plate.
  • the plate is incubated overnight at 4 C, aspirated and then the procedure for coating the plates is as described in Example 2.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention se rapporte à un nouveau dosage destiné au diagnostic de la syphilis utilisant du plasma ou du sérum humain. La syphilis est une maladie sexuellement transmissible par le micro-organisme spirochète Treponema pallidum. Des anticorps sont produits chez des patients infectés. Cette invention constitue un moyen nouveau et efficace pour détecter ces anticorps et, par conséquent, la présence d'une infection. L'invention se rapporte à un procédé de test permettant de détecter la présence d'anticorps dirigés contre l'espèce de Treponema dans le plasma ou le sérum sanguin, caractérisé par l'addition des éléments suivants, selon n'importe quelle séquence, dans un récipient de réaction: un échantillon sensiblement non dilué du plasma ou du sérum de test, des érythrocytes enrobés de constituants antigéniques de l'espèce cible Treponema, et des réactifs permettant de neutraliser les effets d'anticorps dirigés contre des antigènes autres que ceux du Treponema, ou des anticorps dirigés contre des espèces de Treponema autres que l'espèce cible. On mélange ces éléments après l'addition finale et on évalue le niveau d'agglutination des érythrocytes, le récipient de réaction étant revêtu d'un agent de liaison qui combat l'interaction entre la surface du récipient et l'échantillon et/ou les érythrocytes, une telle interaction pouvant entraîner des résultats d'agglutination faux positifs ou faux négatifs.
PCT/GB1994/001486 1993-07-07 1994-07-07 Nouveau dosage diagnostique destine a la detection de la syphilis WO1995002186A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU71285/94A AU7128594A (en) 1993-07-07 1994-07-07 New diagnostic assay for detection of syphilis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB939314011A GB9314011D0 (en) 1993-07-07 1993-07-07 New diagnostic assay for detection of syphilis
GB9314011.9 1993-07-07

Publications (1)

Publication Number Publication Date
WO1995002186A1 true WO1995002186A1 (fr) 1995-01-19

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PCT/GB1994/001486 WO1995002186A1 (fr) 1993-07-07 1994-07-07 Nouveau dosage diagnostique destine a la detection de la syphilis

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AU (1) AU7128594A (fr)
GB (1) GB9314011D0 (fr)
WO (1) WO1995002186A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077486A2 (fr) * 1999-06-14 2000-12-21 The Government Of The United States Of America Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention Compositions et procedes pour la detection du $m(f)i$m(g)treponema pallidum$m(f)/i$m(g)
EP2491394A2 (fr) * 2009-10-20 2012-08-29 Sanofi Pasteur Vaxdesign Corp. Tests d'hémagglutination et d'inhibition d'hémagglutination effectués en surface
WO2019097009A1 (fr) * 2017-11-17 2019-05-23 Hipra Scientific, S.L.U. Compositions vaccinales destinées à être utilisées contre la dermatite digitée chez un mammifère

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5244229A (en) * 1975-10-04 1977-04-07 Fujirebio Inc Method of making serological reagent for syphilis
GB1577131A (en) * 1977-03-18 1980-10-22 Whitley D Serological testing
EP0079145A1 (fr) * 1981-10-23 1983-05-18 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Réactif utile dans le diagnostic de la syphilis et sa préparation
EP0101166A1 (fr) * 1982-07-14 1984-02-22 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Procédé pour le dosage d'anticorps d'une maladie infectieuse
EP0447322A2 (fr) * 1990-03-16 1991-09-18 Sekisui Chemical Co., Ltd. Procédé de préparation d'un antigène purifié de tréponéma et son utilisation
JPH03218465A (ja) * 1989-11-17 1991-09-26 Sekisui Chem Co Ltd 梅毒診断試薬の製造方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5244229A (en) * 1975-10-04 1977-04-07 Fujirebio Inc Method of making serological reagent for syphilis
GB1577131A (en) * 1977-03-18 1980-10-22 Whitley D Serological testing
EP0079145A1 (fr) * 1981-10-23 1983-05-18 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Réactif utile dans le diagnostic de la syphilis et sa préparation
EP0101166A1 (fr) * 1982-07-14 1984-02-22 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Procédé pour le dosage d'anticorps d'une maladie infectieuse
JPH03218465A (ja) * 1989-11-17 1991-09-26 Sekisui Chem Co Ltd 梅毒診断試薬の製造方法
EP0447322A2 (fr) * 1990-03-16 1991-09-18 Sekisui Chemical Co., Ltd. Procédé de préparation d'un antigène purifié de tréponéma et son utilisation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 7720, Derwent World Patents Index; Class B04, AN 77-35388Y *
DATABASE WPI Section Ch Week 9145, Derwent World Patents Index; Class B04, AN 91-328419 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000077486A2 (fr) * 1999-06-14 2000-12-21 The Government Of The United States Of America Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention Compositions et procedes pour la detection du $m(f)i$m(g)treponema pallidum$m(f)/i$m(g)
WO2000077486A3 (fr) * 1999-06-14 2002-07-11 Us Health Compositions et procedes pour la detection du $m(f)i$m(g)treponema pallidum$m(f)/i$m(g)
AU775526B2 (en) * 1999-06-14 2004-08-05 Government of the United States of America, as Represented by the Secretary, Department of Health and Human Services, Centers for Disease Control and Prevention, The Compositions and methods for detecting treponema pallidum
US7005270B2 (en) 1999-06-14 2006-02-28 The United States Of America As Represented By The Department Of Health And Human Services Compositions and methods for detecting Treponema pallidum
US7335736B2 (en) 1999-06-14 2008-02-26 The United State Of America As Represented By The Department Of Health And Human Services Compositions and methods for detecting Treponema palidum
EP2491394A2 (fr) * 2009-10-20 2012-08-29 Sanofi Pasteur Vaxdesign Corp. Tests d'hémagglutination et d'inhibition d'hémagglutination effectués en surface
EP2491394A4 (fr) * 2009-10-20 2013-12-25 Sanofi Pasteur Vaxdesign Corp Tests d'hémagglutination et d'inhibition d'hémagglutination effectués en surface
US8962256B2 (en) 2009-10-20 2015-02-24 Sanofi Pasteur Vaxdesign Corp. Surface-assisted hemagglutination and hemagglutination inhibition assays
WO2019097009A1 (fr) * 2017-11-17 2019-05-23 Hipra Scientific, S.L.U. Compositions vaccinales destinées à être utilisées contre la dermatite digitée chez un mammifère
US11331381B2 (en) 2017-11-17 2022-05-17 Hipra Scientific, S.L.U. Vaccine compositions for use against digital dermatitis in a mammal

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Publication number Publication date
AU7128594A (en) 1995-02-06
GB9314011D0 (en) 1993-08-18

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