Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1995-12-06
1998-03-24
Spiegel, Carol A.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 736, 435 18, 435 23, 435870, 435871, 435961, 435975, 435 794, 436514, 436525, 436528, 436530, 436531, 436541, 436175, 436808, G01N 33554
Patent
active
057311625
DESCRIPTION:
BRIEF SUMMARY
This application is a rule 371 continuation of PCT/EP94/01723 filed May 27, 1994.
BACKGROUND OF THE INVENTION
The present invention concerns a method and an analytical device for the simultaneous detection of Chlamydia trachomatis (CT), Neisseria gonorrheae (NG) and Mycoplasma (M) from a single specimen.
The diagnosis of the pathogenic agents responsible of sexually transmitted diseases (STD) such as gonorrhea, urethritis and similar illnesses, currently requires a specimen taken from the infection district as well as a separated analysis for each pathogen, the presence of which has to be verified.
Sampling of the material for the analysis is carried out by swabbing of the vaginal or urethral district.
This application, if repeated, can cause such inconveniences as inflammations or irritations of the mucosae which the swab comes in contact with and is unpleasant for the patient.
As the above mentioned pathogens infect the same organs and can be associated (coinfections), it is clear that the availability of a method and an analytical device which allows the simultaneous detection of CT, NG, M in a single endocervical or urethral specimen would be a welcomed improvement.
This also avoids the discomfort of taking several swabs from the same patient.
PRIOR ART
The European Patent application No. 0 264 036 (Abbott 20.4.88) describes a device and an immunologic method to detect the presence of CT and NG separately.
The European Patent Application No.0444 303 A2 (Becton Dickinson 04.09.91) describes an immunological method to detect Chlamydia trachomatis based on a flow-through system involving an extraction step with Proteinase K as extractive agent.
WO 87/06620 (05.11.87) discloses a kit for the contemporaneous detection of different agents responsible of sexually-transmitted diseases, comprising a device having at least one monoclonal antibody selective for each agent to be detected, absorbed on spaced apart reaction zones. The biological sample containing the infectious agent(s) is directly contacted, according to this prior art document, with the device on which the various monoclonal antibodies are immobilized, decreasing thereby the overall diagnostic sensitivity.
In fact, only cell surface antigens would be detectable and it is known that these antigens are liable to easily undergo substantial mutations in response to a variety of conditions, with consequent changes in the binding affinity to the antibodies used for the analysis. Moreover, it is generally accepted as desirable to extract the antigens from the organisms in order to increase assay sensitivity. See, for istance, U.S. Pat. No. 5,089,389.
For these reasons, and in view of the considerable differences existing in the chemical and structural characteristics of the antigens of the organisms responsible of sexually-transmitted disease, the prior-art methods do not allow a sensitive and reliable diagnosis of multiple antigens with only one sampling operation.
SUMMARY OF THE INVENTION
It has now been surprisingly found that antigens from genetically and immunologically different microorganisms such as Chlamydia trachomatis, Neisseria gonorrheae and Mycoplasma can be simultaneously detected in a biological sample, obtained for instance by scraping or swabbing the genital zone, by subjecting said sample to an incubation step in a suitable reaction media comprising Proteinase K and a lipase, followed by a solid-phase immuno-assay.
The extraction media containing Proteinase K and a lipase effectively releases the antigens of such diverse microorganisms as CT-NG-M from their respective membranes, without modifying their structure thus allowing recognition and binding of these chemically different antigens by their respective antibodies.
The pH of the extraction medium is suitably buffered at values ranging from 7.5 to 9.0.
DETAILED DESCRIPTION OF THE INVENTION
The present invention therefore provides an extraction reagent for extracting antigens of Chlamydia trachomatis, Neisseria gonorrheae and Mycoplasma from a biological sample comprising
REFERENCES:
patent: 4378344 (1983-03-01), Zahradnik et al.
patent: 4514508 (1985-04-01), Hirschfeld
patent: 5089389 (1992-02-01), Pelanek et al.
patent: 5415994 (1995-05-01), Imrich et al.
International Publication No. WO 87/06620 published Nov. 5, 1987.
Schmitt et al., "Surface Exposed Antigenic Cleavage Fragments of Neisseria gonorrhoeae Proteins IA and IB," Infection and Immunity 54(3):841-845 (1986).
Whipple et al., "Isolation and Analysis of Restriction Endonuclease Digestive Patterns of Chromosal DNA from Mycobacterium . . . ," J. of Clin. Micro. 258) 1511-1515 (1987).
Chemical Abstract 101:87322 (1984).
Chemical Abstract 111:219263 (1989).
Arcioni Laura
Gatti Guido
Boehringer Mannheim Italia S.p.A.
Spiegel Carol A.
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