WO2002012898A1 - Procede ameliore pour la preparation de complexes immunochimiques, utile a la detection d'agents pathogenes et a la determination de la sensibilite aux antibiotiques desdits complexes - Google Patents

Procede ameliore pour la preparation de complexes immunochimiques, utile a la detection d'agents pathogenes et a la determination de la sensibilite aux antibiotiques desdits complexes Download PDF

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Publication number
WO2002012898A1
WO2002012898A1 PCT/IN2001/000140 IN0100140W WO0212898A1 WO 2002012898 A1 WO2002012898 A1 WO 2002012898A1 IN 0100140 W IN0100140 W IN 0100140W WO 0212898 A1 WO0212898 A1 WO 0212898A1
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WO
WIPO (PCT)
Prior art keywords
solution
membrane
immunochemical
washing
buffer
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PCT/IN2001/000140
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English (en)
Original Assignee
University Of Pune
Department Of Biotechnology
Deobagkar, Deepti, Dileep
Limaye, Veena, Abhay
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Application filed by University Of Pune, Department Of Biotechnology, Deobagkar, Deepti, Dileep, Limaye, Veena, Abhay filed Critical University Of Pune
Publication of WO2002012898A1 publication Critical patent/WO2002012898A1/fr

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    • 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
    • G01N33/531—Production of immunochemical test materials

Definitions

  • This invention relates to an improved process for preparation of immunochemical complex useful for the detection of pathogens and determination of their antibiotic sensitivity. More particularly it relates to the process of detection of the gram negative bacterial pathogens, which cause various types of infections in plants, animals and human beings, The immunochemical complex of the present invention are also useful for detection of antibiotic sensitivity of these pathogens.
  • the pathogens are organisms, which cause various types of infections, such as urinary tract infection (hereinafter referred to as UT ⁇ ). Eseherichia coli is one of such most frequently occuring pathogen causing various types of bacterial infections.
  • the rate of isolation of E.coli differs from 48% to 80-90%.
  • Other coli-forms such as Klebsiella, Hnterobacter and Proteus are isolated at the rate of 10-20%.
  • Pseuuonionas and some Gram positive isolates are emerging as important as other pathogens in the hospital acquired infections causing infections such as UTI.
  • Organisms like coagulase negative staphylococci and Staphylococcus aureus are isolated from l-2% UT ⁇ s.
  • Salmonella, Shigella, Haermophilus influenzae, Coryne bacterium group D2 and a typical Iviyeobacte ⁇ a are other pathogen, which are also very rarely isolated from infections.
  • fungi Candida are commonly and Coccidoides and Blastomyces are other rarely found in UTI.
  • Changing etiology of various types of infections and increasing anti-microbial resistance of the pathogens is the serious problem.
  • Inappropriately prescribed antibiotics, frequent use of invasive devices, growing awareness about antibiotics among general public, their free availability and misuse are the major factors contributing to increasing resistance pattern among such pathogens especially the uropathogens.
  • E.coli showed decreased susceptibility to many antibiotics such as co-trimoxazole, norfloxacin and it is also observed that ulti drag resistant strains can be obtained from the faeces of the patients two months o after the discontinuation of anti niicrobial therapy (Didier Lepelleie, Nathalie Caroff,
  • Prompt treatment depends upon the diagnosis and quick availability of the anti-microbial sensitivity pattern of the uropathogens.
  • Clinical laboratories require the rapid screening tests for rapid elimination of negative samples to reduce the labor and for more efficient laboratory flow.
  • Lumac, MS-2 etc. are capable of detecting bacteria within 30 minutes to 9 hours, but these systems are sophisticated and expensive and cannot be used as routine diagnostic methods more particularly in India wherein the need is for a quick, reliable test using cheaper reagents affordable by the patients).
  • the process for the o preparation of immunochemical complex provided by the present invention, useful for pathogen detection is one such effort made in this direction.
  • the primary object of the present invention is to propose a process for the preparation of immunochemical complex useful for detection of pathogens.
  • Another object of this invention is to propose a process for the preparation of immunochemical complex, and which allows a faster detection of the pathogens, more reliable and at much lower costs.
  • Another object is to provide the process for the preparation of immunochemical complex, which could be effectively used for determination of antibiotic sensitivity of these pathogens.
  • a process for the preparation of immunochemical complex which comprises spotting the sample to be tested on a o membrane, blocking the membrane with a blocking solution, washing the membrane with a washing buffer, treating the washed membrane with an antibody solution, washing with buffer solution, further treating with immunochemical solution, washing further with washing buffer solution, staining the membrane with a developer to obtain the immunochemical complex.
  • the blocked membrane is washed and then treated with 2 to 10 ml of an antibody solution for 20 to 60 minutes.
  • the treated membrane is again washed with 5 to 15 ml of a buffer solution, and then treated with 2 to 5 nil of said immunochemical solution for 20 to 60 minutes.
  • the samples may be in liquid o io ⁇ n or may be extracted in solvents such as pK 6 to 8 phosphate buffer saline, normal saline, tris-buffer in pathogen free water.
  • the membrane may be celluloses such as nitrocellulose, nytran, hybond, cellulose acetate or poly vinyl di-fluoride preferably nitrocellulose.
  • the antibody solution comprises commercially available monoclonal, polyclonal, or
  • the wash solution is mixture of 6 to 8 pH buffer and commercially available detergents such as tween 12, tween 80, or commercially available triton X-100 preferably tween 20.
  • the concentration of the detergent in the wash solution may be 0.01 to 0.2%
  • the immunochemical solution may be goat or mouse antirabbit antibody tagged with peroxidase, alkaline phosphatase, luciferase, fluroein isothionate, ⁇ galactosidase preferably goat antirabbit antibody tagged peroxidase.
  • the developer is selected from Nifro Blue tetrazolium, 5-bromo-4-ch1oro-3-indoly1- B-d- g jactopyranoside, Adenosine t ⁇ phosphate, lucife ⁇ n or a mixture of hyurogen peroxide and diamino benzidine chloride, or o-phenylene diamine dihydrochloride or, 4-chloro-l- o naphthol. 5-amino salicylic acid.
  • the concentration of oxidising agent may be 0.05 to 0,5%.
  • the preparation of the immunochemical complex of the present invention can be effectively employed for determination of the antibiotic sensitivity of the pathogens.
  • the formation of immunochemical complex depends upon the presence of pathogen in the , sample and which is mamfest from the formation of immunochemical complex visible by typical colour depending upon the developer used.
  • the sensitivity of the pathogen can thus be related to the intensity of the coloration of the immunochemical complex. The higher sensitive the pathogen is to a particular antibiotic , the lower is the coloration obtained for the pathogen.
  • the urine sample was diluted 100 times, 1 ⁇ l of sample was spotted on a nitrocellulose membrane and was allowed to dry for 5 minutes.
  • the membrane was then treated with 1% suspension of milk powder and washed through with wash buffer solution comprising a mixture of pH 7.2 buffer having 0.1 % Tween20 detergent.
  • the membrane was then treated with a solution of 0,2% solution of immunoglobulin for a period of 30 minutes.
  • the treated membrane was then washed thoroughly again by 10 m ⁇ of washing buffer solution and treated with 5 ml of 0.2% goat antirabbit antibody tagged with peroxidase, solution for 10 minutes.
  • the membrane was then washed thoroughly by 15 ml. of wash buffer solution.
  • the membrane was then treated with a mixture of 0, 1 % diamino benziuinechlo ⁇ de with 0.02% hydrogen peroxide to obtain the brown coloured immunochemical complex.
  • the urine sample was diluted 10 times, 1 ⁇ l of sample was spotted on a nitrocellulose membrane and was allowed to dry for 5 minutes. The membrane was then treated with
  • wash buffer solution comprising a mixture of pH 7.2 buffer having 0.1% Tween80 detergent.
  • the membrane was then treated with a solution of 0,2% solution of immunoglobulin for a period of 30 minutes.
  • the treated membrane was then washed thoroughly again by 15 ml of washing buffer solution and treated with 5 ml of 0.4% of mouse antirabbit antibody tagged with alkaline phosphatase solution for 20 minutes.
  • the membrane was then washed thoroughly by 15 ml. Of wash buffer solution and treated with 5ml of Nitro Blue tetrazolium to obtain the blue coloured immunochemical complex.
  • This example illustrates the use of the immunochemical complex for detection of broth and distributed in eight tubes containing one control and seven with different antibiotics with concentration as mentioned in Table-1 below.
  • the tubes were incubated for at 37°C for eight hours. 1 ⁇ l sample was spotted from each tube on a nitrocellulose membrane after every two hours starting from the time of incubation and the formation of immunochemical complex was carried out as follows. All the spots were allowed to dry for 5 minutes.
  • the membrane was then treated with 1% milk powder and washed thoroughly with wash buffer solution comprising a mixture of pH 7.2 buiier having 0.1% Tween 20 detergent.
  • the membrane was then treated with a solution of 0.2% solution of immunoglobulin for a period of 30 minutes.
  • the treated membrane was then washed thoroughly again by 15 ml of washing buffer solution and treated with 0.4% solution peroxidase tagged goat antirabbit antibody for 30 minutes.
  • the membrane was then washed thoroughly by 15 ml. of wash buffer solution.
  • the membrane was then treated with 10 ml of mixture of 0.2% diamino benzede hydrochloride and 0.02% hydrogen peroxide for 5 minutes.
  • the pattern of immunochemical complex developed was
  • the brown colour formation indicates the presence of immunochemical complex.
  • Positive pattern means formation of brown colour.
  • Negative pattern means absence of brown colour.
  • Positive pattern denotes the resistance to the antibiotic.
  • Negative pattern denotes the sensitivity to the antibiotic.
  • the preparation of the immunochemical complex can be effectively utilized for the detection of the presence or absence of the pathogen.
  • the formation of the complex depends on the presence of pathogen, which is mamfest by brown coloration. If the coloration is not formed the infection is absent.
  • the immunochemical complex prepared by the present invention can also be used for determination of the antibiotic sensitivity of the pathogen.
  • the process for the preparation of the immunochemical complex is simple, fast and accurate and can be carried out using cheaper reagents and sophisticated instruments.
  • the method can be effectively used for the detection of pathogens within a very short period of 2-4 hours and the antibiotic sensitivity can be ascertained within 8 to 10 hrs.
  • the formation of the complex can be carried out at room temperature and no need of special arrangements for lower temperature etc. are necessary.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de préparation de complexes immunochimiques, lequel procédé est utile à la détection d'agents pathogènes et à la détermination de la sensibilité aux antibiotiques desdits complexes. Au moyen de ce procédé, l'agent pathogène peut être détecté dans une plage de temps de 2 à 4 heures et la sensibilité aux antibiotiques dans une plage de temps de 4 à 8 heures. Ce procédé est simple, bon marché et rapide. Ce procedé possède un large champ d'applications pour la détection d'un grand nombre d'agents pathogènes, d'agents, de virus ou d'autres organismes infectieux.
PCT/IN2001/000140 2000-08-04 2001-08-01 Procede ameliore pour la preparation de complexes immunochimiques, utile a la detection d'agents pathogenes et a la determination de la sensibilite aux antibiotiques desdits complexes WO2002012898A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN728MU2000 2000-08-04
IN728/MUM/00 2000-08-04

Publications (1)

Publication Number Publication Date
WO2002012898A1 true WO2002012898A1 (fr) 2002-02-14

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PCT/IN2001/000140 WO2002012898A1 (fr) 2000-08-04 2001-08-01 Procede ameliore pour la preparation de complexes immunochimiques, utile a la detection d'agents pathogenes et a la determination de la sensibilite aux antibiotiques desdits complexes

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0063810A1 (fr) * 1981-04-29 1982-11-03 Ciba-Geigy Ag Dispositif et trousses pour les analyses immunologiques
US4855227A (en) * 1985-06-07 1989-08-08 Institute For Medical Research Rapid detection of mycoplasmas
US5200312A (en) * 1989-01-30 1993-04-06 The United States Of America As Represented By The Secretary Of The Navy Membrane based dot immunoassay and method of use
WO1994011736A1 (fr) * 1992-11-18 1994-05-26 Calypte, Inc. Dosages immunologiques d'anticorps presents dans l'urine diriges contre des micro-organismes associes a des maladies sexuellement transmissibles

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0063810A1 (fr) * 1981-04-29 1982-11-03 Ciba-Geigy Ag Dispositif et trousses pour les analyses immunologiques
US4855227A (en) * 1985-06-07 1989-08-08 Institute For Medical Research Rapid detection of mycoplasmas
US5200312A (en) * 1989-01-30 1993-04-06 The United States Of America As Represented By The Secretary Of The Navy Membrane based dot immunoassay and method of use
WO1994011736A1 (fr) * 1992-11-18 1994-05-26 Calypte, Inc. Dosages immunologiques d'anticorps presents dans l'urine diriges contre des micro-organismes associes a des maladies sexuellement transmissibles

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