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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
  • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

• the invention relates to a reagent for ante-mortem screening for CNTA.
• ATNC Unconventional transmissible agents
• Brain biopsy possible in humans for ante-mortem diagnosis, is no longer done today for the risk of contamination that the gesture poses to actors.
• the problem which the invention proposes to solve is to propose a reagent intended for ante-mortem screening for ATNC, which presents no risk of contamination.
• the invention therefore provides a reagent intended for ante-mortem screening for ATNC capable of being obtained by a process according to which:
• all or part of the protein 14-3-3 or the anti-protein antibody 14-3-3 is brought into contact with an aqueous aldehyde solution to obtain a final dilution of aldehyde of between 1 and 2%,
• the invention consists of a reagent consisting essentially of supports on which are fixed either all or part of the anti-protein 14-3-3 antibodies or either all or part of the proteins 14-3-3 , commercially available today and artificially synthesized.
• the aldehyde fulfills a different function in each step, respectively: - in the first step, a function of increasing the efficiency of the titration and hence improving the sensitivity of the test without altering the antigenic character of the proteins or antibodies anti-proteins,
• the contact time between the abovementioned elements and the aldehyde solution in the first step is advantageously less than 1 hour, preferably more than 15 minutes.
• the aldehyde used in practice is glutaraldehyde.
• the supports intended to support either the anti-protein 14-3-3 or the protein 14-3-3 are red blood cells, in particular avian or even artificial membranes.
• the concentration of the aldehyde in the aqueous solution used in the second step is between 0.01 and
• a step of washing the product obtained at the end of the first step is inserted between the first and the second step.
• the invention also relates to the method of ante-mortem screening for ATNC in a biological sample.
• This process consists in placing in the presence of a biological sample the reagent as described above, then in detecting by agglutination and / or inhibition of agglutination, the presence or absence of all or part of anti-protein antibodies 14-3-3 or 14-3-3 protein in said sample.
• the biological sample is advantageously chosen from biological liquids chosen from the group comprising CSF, blood (serum, plasma, leukocyte concentrate), lymph, urine, and milk.
• the presence of agglutination by anti-protein 14-3-3 antibodies indicates the absence of antigen in the blood, that is to say the absence of infection.
• the absence of agglutination signals the presence of antigens in the biological sample, and therefore that of the infection.
• the absence of agglutination may be due to the fact that the marker is not in sufficient quantity in the sample. In this case, we add an excess of proteins
• the invention also relates to a method of ante-mortem determination of ATNC in a biological sample, according to which said sample to be studied is mixed at different dilutions with the reagent described above, it is then observed at which dilution there is agglutination and / or agglutination inhibition, then the concentration of the protein 14-3-3 or anti-protein 14-3-3 antibody is determined by reference to a standard.
• a suspension of protein 14-3-3 is prepared in a PBS 15 buffer solution containing glutaraldehyde to obtain a final dilution of 1.5%.
• the contact time between the glutaraldehyde solution and the proteins is 20 minutes.
• turkey red blood cells are suspended in a PBS buffer of pH 7.4, in a proportion of 5 ml of red blood cells in a volume of 100 ml of PBS.
• the red blood cells, glutaraldehyde and protein are left to act for
• the sensitized red blood cells obtained are then washed by centrifugation, then resuspended in a PBS buffered solution. A solution of lysine is then added to the suspension and it is left to react for 15 to
• the sensitized red blood cells thus treated are then recovered by centrifugation, then washed before being resuspended in PBS containing 0.7 / 10,000 (weight / volume) of sodium azide.

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• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Hematology (AREA)
• Chemical & Material Sciences (AREA)
• Urology & Nephrology (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Neurosurgery (AREA)
• Neurology (AREA)
• Food Science & Technology (AREA)
• Cell Biology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Peptides Or Proteins (AREA)

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• 2001
  • 2001-02-28 FR FR0102744A patent/FR2821431B1/fr not_active Expired - Fee Related
• 2002
  • 2002-02-28 EP EP02706909A patent/EP1364214A1/de not_active Withdrawn
  • 2002-02-28 WO PCT/FR2002/000728 patent/WO2002068961A1/fr active Application Filing
  • 2002-02-28 CA CA002439134A patent/CA2439134A1/fr not_active Abandoned
  • 2002-02-28 JP JP2002567828A patent/JP2004525364A/ja active Pending
• 2003
  • 2003-08-27 US US10/651,020 patent/US20050054002A1/en not_active Abandoned

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