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
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements

Definitions

• the invention relates to an indication strip for the detection of antibodies in the blood serum of patients with one or more fields in which antigens for one or more diseases are fixed, the corresponding antigen holding the antibodies corresponding to the disease and the antibodies by a subsequent one when patient serum is added Reaction can be made visible as a color spot.
• Such an indication strip for a test for antibodies in the blood also called enzyme immunoassay, is already known from DE 3346795 AI and forms the preamble of claim 1.
• the test described there serves for the serological detection of diseases caused by viruses, bacteria or the like for example infections.
• the test takes advantage of the fact that when there is a disease, the body forms antibodies in its (blood) serum that correspond exactly to the pathogen antigen (dock on to it and render it harmless). If one can detect such disease-specific antibodies in the blood serum, indirect proof is given that the patient has or had exactly this disease. Because of its mechanism of action, this test is one of the so-called indirect measurement methods, since it does not detect the pathogens (antigens) per se, but rather the specific antibodies formed by the body in response to the pathogens.
• antigens ie certain disease-causing proteins and various diseases
• a nitrocellulose or polyamide film is first applied and fixed to the solid phases of several test fields, in particular onto a nitrocellulose or polyamide film. Then the adsorption sites of the film not occupied by the antigens saturated.
• Patient serum is instilled to perform the test.
• the pathogen-specific antibodies contained in the patient's serum if present, enter into a specific binding with the antigen corresponding to the disease, namely an antigen-antibody reaction, Ag-Ak or immune reaction.
• the antibodies reacting to the antigen are then visualized by a subsequent reaction, such as treatment with a specific enzyme conjugate and the subsequent addition of a substrate for the enzyme contained in the conjugate and a dye precursor.
• the method described and the measuring strip used thereby enable qualitative, but not quantitative, detection of pathogen-specific antibodies (and thus diseases) in a serum. It would be desirable not only to know whether a disease is or was present, but to know how many antibodies are present, that is, to know the intensity or topicality of the disease.
• the invention is therefore based on the object of specifying an indication strip which also allows a statement to be made about the quantity of antibodies present in the blood.
• the indication strip according to the invention is characterized in that further fields in which human reference antibodies are in different concentrations are fixed, are provided. These fields become discolored in the (enzyme) reaction known per se following the immune reaction in accordance with the number of antibodies and thus allow a statement about the amount of antibodies in the Serum, i.e. about the strength and topicality of the disease.
• the invention makes use of the surprisingly found fact that the staining reaction equally labels both the body's own antibodies fixed on the solid phase from the liquid to be examined and the reference antibodies applied to the solid phase.
• the enzyme-substrate color reaction causes a darkening of both the body's own antibodies and the reference antibodies.
• the reference antibodies are applied in different concentrations to the solid phase of the test strip and the enzyme-substrate color reaction forms a dye that is characteristic of the respective concentration of the reference antibody, a color comparison scale and thus a comparison standard for the concentration of the antibodies on the Solid phase generated.
• a visual color comparison between this comparison standard and the respective darkening of the body's own antibodies enables a direct statement about the quantity of the antibodies, since in the case of color equality, the quantity can be indicated by reading the concentration of the deposited reference antibodies.
• the quantity of the body's antibodies can be read as an intermediate value between the two concentrations. This is a check of immunity, ie the acquired sensitivity to pathogens, and a statement about the condition or the Strength of the underlying diseases, such as. Infectious diseases, auto-immune diseases, such as rheumatism, allergies, tumors and the like.
• the invention also makes it possible, for example, to better follow the dynamics of the course of a disease, since the determination of the antibody concentration according to the invention, the effectiveness of a drug and / or a drug dose against a specific pathogen can be checked quantitatively after the incubation period.
• a measuring instrument for the quantitative detection of antibodies in a liquid is created, which can be read off directly and can be used anywhere and at any time and is inexpensive to produce.
• the specific antigens are preferably applied to a solid phase. This ensures that the antibodies bind to the solid phase.
• Several different antigens can also be applied to the solid phase to check different diseases, for example different infections, at the same time.
• An advantageous embodiment of the invention consists in that the reference antibodies with the same subtypically specific class, such as, for example, IgG, IgA, IgM or the like, are applied.
• a specific subtypically specific class for the reference antibody provides the necessary meaningful criterion for the detection of the disease.
• the use of different subtypically specific reference antibodies in the comparison standard also enables the determination of the subtypically specific antibody classes present in the body fluid.
• the solid phase is treated with an enzyme conjugate such as a subtypically specific immunoglobin type G, type A or type M (IgG, IgA, IgM) or the like to detect the antibodies and the reference antibodies. This ensures that only those antibodies and reference antibodies are labeled which have the same subtypically specific class compared to the enzyme conjugate used.
• the reference antibody and the enzyme conjugate in the same subtypically specific class are used to determine the existing antibody classes in the liquid to be examined.
• An advantageous embodiment of the invention consists in that an enzyme substrate, preferably based on tetramethylbenzidine, is used as the substrate for a precipitation coloration.
• an enzyme substrate preferably based on tetramethylbenzidine
• a well-adhering and stable dye with high sensitivity is formed on the solid phase, both at the locations where the labeled antibodies and the labeled reference antibodies are located.
• Both organic, in particular proteins, carbohydrates, fats, nucleic acids or the like, and also inorganic substances can be used as antigens. It is thereby achieved that antibodies caused by foreign organic and inorganic substances in the body are bound to the solid phase, so that, for example, the immunity to chemicals and the course, for example, of blood poisoning by chemical toxins, can be checked quantitatively by means of the indicator strip according to the invention.
• tracers are used as fluorescent or radioactive labeling substances. This is, in particular, a numerical quantitative evaluation of the test strips Measured antibody concentrations in a specially designed laboratory allows, spectrophotometers, electrophoresis devices and / or radiometric devices are used in the evaluation.
• an advantageous embodiment of the indication strip according to the invention is that the solid phase is designed as an adsorption film. As a result, the attachment of the antigen and the reference antibody to the surface of the adsorption film is achieved. These deposits are so stable that the measuring strip according to the invention can be stored at an ambient temperature of preferably 4 ° C. for several months.
• the reaction fields and / or the reference fields are assigned, for example printed, data fields, for example names of the pathogen to be examined, control characters, symbols and the like.
• This enables direct detection of one of the diseases to be examined when evaluating the measuring strip, in which case the associated reaction field then has a color.
• the reference fields are labeled with a control symbol, for example, in order to ensure a reconstruction of the different concentrations of the reference antibody applied to the reference fields, a direct statement about the severity of the disease is also possible during the evaluation.
• the dark colors caused by the enzyme-substrate color reaction on the reaction fields and reference fields are to be compared with one another.
• Figures 1 and 2 possible embodiments of a measuring strip according to the invention for the detection of antibodies.
• nitrocellulose film material 2 with a pore size of 0.45 ⁇ m.
• the size of the measuring strip 1, an outer border line 3 and an identification field 4 as well as several antigen fields 5 and antibody fields 6 arranged one below the other are printed in a left and right column.
• the name of the pathogen to be examined is printed above the left-hand antigen fields 5 and a control symbol, for example the letters A to H, for the different concentrations is printed above the right-hand antibody fields 6.
• these are on the left column influenza viruses A and B, adenovirus, Mycoplasma pneumoniae, Bordetella pertussis, Parainfluenza virus 1 to 3, RSV, HSV and Coxsacki virus.
• the indicator strip according to the invention is produced, for example, in such a way that the nitrocellulose film 2 is moistened in a phosphate-buffered saline solution (PBS buffer). Furthermore, protein antigen solutions with a concentration of 5-15 ng / ⁇ l are prepared from commercially available protein antigens for the pathogens to be examined in a known manner. 8th
• nitrocellulose film 2 is then clamped in a conventional dot blot apparatus, for example a "SlotBlotter" from Hofer.
• the diluted protein antigen solutions (corresponding to approximately 500 ng to 1.5 ⁇ g protein) are applied to the antigen fields 5 belonging to the left column of the nitrocellulose film 2.
• solutions with human antibodies are applied to the antibody fields 6 of the right column of the nitrocellulose film 2 with a continuously increasing concentration.
• the nitrocellulose film 2 is washed in a PBS buffer.
• the nitrocellulose film is subsequently treated with a milk powder solution in order to saturate unoccupied adsorption sites on the film.
• the nitrocellulose film 2 is then washed three times in succession with a PBS buffer and then dried under vacuum at room temperature.
• the measuring strips 1 according to the invention are punched out outside the border line 3.
• the measuring strip 1 thus produced is sealed together with a desiccant in foil and stored at 4 ° C. for later use.
• the measuring strip 1 is used by the doctor or a laboratory, for example, in such a way that first the measuring strip 1 is treated with the milk powder solution already mentioned for regeneration. Thereafter, the patient sera to be examined are diluted in a powdered milk solution according to an already known method and applied to the Measuring strip 1 applied. This causes immune reactions by the pathogen-specific antibodies contained in the serum entering into a specific bond with the antigens deposited in the antigen fields 5 on the left column.
• the unbound serum components are then suctioned off and the measuring strip 1 is washed three times with a known PBS-Tween detergent.
• Staining is carried out using 1 ml per film of a commercially available substrate based on tetramethylbenzidine from Kirke-Gaard and Perry with product code 50-77-02.
• the coloring is ended by washing the measuring strip 1 intensively with distilled water.
• the antigen fields 5 arranged on the left column of the measuring strip 1 show different color spots, the color intensities of which depend on the respective quantity of the pathogen-specific antibodies contained in the serum.
• the concentration also corresponds to that in the patient serum contained antibodies of the herpes simplex virus (HSV virus) a value between the two concentrations of the corresponding fields 6 D and E.
• HSV virus herpes simplex virus
• Fig. 2 shows a very narrow measuring strip la with an identification field 4a and several mutually arranged antigen fields 5 and antibody fields 6, which are particularly small here, in the example 5 x 3 mm, so that only a very small amount of patient serum is required for a test .
• the names of the pathogen to be examined are printed above each of the antigen fields 5 and a control character is printed above each of the antibody fields 6, for example the letters A to D for the different concentrations and a "-" for a field for the countercheck (no antibodies).

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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)
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• Biochemistry (AREA)
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• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Microbiology (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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Citations (4)

• 1999
  • 1999-04-26 JP JP2000548728A patent/JP2002514764A/ja active Pending
  • 1999-04-26 CN CN 99805983 patent/CN1300366A/zh active Pending
  • 1999-04-26 EP EP99919270A patent/EP1078262A1/de not_active Withdrawn
  • 1999-04-26 WO PCT/EP1999/002808 patent/WO1999058976A1/de not_active Application Discontinuation

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