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
  • G01N33/6854—Immunoglobulins
• • 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
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

• the invention relates to a diagnostic procedure for the detection or quantitative determination of antibodies in biological fluids in a main test by means of an immunological reaction, in which the sought antibodies are incubated with the corresponding antigen, one portion of which antigen is bound, or is to be bound, to a water-insoluble carrier, and the other portion of which is provided, or is to be provided, with a label, subsequently separating the solid phase from the liquid phase and measuring the extent of the label either in the solid or in the liquid phase.
• a procedure of this kind is known from Maiolini et al. J.
• the result may be invalidated, since an unspecific reaction must be expected when using biological fluids.
• Said reaction may occur in that components of the analytical sample bind labelled antigen to the water-insoluble carrier in an unspecific manner, thereby erroneously indicating the presence of antibodies, thus leading to incorrect positive results.
• Object of the present invention is to obtain valid results or to detect the undesirable effects of certain components of the analytical sample during the detection or the quantitative determination of the sought antibodies, in order to verify the end result in a corresponding manner.
• a further object is to specify antibodies, which are to be detected or quantitatively measured by the diagnostic procedure and are directed against an antigen, which is also be to specified and which, in turn, exercises an antibody-function against human T-lymphocytes.
• the first object is met in that, prior to, during or after the main test, the same reaction as that of the main test
• SUBSTITUTE SHEET is carried out in a separate control test, but the incubation is carried out in the presence of an excess of non-bound and non-labelled antigen, the solid phase is separated from the liquid phase, the extent of the label is measured and is also taken into consideration during the qualitative evaluation or the quantitative determination of the antibodies.
• the procedure according to the invention is developed further such that the antibodies, which are to be detected or to be measured, are incubated jointly, from the outset, with the bound antigen and, in the control test, with the excess of non-bound and non-labelled antigen.
• the procedure according to the invention is preferably directed toward human antibodies against rabbit globulin as the antibodies to be detected or to be measured.
• the further object is met in that the procedure is carried out with human antibodies to rabbit globulin and with rabbit globulin as the antigen.
• the proposed procedures are particularly suitable for the purpose of, prior to, during and after the therapy with antibodies from an immune
• SUBSTITUTE SHEET serum against human T-lymphocyte globulin such as that which is used during organ transplants, e.g. in kidney transplants, in order to ensure immune suppression, controlling the presence or the formation of anti-antibodies which react with the antibodies of the immune serum.
• the procedures according to the invention can be used, in particular, for the detection and for the quantitative determination of human antibodies to rabbit globulin.
• the procedure according to the invention is thus hereinafter described in more detail with reference to human antibodies to rabbit globulin.
• the antibodies to rabbit globulin of the analytical sample as well as those of the conjoint standard solutions with the rabbit globulin which is, on the one hand, adsorptively bound to the solid phase and, on the other hand, is added to the test medium as a soluble rabbit globulin peroxidase conjugate compound, are preferably reacted during a single incubation stage. It is, however, also possible to react them in succession, with an interposed washing stage.
• the non- bound material is subsequently separated off in a washing stage, and the extent of the labelling is determined either in the solid or in the liquid phase.
• the colour intensity produced by this enzymatic indicator reaction is measured by photometric means and is directly proportional to the concentration of rabbit globulin antibodies used.
• the rabbit globulin antibodies present are completely neutralized, such that they cannot bind the rabbit globulin enzyme conjugate compound to the rabbit-globulin-sensitized carrier. Accordingly, the added substrate remains colourless during the subsequent analysis by means of the above-mentioned enzyme reaction.
• Antibodies of all globulin classes can be analyzed by means of the procedure according to the invention, irrespective of whether they have been formed in human beings or in an animal species.
• the main test and the control test are conducted simultaneously, using identical samples and identical reagents. These two tests are based on the solid phase 'double antigen' sandwich principle and are carried out in the 'one-step' method.
• a conventional microtitre plate of polystyrene is used as the water-insoluble carrier.
• test dilution buffer composed of 0.2 mol/l sodium phosphate, pH 7.0, 50 ml/1 foetal calf serum (from Boehringer Mannheim), 1.0 g 1 phenol and 0.2 g/1 kathon MW/WT (from Christ, Aesch, Switzerland).
• a rabbit globulin solution and a rabbit globulin peroxidase conjugate solution are used as the test reagents.
• the first reagent is composed of: 1.0 mg/ml of rabbit globulin in 20 mmol/l of TRIS/HC1, pH 7.5, 150 mmol 1 of NaCl and 0.2 ml 1 of kathon MW/WT.
• the second reagent is composed of: 50 ⁇ g/ml of rabbit globulin peroxidase conjugate compound in 0.1 mol l TRIS/HC1, pH 7.5, 5.0 g 1 bovine serum albumin, 1.0 g 1 phenol and 0.2 ml l kathon MW/WT.
• the phenol and the kathon MW/WT serve to stabilize the second reagent.
• Said second reagent must be diluted to 1:50, using the test dilution buffer described above, in order that it can be used as 'the peroxidase test solution ready for use'.
• the standard solution antibodies to rabbit globulin' is composed of 10
• TMB H2O2 solution composed of 10 mmol 1 of 3,3',5,5'-tetramethyl benzidine, 100 ml/1 of acetone, 900 ml/1 ethanol and 80 mmol/1 H2O2, and a substrate buffer solution, composed of 30 mmol/1 potassium citrate buffer, pH 4.1, and 0.15 ml/1 kathon MW/WT, are also used.
• the 'TMB / H2O2 / substrate buffer solution ready for use' is produced by mixing 1 part by volume of TMB / H2O2 solution and 20 parts by volume of substrate buffer solution.
• An appropriate number of wells of the microtitre plate are sensitized with the first reagent, in that a corresponding quantity of rabbit globulin solution from the first reagent is diluted with a sensitizing buffer, which has a pH value of 9.5 and contains 100 mmol 1 of sodium hydrogen carbonate, such that a sensitizing solution with 1.0 ⁇ g/ml of rabbit globulin is produced.
• a sensitizing buffer which has a pH value of 9.5 and contains 100 mmol 1 of sodium hydrogen carbonate, such that a sensitizing solution with 1.0 ⁇ g/ml of rabbit globulin is produced.
• 0.2 ml are pipetted into the corresponding wells of the microtitre plate.
• the microtitre plate is covered and allowed to stand for 16 to 24 hours at 15 to 25°C Subsequently, the sensitizing solution is removed from the microtitre plate and is discarded.
• microtitre plate which has been sensitized with the rabbit globulin, is then washed three times with de-ionized water.
• 0.2 ml of saturation buffer which is composed of 0.2 mol/l TRIS/HCl, ph
• the standard solution Prior to conducting the tests with test dilution buffer, the standard solution is diluted such that the following dilution series of (goat) rabbit antibodies is produced:
• the 'ready-for-use peroxidase test solution for the main test' is produced, in that the rabbit globulin peroxidase conjugate compound of the second reagent is diluted with the dilution buffer to 1:50.
• the 'ready-for-use peroxidase test solution for the control test' is prepared, in that the rabbit globulin peroxidase conjugate compound of the second reagent is diluted to 1:50 and such a quantity of rabbit globulin from the first solution is, additionally, added to this solution such that a concentration of 10 ⁇ g/ml of free rabbit globulin is provided.
• the saturation buffer is removed from a corresponding number of wells of the sensitized microtitre plate. Without a further washing stage, 200 ml of the individual solutions of
• the colour intensity in the individual wells is measured, using a multichannel photometer, at a wave length of 450 nm.
• the standard curves as they are shown in Figure 1, the absorption values obtained in respect of each concentration of (goat) anti-rabbit-globulin antibodies are recorded.
• the unknown contents of anti-rabbit-globulin antibodies of the analytical samples are then read off the corresponding standard
• the analytical sample in both tests shows an absorption in the region of the zero value, which signifies that the sample contains no antibodies;
• the analytical sample shows a distinct absorption and, in the control test, an absorption in the region of the zero value. This means that the analytical sample contains antibodies.
• anti-rabbit-globulin antibodies can be quantitatively analyzed on the corresponding standard curve
• the analytical sample shows a substantially identically high rate of absorption in the main test and in the control test, which signifies that the sample is negative with regard to content of antibodies, since the absorption results, exclusively, from an unspecific activity;
• the analytical sample shows an increased rate of absorption in the main test and in the control test, the rate of absorption of the main test being considerably higher than the rate of absorption of the control test, which means that the sample is positive with regard to content of antibodies, it being possible to read and compute the concentration of rabbit-globulin antibodies from the absorption difference.
• the sample does, however, also show a certain additional unspecific activity.
• results can either be placed in relationship with the corresponding dilutions of the concentrations of the (goat) anti-rabbit-globulin antibodies as used, or they can be given as titration stages.
• the two tests must be repeated, using a correspondingly stronger dilution of the analytical sample.
• FIG. 2 shows the progress of the development of the ATG antibodies
• FIG. 2a shows the level of the human anti-rabbit antibodies
• Figure 2b shows the ATG level. It results, from a comparison of the two Figures, that the ATG administered on a daily basis is completely neutralized as a result of the powerful development of human anti-rabbit antibodies.
• Figure 3 shows the typical progress of the ATG level in plasma during the ATG thereby in a patient where no human anti-rabbit antibodies have formed.
• the main test and the control test are conducted in the same manner as described in detail in Example 1. Instead of a standard series of different but precisely defined quantities of (goat) anti-rabbit Ig antibodies, a negative, a weakly positive and a distinctly positive control are used in both tests.
• the weakly positive control contains 10 ng/ml

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• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 1993
  • 1993-07-27 TW TW082106006A patent/TW274582B/zh active
  • 1993-07-30 CA CA002120348A patent/CA2120348A1/en not_active Abandoned
  • 1993-07-30 JP JP6504999A patent/JPH07505479A/ja active Pending
  • 1993-07-30 EP EP93917713A patent/EP0606455A1/en not_active Ceased
  • 1993-07-30 WO PCT/EP1993/002045 patent/WO1994003808A1/en not_active Application Discontinuation
  • 1993-07-30 AU AU47053/93A patent/AU665271B2/en not_active Ceased
  • 1993-07-30 RU RU94020983/14A patent/RU94020983A/ru unknown
  • 1993-08-03 ZA ZA935616A patent/ZA935616B/xx unknown
• 1994
  • 1994-03-25 NO NO941094A patent/NO941094L/no unknown
  • 1994-03-30 FI FI941502A patent/FI941502A/fi not_active Application Discontinuation

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