CA2120348A1 - Immunological method of analysis - Google Patents
Immunological method of analysisInfo
- Publication number
- CA2120348A1 CA2120348A1 CA002120348A CA2120348A CA2120348A1 CA 2120348 A1 CA2120348 A1 CA 2120348A1 CA 002120348 A CA002120348 A CA 002120348A CA 2120348 A CA2120348 A CA 2120348A CA 2120348 A1 CA2120348 A1 CA 2120348A1
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- CA
- Canada
- Prior art keywords
- antigen
- antibodies
- bound
- measured
- rabbit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
In a diagnostic procedure for the detection or quantitative determination of antibodies in biological fluids by means of an immunological reaction, in which the sought antibodies are incubated with the corresponding antigen, one portion of which antigen is bound to a water-insoluble carrier, and the other portion of which is provided with a label, after the separation of the phases measuring the extent of the label either in the liquid or in the solid phase, it is possible for components of the analytical sample to bind a labelled antigen to a water-insoluble carrier in an unspecific manner, thus producing incorrect positive results. In order to obtain valid results, the same reaction is conducted but incubation takes place in the presence of an excess of non-bound and non-labelled antigen and, after separation of the phases, the measured extent of the marking is also taken into consideration in the qualitative evaluation or the quantitative analysis of the antibodies. The procedure is particularly suitable for the analysis of human antibodies to rabbit globulin, the antigen being a rabbit globulin.
Description
wo 94/03808 2 1 ? Q 3 ~ 8 PCI'/EP93/021~45 IMMVNOLOGICAL METHOD OF ANALYSIS
The invention relates to a diagnostic procedure for the detection or quantitativs determination of antibodies in biological fluid~ 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 iB 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 liquit phase and measuring the e~tent of the label either in the solid or in the liquid 5 ~ phase. A procedure of this kind is known from ~i~i~C~L
Immunol. Meth, Vol. 20 (1978) pp. 25-34. In addition, the invention, with application of this procedure, i8 directed to the detection or the - quantitative determination of certain alltibodies agai~t a given antigen.
In a procedure of this kind, the result may be invalidated, since an unspecific reaction must be e~pected 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, :E; thereby erroneously indicating the presence of antibodies, thus leading to incorrect positive results.
Object of the present invention is to obtain valid result~ or to detect the undesirable effects of certain components of the analytical sample 30 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 3s against an antigen, which i8 also be to specified and which, in turn, e~ercises an antibody-function against human T-lympbocytes.
.
According to the invention, 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
WO 94/03808 PCr/EP93/02045 2~2~3~8 -2-is carried out in a separate control test, but the incubation i8 carried out in the presence of an exces3 of non-bound and non-labelled antigen, the solid phase iB Beparated from the liquid phase, the extent of the label is measured and is also taken into consideration during the qualitative 5 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 detectsd or to be measured, are incubated jointly, from the outset, with the bound antigen and, in the control test, ~D with the e~cess of non-bound and non-labelled antigen.
It is, however, also possible to carry out the procedure according to the invention such that the incubation of the antibodies, which are to be detectsd or to be measured, with the bound antigen and the labelled 5 antigen is, in each case, carried out in two separate 6tages with an interposed waahing stage, and in the control test the incubation stage with the bound and/or labelled antigen i8 carried out in the presence of an e~cess 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 detectsd or to be measured.
It has, in addition, been found that it is advantageous, when carrying out the procedure according to the invention, if an enzyme, preferably horseradish pero~dase, is used for the labelling of the antigen.
The further object is met in that the procedure is carried out with human antibodies to rabbit globulin and with rabbit globulin as the 30 antigen. L~ this regard, it is possible to conduct the incubation of the human antibodies to rabbit globulin and the rabbit globulin as the - antigen jointly from the outset, or the incubation of the antibody with the bound and the labelled antigen can be carried out, in each case, in two separate stages with an interposed washing stage, although the ~i first-mentioned method i8 preferred.
The proposed procedures are particularly suitable for the purpose of, prior to, during and a~er the therapy with antibodies from an immune SUBSTITUTE S~JEET
~?' WO 94/03808 2 1 2 ~ 3 1 ~ PCr/EP93/02045 serum against human T-lymphocyte globulin such a~ 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 5 serum.
, ~;
Accordingly, the procedures according to the invention can be used, in particular, for the detection and for the quantitati~re 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 i8, on the one hand, sdsorptively bound to the solid phase and, on the other hand, is added to the test medium as a 5 soluble rabbit globulin pero~idase 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 washi~g stage, and the extent of the labelling is dete~nined either in tbe solid or in the :~ liquid phase. The colour intenuty produced by this enzymatic indicator reaction i~ mea~ured by photometric means and is direcl Iy proportional to the concentration of rabbit globuUn antibodies used. As a result of the addition to the test medium of a substantial escess of free rabbit globulin, which is not adsorbed on a water-insoluble carrier and is also not labelled with an enzyme, the rabbit globulin antibodies present are completely neutralized, such that they cannot bind the rabbit globulin enzyme conjugate compound to the rabbit-globulin-sensitized calTier.
Accordingly, the added substrate remains colourless during the subsequent analysis by means of the above-mentioned enzyme reaction.
30 In the event that a colour reaction does, however, occur, this is an indication that the substrate has been reacted, which is attributablè to an unspecific binding of the rabbit globulin enzyme co~uugatecompound to the water-insoluble carrer. The correspondingly reacted quantity of substrate must then be taken into consideration when the 3~ results are evaluated, in order to arrive at an accurate result.
In order to achieve the highest possible degree of test sensitivity, an incubation period of 16 to 24 hours at 15- to 25-C is proposed for the SUBSTITUTE SHEET
w o 94/03808 PC~r/EP93/02045 2 1 2~348 ^ 4 -immunological reaction.
It has been found that the consistency within a test series is 3 to 7% and the consistency from day to day i8 5 to 1596.
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.
F~ther advantages, features and details of the procedure according to the invention are set out in the description of the following Examples.
~5 Quantitative analvzin~ T)rocedure with a control test The main teat 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 camer. Plasma from kidney-transplant patients, which plasma had been treated with (rabbit) anti-human T-lymphocytes (ATG, from tbe company Fresenius, Frankfurt), is used as the samples. Prior to the tests, the samples were diluted to 1:5 with the test dilution buffer, composed of 0.2 mol/l sodium phosphate, pH 7.0, 50 ml~l foetal calf serum (from Boehringer Mannheim), 1.0 gll phenol and 0.2 gll 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 mmolll of TRIS/HClt pH 7.5, 150 mmolll of NaCl and 0.2 mlll of kathon MW/WT. The second reagent is composed of: 50 ~g/ml of rabbit globulin pero~cidase conjugate compound in 0.1 moll T~IS~H Cl, p H 7.5, 5.0 g~ bovine serum albumin, 1.0 g/l phenol and 0.2 mlll 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 descnbed above, in order that it can be used as 'the pero~cidase test solution ready for use'.
The standard solution 'antibodies to rabbit globulin' is composed of 10 ~;UE~STITIJTE Sll~ET
W0 94/03808 2 I 2 ~ 3 i7`; 8 PCI`/EP93/0204~ :
~g/ml (goat) anti-rabbit Ig antibodies (from Nordic, Tilburg, The Netherlands) in 0.1 molll of sodium phosphate, pH 6.5, 5.0 g/l of bovine semm albumin and 0.2 ml/l kathon MW/WT. A tetramethyl benzidine (TMB) H22 solution, composed of 10 mmolA of 3,3',5~5'~tetramethyl benzidine, 100 ml/l of acetone, 900 mlll ethanol and 80 mmolll H202, and a substrate buffer solution, composed of 30 mmoVI potassium citrate buffer, pH 4.1, and 0.15 mlll kathon MW/WT, are also used. The ~ / H22 / substrate buffer solution ready for u~e' i8 produced by minng 1 part by volume of T~ / H22 solution and 20 parts by volume 0 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/l of sodium hydrogen carbonate, such that a sensitizing solution with 1.0 ~ml of rabbit globulin is produced. Immediately after the production of the sensitizing solution, 0.2 ml are pipetted into the corresponding wells of the microtitre plate. The microtitre plate is covered and aUowed to :~ stand for 16 to 24 hours at 15 to 25 C. Subsequently, the sensitizing solution is removed from the microtitre plate and i8 discarded. The microtitre plate, whicb has been sensitized with the rabbit globulin, i8 then washed three times with d~ionized water. In order to saturate the remaining protein binding capacity of the sensitized microtitre plate, 0.2 ml of saturation buffer, which ia composed of 0.2 molll TRISJHCl, ph 7,5, 10 g/l of bov~ne serum albumin, 0.2 ml/l kathon MW/WT, i8 added to each of the wells. The microtitre plate i8 sealed with self-adhes*e foil and allowed to stand for st least one day at 15 to 25 C, prior to further use.
:
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 prQduced:
- 100 ng/ml anti-rabbit Ig antibodies 3650 ng/ml anti-rabbit Ig antibodies 25 n&~ml anti-rabbit Ig antibodie~
O ng/ml anti-rabbit Ig antibodies = test dilution buffer SUBSTITUTE SHEET
WO 94/03808 PCr/EP93/02045 2.~203~18 -6-Also prior to conducting the tests, the 'ready-for-use perosidase test solution for the main test' is produced, in tbat the rabbit globulin peroxidase conJugate compound of the second reagent is diluted with the dilution buffer to 1:50.
In parallel, the 'ready-for-use peroxidase test solution for the cont ol test' i8 prepared, in that the rabbit globuliD perosida~e co~ugate compound of the second reagent is diluted to 1:50 and such a quantity of rabUt globulin from the first solution is, additionally, added to this 0 solution such that a concentration of 10 ~dml of free rabbit globulin is provided.
For the purpose of conducting the te~t, tbe saturation buffer is removed from a corresponding number of wells of the sensitized microtitre plate.
5 Without a further washing stage, 200 ml of the individual solutions of (goat) anti-rabbit antibodies (4 values) and of the diluted analy-tical samples (4 values) are pipetted into the corresponding wells. Into, in eac}~ case, 2 wells (2 values), 50 ~1 'ready-for-use pero~idase test solution for the main test' are mi~ed and into, in each case, 2 wells (2 values), 50 111 of 'ready-for-use pero~ide test solution for the control test' are ~ed. The microtitre plate is then again sealed with the self- ~
adhesive foil, and is incubated for 16 to 24 hours at 15 to 25 C. The test solution is subsequently removed and the wells are washed 6 to 8 times u3ing de-ionized water. 200 ml of the ready-for-use TMB / H22 /
2~ substrate buffer solution, which has been prepared immediately prior to the enzyme reaction, are pipetted into the corresponding wells and incubated at 15 to 25 C for 10 minutes. In order to stop the peroxidase activity and to intensify and stabilize the colour intensity produced, 100 111 stopping solution (1 moVl H2SO4) are mixed into the comsponding 30 wells.
Within half an hour of stopping the enzymatic reaction, the colour intensity in the individual wells is measured, using a multichannel photometer, at a wave length of 450 nm. When plotting the standard 36 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 an~-rabbit-globulin antibodies of the analytical samples are then read off the corresponding standard SUE~STITUl~E SHEET
W0 94/0380~ 2 1 2 ~ 3 4 8 PCl/EP93/02045 curve and multiplied by the corresponding dilution factor. In this connection, four different situations may arise:
- l'he analytical sample in both tests shows an abso2ption 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 ~ralue.
~D This means that the analytical sample contains antibodies.
The content of 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 m~un test and in the control test, which signifies that the sample is negative with regard to content of antibodies, since the absorption results, esclusively, from an unspeciSc activity, - the analytical sample ~hows 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 ~i 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.
The results can either be placed in relationship with the corresponding3D dilutions of the concentrations of the (goat) anti-rabbit-globulin antibodies as used, or they can be given as titration stages.
If the measured value of an ~alytical ~smple falls outside the measured range determined by the standard cur~e, the two tests must ~5 be repeated, using a correspondingly stronger dilution of tbe analytical sample.
Figure 2 shows the progress of the development of the ATG antibodies SUBSTITUTE SHEET
WO 94/03808 PCl`/EP~3/02045 21203~8 8 :
during an ATG therapy in a kidney-transplant patient. Figure 2a shows tbe level of the human anti-rabbit antibodies ant Figure 2b sbows tbe ATG level. It results, from a comparison of the two Figures, ~ ~
that the ATG administered on a daily basis iB completely neutrslized as ~-s a result of the powerfbl development of human anti-rsbbit antibodies.
The progress illustrated occurs fairly infrequently.
Figure 3 shows the typical progress of the ATG level in plasma during tbe ATG thereby in a patient where no human anti-rabbit antibodies ~o have formed. --nple 2 Qualitative method analvsis The main test and the control test are conducted in the same manner as described in detail in E~ample 1. Instead of a standard se ies of different but precisely defined quantities of (goat) anti-rabbit Ig antibodies, a negative, a wealdy positive and a distinctly positive control are u~ed in both pests~ 'rhe wealdy positive control contains 10 ndml (goat) anti-rabbit Ig antibodies and the distinctly positi~e control contains 100 ndml (goat) anti-rabbit Ig antibodies.
The evalualion of the results of the main test and of the control test are :~ carned out in an analogous manner, as set out in Example 1.
: ~ SUBSTITUTE SHEET
The invention relates to a diagnostic procedure for the detection or quantitativs determination of antibodies in biological fluid~ 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 iB 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 liquit phase and measuring the e~tent of the label either in the solid or in the liquid 5 ~ phase. A procedure of this kind is known from ~i~i~C~L
Immunol. Meth, Vol. 20 (1978) pp. 25-34. In addition, the invention, with application of this procedure, i8 directed to the detection or the - quantitative determination of certain alltibodies agai~t a given antigen.
In a procedure of this kind, the result may be invalidated, since an unspecific reaction must be e~pected 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, :E; thereby erroneously indicating the presence of antibodies, thus leading to incorrect positive results.
Object of the present invention is to obtain valid result~ or to detect the undesirable effects of certain components of the analytical sample 30 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 3s against an antigen, which i8 also be to specified and which, in turn, e~ercises an antibody-function against human T-lympbocytes.
.
According to the invention, 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
WO 94/03808 PCr/EP93/02045 2~2~3~8 -2-is carried out in a separate control test, but the incubation i8 carried out in the presence of an exces3 of non-bound and non-labelled antigen, the solid phase iB Beparated from the liquid phase, the extent of the label is measured and is also taken into consideration during the qualitative 5 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 detectsd or to be measured, are incubated jointly, from the outset, with the bound antigen and, in the control test, ~D with the e~cess of non-bound and non-labelled antigen.
It is, however, also possible to carry out the procedure according to the invention such that the incubation of the antibodies, which are to be detectsd or to be measured, with the bound antigen and the labelled 5 antigen is, in each case, carried out in two separate 6tages with an interposed waahing stage, and in the control test the incubation stage with the bound and/or labelled antigen i8 carried out in the presence of an e~cess 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 detectsd or to be measured.
It has, in addition, been found that it is advantageous, when carrying out the procedure according to the invention, if an enzyme, preferably horseradish pero~dase, is used for the labelling of the antigen.
The further object is met in that the procedure is carried out with human antibodies to rabbit globulin and with rabbit globulin as the 30 antigen. L~ this regard, it is possible to conduct the incubation of the human antibodies to rabbit globulin and the rabbit globulin as the - antigen jointly from the outset, or the incubation of the antibody with the bound and the labelled antigen can be carried out, in each case, in two separate stages with an interposed washing stage, although the ~i first-mentioned method i8 preferred.
The proposed procedures are particularly suitable for the purpose of, prior to, during and a~er the therapy with antibodies from an immune SUBSTITUTE S~JEET
~?' WO 94/03808 2 1 2 ~ 3 1 ~ PCr/EP93/02045 serum against human T-lymphocyte globulin such a~ 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 5 serum.
, ~;
Accordingly, the procedures according to the invention can be used, in particular, for the detection and for the quantitati~re 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 i8, on the one hand, sdsorptively bound to the solid phase and, on the other hand, is added to the test medium as a 5 soluble rabbit globulin pero~idase 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 washi~g stage, and the extent of the labelling is dete~nined either in tbe solid or in the :~ liquid phase. The colour intenuty produced by this enzymatic indicator reaction i~ mea~ured by photometric means and is direcl Iy proportional to the concentration of rabbit globuUn antibodies used. As a result of the addition to the test medium of a substantial escess of free rabbit globulin, which is not adsorbed on a water-insoluble carrier and is also not labelled with an enzyme, the rabbit globulin antibodies present are completely neutralized, such that they cannot bind the rabbit globulin enzyme conjugate compound to the rabbit-globulin-sensitized calTier.
Accordingly, the added substrate remains colourless during the subsequent analysis by means of the above-mentioned enzyme reaction.
30 In the event that a colour reaction does, however, occur, this is an indication that the substrate has been reacted, which is attributablè to an unspecific binding of the rabbit globulin enzyme co~uugatecompound to the water-insoluble carrer. The correspondingly reacted quantity of substrate must then be taken into consideration when the 3~ results are evaluated, in order to arrive at an accurate result.
In order to achieve the highest possible degree of test sensitivity, an incubation period of 16 to 24 hours at 15- to 25-C is proposed for the SUBSTITUTE SHEET
w o 94/03808 PC~r/EP93/02045 2 1 2~348 ^ 4 -immunological reaction.
It has been found that the consistency within a test series is 3 to 7% and the consistency from day to day i8 5 to 1596.
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.
F~ther advantages, features and details of the procedure according to the invention are set out in the description of the following Examples.
~5 Quantitative analvzin~ T)rocedure with a control test The main teat 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 camer. Plasma from kidney-transplant patients, which plasma had been treated with (rabbit) anti-human T-lymphocytes (ATG, from tbe company Fresenius, Frankfurt), is used as the samples. Prior to the tests, the samples were diluted to 1:5 with the test dilution buffer, composed of 0.2 mol/l sodium phosphate, pH 7.0, 50 ml~l foetal calf serum (from Boehringer Mannheim), 1.0 gll phenol and 0.2 gll 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 mmolll of TRIS/HClt pH 7.5, 150 mmolll of NaCl and 0.2 mlll of kathon MW/WT. The second reagent is composed of: 50 ~g/ml of rabbit globulin pero~cidase conjugate compound in 0.1 moll T~IS~H Cl, p H 7.5, 5.0 g~ bovine serum albumin, 1.0 g/l phenol and 0.2 mlll 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 descnbed above, in order that it can be used as 'the pero~cidase test solution ready for use'.
The standard solution 'antibodies to rabbit globulin' is composed of 10 ~;UE~STITIJTE Sll~ET
W0 94/03808 2 I 2 ~ 3 i7`; 8 PCI`/EP93/0204~ :
~g/ml (goat) anti-rabbit Ig antibodies (from Nordic, Tilburg, The Netherlands) in 0.1 molll of sodium phosphate, pH 6.5, 5.0 g/l of bovine semm albumin and 0.2 ml/l kathon MW/WT. A tetramethyl benzidine (TMB) H22 solution, composed of 10 mmolA of 3,3',5~5'~tetramethyl benzidine, 100 ml/l of acetone, 900 mlll ethanol and 80 mmolll H202, and a substrate buffer solution, composed of 30 mmoVI potassium citrate buffer, pH 4.1, and 0.15 mlll kathon MW/WT, are also used. The ~ / H22 / substrate buffer solution ready for u~e' i8 produced by minng 1 part by volume of T~ / H22 solution and 20 parts by volume 0 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/l of sodium hydrogen carbonate, such that a sensitizing solution with 1.0 ~ml of rabbit globulin is produced. Immediately after the production of the sensitizing solution, 0.2 ml are pipetted into the corresponding wells of the microtitre plate. The microtitre plate is covered and aUowed to :~ stand for 16 to 24 hours at 15 to 25 C. Subsequently, the sensitizing solution is removed from the microtitre plate and i8 discarded. The microtitre plate, whicb has been sensitized with the rabbit globulin, i8 then washed three times with d~ionized water. In order to saturate the remaining protein binding capacity of the sensitized microtitre plate, 0.2 ml of saturation buffer, which ia composed of 0.2 molll TRISJHCl, ph 7,5, 10 g/l of bov~ne serum albumin, 0.2 ml/l kathon MW/WT, i8 added to each of the wells. The microtitre plate i8 sealed with self-adhes*e foil and allowed to stand for st least one day at 15 to 25 C, prior to further use.
:
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 prQduced:
- 100 ng/ml anti-rabbit Ig antibodies 3650 ng/ml anti-rabbit Ig antibodies 25 n&~ml anti-rabbit Ig antibodie~
O ng/ml anti-rabbit Ig antibodies = test dilution buffer SUBSTITUTE SHEET
WO 94/03808 PCr/EP93/02045 2.~203~18 -6-Also prior to conducting the tests, the 'ready-for-use perosidase test solution for the main test' is produced, in tbat the rabbit globulin peroxidase conJugate compound of the second reagent is diluted with the dilution buffer to 1:50.
In parallel, the 'ready-for-use peroxidase test solution for the cont ol test' i8 prepared, in that the rabbit globuliD perosida~e co~ugate compound of the second reagent is diluted to 1:50 and such a quantity of rabUt globulin from the first solution is, additionally, added to this 0 solution such that a concentration of 10 ~dml of free rabbit globulin is provided.
For the purpose of conducting the te~t, tbe saturation buffer is removed from a corresponding number of wells of the sensitized microtitre plate.
5 Without a further washing stage, 200 ml of the individual solutions of (goat) anti-rabbit antibodies (4 values) and of the diluted analy-tical samples (4 values) are pipetted into the corresponding wells. Into, in eac}~ case, 2 wells (2 values), 50 ~1 'ready-for-use pero~idase test solution for the main test' are mi~ed and into, in each case, 2 wells (2 values), 50 111 of 'ready-for-use pero~ide test solution for the control test' are ~ed. The microtitre plate is then again sealed with the self- ~
adhesive foil, and is incubated for 16 to 24 hours at 15 to 25 C. The test solution is subsequently removed and the wells are washed 6 to 8 times u3ing de-ionized water. 200 ml of the ready-for-use TMB / H22 /
2~ substrate buffer solution, which has been prepared immediately prior to the enzyme reaction, are pipetted into the corresponding wells and incubated at 15 to 25 C for 10 minutes. In order to stop the peroxidase activity and to intensify and stabilize the colour intensity produced, 100 111 stopping solution (1 moVl H2SO4) are mixed into the comsponding 30 wells.
Within half an hour of stopping the enzymatic reaction, the colour intensity in the individual wells is measured, using a multichannel photometer, at a wave length of 450 nm. When plotting the standard 36 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 an~-rabbit-globulin antibodies of the analytical samples are then read off the corresponding standard SUE~STITUl~E SHEET
W0 94/0380~ 2 1 2 ~ 3 4 8 PCl/EP93/02045 curve and multiplied by the corresponding dilution factor. In this connection, four different situations may arise:
- l'he analytical sample in both tests shows an abso2ption 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 ~ralue.
~D This means that the analytical sample contains antibodies.
The content of 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 m~un test and in the control test, which signifies that the sample is negative with regard to content of antibodies, since the absorption results, esclusively, from an unspeciSc activity, - the analytical sample ~hows 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 ~i 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.
The results can either be placed in relationship with the corresponding3D dilutions of the concentrations of the (goat) anti-rabbit-globulin antibodies as used, or they can be given as titration stages.
If the measured value of an ~alytical ~smple falls outside the measured range determined by the standard cur~e, the two tests must ~5 be repeated, using a correspondingly stronger dilution of tbe analytical sample.
Figure 2 shows the progress of the development of the ATG antibodies SUBSTITUTE SHEET
WO 94/03808 PCl`/EP~3/02045 21203~8 8 :
during an ATG therapy in a kidney-transplant patient. Figure 2a shows tbe level of the human anti-rabbit antibodies ant Figure 2b sbows tbe ATG level. It results, from a comparison of the two Figures, ~ ~
that the ATG administered on a daily basis iB completely neutrslized as ~-s a result of the powerfbl development of human anti-rsbbit antibodies.
The progress illustrated occurs fairly infrequently.
Figure 3 shows the typical progress of the ATG level in plasma during tbe ATG thereby in a patient where no human anti-rabbit antibodies ~o have formed. --nple 2 Qualitative method analvsis The main test and the control test are conducted in the same manner as described in detail in E~ample 1. Instead of a standard se ies of different but precisely defined quantities of (goat) anti-rabbit Ig antibodies, a negative, a wealdy positive and a distinctly positive control are u~ed in both pests~ 'rhe wealdy positive control contains 10 ndml (goat) anti-rabbit Ig antibodies and the distinctly positi~e control contains 100 ndml (goat) anti-rabbit Ig antibodies.
The evalualion of the results of the main test and of the control test are :~ carned out in an analogous manner, as set out in Example 1.
: ~ SUBSTITUTE SHEET
Claims (11)
1. 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, characterized in that, prior to, during or after the main test, the same reaction as that of the main test 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 labelling is measured and is also taken into consideration during the qualitative detection or the quantitative determination of the antibodies.
2. Procedure according to claim 1, characterized in that the antibodies which are to be detected or which are to be measured are jointly incubated from the outset with the bound antigen and the labelled antigen and, in the control test, with the excess of non-bound and non-labelled antigen.
3. Procedure according to claim 1, characterized in that the incubation of the antibody, which is to be detected or to be measured, with the bound antigen and the labelled antigen is carried out, in each case, in two separate steps with an interposed washing step and, in the control test, the incubation stage with the bound and/or with the labelled antigen is carried out in the presence of an excess of non-bound and non-labelled antigen.
4. Procedure according to one of claims 1 to 3, characterized in that the antibodies which are to be detected or which are to be measured are human antibodies to rabbit globulin.
5. Procedure according to one of claims 1 to 4, characterized in that the antigen is a rabbit globulin.
6. Procedure according to one of claims 1 to 5, characterized in that an enzyme is used as means to label the antigen.
7. Procedure according to claim 6, characterized in that the enzyme is horseradish peroxidase.
8. Diagnostic procedure for the detection or the quantitative determination of antibodies in biological fluids by means of an immunological reaction, in which the antibodies to be detected or measured are incubated with the corresponding antigen, one portion of which antigen is bound to a water-insoluble carrier, and the other portion of which is 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, characterized in that the antibodies which are to be detected or which are to be quantitatively measured are human antibodies to rabbit globulin and the antigen is a rabbit globulin.
9. Procedure according to claim 8, caricatured in that the incubation of the antibodies, which are to be detected or which are to be measured, with the bound antigen and the labelled antigen, is carried out jointly from the outset.
10. Procedure according to claim 8, characterized in that the incubation of the antibodies, which are to be detected or which are to be measured, with the bound antigen and the labelled antigen, is carried out, in each case, in two separate steps with an interposed washing step.
11. The invention as described above.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH2432/92 | 1992-08-03 | ||
| CH243292 | 1992-08-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2120348A1 true CA2120348A1 (en) | 1994-02-17 |
Family
ID=4233675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002120348A Abandoned CA2120348A1 (en) | 1992-08-03 | 1993-07-30 | Immunological method of analysis |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0606455A1 (en) |
| JP (1) | JPH07505479A (en) |
| AU (1) | AU665271B2 (en) |
| CA (1) | CA2120348A1 (en) |
| FI (1) | FI941502A (en) |
| NO (1) | NO941094D0 (en) |
| RU (1) | RU94020983A (en) |
| TW (1) | TW274582B (en) |
| WO (1) | WO1994003808A1 (en) |
| ZA (1) | ZA935616B (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4595661A (en) * | 1983-11-18 | 1986-06-17 | Beckman Instruments, Inc. | Immunoassays and kits for use therein which include low affinity antibodies for reducing the hook effect |
| US4788138A (en) * | 1987-04-30 | 1988-11-29 | Beckman Instruments, Inc. | Method to achieve a linear standard curve in a sandwich immunoassay |
| US5223400A (en) * | 1989-06-01 | 1993-06-29 | The Ontario Cancer Institute | Immunoassay method for determining the specificity of binding of a monoclonal antibody to an antigen |
-
1993
- 1993-07-27 TW TW082106006A patent/TW274582B/zh active
- 1993-07-30 JP JP6504999A patent/JPH07505479A/en 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 CA CA002120348A patent/CA2120348A1/en not_active Abandoned
- 1993-07-30 AU AU47053/93A patent/AU665271B2/en not_active Ceased
- 1993-07-30 RU RU94020983/14A patent/RU94020983A/en unknown
- 1993-08-03 ZA ZA935616A patent/ZA935616B/en unknown
-
1994
- 1994-03-25 NO NO941094A patent/NO941094D0/en unknown
- 1994-03-30 FI FI941502A patent/FI941502A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| FI941502A (en) | 1994-05-26 |
| FI941502A0 (en) | 1994-03-30 |
| JPH07505479A (en) | 1995-06-15 |
| TW274582B (en) | 1996-04-21 |
| ZA935616B (en) | 1994-02-03 |
| RU94020983A (en) | 1996-11-27 |
| EP0606455A1 (en) | 1994-07-20 |
| AU665271B2 (en) | 1995-12-21 |
| WO1994003808A1 (en) | 1994-02-17 |
| NO941094L (en) | 1994-03-25 |
| AU4705393A (en) | 1994-03-03 |
| NO941094D0 (en) | 1994-03-25 |
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