WO1994003808A1

WO1994003808A1 - Immunological method of analyses

Info

Publication number: WO1994003808A1
Application number: PCT/EP1993/002045
Authority: WO
Inventors: Harald Gallati
Original assignee: Fresenius Ag
Priority date: 1992-08-03
Filing date: 1993-07-30
Publication date: 1994-02-17
Also published as: JPH07505479A; AU4705393A; CA2120348A1; TW274582B; FI941502A0; NO941094L; ZA935616B; FI941502A; RU94020983A; AU665271B2; EP0606455A1; NO941094D0

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

TMMUNOLOGICAL METHOD OF ANALYSIS

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.

Immunol. Meth.. Vol. 20 (1978) pp. 25-34. In addition, the invention, with application of this procedure, is directed to the detection or the quantitative determination of certain antibodies against a given antigen.

In a procedure of this kind, 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.

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 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.

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 detected or to be measured, with the bound antigen and the labelled antigen is, in each case, carried out in two separate stages with an interposed washing stage, and in the control test the incubation stage with the bound and/or labelled antigen is carried out in the presence of an 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.

It has, in addition, been found that it is advantageous, when carrying out the procedure according to the invention, if an enzyme, preferably horseradish peroxidase, 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 antigen. In 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 first-mentioned method is preferred.

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.

Accordingly, 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. As a result of the addition to the test medium of a substantial excess 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 carrier. Accordingly, the added substrate remains colourless during the subsequent analysis by means of the above-mentioned enzyme reaction. In the event that a colour reaction does, however, occur, this is an indication that the substrate has been reacted, which is attributable to an unspecific binding of the rabbit globulin enzyme conjugate compound to the water-insoluble carrier. The correspondingly reacted quantity of substrate must then be taken into consideration when the 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 immunological reaction.

It has been found that the consistency within a test series is 3 to 7% and the consistency from day to day is 5 to 15%.

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.

Further advantages, features and details of the procedure according to the invention are set out in the description of the following Examples.

Example 1

Quantitative analyzing procedure with a control test

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. Plasma from kidney-transplant patients, which plasma had been treated with (rabbit) anti-human T- lymphocytes (ATG, from the 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/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 μg/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

SUBSTITUTE SHEET μg/ml (goat) anti-rabbit Ig antibodies (from Nordic, Tilburg, The Netherlands) in 0.1 mol l of sodium phosphate, pH 6.5, 5.0 g/1 of bovine serum albumin and 0.2 ml 1 kathon MW/WT. A tetramethyl benzidine (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 μg/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 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. The microtitre plate, which has been sensitized with the rabbit globulin, is then washed three times with de-ionized water. In order to saturate the remaining protein binding capacity of the sensitized microtitre plate, 0.2 ml of saturation buffer, which is composed of 0.2 mol/l TRIS/HCl, ph

7,5, 10 g/1 of bovine serum albumin, 0.2 mM kathon MW/WT, is added to each of the wells. The microtitre plate is sealed with self-adhesive foil and allowed to stand for at least one day at 15 to 25^βC, 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 produced:

100 ng/ml anti-rabbit Ig antibodies 50 ng/ml anti-rabbit Ig antibodies

25 ng/ml anti-rabbit Ig antibodies 0 ng/ml anti-rabbit Ig antibodies = test dilution buffer

SUBSTITUTE SHEET Also prior to conducting the tests, 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.

In parallel, 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 μg/ml of free rabbit globulin is provided.

For the purpose of conducting the test, 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

(goat) anti-rabbit antibodies (4 values) and of the diluted analytical samples (4 values) are pipetted into the corresponding wells. Into, in each case, 2 wells (2 values), 50 μl 'ready-for-use peroxidase test solution for the main test' are mixed and into, in each case, 2 wells (2 values), 50 μl of 'ready-for-use peroxide test solution for the control test' are mixed. 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 using de-ionized water. 200 ml of the ready-for-use TMB / H2O2 / 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 μl stopping solution (1 mol/l H2SO₄) are mixed into the corresponding 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 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

SUBSTITUTE SHEET curve and multiplied by the corresponding dilution factor. In this connection, four different situations may arise:

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.

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 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.

The 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.

If the measured value of an analytical sample falls outside the measured range determined by the standard curve, the two tests must be repeated, using a correspondingly stronger dilution of the analytical sample.

Figure 2 shows the progress of the development of the ATG antibodies

SUBSTITUTE SHEET during an ATG therapy in a kidney-transplant patient. Figure 2a shows the level of the human anti-rabbit antibodies and 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.

The progress illustrated occurs fairly infrequently.

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.

Example 2

Qualitative method analysis

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

(goat) anti-rabbit Ig antibodies and the distinctly positive control contains 100 ng/ml (goat) anti-rabbit Ig antibodies.

The evaluation of the results of the main test and of the control test are carried out in an analogous manner, as set out in Example 1.

SUBSTITUTE SHEET

Claims

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.

SUBSTITUTE SHEET

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, 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 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.

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SUBSTITUTE SHEET