ANTI-DRUG ANTIBODY ASSAY

Abstract

The invention provides a method for the immunological determination of an antibody against a drug antibody in a sample using a double antigen bridging immunoassay comprising a capture drug antibody and a tracer drug antibody, characterized in that the capture drug antibody is a mixture of said drug antibody conjugated to the solid phase at at least two different antibody sites and the tracer drug antibody is a mixture of said drug antibody conjugated to the detectable label at at least two different antibody sites.

Claims

1. Method for the immunological determination of an antibody against a drug antibody in a sample using a double antigen bridging immunoassay comprising a capture drug antibody and a tracer drug antibody, characterized in that i) the capture drug antibody is a mixture of said drug antibody comprising at least two of said drug antibodies that differ in the antibody site at which they are conjugated to the solid phase; and ii) the tracer drug antibody is a mixture of said drug antibody comprising at least two of said drug antibodies that differ in the antibody site at which they are conjugated to the detectable label.

2. Method according to claim 1, characterized in that conjugation of the drug antibody to its conjugation partner is performed by chemically binding via N-terminal and/or s-amino groups (lysine), s-amino groups of different lysines, carboxy-, sulfhydryl-, hydroxyl- and/or phenolic functional groups of the amino acid backbone of the drug antibody and/or sugar alcohol groups of the carbohydrate structure of the drug antibody.

3. Method according to claim 1 or 2, characterized in that the tracer drug antibody mixture comprises the drug antibody conjugated via an amino group and via a carbohydrate structure to their conjugation partner.

4. Method according to any one of claims 1 to 3, characterized in that the capture drug antibody mixture comprises the drug antibody conjugated via an amino group and via a carbohydrate structure to their conjugation partner.

5. Method according to any one of claims 1 to 3, characterized in that conjugation of the capture drug antibody to the solid phase is performed by passive adsorption.

6. Method according to any one of claims 1 to 5, characterized in that the ratio of capture drug antibody to tracer drug antibody is 1:10 to 50:1 (ratio means ratio of antibody molecules irrespective of the molecular weight of the conjugates which can be different).

7. Method according to any one of claims 1 to 6, characterized in that the ratio of amino conjugated drug antibody (either tracer or capture drug antibody) to carbohydrate conjugated drug antibody (either tracer or capture drug antibody) in such a mixture is 1:10 to 10:1 (ratio means ratio of antibody molecules irrespective of the molecular weight of the conjugates which can be different).

8. Method according to any one of claims 1 to 4 and 6 to 7, characterized in that the capture drug antibody is immobilized via a specific binding pair.

9. Method according to claim 8, characterized in that the capture drug antibody is conjugated to biotin and immobilization is performed via immobilized avidin or streptavidin.

10. Method according to anyone of claims 1 to 9, characterized in that the tracer drug antibody is conjugated to the detectable label via a specific binding pair.

11. Method according to claim 10, characterized in that the tracer drug antibody is conjugated to digoxigenin and linking to the detectable label is performed via an antibody against digoxigenin.

Description

DESCRIPTION OF FIGURES

[0043] FIG. 1 Bridging assay for detection of anti-drug antibodies: [0044] The biotinylated drug antibody (Capture-BI) is bound to a streptavidin-coated microtiter plate (SA-MTP). The anti-drug antibody bridges the capture drug antibody (Capture-BI; BI=biotinylated) with digoxigenin-labeled tracer drug antibody (Tracer-DIG; DIG=digoxinylated). The immobilized complex is detected by polyclonal anti-digoxigenin horse-radish peroxidase conjugate (pAB<DIG>-POD). Polyclonal rabbit anti-drug antibody (rpAB) is used as standard.

[0045] FIG. 2 Standard curve of bridging ELISA variant 1 using conjugates of example 1 and 4: [0046] The optical densities (ODs) are given for the various concentration of rpAB as diluted in PBS-T-buffer (phosphate buffered saline, 0.05 Vol % Tween® 20) with 5% human serum.

[0047] FIG. 3 Standard curve of bridging ELISA variant 2 using conjugates of example 2 and 5: [0048] The optical densities (ODs) are given for the various concentration of rpAB as diluted in PBS-T-buffer with 5% human serum.

[0049] FIG. 4 Standard curve of bridging ELISA variant 3 using conjugates of example 3 and 6: [0050] The optical densities (ODs) are given for the various concentration of rpAB as diluted in PBS-T-buffer with 5% human serum.

[0051] FIG. 5 Standard curve of bridging ELISA variant 4 using conjugates of examples 1, 3 and 4, 6: [0052] The optical densities (ODs) are given for the various concentration of rpAB as diluted in PBS-T-buffer with 5% human serum.

[0053] FIG. 6 Standard curve of bridging ELISA variant 5 using conjugates of example 1, 2, 3 and 4, 5, 6: [0054] The optical densities (ODs) are given for the various concentration of rpAB as diluted in PBS-T-buffer with 5% human serum.

[0055] FIG. 7 Standard curve of bridging ELISA variant 1 using passive adsorption for solid immobilization: [0056] The optical densities (ODs) are given for the various concentration of rpAB as diluted in PBS-T-buffer with 5% human serum.

EXAMPLES

Example 1

[0057] Biotinylation of Antibody mAB IL-6R with D-biotinoyl-aminocaproic Acid-N-hydroxysuccinimide Ester

[0058] Antibody against IL-6 receptor (mAB IL-6R) has been dialyzed against buffer (100 mM potassium phosphate buffer (in the following denoted as K—PO.sub.4), pH 8.5). Afterwards the solution was adjusted to a protein concentration of 10 mg/ml. D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:5. After 60 minutes the reaction was stopped by adding L-lysine. The excess of the labeling reagent was removed by dialysis against 25 mM K—PO.sub.4 supplemented with 150 mM NaCl, pH 7.5.

Example 2

[0059] Biotinylation of mAB IL-6R with D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide Ester After Treatment With Citraconic Acid Anhydride

[0060] mAB IL-6R has been dialyzed against 100 mM K—PO.sub.4, pH 8.4. Afterwards the solution was adjusted to a protein concentration of 20 mg/ml. Citraconic acid anhydride was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:5. After 120 minutes the reaction was stopped by chromatography on a column with Sephadex® G25 equilibrated with 100 mM K—PO.sub.4, pH 8.4. The antibody solution was adjusted to a protein concentration of about 4 mg/ml. D-biotinoyl-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:5. The reaction was stopped after 60 minutes by adding L-lysine. The surplus of the labeling reagent was removed by dialysis against 200 mM sodium acetate buffer, pH 5.0. The antibody solution was transferred to a 25 mM K—PO.sub.4 supplemented with 150 mM NaCl, pH 7.2, by chromatography on a column with Sephadex® G25.

Example 3

[0061] Biotinylation of mAB IL-6R with Biotin Hydrazide

[0062] mAB IL-6R has been dialyzed against 100 mM sodium acetate buffer, pH 5.5. Afterwards the solution was adjusted to a protein concentration of 20 mg/ml. Sodium periodate was dissolved in 100 mM sodium acetate buffer, pH 5.5, and was added to the antibody solution to a final concentration of 10 mM. The reaction was stopped after 30 minutes by chromatography on a Sephadex® G25 column equilibrated with 100 mM sodium acetate buffer, pH 5.5. The antibody solution was adjusted to a protein concentration of about 5 mg/ml. Biotin hydrazide was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:50. The reaction was stopped after 120 minutes by adding sodium borohydride to a final concentration of 15 mM. After 30 minutes the antibody solution was dialyzed against 25 mM K—PO) supplemented with 150 mM NaCl, pH 7.2

Example 4

[0063] Digoxigenylation of mAB IL-6R with Digoxigenin 3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide Ester

[0064] mAB IL-6R has been dialyzed against digoxigenylation buffer (100 mM K—PO.sub.4 pH 8.5). Afterwards the solution was adjusted to a protein concentration of 10 mg/ml. Digoxigenin 3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:5. After 60 minutes the reaction has been stopped by adding L-lysine. The surplus of labeling reagent was removed by dialysis against 25 mM K—PO.sub.4 supplemented with 150 mM NaCl, pH 7.5.

Example 5

[0065] Digoxigenylation of mAB IL-6R with Digoxigenin 3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide Ester after Treatment with Citraconic Acid Anhydride

[0066] mAB IL-6R has been dialyzed against 100 mM K—PO.sub.4, pH 8.4. Afterwards the solution was adjusted to a protein concentration of 20 mg/ml. Citraconic acid anhydride was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:5. The reaction has been stopped after 120 minutes by chromatography on a column with Sephadex® G25 equilibrated with 100 mM K—PO.sub.4, pH 8.4. The antibody solution was adjusted to a protein concentration of about 4 mg/ml.

[0067] Digoxigenin 3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:5. The reaction has been stopped after 60 minutes by adding L-lysine. The surplus of the labeling reagent was removed by dialysis against 200 mM sodium acetate buffer, pH 5.0. The antibody solution was transferred to a buffer with 25 mM K—PO.sub.4 and 150 mM NaCl, pH 7.2, by chromatography on a column with Sephadex® G25.

Example 6

[0068] Digoxigenylation of mAB IL-6R with Digoxigenin-X-Hydrazide

[0069] mAB IL-6R has been dialyzed against 100 mM sodium acetate buffer, pH 5.5. Afterwards the solution was adjusted to a protein concentration of 20 mg/ml. Sodium periodate was dissolved in 100 mM sodium acetate buffer, pH 5.5, and was added to the antibody solution to a final concentration of 10 mM. The reaction has been stopped after 30 minutes by chromatography on a Sephadex® G25 column equilibrated with 100 mM sodium acetate buffer, pH 5.5. The antibody solution was adjusted to a protein concentration of about 5 mg/ml. Digoxigenin-X-hydrazide was dissolved in DMSO and added to the antibody solution in a molar ratio of 1:50. After 120 minutes the reaction has been stopped by adding sodium borohydride to a final concentration of 15 mM. After 30 minutes the antibody solution was dialyzed against 25 mM K—PO.sub.4 supplemented with 150 mM NaCl, pH 7.2

Example 7

Bridging ELISA for Detection of Antibodies Against mAB IL-6R

[0070] Biotinylated mAB IL-6R has been conjugated to (bound onto) the wells of a streptavidin-coated microtiterplate (SA-MTP) in the first step. Not conjugated (unbound) antibody was removed by washing with universal buffer. Afterwards the samples and the reference standards (polyclonal rabbit anti-mAB IL-6R antibody spiked in 5% human serum) have been incubated in the wells. Anti-mAB IL-6R antibody bound to the immobilized mAB IL-6R. After having washed away unbound substances the bound anti-mAB IL-6R antibody was detected with digoxigenylated mAB IL-6R followed by incubation with a horse-radish peroxidase labeled anti-digoxigenin-antibody (see FIG. 1). The antibody-enzyme conjugate catalyzed the color reaction of the ABTS® substrate. The signal was measured by ELISA reader at 405 nm (reference wavelength: 490 nm). Absorbance values of each serum sample were determined in triplicate.

[0071] Five different variants of the bridging ELISA have been performed: [0072] variant 1 using conjugates of example 1 and 4 [0073] variant 2 using conjugates of example 2 and 5 [0074] variant 3 using conjugates of example 3 and 6 [0075] variant 4 using mixed conjugates of examples 1 and 3 and mixed conjugates of examples 4 and 6 [0076] variant 5 using mixed conjugates of examples 1-3 and mixed conjugates of examples 4-6.

[0077] Reference standard signals and curves obtained in the different ELISA variants are shown in Table 1 and FIGS. 2-6.

TABLE-US-00001 TABLE 1 Reference standard signals in the different ELISA variants. ref. conc. Signal Signal Signal Signal Signal [ng/ml] variant 1 variant 2 variant 3 variant 4 variant 5 0.00 0.031 0.033 0.048 0.039 0.039 0.78 0.064 0.066 0.077 0.072 0.068 1.56 0.097 0.102 0.106 0.102 0.097 3.13 0.162 0.168 0.166 0.164 0.160 6.25 0.288 0.295 0.287 0.287 0.279 12.50 0.545 0.567 0.538 0.544 0.529 25.00 1.055 1.069 1.048 1.065 1.035 50.00 2.092 2.087 2.140 2.085 2.030

[0078] Sample analysis with the different standard curves is shown in Table 2.

TABLE-US-00002 TABLE 2 Serum Sample analysis. concentration pAB anti-mAB IL-6R equivalents [ng/ml] Sample-Id variant 1 variant 2 variant 3 variant 4 variant 5 F262760-16 54.46 53.78 88.02 76.26 68.50 F825050-26 10.21 11.51 4.57 7.88 9.71 F963840-22 21.92 23.04 23.53 23.63 24.82 E597480-16 76.02 76.29 55.18 N/A N/A

[0079] As Table 1 shows, all conjugates can be used for detection of anti-mAB IL-6R antibodies. Using the same rabbit polyclonal anti-mAB IL-6R antibody the reference standard curves for all assay variants are very similar (FIGS. 2-6).

Example 8

Bridging ELISA for Detection of Anti-mAB IGF-1R Antibodies Using Streptavidin/Biotin Interaction for Immobilization at Solid Phase

[0080] Biotinylated antibody against IGF-1R (mAB IGF-1R, drug antibody) has been conjugated to (bound onto) the wells of a streptavidin-coated microtiterplate (SA-MTP) in the first step. Unconjugated (unbound) antibody was removed by washing with universal buffer. Afterwards the samples and the reference standards (polyclonal rabbit anti-mAB IGF-1R antibody spiked in 5% human serum) have been incubated in the wells. Anti-mAB IGF-1R antibody bound to the immobilized mAB IGF-1R. After having washed away unbound substances the bound anti-mAB IGF-1R antibody has been detected with digoxigenylated mAB IGF-1R followed by incubation with a horse-radish peroxidase labeled anti-digoxigenin-antibody. The antibody-enzyme conjugate catalyzed the color reaction of the ABTS® substrate. The signal was measured by ELISA reader at 405 nm wavelength (reference wavelength: 490 nm). Absorbance values of each serum sample were determined in triplicates.

[0081] Three different variants of the bridging ELISA have been performed: [0082] variant 1 using conjugates made according to example 1 and 4 [0083] variant 2 using conjugates made according to example 2 and 5 [0084] variant 3 using mixed conjugates made according to examples 1 and 2 and mixed conjugates made according to examples 4 and 5.

[0085] All reagent variants of biotinylated and digoxygenylated mAB IGF-1R have been synthesized as described above for mAB IL-6R (examples 1, 2, 4 and 5).

[0086] Reference standard signals obtained in the different ELISA variants are shown in Table 3.

TABLE-US-00003 TABLE 3 Reference standard signals in the different ELISA variants. ref. conc. Signal Signal Signal [ng/ml] variant 1 variant 2 variant 3 0.00 0.126 0.056 0.110 0.31 0.161 0.092 0.147 0.63 0.205 0.131 0.191 1.25 0.285 0.203 0.267 2.50 0.440 0.348 0.425 5.00 0.772 0.660 0.744 10.00 1.349 1.223 1.321 20.00 2.113 2.060 2.133

[0087] Sample analysis with the different standard curves is shown in Table 4.

TABLE-US-00004 TABLE 4 Serum sample analysis. concentration pAB anti-mAB IGF-1R equivalents [ng/ml] Sample-Id variant 1 variant 2 variant 3 840 h_ female 2.04 10.64 7.27 1008 h_ female 11.38 24.05 14.05 504 h_ male 51.17 67.76 60.49

[0088] Table 3 shows that all conjugates can be used for detection of anti-mAB IGF-1R antibodies. Using the same rabbit polyclonal anti-mAB IGF-1R antibody the reference standard values for all assay variants are very similar (Table 3).

Example 9

Bridging-EISA for Detection of Anti-mAB IGF-1R Antibodies Using Passive Adsorption for Conjugation (Immobilization) at a Solid Phase

[0089] A microtiter plate (MTP) (Maxisorb®, Nunc) has been coated with mAB IGF-1R in carbonate buffer (pH 9.6), at room temperature (RT) for 1 hour. After washing three times with PBS-Tween®20, all wells of the MTPs were blocked with PBS/3% (w/v) BSA (bovine serum albumine) at room temperature for 1 hour and then washed again. Afterwards the samples and the reference standards (polyclonal rabbit anti-mAB IGF-1R antibody spiked in 5% human serum) have been incubated. Anti-mAB IGF-1R antibody bound to the immobilized mAB IGF-1R. After having washed away unbound substances the bound anti-mAB IGF-1R antibody has been detected with digoxigenylated mAB IGF-AR followed by incubation with a horse-radish peroxidase labeled anti-digoxigenin-antibody. The antibody-enzyme conjugate catalyzed the color reaction of the ABTS® substrate. The signal has been measured by ELISA reader at 405 nm wavelength (reference wavelength: 490 nm). Optical densities of each serum sample have been determined in triplicates.

[0090] Three different variants of the bridging ELISA have been performed: [0091] variant 1 using conjugates made according to example 4 [0092] variant 2 using conjugates made according to example 5 [0093] variant 3 using mixed conjugates made according to examples 4 and 5.

[0094] All reagent variants of digoxigenylated mAB IGF-1R have been synthesized as described above for mAB IL-6R (examples 4 and 5). Reference standard signals in the different ELISA variants are shown in Table 5 and FIG. 7.

TABLE-US-00005 TABLE 5 Reference standard signals. ref. conc. Signal Signal Signal [ng/ml] variant 1 variant 2 variant 3 0.00 0.124 0.113 0.113 8.00 0.163 0.138 0.158 16.00 0.238 0.166 0.189 32.00 0.305 0.289 0.337 64.00 0.598 0.510 0.558 128.00 1.023 0.990 1.032 256.00 2.097 1.864 1.990

[0095] As Table 5 shows the bridging assay according to the invention using passive adsorption for conjugation (immobilization) of mAB IGF-1R on the solid phase can be conducted for detection of anti-mAB IGF-1R antibodies. Using the same rabbit polyclonal anti-mAB IGF-1R antibody the reference standard values for all three assay variants are very similar (Table 5).