SPECIFIC DETECTION OF RAT ANTIBODIES IN MOUSE SERUM

Abstract

One aspect as reported herein is a method for detecting a rat antibody in a serum or plasma sample (obtained) from a mouse comprising the steps of a) providing the sample to be analyzed, b) incubating said serum or plasma sample with an antibody that specifically binds to rat IgG and that does not specifically bind to mouse IgG, wherein the antibody is i) a mixture of an antibody binding to rat kappa light chain and an antibody binding to rat lambda light chain, or ii) a mixture of an antibody binding to rat IgG1 with an avidity of 4.1×10.sup.10 M.sup.−1 or more, an antibody binding to rat IgG2a with an avidity of 8.6×10.sup.9 M.sup.−1 or more, an antibody binding to rat IgG2b with an avidity of 6.4×10.sup.10 M.sup.−1or more and an antibody binding to rat IgG2c with an avidity of 9.5×10.sup.10 M.sup.−1 or more, c) optionally incubating said sample with a reagent appropriate for the selective detection of total, active or antigen-bound rat antibody, and d) correlating the complex formed in (b) or (c) to the concentration of the rat antibody in the sample.

Claims

1. Method for detecting a rat antibody in a serum or plasma sample obtained from a mouse comprising the steps of a) providing the sample to be analyzed, b) incubating said serum or plasma sample with an antibody that specifically binds to rat IgG and that does not specifically bind to mouse IgG, wherein the antibody is i) a mixture of a monoclonal antibody binding to rat kappa light chain and a monoclonal antibody binding to rat lambda light chain, or ii) a mixture of a monoclonal antibody binding to rat IgG1 with an avidity of 4.1×10.sup.10 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2a with an avidity of 8.6×10.sup.9 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2b with an avidity of 6.4×10.sup.10 M.sup.−1or more and a monoclonal antibody binding to rat IgG2c with an avidity of 9.5×10.sup.10 M.sup.−1 or more, and c) correlating the complex formed in (b) to the concentration of the rat antibody in the sample.

2. Method for detecting a rat antibody in a serum or plasma sample obtained from a mouse comprising the steps of a) incubating said serum or plasma sample with a capture antibody conjugated to a solid phase and thereby forming a capture antibody-rat antibody complex, b) incubating the complex formed in (a) with a tracer antibody and thereby correlating the complex formed in (a) to the presence or concentration of said rat antibody, wherein the capture antibody specifically binds to rat IgG and that does not specifically bind to mouse IgG and is i) a mixture of a monoclonal antibody binding to rat kappa light chain and a monoclonal antibody binding to rat lambda light chain, or ii) a mixture of a monoclonal antibody binding to rat IgG1 with an avidity of 4.1×10.sup.10 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2a with an avidity of 8.6×10.sup.9 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2b with an avidity of 6.4×10.sup.10 M.sup.−1or more and a monoclonal antibody binding to rat IgG2c with an avidity of 9.5×10.sup.10 M.sup.−1 or more.

3. Method for detecting a rat antibody in a serum or plasma sample obtained from a mouse comprising the steps of a) incubating said serum or plasma sample with a capture antibody conjugated to a solid phase and thereby forming a capture antibody-rat antibody complex, b) incubating the complex formed in (a) with a tracer antibody and thereby correlating the complex formed in (a) to the presence or concentration of said rat antibody, wherein the tracer antibody specifically binds to rat IgG and that does not specifically bind to mouse IgG and is i) a mixture of a monoclonal antibody binding to rat kappa light chain and an antibody binding to rat lambda light chain, or ii) a mixture of a monoclonal antibody binding to rat IgG1 with an avidity of 4.1×10.sup.10 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2a with an avidity of 8.6×10.sup.9 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2b with an avidity of 6.4×10.sup.10 M.sup.−1or more and a monoclonal antibody binding to rat IgG2c with an avidity of 9.5×10.sup.10 M.sup.−1 or more.

4. The method according to claim 2, characterized in that the tracer antibody and the capture antibody are independently of each other i) a mixture of a monoclonal antibody binding to rat kappa light chain and a monoclonal antibody binding to rat lambda light chain, or ii) a mixture of a monoclonal antibody binding to rat IgG1 with an avidity of 4.1×10.sup.10 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2a with an avidity of 8.6×10.sup.9 M.sup.−1 or more, a monoclonal antibody binding to rat IgG2b with an avidity of 6.4×10.sup.10 M.sup.−1 or more and a monoclonal antibody binding to rat IgG2c with an avidity of 9.5×10.sup.10 M.sup.−1 or more.

5. The method according to any one of claims 1 to 3, characterized in that the method is an antigen bridging immunoassay.

6. The method according to any one of claims 1 to 3, characterized in that the method is a sandwich immunoassay.

7. The method according to claim 2 or 3, characterized in that the capture antibody is conjugated to biotin and immobilization is performed via immobilized avidin or streptavidin.

8. The method according to claim 2 or 3, characterized in that the tracer antibody is conjugated to digoxygenin and linking to the detectable label is performed via an antibody against digoxygenin.

9. The method according to any one of claims 1 to 3, characterized in that said rat antibody is a Fab.

Description

DESCRIPTION OF THE FIGURES

[0116] FIG. 1 Reactivity of different Anti-Rat IgG antibodies with serum of several animal species; left bar: MAR(K+L), right bar: Pab anti-rat-HRP (Cell Signaling Technology).

[0117] FIG. 2 Schematic assay setup of an exemplary SA-MTP ELISA for quantification of total therapeutic antibody using the method as reported herein.

[0118] FIG. 3 Comparison of MARGCOC-1 and MAR(K+L) as an example of a mixture of monoclonal antibodies with a polyclonal antibody for quantification of total therapeutic antibody; upper curve (triangles): Pab anti-rat IgG-HRP (Cell Signaling Technology); lower curve (diamonds): MAR(K+L).

EXAMPLE 1

[0119] Reactivity of Different Anti-Rat IgG Antibodies with Serum of Several Animal Species in an MTP-ELISA

[0120] A microtiter plate (MTP) (Maxisorb®, Nunc) was coated with serum of different species diluted to 10% in phosphate buffered saline at room temperature (RT) for 1 hour, respectively. Serum from rat, mouse, rabbit, cynomolgus, hamster and guineapig was used. After washing 3 times with PBS-Tween®20, all wells of the MTPs were blocked with PBS/3% BSA at room temperature for 1 hour. Then the wells of the MTPs were incubated (1 h; RT) with different anti-rat IgG antibodies (digoxigenin or horseradish peroxidase conjugates (HRP) (POD)). The anti-Rat antibodies were used as recommended by the corresponding manufacturer.

[0121] Wells were washed three times as above. Wells incubated with POD-conjugates were directly processed for enzymatic reaction/detection of bound anti-rat immunoglobulin. The other wells were incubated (1 h; RT) as appropriate with anti-DIG conjugates (all reagents from Roche Diagnostics, Germany) followed by a washing step. The POD comprised in the POD-conjugates catalyzes the color reaction of ABTS substrate. The signal was measured by an ELISA reader at a wavelength of 405 nm (reference wavelength: 490 nm) (see FIG. 1). For every anti-rat IgG antibody the ratios of the signal against rat antibodies to the signal of the other sera were calculated. These values were used for evaluation of the specificity of the anti-Rat IgG antibodies. A high ratio translates to a strong reactivity with rat immunoglobulin and at the same time to a low (cross-) reactivity with immunoglobulin from other species (see table below).

TABLE-US-00001 TABLE MAR(K + L)-DIG pAb anti-rat-HRP - Signal Signal Ratio Ratio Signal Rat/other Signal Rat/other [OD405 nm] species [OD405 nm] species Rat Serum 3.074 1.0 2.292 1.0 Mouse Serum 0.061 50.8 0.261 8.8 Rabbit Serum 0.062 50.0 0.099 23.3 Hamster Serum 0.055 56.4 0.470 4.9 Guineapig Serum 0.094 32.9 0.266 8.6 Cynomolgus Serum 0.074 41.8 0.279 8.2

EXAMPLE 2

Use of Mixtures of Monoclonal Antibodies as Capture and Tracer Antibodies for Quantification of Total Therapeutic Antibody

[0122] Biotinylated MARGCOC-1 (as example of a mixture comprising 4 monoclonal antibodies binding to different epitopes) was bound to streptavidin-coated microtiter plates (SA-MTP) in the first step. The excess of unbound antibody was removed by washing. Then, samples/standards, e.g. Rat anti-CD47 antibody spiked in Mouse serum, were added and incubated for 1 hour. After washing, the wells were incubated with digoxigenylated MAR(K+L) (as example of a mixture comprising 2 monoclonal antibodies binding to different epitopes) or HRP labeled pAb anti-Rat-IgG (Cell Signaling Technology). After washing the bound digoxigenylated MAR(K+L) was detected with an anti-digoxigenin antibody HRP conjugate. The HRP of the antibody-enzyme conjugates catalyzes the color reaction of ABTS substrate. The signal is measured by ELISA reader at 405 nm wavelength (reference wavelength: 490 nm) (see FIG. 3). Absorbance values of each serum sample were determined in triplicates.

EXAMPLE 3

Use of the Same Mixture of Monoclonal Antibodies as Capture and Tracer Antibodies for Quantification of Total Therapeutic Antibody

[0123] A microtiter plate (MTP) (Maxisorb®, Nunc) was coated with 10 μg/mL of MAR(K+L) and Pab anti-rat-IgG (Molecular Probes #A10536) in the first step. The excess of unbound antibody was removed by washing. Then, samples/standards, e.g. mAb anti-CD47-rat-IgG spiked in mouse serum and also diluted in buffer were added to and incubated for 1 hour. After washing, the wells were incubated with digoxigenylated MAR(K+L) or HRP labeled Pab anti-rat-IgG (Molecular Probes #A10549). After washing, the bound digoxigenylated MAR(K+L) was detected with an anti-digoxigenin-antibody HRP conjugate. The other detection antibody already had the HRP label, so no second detection antibody was needed prior to Substrate incubation. The HRP of the antibody-enzyme conjugates catalyzes the color reaction of ABTS substrate. The signal is measured by ELISA reader at 405 nm wavelength (reference wavelength: 490 nm). Absorbance values of each serum sample were determined in triplicates.